INTRODUCTION

Welcome to this course on VGO 411 Veterinary Gynaecology! This course has been
prepared with the objective of providing the veterinary student a concised and practical
information on diagnostic and therapeutic techniques in large and small animal
reproduction.

The emphasis in this course is directed mainly towards diagnosis and therefore the
approach is clinical, while a more detailed review of the anatomy, histology, physiology
and pathology has been kept in breif which can be obtained from other courses. While
all attempts have been made to present information documented by various text books
in a concised manner supplemented with visual aids, the learner has to understand that
this content is designed not to substitute for but to supplement the recommended
textbooks in theriogenology. The list of additional reading material given at the end of
this course will enable the learner to have access to a more detailed information on each
topic covered.
Finally, as theriogenologists our ultimate aim is to protect animal health and relieve
animal suffering by learning normal reproductive physiology, understanding
pathogenesis and treatment of reproductive disease, promoting sound reproduction in
some and preventing reproduction safely in others. We believe that every young one
born should have the best opportunity for a good life, and so let us strive to be a part of
profession that works to those ends.

MODULE-1
CLINICAL EVALUATION AND ABNORMALITIES OF
REPRODUCTIVE TRACT IN DOMESTIC ANIMALS
A thorough examination of the female and her reproductive tract should be done before
purchasing for breeding purpose or prior to each breeding season. It is essentially being
carried out for the following reasons

 Diagnosis of pregnancy
 Estimation of the gestational age
 Characterization of reproductive physiological and pathologic status
 Allows the clinician to predict important events to come such as
o estrus
o ovulation
o parturition
o abortion
 Allows for a rational approach to therapy
 Allows for establishing a prognosis of conditions of the uterus, uterine tubes,
ovaries and supporting structures

CLINICAL EVALUATION OF COWS AND HEIFERS

HISTORY

History taking can be done simultaneously while the animal is being examined. The
important issues that need to be addressed include the following:

 Parity (virgin heifer, pregnant heifer, uniparous or multiparous cow).

 Age (including age at first calving).
 Cyclic history (normal or abnormal cycle lengths, anestrus, nymphomania).
 Calving dates and comments (dystocia, twins, retained placenta, surgical or
mechanical intervention, viability of calf).
 Breeding dates and methods (artificial insemination or natural service, estrus
detection methods and personnel, semen supplier and quality, previous record of
bull fertility, including examination for venereal disease).
 Previous treatments (drugs, dosages and routes, treatment intervals, clinical
outcome, drug withdrawal disease).
 Nutritional program (periparturient supplementation of beef cows, dry-period
feeding of dairy cows, body conditions of cows at calving, milk production levels).

PHYSICAL EXAMINATION

Visual Inspection

Visual inspection gives valuable information about the individual animal and will be an
aid to genital examination. Visual inspection involves the following:

 General conformation
 Conformation of the external genitalia
  Vulvar discharges
 Condition of the mammary gland
 General behavior of the animal

General Conformation

 Cows with a masculine appearance of the head and shoulder region may be
suffering from Cystic ovarian degeneration Freemartin heifers may exhibit steer-
like appearance.

Conformation of the External Genitalia

Physiological alterations

 The vulval labia are normally covered with soft, thin skin and are symmetrical
and closely opposed to ensure closure of the vestibule and vagina
 There is high tonicity of the pelvic diaphragm as well as the vulva in a non-
pregnant or early pregnant cow manifested by firmness of the diaphragm and
relatively small and wrinkled appearance of the vulval lips
 During the last trimester of pregnancy these structures undergo gradual but
continuous relaxation.
 The relaxation becomes marked and the vulva nearly doubles in size during the
last 10 to 14 days prior to parturition and this extreme relaxation of the pelvic
ligaments causes the tail head to become elevated
 Within 2 weeks following parturition the vulva returns to normal size

Pathological alterations

Pathological alterations involve the following

 Extreme relaxation of the pelvic diaphragm, enlargement of the vulva and

elevation of the tail head in Cystic ovarian degeneration
 Relaxation of the vulval lips alone in cows with long standing cases of metritis
 Extreme swelling of the vulva associated with edema, but with increased tension,
is found as the first sign of Infectious Pustular Vulvovaginitis (IPV)
 Edema of vulva in a cow with Linearly arranged lymph follicles
cystic follicular degeneration in infectious pustular
vulvovaginitis

Discharges from the Vulva

Discharges observed in normal animals

 During estrus, the vulva may appear edematous with presence of characteristic
clear, elastic mucus that hangs from the ventral commissure. In many cases the
mucus may be present adhering to the tail
 Blood stained mucus may be present in some heifers and cows during the first
few days after the end of estrus referred to as metestrual bleeding
 Reddish grey discharge, consisting of blood elements and debris of endometrium
referred to as lochia, is observed in post parturient cows and increases in amount
reaching peak quantities around the third week of the post partum period

Discharges from the Vulva

Discharges observed in normal animals

 During estrus, the vulva may appear edematous with presence of characteristic
clear, elastic mucus that hangs from the ventral commissure. In many cases the
mucus may be present adhering to the tail
 Blood stained mucus may be present in some heifers and cows during the first
few days after the end of estrus referred to as metestrual bleeding
 Reddish grey discharge, consisting of blood elements and debris of endometrium
referred to as lochia, is observed in post parturient cows and increases in amount
reaching peak quantities around the third week of the post partum period

Discharges associated with pathological conditions

 The presence of a mucopurulent (clear mucus discharge with pus flakes) to

purulent discharge indicates
o inflammation of any segment of the reproductive tract
o infection of the urinary system
 It should be noted that apparent purulent discharge may be absent in cases of
pyometra while a heavy purulent discharge may be present in animals with
normally progressing pregnancy
 Greyish discharge not containing apparent pus has been observed in certain cases
of cystic ovaries

Appearance of the Udder

 Edema and enlargement of the mammary gland are normally found in the pre
parturient and post parturient period
  Cows that have failed to conceive over long periods may have a small shrunken
vulva

General Behaviour

 The general behavior of the animal can be observed only when the animal is not
confined
 Signs of estrus, hyperestrus, bellowing and pawing can be observed

VAGINAL EXAMINATION

 Supplemental information obtained by vaginal examination helps to refine the

tentative diagnosis made after rectal examination of postpartum cows. However,
it is seldom employed in the cow.

Manual Examination

 Manual examination of the vagina and cervix of the early postpartum cow will aid
in the diagnosis of the following conditions
o Vaginal/cervical trauma
o Retention of fetal membranes
o Patency of the cervical canal
 The cow’s vulva and perineum should be carefully washed with a mild
disinfectant soap, and a lubricated disposable plastic sleeve should be worn by
the examiner to perform vaginal examinations

Vaginoscopic Examination

 After washing the vulva and perineum, the speculum is inserted first in a dorsal-
cranial direction until the ischial symphysis has been passed, then in a cranial
direction
 Slight resistance will be noticed at the vestibulovaginal junction which can be
easily overcome by gentle pressure
 With a torch light, the vaginal vault should be examined for
o the location of the cervix,
o cervical/vaginal color and secretions,
o cervical anomalies,
o trauma and discharges
 VAGINOSCOPIC FINDINGS

 Normal findings

Findings Condition
Large quantities of stringy, water-clear mucus Proestrus or estrus
phase
Bloody discharge through cervix into a mucus pool in the Metestrus phase
anterior vagina
A pale mucosa with scant amounts of sticky mucus Diestrus phase

 Pathological findings

Findings
Pus in the external os of the cervix or
on the floor of the anterior vagina
Pool of urine mixed with mucus in
the anterior vagina
Presence of papules, pustules or
ulcers in the vagina and vestibule
Condition
Endometritis
Urovagina. May temporarily result in an
irritated, hyperemic vaginal and cervical mucosa
Infectious Pustular Vulvovaginitis

RECTAL EXAMINATION

 At present, the most cost-effective and accurate method of examination of the

reproductive tract is per rectal palpation of the cervix, uterus, ovaries and
supporting structures. The technique of examination of reproductive tract by
rectal palpation has been dealt with in detail under the practicals.
 Rectal examination involves
o Examination for pregnancy
o Examination for non pregnancy

EXAMINATION FOR PREGNANCY

 Pregnancy diagnosis is based on detection of the physiological changes of the

genital organs associated with pregnancy.
 The uterus is the organ mainly involved and the positive signs of pregnancy
include
o Palpation of amniotic vesicle
o Palpation of fetal membrane slip
o Palpation of placentomes
o Palpation of fetus

 NORMAL NON-PREGNANT REPRODUCTIVE TRACTThe size, muscular

tone and contents of the uterus should be assessed. This can be done
 simultaneously with the “membrane slip” for pregnancy determination.
Commonly used terms for characterizing uterine tone include the following:
o Estrus tone: a turgid, contracted uterus that is often curled into a rather
tight configuration
o Diestrus (“normal”): a relaxed muscular uterus
o Edematous: a somewhat turgid uterus but without muscular
contraction; may be palpable for a few days after estrus
o Flaccid: a limp, soft, usually thin-walled uterus that does not contract in
response to palpation
o Thickened (“doughy”): a pathologic description, indicating thickening
of the endometrium and possibly the myometrium as well
o Fluctuant: uterus in which there is intra luminal fluid

Post Parturient Involution

 Pregnancy and involution represent the only two clinically appreciable

physiological alterations of size. In pregnancy, the size progressively increases
while during the involution period, the size of the uterus regresses and returns to
the non pregnant state

ABNORMALITIES INVOLVING UTERUS

Palpation for Uterine Disorders

 During routine post partum examinations in cases in which pregnancy diagnosis

is negative or in examination of “problem cows,” the reproductive tract should be
examined for palpable abnormalities. The essential questions for the examiner to
answer are the following:
o Is the uterus symmetrical and approximately the size and tone of the non
gravid tract?
o Is there a corpus luteum or an ovarian follicle associated with corpus
luteum or an ovarian follicle associated with increased uterine tone that is
indicative of cyclicity?
o Are there any palpable lesions of the reproductive tract?

Uterine Inflammation

 It is generally possible to diagnose moderate to severe endometritis, acute

metritis or pyometra by rectal examination.

Adhesions
  On rectal examination it would be possible to detect the presence of uterine or
utero-ovarian adhesions that would interfere with normal retraction of some part
of the tract.
 Commonly, the uterus will adhere to the rumen, the omentum or the ovarian
bursae.
 Prognosis depends on the severity of adhesions and the degree of involvement of
the oviducts and fimbriae.

Abscesses

 Uterine abscesses can occur

o following dystocia
o as a sequel to the improper use of an intrauterine pipette.
 Location and size of the abscess varies depending on the degree of mechanical
insult in the former and the degree of endometrial/ myometrial insult in the
latter.
 Abscess is most often located in the area of the uterine body and is approximately
the size of a golf ball and in either case the abscess is firm and raised and may
cause discomfort when palpated.
 Adhesions of the abscessed portion of the uterus to other abdominal or pelvic
organs are common.

Tumors

 Tumors of the bovine uterus are not common but when seen occur
predominantly in older cows. Uterine lymphosarcoma, leiomyoma and rarely
carcinoma have been diagnosed.
 Lymphosarcoma may be detectable as multiple smooth nodular enlargements of
the uterine wall, often with concurrent enlargement of the deep inguinal and iliac
lymph nodes.

Fetal Remnants

 Occasionally, a fragment of an autolyzed term fetus may remain in the uterine

lumen following parturition
 Can be detected as a moveable firm mass in the lumen of an involuting uterus. A
foul vaginal discharge will often be noted.
 Cows or heifers that do not calve at the expected time following a positive
pregnancy diagnosis may have either a mummified or macerated fetus.
 In cases of fetal maceration, a distended uterus with palpably crepitant fetal
bones can be felt. An ipsilateral CL may be present, as well as a fetid vaginal
discharge.
 The prognosis for future fertility of such cows is grave due to severe damage to
the endometrium .

Freemartinism
  In Freemartinism the cervix is rudimentary while the uterus is underdeveloped
and characterized by the presence of two thin walled, very narrow tubes
occupying the sites of the normal horns, suspended in ligamentous sheets
resembling the broad ligaments
 Lateral exploration along the edge of the broad ligaments leads to location of
barely perceptible thickening indicating the rudimentary ovary
 Failure to locate the normal cervix during the course of rectal examination should
always be followed by a thorough exploration for signs of freemartinism.

White Heifer Disease

 Also known as segmental aplasia of the Mullerian duct

 The extent of aplasia and the number of the missing segments is variable
 Secretion of the normal segments becomes entrapped between the missing
segments or anterior to the missing part, resulting in marked distension of the
normal segment associated with thinning of the wall. Persistence of the “hymen,”
one of the forms of white heifer disease, results in accumulation of secretion in
the anterior part of the vagina, with consequent dilation which elicits tenesmus.

Uterus Unicornis

 This relatively rare abnormality has been found in practically all breeds.
 The horn that is present is functionally normal and conception is possible only
during an estrus when the follicle develops and ovulates occurs in the ovary on
the side of the normal horn
 Reduced fertility can be anticipated.

Cervix Duplex or Double Cervix

 This also is a rare abnormality. The presence of two cervices, resulting in two
single tube genital tracts anterior to the vagina, might cause temporary confusion
in the examiner’s mind. Diagnosis, however, is easy .
 Fertility of the affected animal does not have to be impaired if natural breeding is
employed. Artificial insemination might result in failure to conceive unless the
ovary carrying the follicle ready to ovulate is detected and the semen is deposited
in the cervix of the same side.
 Bilateral insemination in the double cervices might also be recommended.

Pyometra

 Characerised by accumulation of pus in the uterus and may occur due to lack of
sufficient relaxation of the cervix or to the presence of cervicitis combined with
atony of the uterus and consequent lack of expulsive force.
 The amount of exudates varies from 25cc., which is barely felt upon examination,
to several liters.
 The uterine walls are thinner than in the non-pregnant uterus, but thicker than
the pregnant uterus
  The condition has to be differentially diagnosed from pregnancy

Mucometra or Hydrometra

 Both mucometra and hydrometra are similar except for the degree of hydration
of mucin present in the uterus which may vary from a watery fluid to a semisolid
mass.
 Condition is observed in heifers or cows following
o arrest in the development of mullerian duct system.
o persistence of hymen
o prolonged hormonal stimulation with estrogens or progestogens
 Cows with mucometra or hydrometra due to defects of genital tract are sterile.
 Cows with pyometra do not cycle, while cows with a hydrometra do.

Chronic Nonproductive Metritis

 This condition is often referred to as chronic endometritis

 On rectal palpation
o the uterus lacks tone,
o has a thin wall, especially in the intercaruncular spaces,
o caruncles, therefore, appear more prominent, and the endometrial surface
feels wavy and uneven.

 Diagnosis is by vaginal examination and histological examination of biopsies.

ABNORMALITIES INVOLVING THE OVARIES

Smooth Ovaries

 Ovaries are smooth

 Repeated palpation confirms the presence of smooth ovaries. Systemic or local
causes should be investigated.

The detection of smooth ovaries at a single examination in cycling cows, especially

during the first few days following ovulation when the developing CL is not palpable is
perfectly normal.

Ovarian Cysts

 Ovarian cysts are fluid-filled structures greater than 2.5 cm in diameter.

Should be differentiated from parovarian cysts, which do not involve the ovary but
rather involve remnants of the mesonephric or paramesonephric duct systems.

Ovarian Hypoplasia
  Ovarian hypoplasia is found in all breeds and may be bilateral or unilateral.
 Bilateral hypoplasia is found in heifers only and is always associated with sterility
.
 Cows and heifers which are affected unilaterally might reproduce relatively
normally.
 The degree of hypoplasia varies and the affected ovary might be recognized as a
barely distinguishable thickening of the mesovarium. In other cases, the ovary
might be slightly larger.
 Whenever the dimensions of the ovary are found to be less than 2 cm x 0.5 cm x
0.5 cm this disease should be suspected. The affected gonads are are hard and
static.
 Functional structures such as follicles or corpus luteum are not present.
 In cases of doubt, re-examination should be recommended, especially for
differentiation from ovarian atrophy.
 Hypoplasia of ovaries has been found to be hereditary in nature, and it is very
important to detect the unilateral cases which might reproduce and transmit the
disease to offspring.

Underdevelopment of Ovaries in Heifers

 Usually bilateral and is found primarily in poorly fed and managed heifers
 Most commonly heifers of the same age are affected
 The ovaries are small and static, consistency varies from flaccid to fibrotic while
there is atrophy of the uterine wall
 It is difficult to differentiate this condition from ovarian hypoplasia based on
single examination. Re-examination, preferably after correction of feeding
practices, might be necessary

Ovaritis or Oophoritis

 Inflamation or infection of the ovary also known as ovaritis or oophoritis occurs

o secondary to trauma
o to infection from the uterus that passes through the oviducts
o by extension of infection through the uterine walls
 Associated with marked enlargement of the ovary.
 In acute ovaritis, enlargement is due to edema.
 Diagnosis of chronic ovaritis is based on enlarged fibrotic ovaries and presence of
organized adhesions to the surrounding structures, primarily to the mesosalpinx.

Miscellaneous Ovarian Conditions

 Include abscesses and tumors. Both of these conditions result in a greatly

enlarged, usually firm ovary and may be associated with bursal and uterine
adhesions
 Abscessed ovaries may have a softened area within the firm mass and may cause
pain when palpated
 Unaffected ovary may function normally so that cyclic structures may be palpated
 ABNORMALITIES OF THE OVIDUCT

 Only those associated with enlargement of the oviduct are detectable clinically.

Hydrosalpinx

 Hydrosalphinx is the local or general enlargement of the oviduct

 Manifests itself in the form of enlarged segments of varying length
 Local enlargements may resemble ovaries in size. When the entire oviduct is
involved, it appears as a conglomerate of a fluctuating tube
 The width of the enlarged oviduct varies from case to case and may reach 2 cm. in
diameter
 Impossible to pathologicaly differentiate serosalpinx, pyosalpinx and
hemosalpinx by clinical examination
 Adhesions may be present between the loops of the enlarged oviducts

Pyosalphinx

 Follows severe uterine infection and is less commonly reported than

hydrosalphinx
 Associated with severe adhesions of the mesosalphinx and mesovarium.
 May also follow
o removal of retained corpus luteum
o injection of large doses of estrogen

ABNORMALITIES INVOLVING THE MESOSALPHINX AND

OVARIAN BURSA

 Clinical differentiation between parasalpingitis, perisalpingitis and ovarian

bursitis is practically impossible.
 For clinical purposes, perisalpingitis appears to be the most correct term to
describe the inflammation and the consequent thickening and adhesion
formation involving mesosalpinx, mesovarium and salpinx. Other structures in
the area, such as the ovaries, the horns of the uterus and others, might also be
embedded in the adhesions.
 Very fine adhesions between the ovary and fimbria-the fringes of the edge of the
infundibulum-are present in numerous animals, especially immediately after
ovulation. These do not appear to interfere with the normal function of the
oviduct.

ALTERNATIVE METHODS OF EXAMINATION

Laparoscopy
  The reproductive tract can be directly visualized by laparoscopy/endoscopy

Ultrasonographic Examination

 The uterus and ovaries can be indirectly examined by ultrasonographic

techniques
 Real-time ultrasound, in which a two-dimensional “sonic picture” is generated
from echoes
 The use of ultrasonography to diagnose pregnancy, normal ovarian structures,
uterine and ovarian pathology are described in detail in practical exercise.
 CLINICAL EVALUATION OF MARES
 HISTORY

As a rule, there is less recorded history available about mares than cows, and the
information which might be obtained is less complete. If possible, information should be
acquired with regard to the following:

 Age of the animal

 Duration of the present ownership
 Previous pregnancies and foalings, if any
 Date of the last foaling and the rate of growth and development of the foal
 History of any infections
 The month of commencement of the breeding season as well as its length
 Intensity of estrus, the length of the estrus period and the length and regularity of
the estrous cycle
 The client should be asked about the method employed for observing heat

PHYSICAL EXAMINATION

Visual Examination

 Conditions that alter the general conformation and the external appearance of
the cow are simply not encountered in the mare. Changes in the appearance of
the “tailhead” are not as apparent in the mare.
 Relaxation of the pelvic diaphragm and vulva associated with gestation is
observed during the last 2 to 5 days of pregnancy, and then not in all animals.
 Copious discharge of estrus mucus is seldom observed in the mare. Thus,
information suggesting physiological events cannot be obtained by visual
inspection.
 Significant information, however, is gained from observing conformation changes
involving
o External genital organs
o Abnormal vaginal discharge
o Hoof and leg infirmities such as a rotated third phalanx following acute
laminitis that may make a mare reluctant to stand for breeding or may
make her unfit to carry a pregnancy to term
 o Pelvic injuries or abnormalities that may predispose a mare to dystocia
o Small stature
o Hirsutism associated with a pituitary tumor may be the cause of a mare’s
unseasonal anestrus
o Hypertrophic pulmonary osteopathy has been associated with certain
types of ovarian tumors in mares

Examination of Genitalia

 After completing the general physical examination, a detailed evaluation of the

reproductive organs should be undertaken with the tail wrapped in gauze and
tied out of the way
 Mammary glands should be examined and palpated for signs of mastitis,
abscessation, neoplasia or injury.
 Vulva should be examined for conformation, apposition, tone and evidence of
discharges.
o Mal apposition of the vulvar lips or poor vulvar conformation may lead to
pneumovagina and fecal contamination of the vaginal vault
o Examination of the vulvar area should continue with the examination of
the clitoral fossa and clitoris that harbors the contagious equine metritis
organism Hemophilus equigenitalis.
 The examination then continues with either rectal or vaginal palpation.

PHYSICAL EXAMINATION FINDINGS

Pneumovagina

 Pneumovagina or” windsucker” condition is the most frequent conformation

change observed in mares.
 In normal mares the long axis of the vulva occupies an almost vertical position. In
pneumovagina it forms an acute angle with the horizontal plane. In extreme cases
the vulva might be found in an almost horizontal position
 The vulvar lips are relaxed, resulting in partial exposure of the vestibulum and
clitoris. Scar tissue and consequent deformation of the vulva lips are frequently
observed in mares which have experienced dystocia and lacerations
 The anus is sunken and enhances the appearance of a more or less horizontally
directed vulva. The relaxed vulva and its position facilitate contamination of the
vagina with fecal material

The relaxation permits aspiration of air into the vagina and also into the uterus,
especially during estrus thus allowing saprophytic and pathogenic bacteria to gain
entrance into the genital tract. Treatment should be directed at correcting the cause of
pneumovagina, and concurrently treating the resulting acute endometritis. The former
can be done surgically by Caslick’s operation. Rectovaginal Fistula
  The trauma which results in rectovaginal fistula affects the perineum, vulva,
vestibule and rectum. Localized internal fistulas are rare.
 The lesions are easy to see, and the diagnosis of either type of fistula does not
present any difficulties. This abnormality leads to contamination and consequent
infection of the genital tract.

Abnormal Development of the Vulva

 The vulva might be too small when compared with general body size
 Asymmetry of the vulva and overlapping of vulvar lips are abnormalities that
result in improper closure of the vestibule and permit aspiration of air

Discharge from the Vulva

 During estrus the vulva is moist, but the copious discharge which is a sign of
estrus in the cow is not present
 The very viscid discharge present in practically all cows in advanced pregnancies
is absent in the mare
 Lochia, the grayish red discharge in the postparturient animal, appears in much
lesser amounts and only for a few days in the postparturient mare
 Postestrual hemorrhage is not seen in the mare. The appearance of an abnormal
discharge is, however, significant
 Abnormal discharges include haemorrhagic and purulent discharge

Haemorrhagic discharge

 Bloody discharge from the vulva is always serious. In the recently serviced mare,
it indicates service injuries
 Bloody discharge from a pregnant animal practically always indicates threatened
or completed abortion

Purulent discharge

 Purulent material in the vaginal discharge may be observed directly, or noticed as

crusts on the thighs and a loss of hair between the thighs if the exudates has been
discharging for a longer period of time.
 Indicates the presence of inflammation in the genital or in the urinary tract,
especially in the bladder.
 Determination of the site of inflammation requires performing a vaginal
examination and occasionally doing a rectal examination and urine analysis.


 Almost all animals with pneumovagina are infertile and show signs of genital
infection

VAGINAL EXAMINATION
  The vaginal speculum or vaginoscope is more frequently used for vaginal
examination in the mare than in the cow and is always indicated whenever an
abnormal discharge has been observed.

PREPARATION AND EXAMINATION TECHNIQUE

Preparation

 Vulva and perineum are thoroughly cleaned

 The tail is wrapped and tied out of the way
 The area is disinfected using a mild surgical scrub
 Caution is taken to prevent forcing fluid through the vulvar cleft into the vagina

Examination

 After drying the area the vulvar lips are separated and a sterile vaginal speculum,
either tubular or the three-pronged Caslick speculum, is introduced into the
vestibular area
 With the aid of a light, the speculum is used to examine
o the cervical os for color and tone as soon as possible, since changes occur
as cool air enters through the speculum
o the vaginal wall for color, evidence of congestion or inflammation, tumors,
lacerations and scars
o the vaginal floor for evidence of exudates or fluid accumulation or injury
o the dorsum of the vagina for evidence of injury or fistulation into the
rectum.
 Endometrial cultures can be obtained during speculum examination

VAGINAL EXAMINATION FINDINGS

Physiological Findings During Vaginal Examination

Estrous cycle

 Diestrum
o In the luteal phase vaginal mucosa is pale pink and rather dry.
o Speculum examination results in influx of air and ballooning of the vagina
thus favouring visibility. However, exposure to air causes the mucosa to
become congested and hence, the color of the mucosa should be noted
immediately after insertion of the speculum.
o Secretion is absent.
 Estrum
o Vaginal mucosa appears deep pink and glistening with a small amount of
clear secretion on the floor of the anterior vagina
 o The external os is relaxed and lies limp on the floor of the vagina but is,
however, extremely sensitive to touch and responds quickly by becoming
erect.
o The appearance of the cervix is greatly helpful in determining whether a
mare is in estrus or not. The erect cervical os during the luteal phase has
been described as a “rosebud,” whereas the relaxed external os during
estrus has been described as “wilted rose.”

Pregnancy

 The vaginal mucosa appears dull, anemic, rough and extremely dry, and insertion
of the speculum might be difficult.
 The external os is more or less relaxed and covered with a sticky, grayish
secretion.
 The stickly mucus sticks to the vaginoscope and to the vaginal mucosa and
resembles rubbery glue during manual examination of the vagina.
 These findings are encountered in diestrus mares also

Abnormalities Detected During Vaginal Examination

Persistent hymen

 Easily diagnosed during the process of insertion of the speculum

 Septum may be partial, and might be brushed aside during introduction of the
speculum
 If complete, it might lead to a condition resembling white heifer disease in cattle.
This is more pronounced in maiden mares which have experienced estrus
 The cervical and vaginal secretion produced during estrus accumulate anterior to
the obstruction formed by the persistent hymen and cause distention of the
vagina
 Prolapse of the vagina and marked straining are common observed

Inflamation of the vagina and cervix

 Recognized by the presence of purulent exudates on the floor of the anterior

vagina
 The cervix is open and often discharges purulent material during vaginal
examination
 The mucosa of the external os of the cervix and the vagina has an unhealthy red
appearance.
 The observations described above, however, always accompany pneumovagina.

Scars, abrasions, ulcers and other defects of mucosa of the vagina

 Observed as complications after service and parturition injuries.

 Diagnosis, as a rule, is easy.
Retention of urine in the anterior vagina

 The history is that of an intermittent discharge of grayish material.

 Vaginal examination reveals vaginitis and cervicitis which is secondary, since the
fluid retained in the anterior vagina is primarily urine with admixture of
epithelial debris from the mucosa.
 The downward and forward slope of the vaginal floor which might be the primary
cause of the condition.

RECTAL EXAMINATION

 Rectal examination is done to rule out pregnancy. If the mare is pregnant, the
procedures that follow will be altered
 Due to the friable nature of mare’s rectum cre is taken when conducting a rectal
examination
 A well lubricated, gloved arm and hand is introduced one finger at a time through
the anal sphincter into the rectum. With the fingers held together, the cupped
hand should clean out the feces as far cranial as possible prior to searching for
the reproductive tract.
 One may locate the ovary first by reaching up into the sublumbar area ventral to
the fourth or fifth lumbar vertebrae or the uterus first.

EXAMINATION FOR PREGNANCY

 Pregnancy examination should precede all other examinations and should form
the first and basic step of the approach to any form of infertility problem.
 The earliest time during the gestation period when the laboratory tests for
pregnancy are reliable is 60 to 80 days after conception. i.e., between the third
and fourth expected estrous periods after service
 Pregnancy diagnosis might be made by rectal examination as early as 30 to 35
days of pregnancy, thus providing ample time for treatment, if indicated, and
rebreeding a few days later on the second expected estrus after service

NORMAL CHANGES IN THE REPRODUCTIVE TRACT

 Unlike seen in the cow, no dramatic and consistent changes in uterus

are observed in the mare
 During diestrus and proestrus, certain mares have a well defined, slightly
contracted uterus.
 During the estrus period, the uterus appears edematous and firm. This
consistency persists until ovulation, after which the uterus becomes limp and
flaccid.
Seasonal Changes

Stage Findings Terminology

Anestrus The uterus is flaccid, thin walled and quiescentThe CX 1

ovaries are small and firm the vagina is pale and
dry . The cervix is usually in the upper one third of
the vaginal vault, pale and dry and tight. Passage
of a finger through the cervix is difficult, and some
time should be spent allowing it to soften and to
dilate.
transition from Estrogens from the follicles cause the uterus to CX 2
anestrus to the become more edematous, congested and heavier.
normal The cervix changes from a CX 1 to a CX 2 in which
breeding case the cervix is pinkish, softer, moister and lower
season in the vaginal vault. Also the cervix develops folds
of tissue extending from the external os towards
the vaginal floor and will readily admit one to two
fingers.
Estrus Increasing estrogen levels makes the cervix very CX 3
soft and pink. It is usually located in the lower
third of the anterior vaginal wall and is very
edematous, and glistens with moisture.

The edematous folds of the external os actually

touch the floor of the vagina, yet the cervix is still
recognizable. Two to three fingers may easily be
introduced through the cervix
Ovulation Estrogen levels have peaked, the cervix is at its CX 4
softest, salmon pink in color, very moist and
edematous and often appears as a mass of
edematous folds on the floor of the vagina.
ABNORMALITIES INVOLVING UTERUS

 Uterine abnormalities can be subdivided into

o Abnormalities associated with a uniformaly enlarged uterus
o Abnormalities associated with discrete abnormalities within the uterus
o Parauterine abnormalities

Uniformaly Enlarged Uterus

Must be differentiated from pregnancy and a postpartum uterus. Involution of the

mares uterus occurs very rapidly after foaling in comparison to the cows. By the
beginning of foal heat, it should be no more than two to three times its normal size.
Other causes of a uniformly enlarged uterus are pyometra and pneumo uterus.

Discrete Uterine Enlargements

 Must be differentiated from early pregnancy by identifying the embryo as a

discrete bulge in the uterine horn.
 Other enlargements include
o endometrial cysts (result from blocked and dilated endometrial glands),
o lymphatic lacunae (which result from blocked lymph channels),
o abscesses in the uterine wall and corneal dilatation (following atrophy of
the uterine mucosa in older mares)

Parauterine Abnormalities

 Parauterine abnormalities include hematomas in the broad ligament of the

uterus that are usually associated with parturition.
 A fresh hematoma should not be disturbed, and the mare should be treated with
systemic antibiotics to prevent abscessation.
 A chronic hematoma rarely causes a fertility problem and usually regresses over
time.

ABNORMALITIES INVOLVING OVARIES

 Ovarian abnormalities identified on physical examination can be divided into

small ovaries and large ovaries

Small Ovaries

 These may be either normal or abnormal. Prepubertal or juvenile ovaries are

small; therefore, the age and previous cyclic history of the mare is important
 In anestrus the ovaries are inactive and one half the size they will attain during
the breeding season. In some small, docile, chronically anestrus mares a
chromosomal anomaly called XO Gonadal dysgenesis may be the cause of small
ovaries. An endometrial biopsy from these mares often demonstrates glandular
insufficiency
 “True” nymphomaniac mares also contain smal ovaries. These mares act as if
they are in persistent estrus, yet often they will not allow mounting; some of these
mares will demonstrate male like behavior. Other than small, firm ovaries, no
other abnormalities of the genital tract are noted in the nymphomaniac mare

Enlarged Ovaries

 May be a seasonal phenomenone. During the transitional periods, follicles may

grow to abnormally large sizes and persist for various lengths of time before
ovulating or regressing
  They usually do not suppress activity in the other ovary and resolve themselves
and cause no permanent problem
 In the early transition period they can be treated with 1000 to 5000 IU of Human
Chorionic Gonadotrophin but results are variable. These persistent follicles are
often diagnosed as cystic ovaries by practitioners unaccustomed to palpating the
mare’s ovaries
 Cystic ovaries, such as those that occur in cows, do not occur in mares. The
biggest problem in dealing with persistent follicles in mares is differentiating
them from certain types of ovarian tumors

Ovarian tumors

 In the mare ovarian tumors are usually classified according to the main type of
cell making up the tumor. Most of these tumors are unilateral and rarely
malignant
 The cystadenoma must be differentiated from persistent follicles. These usually
enlarge over a period of time unlike the persistent follicle that remains the same
or regresses. They probably arise from the surface epithelium of the ovary or the
rete ovarii and have one or several large fluid-filled cavities within them.
Diagnosis is by ultrasound treatment is only ovariectomy

Granulose-theca cell tumors

 Most common ovarian tumor in the mare

 Grow to very large sizes and are usually unilateral and benign
 Produce a variety of hormones and usually suppress activity of the opposite ovary
 In addition to secreting inhibin, these tumors frequently secrete testosterone
causing the mare to exhibit stallion-like behavior.
 Hormonal analysis and endometrial biopsy may also be helpful in diagnosing this
tumor.
 Clinical signs vary from anestrus to nymphomania and even to virility depending
on the predominant hormone produced by these tumors.
 Treatment is removal of the affected ovary, and the prognosis for fertility is fair to
good, depending on the length of time that the tumor has existed and the degree
of suppression of the opposite ovary. Resumption of cycle occurs 1 to 4 month
after the tumor is removed.
 Much less common ovarian tumors are teratoma and the dysgerminoma.

Teratoma

o The teratoma is a multiple tissue type tumor that usually has epithelial
structures including cartilage, bone, hair and glandular epithelium.
o Are usually benign and produce no hormones, so the contralateral ovary
usually remains functional, and the mare may continue to cycle. As in the
other ovarian tumors, ovariectomy is indicated.

Dysgerminoma
 o Unlike the previously discussed ovarian tumors, the dysgerminoma can be
malignant.
o It arises from the germinal epithelium of the ovary and can become very
large. The tumors may be solid or contain fluid-filled multiple cysts.
Ovariectomy is the treatment of choice.

Non-Neoplastic Ovarian Enlargements

 Other causes of ovarian enlargement such as non-neoplastic ovarian abscesses

and hematoma are common and are difficult to differentiate.
 The mare’s temperature and white blood cell count may help identify the ovarian
abscess, yet these abscesses are often encapsulated within the ovary and do not
produce a systemic reaction after they become chronic.
 Ovarian hematomas often feel similar to ovarian abscesses. In both cases the
opposite ovary usually remains functional and the mare continues to cycle.
Ovarian hematomas usually regress over a period of time and cause no fertility
problems.
 Hormone stimulation tests may differentiate these from ovarian tumors. An
ultrasound examination may be of some help in differentiating them.
 The last cause of ovarian enlargement that should not be overlooked is the
unusually large, normal cyclic follicle. Most cyclic follicles range in size from 2.5
to 6 cm in diameter prior to ovulation.
 Occasionally, one or several large follicles grow to 10 cm or more before
ovulation. In this and all cases of ovarian enlargement, several examinations over
a 15 to 30 day period are a valuable means of differentiating these ovarian
abnormalities.

ABNORMALITIES INVOLVING OVIDUCT

 The incidence and abnormalities of salphingitis and hydrosalpinx seems to be

very low when compared with cattle.
 Fimbrial cysts are not a rare finding in the mare. These are usually small and
inconsequential. Occasionally, they may grow large enough to interfere with the
collection of the ovum by the fimbria.

ABNORMALITIES INVOLVING CERVIX, VAGINA AND VULVA

Cervical Abnormalities

 When evaluating the cervix, the normal pinkness of estrus must be differentiated
from the redness of inflammation.
 Cervicitis may be caused by contagious equine metritis, endometritis or vaginitis
or may be secondary to pneumo vagina or recto vaginal fistula
 The most common non-infectious abnormalities noted are cervical adhesions and
scars secondary to foaling or breeding problems. These abnormalities may
prevent the cervix from opening and/or closing properly dilate thereby
 o preventing the stallion from ejaculating into the uterus
o making delivery of a foal difficult
o predisposing the mare to endometritis which may prevent her from
carrying a foal to term.
 Other abnormalities of the cervix include leiomyoma of the cervix and squamous
cell carcinomas.

ALTERNATIVE METHODS OF EXAMINATION

Ultrasonography

 Can be used to identify the amniotic vesicle as early as day 15 of gestation and
is valuable in predicting the presence of twins early enough in gestation to correct
the problem without danger to the mare.
 Also helpful in producing an image of various vaginal, uterine and ovarian
masses to determine if they are solid or fluid filled.

CLINICAL EVALUATION OF SOWS AND GILT

PHYSICAL EXAMINATION

 Careful physical examination is required for

o selecting potentially fertile breeding animals
o culling gilts with structural or genital abnormalities prior to breeding
o along with a herd history helpful in the diagnosis of reproductive failure in
individual animals or in breeding groups within a herd
 When indicated, internal reproductive organs should be recovered from
slaughtered animals for a thorough examination as useful information can be
obtained when other procedures fail

Structural Soundness

 Soundness in replacement gilts is especially significant, since most structural

faults and weaknesses are aggravated with age and confinement rearing.
 Special attention should be given to selecting gilts free from foot, leg and joint
problems, which may impair their future reproductively.
o A moderate slope to the pasterns provides the animal with a cushion to the
foot and leg joints, enabling her to cope with solid surfaces in
confinement. Gilts and sows with hoof cracks, sole bruises or other foot
problems should be culled because attempted treatments are often
unsuccessful. Such problems may arise from abrasive or damp, slick
flooring
 Too much slope in the rump area tends to make the animal more prone to
unsoundness as she matures. A steep rump also displaces the vulva to a low
position and angle so that boars often experience difficulties in entering the sow
during mating.
  Extreme muscling leads to delayed puberty, low conception rate, farrowing
difficulty and poor mothering ability.

Examination of External Genitalia

 Observing the vulva of replacement gilts at 5 ½ to 6 months of age can help

detect potentially sterile or slow-breeding females.
 The most commonly observed abnormality is the
o infantile vulva which is usually accompanied by small, prepubertal ovaries
and uterine horns.
o dorsally “tipped vulva”. Boars may experience difficulty in servicing gilts
having this trait.
o Injures of the vulva may occur from fighting or at parturition. Unless they
are severe, they generally do not contribute to future reproductive
problems.
o Atresia ani, or imperforate anus, is a congenital defect observed in all
breeds.
 In gilts the rectum and vagina may be joined, forming a recto
vaginal fistula just anterior to the vulva.
 Males die because they are unable to defecate. Gilts defecate via the
vulva opening.
o Occasionally, an unusually large percentage of females within a group is
observed to have red, swollen vulvas, typical of females in estrus. This
observation, when coupled with mammary development in non pregnant
females and barrows, indicates the presence of exogenous estrogenic
substances in the feed

Examination of Mammary System

 A sound underline with atleast six functional, well developed and evenly spaced
teats on each side, with three in front of the navel is prefered
 Gilts with a blind teat that does not fully develop, a pin nipple or an inverted
nipple should not be considered as replacement animals

INTERNAL EXAMINATION OF THE REPRODUCTIVE TRACT

 In herds with a high incidence of reproductive failure that are not diagnosed by
other methods, examination at slaughter is recommended. Females should be
tattooed prior to slaughter for identification so that the individual animal’s
reproductive history can be related to the observed reproductive tract disorder.
Observe for
o the presence and size of follicles, corpora lutea and cysts in each ovary
o any adhesions surrounding the ovaries or within the ovarian bursa
o size of the uterine horns
 Externally, each oviduct and uterine horn to the vagina should be traced, for
occlusions, missing parts or adhesions.
  After gross examination the tract is opened and is examined and noted for the
presence and characteristics of fluid or embryonic tissue.

ABNORMALITIES OF THE REPRODUCTIVE TRACT

Anatomical Abnormalities

Hydrosalpinx and Pyosalpinx

 Hydrosalpinx and pyosalpinx refer to distention of oviduct with clear fluid and
pus like material, respectively. Occur more frequently in gilts than in sows
 Result from abnormal embryonic development and may be hereditary.
 Affected females have regular estrous cycles but are prone to repeat breeding and
reduced litter size. If lesions are bilateral, affected females are sterile.

Segmental Aplasia

 Segmental aplasia may occur at any position along the uterine horn but most
commonly occurs near the uterine body. Occasionally, an entire uterine horn may
be absent.
 Afflicted females will cycle normally. It is possible to have pregnancy in the
patent side, but litter size is usually reduced.

Blind, Double and Missing Cervix

 These abnormalities occur infrequently. Females with either condition cycle

normally.
 If part or the entire cervix is missing, the female is sterile.
 Pregnancy can be achieved in females with a double cervix.

Infantilism

 This is a common abnormality and is generally but not always associated with
confinement-reared gilts wherein the presence of a very small vulva and the
absence of estrus are suggestive of this condition.
 The infantile tract is approximately 30 per cent of the size of a tract from
normally cycling gilt.
 The ovaries are hypoplastic and nonfunctional with numerous small follicles and
no corpora lutea. This condition is common in gilts with delayed puberty or gilts
less than 6 months of age.

Adhesions

 Adhesions have been observed in all areas of the reproductive tract but more
frequently in the oviduct and ovarian bursa.
 Cyclic activity is normal. Reduced litter size and infertility are frequently noted.
Intersexuality

 More predominant in the Yorkshire breed in which a portion of the female

reproductive tract has differentiated into its male homolog.
 Mostly inherited, inheritance is thought to be autosomal recessive accompanied
by modifier genes.
 Characterised by presence of an ovotestis, which may be internal or external,
whereas others may have a prominent clitoris and “sky hood” vulva.
 Some intersexes show male characteristics such as tusk development and
mounting behavior.

Cystic Ovaries

 Cystic ovaries originate in a complete or partial failure of ovulation. Therefore,

cystic follicles may appear on the same ovary as normal appearing corpora lutea.
 Cysts may vary in size from 12 to 50 mm.
 Affected females may be anestrus or exhibit near normal estrus cycle patterns.
 Most attempts at treatment are ineffective.

CLINICAL EVALUATION OF SHEEP AND GOAT

HISTORY

 History is an essential component of the clinical examination, particularly in

sheep and goats due to inaccessibility of the majority of the reproductive tract to
palpation or observation.

Season

 Degree of seasonality varies with breeds of sheep and goats and whether a male is
present or not
 A prolonged breeding season indicates increased or earlier months of transition
 Meat goats as a breed are known for their ability to breed more than once per
year
 Introduction of a buck or ram during periods of transition hlps in advancing the
onset of the breeding season by 4 to 6 weeks

Nutrition and Size

 Young females should be two thirds of their expected adult body weight before
they are bred
 Thin animals with dull hair coats may take months to recover from poor
nutritional conditions.

Social Order
  To the extent possible, the “comfort” of the animal should be determined in terms
of whether it is a dominant or submissive animal in the group.
 Amount of feeding space, access to shelter for all animals and the opportunity for
dominant does to keep timid ones from seeking the buck should be investigated.

CLINICAL EVALUATION OF SHEEP AND GOAT

HISTORY

 History is an essential component of the clinical examination, particularly in

sheep and goats due to inaccessibility of the majority of the reproductive tract to
palpation or observation.

Season

 Degree of seasonality varies with breeds of sheep and goats and whether a male is
present or not
 A prolonged breeding season indicates increased or earlier months of transition
 Meat goats as a breed are known for their ability to breed more than once per
year
 Introduction of a buck or ram during periods of transition hlps in advancing the
onset of the breeding season by 4 to 6 weeks

Nutrition and Size

 Young females should be two thirds of their expected adult body weight before
they are bred
 Thin animals with dull hair coats may take months to recover from poor
nutritional conditions.

Social Order

 To the extent possible, the “comfort” of the animal should be determined in terms
of whether it is a dominant or submissive animal in the group.
 Amount of feeding space, access to shelter for all animals and the opportunity for
dominant does to keep timid ones from seeking the buck should be investigated.

PHYSICAL EXAMINATION

 Physical examination should be done to

o evaluate the current body condition and femininity of the animal
o determine the age by examining the dentition in order to avoid
mistaking under grown infertile adults for young ones.
o Determine whether the animal is polled or horned when the potential for
the intersex condition is considered. Intersex animals can present with a
wide array of clinical features.
Examination of External Genitalia

 Includes evaluation of the anogenital distance and whether the clitoris is visible
without parting the lips of the vulva
 Vulva should be examined for possible abnormalities such as pox or herpesvirus
lesions, tumors, pustular dermatitis, and ectopic mammary tissues
 Includes the palpation of abnormal lumps or swellings in the inguinal region
 Vaginal speculum examination or, alternatively, an endoscopic examination to
rule out any membranes or adhesions present
 Presence of any discharges from the cervix or vagina should be noted
 It should be remembered that the normal caprine vaginal discharge turns from
clear mucus early in standing estrus to thick ”cheesy” exudate late in standing
estrus. This type of discharge at this time of the estrous cycle is normal and does
not require treatment

ABNORMALITIES OF THE REPRODUCTIVE TRACT

 Pathological lesions of reproductive system in sheep and goats are similar in most
respects to those in calttle.
 Vulvitis, vaginitis, cervicitis, metritis, pyometra, perimetritis, and salphingitis
may occcur in sheep and goats as a sequelae to dystocia, embryotomy, difficult
parturition, retained placenta, and delayed involution of the uterus.
 Tumours of the ovaries and genital tract of the ewe are rare.

ULTRASONOGRAPHY

 Transabdominal ultrasonography can be used to examine the animal for

pregnancy, pseudopregnancy (hypoechoic uterine fluid but no cardinal signs of
pregnancy), pyometra, fetal death, fetal maceration or resorption of the
pregnancy.
 Clipping the hair in the inguinal region prior to the examination permits the best
quality images to be obtained. Transrectal imaging with 5 to 7.5 MHz linear
probes often allows visualization of the nonpregnant caprine uterus and ovaries,
or early cases of the previously mentioned conditions.
 The quality of transrectal scanning depends to some extent on the size of the
animal and whether the reproductive tract has descended ventrally along the
body wall.
 Holding animals off feed for 24 hours may improve the image quality in some
case.

LAPAROSCOPY, LAPAROTOMY AND NECROPSY

 A definitive diagnosis of infertility sometimes cannot be made without

examination and palpation of the reproductive tract. Laparoscopy is less invasive
and preferred over laparotomy when the equipment is available.
  The gonads may be aberrantly positioned in intersex animals, or various degrees
of maldevelopment of the three tubular portions of the reproductive tract may be
present.
 In animals that have had peritonitis, abdominal surgery, or embryo collections
performed, the presence of abdominal abscesses or adhesions involving the
reproductive tract may be identified.
 Ovarian cysts or reproductive tract tumors may also be diagnosed by these
methods.

CLINICAL EVALUATION OF THE BITCH

HISTORY

 The differential diagnosis for most infertility disorders is established by obtaining

a thorough history from the owner
 The initial history should include information regarding
o how well the owners know the bitch and does she live indoors with them
or away.
o is she hosed alone, with another bitch that recently completed ovarian
cycles, with ovariohysterectomized bitch or with males?
o Is she normal in height and weight for her breed and for her line?
o Is she receiving any medication and is she well or ill?
 Onset of the pubertal estrus occurs at ages ranging from 6.3 to 23 months
 Toy poodles may benefit from evaluation earlier in life than Bull Mastiffs

PHYSICAL EXAMINATION

 The problem area should always be examined last to mk sure that each bitch
receives a complete physical examination prior to an evaluation of the
reproductive tract.

Vulva

 Vulva should be examined to check for size and conformation and for presence of
any discharge.
 Small immature vulva or one that is recessed under a fold of tissue owing to body
type or obesity interfere with normal breeding while an obese bitch is prone to
perivulvar dermatitis.
 A swollen, turgid vulva is suggestive of proestrus while a swollen and flaccid one
can be consistent with estrus or approaching parturition.

Vaginal Discharges

 The bitch in anestrus or diestrus usually has no vaginal discharge.

. Digital Examination of the Vestibule and Vagina

  A digital examination of the vaginal vault should be performed routinely and
should follow culture and cytology. Masses, foreign bodies, strictures, painful
vaginitis, or abnormal tissue bands all prevent easy and painless examination
 If the digital examination is abnormal but inconclusive, vaginoscopy provides a
more thorough evaluation

Mammary Glands

 The mammary gland should be palpated for the presence of mammary tumors,
for evidence of lactation, mastitis, inverted teats, or benign nodules
 The ventral midline can also be checked for evidence of a previous surgical
incision, which might be a clue suggesting that the bitch has undergone
ovariohysterectomy.

Rectal Examination

 A rectal examination ensures that the pelvic canal has been assessed for previous
fractures or other unsuspected abnormalities as compression of the pelvic canal
is a potential cause of dystocia.

Abdominal Palpation

 The abdomen should be palpated in an effort to identify and characterize the

uterus. However, except in pregnancy and pyometra, the uterus almost never can
be evaluated with confidence on abdominal palpation.

ABNORMALITIES INVOLVING UTERUS

 Abnormal development of the uterus or uterine tubes may occur in intersex

animals
o Male pseudohermaphrodites are animals with testes and female external
genitalia
o Female pseudohermaphrodites are animals with normal uterus and
ovaries and male external genitalia
o XX sex reversed cocker spaniels are genetic females with male gonads and
abnormal male external genitalia
 Hydrometra and mucometra are accumulations of sterile serous or mucoid fluid
in the uterus
o Incidental findings either at the time of elective OHE or in aged intact
female dogs undergoing diagnostic work-ups for unrelated disorders, such
as congestive heart failure or mammary neoplasia
o Cystic endometrial hyperplasia frequently is associated with hydrometra
and mucometra.
o Pathogenesis is likely to include that of CEH
 o Presumptive diagnosis of hydrometra and mucometra is based on
presence of uterine enlargement, documented by abdominal palpation,
radiography or ultrasound and lack of a systemic inflammatory response
o The primary differential diagnoses are pyometra and pregnancy
o Definitive diagnosis requires cytology and culture of the intrauterine fluid
o The fluid varies in character from serous to mucoid and in color from
straw colored to serosanguineous
o The treatment of choice is OHE, especially in bitches not intended for
breeding. Hematometra is sterile accumulation of blood within the uterus
 Cystic endometrial hyperplasia – pyometra complex is an acute or chronic post-
estrual disease of adult intact bitches leading to inflammatory exudates in the
uterus that is associated with variable clinical and pathologic signs. It also is
called pyometritis, pyometra complex, catarrhal endometritis, purulent
endometritis, chronic cystic endometritis, and chronic purulent endometritis

ABNORMALITIES INVOLVING OVARIES

Congenital Abnormalities

 Complete absence of one or both ovaries known as ovarian agenesis may be

associated with other abnormalities of the reproductive tract, such as uterus
unicornis.
 Ovarian hypoplasia has been reported in female dogs with abnormal
chromosome number.

Female dogs with anomalous numbers of sex chromosomes and abnormal ovaries are
usually infertile and include

 True hermaphrodites
 Pseudohermaphrodites
o Male Pseudohermaphrodities
o Female Pseudohermaphrodites

Ovarian Cysts

 Ovarian cysts are fluid-filled structures with a distinct wall that develop within
the ovary.
 Parovarian cysts are similar in morphology to ovarian cysts but lie next to the
ovary.
 Follicular cysts are thin – walled structures containing clear, serous fluid, may be
single or multiple and if multiple cysts are present on one ovary, the cysts do not
communicate. More about follicular cyst in dogs

ther Ovarian Cysts

  Other ovarian cysts include luteal cysts, germinal cysts, cystic corpora lutea,
cystic rete ovarii or rete cysts (are small masses of irregular, anastamosing
tubules with cystic changes in the hilus region of the ovary) and parovarian cysts
(cystic structures in remnants of the mesonephric and paramesonephric tubules
surrounding the ovary

Ovarian Remnant Syndrome

 Ovarian remnant syndrome occurs when a retained piece of ovarian tissue

revascularizes and becomes functional. The condition may occur following
surgeon’s error in incomplete removal of the ovary. The most common
presentation of ovarian remnant syndrome is recurrent estrus after OHE. More
about ovarian remnant syndrome

Oophoritis

 Oophoritis is diffuse infiltration of the ovary with mononuclear inflammatory

cells, with subsequent degeneration of germ cells and fibrosis of surrounding
tissues wherein an autoimmune pathogenesis is hypothesized.

Ovarian Neoplasia

 The three general categories of primary ovarian neoplasms are those arising from
epithelial cells, those tumors of sex cord / stromal origin, and those arising from
germ
 May be palpable per abdomen, and often are visible on radiographs or
sonograms.
 Secondary tumors of the canine ovary include lymphosarcoma.

CLINICAL EXAMINATION OF THE QUEEN

NORMAL TRACT

Ovaries

 The ovaries of the adult queen are

o oval structures
o approximately 1.0 x 0.3 x 0.5 cm in size
o 220 mg in weight
o located in the dorsal abdomen caudal to the kidneys attached to the
diaphragm by the suspensory ligament, to the dorsal body wall by the
mesovarium, and to the end of the uterine horn by the short, thick proper
ligament of the ovary
o enclosed by the ovarian bursa that has a small slit-like opening on the
medial side
 The queen is an induced ovulator.
 o Copulation, vaginal stimulation, or gonadotropin administration induces
ovulation within approximately 24 to 32 hours.
o Corpora lutea, which form after ovulation, appear orange-yellow grossly
and may reach 4.5 mm in diameter, peaking in size about 16 days after
ovulation.

Oviduct

 The uterine tube (oviduct) of the adult queen is 5 to 6 cm in length

 The wall of the uterine tube is thin, and the lining is thrown up into longitudinal
folds or ridges.

Uterus

 The uterus of the adult queen is a Y-shaped organ consisting of a 2-cm-long body
lying between the descending colon dorsally and the urinary bladder ventrally
and two 10-cm uterine tubes (oviducts). The cervix is the thick-walled neck of the
uterus, connecting it to the vagina.

Vagina

 The vagina and the vestibule are each about 2 cm long , so that the cervix is
located about 40 to 45 mm cranial to the vulva; in pregnancy the vagina is
stretched cranially by the weight of the uterus.

Vestibule

 The vestibule extends from just cranial to the external urethral cranially, to the
vulva caudally, a distance of about 2 cm.
 The external urethral orifice in the cat opens into a mucosal groove located on the
floor of the vestibule just caudal to a transverse fold of mucosa that represents
the hymen.

Vulva

 The vulva of the adult queen consists of two small, round labia located just below
the anus, which unite at dorsal and ventral commissures
 The labia are similar in spayed than in intact cats. During estrus the labia are
slightly edematous and reddened, vulvar discharge is negligible

Mammary Glands

 The queen has four pairs of mammary glands, arranged in two bilaterally
symmetrical rows from the ventral thoracic to ventral abdominal region.
 They have been designated as
o the right and left axillary
o thoracic
 o abdominal
o and inguinal mammary glands
 Also designated as right and left mammary glands 1,2,3 and 4 when counting
from cranial to caudal

ABNORMALITIES INVOLVING OVARY

Ovarian Dysgenesis

 Ovarian dysgenesis refers to underdevelopment of the ovary; ovarian hypoplasia

or to hermaphroditic and streak gonads usually associated with an abnormal sex
chromosome complement, such as XO monosomy or mosacicim.

True Hermaphroditism

 Rare in cats, and has not been reported in phenotypic females where both gonad
histology and chromosome complement are known.
 Reported in cats that are phenotypically male, where both gonad histology and
chromosome compliment are known, suggesting that presence of testicular tissue
in the embryo induces development of (male phenotype) secondary sexual
characteristics, regardless of presence of ovarian tissue or karyotype.
 Diagnosis of ovarian anomalies is based on history of primary anestrus (ovarian
agenesis or dysgenesis) on careful gross evaluation of internal and external
genital organs, on histologic examination of the ovary, and on karyotype of
affected queens

Ectopic Adrenocortical Paraovarian Nodules

 Ectopic adrenal gland nodules occur in the broad ligament of the ovary, within 1
to 4 cm of the ovary, as single, unilateral nodules, as bilateral nodules, or as two
nodules on a single side and range in size from 2 to 5 mm in diameter.

Ovarian Cysts

 Follicular cysts that arise from mature or atretic follicles are common and
affected queens may be asymptomatic or may exhibit prolonged estrus if cells
lining the cyst secrete estrogen.
 Prolonged estrus may be hard to distinguish from normal estrus, because the
normal queen may cycle in and out of the follicular phase as frequently as every 4
to 7 days.

The Ovarian Remnant Syndrome

 The ovarian remnent syndrome describes presence of ovarian tissue and signs of
estrus in a female cat after OHE. The causes are
o may be failure to remove all or a normal ovary at OHE
 o presence of a partial or complete separation of a portion of normal ovary
during development (the fragment may be located near the ovary or in the
broad ligament) that is not detected at OHE
o supernumerary ovary although rare may also be considered as the cause of
estrus signs after bilateral OHE
 Affected queens demonstrate normal signs of estrus, and may allow copulation,
but do not become pregnant if bred.
 Diagnosis is based on confirmation of estrus, on detection of serum progesterone
concentrations exceeding 2 ng/ml 2 to 3 weeks after induction of ovulation at
estrus in a neutered cat.
 Treatment is exploratory laparotomy within 3 to 6 weeks of induction of
ovulation at which time presence of corpora lutea in a “grape cluster” appearance
on the surface of the ovarian remnant may make small remnants easier to
identify.

Ovarian Neoplasia

 The granulose cell tumor of sex cord-stromal origin is the most common primary
ovarian tumor in the cat.
 Presence of palpable mass in the cranial or mid abdomen. Abdominal and
thoracic radiographs and abdominal ultrasonography are indicated in all cases to
assess tumor size and location, and evidence, if any, of the presence of
metastases. A vaginal cytology specimen should be examined for cornification as
evidence of estrogen secretion in suspect queens. Measurements of serum
estrogen, testosterone, and progesterone are of interest if functional tumors are
suspected based on clinical signs of prolonged estrus, virilization, or pyometra.
Evaluation of the hemogram and serum chemistry profile is indicated prior to
exploratory surgery.
 Adenoma/Cystadenoma, Adenocarcinoma, Dysgerminoma have also been
reported in cats.

ABNORMALITIES INVOLVING FELINE UTERUS AND OVIDUCT

Hyperplasia of the Uterus and Uterine Tubes

 Multiple, broad-based or pedunculated hyperplastic endometrial polyps have

been reported in cats ranging in age from 4 to 15 years and protrude into the
uterine lumen.

Hydrometra/ Mucometra

 Hydrometra and mucometra, the accumulation of non-inflammatory, clear to

slightly cloudy, watery to viscid, sterile fluid in the uterine lumen, occurs
occasionally in the cat and are caused by
o lack of patency of vulva, vagina, cervix, or uterus resulting from congenital
anomaly,
 o neoplasia
o inflammation,
o scarring,
o accidental ligation.
 Fluid volume in the uterine lumen may reach 500 ml, and distention of the
uterine body and/or horns may be diffuse or segmental.
 Treatment is OHE.

Cystic Endometrial Hyperplasia/Pyometra Complex

 Pyometra in cats is a uterine inflammatory disorder characterized by cystic

endometrial hyperplasia (CEH).
 Clinical signs include purulent vulvar discharge, anorexia, dehydration,
lethargy,pyrexia,vomiting, polyuria/polydipsia, and weight loss. The uterus
becomes palpably enlarged.
 Diagnosis in the intact queen is based on
o signalment,
o history of previous estrus and clinical signs,
o physical examination,
o hemogram,
o presence of a purulent vulvar discharge and /or enlarged uterus in the
nonpregnant animal.
o Abdominal radiography or ultrasonography is indicated to define uterine
size and shape for initial diagnosis, to rule out pregnancy
(ultrasonography, after 21 days following estrus).
 Recommended treatment for CEH/pyometra in the queen is OHE with
concurrent fluid and antibiotic therapy.
 In females with reproductive value and an open-cervix pyometra (diagnosed by
the presence of a purulent vulvar discharge), uterine evacuation can be attempted
with
o PGF2 alpha at a dose rate ranging from 0.05 to 0.5 mg/kg subcutaneously
(SC) once or twice daily for 2 to 5 days until uterine size decreases to
normal.
o Prostaglandin analogues should not be used in the cat, because safe and
effective does have not been established.
o Within 1 to 60 minutes of drug injection, panting, restlessness, grooming,
tenesmus, salivation, vomition, defecation, or diarrhoea.

Salpingitis

 Inflammation of the feline uterine tube, salpingitis, usually is purulent, and

occurs secondary to uterine inflammation

Neoplasia of the Uterus/Uterine Tubes

  Uterine tumors constitute 1 to 2 per cent of tumors of the female reproductive
organs of the cat including mammary glands), or 0.2 to 0.4 per cent of all feline
tumors and include uterine leiomyomas and leiomyosarcomas.
 Clincal signs of uterine adenocarcinomas depend on tumor size and pattern of
metastatsis and include ascitis, anorexia, weight loss, purulent or hemorrhagic
vulvar discharge, vomiting, constipation, dysuria, and presence of a palpable
abdominal mass.
 Diagnosis is based on uterine palpation, abdominal and thoracic radiographs,
surgical exploration, and histopathologic examination of tumor tissue.
 Ultrasonography has been used to detect uterine neoplasia in the diffusely
enlarged uterus with pyometra.
 The recommended treatment for primary uterine neoplasia without metastasis is
OHE.
 Tumors of the uterine tubes have not been reported in the queen.

DISORDERS OF THE FELINE VAGINA, VESTIBULE AND VULVA

 Anomalies of the vagina and vulva that have been described in the cat include
o segmental aplasia of the cranial vaginal (mullerian duct system),
o presence of a common vulvovestibular-anal opening,
o rectovaginal fistula.

Neoplasia

 The most common primary vaginal tumor type in the cat is the leiomyoma,
which may measure up to 7 x7 x 8m.
 Clinical signs of vaginal tumors include
o bulging of the perineal region,
o prolapse of tumour tissue from the vulva,
o dysuria,
o pollakiuria,
o constipation.
 Initial diagnosis is based on palpation and on retrograde vaginography and/or
cystourethrography to characterize size and extent of the mass. Abdominal and
thoracic radiography to look for tumor metastasis should be performed prior to
surgical excision. Exfoliative cytology may be diagnostic and should be performed
on accessible masses of the vagina and vestibule. Final diagnosis is based on
histopathologic examination after core or excision biopsy.
MODULE-2

 DELAYED PUBERTY AND SEXUAL MATURITY
 Puberty represents the initiation of estrous cycle and is defined as the age or
time at which the female gonads are able to produce gametes and reproduction
may occur. It is not a single event, but a process. In the female, puberty is
characterized by the exhibition of estrum and ovulation.
  Onset of puberty normally occurs at a certain age relative to the animal’s body
weight. Heifers must attain approximately two thirds their adult sizes before they
reach puberty.
 The word puberty originated from latin word "pubscere" that means "to be
covered with hair". The original definition that relates to the presence of hair in
certain anatomical regions, obviously does not hold good to other animals.

HYPOTHALAMUS DEFEMINIZATION MECHANISM

 It is important to know the fundamental differences in the hypothalamus of the

male and female in order to have a clear understanding of the puberty in both the
sexes. To address this issue, one should first neccessarily understand

Why GnRH surge centre develops only in female and not in males?

 In the male..
o During prenatal development, defeminization of the brain occurs due to
testosterone from the fetal testis.
 In the female...
o Since there is no testis to produce testosterone, GnRH surge centre
develops in the hypothalamus.
o In order to defeminize the hypothalamus, it is most important that, first
testosterone has to be converted into estradiol.

Why then the estradiol produced by the female fetal ovaries does not cause
defeminization?

 In order to cause defeminization, the estradiol produced by the fetal ovaries has
to cross the blood-brain barrier and gain access to the hypothalamus. A protein
called, alpha-fetoprotein binds to the estradiol thus preventing it from
crossing the blood-brain barrier.
 Alpha-fetoprotein, is a glycoprotein synthesized by the embryonic yolk sac and
later the fetal liver and serves as a fetal blood osmotic regulator and a carrier of
fatty acids.

CRITERIA USED TO DEFINE PUBERTY

Age at First Estrus (Heat)

 Female becomes sexually receptive and displays her first heat

 Exhibit behavioural signs of sexual receptivity
  In heifers and ewes, silent ovulation is common and generally not accompanied
by behavioural estrus
 Age at first estrus may not reflect true acquisition of puberty

Age at First Ovulation

 Age at which first ovulation occurs.

 In bovine, it can be assessed by rectal palpation, ultrasound and laparoscopy.
Requires frequent observations for precise determination.
 Although good criterion, often difficult to determine.

Age at which Pregnancy can Occur without Deleterious Effects

 Applicable to all domestic animals.

 Generate highest number of offspring in the shortest time interval without
compromising the well being of the dam or the neonate.
 In biological sense, females cross a “metabolic threshold” before puberty occurs.

MECHANISM OF PUBERTY

Differences in the LH secretory patterns in the male and female

 There are fundamental differences in the pattern of LH secretion in postbertal

male and female. LH does not surge in the male, but maintains a relatively
consistent day -in and day-out episodic pattern of secretion with episodes
occuring every 2-6 hrs. which in turns results in a steady pulse of LH and in turn
testosterone. In contrast LH and Testosterone surge every 21 days in the female.
Between these surges, low amplitude repeated LH pulses are present.

Changes in hypothalamic secretion of GnRH before and after puberty

 Before puberty in the female, the GnRH neurons in both the tonic and surge
center of the hypothalamus release low amplitude and low frequency pulses of
GnRH
 After puberty, the tonic center controls basal levels of GnRH but they are higher
than in the prepubertal female because the pulse frequency increases. The surge
center controls the preovulatory surge of GnRH.

Mechanism of Puberty

 The onset of puberty is not affected by the performance of gonads or the anterior
lobe of the pituitary
 The failure of the hypothalamus to produce sufficient quantities of GnRH to
cause gonadotropin release is the major factor limiting pubertal onset.
 Prior to onset of puberty the following events take place
 o The tonic centre of the hypothalamus produces GnRH in a pulsatile
fashion. However, the frequency of GnRH pulses are much lower than in
the post pubertal female.
o The low frequency GnRH pulses are not sufficient enough to cause
production of FSH and LH from the anterior pituitary in high levels
o Thus,follicle development (even though it occurs before puberty) connot
result in high levels of estrogen secretion.
o Although estrogen production is low, the hypothalamus is highly sensitive
to the negative feed back effects of low estrogen.
o GnRH and gonadotropin levels are low
 Onset of puberty is characterized by two important events
o Decrease in sensitivity of the hypothalamus to the negative feed back
effects of estrogen
o Higher and higher amounts of GnRH and gonadotropins are produced
that stimulate the ovary to produce more and more of estrogen
o When estrogen levels reach a threshold, they exert a positive feedback on
the surge center
o Ovulation can take place and puberty ensues.
 Thus, the triggering mechanism for onset of puberty in the female is the decrease
in sensitivity of the hypothalamus to negative feed back

What stimulates GnRH neurons to change its secretory pattern?

 There is a possible influence of metabolic signals upon GnRH neurons

 Adipocytes produce leptin that enters the blood and may stimulate neuropeptide
Y neurons or directly stimulate GnRH neurons. Blood leptin reflects the
nutritional status of the animal because greater the amount of fat, greater the
amount of leptin.
 Blood glucose levels, another indicator of metaboloic stimulus probably stimulate
glucose sensing neurons that in turn stimulate GnRH neurons.
 Blood fatty acids may stimulate neurons that in turn stimulate the GnRH
neurons. Blood fatty acids would be an indicator of nutritional status of the
animal.

ONSET OF PUBERTY AND OPTIMUM BREEDING AGE (IN

MONTHS) IN DIFFERENT SPECIES

Animal Onset of puberty Optimum breeding
age
Mare 10-24 (Av.18) 24-36
Cow 6-18 14-22
Ewe & Doe 6-12 12-18
Sow 5-8 8-9
Bitch 6-12 12-18
 Queen 5-18 12-18
FACTORS AFFECTING PUBERTY

 Interaction with the opposite sex.

 Influence of sex: Female attains puberty at an earlier age.
 Breed: Small breeds attain puberty at an earlier stage. eg. Nine months in Jersey
and 11 months in Holstein Friesian.
 Climatic influence: Animals in the tropics attain puberty at an early age.
 Seasonal influence: Breeding season affects onset of puberty. Eg. Ewe lambs born
in early spring attain puberty in the fall i.e., within 180 days. But ewe lambs born
in late spring and early summer attain puberty only in the fall of the next
breeding season i.e., only after 400 days.
 Plane of nutrition: High plane of nutrition leads to early puberty and
undernourishment delays onset of puberty.
 Body weight: Sheep attain puberty after reaching a body weight of 40 kg.

DELAYED PUBERTY AND SEXUAL MATURITY IN CATTLE

 With good nutritional management, most cattle reach puberty between 8 and 13
months of age. Failure to exhibit estrus beyond this time is called prepubertal
anestrus.
 To begin with, the history of the individual or the group of heifers will provide the
key to the etiology.
 If the problem exists in a single heifer of the same age as rest of the cycling
heifers in the group it is probably related to abnormal reproductive tract such as
o freemartinism,
o hermaphrodite
o aplasia of the mullerian duct.
 Cyclicity in rest of the herd mates indicates that the problem has not affected the
entire group.
 Similarly, any debilitating disease such as chronic pneumonia can delay puberty
by decreasing rate of gain, which appears to be a functional dietary problem.
 If delayed puberty occurs in a group of heifers of the same ages or a group of
mixed ages then the problem is one of management.
 Since, the onset of puberty is influenced by the available nutrition heifers of the
same age that are fed a suboptimal energy diet will show a prolonged prepubertal
anestrus period.
 Similarly, when a group of heifers of different ages are housed together and given
a balanced ration, the larger or more aggressive herd mates will consume a
 greater portion of the available nutrition and tend to attain puberty at an earlier
age while others may remain acyclic. However, in due course the entire
population begins to cycle. The inherent danger of breeding these late heifers
before they have developed adequate body size must be borne in mind as they
tend to have more dystocia problems and long postpartum anestrus periods
 Infectious diseases like blue tongue, and bovine diarrhoea virus may result in
acute ovaritis and subsequently ovarian atrophy thus causing anestrus. Animals
may respond to hormonal therapy but promptly return to anestrus once therapy
is withdrawn.
 The use of growth stimulation implants in prepubertal heifers can delay pubertal
estrus and affect future fertility.

DELAYED PUBERTY AND SEXUAL MATURITY IN GILTS

 In hybrid gilts, puberty usually occurs at around 160 days or age but it is breed
dependent.
 Puberty may be delayed by poor environmental conditions, cold, sunburn and
poor light.
 Overcrowding and the associated bullying and stress may result in delayed
puberty.
 Poor nutrition acts by reducing growth rates or by causing deficiencies in
particular nutrients.
 Disease may affect the gilt by reducing body condition or by causing pain.
Lameness reduces bodily condition and precludes behaviour associated with
estrus.
 Finally, poor management of peer group contacts may delay puberty.
 Puberty may be delayed by housing with young boars or by contact with old boars
too early. As the expected time of first estrus approaches, inadequate boar
contact or contact with board with low levels of boar odour may delay puberty.
 Clinical signs of delayed puberty consist simply of the failure of gilts to show
estrus (enlargement of the vulva, reddening of the vulva, remaining still for back
pressure, clustering round a boar) by the time they would be expected to have
reached puberty based on previous experience with the breed or hybrid on the
farm concerned, or industry norms. For this to be ascertained, records of the
chronological age of the animals must be available, or an estimate of their age
made based on weight, size for age, or time since selection for the breeding pool.
Anatomical evidence of hermaphroditism may be obvious Delayed puberty may
occur in individual animals in a group or in whole groups of animals. If estrus has
not occurred or been noted by 240 days of age, it is unlikely that it will occur.
 For the accurate diagnosis, the age of the animals under examination must be
known and individual animals must be identified or removed from the group
when estrus is first detected.
 Daily inspection of the group in adequate light for physical signs of estrus, signs
of mounting and should include behaviour towards boars.
  Prevention of delayed puberty depends upon correcting the management factors.
o Gilts should be reared to 5-6 months of age in groups of 6-30, isolated
from boars, with 12-16 hours light of adequate intensity at a temperature
of about 20°C and given food. Disease should be controlled.
o Gilts which have reached 160-210 days of age should be exposed to
vasectomised boars or housed in sight, smell and touch of a smelly older
boar. Estrus should then be observed. If not, then gilts can be tested with
another boar daily, taking the gilt to the boars.
o Treatment with gonadotrophic hormones induces estrus in pre-pubertal
gilts. Gilts should be reared to 5-6 months of age in isolation from boars,
injected with gonadotrophic hormone preparations and then allowed
contact with boars for 15-20 minutes per day. Ninety percent of gilts will
develop estrus within 5-7 days. They should not be mated at this estrus.

MODULE-3
ESTRUS DETECTION IN DOMESTIC ANIMALS

The single most important problem limiting high reproductive efficiency in a herd is
poor detection of estrus. Thus, effective estrus detection is the key to maximize
reproductive efficiency through AI.

Herds, with poor estrus detection efficiency, are characterized by one or more of the
following:

 Prolonged calving to first service interval. The interval should be no more than 15
to 18 days longer than the farmer’s goal and/or average no more than 70 days.
 Prolonged intervals between breedings, especially when many are multiples of a
normal 18 to 24 –day when many are multiples of a normal 18 – to 24- day cycle.
 Veterinary examinations confirming that cows are cycling normally, although
estrus is not observed.
 More than 10 to 15 per cent of the cows confirmed to be open at a 35 – to 50- day
pregnancy check.
 Heat detection index less than 0.05 or less than 50 per cent of the eligible cows
observed in estrus within a period of 3 to 4 weeks.

There are several aids to detect the heat in animals however they cannot substitute
visual observation.

EVALUATING ESTRUS DETECTION EFFICIENCY

Complete and accurate herd records are essential for evaluating the efficiency and
accuracy of estrus detection. Calving dates, heat and breeding dates and the results of
prebreeding and pregnancy examinations are needed. From these data the following
measures of reproductive efficiency relating to estrus detection can be determined.

 Percentage of cows observed in estrus within 60 days after calving.

 Interval from calving to first breeding.
 Intervals between breeding.
 Estrus detection index, which is defined as the average number of breeding per
cow.

ESTRUS DETECTION PROBLEMS AND THEIR CAUSES

 Basically, there are two estrus detection problems:

o Missed or unobserved estrous periods
o Estrus detection errors.
 The latter results in the insemination of cows that are not in the proper stage of
the estrous cycle for conception to occur. Some are near estrus but are bred 1 to 2
days too early or too late, some are open but in the luteal phase of the cycle and
some are pregnant.

UNOBSERVED ESTRUS

 If in most herds essentially all cows are cycling normally, why does the dairy
farmer have difficulty “catching the cycling cow”?. There are several reasons:
some represent “people problems,” but there are several “cow factors” that make
estrus detection difficult. The major factors contributing to poor heat detection
efficiency are:
o Failure to spend sufficient time on a daily basis for estrus detection.
o Most mounting activity occurs at night in loosely-oused herds.
o Heat periods are short.
o Low levels of estrus activity when few cows are in heat. This can be a
significant problem in small herds and in groups of cows in large herds in
which many cows are either pregnant, not cycling or in the luteal phase of
their estrous cycles.
o Mounts last 10 seconds or less. Farmers must concentrate on estrus
detection and should avoid combining it with other activities.
o Feet and leg problems, slippery floors, summer heat, winter cold and other
environmental factors reduce estrus activity.
 Heat detection programs that limit the effects of these “people” and “cow” factors
must be developed in order to maximize heat detection efficiency.

ESTRUS DETECTION ERRORS

 Estrus detection errors must be avoided. Breeding pregnant cows can cause
abortion. Breeding nonestrus, open cows wastes time, semen and money.
 In order to reduce the number of errors, potential causes of the problem must be
identified. In most herds errors result from
o misidentification of cows,
o misinterpretation of the signs of heat,
o misuse or misinterpretation of the signs of heat
o misuse or misinterpretation of heat detection aids
o cows transmitting the wrong signals (up to 10 per cent of pregnant cows
may stand to be mounted).

ESTRUS DETECTION AIDS

 Estrus detection is difficult, so estrus detection aids are needed in many herds to
identify effectively all the cows that must be inseminated.
 The most important consideration for farmers is to remember that they are only
aids.
 For best results aids must be used in conjunction with good visual detection
programs, not as a substitute for visual detection.
 The various estrus detection aids are as follows
o Wall charts, Breeding wheels, Herd monitors and Individual cow records.
o Secondary signs of estrus
o Palpation of reproductive organs
o Mount detection
 o Heat detector animals
 Lateral deviation of penis
 Caudal epididymectomy
 Dorsal ligament resection
o Vaginal electrical resistance
o Heat check report system
o Prostaglandins
o Pedometers
o Tricks of the trade

WALL CHARTS,BREEDING WHEELS, HERD MONITORS AND

INDIVIDUAL COW RECORDS

 These systems are the least expensive and aim at detection of the next heat
period. If the farm workers know when the next possible heat period is they could
closely observe individual cows for signs of estrus. Thus, more short-or weak-heat
periods can be identified. The key to successful use of these management aids are
o The accurate recording of every heat beginning with the first after calving.
o Their daily use to identify those cows that are due to return to estrus.

SECONDARY SIGNS OF ESTRUS

 Secondary signs indicate that a cow is in or near heat.

 They should be used primarily to identify cows that need careful observation for
standing estrus.
 A twice-daily walk behind the cows when most of them are lying down provides a
good opportunity to check for the secondary signs of estrus.

PALPATION OF THE REPRODUCTIVE ORGANS

 Routine rectal examination of all cows between 30 and 40 days after calving and
of individual problem cows that have not been inseminated within 70 days after
calving should be encouraged to confirm that the reproductive tract is normal
and to predict when the next estrus will occur or to identify cows for
prostaglandin treatment when estrous cycles are occurring, but estrus has not
been detected.

MOUNT DETECTION

 Two methods are widely used for mount detection

o pressure – sensitive devices
 paint stick, chalk or paint on the tail head.
 Kamar Heat Mount Close-up of Dye Capsule Paint on Tail Head Chalk on Tail Head
Detector
Red dye is contained in a soft Tail head painting as an aid Tail head painting with an
A pressure sensitive heat compressible capsule that is to estrus detection. Oil based oil based paint as an aid to
mount detector patch connected by a hairline paint is used, subsequently to estrus detection.
(KaMar) is glued to the channel via a hard be covered with a layer, of a Subsequently a second layer
tailhead of the cow cylindrical tube to an outer different color, of chalk. of a different color is applied
compartment (not shown). with a crayon.
Sustained pressure, as from
a mounting animal, is
required to express the
contents which are then
readily visible in a
compartment glued to the
tailhead of the cow.

 When animals are in estrus, mounting activity changes the color of the detector
or erases the chalk or paint stick markings. With good management and proper
interpretation, pressure-sensitive mount detectors provide excellent results.
However, care must be taken to position the detectors properly and to minimize
the opportunities for false activation of the devices.
 False activation of mount detectors can be reduced by removing cows that are in
estrus from the herd. The disadvantage of this practice is that it removes sexually
active cows that stimulate increased mounting behavior in others that may be in
estrus but are less active.
 Recent studies show that the accuracy of mount detectors, when used as the sole
method of heat detection, may be as low as 30 to 50 per cent. These results
strongly suggest that mount detectors should be used only to identify cows that
require additional observation. Breeding on the basis of activated mount
detectors without additional signs to confirm that cows are in estrus should be
discouraged.
 Chalking the tail head is a less expensive alternative for mount detection. False-
positive are sometimes a problem, and animals must be restrained and marked
every few days, since mud and manure may obscure the chalk or paint stick
marking. Paint can be used instead of chalk or paint stick. When the paint dries,
it becomes brittle and flakes off when the cow is mounted.
 Tail Head Painting Tail Head Chalking Tail Head Marking with Tail Head Marking with
Crayon Crayon
Tail head painting as an aid Tail head marking with a
to estrus detection. Oil based crayon or "chalk" as an aid Tailhead marked with With cows in lock-up
paint is used, subsequently to to estrus detection. crayon to facilitate estrus stanchion tail heads are
be covered with a layer, of a detection. easily marked with a crayon
different color, of chalk. and inspected for scuff
marks.

HEAT DETECTOR ANIMAL

 Sexually active animals can be used to identify estrus cows. They may be fitted
with halters containing ink-filled reservoirs and ball point pen type devices that
wil mark animals that are mounted, or they can be used without these devices to
increase sexual activity and make visual detection programs more effective.

Chinball Marking Harness Chinball Marking Harness in Marked Heifer

Filling of the chinball marking Place Heifer marked by a teaser bull.
harness with marking fluid or Chinball marking harness fitted on a
paint. The spring loaded ball is teaser bull
depressed and paint is poured or
squirted in
  Bulls, “cystic” cows, hormone-treated steers and hormone-treated cows and
heifers have been used. Cows with chronic follicular cysts are inconsistent, and
there appears to be variation in effectiveness among hormone- treated steers.
 The marker bull is the most effective detector animal. Copulation must be
prevented even in sterilized animals to ensure against the spread of veneral
diseases.
 Use of surgical techniques that prevent sexual contact is preferred. Mechanical
devices that prevent copulation are less desirable because they sometimes fail,
cause infection and tend to reduce the sex drive of the bull.
 Bulls are dangerous. Injuries to cows and farm workers can and do occur. For this
reason, other bulls must be available so that bulls can be replaced when they
become too aggressive.
 Hormone-treated heifers and cows are more docile although they may be slightly
less effective, they are the animal of choice on most farms.
 When marker animals are used, cows should be removed from the herd as they
come into estrus. This will stimulate the marker animal to seek out and identify
additional cows that may be in heat. The ratio of cows to markers should be no
greater than 40:1.

Disadvantages

 Some cows may be marked when they are not in estrus.

 Others that are coming into estrus may be marked before they stand to be
mounted. Therefore, care must be exercised when interpreting the marks. For
these reasons, the best results are obtained when marker animals are used in
addition to a good visual detection program.
 Also, marker animals tend to become too fat if feed intake is not restricted. A
possible solution to the latter problem in loose-housed herds is to put the marker
with the herd only at night or other periods during the day when visual
observation is limited.

VAGINAL ELECTRICAL RESISTANCE

 This method is based on the concept that the electrical resistance (ER) of vaginal
fluids decreases during proestrus and through the estrus due to increase in the
volume and ionic composition of the cervical and vaginal fluids.
 The estrus probe is designed to monitor these changes wherein “low” probe
readings are associated with estrus. However, this tool is labour intensive since
cattle must be probed frequently to detect significant changes in ER. Care must
be taken to wash the probe in disinfectant and thoroughly rinse and dry it before
using in another cow.

HEAT CHECK REPORT SYSTEM

 A heat check report system for herds experiencing estrus detection problems has
been developed by Eastern AI Cooperative and Cornell University.
 It has been particularly useful in herds in which more than one person routinely
reports estrus cows. In these herds the best “cow person” is given responsibility
for the estrus detection and breeding programs.
 Workers return heat reports to the person in charge, who then makes the
decision on whether or not to breed the cow

PROSTAGLANDINS

 One of the greatest potential uses of prostaglandins is as estrus detection aid in

dairy cows in which estrus has not been observed.
 Research has shown that prostaglandin treatment of cows with functional
corpora lutea will induce a fertile estrus within 2 to 7 days.
 Approximately 50 per cent will be observed in estrus within 80 hours after
treatment and will demonstrate normal fertility.
 For best results, insemination should be based on estrus observation, but
insemination at 80 hours after treatment for cows that have not been observed in
estrus by that time has been recommended.
 In these cases estrus detection efforts should continue because some will come
into estrus after the “80 hour breeding” and will have to be inseminated again.

PEDOMETERS

 Because cows become more active when they are in estrus, activity monitoring
through the use of pedometers is a potentially valuable method of identifying
estrous cows.
 Studies have shown that the cow activity measured by pedometers strapped to
the cows’ rear legs increased approximately 400 per cent in cows housed in free
stalls.

TRICKS OF THE TRADE

 In certain management situations various tricks can be used to improve estrus

detection.
o First, cows in heat can be left with the herd to stimulate activity. Studies
have shown that mounting activity increased 3-to 5-fold when more than
one cow was in heat. However, an argument for removing estrus cows is
that animals who are actively mounting sometimes choose favorites. This
can reduce the chances of detecting additional cows that are in heat but
less aggressive.
o Second, questionable cows can be placed with strage animals to stimulate
activity.
 o Third, simply moving cows as a group form one area to another, such as
from concreate to a dirt lot, sometimes stimulates activity. Heat checking
should always include getting all cows up and moving them if they are in
free stalls or outside. These tricks will not be feasible in all operations, but
for those in which they can be used more heats may be accurately detected.
MODULE-4
 ABERRATIONS OF ESTRUS AND ESTROUS CYCLE IN
DOMESTIC ANIMALS

After puberty the female enters a period of reproductive cyclicity, which continues,
throughout most of her productive life.

Estrous cycle is the rhythmic sexual behavioural pattern that is exhibited by the
female beginning at one estrus (heat) and ending at subsequent estrus. The word
"Estrous" is derived form a Greek word "Oistros" meaning "Gad fly" - used to
describe the behaviour of cows when attacked by such flies. They continue throughout
the adult female’s life and are interrupted by pregnancy, nursing and by season in some
species.

Estrous cycles provide females with repeated opportunities to copulate and become
pregnant. If conception fails, another estrous cycle begins, providing the female with
another opportunity to mate and conceive. When conception occurs, the female enters a
period of anestrus during pregnancy, which ends after parturition (giving birth) and
uterine involution (repair and returning to normal size).

CLASSIFICATION OF ESTROUS CYCLE

 Animals could be categorized based on the occurrence of estrous cycle as:

PHASES OF ESTROUS CYCLE

 In bovine, the estrous cycle can be divided into two phases depending upon the
dominant structure present on the ovary.

Follicular Phase

 It is the period from the regression of corpora lutea to ovulation. The primary
ovarian structure is the Graafian follicle and the primary reproductive hormone is
estrogen.

Luteal Phase
 The luteal phase is much longer than the follicular phase and extends from
ovulation to luteal regression. Predominant ovarian structure is the corpora lutea
and the primary reproductive hormone is progesterone.
 Even though the luteal phase is dominated by corpus luteum, follicles continue to
grow and regress during this phase. However, these follicles do not produce
sufficient amount of estrogen to bring about estrus and ovulation


o Monoestrus: Only one estrous cycle per year. eg. Wild animals.
o Polyestrus: Periodic estrous cycles throughout the year. eg. cow and sow.
o Seasonally polyestrus: Periodic estrous cycles only during a particular
season. eg. Sheep and mare.
 Depending on the ovarian activity again estrous cycle is classified as:
o Regular estrous cycle: Characterised by ovulation and formation of corpus
luteum. eg. Cow, sheep, mare, bitch and sow
o Spontaneous ovulators: Ovulations are spontaneous but the corpus
luteum formed will not be functional until mating has occurred. eg., rat or
mouse
o Induced ovulators: Ovulation and corpus luteum formation depends on
whether mating has occurred or not. eg. Cat, rabbit, mink.

CHANGES FOLLOWING OVULATION

 Following ovulation the theca interna and the granulosa cells of the follicle
undergo luteinisation, a process governed by LH.
o Immediately after ovulation the walls of the follicle collapse into many
folds.
o These folds begin to interdigitate, allowing thecal cells and granulosa cells
to mix.
o The luteal tissue consists of large and small luteal cells. The large cells
originate from granulosa cells and secrete oxytocin and progesterone.
The small cells originate from cells of theca interna and secrete
progesterone only.
 The functions of progesterone are
o stimulates hypertrophy of endometrial glands,
o increases uterine milk secretions,
o inhibits contractions of uterus
o prevents neutrophilic infiltration,
o plays an important role in endocrine control of reproduction because
 it exerts a strong negative feed back on the hypothalamus thereby
preventing development of preovulatory follicles (follicles develop
during luteal phase but do not reach preovulatory stage unless
progesterone decreases),
 production of estrogen,
  behavioural estrus,
 preovulatory surge of GnRH and LH.

If the animal is not pregnant, the CL undergoes luteolysis. Luteal oxytocin and PGF 2 alpha from
endometrium cause luteolysis. Estrogen from the follicular waves causes formation of oxytocin
receptors in the uterus. The oxytocin from the CL binds to oxytocin receptors in the
endometrium and activates the enzyme system necessary for formation of PGF 2 alpha . The
PGF2 alpha reaches the ovaries by a vascular counter current exchange system.

Mechanism of Luteolysis

 Luteolysis is brought about by

o Reduction in blood flow to the CL by vasoconstriction.
o PGF2 alpha binds to specific receptors on large luteal cells causing influx of
Ca ions. High intracellular Ca ions are thought to cause apoptotic effects
(programmed cell death).
o PGF2 alpha receptor complex also activates protein kinase C (PK-C) that
inhibits progesterone synthesis.

REPRODUCTIVE CHARACTERISTICS IN BOVINES


Reproductive Characteristics of Cattle and Buffaloes
Parameter Cattle(mean) Buffalo (mean)
Sexual Season Polyestrus Polyestrus
Age at Puberty (months) 15 (10-24) 21 (15-36)
Estrous Cycle

Length (days) 21 (14-29) 21 (18-22)

Estrus (hour) 18 (12-30) 21 (17-24)

Ovulation

Type Spontaneous Spontaneous

Time of Onset (hour) 30 (18-48) 32 (18-45)

Number of Eggs Shed 1 1

Life Span of CL (days) 16 16

ESTROUS CYCLE IN THE MARE

 The normal estrous cycle in mare is 21-22 days.
 Estrus, the period when the mare displays behavioural signs of sexual receptivity
to the stallion, lasts for 5 to 7 days
 During diestrus, the luteal phase that lasts for 14-15 days, the mare displays
sexual rejection to the stallion.
 Duration of estrus varies among individuals and also among estrous cycles of the
same mare.
 Long duration of estrus in the mare may be due to the fact that :
o The ovary is surrounded mostly by a serous coat and some follicles have to
migrate to reach the ovulation fossa to rupture.
o The ovary is less sensitive to exogenous FSH than other species (eg. cattle
and sheep), so that the preovulatory follicle requires a longer time to reach
maximal size.
o The level of LH is low compared with FSH and this delays ovulation.
 The intensity of behavioural estrus varies both throughout the estrus period and
among individual mares at comparable stages of the period.
 Old mares, mares underfed during the early part of the breeding season and
during twin ovulations have a longer duration of estrus.

SYMPTOMS OF ESTRUS IN MARE

 Restless and irritable.

 Frequently adopts the micturition posture and voids urine with repeated
exposure of the clitoris by prolonged rhythmic contractions. This is known as
winking of clitoris.
 Introduction of a stallion or teaser, these postures are accentuated.
 Raises the tail to one side and leans her hind quarters.
 Vulva becomes large and swollen.
 Labial folds are loose and readily open for examination.
 Vulva becomes scarlet or orange, wet, glossy and covered with a film of
transparent mucus.
 Variable amount of mucoid discharge.
 If the mare is in estrus, the stallion will usually exhibit “Flehmen”.
 Vaginal mucosa is highly vascular, and thin watery mucus may accumulate in the
vagina.
 During estrus cervix dilates enough to admit 2-4 fingers, during diestrus only one
finger can be inserted.
 If the mare is in estrus, the stallion will usually exhibit “Flehmen”.

ESTROUS CYCLE IN SOW

 Sow is polyestrus.
 The average length of the cycle is 21 days and may vary from 18-23 days.
 The estrogenic phase of the cycle is 6-7 days.
 The luteal phase is 14 days with corpus luteum regression beginning on day 16.
 The duration of estrum is 1-4 days, with an average of 2-3 days.
 Silent heats occur in about 2% of porcine cycles.
 Senility (after 6-10 years) may affect estrous cycle.

SYMPTOMS OF ESTRUS IN PIG

 Estrus symptoms are definite and marked and include

o Reduced appetite
o Restless and nervous
o Often pace back and forth by the fence
o Salivation
o Champing of the jaws
o Sow, if suckling, may ignore her piglets, may try to escape or if free will
seek out the boar and stand for service
o Vulval lips swollen and congested
o Mucous membrane pinkish-red
o Mucous discharge
o Mounting other animals
o Grunting and standing motionless for long periods in a position for service
is the most noticeable feature. This peculiar immobilizing reflex or stance
(lordosis response) can be produced by applying pressure of the hands on
the sow’s back or by straddling the sow.

ESTROUS CYCLE IN SHEEP AND GOAT

 The normal estrous cycle length in sheep is 17 days and in goats is 21 days.
 Photoperiodism is a characteristic of the reproductive cycle or sexual receptivity
in sheep and goats. A reduction in daylight to about 10 or 11 h, whether it occurs
naturally or whether it is induced artificially by penning the ewes in a darkened
area during part of the day, will induce the onset of estrous cycle in anestrus
ewes.
 In both sheep and goats a considerable variation in the cycle length occurs due to
o Breed differences
o Stage of breeding season, and
o Environmental stress
 During early breeding season, premature regression of corpus luteum or
anovulation results in abnormally short cycles.
 Estrus lasts for 24-36 h in ewe and 24-48 h in the doe.
 Duration of estrus is inflenced by
o both species,
o breed,
o age,
o season and

SYMPTOMS OF ESTRUS IN SHEEP AND GOAT

Doe

 Estrus symptoms are more conspicuous in does and include

o Restless
o Frequent bleating
o Tail wagging from side to side and up and down (most reliable sign)
o Reduced appetite
o Decreased milk production
o Vulva edematous and congested
o Clear mucous discharge from the vagina
o Occasionally does exhibit homosexual behaviour
o

Ewe

 Estrus symptoms are relatively inconspicuous, and is not evident in the absence
of ram.
o Ewes seek the ram, and together form a following “harem”
o Vulva edematous and congested
o Clear mucous discharge from the vagina
o Waggles her tail and moves it laterally

ESTROUS CYCLE IN BITCHES

 Dogs experience ovarian cycles twice yearly. Breeding seasons depend on both
genetic and management factors. Breeds enter their first heat between 6 and 10
months of age. Some dogs may not begin to cycle until 18 to 24 months of age.
The inter-estrus interval on an average is 7 months and ranges from 3.5 months
to 13 months. However, the Basenji breed cycles once a year only.

Phases of Estrous Cycle

 There are four phases namely

o Proestrus
o Estrus
o Diestrus
o Anestrus

Proestrus

 Begins with the appearance of vaginal bleeding (spotting) and ends when the
bitch allows a male dog to mount and breed. Duration is 9 days (average and
ranges from 2-3 to 25 days).
 Hormonal changes
 Proestrus is under the influence of estrogen, which is synthesized by the
developing follicles. Early proestrus is associated with estradiol concentration of
 25 pg/ ml which may increase to 60 –70 pg/ml in late proestrus. Thereafter
estrogen levels start declining to reach basal levels (5 – 15 pg/ml) at the onset of
diestrus.
 Progesterone concentration start increasing from basal levels (< 1ng/ml) at late
proestrus to reach levels of 3 ng/ml at the onset of estrus and 5 ng/ml at the start
of ovulation. This progesterone is secreted by follicle which becomes partially
luteinised prior to ovulation and developing CL.

Estrus

 Begins with the first acceptance of the male and ends with the refusal. The
duration is 9 days but ranges from 1-2 days to 18-20 days.
 Hormonal changes
o The bitch is unique in that standing estrus is exhibited when estrogen
levels have started to decline and progesterone levels are increasing. This
triggers two events namely
1. Exhibition of maximal estrus behaviour
2. Initiation of LH surge in the bitch
 Ovulation
o Ovulation occurs 24 to 48 h after the LH surge and occurs over a span of
72 to 96 h. The eggs are released as primary oocytes and require 24 to 72 h
to mature and become fertile. Once mature, their lifespan is 2 to 4 days. At
the time of ovulation the serum progesterone concentration is typically in
the range of 4 to 10 ng/ml.

Diestrus

 Begins with the cessation of standing heat and ends when blood progesterone
levels fall to basal levels. Progesterone levels continue to rise to reach levels of 50
to 60 ng/ml 20 to 30 days after ovulation. The bitch is unique in that whether the
bitch is pregnant or not, the CL continues to remain functional for a particular
period of time. There is no pregnancy recognition system in dogs. Therefore CL
functions throughout normal gestational period regardless of the presence or
absence of fetuses.
 In a pregnant bitch the luteal phase ends with the onset of parturition
(approximately 65 days) while in a non pregnant bitch the CL functions for a
longer period. ie., 75 to 100 days and regression is brought about by aging of CL.
Thus the uterus is not involved in the regulation of the lifespan of the CL. Apart
form LH; prolactin is the major luteotrophic factor especially in the second half of
the luteal phase.

Anestrus

 Anestrus is the phase in which uterus involutes. Average duration is 4.5 months
but may be variable.
 FSH levels are quiet high as equal at levels at proestrus but since follicles never
fully mature but regress, estrus behaviour is not exhibited.
 SYMPTOMS OF ESTRUS IN BITCH

Proestrus

 The bitch is playful

Appears sexually attractive to the male but refuses mounting attempts by the

male by moving away, baring of the teeth and snapping.
 The bitch may keep her tail tight against the perineum and cover her vulva. This
behaviour changes as proestrus progresses.
 The female becomes more receptive, seeks males, playing and teasing but still
refuses the male by crouching or lying down.
 In late proestrus, the bitch may sit or stand passively when mounted.
 The bloody discharge fades and becomes transparent to straw coloured and the
vulva which is swollen and hard during proestrus, now becomes small and soft as
estrus approaches.

Estrus

 The bitch may crouch and elevate the perineum toward the male.
 The bitch attracts males over long distances due to the presence of potent
pheromones.
 The vulva is soft and flaccid and the vaginal discharge is often straw coloured or
pink.

ESTROUS CYCLE AND SYMPTOMS IN QUEEN

Reproductive Cycle

 Cats are polyoestrus

 Several estrous cycles are noticed during each of its 2 or 3 seasons per year

Short haired breeds come to cycle through out the year. Use of artificial light from
September to March to lengthen the `day light hours’ will make the females to cycle all
year around Estrous Cycle

Anestrum

 Females may rebuff approaching Tom cat by hissing and striking out. If she
accepts the tomcat she will flex her spine when he mounts, covers the perineum
tightly with her tail, almost achieving a sitting position instead of lardosis in
estrus
 Olfactory signals from vulvar area are repulsive to some tom cats that turn away
after smelling her perineum
Proestrum

 This is a short phase of one to three days

 Increased rubbing against objects with head and neck (may be misjudged by
owners to be increased friendliness)
 Rubbing is very marked in 36 h of onset of proestrum. Progresses to rolling
(gently or violent) with purring, rhythmic opening and closing of the claws,
squirming and scratching
 The female begins to call a male using the `heat cry’ which is unique to proestrus
and estrus (monotone howling which lasts for three minutes at one time) and is
more prevalent in Siamese females. The female sprays the urine so that both
urine and sebaceous secretions left by rubbing will attract the males

Estrus

 Change in behaviour towards females is noticed

 The females still roll and rub and do not refuse the males. Attempts to mount and
assumes a crouching lardosis (thorax and abdomen touch the floor with
perineum elevated)
 Copulatory stance can also be induced by stroking queens back, thighs or neck
 Tail is laterally displaced and slight amount of serosanguinous discharge on the
vulva is observed
 Lardosis is necessary if intromission is to occur. It is stimulated by the treading of
the mounted male
 While the male performs copulatory thrust the female adjusts position slightly by
alternate treading with hind limbs
 Facial expression is intense and is similar to that seen in aggressive cats. Lasts for
10 seconds to five minutes
 Post mating behaviour is characteristically dramatic
o As the male starts to withdraw his penis following ejaculation, the female’s
pupils suddenly dilate
o As she is freed she utters a copulatory cry- a small piercing vocalization
o She turns aggressively on the male, striking out and hissing.
o The female proceeds into the `after reaction’- violently rolls on the ground
and licks her vulva
o Mating resumes in 11 to 95 minutes.
o Mate as frequently 8 times in 20 minutes or 10 times in one hour
 Another feature useful in reproductive management of cat is that cats are induced
ovulators. Estrus female does not ovulate unless mating occurs. Ovulation can
also be induced by stimulation with males penile spines or by artificial means
such as a glass rod (several insertions – 10 seconds duration 5 –10 minutes apart
over 48 h periods). Successful stimulation will result in aggressive after reaction
 Female will be in estrus for 4-6 days. Most females are receptive on 3 rd and 4th
day. Estrus ends abruptly within 24 h after coitus. If pregnant she will not return
to estrus until next seasonal peak or the next year. Ten per cent pregnant queens
display estrus behaviour (3 – 6th week of gestation). Mating at such times will
result in superfetation
  If a sterile mating occurs during estrus, ovulation and CL formation are induced.
This luteal phase can be termed as diestrus. Therefore there are three possibilities
for a feline estrous cycle:
o Proestrus, estrus (nonbred), interestrus
o Proestrus, estrus (sterile mating ),diestrus , interestrus
o Proestrus ,estrus (fertile mating), pregnancy
 If no tomcat is present, female is in estrus for 10-14 days then, returns to estrus in
2-3 weeks. Estrous cycle averages 29 days long

Metestrus

 Metestrus is actually an interestrus period between two estrous periods if

breeding does not occur.
o A queen in estrus does not always mate even when this is desired by the
owner. There are a number of factors that may prevent mating such as:
 Size incompatibility: A small male may have difficulty mating a
large female
 Unfamiliar surroundings: A tom brought to a new area may be
more interested in `marking’ the territory than in breeding
 Personality: Shy or timid females may reject can aggressive male.
The owner can pet the queen and stimulate her to posture for the
male. Occasionally a queen will reject one male but accept another.

LENGTH OF VARIOUS PERIODS OF ESTROUS CYCLE IN

DOMESTIC SPECIES

Length of Estrous Cycle in Different Species
Species / Stage Estrus Metestrus Dioestrus Proestrus
Cow 12-24 h 3-5 13 3
Mare 4-7 3-5 6-10 3
Sow 2-4 3-4 9-13 3
Ewe 1-2 3-5 7-10 2
Bitch 9 - 75-90

FACTORS AFFECTING ESTROUS CYCLE

Nutrition
  Inanition or starvation caused by lack of energy or nutritional deficiency impairs
or prevents secretions of gonadotropic hormones from pituitary gland thereby
preventing the occurance of cycles.

Seasonal Influences and Light

 Seasonal breeding is regulated by light through the medium of eyes or the

hypothalamus and pituitary gland. The total daily amount of light is important in
control of the onset of estrous cycle. This is called as sexual photoperiodicity.
 In sheep and goat decreased day light influences onset of estrus.
 In mares increased total daily hours of light will hasten the onset of estrous cycle.

Temperature

 Excessive heat during summer months causes decreased thyroid activity which
directly reduces reproductive efficiency.
 In cattle under hot conditions length of the estrous cycle is increased to 25 days
as compared to 20-22 days in cool weather.

Age

 In cattle and swine the young female usually has a slightly shorter length of
estrous cycle than the adult animals.

Systemic Diseases

SUB ESTRUS/SILENT HEAT/QUIET OVULATIONS

 Ovulations occur without behavioural manifestation of estrus. The first and

second ovulation postpartum are often not preeced by behavioural signs of estrus
and are thus are truly 'Silent heats'.
 In heifers, the estrus associated with first ovulation is usually silent.

Causes

 A sub threshold of hormone production or an imbalance between hormones

 Nutritional defficiencies such as defficiency of ß Carotene, Phosphorous, Copper,
Cobalt etc

Diagnosis

 Diagnosis is based on
o clinical history
o rectal palpation of the genital system.

Treatment
  If a mature Cl is present, PGF2α or an analogue followed by Timed Artificial
Insemination (TAI) can be carried out. If a CL is at refractory stage, double
injection PG regimen at 11 days interval could be used.
 Alternatively PRID or other progesterone implants could be used folllowed by
fixed time insemination.

 Severe chronic wasting diseases cause debility and emaciation resulting in

cessation of estrous cycle.

Pathology of Uterus and Cervix

 In cattle, conditions like pyometra, foetal maceration, mummification causes

persistence of corpus luteum and cessation of estrous cycle.

Endocrine Disturbances

 Endocrine disturbances leading to cystic ovaries or pituitary, ovarian and adrenal

tumors affect the length of the estrous cycle.

Miscellaneous Causes

 Pregnancy causes physiological cessation of estrous cycle.

 The presence of males hastens onset of estrus.
 Infusion of iodine compounds into the uterus shortens the estrous cycle.

ANESTRUM

 Failure of estrum or anestrum is the principle symptom of many conditions that

may affect the estrous cycle. Anestrum is observed most commonly either
o After parturition as post partum or Pre service anestrum
o Following service- Post service anestrum (when conception does not
occur).
 It is due to multiple causes and often not directly related to endocrine system.
 Diagnosis is based on
o history
o careful clinical examination of the genital tract and ovaries per rectum and
vagina by a speculum
o physical examination of the cow for accurate differential diagnosis
 The treatment of anestrum has been dealt with in detail under the Module on
Anestrum.

DELAYED OVULATION
 If ovulation occurs more than 18 hrs after the end of heat, then ovulation is said
to be delayed.
 Caused by
o endocrine defficiency or imbalance,
o failure of development of hormone receptors at the target tissue
o mechanical factors.
 Delayed ovulation is generally assumed to be one of the causes for failure of
conception in Repeat Breeders and is diagnosed by palpation of mature follicle on
the ovary more than 24-48 hours after the end of estrum.
 Treatment of delayed ovulation has been discussed in detail under the Module on
ovulatory defects.

IRREGULARITIES OF ESTROUS CYCLE IN MARES

 Irregularities of the estrous cycle in mares may be associated with

o ovarian pathology,
o uterine pathology
o apparent functional abnormalities of the ovarian hypophyseal axis.
 Abnormalities of endocrinology, cyclicity, or ovulation are uncommon in mares
with normal reproductive tracts, and hence, other causes of infertility should be
ruled out before abnormal cyclicity is implicated as a cause of infertility.
 Cystic ovaries, as seen in cattle, do not occur in mares.
 Apparent estrous cycle irregularities that are not associated with pathology
include
o prolonged anovulatory estrus during the transition period,
o “silent heat”,
o prolonged luteal activity,
o estrus during pregnancy,
o “persistent estrus” or behavioral changes attributable to estrus.
 Diagnosis of the cause of estrous cycle irregularities in mares is based on
o history,
o teasing records,
o findings on palpation
o ultrasonography
o determination of hormone concentrations

ASSESSMENT OF ESTROUS CYCLE AND OVULATION

 An important factor to remember when evaluating apparent cycle abnormalities

is the normal variation in length of estrus.
o The normal estrous cycle length ranges from 2 to 12 days.
o The length of cycle is generally repeatable within mares, but is longer at
the beginning and end of the breeding season.
 Teasing is a major determinant of apparent cyclicity.
o Individual teasing with an active stallion is the best method of heat
detection.
 o Should be performed at least three times weekly. The mare’s behavior
should be scored by a knowledgeable individual.
o “Silent heat” may occur in normally cyclic mares.
 Knowledge of the estrus and diestrus reactions of each individual mare is crucial;
a mare in heat may be less demonstrative than another mare in diestrus.
o Some mares may show signs of estrus immediately on contact with the
stallion.
o Some mares may require teasing for 3 to 4 minutes before they respond.
 A mare that shows no change in behavior at all during her cycle should be
examined regularly by transrectal palpation and ultrasonography to detect
estrus.
 Estrus behavior can also be seen in mares
o during seasonal anestrus
o in mares that have been ovariectomized
o in mares with gonadal dysgenesis.
 The length of diestrus is more repeatable among mares than is the length of
estrus, at 15-20 days.
 A pattern of high progesterone for about 15 days followed by low progesterone
four more days is strongly indicative of normal cyclicity.

Ovarian and Uterine Characteristics of Normal Cyclicity

 In assessing the normality of follicle growth and ovulation, the large variation in
follicle size at ovulation should be recognized.
o The size of follicles at ovulation is commonly 35 to 45 mm diameter, but
mares can ovulate much smaller follicles or larger follicles, with normal
fertility.
o Follicle size at oulation is often repeatable for a given mare.
o Follicle size at ovulation also decreases toward the middle of the breeding
season.
o Numerous large follicles and corpora lutea are normally present on the
ovaries of pregnant mares, especially between 30 and 120 days. These are
sometimes mistaken for ovarian pathology.
 Transrectal palpation and ultrasonography are indispensable tools in the
evaluation of the estrous cycle.
 Uterine changes detectable on ultrasonographic examination are also helpful in
estimating the stage of the estrous cycle in mares with questionable estrus
behavior.

PHYSIOLOGICAL ESTRUS IRREGULARITY: THE

TRANSITIONAL PERIOD

 The mare is a seasonal, long-day breeder

 Mares enter anestrus during the winter months.
 The period between late anestrus and the first ovulation of the year is termed the
transitional period.
 Follicular growth occurs due to sufficient Pituitary gonadotropin output but
normal follicle maturation and ovulation do not take place.
 Palpation and Ultrasonography may not be helpful in distinguishing from
normal follicles; however, they may be structurally and hormonally abnormal.
 During the transitional period, mares may exhibit constant or irregular estrus,
as a response to rising and falling estrogen from waves of non ovulatory follicles.
 When estrus behavior does subside, it may recur within days; there is no normal
diestrus interval.
 Diagnosis is based on the season of the year, lack of evidence of ovulation and
multiple small to large follicles present on both ovaries.

CYCLE IRREGULARITIES ASSOCIATED WTH AGING

 Mares cycle less efficiently after about 20 years of age.

 Cycles may cease over 25 years .
 These mares may be presented because of erratic or constant heat during the
breeding season, or because multiple breeding have not resulted in pregnancy.
They may also be apparently anestrus during the breeding season.
 Diagnosis is based on the mare’s age; these changes are not usually seen until the
mare is near 20 years of age.
 Breedings during this time will not result in pregnancy because the mare does not
ovulate
 Treatment with pulsatile gonadotrophin- releasing hormone may be effective in
inducing follicular growth and ovulation.
 Fertility is reduced due to
o decreased oocyte viability and uterine changes,
o longer follicular phase and
o fewer ovulations per year.
 Mares over 25 years of age may cease cycling altogether. These mares may be
presented because of erratic or constant heat during the breeding season, or
because multiple breeding have not resulted in pregnancy. They may also be
apparently anestrus during the breeding season.

PROLONGED LUTEAL ACTIVITY

 A prolonged luteal phase is common in mares.

o normally occurs in 4 to 18 per cent of cycles
o severe damage to the endometrium, as seen in cases of pyometra. If the
damage is severe enough that prostaglandin production is impaired,
retention of the primary CL results.
 Differential diagnosis includes
o pregnancy,
o silent heat,
o poor estrus detection,
o short heats that are missed, especially near the middle of the breeding
season.
 Diagnosis is based on finding a normal non-pregnant diestrus reproductive tract
associated with failure to show estrus or failure during examination and to find
changes consistent with estrus for more than 2 weeks after ovulation.
Progesterone concentrations will be high for more than 2 weeks.
 Treatment involves administration of prostaglandin F2α. To assure a response,
the prostaglandin should be given at least 5 days after the most recent
ovulation. Return to heat occurs in about 3 days.

SHORTENED LUTEAL PHASE

 A decrease in the length of diestrus may be indicative of premature luteolysis.

 May be associated with
o endometritis;
o prostaglandin production associated with uterine inflammation or
bacterial endotoxin production, or both
 If a shortened luteal phase is detected, an endometrial culture and biopsy should
be obtained to determine if endometritis is present, and, if so, which organism
may be responsible.
 Resolution of endometritis should result in return of normal diestrus intervals.

PERSISTENT ESTRUS

 “Persistent estrus” is a fairly common complaint in working and racing mares.

 When evaluating cases of persistent estrus, the normal variation of estrus length,
from 2 to over 10 days, should be considered.
 A major confusing issue in these mares is what is considered to be estrus
behavior. Such mares may be characterized by their owner as being irritable,
kicking when their sides are touched, leaning on the handler, striking, urinating,
or wringing their tails interpreted as heat; however, they may be simply signs of
agitation.
 To diagnose the cause of the apparent estrus behavior, findings on palpation and
ultrasonography of the reproductive tract are evaluated along with the history,
and the mare should be teased with an active stallion.
 Mares with large follicles may respond to administration of human chorionic
gonadotrophin by ovulating approximately 2 days late and going out of heat in
another 1 to 2 days.
 Mares that have constant agitated behavior, rather than true estrous behavior,
may be in any stage of the estrous cycle at the time of examination. When
evaluated critically, although some signs associated with estrus may be present
the behavior of the agitated mare is anxious or guarding. She is resentful of the
approach of a stallion when teased and may lean away from the stallion or appear
fearful. This is in contrast to the mare in true estrus who; during teasing, is
typically calm, submissive, and interested in maintaining contact with the
stallion.
 Abnormal estrus-type or aggressive behavior may be associated with granulose
cell or other ovarian tumors
  Signs of persistent estrus may be attributable to vaginal inflammation due to
aspiration of air into the vagina when the mare is working. These mares lack
perineal fat, which affects tone of the vulvar lips, the angle of the vulva, and the
weight of the perineal body.
 The mare has signs of vaginitis such as frequent urination, hunching the back,
dragging the hind feet, and wringing the tail. Racing mares commonly have a
history of “stopping” or “pulling up” part way through training periods; this may
be due to pneumovagina incurred during work.
 Examination of the tract shows the mare at any stage of the cycle; bright
echogenic particles representing air may be seen in the vagina or uterus or both
during ultrasonographic examination.
 Vaginoscopy may reveal inflammation and the mare may show extreme signs of
irritation after the speculum is inserted or removed. This problem may occur
even after Caslick’s surgery has been performed.
 Abnormalities of the bladder or urethra, such as cystitis or urethral masses, may
result in frequent urination that is interpreted as persistent estrus.

CYCLIC BEHAVIOUR PROBLEMS ATTRIBUTED TO ESTRUS

 Some mares become hard to manage, perform irregularly, or even appear lame
when in heat; this behavior is intermittent and corresponds to specific stage of
the estrous cycle.
 Altrenogest is commonly used to suppress problem estrus behavior in mares and
may be effective.
 Ovariectomies for mares should be reserved only for cases in which the mare has
cyclic behavioral problems corresponding to a specific part of the estrous cycle;
this behavior should seem to improve over the winter.
 Occasionally a mare is presented that has vicious outbursts of aggressive
behavior. Such mares should be approached with caution. Little information is
available on the cause of this behavior but it is unlikely to be related to the
reproductive tract.

CYCLIC IRREGULARITIES ASSOCIATED WITH OVARIAN

PATHOLOGY

Gonadal Dysgenesis

 Gonadal dysgenesis refers to congenital lack of development of the ovaries.

 Only a “streak” gonad is present, with no follicular activity. The remainder of the
tract is intact but juvenile because no ovarian steroids are present to induce
secondary development.
 Condition is most commonly associated with defects of the X chromosome,
including XO and XXX; however. It may be seen in mares with apparently
normal karyotypes.
 Presenting signs include anestrus, erratic estrus, or constant estrus. Exhibition of
estrus behavior is due to lack of progesterone.
  Diagnosis of gonadal dysgenesis is based on history, karyotype and repeated
palpation and ultrasonography or progesterone determination. Mares with
gonadal dysgenesis have never foaled and have never been pregnant. Palpation
and ultrasonography reveal very small or apparently absent ovaries; if ovaries are
present, no follicular activity is seen.
 A chromosomal abnormality on karyotype supports the diagnosis of gonadal
dysgenesis. No treatment is possible for these mares.

ANOVULATION OR FAILURE OF OVULATION

 Failure of ovulation is often preceded by an anovulatory estrus, with follicle

regressing and become atretic. Sometimes however a follicle does not regress,
but having reached its maximum size of 2.0-2.5 cm in diameter the walls become
luteinized. This structure behaves the same way as CL either regressing after 17-
18 days or frequently much earlier so that the cow returns to estrus at a shorter
than normal interval.
 Diagnosis of anovulation can only be made retrospectively by noting on rectal
palpation that a follicle persists longer than one would have suspected. In cases of
lutenized follicle, it will remain for 17-19 days before regressing, the ovary
containing it will be rounded, smooth, fluctuating rather than irregular and solid
as it is with a CL.
 Treatment is directed towards ensuring that ovulation occurs at the next estrus.
Hence, hCG or GnRH is given. If ovarobursal adhesions are present, there is no
treatment.

ABNORMALITIES OF ESTRUS

SPECIES ABNORMALITY CAUSES PHYSIOLOGIC MECHANISM
CATTLE Anestrus Pyometra, Maintenance of CL
mummification
Lactation Suckling stimulus inhibits
gonadotropin release
Cystic Ovaries Deficiency of LH and/or GnRH
Ovarian Failure to produce ovarian
hypoplasia and estrogens
Freemartinism
Nutritional and Gonadotropin production by
vitamin anterior pituitary
deficiencies
Subestrus, Silent High lactation
estrus
Nymphomania Cystic ovaries Endocrine imbalance
SHEEP Anestrus Season, Lactation Effect of photoperiod on
gonadotropin secretion
SWINE Anestrus Lactation As for cattle
HORSE Anestrus Season, diet, As for sheep
ovarian
hypoplasia
Prolonged estrus Early in breeding Failure of follicle beyond 2 cm to
season develop that is due to inadequate
endocrine stimulus
Split estrus, silent Pseudopregnancy Early pregnancy failure with
estrus, lack of persistence of corpus luteum
estrus Prolonged Persistence of corpus luteum
diestrus after
foaling

MODULE-5
SEASONAL BREEDING

 In most wild animals the breeding season is initiated at a time when the
environment is suitable for the maximum survival of the young at their birth.
 Some species have only one period of estrus each year (monoestrous) while some
have series of estrous cycles limited to a portion of the year (Seasonally
polyestrous).
 True seasonal breeding are inherent in ewe, does and mares.

SHEEP AND GOATS

 Most breeds of sheep and goat exhibit seasonal breeding pattern. However, those
in the tropics as an exception will cycle throughout the year.
 Sheep are short day breeders
 Breeding season is initiated with decreasing length of daylight and ends when
increasing day length reach a ratio of nearly equal daylight and darkness.
 Ewe-lambs and yearling ewes have shorter breeding seasons than older ewes.
 The onset of breeding season can be advanced by
o Artificial manipulation of the photoperiod and by use of hormonal agents.
o Introduction of rams into a flock during the transition from anestrus to
estrus will result in high degree of synchrony in first mating with estrus
peaking 15-20 days after introduction of the male.
 As with sheep, goats are short day breeders with cyclic activity occurring between
late June and early April. Placement of bucks with does just before start of the
breeding season will stimulate estrus and result in good synchrony.
 Both rams and bucks are affected by photoperiod showing highest breeding
activity and fertility in the fall. The day length pattern has a dominant controlling
influence on initiation and termination of breeding season.
 HORSES

 Mares are long day breeders whose seasons are initiated as the ratio of daylight to
darkness increases and ends during decreasing day length.
 Behavioural estrus that occurs during the short day months from spring to
autumn is not frequently accompanied by ovulation.
 The seasonal breeding pattern is not as well defined for stallion. Fertile semen
can be collected throughout the year.
 However, during months of short photoperiod there is a decline in sexual activity
and semen production.


PHOTO PERIOD ACTION

 The role of photo period in regulating seasonal breeding activity is well

established. As breeding season approaches there is an increase in frequency and
amplitude of episodic surge of LH.
 The sensor of photo periodic response change in mammals is the retina of the
eye. The nerve impulse from these photic signals is transmitted from the retina
along the retino hypothalamic tract to suprachiasmatic nuclei, located anterior to
the hypothalamus, and then to the superior cervical ganglia near base of the brain
from which arise the sympathetic nerves that innervate the pineal gland.
 The diurnal rhythm of secretory activity of the pineal gland is generated by these
suprachiasmatic nuclei. Darkness causes increased sympathetic activity of pineal
activity which increases the secretion rate of melatonin whose secretion has been
demonstrated only in seasonal breeders.
MODULE-6 
 PREGNANCY DIAGNOSIS IN FARM AND COMPANION
ANIMALS: PART I
 Accurate and early pregnancy detection is an important part of monitoring or
controlling herd fertility
 Early pregnancy diagnosis will help to detect those that are not pregnant so that
they can be inseminated again or culled from the herd

HORMONE PATTERNS IN BLOOD OF DOMESTIC ANIMALS

Cow

(Cycle 21-22 days; Pregnancy 277-300 days)

 Progesterone
o Values vary from 0.44 ± 0.17 ng/ml at estrus to a peak of 6-7 ng/ml from
days 9-16 of the estrous cycle and then falls to base line.
o During first 14 days of pregnancy, values are the same as during 14 days of
proestrus.
 o Cycling animals show a decline, while pregnant animals attain values at or
somewhat above cycling maxima which is then maintained for most of
pregnancy.
 Estrogens
o The major estrogens are estradiol-17 alpha, estrone and estradiol-17 beta.
o Mated and pregnant cows show values less than 5 pg/ml from day 3-39.
o Between days 140 and 245, values were reported to below or about 100
pg/ml which then rises after day 250.

Sheep

(Cycle 16.4-17.5 days; Pregnancy 144-152 days)

 Progesterone
o Serum values vary from 0.12 ng/ml (equivalent to anestrus) to 2.0 ng/ml
from 10-14 days of cycle.
o After mating, pregnant and non-pregnant values do not differ till day 16.
o Values rise in pregnant ewes to 2.5 ng/ml at day 50, 12 - > 20 ng/ml at
days 125-130 reach a plateau, and finally fall steeply to the day of lambing.
 Estrogens
o Pregnancy levels were low (in comparison with the goat), remaining less
than 50 pg/ml for most of the period and rising to 100 pg/ml just 1 day
before birth.
 Luteinizing hormone
o Levels are less than 1 ng/ml until 20 th day of pregnancy. At days 123-128,
no maternal blood LH can be detected
 Prolactin
o Levels in blood of pregnant ewes range between 20-80 ng/ml during the
first 20 days.
o At 2 days prepartum a sharp rise begins, reaching 400 ng/ml on the day of
parturition.

Pig (Sow)

(Cycle 20-22 days; Pregnancy 112-115 days)

 Progesterone
o Levels increase from 1 ng/ml to a peak of 35.4 ng/ml on day 12 of
pregnancy, then decline to 17.2 ng/ml on day 24.
o In the final 20 days before birth, the levels are 8-14 ng/ml, with possibly a
tendency to decrease in the last few days, and a fall to less than 1 ng/ml at
birth.
 Estrogens
o Estrogens remained around 20 pg/ml for the first 24 days of pregnancy
o Between the 20 th and 10 th day prepartum, the level was about 100 pg/ml,
peaked to 300 pg/ml at days -2 to -1, and fell to zero after delivery of
piglets.
Goat (Doe)

(Cycle 21 days; Pregnancy 149 days)

 Progesterone
o Levels rise gradually to 33 ng/ml by the 90 th day and then fall to 7 ng/ml
on the 140 th day, 3 or 4 days before parturition.
 Estrogens
o Both estrone and estradiol 17- beta are present throughout pregnancy.
o Levels are 5 pg/ml on days 0-30; 47±15 pg/ml on days 39-48; 272±35
pg/ml on days 79-88; 451±70 pg/ml on days 119-128 and 622±78 pg/ml
on days 139 - 148 (term).

Horse (Mare)

 Progesterone
o Beginning with the day of estrus and insemination, progesterone
values are as follows: day 0, 1.1 ng/ml; day 8, 7.5 ng/ml; day 28, 4.9
ng/ml; days 52-120, plateauing at 11-14 ng/ml with a maximunm of 15.2
ng/ml on day 64; day 150, 7.0 ng/ml; day 180, 2.0 ng/ml. Following 180
days, with minor fluctuations, values remained below 1 ng/ml until the
final 30 days of gestation.
 Estrogens
o For group E-1, constituted by estrone, equilin and equilenin, amounts
observed till day 80 are 10-20 pg/ml; day 90, 40 pg/ml; day 120, 275
pg/ml; day 210, a peak of 828 pg/ml.
o A fall in level to 370 pg/ml on day 300 and about 150 pg/ml close to
parturition is observed.
o Values for group E-2, constituted by estradiol-17 alpha and estradiol-17-
beta, a similar time course at about 10% of the amounts of E-1 are
observed

 .

DIFFERENT METHODS OF PREGNANCY DIAGNOSIS

Different methods of pregnancy diagnosis in Cattle, Sheep and Goats have been dealt
with in detail under the following headings

COWS SHEEP AND GOATS

 Management Method  External Palpation

 Abdominal Ballotment
 Physical Method  Recto Abdominal
 Laboratory Method Palpation
 Ultrasonography and  Radiography
 Fetal  Ultrasonography
Electrocardiography  Hormonal Assay
 Prostaglandin Induced
Milk Flow Test
(PGIMFT)

PREGNANCY DIAGNOSIS IN CATTLE

SIGNIFICANCE

 Dairy cows should calve for the first time approximately 24 months of age and
should deliver subsequent calves at intervals of approximately 13-13.5 months.
Thus, dairy cows should conceive within approximately 4 months or less of
calving in order to get the maximum economic returns.
 An ideal test would accurately detect pregnancy before the first expected estrus
after insemination (about 21 days) so the cow could be re-inseminated without
further loss of time.
 Unfortunately no tests are currently available that are practical to use and allow
detection of pregnancy in cows prior to the first expected estrus.

MANAGEMENT METHODS

 A presumptive pregnancy diagnosis by cattle owners is based on history and

clinical signs.

a) Exposure to a Bull or Artificial Insemination

 History of a cow with a bull or the observation of mating or AI is not a reliable

indicator of pregnancy status and may sometimes be deceptive. Reasons are
o even with high fertilization rates only about 50% of the inseminations
result in detectable pregnancy
o due to unobserved, unplanned, or unrecorded matings, many cows
presented with a complaint of infertility are found to be pregnant on rectal
palpation

b) Cessation of the Estrous Cycle

 Around day 15 to 17 after ovulation, bovine embryos signal their presence as a

result the corpus luteum is maintained, and the maternal estrous cycle is
 suspended. Thus, failure to return to estrus approximately 18 - 24 days after
mating, suggests that conception has occurred.

PHYSICAL METHODS

RECTAL PALPATION

When to Palpate for Pregnancy?

 First palpation should be made between 35 - 42 days after artificial insemination.

 All cows that have not returned to estrus by 60 days post breeding should be
palpated a final time.
 After about 60 days, fetal death rates are low and most, but not all; cows that are
found to be pregnant after this proceed to term and deliver calves.

Palpable Changes Per Rectum

 Although number of changes occur in the size, texture, location and content of
the uterus during pregnancy, there are four positive signs of pregnancy
that are detectable by rectal palpation, and examiner must detect at least one of
these four signs before declaring the cow pregnant.
 The four positive signs of pregnancy in cows are:
o Palpation of the fetal membrane slip
o Palpation of amniotic vesicle
o Palpation of placentomes
o Palpation of fetus

PALPATION OF FETAL MEMBRANE SLIP

 It is best performed from 35-90 days of gestation.

 It is valuable in the differential diagnosis of pregnancy from uterine diseases
characterized by fluids causing uterine distension, eg. pyometra or mucometra.

Double Slipping can be performed by two techniques:

 Fincher Technique consists of gently picking up and pinching or compressing

either horn of the uterus and feeling the fetal membranes, the allantois chorion,
slip between the thumb and the fingers before the uterine wall escapes from
between the fingers.
 Zemjanis Technique is grasping the entire horn and letting it slip through the
fingers so that the connective tissue band allantoic vessels on the lesser curvature
of the uterus which are three or four times thicker than the chorioallantoic
membrane could be palpated.

PALPATION OF AMNIOTIC VESICLE

  The amnion contains the developing conceptus and the amniotic fluid and is
palpable as early as 28 days after conception in heifers and by 32-35 days in
pluriparous cows.
 The vesicle is recognized as a nearly spherical, turgid, fluid filled structure that is
approximately 1 cm in diameter at 28 days and increases in size as pregnancy
advances.
 It is detected by encircling the uterine horn with the thumb on one side and the
fingers on the other.
 The vesicle is free floating within the uterus but is most commonly found at the
cranial edge of the inter-cornual ligament.
 The amniotic vesicle becomes progressively less turgid and is difficult to
recognize by about day 65 of gestation. At that time the vesicle softens and the
fetus becomes palpable.
 In a bovine conceptus, the heart is external until approximately day 42; therefore,
caution must be exercised when attempting to detect early pregnancies and
undue pressure must not be applied to the amniotic vesicle, as rupture of the
embryonic heart or other fragile organs may result.

PALPATION OF FETUS

 Fetus becomes palpable at approximately 65 days when the amniotic membrane

loses its turgidity and remains theoretically palpable for the balance of gestati
 In early stages of gestation fetus can be grasped directly. Later, the fetus is
detected by ballottement; the examiner sets the fetal fluids in motion by rocking
the hand against the uterine wall and recognizes the fetus as it rebounds against
the hand
 The fetus is easily palpable as a free floating firm object within the fluid-filled
uterus during the first 4 months of gestation
 As pregnancy advances, increased weight of the fetus and fluids pulls the uterus
ventrally and cranially until the fetus comes to rest on the abdominal floor during
the fifth and sixth months. Continued growth of the fetus positions it closer to the
maternal pelvis during the last trimester (period of ascent) and palpation of the
fetus is facilitated.

SUPPORTING SIGNS OF PREGNANCY

 Asymmetry of the uterine horns

 Resilience and fluctance of the uterine wall
 Fixation of the cervix

LABORATORY METHODS

Hormone Estimation

 Progesterone in milk and plasma

  Estrone sulfate in milk

Proteins

 Bovine Pregnancy Specific Protein -B

 Immunosuppressive Early Pregnancy Factor

PROGESTERONE IN MILK AND PLASMA

 Robertson and Sarda (1971) described a method of diagnosing pregnancy by

determination of progesterone concentration in the plasma of cows.
 Measured using Radioimmunoassay (RIA) or by various assay kits that are
available for on-farm use.

Basis

 In cows, progesterone (P4) from corpus luteum is required to maintain

pregnancy. If pregnant, the cow does not return to estrus and P 4 concentrations
in blood and milk are elevated at 20-24 days post insemination. Conversely, if
pregnancy fails, P4 concentration is elevated until approximately day 17 when
luteolysis is followed by a sharp decline in P4 concentration by day 20 and return
to estrus.
 Optimum time for collecting the milk sample: 24 days after breeding
 If P4 concentrations are low in blood or milk samples assumed to be non-
pregnant, if elevated assumed to be pregnant.

Cow Side Tests

 Developed to obtain results within 1 h of collecting milk sample.

 All necessary reagents and equipment are provided in kit form.
 Semi quantitative or fully quantitative tests are available which requires
minimum equipments and some expertise.
 Both tests are based on the enzyme linked immunosorbent assay (ELISA).

Assay Procedure

 Milk sample to be tested (containing unlabelled P4 if the cow is pregnant or in

dioestrus) is added to the plastic well of the microtitre plates precoated with a
specific P4 antibody, together with a fixed quantity of P4 labelled with an enzyme
(usually alkaline phosphatase).
 After a period of incubation, the contents of the wells are washed; however, P 4
will remain bound to the antibody in the well.
 A substrate reagent is then added to the well which, after the second incubation
period, reacts with the enzyme-labelled P4 to produce a colour reaction.

Interpretation
  Colour is assessed visually or using a spectrophotometer by comparison with
those produced by known standard solutions of P4.
 The amount of labelled P4 that remains bound to the antibody on the wells is
inversely proportional to the amount of unlabelled P 4 in the milk sample.
 Light colour reaction indicates high the concentration of P4 in the
unknown milk sample.
 Most intense colour reaction indicates Zero P4 in the unknown milk
sample

Accuracy

 Pregnant: 80 and 88%

 Non- pregnant: Nearly 100%.

Limitations in Using the Assay On-Farm

 Instructions are not readily understood by non-technical personnel’s.

 Requires skill and ease in using simple equipments.
 Timing of incubation and quantities of reagents should be strictly followed.
 Kits should be stored at 4 °C and before use allowed to warm to room
temperature, and not be heated.
 Interpretation of the colour differences can be difficult for some persons.
 Milk samples should be kept at 2-8 °C until assayed and the recommended
preservative tablets must be

ESTRONE SULPHATE IN MILK

 Estrone sulfate is a product of the placenta and is present in the milk of pregnant
cows in concentrations sufficient to differentiate between the pregnant and non-
pregnant cows after approximately day 100 of gestation.
 Practically, however, assays for estrone sulfate are not useful for early detection
of pregnancy and offer no substantial advantage over other methods except in the
case of a few cows in which rectal palpation cannot be performed.

BOVINE PREGNANCY SPECIFIC PROTEIN-B (bPSPB)

 In bovines a pregnancy-specific protein (bPSPB) secreted by the trophoblastic

cells has been isolated and purified.
 RIA for measurement of bPSPB have been developed and used to differentiate
pregnant from non-pregnant cows.
 Concentrations of bPSPB are detectable in a few cases as early as 15 days after
insemination and in nearly all pregnant cows by 24 days after insemination.
 The protein increases in concentration as gestation advances and is detectable
until parturition.
  Concentrations of bPSPB are higher in twin pregnancies than in single
pregnancies, but individual variations did not permit accurate prediction of fetal
numbers.

ULTRASONOGRAPHY AND FETAL ECHOCARDIOGRAPHY

ULTRASONOGRAPHY

 In animals, transducers of 5 MHz and 7.5 MHz frequencies are most widely used
for transrectal ultrasonography.
 Lower frequency transducers are capable of penetrating greater depths of tissue
but are not capable of resolving small structures.
 Higher frequency transducers are capable of resolving smaller structures but do
not penetrate deeply through tissues.
 Under practical conditions, ultrasonography with 5 MHz transducer is an
accurate method for pregnancy diagnosis after approximately day 24. A 3.5 MHz
transducer is found to be reliable after day 30.

FETAL ECHOCARDIOGRAPHY

 It is not applicable before 5 months of gestation, but might have application for
the diagnosis of multiple pregnancies. Refer to practical module on
Ultrasonography for further details on instuments, technique etc.

PROSTAGLANDIN INDUCED MILK FLOW TEST [PGIMFT]

 Labussiere et.al. (1992) reported diagnosis of pregnancy in cows based on the

observation of milk ejection which in the case of CL maintenance results from the
release of luteal oxytocin induced by intravenous administration of a non-
luteolytic dose of PGF2 alpha .
o Examine all cows per rectum on day 18 post insemination to assess
ovarian status.
o Perform PG-IMF test on the same day 3 hours after evening milking.

Preparation of Non-Luteolytic Dose of PGF 2 alpha

 One ml of PGF2 alpha (lutalyse) which contains 5000 micro gram is reconstituted
in 39 ml of distilled water to arrive at a final concentration of 125 micro gram/ml.

Protocol

 After washing the udder and teat, a sterile cannula is placed in the left fore teat to
empty the cistern milk.
 Subsequently a non-luteolytic dose of 125 micro gram of PGF2 alpha is
injected through the ear vein.
 After the injection the time duration of milk flow is recorded.
Inference

 Elicitation of milk let down reflex with free flow of alveolar milk within a few
seconds after injection and lasting for 3-5 minutes is considered to have a
functional CL/presence of conceptus.
 Absence of milk flow is indicative of non-functional CL.

Reason

 The prostaglandin induced milk let down response observed in pregnant animals
could be attributed to the release of endogenous luteal oxytocin that was actively
synthesised by the luteal tissue and was available for immediate release in
response to PGF2 alpha resulting in alveolar milk ejection immediately by
increasing the intra-mammary pressure within the cistern and enlargement of
cisternal volume.

SUMMARY OF DIFFERENT METHODS


SUMMARY OF METHODS OF PREGNANCY DIAGNOSIS IN COW
Methods Earliest time
(in days)
1. Early pregnancy factor (EPF) / early conception factor (ECF) 3
2. Real-time ultrasound (direct imaging) 13
3. Failure of return to estrus and persistence of corpus luteum 21
4. Progesterone concentration in plasma and milk 21-24
5. Assay of pregnancy specific protein -B 24
6. Palpation of allantochorion (membrane slip) 33
7. Unilateral cornual enlargement and disparity in size, thinning of 35
the uterine wall, fluid filled fluctuation of enlarged horns
8. Palpation of the early fetus when the amnion loses its turgidity 45-60
9. Palpation of the caruncles/cotyledons 80
10. Hypertrophy of the middle uterine artery until presence of 85
fremitus
11. Estrone sulphate in milk 105
12. Palpation of the fetus 120
  .

PREGNANCY DIAGNOSIS IN SHEEP AND GOATS

 Methods Commonly Used for Pregnancy Diagnosis



Clinical Techniques

 External palpation
 Abdominal ballotment
 Recto-abdominal palpation technique (Hulet's technique)

Other Techniques

 Radiography
 Ultrasonography
 Hormonal assays

BIMANUAL PALPATION TECHNIQUE

 This method involves digital palpation per rectum combined with abdominal
manipulation
 Does are examined in the morning before feeding and watering
 Obese animals are fasted over night
 Restrain the animal in standing position by an attendant holding the head
 The urinary bladder is emptied before examination
 Sitting at the level of pelvic region on the right side of the animal, the examiner's
pre lubricated, gloved index finger of the left hand is introduced in to the rectum
 Fecal pellets are removed and a distended urinary bladder is evacuated by gentle
recto-abdominal pressure
 The right palm is held vertically, with the finger tips touching the ventral floor of
the posterior abdomen; it is then lifted upwards to move abdominal organs
forward
 Then, using regulated forward, upward, and backward movements, the
reproductive tract within the pelvic cavity could be held in palm of the right hand
 Examination is performed per rectum using the left index finger assisted by the
fingers of the right hand
 The size, shape, consistency and surface characteristics of the vagina, cervix,

RECTO-ABDOMINAL PALPATION

 This method was described by Hulet and hence the name, Hulet's Technique.
 This technique involves use of a glass or steel rod (50 cms long and 1.5 cms
diameter) inserted in rectum.
  A soap enema is given 5 minutes before examination to evacuate the rectum.
 The ewe or doe is turned on her back.
 The probe is lubricated with paraffin oil and carefully inserted approximately 30
cms inside the rectum.
 Left palm is placed on the abdominal wall and the rod is moved to and fro in a
horizontal plane with the right hand.
 uterine horns and adjoining structures are then assessed
 Palpation of the ovaries is performed in the same manner, pressing the index
finger per rectum against the right fingers
 Once the uterine horns are palpated, the ovaries are easily located and palpated
lateral to the center of the coils as small oval bodies on each side
 Palpation is continued to assess their size, shape, mobility and any other gross
structural abnormalities

nference

 If the rod is palpable as it moves slowly with no obstruction to its passage

across the abdomen from side to side, the ewe is considered non-pregnant. If a
palpable mass is detected with the free hand through the abdominal wall on
one or both sides, the ewe is pregnant and the number of fetuses assessed
according to the size and position of masses, wherever possible.
 The rectum and uterus should be carefully examined for injury or damage
following examination.

ULTRASONOGRAPHY

 Ultrasonographic determination of pregnancy has been well documented in

sheep and has been reported to some extent in goats and has been discussed
under the following headings
o Preparation and restraint
o Speed and accuracy of the procedure
o Transrectal ultrasonography
o Transabdominal ultrasonography
 The choice of the transducer is typically between the linear array and sector
 Transducers of 3 to 5 MHz frequencies are most commonly employed.
 Quite reliable from 30 days of gestation to term with transabdominal use of
sector scanners.
 Identification of single vs multiple fetuses are most accurate from 45 to 90 days
of gestation.

PREPARATION AND RESTRAINT

 Sheep are most commonly scanned in the right inguinal wool-less area in the
standing position.
  Goats have some hair in this area and may need to be clipped for best results.
When large numbers of goats are being checked, acceptable scans can be
obtained without clipping if a thick coupling lubricant is used.
 Dairy goats are usually restrained in stanchions or against wall.
 Large numbers of sheep and goats are usually moved through a chute designed
for pregnancy testing.
 Restrict feed for 12-24 h before they are scanned to reduce gas and ingesta in the
intestinal tract, which may interfere with visualization of pregnant uteri.
 In advanced pregnancy or in marginal nutrition animals, care must be taken not
to induce pregnancy toxemia.

SPEED AND ACCURACY OF THE PROCEDURE

Speed

 Experienced sonologist with good facilities can pregnancy test 300 or more
females per hour
 The separation of females into open, single or multiple groups is more time
consuming, with 60-120 animals per hour
 Sonologist should not sacrifice accuracy for speed

Accuracy

 The accuracy of identifying single vs. multiple fetuses typically approaches or

even exceeds 90% if females are within the 45–90 day range of gestation
 The identification of females carrying three or more fetuses is much less accurate,
usually only about 50%

TRANSRECTAL ULTRASONOGRAPHY

 A small amount of lubricant is applied to the end of the linear - array transducer,
which is then inserted in to the rectum.
 The operator’s index finger maintains contact with the transducer and is used to
orient the direction of the scan.
 The transducer is directed ventrally and then rotated approximately 45° to each
side.
 Some operators like to stiffen the cord behind the transducer when it is used in
this manner by taping a rigid tube around it to provide leverage.
 Urinary bladder is identified as a non-echogenic landmark.
 Gravid uteri are identified by non-echogenic areas, a fluid filled structure anterior
and ventral to the urinary bladder.
 Transrectal imaging allows visualization of pregnancies as early as 15 days post-
breeding; but routinely observed by 19-22 days post-breeding.
 After approximately 50 days of gestation, fetuses are too large to be completely
visualized on the screen. After this stage of gestation, placentomes are the most
easily identified cardinal sign of pregnancy.
 TRANSABDOMINAL ULTRASONOGRAPHY

 Accurate estimations of gestation length can be made by measuring the biparietal

diameter of fetuses between 40-100 days of gestation.
 Gestational age can be estimated within approximately one week by visualization
of the diameter of the uterine lumen, crown-to-rump length of the embryo or
young fetus or thoracic or skull size of the fetus up to approximately 90 days of
gestation.
 Placentome size provides some guidance, but will vary depending on the position
of the placentomes scanned in relation to the umbilicus of the fetus.

MODULE-7
PREGNANCY DIAGNOSIS IN FARM AND COMPANION ANIMALS-
PART II

METHODS OF PREGNANCY DIAGNOSIS

MARE SOW

o Management o Management methods

o Clinical o Transrectal palpation
o Ultrasonography o Ultrasonography
o Laboratory o Laboratory methods

PREGNANCY DIAGNOSIS IN MARE

Management

 Failure of return to estrus is a good sign that a mare is pregnant

 This requires the presence of a teaser stallion
 It is preferable that mares should be accustomed to the teasing routine
 Teasing should commence 16 days after service and continue for a further 6 days

False Positives will Occur:

 If the mare has a silent heat, a common problem when the foal is with mother
 If the mare becomes anestrus as a result of lactation or environmental factors
  If the mare has a prolonged diestrus and yet has not conceived
 If the mare has a prolonged luteal phase associated with embryonic death; this is
referred to as'pseudopregnancy'.

False Negatives will Occur:

 A few mares will show estrus although they are pregnant.

CLINICAL METHODS

Rectal Palpation

 Uterine tone is marked at 17-21 days of pregnancy when the cornua can be
palpated as resilient tubular organs
 Palpation of the conceptus is first possible at 17-21 days when it is a small soft
swelling of 2.4-2.8 cm
 At about 100 days it is often possible to ballot the fetus as it floats in the fetal
fluids of the uterine body
 False positive:
 confused with pyometra
 incomplete involution
 pseudopregnancy
 False negative:
 confusion over service date
 uterus not palpated completely
 A portion of the corpus luteum is only palpable for a few days after ovulation in
the region of the ovulation fossa before it is covered by the dense fibrous ovarian
tunic
 Ovulation occurs commonly, 52-63 % in the left ovary, about 60 % or more of the
fetuses develop in the right horn
 Based on the ovarian changes the gestation period may be divided in to 4 periods
as follows:
o Period-I: (ovulation to 40 days) characterized by the presence of a single
CL of pregnancy and a number of various sized follicles on both ovaries.
o Period-II: (40-150 days) characterized by marked ovarian activity with as
many as 10-15 follicles (over 1 cm in diameter) and formation of 3-5 or
more accessory corpora lutea in each ovary. This ovarian activity with
follicle and corpora lutea formation is probably produced by the high level
of gonadotrophic hormones secreted by the uterine endometrium from
40-120 days of gestation.
o Period-III: (150-210 days) characterized by regression of the corpora lutea
or absence of follicles.
o Period-VI: (210 days to foaling) no corpora lutea or follicles are present.

ULTRASONIC METHOD
  In mares, three types of ultrasound are used for pregnancy diagnosis.
o Ultrasonic fetal pulse detector
o Ultrasonic amplitude depth analyser (A-Mode)
o Brightness Mode (B-Mode) ultrasound
o
 During these latter two periods steroid hormones produced in the placenta
maintain gestation.

LABORATORY METHODS

 Milk or Blood Progesterone.

 Blood Estrogens.
 Immunologic-Gel Diffusion or Haemagglutination-Inhibition Techniques.
 Biological Methods
o There are two tests performed for detecting pregnancy in mares.
 Ascheim-Zondek test
 Mucin Test or Kursowa Method
 Chemical Tests
o Chemical methods are used for detecting the presence of estrogenic
hormone in the urine of pregnant mares.
o There are two tests:
 Cuboni's Test
 Lunaa's Test

ASCHEIM-ZONDEK (AZ) TEST

 Used for detecting the presence of gonadotrophic hormones in the mare serum.
 The gonadotrophic hormone from the endometrial cups is first found in the
serum from 40-120 days of gestation, reaches its maximum between days 50-80,
gradually declines and is absent after 150 days.

Normal Values

 At 40 days : 21,000 IU and 0.1-0.6 IU/ml

 At 80 days : 73,000 IU and 6-296 IU/ml
 At 120 days : 42,000 IU and less than 0.5-106 IU/ml

Materials Required

 About 10-20 ml of blood drawn from the jugular vein, after clotting and
separation of serum at room temperature, the serum should be removed and
refrigerated. Avoid overheating of the sample.
 One or preferably two or three immature female rats of 22 days of age.

Test Procedure
  Two ml of fresh blood or serum from the mare to be tested are injected
intraperitoneally or 0.5 ml of serum can be injected s/c daily for 2-4 days.
 The animals are sacrificed 72 h later (when injected intraperitoneally) or 96-120
h later (when injected s/c).

Result

 In positive cases: Haemorrhagic spots or corpora haemorrhagica on the

ovaries, edema of the uterine horns (2-4 times the normal size).
 In negative cases: No definite changes in the ovaries or uterus.
 False positive cases: May be due to over heating of the serum or storage for
too long a period at room temperature.

Accuracy of the Test

 42-50 days of pregnancy : Good

 50-80 days of pregnancy : High
 80-120 days of pregnancy : Good

Testing before 40 days and after 120 days of gestation may be inaccurate due to low
level of circulating gonadotrophins in the blood.

MUCIN TEST OR KUROSAWA METHOD

 A pregnancy test that utilizes changes in the cervical-vaginal mucus was

developed by Japanese workers in the 1920s and 1930s.
 Applicable over a long period of pregnancy.
 The test is as follows
o Spread a sample of mucin from the cervical os on to a glass slide
o The mucous smear is fixed in alcohol, dried, and stained with methylene
blue or hematoxylin

Inference

 Smears from pregnant mares are thick and dark and contain globules of mucous
and epithelial cells
 Smears from non-pregnant mares are thin and pale and do not contain globules
of mucous
 Kurosawa method must be used with caution during the anestrous season. At
that time the smears contain mucous globules, but differentiation can be made
between anestrus and pregnancy by the absence of epithelial cells in the anestrus
condition
 The reliability of the test in pseudopregnancy has not been determined
 False positives are likely
 CUBONI'S TEST

 To 15 ml of urine add 3 ml of concentrated Hydrochloric acid in a 100 ml conical

flask.
 Place in a boiling water bath for 10 minutes and cool under a tap.
 Add 18 ml of benzene with vigorous shaking for at least half a minute and the
supernatant solution mainly the benzene is poured off.
 Then add 3-10 ml of concentrated sulfuric acid.
 Place in a boiling water bath at 80°C for 5 minutes and shake at intervals during
this time.
 The mixture is then cooled.

Result

 Positive result: Presence of dark, oily green fluorescent colour in the lower layer
of the sulfuric acid.
 Negative result:Absence of fluorescence and presence of brownish colour.

LUNAA'S TEST

 This test is a refinement of Cuboni's test.

o Add 1 ml of urine to 10 ml of distilled water in 100 ml flask.
o Add 15 ml of concentrated sulfuric acid.
o After 3-5 minutes the flask is cooled.
o A strong, narrow, 3-5 mm flash light beam is placed close to the flask in a
dark room.
o Positive: Presence of light green fluorescence.

METHODS OF PREGNANCY DIAGNOSIS IN SOWS

 Different methods have been used to detect pregnancy in the sow. They are as
follows:
o Management Methods
o Clinical Method - Transrectal Palpation
o Ultrasonographic Methods
o Laboratory Methods
 Vaginal biopsy
 Plasma progesterone assay
 Plasma estrogen assay

MANAGEMENT METHOD
  Failure to return to estrus at 18-22 days after service or artificial insemination is
considered as pregnant.
 This may also be due to a reluctance to show signs, anestrus or ovarian cysts.
 Early identification of pregnant sow or gilt is essential so that breeders can certify
before sale, she can be served again, treated or culled.

TRANSRECTAL PALPATION

 Need less restraint

 Preferably to be done when the animal is feeding
 Not possible to perform in gilts because they are too small, and even in large sows
a slender arm is advantageous
 Reliable on per rectum palpable changes of the cervix, uterus and middle uterine
arteries

0-21 Days of Gestation

 Cervix and uterus feel very similar to their state at diestrus

 Bifurcation of the cornua becomes less distinct and the uterus becomes slightly
enlarged, with soft walls
 Middle uterine artery (MUA) increases to approximately 5 mm in diameter
towards the third week. It is located as it passes across the external iliac artery
(the latter can be identified as it runs along the anteromedial border of the ilium
towards the hind leg, ventrally and slightly posteriorly; it is about 1 cm in
diameter in the adult sow) running forwards towards the abdominal cavity.

21-30 Days of Gestation

 Cornual bifurcation is less distinct, the cervix and uterine walls are flaccid and
thin.
 MUA is 5-8 mm in diameter and more easily identified.

31-60 Days of Gestation

 Cervix feels like a soft-walled tubular structure.

 Uterus is ill defined and thin-walled.
 MUA enlarged to about the same size as the external iliac. Fremitus can be first
identified at 35-37 days; the pulse pattern comparable with that of the external
iliac artery.

60 Days to Term

 MUA is greater in diameter than the external iliac and it has strong fremitus; it
crosses the external iliac artery more dorsally than before.
 Only towards the end of gestation it is possible to palpate piglets at the level of
the cornual bifurcation.
Accuracy

 Improves with experience and advancing pregnancy. Between 30-60 days of

gestation:
o Positive: 94% and Negative: 97%
o Positive: 99% and Negative: 86%

ULTRASONOGRAPHIC METHODS

Fetal Pulse Detector (Doppler)

 Earliest diagnosis using a rectal probe - about 25 days of gestation.

 Accuracy
o Pregnant sows: 92-100%
o Non-pregnant sows: 25-100%

Ultrasonic Amplitude-Depth Analysis (A-mode ultrasound)

 More reliable

B-Mode Direct Imaging

 Very successful.
 The transducer probe is applied to the abdominal wall of the standing sow about
5 cm caudal to the umbilicus, to the right of the midline and just lateral to the
teats, and is directed towards the caudal abdomen; a coupling medium is always
required.

LABORATORY METHODS

 Laboratory methods include

o Vaginal Biopsy
o Plasma Progesterone Assay
o Plasma Estrogen Assay

Vaginal Biopsy

 Histological assessment of the number of layers of the stratified squamous

epithelium of the vaginal mucosa obtained by biopsy can be used as a method of
diagnosing pregnancy.
 Between 30-90 days of pregnancy, the accuracy is over 90%.
 Sections taken erroneously from the cervix or posterior vagina are unsatisfactory
for diagnosis.
 Although the technique is a satisfactory one, the big disadvantage is the cost of
the procedure and the time taken to perform the test.
 Histological Assessment
Reproductive status No. of layers Thickness (in μ)
Pregnant 2-4 12-15
Diestrum 4-5 20-24
Estrum Greatly thickened

Plasma Progesterone Assay

 In non- pregnant cyclical animals, from about day 16, there is a decline in
progesterone concentrations in the peripheral blood.
 Estimation of progesterone concentrations from this time after service would be
useful.
 Accuracy at 16-24 days after service: 96%
 Plasma values ≥ 7.5 ng/ml are indicative of pregnancy.
 More reliable to identify non pregnant sows because of the irregularity of the
interestrus interval.
 The major problem is difficulty in obtaining blood samples.

Plasma Estrogen Assay

 In pregnant animals, estrogens are detectable from day 20 of gestation.

 A small volume of blood sufficient for the assay can be collected from the ear
vein.
 Optimum time to perform the assay is about 24—28 days.

PREGNANCY DIAGNOSIS IN CANINES

 In most domestic species, pregnancy interrupts normal cyclicity by increasing the

length of the luteal phase and delaying the return to estrus.
 However, in the bitch the length of luteal phase is similar in both pregnancy and
non-pregnancy. This is the reason why pseudopregnancy is a common and
normal event in bitches. Due to the peculiarities of the estrous cycle of the bitch,
endocrinological methods of pregnancy diagnosis from other species cannot be
simply extrapolated.
 In canines, the following methods are employed for pregnancy diagnosis:
o Absence of Estrus
o Behavioural Changes
o Physical Changes
o Abdominal Palpation
o Identification of Fetal Heart Beats
o Radiography
o Endocrine Tests
o Acute Phase Proteins
o Ultrasonography
 ABSENCE OF ESTRUS

 The failure to return to estrus is not a reliable indicator of pregnancy as the bitch
is not polycyclic.
 Further, the interestrus interval is identical in pregnant and non-pregnant cycles.
o

BEHAVIOURAL CHANGES

 Behavioural changes typical of pregnancy are observed in both pregnant and

non-pregnant bitches.
 Changes not specific for pregnancy are associated with an increase in plasma
prolactin concentration.
 During the second half of pregnancy, there is approximately 50% increase in food
intake. Some bitches may show a brief period of reduced appetite approximately
3-4 weeks after mating.

PHYSICAL CHANGES OBSERVED AT DIFFERENT DAYS OF

PREGNANCY

DAY CHANGES
Approximately one  A slight mucoid, vulval discharge is noticed in pregnant, and
month after mating also noticed in non-pregnant bitches
 Teats become pink and erect.

From day 35 onwards  Body weight begins to increase and may go to up 50% of
normal.

From day 40 onwards  Abdominal swelling may be noticed.

 Appreciable mammary gland enlargement and serous fluid
can be expressed from the glands. Between primigravida and
multigravida, changes may vary considerably.

From day 50 onwards  The abdominal swelling may progress to abdominal

distension. In primigravida or bitches with small litters,
changes not appreciable

During the last 7 days  Colostrum may be present in the teats. Exercise care while
of pregnancy assessing changes in mammary size and secretion, since
pseudopregnant bitches also exhibit similar features.
 ABDOMINAL PALPATION

 Technique of abdominal palpation can be highly accurate if performed between

day 26 and 30 of pregnancy which is at a time when the uterine swellings are
approximately 2 inches in lengthBeyond day 35, pregnancy diagnosis by
abdominal palpation becomes difficult to perform as swellings become larger,
more elongated, nearly confluent, pliable rather than firm.

 Counting the number of fetuses by abdominal palpation is difficult except when

performing an examination at approximately day 28 in a relaxed and thin bitch.
 Difficult to perform in obese or nervous animals, in bitches with tense abdomen, in
bitches carrying single pup or a few pups in cranial abdomen.

Changes Observed by Abdominal Palpation at Different Days of Pregnancy

DAY CHANGES
26-  Conceptuses are spherical in outline.
30  Diameter varies between 15 and 30 mm.
 They are tense fluid filled structures.
 Readily palpated in a relaxed bitch.

35  Conceptuses become elongated, and enlarged.

 Tend to lose their tenseness.
 Less easy to palpate at this time.

45  Uterine horns tend to fold upon themselves, so that the caudal portion of
each horn gets positioned against the ventral abdominal wall, and the
cranial portion of the same horn being positioned dorsally.

55  Fetuses can often be palpated with the forequarters of the bitch elevated
and the uterus manipulated caudally towards the pelvis.
 Difficult to count accurately the number of conceptuses, except at
approximately day 28 in a relaxed and thin bitch.

ENDOCRINE TESTS

 Plasma concentrations of progesterone are not useful for the diagnosis of

pregnancy in the bitch.
 A significant elevation of plasma prolactin occurs in pregnant bitches compared
with non-pregnant bitches, and it is possible that prolactin assays may become
useful as methods of pregnancy diagnosis.
 Measurement of the hormone relaxin is diagnostic of pregnancy.

ACUTE PHASE PROTEINS

  Approximately at the time of implantation, an acute phase response occurs,
appears to be unique to the pregnant bitch.
 Measurement of fibrinogen, C-reactive protein, or other acute phase proteins is
sensitive markers for pregnancy.
 The initial rise occurs from day 20 onwards with a peak at approximately day 40.
Methods appear to be reliable, although false positive diagnoses may result from
inflammatory conditions such as pyometra.
 The rise in fibrinogen concentration is the basis of commercial pregnancy
test. Estimation of plasma fibrinogen level by 17 days after mating can be taken
as an index for detecting pregnancy and pseudopregnancy in bitches.


ULTRASONOGRAPHY

 Diagnostic B-mode ultrasonography can be used for early pregnancy diagnosis.

 It is a non-invasive imaging modality, which is safe both for the operator and the
animal.
 Most accurate time to perform is generally one month after the last mating.
 Sound frequencies in the range of 2-10 MHz are commonly employed in
diagnostic examinations.
o Small dogs (< 10 Kg) : 7.5 or 10 MHz.
o Medium sized dogs : 5.0 MHz
o Large breed dogs : 3.0 MHz or lower frequencies.
 To visualize the entire reproductive tract, multiple positions and scanning planes
may be required

TECHNIQUE

 The dog should be placed in dorsal recumbency, including right or left lateral
recumbency and scanned from the dependent or non-dependent side or with the
animal standing.
 Standing on the floor is advantageous for large or giant-breed dogs.
 Clipping the ventral abdominal hair is the standard protocol to obtain the best
image.
 Application of alcohol or other wetting agents prior to applying acoustic gel to an
unclipped hair coat may improve image quality by reducing air between the
transducer and skin.

CHANGES OBSERVED BY ULTRASONOGRAPHY AT DIFFERENT

DAYS OF PREGNANCY

STAGE CHANGES
From 15 days after  Homogeneous uterus can be identified dorsal to the bladder.
ovulation  Conceptuses may be visualized, and appear as spherical
anechoic structures approximately 2 mm in diameter.

From day 20 after  Conceptus is approximately 7 mm in diameter and 15 mm in

ovulation length and the embryo can be visualized.

Approximately 22 days  Embryonic heart beat can be detected

after ovulation
Between days 32-55  The limb buds become apparent and there is clear
differentiation of the head, trunk and abdomen.

From 40 days onwards  Fetal skeleton becomes evident, fetal bone appears
hyperechoic, and casts acoustic shadows.

In late pregnancy  The head, spinal column and ribs produce intense reflections
and become more easily identifiable.

PREGNANCY DIAGNOSIS IN FELINES

Abdominal palpation

 Satisfactory time to perfom: 16-26 days post breeding

 Conceptuses are readily identifiable as individual turgid spherical swellings
 Conceptuses can be palpated as early as 13 days post breeding, but can be
confused with fecal mass
 After 6 weeks - conceptus swelling increase markedly in size, elongate and merge,
making palpation more difficult
 Abdominal enlargement at this stage will be appreciable

B-MODE ULTRASOUND

 Enlarged uterus can be confirmed as early as first week of pregnancy

 Gestational sacs can be identified from second week which is more reliable
 Fetal viability can be assessed by cardiac activity detected from third week
onwards

DIFFERENT CLINICAL METHODS OF PREGNANCY DIAGNOSIS

 IN FARM ANIMALS

SPECIES DIAGNOSTIC DAYS FROM MATING

TECHNIQUE
Cattle and buffalo EPF First week
Buffalo Failure to return to estrus 21-14
Progesterone 22-24
Real-time ultrasonography 24
Bpspb 24-30
Rectal palpation 35-70
Estrone sulfate 72
Sheep and Goat EPF (sheep) Within 24h
Return to esturs 16-21
Real-time ultrasonography 35-40
P4 (goat) 20
Estrone sulfate 40-50
Horse Real-time ultrasonography 9-16
Return to estrus 16-21
P4 16-22
Rectal palpation 17-25
eCG 40-120
Estrone sulfate 40-100
Pig Return to esturs 18-25
P4 21
Real time ultrasonography 24
Esreone sulfate 26
Fremitus in middle uterine 28
A-mode ultrasonography 30-90

MODULE-9
SUPERFETATION AND SUPERFECUNDATION

Superfetation is a condition that occurs when a pregnant female carrying one or more
live fetuses, comes into estrus, ovulates, is bred again and conception occurs.

Superfecundation occurs when a female ovulates two or more ova during one estrus and
copulates with two or more males during the same estrus with the resultant that the ova
gets fertilized with spermatozoa from each male.
 SUPERFOETATION

 Condition occurs when a pregnant female carrying one or more live fetuses,
comes into estrus, ovulates, is bred again and conception occurs.
 Superfoetation is theoretically not possible in cows for the following reason
o Even though there are chances that cows may be bred during gestational
heat, there are no evidences that ovulations occur during pregnancy.
 However, in mares ovulations do occur in pregnancy, and hence, in this species
superfoetation is theoretically possible.
 In both the species even if ovulation did occur, the spermatozoa would not be
able to pass through a cervix that is closed by a cervical seal and enter the oviduct
through the pregnant uterus; even if the ovum did get fertilized and reached the
uterus, the, endometrial area of both the uterine horns would already be occupied
by the first embryo or fetus. If by chance, the second zygote does develop in the
horn opposite to the one containing the embryo, when the earlier fetus was
expelled, the latter would also be expelled at the same time. Hence, the possibility
of superfetation in uniparous animals is highly questionable.
 In multiparous animals with a poorly defined cervix and cervical seal,
superfetation is more likely to occur. There have been reports of pregnant cats
developing mature follicles and ovulating as late as six weeks after conception. All
the fetuses of one service might develop in one horn and subsequently an estrum
may occur with ova fertilized from the second service. Parturitions could occur at
different times from each horn.
 Although superfetation is almost possible in multipara and rare in unipara, it
appears highly improbable in most reported instances where the reports on
superfetation are obviously incorrectly diagnosed.

SUPERFECUNDATION

 Superfecundation occurs when a female ovulates two or more ova during one
estrus and copulates with two or more males during the same estrus with the
resultant that the ova gets fertilized with spermatozoa from each male.
 One may suspect the condition based on the breeding history. Following
parturition the condition becomes obvious with offspring resembling each sire.
 Superfecundation occurs more commonly in dogs and cats, because these species
have long heat periods, multiple ovulations and opportunities of being served by
different males.
 Condition has also been reported in unipara where there has been birth of twin
horse and mule foals and twin Holstein and Hereford calves.

MODULE-10
 INFERTILITY IN FEMALE FARM AND COMPANION ANIMALS-
PART I

Regular breeding depends upon the normal function of the reproductive system. In
order to breed regularly, the female has to have functional ovaries, display estrus
behavior, mate, conceive, sustain the embryo through gestation, calve, and resume
estrus activity and restore uterine function after calving. Each of these functions can be
affected by management, disease and genetic makeup of the animal. Impairment in
reproductive tract function affects the calf per year programme in bovines.

Fertility

The term fertility as applied to the female denotes the desire and ability to mate, the
capacity to conceive and nourish the embryo and finally the power to expel a normal
young one and fetal membranes.

Infertility

Sometimes considered as synonymous with sterility or it implies a failure or delay in

producing the annual live young one. The term sub fertility is a more appropriate term.

Sterility

Sterility refers to absolute inability to reproduce.

HEREDITARY OR CONGENITAL ANATOMICAL DEFECTS OF

THE REPRODUCTIVE TRACT THAT AFFECT FERTILITY

 Both congenital and acquired abnormalities of the genital system can influence
fertility.
 Anatomical abnormalities usually affect individual cows or heifers and therefore
may not influence the fertility of a herd.
 In some cows, because of the severity of the abnormalities, sterility is manifested
at the time of first service period while in some, where the defect is less severe, it
may not be detected until late in life.

ABNORMALITIES OF REPRODUCTIVE TRACT AND OVARIES

Ovarian hypoplasia

Ovarian hypoplasia caused by a single autosomal gene with incomplete penetration is

a condition in which one or both ovaries are small, narrow and functionless. The
affected ovary may be partially or totally hypoplastic and undergoes incomplete
development as a part or whole lacking the normal number of primordial follicles.
Depending on the severity of the hypoplasia and whether condition is unilateral or
bilateral, infertility or sterility will result.
 Segmental aplasia of the Mullerian ducts and Imperforate hymen

Segmental aplasia of the Mullerian ducts and imperforate hymen are developmental
defects of the Mullerian ducts that lead to various anomalies of the vagina, cervix and
uterus. A single, recessive, sex-limited gene with linkage to the gene for white coat
color is considered to be the cause for this condition.

Congenital lack of endometrial glands

This condition has been observed in few heifers where they exhibited failure of estrus
and a retained or persistent corpus luteum apparently due to a failure of the
endometrium to produce the PGF2α required for regression of the corpus luteum.

Double external os of the cervix

Double external os of the cervix in cattle is due to failure of the Mullerian ducts to fuse.
The lesion often occurs as a band of tissue 0.5 to 2 inches in width and 0.5 to 1 inch in
thickness, caudal to the external os of the cervix. On speculum examination it may
appear as if there were a double os. In other cases there may be a true double external
os of the cervix and the band of tissue separating the 2 openings may extend a short
way into the caudal part of the cervical canal. These seldom interfere with conception.
Affected cows usually conceive and calve normally. Occasionally a portion of the fetus
will pass on either side of the band of tissue and result in dystocia that is easily relieved
by incising the band.

Uterus Didelphys

In true uterus didelphys with a double cervix, conception may be delayed when the
semen is deposited in the cervix opposite of the ovary from which ovulation has to take
place. Since, only one horn takes part in the placentation of the fetus, abortions,
premature births, retained placenta, and infertility are more common.

Abnormal Wolffian or Gartner’s ducts

Multiple cysts may develop along the course of the ducts or the duct may form a long,
sometimes rather coiled, cord 0.5-1.5 cm in diameter distended with fluid.

Intersexuality and Freemartinism

Freemartinism is a distinct form of intersexuality which arises as a result of a vascular

anastomosis of the adjacent chorioallantoic sacs of heterozygous fetuses in twin
pregnancies. As a result, although the external genitalia of freemartin heifers appear
normal the internal genitalia frequently show masculinization.
ACQUIRED DEFECTS OF REPRODUCTIVE TRACT
LESIONS OF THE OVARY

Tumours of the Ovary

Granulosa cell tumours are the commonest neoplasm of the bovine

ovary but carcinomas, fibromas, thecomas and sarcomas have also
been described. Most of the large and cystic neoplasms of the bovine
ovary reported in pregnant as well as non-pregnant cattle are
granulosa cell tumours. In the early stages of the tumour it
presumably secretes estrogen, for the affected animal is often
nymphomanical. Later, most of the tumour tissue undergoes
luteinization and then anestrus usually occurs. Virilism has been
reported in long-standing cases.

Ovaritis, Inflammation or Infection of the Ovary

Inflammation or infection of the ovary may occur secondary to an

ascending infection from the uterus by extension of infection through
the uterine walls causing a peritonitis and perimetritis, trauma
produced by rough handling or massage of the ovary. Enucleating
the corpus luteum or manual rupturing of cysts may lead to
formation of star shaped or transverse scars on the ovary.

Para Ovarian Cysts

Paraovarian cysts, vestiges of the Wolffian or Mullerian duct system

have been occasionally found in the broad ligament of the cow
around the ovary and oviduct but reports are less common in the cow
when compared to dog, sheep and horse.

They may be 0.5 to 2 or more inches in diameter and round or oval in

shape located most commonly near or in the fimbria of the oviduct.
LESIONS OF THE OVIDUCT AND ADNEXA

Ovarobursal Adhesions

Lesions between the ovary and the ovarian bursa are known as
ovarobursal adhesions. The extent of the adhesions may vary and may
consist of fine web-like strands in the depth of the bursa which does not
involve the uterine tube while in others the ovary may be completely
enveloped by the bursa. Conception is unlikely to occur due to ovulations
from the affected side. Where there are extensive adhesions of the bursa
with the ovary, ovulation may not occur and the follicle undergoes
luteinization. In some cases ovarian cysts can develop. The condition is
rarely seen in heifers but its incidence increases with the age of the cow.

Hydrosalphinx and Pyosalphinx

Hydrosalphinx has been reported secondary to segmental aplasia of the

paramesonephric duct or to adhesions of proximal and distal portions of
the oviduct. The oviduct gets distended to a diameter of 0.5 to 1 cm or
more with clear watery mucus and may appear as elongated, coiled,
thin walled and fluctuating on palpation. Pyosalphinx, commonly
associated with extensive adhesions of the mesosalphinx and
mesovarium may follow severe uterine infection. The condition is less
common than hydrosalphinx. In pyosalphinx, extensive perimetritis is
not unusual.
LESIONS OF THE UTERUS

Adhesions of the Uterus and Parturient Trauma of the Tubular

Genital Tract

A troublesome sequel to the caesarean operation is adhesion of the uterus

to the omentum, rectum, intestines or abdominal wall which is frequently
associated with sterility.

Dystocia due to fetal oversize is common in cattle, particularly in the

Friesian breed. Delivery of large calves by heavy traction frequently
damages the birth canal to such an extent that the animal is rendered
sterile.

Endometritis

Endometritis due to uterine infection and secondary inflammation has

been shown to be a cause for infertility in cattle.The condition has been
dealt in detail in the module on specific and non specific infections of the
reproductive tract.

Pyometra

Pyometra in cattle is characterized by accumulation of pus or

mucopurulent material, persistent corpus luteum and failure of estrum.
The corpus luteum persists due to failure of PGF2α secretion as a result of
severe endometritis. The condition usually follows an abnormal
parturition, uterine infection, and delayed involution of the uterus
following abortion, premature birth twin birth, dystocia, retained
placenta, septic metritis, or post partum metritis.
 Mucometra or Hydrometra

Mucometra or hydrometra almost similar in condition are occasionally

seen in cattle with the mucin present in the uterus varying from a watery
fluid to a semisolid mass. The condition usually follows long standing
cases of cystic ovaries, cystic endometrial hyperplasia, arrests in the
development of Mullerian duct system or segmental aplasia of the
paramesonephric ducts and persistent hymen.The genetic or congenital
defects may result in distension of both horns with watery, viscous or even
rather solid coagulated masses of mucus and cellular debris that may be
confused with pregnancy. In these cows the ovaries and endometrium are
normal and estrum therefore occurs normally. Cows with mucometra or
hydrometra are usually sterile unless the case is a uterine unicornis where
pregnancy can occur in the normal horn, or a simple imperforate hymen
that can be opened. In cases that are associated with cystic ovaries
treatment should be aimed to resolve the cystic ovarian condition. Cases
associated with persistent corpus luteum need to be treated with
prostaglandins.
Perimetritis and Parametritis

Perimetritis and parametritis may be occasionally observed in the cow

and may be characterized by adhesions between uterus and broad
ligaments and other pelvic and abdominal organs. The adhesions may
occur due to severe septic metritis, douching and perforation of the uterus
with a catheter releasing irritant material into that area, nonfatal rupture
of the rectum due to carelessness in rectal examination or therapy,
perforation of the vaginal wall by the penis of the bull, occasional trauma,
severe haemorrhage or rupture of the uterus at the time of calving due to
torsion of the uterus, embryotomy, forced extraction, severe cervical or
vaginal lacerations, cesarean section, extrauterine fetus, excessive
bleeding following manual removal of the corpus luteum or excessive
massage or rough handling of the uterus during removal of the placenta.
Lesions may vary from a few thin fibrous strands of connective tissue to
firm adhesions between the uterus, broad ligaments, rumen, omentum,
intestines, rectum, bladder and other organs.
 Abscess of the Uterine Wall

Abscess of the uterine wall is occasionally observed in the cow and may
appear as a round or oval structure that is tense and firm on palpation.
The size may vary from 1-15 cm in diameter changing the normal contour
or outline of the uterine horn. The condition may usually follow severe
metritis, improper removal of the placenta, trauma caused during
insemination or douching. The condition can be easily diagnosed on rectal
palpation and should be differentiated from tumour, cyst or hematoma.
Symptoms are usually absent and in most cases the cow fails to conceive.
Treatment is usually impossible. In rare instances the abscess may
rupture into the rectum, bladder or vagina. Rupturing the abscess into the
uterine lumen may be attempted as the last resort.

LESIONS OF THE CERVIX, VAGINA AND VULVA

Cervicitis and Vaginitis

Cervicitis or inflammation of the cervix of the cow is a condition that is commonly

associated with metritis and follows abnormal parturitions such as abortions,
premature births, dystocia resulting in lacerations or trauma to the cervix during
forced extractions and retained placenta.

Vaginitis or inflammation of the vagina is observed as a primary condition or

secondary to metritis or cervicitis and may follow trauma, lacerations, bacterial, viral
or protozoal infections, abortions, dystocia, fetotomy, retained placenta, prolapsed of
the vagina and postpartum metritis.

Tumours of the Cervix, Vagina and Vulva

Tumours of the cervix are rare in cattle and the few recorded cases have been benign.
Fibropapillomata of the vagina and vulva of cattle have been reported. They do not
cause infertility but may interfere with birth. They are usually pedunculated and may
be removed surgically. There is a possibility that one form of vaginal fibropapilloma is
of viral origin and that it is transmitted venereally. It occurs in young cattle and
undergoes spontaneous resolution. Tumours of the vulva include fibromas, angiomas,
carcinomas, and fibropapillomas. The prognosis on squamous cell carcinomas are
guarded to poor as it affects all the regional lymph nodes. Slaughter is usually
recommended.
 Parturient Laceration or Bruising of the Vulva

Condition may be followed by cicatrization and distortion with imperfect closure of the
vulval sphincter and aspiration of air.

The sequel is similar to but less severe than those of rupture of the perineum. Some of
these cows are infertile to natural service but conceive to intrauterine insemination.
Dystocia owing to fibrosis of the vulva may arise at subsequent parturition.

Rupture of the Perineum

A third-degree perineal rupture may occur at calving following dystocia wherein the
whole thickness of the vagina and rectal wall ruptures so that the rectum and vagina
are confluent. Healing may not occur as a result of which air and faeces are aspirated
into the vagina causing vaginitis and metritis.

Affected cows have a chronic mucopurulent vulval discharge without impairment of

general health. Normal cyclic behaviour resumes but conception may not occur
because of the metritis. Surgical reconstruction of the perineum may be the choice of
treatment. Rupture of the perineum may be prevented by sound obstetric technique,
including episiotomy.

Other Conditions of Cervix, Vagina and Vulva

Cirrhosis of the Cervix

 Rarely, parturient laceration of the cervix is followed by fibrosis and obstruction

of the cervical canal, with infertility. Occasionally, cirrhosis of the cervix may
prevent proper dilation of the organ at parturition, but most cases of failure of
cervical dilatation are of functional origin.
 Prolapsed of one or more of the cervical folds is commonly seen in the
pluriparous cow. It is a physiological hazard of parturition and is not a cause of
infertility.

Gross Fibrosis of the Vagina

 This may follow laceration and pyogenic infection causing a narrowing of the
birth canal and dystocia. Caesarean section may then be required.

Anestrum

 Following puberty, the cow should cycle regularly at approximately 21-day

intervals unless interrupted by pregnancy and for a short period in the
puerperium. The occurrence of estrus signs is the only way to tell that the animal
 is cycling. It is possible that estrus signs are occurring but are not being observed
which is purely a management problem. The other possible causes why the
animal may not exhibit estrus signs are.
o The ovaries may be quiescent and inactive; this is referred to as true
anestrus.
o There may be cyclic ovarian activity but the cow is not showing the normal
behavioural signs; this is described as sub estrus or silent heat.
o There may be a progesterone-producing structure in the ovary which is
exerting an inhibitory effect upon the hypothalamus and anterior
pituitary; this may be a persistent corpus luteum or a cyst.

True Anestrus

 In true anestrus the ovaries are quiescent and nonfunctional. The reasons for the
failure of cyclicity may be insufficient release or production of gonadotrophins to
cause follicle development, or it may reflect the failure of the ovaries to respond,
but the latter is unlikely.

 The clinical history will be

o That a cow or heifer has not been seen in estrus;
o Rectal palpation will reveal small, flat and smooth ovaries and absence of
either a developing, mature or regressing corpus luteum. In buffaloes the
ovaries may appear spindle like.
o Re-examination of the cow per rectum after 10 days will help confirming
the case where
 in true anestrus ovaries will be virtually unchanged;
 in late diestrus or early diestrus (metestrus) the ovaries will have a
distinctly palpable corpus luteum.
o Milk or blood progesterone determinations are helpful in confirming
diagnosis; two samples can be taken at 10-day intervals where
 low-low values are indicative of anestrus
 high-low or low-high values are indicative of diestrus

The etiology, diagnosis and treatment of True Anestrus has been dealt in detail in
Module 12.

Anestrum due to Subestrus or Silent Estrus

 Subestrus or silent estrus most commonly occurs when the first and second
ovulations postpartum are not preceded by behavioural signs of estrus.
o The causes for silent estrus or subestrus could be due to
 improper heat detection,
 genetic predisposition,
 climates where incidence is more common in temperate climates
and more common in the winter than in the summer months,
 nutritional deficiencies such as deficiency of ß-carotene,
phosphorus, copper cobalt and
  overweight.
o Diagnosis is based on the clinical history and rectal palpation of the genital
system.
o One cannot differentiate from unobserved estrus, since the clinician will
be checking for evidence of cyclic ovarian activity.
o The corpus luteum must be differentiated from a cyst; it may be persistent
or the cow may be pregnant. In the event of doubt then a re-examination
done in 10 days would help in confirmation.
o Treatment involves administration of prostaglandin F2 α or an analogue
followed by fixed-time insemination if a mature corpus luteum is present
and the cow is not pregnant. If the corpus luteum is at a refractory stage
(refer module on estrus synchronization) a double injection prostaglandin
regimen at an 11 day interval could be used. Alternatively a PRID or other
progestogen implant could be used followed by fixed-time insemination.

 The clinical history will be

o That a cow or heifer has not been seen in estrus;
o Rectal palpation will reveal small, flat and smooth ovaries and absence of
either a developing, mature or regressing corpus luteum. In buffaloes the
ovaries may appear spindle like.
o Re-examination of the cow per rectum after 10 days will help confirming
the case where
 in true anestrus ovaries will be virtually unchanged;
 in late diestrus or early diestrus (metestrus) the ovaries will have a
distinctly palpable corpus luteum.
o Milk or blood progesterone determinations are helpful in confirming
diagnosis; two samples can be taken at 10-day intervals where
 low-low values are indicative of anestrus
 high-low or low-high values are indicative of diestrus

The etiology, diagnosis and treatment of True Anestrus has been dealt in detail in
Module 12.

Anestrum due to Subestrus or Silent Estrus

 Subestrus or silent estrus most commonly occurs when the first and second
ovulations postpartum are not preceded by behavioural signs of estrus.
o The causes for silent estrus or subestrus could be due to
 improper heat detection,
 genetic predisposition,
 climates where incidence is more common in temperate climates
and more common in the winter than in the summer months,
 nutritional deficiencies such as deficiency of ß-carotene,
phosphorus, copper cobalt and
 overweight.
o Diagnosis is based on the clinical history and rectal palpation of the genital
system.
 o One cannot differentiate from unobserved estrus, since the clinician will
be checking for evidence of cyclic ovarian activity.
o The corpus luteum must be differentiated from a cyst; it may be persistent
or the cow may be pregnant. In the event of doubt then a re-examination
done in 10 days would help in confirmation.
o Treatment involves administration of prostaglandin F2 α or an analogue
followed by fixed-time insemination if a mature corpus luteum is present
and the cow is not pregnant. If the corpus luteum is at a refractory stage
(refer module on estrus synchronization) a double injection prostaglandin
regimen at an 11 day interval could be used. Alternatively a PRID or other
progestogen implant could be used followed by fixed-time insemination.

Anestrum due to Persistent Corpus Luteum

 Persistent corpus luteum occurs when there is failure in the production or release
of endogenous luteolysin. Conditions that result in persistence of corpus luteum
are
o Pregnancy
o Pyometra
o Mucometra or hydrometra
o Mummification
o Maceration
 It is important to remember that persistent corpus luteum does not occur in the
presence of a normal non-pregnant uterus. Many veterinarians tend to call
wrongly a cyclic CL as persistent CL.
 The condition, once diagnosed, can be readily treated with PGF2 α or a synthetic
analogue, provided that the clinician is confident that the cow is not pregnant;
estrus will occur in 3-5 days.

MANAGERIAL CAUSES OF INFERTILITY

 Managerial deficiencies are a common cause for lowered infertility and repeat
breeding in herds and must be differentiated from infectious form of infertility
caused by Vibriosis and Trichomoniasis which are also herd problems. The
common managerial deficiencies leading to infertility are
o Poor heat detection
o Improper time of insemination
 The average length of the estrus period is 18 to 24 hrs and ovulations occur 12 hrs
after the end of estrus period. Since, the sperm survivability is 48 hrs and the
ovum survivability is only for 12-24 hrs, the sperms should be present in the
female reproductive tract about six hours before ovulation for optimum
fertilization. Therefore, the best time to do AI is 12 to 18 hrs after onset of estrus.
Improper Artificial Insemination Technique

In natural service the bull deposits the semen in the proper site, while in artificial
insemination there are many possibilities for man to render infertile the bovine sperm
cells. These include

 Improper extension, freezing and storage of fertile semen.

 Improper thawing of frozen semen.
 Insemination should take place promptly, within several minutes after thawing.
Thawing semen and keeping it at 5 ° C or 40 ° F for an hour or more is very
detrimental to sperm cells.
 Proper insemination technique should be followed
 Palpation of the ovaries during estrus should be avoided as it may result in
rupture of the graffian follicle. Extreme caution should be taken to perform
palpation in a gentle, skilled manner so as to avoid manual rupturing of the thin
walled follicle.

EFFECTS OF NUTRITION UPON REPRODUCTION

 The major contributors to infertility and poor reproductive performance are

o deficiencies of various trace minerals
o inadequate vitamin intakes
o energy protein imbalances
 o excessive protein intakes
 Infertility or sterility due to nutritional causes is usually characterised by
o a failure of estrum or a cessation of estrous cycle
o a failure of conception or early embryonic death
 The effects of nutrition on fertility has been dealt with in detail under the following
headings
o Effects of Energy Intake on Fertility
o Effects of Protein Intake on Fertility
o Effects of Vitamins on Fertility
o Effects of Micronutrients on Fertility

EFFECTS OF ENERGY INTAKE UPON FERTILITY

 Inadequate energy intake in heifers and early lactation cows reduces reproductive
performance.
o inadequate amounts of energy in heifers delays onset of puberty
o If energy deficient rations are fed to heifers that have begun to have
normal estrous cycles, they may stop cycling.
o Negative energy balance during early lactation affects reproduction
because they cannot consume adequate feed to meet the nutrient
requirements for high levels of milk production. Energy stores in body
tissues are mobilized and weight losses occur.
 Excessive energy intake during late lactation and the dry period can cause “fat
cow” problems which lower reproductive efficiency in the next lactation.
 The most severe consequence of inadequate nutrition is
o the cessation of cyclical activity (anestrus)
o silent estrus
o ovulatory defects
o fertilization failure
o embryonic or fetal death.
 The two main ways in which energy deficiency is believed to affect pregnancy
rates is via
o GnRH system
o metabolic regulators of ovarian function.
 Negative energy balance does not affect FSH secretion but LH secretion is
impaired.
 Circulating concentrations of glucose, insulin and insulin like growth factor 1 are
lower in cows in negative energy balance than in fully fed animals, whilst
concentrations of non-esterified fatty acids are higher. All of these might be
expected to affect follicle development.

Fatty Liver Syndrome

 Excessive energy intakes during the late lactation and dry periods can lead to “fat
cow” problems.
 Over conditioned cows have a higher incidence of retained placenta, more uterine
infections, and more cystic ovaries.
  Cows have poor appetite and body fat reserves are mobilized to meet the energy
deficit for lactation.
 Inadequate supplies of endogenous and exogenous protein exacerbate the
syndrome.
 The liver becomes infiltrated with fat and the cow often develops ketosis.

Diagnosis

 Biopsy
 Estimation of blood parameters to diagnose impaired liver function.
 Eight weeks before calving
o There is an increase in
 non-esterified fatty acids
 bilirubin
 aspartate aminotransferase
 ß-hydroxybutyrate concentrations
o There is a decrease in
 glucose
 cholesterol
 albumin
 magnesium
 insulin
 Impaired liver functions will affect albumin production, whilst if fat has replaced
glycogen in the liver parenchyma total glycogen reserves will be reduced.
 In cows with fatty liver
o basal concentrations of LH are lower
o fewer pulses of LH in affected
o pre ovulatory concentrations of LH are lower in cows with fatty liver
o the LH response to administered GnRH is lower
o luteal progesterone concentrations are lower than in normal cows

Treatment

 Treatment is not possible, and usually there will be eventual recovery. Attempts
to prevent the disease can be made by ensuring that cows are not excessively fat
at calving and receive adequate energy thereafter to exclude the need for excess
fat mobilization.

EFFECT OF VITAMINS ON FERTILITY

 Adequate intake should be ensured by feeding vitamins and minerals in small

amounts of low energy concentrates or mixing them in a complete dry cow
ration. Vitamin deficiencies in cattle are mainly limited to vitamin A
while deficiencies of other vitamins are not likely to cause any reproductive
failure.
Vitamin A

 Vitamin A is required for maintaining healthy tissue in the reproductive tract.

Deficiency in cattle causes delayed sexual maturity, abortion, birth of dead or
weak calves, retained placenta and metritis. The recommended daily
supplementation for dairy cows is 30,000-50,000 units. Dry cows fed only poor
quality hay for extended periods without additional supplementation may benefit
from vitamin A injections.
 β-carotene is a substance found in many plants. The cow converts this into
vitamin A. It is known to be in high concentrations in fresh green roughages
while grains contain relatively low amounts. Silages, especially alfalfa, contain
moderate levels while corn silage is a poor source. Dry hays, especially alfalfa, are
excellent sources of carotene. Despite high levels at harvest, β-carotene levels
decrease during storage, with the extent of destruction being dependent on
storage conditions.
 Deficiency of β-carotene in diet causes
o Delayed uterine involution
o Delayed first estrus after calving
o Delayed ovulation
o Increased incidence of cystic ovaries
o Early embryonic death and abortion

Vitamin B – Complex

 Deficiency of vitamin B has been demonstrated in animals to produce the same

inhibitory effects on reproduction as reduced food intake or starvation. However,
as B complex vitamins are synthesised in the rumen, cows are in no danger of
suffering from vitamin B deficiencies. Vitamin B12 requires cobalt synthesis and
therefore cows in cobalt deficient areas may suffer from severe inanition due to
lack of appetite and insufficient intake of feed brought about by cobalt or B12
deficiency.

Vitamin D

 Vitamin D is required for normal calcium and phosphorus metabolism. However,

deficiencies are seldom encountered as it is present in roughage of any quality.
Cows receiving a normal amount of natural light manufacture their own Vitamin
D. Most commercial concentrates contain supplemental vitamin D in amounts
sufficient to meet the cow’s requirement of 10,000 IU per day.

Selenium and Vitamin E

 Selenium deficiency in dry cows has been reported to cause retained placenta,
abortions, a high incidence of embryonic-fetal loss, poor fertility, and increased
incidence of metritis, a higher level of general infection and the birth of dead or
weak calves in some problem herds. It is difficult to separate the effects of
selenium and/or vitamin E deficiency since both have a ubiquitous antioxidant
 function which protects a wide range of biological systems from oxidative
degradation.
 Deficiency occurs when soils contain < 0.5 mg/kg, or diets < 0.05mg/kg
selenium. Vitamin E deficiency occurs when animals graze post-mature pasture,
receive other diet components that contain < 0.7 mg/kg of the vitamin, or are fed
diets that are high in polyunsaturated or rancid fats. Vitamin E deficiency does
not affect the estrous cycle or ovarian function.
 Diagnosis of selenium deficiency can be made by measuring circulating
concentrations of selenium or, better, by measuring selenium stores in the liver.
Measurement of levels in feed, pasture or soil is often also indicated.
Supplementation is widely practiced, especially in areas where soils are known to
be marginal or deficient. However, it should be remembered that excessive
selenium is toxic, especially where it has been given by injection.

EFFECTS OF MICRONUTRIENTS UPON FERTILITY

 Micronutrient deficiencies affect reproduction by

o depressing the activity of rumen microflora;
o reducing enzyme activity
o affecting energy and protein metabolism and the synthesis of hormones;
o altering the integrity of rapidly dividing cells within the reproductive
system.

Cobalt

 Cobalt deficiency causes

o anaemia
o inappetance
o poor bodily condition
o thriftiness
o loss of condition
 Fertility is affected by
o increased number of ‘silent’ estruses
o poor pregnancy rates
o irregular inter estrus intervals.
 Deficiency occurs when diets contain < 0.07 mg/kg D.M. cobalt and is due to
failure of vitamin B12 synthesis, which is an essential co-factor for carbohydrate
metabolism.

Copper

 Copper deficiency results in

o anestrus
o sub estrus
o poor pregnancy rates.
  When this occurs in association with other signs of hypocuprosis, such as
anaemia, poor growth, bleached coat colour and diarrhoea, a diagnosis is likely.
Hypocuprosis can be either direct or indirect.
 Indirect deficiency occurs due to excessive molybdenum, iron or sulphur intake
and, possibly, calcium or zinc. Copper deficiency occurs when cattle are fed diets
containing <3mg/kg copper, if the molybdenum content is <3mg/kg; 3 to 10
mg/kg; or >10mg/kg if the molybdenum content is >10 mg/kg.

Iodine

 Reproductive failure resulting from iodine deficiency is invariably related to

impaired thyroid function in the dam, embryo or fetus, which in the last two can
cause embryonic death, abortion, stillbirth or weak goitrous calves.
 There is good evidence that treatment with iodised oil injection can improve the
deficient status. Simple iodine deficiency can occur because of an intake below
0.8 mg/kg D.M. considering a level of 2.0 mg/kg D.M. to be the threshold for
deficiency.
 Disturbance of thyroid function can also be due to goitrogenic substance present
in lentils, soya bean and linseed. High levels of goitrogenic substance can produce
anestrus in heifers. Since iodine is needed for thyroxine synthesis, iodine
deficiency is largely manifested through the effects of a lack of thyroxine.
 Thyroxine is a general metabolic regulator and, in particular, a regulator of
mitochondrial activity. Thyroxine deficiency is associated with non-specific signs
of poor growth and poor ‘doing’ together with loss of libido and inhibition of
estrous behaviour.

Manganese

 Manganese has a ubiquitous role in reproductive function, being involved in

steroid synthesis. Both the pituitary gland and ovaries are relatively rich in this
trace element.
 A variety of reproductive disorders which depress fertility in cows have been
blamed on manganese deficiency; these include anoestrus, poor follicular
development, delayed ovulation, silent estrus and reduced conception rates it
also causes joint and limb deformities in calves.
 Under normal circumstances it is likely that normal pasture will provide the
necessary requirement of 80 mg/kg D.M. in the food although some foods are
low in manganese. In addition, there is an interaction with the calcium:
phosphorus ratio in the diet, with some evidence that high liming of pasture can
cause manganese deficiency.
 Manganese is a cofactor in a number of enzymes that are responsible for
gluconeogenesis and has a significant role as an antioxidant. Manganese is also
involved in cholesterol synthesis and, hence, affects steroidogenesis.

Phosphorus
  It has been estimated that the normal requirements for phosphorus in the cow for
the maintenance of pregnancy are about 13 g/day, with about 7 g extra for each
4.5 liters of milk. Providing that forage contains adequate levels of phosphorus,
normal diets should contain adequate phosphorus to ensure normal fertility.
 However, deficiencies can occur where forages have inadequate levels and,
perhaps, because of the interaction between calcium and phosphorus. However,
phosphorus – deficient pastures are often deficient in many other micronutrients
making assessment of the role of phosphorus difficult.
 The evidence for the importance of hypo phosphataemia as a cause of infertility is
conflicting. The provision of supplementary phosphorus has been shown to
improve the breeding performance of grazing cattle.
 A number of authors have described infertility, which was characterized by
anoestrus, sub estrus, irregular cycles and low conception rates in the absence of
other clinical signs of phosphorus deficiency. If hypo phosphataemia is
suspected, a rapid response can follow the feeding of dicalcium phosphate or
bone meal. It is important to ensure that the ratio of calcium to phosphorus is 1:1.

Zinc

 Zinc deficiency has been shown to have an adverse effect upon reproductive
function in the male of many species. Its influence on reproductive function in
the cow and heifer is not clear.
 Uptake of zinc is impaired by copper, calcium, iron, molybdenum and cadmium.
Excessive levels of zinc supplementation can lead to perturbation of essential
fatty acid metabolism, which affects prostaglandin synthesis. Its potential role as
an antioxidant is considered below.

Phyto-Estrogens

 When cows ingest large quantities of these substances they become anestrous,
with large ovarian cysts, vulval and cervical enlargement and poor conception
rates Such substances are found in subterranean clover, certain strains of red and
white clover and Lucerne.

SPECIFIC INFECTIOUS DISEASES CAUSING INFERTILITY IN

CATTLE

 Infectious diseases of cattle adversely affect reproductive performance, either

directly or indirectly.
 Direct effects upon the embryo include infections that result in early embryonic
death, and those that infect the more advanced fetus or its placenta, resulting in
abortion, stillbirths or the birth of weak calves.
 Indirect effects upon embryo survival include infections that adversely affect
uterine function and infect the maternal component of the placenta leading to
embryonic death and fetal stillbirth.
  Reproduction is also affected by systemic illness causing fetal losses or via a
direct impairment of reproductive cyclicity.
 The various infectious diseases that affect cattle have been dealt with in detail in
the module on Specific Infectious Diseases Causing Infertility.

INFERTILITY IN SHEEP

 Fertility can be defined as the number of lambs born per 100 ewes put to the ram
(i.e. true lambing percentage).
 The introduction of rams marks the begening of breeding season, and all physical
and financial performance should be calculated from this point, taking into
consideration ewes that die, those that are culled and those that abort or are
barren.
 The three factors that influence the number of lambs sold are
o Fertility, i. e. whether the ewes are pregnant and lamb
o Fecundity, i.e. the number of lambs born per pregnancy
o Survival rate to weaning
 Ewes are generally allowed to run with the ram during the breeding season and
not segregated; thus estrus detection problems are not encountered.
 Most breeds of sheep remain acyclici for longer periods after parturition than the
cow, thus allowing the reproductive system time to recover from the effects of
pregnancy.
 The main factors responsible for infertility in sheep are specific infectious agents
that usually result in abortion.
 Structural, functional and management factors are of limited importance.

Structural Defects

 Structural defects of ovine genital organs are uncommon. Most defects involve
the ovaries and their associated bursae, with fibrin tags and paraovarian cysts
being most frequently identified.
 Other lesions identified that would have caused infertility or sterility are ovarian
aplasia, ovarian hypoplasia, bilateral hydrosalpinx, aplasia of the para
mesonephric ducts, freemartinism and hermaphroditism. Owing to the rarity of
anastomoses of the adjacent allantoic vessels of twins, the freemartin condition is
likely to be rare.
 Cases of intersexuality are seen, mainly at lamb castration. They are male
pseudohermaphrodites referred to by shepherds as ‘wilgils’. The fact that several
may be seen at once in a flock tends to point to a possible hereditary cause.

FUNCTIONAL CAUSES OF INFERTILITY

 Except in the case of unthrifty ewes (which are usually culled), anestrus is
uncommon is sheep. In fact, when there rams are turned out with the flock it is
usual for most of the ewes to be mated within a month.
  The first estrus of the breeding season in some ewes is anovulatory and, more
frequently ewes fail to become fertilized at these early matings compared to later
one.
 Ovarian follicular cysts are occasionally seen while luteal cysts are rare.
 Embryonic death, or resorption, is a conspicuous feature of sheep infertility.
Early embryonic death has been associated with infectious diseases such as
toxoplasmosis and Border disease.
 Abortion and of fetal mummification are occasionally seen.
 A specific environmental cause of sheep infertility, due to grazing on pastures of
subterranean clover which contains large amounts of the estrogenic substance
genistein, is the cystic degeneration of the endometrium and permanent sterility.
 Asynchrony or imbalance of the hormonal changes that occur around the time of
estrus and during the early luteal phase probably results in embryonic death.

MANAGEMENT FACTORS

Estrus Detection and Artificial Insemination

 The best method of estrus detection is with a raddled, vasectomised ram.

Artificial insemination in sheep has not assumed the popularity achieved in
cattle. A number of factors have been responsible, notably the disappointing
results using frozen/thawed semen deposited intracervically. The spermatozoa
are unable to colonize or traverse the length of the cervix and are rapidly lost
from the ewe’s reproductive tract. However, the use of intrauterine insemination
by laparoscopy has been much more successful. Artificial insemination is best
used in mid estrus, or 12-14 hours after its onset.

Teasing

 The introduction of vasectomised teasers into the flock, before fertile rams, had
no effect on pregnancy (conception) rates but had a profound effect upon the
onset of cyclical activity and hence a compact lambing season. Teasing caused
ewes to exhibit estrus in the first 16 days after exposure to the fertile ram, whilst
two cycles were required for the unteased ewes to show comparable activity.

Ram: Ewe Ratio

 The number of rams per ewe will vary depending upon a number of factors: age
of the ram; age of the ewes; whether more than one ram is to be used with the
group of ewes; and terrain and size of the enclosure. Ram: ewe ratios of 1:25 to
1:40 are suitable in non-synchronized flocks. However, where synchronization is
attempted, a ratio of at least 1:10 should be available.

Nutrition
  It is important that ewes are in good bodily condition at tupping. Increasing the
energy intake several weeks before tupping, so that the ewes are gaining weight
(flushing), will increase the fecundity in those ewes with the genetic potential.
Provided the level of feeding is maintained for a month after mating this should
ensure good pregnancy rates. Some reduction in food intake is reasonable during
the second and third months of gestation, but feeding should be increased in the
last 6-8 weeks before lambing.

Increasing Fecundity

 Increased ovulation rates can be achieved by the administration of equine

chorionic gonadotrophin (eCG) on the 12th or 13th day of the estrous cycle. Good
results have been obtained by immunization against androstenedione.

INFECTIOUS AGENTS

 Non-specific infections of the genital tract, especially the uterus, are of minimal
importance in ewes, probably because in most breeds of sheep there is a long
period of anestrus following lambing. In the small number of ewes in which
bacterial contamination occurs at lambing or postpartum, which is less than 20%
they are rapidly eliminated within a week and thus before the genital tract can be
exposed to a period of progesterone influence: this will occur at the next diestrus
which will normally be many months away.
 In the cow, retention of the fetal membranes (RFM) postpartum is quite
common, and this is a major risk factor in the development of endometritis and
subfertility. RFM is relatively uncommon in ewes; where it does occur, attempted
removal by applying traction to the exposed portions of the membranes can be
attempted. If left, they will usually separate and be shed within 5-6 days. If an
affected ewe shows signs of systemic illness due to the development of metritis,
then she should be treated with an appropriate broad-spectrum antibiotic.

Specific infectious diseases that cause infertility in ewes are

 Enzootic Abortion in Ewes (EAE)

 Toxoplasmosis
 Campylobacteriosis
 Salmonellosis
 Listeriosis
 Border Disease
 Leptospirosis
 Brucellosis
 Q Fever
 Ureaplasmosis
 Tick-borne Fever

INFERTILITY IN GOATS
  In the absence of any major infectious cause of abortion, infertility in the goat is
generally not a major problem, normally with only a small number of barren does
remaining at the end of the breeding season.

Structural Defects

 Abnormal sexual differentiation during embryological and fetal development,

resulting in intersexes is relatively common in the goat, especially in breeds such
as the Alpine, Saanen and Toggenburg. It is much more prevalent in polled
individuals, where ‘polledness’ is a simple dominant character with full
penetrance, but it is also associated with a recessive hermaphrodite effect with
incomplete penetrance. Intersexes can also occur as a result of Freemartinism,
where placental fusion occurs in twins of dissimilar sex. However, the incidence
of hermaphroditism appears to be higher than that of freemartinism in this
species.
 Hermaphrodites that have been described in goats are mainly male
pseudohermaphrodites, having testes and the accessory reproductive organs of
the female; they are genetic females.
 Intersexes vary in the degree of external structural abnormality. Most are
generally female-like in appearance at birth but, as they grow and mature, there
will be evidence of an enlarged clitoris, perhaps testes in the inguinal region and
the development of male secondary sex characteristics, including the typical male
odour.

FUNCTIONAL FACTORS

 The goat is a seasonal breeder responding to the effects of declining day length. It
is not unusual to have irregular estrous cycles at the beginning and end of the
breeding season, especially in goatlings, with short cycles of between 5 and 7
days.
 Anestrus may be due to starvation, parasitism or mineral deficiencies. In the case
of the latter, phosphorus and the trace elements copper, iodine and manganese as
well as vitamin E have been implicated. It can also be influenced by chronic
debilitating diseases.

HYDROMETRA OR PSEUDOPREGNANCY ("CLOUD BURST")

 Hydrometra is the accumulation of sterile secretions within the uterine lumen.

The etiology of the condition is not known precisely, but it is always associated
 with high progesterone levels secreted by a persistent, functional CL, cessation of
cyclical activity, and variable degrees of abdominal distension. The incidence of
the disease varies between herds and within the same herd from year to year. One
possible cause of hydrometra is early, embryonic loss. However not all animals
will have been mated by the buck prior to pseudopregnancy.
 Two types of pseudopregnancy occur:
o After mating there is fertilization, followed by early embryonic death, the
CL persists and the doe acts as if pregnant. The abdomen becomes
enlarged, and in some there is a degree of udder development if not the
onset of lactogenesis. Those that are lactating may have a fall in yield. This
type of false pregnancy generally lasts for the duration of the gestation
period, or even longer, until the CL has regressed spontaneously. The term
‘cloud burst’ is used to describe the voiding of large volumes of fluid from
the uterus as the pseudopregnancy is terminated. Following this, the
abdominal distension disappears; some does may search for the ‘missing’
kids.
o Following estrus, when the doe was not mated, there is cessation of
cyclical ovarian activity but there is no marked hydrometra. At the end of
the period of acyclicity, affected does expel a bloody discharge.Therefore,
any unbred does that do not return to estrus after their first estrous cycle
in the autumn, should be treated with PGF 2α for possible
pseudopregnancy.
 Differentiation of hydrometra from normal pregnancy can be made using
transabdominal B-mode ultrasound imaging, demonstrating the presence of a
fluid-filled uterus in the absence of a fetus or placentomes. After 50 days of
anestrus, pregnancy and pseudopregnancy can be differentiated on the basis of
serum estrone sulphate levels. Treatment with 2.5 mg PGF2α will be followed by
expulsion of the fluid and estrus will occur in approximately 4 days. By using a
second injection 12 days after the first, good levels of fertility can be achieved
with 85% conceiving, compared with 95% of unaffected animals.

CYSTIC OVARIAN DISEASE

 Cystic ovarian disease has been described in dairy breeds and has been
particularly evident where they have grazed estrogenic clovers and legumes.
 A history of nymphomania may suggest follicular degeneration, and the typical
clinical sings are those of continuous estrus and short inter estrus intervals with a
failure to conceive. They should be treated with 1500 – 2500 IU of Human
chorionic gonadotrophin (hCG); Gonadotrophin-releasing hormone or
Progesterone for 18 days

MANAGEMENT FACTORS

Timing of Service or Artificial Insemination

  Optimum pregnancy rates are obtained when does are mated towards the end of
estrus (which lasts 12-36 hours) and just before ovulation.
 Some goat owners serve their does only in the first 12 hours of estrus supposedly
to increase the number of female kids but with a consequential reduction in
pregnancy rates.

Nutrition

 Vitamin A, certain minerals (manganese and iodine) and energy deficiencies

reduce fertility, and may be associated with abortion when the deficiency is
chronic.

Stress

 Stress-induced abortion has been described above in Angora goats. However,

there is evidence that other breeds of goat will abort if subjected to stress. This
can result from being chased by dogs, inadequate feeding, transportation and
adverse weather, particularly during the fourth month of gestation.

INFECTIOUS AGENTS

Non-specific infections appear to play a minor role in causing infertility in does,

probably for similar reasons discussed above for the ewe. However, specific infectious
agents as listed below are important in causing abortion and have been discussed in
detail under the Module on Specific Infectious Diseases.

 Brucellosis
 Campylobacteriosis
 Chlamydial (enzootic) abortion
 Leptospirosis
 Listeriosis
 Salmonellosis
 Toxoplasmosis
 Q- Fever
 Mycoplasmosis

MODULE-11
INFERTILITY IN FEMALE FARM AND COMPANION ANIMALS-
PART II
INFERTILITY IN THE SOW AND GILT

 People working in pig production expect very high levels of fertility, and any
shortfalls represent a serious economic loss. The efficiency of a pig operation is
always described in terms of the number of pigs sold per sow per place per year,
or the number of kilograms of pig meat sold per square metre of pig unit.
However, there are certain fertility parameters that determine the efficiency of a
pig industry. They are
o Farrowing Rate: The number of sows that farrow to a given number of
services, normally expressed as a percentage.
o Farrowing Index: The number of farrowings per sow per year.
o Conception Rate (or non-return rate): The number of sows that conceive
to service expressed as a percentage of those served. The conception rate is
usually estimated as the non-return rate to estrus (28days after service) or
is identified by pregnancy diagnosis at 30 days or more, after service. This
term does not necessarily equate to the farrowing rate, as pregnancy can
end at any time, but it can provide an earlier warning of a problem.
o Non-Productive or Empty Days: The number of days in which a sow is not
pregnant. There are, of course, days during which it is not possible for a
sow to be pregnant (e.g. in lactation, and during the weaning to
estrus interval), which should be taken into account.
o Piglets Born Per Sow Per Year: This figure can be divided into two
components: total numbers born, and numbers born live.
 All fertility parameters interrelate and each producer must establish targets for
reproductive performance. Any discrepancy between the targets and the reality
represents an economic loss resulting from suboptimal fertility. Targets set for a
particular unit must take into account all management factors that influence
fertility.

FACTORS THAT AFFECT FERTILITY

 An investigation of a fertility problem in the pig is rarely, if ever, considered on

an individual sow basis. While attempting to solve a fertility problem in sows, the
importance of management and stockmanship should be borne in mind. Any
investigation of herd infertility should take into account management factors
before making a detailed study of other issues. The quality of stockmanship will
be reflected in such basic procedures as
o Estrus detection
o Supervision of service
o General hygiene
o Record-keeping
 Good stockmanship should involve a combination of
o Sound basic knowledge
 o Patience
o Empathy
o Sensitivity
o Organizational skills
o An appreciation of priorities.
 As units grow larger and as management systems change in response to
consumer demands, stockmanship characteristics and requirements may have to
be adjusted, but they remain, none the less, of paramount importance.
 Sows that deviate from the normal or required parameters of reproductive
performance are invariably removed from the herd rather than being treated or
being allowed to continue in a sub fertile way. Any investigation of herd infertility
must begin with an evaluation of the problem.
 Parameters of relevance to the investigation include:
o Herd size
o Age profile replacement rate
o Return rate/conception rate
o Distribution of return intervals
o Weaning-to-estrus interval
o Farrowing rate
o Total numbers born
o Total numbers born live
o Lactation length
o Number of non-productive (empty) days.
 Examination of records should provide a definition of the nature and extent of
the problem. This exercise should be followed by a clinical appraisal of the stock,
post-mortem examination and laboratory diagnosis. At the outset it should be
recognized that the route of a fertility problem is often multi factorial
 The problem will probably fall into one or more of the following categories:
o Anestrus
o Conception Failure
o Pregnancy Failure

ANESTRUS
(Investigation and Treatment)

 Anestrus is one of the most common reproductive disorders in sows and is defined as
‘the absence of estrus behavior (standing to a boar or to a riding test) but excludes the
normal interval (diestrus) between two successive estrus periods’. By definition, delayed
estrus is also included in this category.
 Anestrus is inevitable at certain stages in a sow’s life (e.g. before puberty, and during
pregnancy and lactation), and this should be taken into account in any investigation.
 The term ‘sub estrus’ refers to a condition in which cyclic animals show no obvious
external signs of estrus and is characterized by the presence of corpora lutea. On rectal
palpation the cervix is relatively small and firm in anestrus and softer in sub estrus under
the influence of this luteal tissue.
  Firstly, it is important to establish that the problem is truly one of anestrus and not
simply improper heat detection. Accurate estrus detection involves time and effort, and
strategic use of boar presence, in conjunction with good record keeping.

Investigation

 Once it is established that the problem on hand is due to anestrus, it is necessary

to investigate the situation by either
o ovarian function tests,
o post mortem examination or
o real time ultrasound.
 Ovarian function tests
o The ovaries of sows that fail to exhibit signs of estrus may be truly inactive
(anestrus), active with in apparent (sub estrus) estrus or may have CL due
to pregnancy.
o Tests for ovarian function such as changes in plasma progesterone
concentration will differentiate between these conditions. Progesterone
may be measured in plasma or whole sow’s blood by enzyme-linked
immunoassay (ELISA) and weekly blood progesterone assays will help
differentiate between truly inactive, pregnant and normally cyclical
ovaries.
 Post-mortem examination
o Ovarian function may be assessed by post-mortem examination of the
reproductive tracts of sows culled from the herd under investigation and
can reveal the following abnormalities.
 Acyclic ovaries: inactive, with some small follicles (<5mm
diameter), absence of corpora lutea.
 Cystic ovaries: Multiple large cysts (usually <14 mm in diameter),
generally containing some luteal tissue that produces progesterone.
Cysts may regress, but some persist and can inhibit estrus.Multiple
small cysts that often produce estrogens and result in sows having
markedly irregular cycle lengths and exhibiting intense signs of
estrus (nymphomania). Single cysts which rarely affect sow fertility
and tend to be incidental findings at post-mortem.
 Real time ultrasound
o This technique can be used to observe changes in the size and shape of the
ovaries by a non-invasive method in the live animal. Such investigations
are time-consuming; require expensive equipment and a considerable
level of expertise.

TOP

Treatment

 Anestrus is of economic significance and must be treated promptly by identifying

and remedying all contributing factors.
 o Injection of a preparation containing 200 IU hCG and 400 IU of eCG
induces estrus within 3-8 days.
o In sows that have normal cyclical ovaries, but are apparently sub estrus,
the condition may result from inadequate estrus detection. Hence, ideally,
the producer should be encouraged to observe for estrus in the presence of
the boar at least once (and preferably twice) each day from the day of
weaning.

CONCEPTION FAILURE

 Failure of conception is recognized by an increased number of regular returns to

service (i.e. returns at 18-24 days after service). The conception rate (or non-
return rate) in breeding herds should be at least 90% and an incidence of return
of higher than 10% should be regarded as abnormal and unacceptable.
Conception failure suggests that viable ova did not come into contact with viable
spermatozoa at the appropriate time. The result of this will be either total
conception failure (i.e. regular return to service) or partial conception failure (i.e.
reduction in litter size). Assuming that ovulation has taken place, conception
failure must be due to one or more of the following factors:
o Timing of Service
o Quality of Service
o Semen Quality

Timing of Service

 High embryonic survival rate and large litter size at birth can be ensured by a
single mating at the appropriate stage of estrus resulting in a high proportion of
ova fertilized at the optimum time. However, identifying this ideal time is most
difficult.
o The sow ovulates, on average, 36-44hours after the onset of standing
estrus
o Spermatozoa can survive for approximately 24 hours inside the sow’s
reproductive tract
o A service regimen must take all these factors into account and aim to
ensure that the uterus contains viable spermatozoa prior to the arrival of
the ova
o Inappropriate timing of service results in conception failure
 Keeping this in mind, the service management regimen should ensure that each
sow is served on the day of onset of standing estrus and at least once more, 18-24
hours later.

Quality of Service

 Service pen design, particularly with reference to the floor surface, is of direct
relevance to the quality of service. Young boars need to be trained to natural
service, and should be well supervised each time they mount a sow or gilt.
Semen Quality

 Semen quality can be affected in terms of ejaculate volume, sperm count, sperm
motility or morphology by a wide range of factors such as age, environmental
temperature, frequency of use and disease.
 Where boars are used for natural service, it may be that sub fertile or infertile
individuals remain unidentified. The physical breeding soundness, paying
particular attention to feet and leg conformation of the boar should also be taken
into account.

PREGNANCY FAILURE

 Pregnancy failure may be divided into two main sections:

o Failure to Establish Pregnancy
o Failure of an Established Pregnancy

Failure to Establish Pregnancy

 Unattached conceptuses within the uterus are susceptible to damage by many

factors. Where pregnancy fails around the time of maternal recognition of
pregnancy (i.e. around days 12-13) sows tend to return to estrus outside the
normal range for ‘regular returns’
 The problem may be associated with failure of maternal recognition of pregnancy
related to
o Stress
o Infectious challenge
 Failure of pregnancy at this stage may be total (resulting in an irregular return to
service) or partial (resulting in resorption of some embryos, and consequent
reduction in litter size)

Failure of an Established Pregnancy

 Death of conceptuses during the embryonic stage tends to result in resorption if

abortion does not occur
 Dissolution of embryos in the absence of anaerobic bacteria is an aseptic,
autolytic process resulting in complete disappearance of the products or a vaginal
discharge.Reduction in numbers born due to partial resorption of litter will be
the only presenting sign
 Once pregnancy has been confirmed (e.g. by the Doppler ultrasound technique
on days 28-35), fetal death is more likely to result from an infectious disease
 Beyond 35 days, fetal death will result in mummified fetuses at farrowing.
Mummification is the most common clinical manifestation of a viral infection
(e.g. Aujeszky’s disease, porcine parvovirus, porcine reproductive respiratory
syndrome or swine fever) at this time, although only a proportion of the fetuses
may be affected.
 INFECTIOUS FORMS OF INFERTILITY

 An infectious form of infertility can be of great economic significance to a unit. It

is vital, however, to ensure that management and stockmanship are adequate
before searching for an infectious agent in any investigation into infertility. A
whole range of management factors (e.g. environment, stress and nutrition) may
lower the natural defense mechanisms, rendering an animal population more
susceptible to disease.
 Various infectious causes of infertility have been dealt with in detail under the
Module on Specific Infectious Diseases Causing Infertility.

VULVAL DISCHARGES

 Vulval discharges are the most obvious clinical sign of bacterial genital infections
 Return intervals are not usually affected. In late pregnancy uterine infection can
lead to abortion
 Investigation of an outbreak of vulval discharge involves identification of the
source of the discharge by
o speculum examination per vaginam. Discharges may originate from the
vestibule, the vagina, the uterus or the bladder
o cytological examination
 The consistency of the discharge may be
o thin pale yellow fluid without blood or mucus or
o necrotic debris and mucus with or without blood

Differential Diagnosis

 Abnormal vaginal discharges should be differentiated from

o watery or slightly cloudy discharge of proestrus and estrus
o seminal fluids, including gel expelled after mating
o discharge during pregnancy
o lochial discharge following parturition that will normally persist for up to 5
days

Treatment

 Treatment involves
o improved hygiene, particularly in the service house
o antibiotic injection of sows at weaning
o a programme of in-feed medication

STRUCTURAL ABNORMALITIES OF THE FEMALE

REPRODUCTIVE TRACT
  Anatomical defects of the female genitalia include intersexuality, gonadal
hypoplasia and other miscellaneous abnormalities.

Inherited Hypoplasia of the Gonads

 Both sexes may be affected but it is more readily apparent in the male.

Intersexuality

 Such abnormalities are common and appear to be a hereditary condition

determined by recessive genes. Male pseudohermaphrodites are more common
and have testes that may be subanal or intra-abdominal. Externally, intersexes
resemble the female and micturate through the vulva, although a phallus may be
present. The animal may be considered a gilt until puberty, when it starts to
demonstrate male behavior.

Bilateral Uterine Tubal Lesions

 Structural sterility resulting from bilateral tubal lesions (e.g. hydrosalpinx,

pyosalpinx and ovarobursal adhesions) has been shown to occur in up to 33.3%
of sows and gilts that failed to breed.
 Apart from the uterine tubes, other parts of the tubular genital tract may show
aplasia or duplication but only when the whole tubular system is aplastic, or
when the vagina, cervix or uterine body is imperforate, will sterility result. The
condition of uterus unicornis will lead only to lowered fecundity.

SEASONAL INFERTILITY

 Reduction in fertility in pigs in the summer and early autumn has been reported
in many countries and appears to manifest as a range of problems including
delayed puberty in gilts, delayed post-weaning estrus in sows, regular and
irregular returns to estrus, delayed return to estrus, reduction in the farrowing
rate, embryonic death, ovarian cysts and silent estrus.
 Autumn abortion syndrome may also be connected to seasonal infertility.
 It has been suggested that heat stress is particularly damaging during the first 8-
14 days post mating.
 Improved management of sows to avoid stressful and overheated conditions
during the hot summer months can reduce the problem.

INFERTILITY IN THE MARE

 The objective of the veterinarian working in any horse-breeding enterprise, regardless of

size; should be to produce the maximum number of live, healthy foals from the mares
bred during the previous season. However, the biggest difficulty in reaching this aim is
the infertile or problem breeding mare.
  Very few mares are permanently and completely infertile, but subfertility of varying
degrees is a major problem.
 It might take several cycles to establish a pregnancy, and even then, there is an increased
possibility of pregnancy failure.
 Commitment from both mare owner and veterinarian is needed; the owner should be
made aware of this at the outset and be given a realistic expectation as to the chance of
success.
 Infertility in Mares has been dealt under the following headings
1. Protocol for clinical evaluation of a mare
2. Causes of infertility and sub-fertility
3. Structural abnormalities of the female reproductive tract
4. Functional forms of infertility
5. Puerperal metritis
6. Pyometra
7. Retained fetal membranes
8. Endometritis
9. Diagnostic aids used in equine endometritis

PROTOCOL FOR CLINICAL EVALUATION OF A MARE

 The clinician should be aware of how to investigate the problem breeding mare. A
protocol for such an investigation of an infertile or subfertile mare is outlined in
table given below.

Outline of a Step - by Step Protocol of the Clinical Examination of

an Infertile Mare

Steps Instruction
1 Obtain the mare's previous breeding history
2 Assess her physical condition, general health and perineal conformation
3 Culture swab samples collected from the vestibule, clitoral fossa and sinuses
4 Examination per vaginum using a speculum and collection of endometrial swabs
for bacterial culture and stained cytological smear.
5 Manual vaginal examination
6 Examine the reproductive tract by rectal palpation
7 Transrectal real time ultrasound examination of the reproductive tract
8 Endometrial biopsy
9 Endoscopic examination of the endometrium
10 Peripheral venous blood sample for hormone analysis
11 Peripheral venous blood sample or hair follicle for chromosome analysis.

CAUSES OF INFERTILITY AND SUBFERTILITY

  Many factors, either alone or in combination with others, cause infertility or subfertility
and can broadly be categorized into
o Infectious (Refer to Module on Specific Infectious Causes of Infertility)
o Non-infectious factors
 Structural Abnormalities
 Functional Aberrations

STRUCTURAL ABNORMALITIES OF THE VULVA, VULVO-

VAGINAL CONSTRICTION AND HYMEN

Vulva

 In the normal mare, the vulva provides the first effective barrier to protect the
uterus from ascending infection. The `normal' mare has three functional genital
seals forming a barrier between the external environment and the uterine lumen:
o the vulva,
o the vulvo-vaginal constriction
o the cervix.
 During estrus, the vulva and cervix relax, leaving the vulvo-vaginal constriction
as the only seal.
 The vulval lips should be full and firm and meet evenly in the mid line with 80%
or more of the vulval opening below the brim of the pelvis.
o If the vulval seal is high (more than 4 cm of length dorsal to the pelvic
floor) in relation to the pelvic brim, the vestibular seal is incompetent and
there will be aspiration of air with bacteria and contaminated material into
the vagina (pneumovagina ; also called `windsucking'). The initial
vaginitis may lead to cervicitis and acute endometritis resulting in sub
fertility.
o Contamination of the caudal reproductive tract with bacteria during
pregnancy can result in embryonic death, and in late pregnancy can result
in the development of placentitis and lead to abortion.
o Furthermore, the penumovagina may lead to urovagina (urine pooling
within the vagina) when the vestibule and urethral opening are displaced
cranially.
o The more severe conformational abnormalities are more likely to result in
failure of the vulval seal, and to increased faecal contamination since the
vulva forms a shelf on to which feces may collect. The vulval lips may be
angled at 25 or even 50° to the vertical in these cases.
 Defective vulval confirmation can be congenital, which is very rare, or acquired,
which is seen in (1) vulval stretching following repeated foalings, (2) injury to
perennial tissue, or (3) poor bodily condition (old, thin mares).
 Older multiparous mares are more commonly affected with pneumovagina.
However, young mares that are in work and have little body fat and / or poor
vulval conformation can develop pneumovagina. In some mares, pneumovagina
may only occur during estrus when the perineal tissues are more relaxed. Some
 mares make an obvious noise whilst walking, but in other mares the diagnosis
may be more difficult.
 Diagnosis is by
o The presence of frothy exudate in the anterior vagina on examination with
a speculum
o Rectal palpation of a ballooned vagina or uterus from which air can be
expelled confirms the diagnosis.
o Real time ultrasound examination of uterus may reveal the presence of air
as hyperechoic (white) foci sometimes seen as a line at the opposed
luminal surfaces.
o Cytological and histological examination of the endometrium may
demonstrate significant numbers of neutrophils indicative of an
endometritis. Rarely, eosinophils are also found in association with
pneumovagina.
o Treatment involves Caslick's Vulvoplasty Operation.

Vulvo-Vaginal Constriction

 Immediately in front of the external urethral opening is the vulvo-vaginal

constriction or vestibular seal. In genitally healthy mares this forms the second
line of defense against aspirated air and faecal material.

Hymen

 Manual vaginal examination of maiden mares often reveals the presence of

hymen tissue which generally breaks down with pressure. A complete persistent
hymen can also occur, which can result in the accumulation of fluid within the
vagina and uterus due to impaired natural drainage. Sometimes the hymen may
be so tough that it can only be ruptured using a guarded scalpel blade or scissors.
The small incision can then be enlarged using the fingers and hand. Rarely,
failure of proper fusion of the Mullerian ducts may result in the presence of dorso
ventral bands of fibrous tissue in the anterior vagina and fornix. They do not
interfere with fertility and are easily broken down manually.

VAGINA, PERINEAL LACERATIONS AND RECTO- VAGINAL

FISTULAS

Vagina

 Urovagina urine pooling, is the retention of incompletely voided urine in the

cranial vagina due to an exaggerated downward cranial slope of the reproductive
tract. Pneumovagina from a defective vulval conformation also predisposes to the
condition. Transient urine pooling, which is sometimes found in postpartum
mares, usually resolves after uterine involution has occurred. Clinical signs can
include urine dripping from the vulva, urine scalding and a history of failure to
conceive.
  Diagnosis is easiest using a speculum examination during estrus to detect urine
in the cranial vagina. Uterine infection with an accumulation of exudates in the
vagina can be confused with the condition.
 In severe cases, urine pooling should be surgically corrected by vaginoplasty
(perhaps more correctly termed caudal relocation of the transverse fold; as
surgical intervention is in the vestibule), urethral or perineal resection.
 Vaginal bleeding from varicose veins in the remnants of the hymen at the dorsal
vestibulovaginal junction is occasionally seen in older mares, particularly during
estrus and the second half of pregnancy. Although diathermy can be used,
treatment is not usually necessary as the varicose veins normally shrink
spontaneously.

Third - Degree Perineal Lacerations and Recto-Vaginal Fistulas

 Both conditions are most often seen in young, primiparous mares where the
rigidity of the birth canal, especially the vulvo-vaginal junction, is important in its
pathogenesis.
 In most cases, the veterinarian becomes involved only after the foal is born and
the damage already exists. For treatment in the acute situation, it is difficult to
estimate the amount of devitalized tissue. Even though the edges of the wound
may look fresh and clean, much more tissue is damaged and bruised. This is why
immediate repair is not performed, unless one is present within 2 hours of the
injury, and even then most clinicians advise delaying surgery.
 First aid treatment should include:
o debridement of non-viable tissue
o provision of haemostasis and general cleaning of the area
o parenteral broad-spectrum antibiotics for 5 days
o NSAIDs and tetanus prophylaxis
o daily cleaning
o monitoring of uterine involution.
 Elective surgery is performed after atleast 10 weeks, and if the foal survives, the
operation is best performed after weaning.

STRUCTURAL ABNORMALITIES OF CERVIX

Cervix

 The cervix is the important third (and last) protective physical barrier to protect
the uterus from the external environment. The cervix must also relax during
estrus to allow intrauterine ejaculation and drainage of uterine fluid. An
inflammation of the cervix is usually associated with endometritis and / or
vaginitis.
 Anatomically, the cervix is a thick-walled sphincter. Expansion and contractions
are possible due to the action of the longitudinal and circular smooth muscle,
which is rich in elastic fibres. A distinctive feature of the equine cervix is its
dilatability, and the absence of rigid, annular constricting rings seen in farm
 animals. This means that the uterine body can be entered by a relatively large-
diameter instrument.
 Often an older maiden mare has an abnormally tight cervix due to fibrosis. The
cervix fails to relax properly during estrus, so that fluid is unable to drain and
accumulates in the uterine lumen. In many cases this fluid is sterile and contains
no neutrophils. Once the mare is bred, the fluid accumulation will be exacerbated
due to poor lymphatic drainage and impaired myometrial contraction
compounded by the tight cervix.
 Failure of the cervix to open during estrus can lead to unwillingness of the
stallion to complete mating or ejaculate intravaginally. Artificial insemination has
been used successfully in mares with an abnormally narrow cervix. Mares with a
fibrosed cervix that become pregnant do not normally have any difficulties at
foaling.
 Failure of the cervix to close during diestrus can lead to persistent endometritis
and failure to conceive, or early embryonic death. Failure to maintain closure
during pregnancy can lead to gestational failure.
 Assessment of the cervix must form a part of the routine pre breeding
examination of a mare, either directly using a speculum per vagina and / or by
digital exploration, preferably during diestrus when it is more tightly closed
under the influence of progesterone.
 Injury, resulting in cervical incompetence or fibrosis, most often occurs during
parturition when fetotomy is performed by an inexperienced clinician, or without
adequate instrumentation., during vigorous mating by an oversized stallion,
especially if the mare was not in full physiological and behavioural estrus,
although usually it is not too severe, or by irritant chemicals such as povidone-
iodine.
 If severe, cervical lacerations may need surgical repair to restore normal cervical
shape and function.
 Developmental abnormalities of the cervix include aplasia and a double cervix.

UTERUS

Uterine Cysts

 Uterine cysts are the most common type of uterine lesion identified in the mare.
The two distinct morphological types are
o endometrial cysts, which are usually 2 cm or less in diameter
o lymphatic cysts, which are generally larger
 Cysts can be confused with an early conceptus and give rise to false positive early
pregnancy diagnosis or the incorrect diagnosis of twin pregnancies during
ultrasound scanning. Differentiation is based on
o previous cyst mapping
o early mobility of the conceptus
o the conceptus's spherical appearance
o growth rate
  Some cysts can be very difficult to distinguish from pregnancies. Reassessment of
the irregular structure based on ultrasound will confirm that the fluid is
contained, and does not extend up or down the horn as would be found with free
fluid. The appearance of an embryo around 22-24 days of pregnancy provides a
definitive diagnosis. Thorough identification of cysts at the beginning of the
breeding season minimizes the chance of false positive pregnancy diagnosis
 Larger lymphatic cysts may interfere with the mobility phase of the early
conceptus and thus prevent luteolysis (failure of maternal recognition of
pregnancy). Later in pregnancy, the absorption of nutrients and the development
of chorionic villi may be diminished in places of contact between cysts and fetal
membranes leading to an increased risk of embryonic death
 The need for treating endometrial cysts is uncertain
o If a mare is found at the beginning of the breeding season with a large
number of cysts, it is generally best to continue to attempt to get the mare
in foal that reason
o If she fails to become pregnant, some form of treatment should be
attempted and an endometrial biopsy should be taken to help determine
the likelihood of her carrying a foal to term because of risks such as uterine
haemorrhage
o Larger cysts can be punctured using an endometrial biopsy instrument or
manually if the cervix allows passage of one hand
o Chemical curettage has equivocal results; the cysts may disappear but scar
tissue may form
o Thermocautery, in conjunction with endoscopy involving looping and
subsequent burning of cysts, is possible. Wounds after cautery appear to
heal very quickly, usually within -6 weeks. Because endoscopy should be
done while the mare is in diestrus when the cervix is relatively closed,
prostaglandin F2α should be given after cauterization and the uterus
should be lavaged with saline
 Most uterine cysts involve the endometrium. Occasionally an extra luminal cyst
lying external to the endometrium can be identified on ultrasound examination.
Its location should be verified by identification of the uterine lumen. Extra
luminal cysts usually have no adverse effects on fertility

Partial Dilatation of the Uterus

 Ventral uterine enlargement originated by one of four mechanisms:

o mucosal atrophy,
o myometrial atony,
o lymphatic lacunae
o endometrial cysts.
 Their precise relationship to subfertility is not clear, but mares that fail to
eliminate the fluid and debris that accumulate in these sacculations after mating
are susceptible to the establishment of chronic endometritis
 Treatment is the same as for mares with defective uterine clearance

Uterine Adhesions
  Uterine adhesions are most frequently diagnosed on endoscopic examinations of
the uterus. Multiple adhesions adversely affect fertility by causing fluid
accumulation or by affecting the mobility of the conceptus
 It is possible to remove the obstruction endoscopically by either cautery or laser
techniques, starting at the thin membranous parts of the obstruction. It is
important not to `burn' too deeply in the uterine wall, as in these cases more
severe damage to the uterine wall might occur. After removing the obstruction,
the uterus should be flushed to remove any debris and the mare treated with
PGF2α
 In addition to an assessment of an endometrial biopsy, the prognosis for future
breeding also depends on the severity of the obstruction and to what extent the
obstruction could be removed

Uterine Foreign Bodies

 Uterine foreign bodies (e.g. fetal remnants) may act as a nidus for the
establishment of chronic endometritis but are uncommon. Other foreign bodies
that have been reported include straws following AI, and the tips of uterine swabs
 Uterine neoplasia, abscesses and haematomata are rarely reported in the mare

Uterine Tubes and Periovarian Structures

 Uterine tube abnormalities are usually due to remains of embryological

structures, and are rarely reported in the mare. The presence of collagenous
masses within the uterine tube that might occlude the lumen has been
documented
 Dye tests are used in cattle to test tubule patency, but this is difficult in the mare,
and occlusion is very rare
 Cysts lying within the ovarian stroma near the ovulation fossa of the ovary arise
from the surface epithelium and are often seen in older mares during
examination of the ovary. They are known as `retention', `inclusion' or `fossa'
cysts and generally have no adverse effect upon fertility
 Periovarian cysts, are not endocrinologically active, do not usually interfere with
the process of ovulation and do not generally affect fertility
 Occasionally, large cysts may be palpated or imaged with ultrasonography and
may be confused with follicles. However, the lack of change in size or appearance
of these structures is usually diagnostic

OVARY

Ovarian Neoplasia

 Reasons for the presence of a large ovary in a mare include

o a normal ovary during the transition or breeding season with large
follicles, as frequently detected during early spring, persistent luteal phase
and early pregnancy;
 o a solid neoplastic lesion, such as a granulose theca cell tumour, teratoma,
dysgerminoma, cystadenoma and carcinoma
o haematomata of the ovary;
o abscesses and
o haemorrhagic and luteinised follicles.
 Ovarian neoplasia is uncommon in the mare although many types of tumour have
been described, with the granulose theca cell tumors (GTCTs) being by far the
common.

Gonadal Dysgenesis

 This condition is not common. However, in a maiden mare, once winter

anoestrus has been eliminated as a cause of acyclicity, XY ovarian dysgenesis
must be considered as a possible cause with small, inactive ovaries and an
immature tubular genital tract. Examination of the reproductive system detects
very small ovaries (<1 cm in diameter) and a poorly developed tubular genital
tract, which is difficult to palpate. This is similar to mares with XO chromosomes
(Turner's syndrome). There is no treatment, and the mare is sterile.

FUNCTIONAL INFERTILITY AS A CAUSE OF SUBFERTILITY

 Mares are seasonally polyestrus, and environmental and other factors can exert a
profound effect on reproductive function, particularly during the transitional period
between winter anestrus and the onset of cyclical activity in the spring. Although
irregularities of follicular development, ovulation and behavioural patterns are also
observed during the normal breeding season, they are not as common. However,
endometritis can also cause cyclical irregularities.
 Functional infertility can be dealt under the following headings:
o Anestrus due to Ovarian Acyclicity
o Anestrus caused by a Prolonged Luteal Phase
o Behavioural Anestrus - Silent Estrus
o Anestrus caused by a Shortened Luteal Phase
o Irregular or Prolonged Estrus
o Ovulatory Dysfunction

ANESTRUS DUE TO OVARIAN ACYCLICITY

Winter Anestrus

 The onset of cyclical activity is stimulated by increased day length. During winter
months mares are normally acyclical
 Diagnosis
o On rectal palpation or transrectal ultrasound imaging both ovaries will be
small (<3 x 2 x 2 cm), and in some mares there will be a number of small
follicles. Plasma progesterone concentrations are > 1 ng/ml
 Treatment
 o Although increasing day length is the primary controlling factor, ensuring
freedom from disease and good body condition by stabling, adequate
nutrition, anti helminthic therapy and attention to dental conditions can
hasten the onset of cyclical ovarian activity. Thus, prolonged anestrus can
be prevented by good management. Progesterone / progestogen
withdrawal therapy has been used successfully
o Progesterone can be administered as an oil-based intramuscular injection,
orally as the synthetic progestogen altrenogest (Equine Regumate) or by
using a silastic progesterone - releasing intravaginal device (PRID).
However, such therapy is effective only in anestrus mares that are already
well into the transitional phase to the resumption of normal cyclical
ovarian activity
o Repeated daily injections of equine pituitary gland extract to mares in
winter anestrus lead to follicular development. In aged mares, the delayed
initiation of normal cyclical ovarian activity may reduce the number of
estrus cycles during the breeding season and, therefore, it is particularly
important to prevent poor body condition from occurring in such animals
 Pituitary abnormalities
o Rarely Cushing's syndrome caused by adenomatous hyperplasia of the
intermediate pituitary has been associated with anestrus in aged mares.
This is presumably due to destruction of the cells secreting luteinising
hormone and follicle stimulating hormone

Lactation - related Anestrus

 Lactation - related anestrus is commonest in mares foaling early in the season.

Affected mares may have a normal postpartum estrus after 6-12 days, but fail to
return to estrus at the end of the first diestrus. Alternatively they may not even
have a normal `foal heat'
 Diagnosis
o The ovaries resemble those of a mare in deep winter anestrus. ie. small
and inactive, the condition can last for several months. Originally it was
thought to be due to prolactin suppressing pituitary gonadotrophin
release, but this is now in doubt. Affected mares should be teased and
examined weekly per rectum to assess their ovarian status
 Treatment
o Treatments similar to those described above for winter anestrus have been
used, but with little success. Twice-daily injections of 0.04mg (10 ml) of a
synthetic GnRH analogue (buserelin ; Receptal) have been found to induce
the development of a follicle within 7-14 days of commencing therapy

ANESTRUS CAUSED BY A PROLONGED LUTEAL PHASE

Persistence of Luteal Activity

  Persistennce of luteal activity in the non-pregnant mare is a major cause of
subfertility. Traditionally, the term `prolonged diestrus' has been used to
describe a condition where the corpus luteum persists beyond its normal cyclical
life span of 15/16 days, resulting in the maintenance of elevated circulating
progesterone concentrations for longer than expected
 These occur in upto 20% of estrous cycles in thoroughbred mares (less frequently
in ponies) and are not accompanied by estrus; the cervix will remain pale in
colour, dry and tightly closed. If diestrus ovulations occur late in the luteal phase,
they will be refractory to the effect of endogenous luteolysins, resulting in a
persistent luteal phase
 True persistence of the corpus luteum occurs in approximately 20% of ovulations.
These mares present great difficulty to the stud manager as they can be assumed
incorrectly to be pregnant
 Diagnosis
o Plasma progesterone profiles are indistinguishable from those of pregnant
animals. The uterus becomes firm and tubular (tonic) and the cervix is
typical of that of pregnancy. Transrectal ultrasound imaging fails to detect
a conceptus
 Treatment
o Failure of synthesis and or release of PGF2 α at the end of diestrus is the
most likely cause of persistence of the corpus luteum. Failure of the corpus
luteum to respond to PGF2 α or failure of PGF2 α to reach the corpus
luteum could also be a reason for CL persistence
o Treatment is by the injection of a luteolytic dose of PGF2 α or a synthetic
analogue. The interval between treatment and ovulation varies
considerably depending upon the size of follicles at the time of treatment.
Therefore it is invisible always to examine mares using ultrasonography
before treatment in order to assess the status of folliculogenesis

Pyometra

 Pyometra is the accumulation of substantial quantities of inflammatory exudates

in the uterus causing its distention. When the endometrium is severely damaged,
there is extensive loss of surface epithelium, severe endometrial fibrosis and
glandular atrophy causing a prolonged luteal phase, presumably due to
interference with the synthesis or release of PGF2α .
 This is in contrast to mild endometritis with the collection of small amounts of
intra luminal uterine fluid, which is more likely to cause premature release of
PGF2 α

Pregnancy and Pseudopregnancy

 Pseudopregnancy is a term used to describe a syndrome in which non-pregnant

mares that have been mated do not return to estrus. It occurs if there is early
embryonic death after 15 days of gestation with persistence of the corpus luteum
veerum resulting in a prolonged luteal phase
  The cervix remains tightly closed, and the uterus is tense and tubular. It is
differentiated from pregnancy by the absence of a conceptus on ultrasound
examination. If early fetal death occurs after endometrial cup formation at 36
days, mares will either become anestrus or come into estrus. However, in the
latter, follicular luteinization without ovulation is thought to occur and therefore
the estrus is not fertile; this will last until the endometrial cups regress
spontaneously at 90-150 days. There is currently no practical way of destroying
endometrial cups prematurely

BEHAVIOURAL ANESTRUS-SILENT ESTRUS

 In silent estrus, mares either do not show estrus, or are slow to show detectable
signs using standard teasing methods despite the fact that ovulation occurs
 The degree of reduced expression of estrus varies from partial (sub estrus) to
complete (anestrus)
 The incidence of silent estrus is higher in maiden mares early in the breeding
season and in mares with a young foal `at foot'
 Other factors that affect estrus behaviour include being at grass with very
dominant mares, and stallion preference.Fillies that are in training and have been
treated with anabolic steroids may be more likely to suffer from the condition due
to `androgenisation'.
 In many cases, it is a failure of the estrus detection system rather than a true
reproductive disorder of individual mares. However, it has been associated with
reduced estradiol concentrations in the peripheral circulation and a shorter
interval from luteolysis to ovulation

Diagnosis

 Rectal and vaginal examinations confirm that the mare is in estrus and has
follicles of an ovulatory size. It is essential to distinguish the condition from a
prolonged luteal phase in which there is also follicular development

Treatment

 The treatment is based on thorough and careful teasing. Frequent and persistent
teasing may persuade the mare to show estrus. Alternatively, placing the mare in
a stable next to a stallion may be helpful
 If permissible, artificial insemination can be used. To breed mares naturally
during a silent estrus, some form of restraint may be necessary; many mares
approaching ovulation accept the stallion when twitched and hobbled
 An intramuscular injection of estradiol benzoate (10-20 mg) 6 hours before
breeding can be tried as a last resort
 The veterinary surgeon must ensure that the mare is physiologically ready to be
bred. In some cases when the mare is not psychologically prepared for breeding,
estrogens are of little value, and tranquilizers may be more appropriate
 SHORTENED LUTEAL PHASE

Endometritis

 At coitus, the mare's uterine lumen becomes contaminated with microorganisms

and debris leading to a transient endometritis that usually resolves spontaneously
within 24-72 hours so that the environment of the uterine lumen is compatible
with embryonic and fetal life
 This endometritis should not be regarded as a pathological condition. However, if
the endometritis persists after day 4 or 5 of diestrus, in addition to being
incompatible with embryonic survival, the premature release of PGF2α may
result in luteolysis and a rapid decline of progesterone and an early return to
estrus
 These mares are referred to as susceptible and they develop a persistent
endometritis


IRREGULAR OR PROLONGED ESTRUS

 True persistent estrus appears to be rare in mares other than during the
transitional period from winter anestrus, or in association with steroid hormone-
producing ovarian tumours. Some cases that are presented as having a persistent
estrus may actually represent normal behavior.
 Frequent urination due to hind limb or back pain, or a urogenital problem may
be mistaken for persistent estrus.

TRANSITIONAL 'SPRING' ESTRUS

 Pressure to breed mares early in the year before onset of their natural breeding
season can pose problems for the veterinarian. Because of considerable variation
in the duration of estrus during the transitional period, efficient handling of the
mare can be difficult. Shortly after winter solstice, changes in the
pineal/hypothalamic pituitary axes result in some follicular growth; however,
follicles remain small, do not ovulate, and regress. Eventually, after a variable
transitional period of upto 2 months, larger follicles (> 35 mm) will develop and
ovulate, usually adding the onset of normal cyclical ovarian activity.

Diagnosis

 The diagnosis is by thorough ultrasonographic examination and rectal palpation,

which can diagnose transitional follicles reaching a preovulatory size of > 30 mm.
Visual identification of a corpus luteum or progesterone levels above 4 ng/ml
indicate that the first ovulation has occurred and the onset of normal ovarian
cyclical activity.
Treatment

 The treatment of mares in the transitional stage is based on progesterone or

progestogens, with or without the addition of estradiol esters. Progesterone can
be administered as an oil-based intramuscular injection, orally the synthetic
progestogen altrenogest or by using a silastic progesterone releasing intravaginal
device.
 Progesterone exerts a negative feedback on gonadotrophin secretion which is
followed by an decreased release of FSH and luteinising hormone (LH). When the
source of progesterone is withdrawn or its effect wanes, because of the
withdrawal of the negative feedback effect there is follicular growth, maturation
and ovulation.
 Progesterone treatment is more effective in mares that are in late transitional
stage and is ineffective in mares with minimal follicular activity, particularly
during deep anestrus.
 Currently, the most effective treatment is the use of in-feed medication with the
potent progestogen altrenogest (Equine Regumate).
o This liquid contains 2.2 mg/ml of the active substance and should be
added to the food once per day at a dose rate of 0.044 mg/kg body weight
for 10 consecutive days.
o Estrus occurs within 6 days and ovulation between 7 and 13 days after the
last treatment.
o Because of the possibility of ovulation occurring during treatment, an
injection of PGF2α on the last day of in-feed medication may be necessary
to cause luteolysis of any corpus luteum that may be present.
 The use of intramuscular injections of progesterone and estradiol-17β in oil for 10
days produces a similar response to altrenogest, but the interval to estrus is
longer due to the suppression of follicular development by the estradiol.
 The use of 0.04 mg of buserelin (Receptal) given twice daily by intramuscular
injection is also quite successful. It is expensive, as treatment is necessary for
atleast 1-2 weeks.
 Regardless of the hormones used, mares undergoing treatment early in the
season need 16 hours of adequate light and good housing and nutrition to ensure
success.
 During the transitional period before the first ovulation of the year, mares
demonstrate erratic estrus behaviour of varying intensity.
o The presence of multiple large follicles, possibly as large as 30 mm, makes
detection of ovulation difficult by palpation alone.
o Even outside this transitional period, misinterpretation of ovulation, even
by experienced clinicians, has been shown to be as high as 50%.
o Visualization of the corpus haemorrhagicum/early corpus luteum
ultrasonographically when the anechoic follicle is replaced by an intensely
echoic area representing the early corpus luteum is much easier.
o It is recommended that the interval between matings should not exceed 2
or 3 days, although there have been no critical studies on the survival time
of sperm in the mares genital tract. It is important not to begin breeding
too early or this will result in the mare being mated many times.
 o The appearance of uterine edema is an indication that the follicle should
ovulate within a few days.

CYSTIC OVARIAN DISEASE AND OVARIAN NEOPLASIA

Cystic Ovarian Disease

 Cystic ovarian disease as comparable to the condition described in the cow does
not occur in the mare. The persistent follicles that occur during the transitional
and other periods are structurally normal; however, their presence may explain
why this condition has been diagnosed in the past.

Ovarian Neoplasia

 This has been considered earlier under structural infertility.

CHROMOSOMAL ABNORMALITIES

 The normal chromosome complement for the domestic horse is 2n = 64. Various
sex chromosome anomalies have been described in the horse, but are not
common. The incidence of chromosomal abnormalities is difficult to assess, but
must be suspected in the maiden mares with small, inactive ovaries and an
immature tubular genital tract once winter anestrus has been eliminated as a
cause of acyclicity. However, some genetically normal young fillies in training can
be acyclic and thus they must be given more time to mature reproductively;
karyotyping must be performed before making a final diagnosis.
 The main karyotypic abnormality of such mares is the 63, XO (Turner’s
syndrome) genotypes. Examination detects very small ovaries (<1 cm in
diameter) and a poorly developed tubular genital tract that is difficult to palpate.
These mares are usually small for their age and do not cycle, although
occasionally they may show passive estrus signs. There is no treatment and the
mare is sterile.
 Other chromosome abnormalities include ovarian hypoplasia and testicular
feminisation. Thee are also rare, but must be considered in female horses with
irregular cycles and small ovaries during the breeding season.

OVULATORY DYSFUNCTION

Anovulatory Haemorrhagic Follicles

 A form of apparent ovulatory failure has been described in the mare wherein
o preovulatory follicle grows to an unusually large size (7-10 cm)
o Fails to rupture and ovulate, but fills with blood and then gradually
regresses.
 o These haematomata persist for a variable period of time, often beyond the
next ovulation and corpus luteum formation and normal cyclic ovarian
activity continues.
o Spontaneously resolve and no treatment is required.
o The condition is known as 'haemorrhagic anovulatory follicle syndrome’.
o The condition can be diagnosed ultrasonographically where the
preovulatory follicle filled with blood is initially recognized during
transrectal ultrasound, by the presence of scattered free-floating echogenic
spots within the follicular antrum. As the blood coagulates, the ultrasonic
appearance varies from honeycomb or `net-like’ to a uniformly echogenic
mass.
o These structures can be as large as 8-10 cm, occasionally much larger, and
develop an outer wall of luteal tissue.
o Functionally, they gradually regress in the same way as a normal corpus
luteum, but they remain visible ultrasonically over subsequent estrous
cycles.
o No treatment is usually necessary. Sometimes they may also fail to regress
around day 14-15 of the cycle and persist.
o Haemorrhagic follicles may be difficult to diagnose.
 The rise in plasma progesterone is not useful for detecting ovulation
since most haemorrhagic follicles tend to luteinise, thus producing
progesterone and hence their alternative name luteinised
unruptured follicle.
 These structures cannot be detected by the behavioural responses of
the mare, since estrogen concentrations are initially elevated, and
subsequently, progesterone concentrations may increase and
terminate estrus behaviour similar to that following ovulation.
 On palpation, they are smooth with varying degrees of firmness.
This can be confusing, since they may feel like preovulatory follicles
or corpora haemorrhagica, or they may increase in size and become
very large.
 The most obvious difference in their appearance is when they are
examined ultrasonographically. Commonly, there are multiple
echoes from within the follicular cavity, giving a net-like
appearance within the follicular fluid.
 The structures may have a similar appearance to that of a Granulose
Theca Cell Tumour (GTCT); the anechoic areas are separated by
trabeculae and are similar to those of a multicystic GTCT.
 The diagnosis of a haemorrhagic follicle may be made on the basis of clinical
signs: namely, maintenance of cyclicity, a normal contralateral ovary, the
presence of an ovulation fossa and speed of enlargement and regression of the
ovary with time.
 Their significance is that the oocyte is not released but remains within the large
unruptured haemorrhagic follicle. The abrupt decrease in follicle diameter
normally associated with ovulation is not noted, but rather a steady increaese in
size and shape; stigma formation due to follicle softening is not seen. However,
 one cannot unequivocally state that they did not form by rapid filling between
examinations.
 The cause of these haemorrhagic follicles is not known. Similar structures are
seen under continued equine chorionic gonadotrophin (eCG) stimulation during
days 40-15 of pregnancy.

Multiple Ovulation

 Double ovulations occur during 8-25% of estrous cycles, the frequency depending
upon the breed and type of the mare (thoroughbreds, highest rate; ponies, lowest
rate).
 Accurate detection of such ovulations is important as twinning is highly
undesirable;
o first, because it often results in abortion and,
o secondly, even if both fetuses survive and are carried to term, many are
dysmature, resulting in a high neonatal mortality rate.
o A further complication is that if embryonic / fetal death occurs after the
formation of the endometrial cups, these latter structures persist until they
spontaneously regress as if pregnancy had been maintained, resulting in
psedopregnancy.
 Rectal palpation alone can be misleading in detecting a double ovulation,
particularly when the two follicles are on the same ovary.
 The use of ultrasound examination of the ovaries, which should routinely be
performed in conjunction with a thorough transrectal palpation helps in
detection of a double ovulation. Sometimes the ovulatory area can appear
indistinct for the first 24 hours; in these cases the mare should be re-examined 2
days later when it can be seen more easily whether there is more than one corpus
luteum.

MANAGEMENT OF TWIN OVULATION

 Multiple ovulations in the mare should not be regarded as a reason for

withholding breeding. Instead, pregnancy rates are improved after twin
ovulation. Although accurate interpretation of the ultrasound image of early
pregnancies in the mare and the technique of crushing a conceptus are skills that
require experience, the advent of B-mode ultrasound imaging has provided a
method of more readily managing a twin pregnancy in the mare
 There are two approaches to dealing with twins :
o If the initial examination of the mare occurs before fixation (day 16/17) the
twin embryos are reduced to a singleton by the manual destruction of one,
either by pressure with the transducer or by the use of the hand. When the
conceptual vesicles are of dissimilar sizes, the smaller one should be
ruptured. This is easier at days 14-16 when they are 14-20 mm in diameter
than days 11-13 when they are 5-11 mm in diameter.
o The disadvantage of this method is that it is more expensive, in that all
mares are scanned before the time of return to estrus.
 o In addition, if ovulations that occur more than 3 days apart have not been
detected, a mistaken diagnosis of a single pregnancy may be made if the
second vesicle is too small to detect
o If initial examination is done after fixation but before day 30, and if both
conceptuses are in one horn, one option is to terminate a pregnancy using
PGF2α.
o It is advisable to re-examine the mare 5 days later in case reduction has
occurred, or transvaginal ultrasound-guided allantocentesis can be
attempted. Management of twin pregnancies after this period is
complicated by the formation of endometerial cups at approximately day
37, 38 of gestation.
o Endometrial cups remain functional until around days 90-130 of gestation
in the presence or absence of viable fetus. Therefore, if twin pregnancies
are not successfully managed before the cups are formed or both embryos
die after ay 37, the mare usually will not return to a fermale iestrus for a
prolonged period of time
 After day 37 of gestation, reduction methods are unreliable. They include dietary
energy restriction, surgical removal of one vesicle, intra-cardiac injection and
transvaginal ultrasound guided needle puncture

PREGNANCY FAILURE

 Pregnancy failure is a source of major economic loss to the equine industry. Embryonic
death occurs before 40 days of gestation when organogenesis is complete, with early
embryonic death (EED) occurring before the maternal recognition of pregnancy. Early
fetal death occurs before 150 days of gestation, and late fetal death occurs after that.
Abortion is defined as expulsion of the fetus and its membranes from day 300 onward.
o Embryonic Death
o Fetal Death and Resorption

EMBRYONIC DEATH

 In normal fertile mares the fertilization rate is more than 90%, which is comparable with
other domestic species, with estimates of the Early Embryonic Death (EED) rate at
between 5 and 24%. In subfertile mares, the rate is higher.
 The period of greatest embryonic death in subfertile mares occurs in the interval before
pregnancy can be detected with ultrasound (day 11), particularly at the time the embryo
enters the uterus. Between days 14 and 40, the rate of embryonic death varies between 8
and 17%. EED is multifactorial, in which external factors such as environment and
management as well as pathophysiological factors are involved. The factors involved in
embryonic death are
o External Factors
o Maternal Factors
o Embryonic Factors

External Factors
  External factors involved in embryonic death include stress, nutrition, season of the year,
climate, sire effects and transrectal palpation
 Maternal stress due to severe pain, malnutrition and transport has been implicated as a
cause of EED. Transporting pregnant mares for a distance of 300 miles (500 km) in less
than 9 hours of traveling time can be stressful, but should not result in embryonic death.
If a longer journey is necessary, the journey should be broken after 8 hours. Waiting
until the fifth week of pregnancy or later to transport brood mares may be advisable
when critical events such as descent of the embryo into the uterus and transition from
the yolk sac to the chorioallantoic placentation have occurred. The common practice of
transporting mares to stud for mating and returning home the same day should not be
detrimental to their fertility, as long as the transport is safe and comfortable
 Far from being avoided, regular exercise is important during pregnancy, although during
the latter half, forced exercise should be decreased. Rectal palpation and ultrasound
examinations should be considered safe procedures when performed correctly and there
is no indication that ultrasound examination is detrimental to the embryo

TOP

Maternal Factors

A number of abnormal maternal factors including hormone deficiencies and

imbalances, uterine environment, age and lactation have been implicated

 Hormonal Deficiencies and Imbalances

o Progesterone is critical for the maintenance of pregnancy in mares. The only
source of progesterone during the embryonic period is the primary corpus luteum
(corpus luteum verum). On the assumption that luteal insufficiency is important
in EED, many mares are given exogenous progesterone or progestogens in an
attempt to prevent it from occurring.Many dosage regimens do not effectively
elevate or maintain plasma progesterone levels. A single injection of 40 µg of the
GnRH agonist buserelin has also been shown to reduce the incidence of EED
when given 10 days after ovulation
 Uterine Environment
o An abnormal uterine environment is detrimental to embryonic survival. Acute
endometritis may result in EED by inducing premature luteolysis, or because of
its direct embryopathic effect
o Severe periglandular fibrosis of the uterine glands may reduce the chances of
embryo survival. Not only is this a response to persistent endometritis, but it also
increases with age. This is one of the reasons for the reduced fertility of mares
over 12 years of age
 Foal Heat Breeding
o Mares normally resume cyclical ovarian activity very shortly after parturition so
that they are sometimes bred as early as 7 to 10 days postpartum (at the foal
heat). There is conflicting evidence about the level of embryonic death if
fertilisation occurs at this time, with some studies showing a higher rate and
others no effect. An advantage of breeding at the first estrus post-partum is that
the foaling-conception interval is significantly shorter. The reason for the
apparent decreased fertility in mares mated at the foal heat is the hostile uterine
environment due to delayed uterine involution or persistent endometritis.
However, pregnancy rates are clearly influenced by how strict the selection
criteria are for mating at the foal heat. Traditionally, such factors as a normal
 foaling, placental expulsion, minimal vaginal bruising and absence of infection
have been used. Endometrial cytology and ultrasonic scanning of the genital tract
of each mare may be more reliable methods on which to base a decision
 Lactation
o More pregnancy failures are detected in lactating than non-lactating (maiden or
barren) mares; this phenomenon also increases with the age of the mare

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Embryonic Factors

 Embryonic abnormalities are also important to consider in- relation to embryonic death.
Embryos recovered from sub-fertile mares are smaller and have more morphological
defects than embryos from fertile mares; however, this may be due to an abnormal
uterine environment
 Ultrasonic scanning has provided a valuable tool in studying embryonic death. Because
pregnancy is often diagnosed at an early stage, it is important to inform owners that not
all pregnancies detected with ultrasound will survive, even in apparently normal mares
 There are certain morphological features detected with ultrasound that are typical of
mares in which embryonic death is occurring. Some of the consistent features include :
o Presence of fluid within the uterine lumen
o Prominent endometrial edema
o Decreased or prolonged conceptus mobility
o Undersized or irregularly shaped conceptus
o Cessation of embryonic heart beat
o Reduced volume of placental fluids
o Disorganization of placental membrances
o Hyperechogenic areas in the embryo and membranes

FETAL DEATH AND ABORTION

 The causes of equine abortion can be broadly divided into non-infectious (70%),
infectious (15%) and unknown (15%). In practice, it is important to distinguish infectious
from non-infectious causes. Vaginal discharge, premature lactation and colic in pregnant
mares may indicate an impending or recent abortion.
 When abortion occurs, the mare should be isolated, a history obtained and the fetus sent
to an approved laboratory for necropsy. If one wishes to perform a post-mortem
examination, small but representative samples of liver, lung, thymus, spleen and
chorioallantois (two samples, one of which is from the cervical star) should be sent in
formal saline for histological examination. In addition, frozen samples of fresh fetal liver
and lung should be stored in a deep freeze at –20°C should viral isolation investigation
be required at a later stage. Paired serum samples from the mare and close companions
should also be taken for serological investigation. Swabs from fetal heart or liver and the
cervical pole of the chorion are used to screen for bacterial infection.
 The fetus and fetal membranes (amnion, chorioallantois and umbilical cord) must be
carefully examined for the presence of abnormalities and areas of discoloration.

Infectious Causes of Abortion

  The causal agents of infectious abortion are viruses, bacteria, fungi and more rarely
mycoplasma and protozoa and have been dealt with in detail under the module Specific
infections that affect the genital tract in mares.

Non-infectious Causes of Abortion and Stillbirth

Twinning

 Historically, twins have been the single most important cause of abortion in
thoroughbreds. However, they are now much less common due to the widespread
use of ultrasonography. The diagnosis of twin pregnancy can be made even if only
one fetus is found as examination of the placenta reveals an area devoid of villi
where the two placentas were in contact. Twins should still be submitted to a
diagnostic laboratory as twin pregnancies are not protected from equine
herpesvires (EHV) infection.

Umblical Cord Abnormalities

 In mares, the umbilical cord is twisted, usually on a clockwise spiral. The normal
length ranges from 36 to 83 cm. Increased cord length has been associated with
excessive cord torsion, which can cause twisting of the umbilical blood vessels.
This twisting causes increased resistance to blood flow on both directions and the
resulting poor placental perfusion can lead to fetal death. This can result in
abortion of an autolysed fetus. Decreased cord length can cause premature
tearing of fetal membranes, leading to fetal asphyxia. Twisting and vascular
compromise currently constitute the commonest single cause of observed non-
infectious abortion.

Premature Placental Separation

 In mares, the interdigitating microvilli are connected by an unidentified electron

–dense material. Placental separation is largely unknown, although maternal
stress and endophyte-contaminated tall fescue have been implicated. When
placental separation occurs shortly before parturition, the thickened placenta
often does not rupture through the cervical star, and the allantochorion bulges
out of the vulva (`redbag’ delivery). The foal can become hypoxic, resulting in the
neonatal maladjustment syndrome.

Body Pregnancy

 In this condition almost the entire chorionic surface of the placenta contained
within the uterine body is without villi, while that contained within the horns is
covered with an excessive number of villi. The portion of the placenta
corresponding to the two uterine horns is small, and the fetus is situated entirely
within the uterine body. The fetus is frequently aborted completely contained
within its placenta; its growth has been retarded. The abortion occurs when the
nutritional demands of the fetus exceed the ability of the placenta to meet them.
Fetal Abnormalities

 Severe developmental anomalies involving the central nervous system and

development of body cavities have been reported in aborted fetuses.

Maternal Disease

 Pyrexia and malnutrition during pregnancy have been implicated as causes of

abortion.

PYOMETRA

 Pyometra is the accumulation of large quantities of inflammatory exudates in the

uterus causing its distention.
 It must be distinguished from the smaller, and intermittent, accumulations of
fuid that can be detected by ultrasonography in acute endometritis.
 Pyometra occurs because of interference with natural drainage of fluid from the
uterus, which may be due to cervical adhesions or an abnormally constricted,
tortuous or irregular cervix. In some cases, the fluid accumulates in the absence
of cervical lesions presumably due to an impaired ability to eliminate the
exudates. Other predisposing factors are chronic infection with P.aeruginosa or
fungi.
 Damage to endometrium leads to
o extensive loss of surface epithelium,
o severe endometrial fibrosis
o glandular atrophy.
 As a result the luteal phase becomes prolonged presumably due to interference
with the synthesis or release of PGF2α. This is in contrast to mild endometritis
with the collection of small amounts of intraluminal uterine fluid, which is more
likely to cause premature release of PGF2α and luteolysis.
 Some clinicians restrict the term `pyometra’ to cases where, in addition to the
accumulation of exudates within the uterine lumen, the corpus luteum persists
beyond its normal life span.
 Some mares with pyometra have normal regular cyclical ovarian activity.
Persistence of the corpus luteum is probably due to failure of the synthesis and or
release of prostaglandins from the uterus.
 Mares that have prolonged luteal phase have the greatest endometral damage.
 The mare with pyometra seldom shows over signs of systemic disease even when
there is upto 60 litres of exudate in the uterine lumen. Very occasionally there is
weight loss, depression and anorexia.
 Pyometra has been classified into two categories in mares. Open and closed.
o In a case of closed pyometra, the fluid accumulates due to a closed cervix.
o In open pyometra, the cervix remains open, but purulent material
accumulates because of impaired uterine clearance. A vulval discharge is
often observed in open pyometra, especially at estrus, which may vary
inconsistency from watery to cream like.
  Although the culture of endometrial swabs can sometimes result in the growth of
mixed organisms or sometimes no bacterial growth at all. In most cases the
organism isolated is S.zooepidemicus.

Diagnosis

 The diagnosis of pyometra is based upon rectal palpation, ultrasonic examination

of an enlarged fluid – filled uterus and analysis of the uterine fluid. Pregnancy
must be eliminated together with rare conditions such as mucometra and
pneumo uterus.
 Due to the lack of systemic illness, cases of pyometra have often become chronic
before treatment is sought. In such cases the prognosis is poor because of severe
endometrial damage, which is unlikely to be able to sustain a normal pregnancy.

Treatment

 The aim of treating pyometra is to expel the purulent material from the uterus.
 In the absence of systemic illness or an unsightly vulval discharge treatment of
chronic pyometra may not be indicated, although some mares can show signs of
discomfort during exercise.
 Many cases can be significantly improved by repeated large volume lavage with
several litres of warm saline via a wide- bore tube such as a nasogastric tube.
 Initially PGF2α can be used to induce luteolysis of the corpus luteum if present,
which should allow the cervix to relax sufficiently for digital exploration for the
presence of any adhesions. Estradiol or PGF2α may also help relax the cervix.
 The broad-spectrum combination of antibiotics and crystalline benzylpenicillin
should be infused after repeated large volume lavage and oxytocin to achieve
drainage of exudates, and an endometrial biopsy is useful in assessing the degree
of endometrial damage.
 Monitoring the uterus by a combination of rectal palpation and ultrasound
provides information on the response to treatment. Even if successfully treated,
the mare must be considered a susceptible mare if she is to be bred and managed
accordingly.
 In non responsive cases, hysterectomy can be performed following aspiration of
the exudates from the uterus although great care has to be taken to prevent
contamination of the peritoneal cavity.

RETAINED FETAL MEMBRANES (RFM)

 Retention of the fetal membranes (RFM) is properly regarded by veterinary

surgeons as a potentially more serious affection than the same condition in cattle.
This has originated from the times when draught horses predominated in the
horse population and was invariably followed by serious sequelae; as a result
early manual removal was the rule.
 Complications include acute metritis, septicaemia, laminitis and even death.
With prompt and effective treatment these sequelae can be avoided.
  In many cases, uterine involution is delayed even if these more serious
complications do not develop. The riding horses and ponies of today are less
likely to suffer form these complications, but RFM should be treated as an
emergency.
 The average time taken for the fresh membranes to be expelled is about 1 hour,
and should not exceed 2 hours, although there is debate amongst equine
clinicians about the latter. RFM is one of the most common peripartum problems
in the mare, with an incidence in the range of 2% to 10%.

Aetiology

 The precise cause of retained placenta remains unclear. The most likely is uterine
inertia due to hormonal imbalance. Oxytocin has an important role in
postpartum uterine contractions, and low levels of this hormone in the
circulation may result in abnormal myometrial activity. This in turn leads to
placental retention.

Clinical Signs

 The most obvious sign of RFM is the presence of a variable portion of tissue
protruding from the vulva; less commonly nothing is visible. Either this means
that no parts of the fetal membranes have been expelled or, more likely, portions
remain attached.

Treatment

 Initially, the protruding membranes should be tied in a knot to prevent them

touching the hocks.
 As uterine contractility plays an important role in the dehiscence of the fetal
membranes, administration of oxytocin is recommended as a first and most
successful method of treatment in up to 90% of cases.
 It is good rule not to wait longer than 6 hours after delivery of the foal; the time
interval should be shorter in heavy breeds. This method of treatment avoids
manipulation within the uterus, with the risk of introducing micro-organisms.
Oxytocin can be given via the intramuscular route (20-40 IU), which can be
repeated after 1 hour if the membranes have not been expelled.Altenatively, slow
intravenous infusion of 50 IU oxytocin in 1 litre of physiologic saline over 1 hour
could help. Symptoms of colic often follow injections of oxytocin and commonly
precede natural expulsion so that pain-relieving drugs and sedation may be
required.
 Only if this treatment fails and the membranes are almost detached but retained
within the uterus should one attempt gentle manual removal. This interference
should be carried out with scrupulous regard to asepsis, and no undue force
should be applied, for even moderate traction on the after birth may cause the
uterus to become inverted and prolapsed.
 In most cases of retention some separation of the allantochorion has occurred
and consequently a variable amount of the after birth hangs down from the vulva.
o The mare is effectively restrained and measures should be taken to protect
the operator from being kicked.
o The tail is bandaged and held to one side by the attendant while the
obstetrician thoroughly washes the perineum and rear of the mare.
o With the hand and arm protected by a clean plastic sleeve, the extruded
mass, or failing that the freed part lying within the vagina, is grasped and
twisted into a rope.
o The gloved hand anointed with lubricant is gently introduced along the
`rope’ to the area of circumferential attachment in the uterus. As the
`rope’ is gently pulled and twisted, the tips of the fingers are pressed
between the endometrium and the chorion.
o The villi are easily detached, and as the allantochorion is gradually freed it
is taken up by further twisting of the detached mass.
o The allantochorionic membrane is gently separated from the endometrium
by moving one of the hands between them. The tightest attachment is
usually at the tip of the horn. The process of separation usually goes quite
smoothly, and the complete sac of allantochorion can be gradually
detached from the pregnant horn. There is a tendency for attachment to be
firmer in the non-pregnant horn, and occasionally retention is confined to
this horn. If it is found impossible to detach the apical portions of the
allantochorionic sac without tearing the membranes it is better to desist
and to try again in 4-6 hours, by which time a successful outcome will be
likely.
o Unwanted side – effects of this manual removal may be serious
haemorrhage, invagination of one of the horns and a higher chance of
retention of microvilli in the endometrium.
o During a difficult manual removal only the central branches of the
chorionic villi are removed while practically all the microvilli are broken
off and retained ; rupture of endometrial and subendometrial capillaries
may also occur.
o The consequences of different removal are increased puerperal exudates,
containing much tissue debris; endometritis and laminitis; uterine spasm
and delayed involution of the uterus.
o A third method described in the literature, and which may be successful
under some circumstances, is the placement of some 10 titre of warm,
sterile saline inside the chorioallantoic membrane. Stretching of the
uterine wall stimulates uterine contractions, via endogenous oxytocin
release, and may assist in the separation of the microvili from their
endometrial crypts. This treatment should be used in combination with
exogenous oxytocin administration.
o After removal, it is always important to examine the membranes for
completeness confirming that al the allantochorion has been removed. If
necessary, the uterus should be flushed and siphoned to remove any fluid
exudates remaining in the uterus by using a stomach tube and funnel.
After care includes (depending on the severity of the case) regular general
clinical examination, particularly the uterus (for involution and contents)
and, if indicated, flushing and siphoning the uterus once or twice daily for
 a few days in combination with further injections of oxytocin. The
rationale for uterine lavage is to remove both debris and bacteria from the
uterus. Warm, sterile physiologic saline should be used in 2-4 litre flushes
(until the recovered fluid is clear).
o Special attention is paid for signs of laminitis, and non-steroidal anti-
inflammatory drugs are given when laminitis is a suspected complication.
o Tetanus antitoxin is recommended and, if indicated, treatment with
antibiotics.
o If there is a risk of the mare developing a toxic metritis, she should be
treated with systemic and intrauterine antibiotics. The dominant infective
organism is often Streptococcus zooepidemicus initially, but infection with
Gram-negative bacteria such as Escherischia coli frequently develops. The
antibiotics chosen should have broad-spectrum activity and should be
effective against endotoxin-producing organisms. Cyclo-oxygenase
inhibitors such as flunixin meglumine should be given to either treat or
minimize the risk of development of endotoxaemia.
 Provided treatment is begun at the correct time and no secondary complications
develop, the prognosis for a case of retained placenta is good.

ENDOMETRITIS

 The term `endometritis’ refers to the acute or chronic inflammatory process involving
the endometrium brought about by microbial infection or may occur due to non-
infectious causes. One of the main obstacles to producing the maximum number of live,
healthy foals from mares bred during the previous season is the mare, which is
susceptible to persistent acute endometritis following breeding.

Cause and Pathogenesis

 The underlying etiology of the specific cause of endometritis determines the type of
treatment to be used, and the following classification system for equine endometritis is
useful:
o Venereal Infection
o Chronic Infectious Endometritis
o Endometriosis (chronic degenerative endometritis)
o Persistent Mating-induced Endometritis (delay in uterine clearance).

VENEREAL INFECTION AND CHRONIC INFECTIOUS

ENDOMETRITIS

 It is generally assumed that the uterine lumen of the normal fertile mare is
bacteriologically sterile or may have a temporary, non-resident microflora. This is
despite the fact that the mare’s reproductive tract is often contaminated with
bacteria from the act of coitus, foaling and veterinary procedures. Mares with a
 defective vulval conformation can also aspirate air and bacteria into the vagina
that can develop into endometritis
 The bacterial species that cause bacterial endometritis are numerous, and can be
classified as follows:
o Contaminants and commensal
o Opportunist
o Venereally transmitted.
 Normally, the vestibular and clitoral area has a harmless and constantly
fluctuating bacterial population. In association with benign saprophytic
organisms, opportunistic organisms such as Streptococcus zooepidemicus, E.coli
and Staphylococcus spp. can be found. The stallion’s penis is colonized by similar
organisms. S. zooepidemicus is the most commonly isolated bacterial species
from acute endometritis, particularly in the initial stages. E. coli is the next most
common isolate.
 The uterus responds to these bacteria with a rapid influx of neutrophils. Normally
these neutrophils phagocitize and kill the bacteria rapidly (<24 hours). The
inflammatory byproducts are then mechanically removed and the endometritis
resolves itself expect when the mare suffers from pneumovagina or is a
‘susceptible’ mare. Susceptible mares have a delay in uterine clearance, and the
inflammatory byproducts accumulate as uterine fluid. Such mares have a reduced
pregnancy rate due to a hostile environment for the early developing conceptus
 In addition to opportunist pathogens, there are three bacteria that are venereally
transmitted: Taylorella equigenitalis (contagious equine metritis organism,
CEMO), Klebsiella pneumoniae and Pseudomonas aeruginosa (some strains)
 Symptomless carriers of both sexes allow persistence within the horse
population. Carrier mares, which may or may not have shown signs of previous
endometritis, harbour the organisms in the vestibular area, particularly the
clitoral fossa and sinuses. Mating or gynaecological examination may result in
their transfer into the uterus. Stallions may harbour the organisms over the
entire surface of the penis and in the distal urethra. Control is by laboratories
experienced in the isolation and identification of these specific organisms

DIAGNOSIS

Venereal Disease Screening

 Before the breeding season, swabs should be taken from the clitoral fossa, clitoral
sinuses (only the central sinus may be obvious ) and the vestibule. The perineal
area of the mare should not be cleaned except for the removal of gross
contamination of the vulva with faeces using a dry paper towel.
 A protective disposable glove should be worn by the veterinary surgeon on the
hand used to evert the ventral commissure of the vulva and expose the clitoris.
The swabs should be placed in transport medium, clearly labeled with the mare’s
name and sent to an approved laboratory.
 ENDOMETRIAL CULTURE AND CYTOLOGY

 A diagnosis of endometritis can be made by collection of concurrent endometrial

swab and smear samples during early estrus for bacteriological culture and
cytological examination, respectively. This allows time for resolution prior to
mating, and maximizes the chances of pregnancy.
 The ideal technique should ensure that the swab enters the uterus and collects
bacteria from the uterine lumen only. It is important to ensure that the method of
swabbing does not introduce bacteria into a previously normal uterus. Two
methods can be used:
o A non-guarded endometrial swab on a sterile extension rod is carefully
passed via a sterile speculum through the cervix into the uterine body and,
after withdrawal, is placed in transport medium. A second swab is taken
immediately afterwards for the endometrial smear.
o A guarded swab is passed into the uterine lumen using a sterile speculum
or enclosed in a disposable plastic arm-length glove. The swab tip is
exposed only when it is in the uterine lumen. A swab for cytological
examination should again be taken. To reduce the risk of contamination,
the use of guarded swabs is advised.
 Swabs for culture should be plated on blood and Mc Conkey agar, and incubated
at 37°C for 48 hours. Cultures should be examined at 24 and 48 hours.
 An air-dried smear is made by gently rolling the second swab on a clean dry
microscope slide.The smear can be differentially stained with a rapid stain such
as Diff-Quick (American Hospital Supplies).The stained smear should then be
examined for the presence of inflammatory and endometrial cells, the latter
confirming contact of the swab with the endometrium. The veterinarian must
ensure that the mare is not pregnant before passing a swab through the cervix.
o A positive culture result, with no evidence of inflammatory cells in the
smear (usually neutrophils), is likely to be due to contamination during
collection.
o Diagnosis of acute endometritis is based on the presence or absence of
significant numbers of neutrophils in the smear. Mares that have > 5
neutrophils / high power field (x40) on a cytology smear should be
considered to have active endometritis.

ENDOMETRIAL HISTOLOGY

 In some cases, endometrial biopsy may be a useful diagnostic aid. The technique
involves the insertion of a biopsy instrument through the cervix and into the
uterus. The instrument most commonly used today is the Yeoman (basket-jawed)
biopsy forceps, ideally 60-70 cm in length, with which tissue specimens 2 x 3 x 1
cm (about 0.2% of the whole endometrial surface) are obtained. If the uterus
appears normal on palpation, the sample should be taken from one of the areas of
embryo fixation, i.e., the uterine horn-body junction on either side. Single
samples are usually representative of the entire endometrium.
  If the uterus is abnormal on palpation per rectum, biopsy samples should be
taken from both the affected area and a normal area. Biopsy specimens should be
fixed in Bouin’s followed by sectioning and staining with haematoxylin and eosin.
The endometrial biopsy sample should be sent to a laboratory that is experienced
in evaluating samples.

UTERINE LUMINAL FLUID

 The detection of uterine fluid during both estrus and diestrus has been reported.
Endometrial secretions and the formation of the small volume of free fluid may
be associated with the same mechanism that causes normal estrual edema
 In many cases, the uterine luminal fluid that accumulates before mating is sterile
and contains no neutrophils. The importance of these sterile fluid accumulations
is that though initially sterile, the fluid may act as a medium for bacteria that gain
entry into the uterus at mating to multiply and may be spermicidal
 The amount of fluid that should be considered significant is not clear and it may
be that quantity is more important than nature. This is particularly true of fluid
appearing during estrus
 The significance depends to some extent on when during estrus the fluid is
observed. Fluid detected early in estrus may have disappeared when the mare is
further advanced in estrus and the cervix relaxes more
 Small volumes of intrauterine fluid during estrus do not affect pregnancy rates, in
contrast to mare with larger (>2 cm depth) collections of fluid. In mares that are
susceptible to endometritis there is an accumulation of more fluid than in non-
susceptible mares
 Generally if there is more than 1 cm of fluid during estrus, some attempt should
be made to remove this before breeding using oxytocin treatment. If the volume
is above 2 cms, the fluid may need to be drained and investigated for the presence
of inflammatory cells and bacteria. The mare may then need to have a large-
volume uterine lavage
 Intrauterine fluid during diestrus is indicative of inflammation, and associated
with subfertility, due to early embryonic death and a shortened luteal phase
 Intraluminal uterine fluid can be graded I to IV according to the degree of
echogenicity. The more echoic the fluid, the more likely the fluid is contaminated
with debris including white blood cells. However, fluid containing cells can
appear relatively anechoic so care is needed in interpretation. Inspissated pus can
be so echoic that it is overlooked. It may be that the actual appearance of the fluid
and the ultrasonographic appearance are not as closely linked as once thought.
Ultrasonographic appearance may be proportional to the size and concentration
of particulate matter within the fluid, rather than the viscosity of the fluid; for
example, purulent exudates can appear non-echogenic. Air has hyperechoic foci,
and fluid with air bubbles appears cellular. Urine in the bladder can appear
echoic, despite being a watery liquid

Detection of Intraluminal Uterine Fluid Using Transrectal Ultrasound

Imaging
 Transrectal ultrasonography provides a rapid, non-invasive method of
assessment of the uterus. In mares that are particularly susceptible to
endometritis and in which vaginal contact should be minimized, endometritis can
often be diagnosed on the basis of intrauterine fluid accumulation. This is more
meaningful when the mare has already been swabbed and cleared of potential
venereal diseases. If fluid is present in the uterus, there is vulvar discharge, or the
mare has abnormally short luteal phases, uterine swabs should be taken to
determine the cause of these symptoms

TREATMENT OF VENEREAL INFECTIONS AND CHRONIC

INFECTIOUS ENDOMETRITIS

 Any mare that is suspected of having a venereal infection must not be bred. In the
case of clitoral or vestibular infections, topical treatment is used. This involves
o Thorough cleaning with chlorhexidine surgical scrub followed by the
application of
 0.2% nitrofurazone ointment for T.equigenitalis,
 0.3% gentamicin cream for K. pneumoniae
 silver nitrate and gentamicin cream for P. aeruginosa
o Clitoral sinusectomy or clitorectomy may have to be used in refractory
cases. A broth culture containing a mixture of growing organisms prepared
from the normal clitoral flora can suppress venereal pathogens in some
cases
o Chronic infectious endometritis is found most frequently in older mares
that have had several foals. Such mares have compromised uterine defense
mechanisms that allow the normal vestibular and vaginal flora to colonise
the uterus, thus inducing a persistent endometritis
o The most favoured approach to treatment has been the infusion of various
antibiotics, dissolved or suspended in water or saline, into the uterine
lumen during estrus
o The intrauterine route is preferable to systemic therapy as most acute
endometritis cases are localized. Systemic treatment alone, or in
combination with local application, is suitable in a few circumstances
o Ideally, the choice of antibiotic of local treatment should be based on in
vitro antibiotic sensitivity tests. However, in many cases this is not
possible and a broad-spectrum combination should be used that is
effective against the mixed aerobic and anaerobic infections that
commonly occur. A particularly successful preparation has been a
buffered, water-soluble mixture of neomycin sulfate (1g), polymyxin B (40
000 IU), furaltadone (600 mg); and crystalline benzyl penicillin dissolved
in 40 ml of sterile water and then instilled through the cervix into the
uterus via a sterile irrigation catheter. A larger volume (upto 100 ml) may
be better in older, pluriparous mares to ensure distribution throughout the
uterus. The use of this extremely broad-spectrum, non-irritant, soluble
preparation has not resulted in super infection with Pseudomonas sp.,
Klebsiella sp., yeasts or fungi. The number of treatments required depends
 on individual circumstances, but daily infusions for 3-5 days during estrus
work well in most cases. The success of this treatment can be monitored
using ultrasonography to identify the presence of intrauterine fluid
o When antibiotics are combined with oxytocin a single daily treatment for 3
days has, in many cases, proved successful. Repeated endometrial
swab/smear examinations may be used to monitor the response to
therapy; however, every time the cervix is breached there is the risk of
introducing more bacteria
o An indwelling intrauterine device has been used that can retain a narrow-
diameter infusion catheter within the cervix ; however, there is a risk of
ascending infection
 In addition to the antibiotic therapy, repeated treatment with PGF2α increases
the frequency of the follicular phases, thus allowing intrauterine therapy to be
used more readily. In addition, it also reduces the duration of the luteal phase
where progesterone increases the susceptibility to infection
 Predisposing causes to the persistent endometritis, such as defective vulval
conformation, should also be attended to

ENDOMETRIOSIS

 Endometriosis is a collective term used to describe a wide range of degenerative

changes (fibrosis and glandular degenerative changes) that can be diagnosed by
endometrial biopsy.
 Successful treatment of endometriosis is difficult. Improved fertility after
endometrial curettage has been reported. This has involved the use of mechanical
and chemical agents (namely povidone-iodine and kerosene) that cause
endometrial necrosis. This treatment apart from being of questionable efficacy
can cause irreversible damage such as adhesions.
 Repeated daily lavage with 2-3 litres of hot (50°C), sterile, isotonic saline has
been suggested as a method of reducing the size of the lymphatics and thereby
the whole uterus.
 The prognosis for fertility remains poor whatever treatment is used.

UTERINE LAVAGE

 The technique of uterine lavage involves the mechanical suction or siphonage of

2-3 litres of previously warmed (to 42°C), sterile physiological (buffered) saline
or lactated Ringer’s solution infused into the uterus via a catheter that has been
retained within the cervix via a cuff. The most convenient is a large-bore (30
French) (80 cm) autoclavable equine embryo flushing catheter. The cuff is useful
as it effectively seals the internal cervical os. The catheter should only be inserted
after thorough cleansing of the perineum. The rationale for such an approach is :
o To remove accumulated uterine fluid and inflammatory debris that may
interfere with neutrophil function and the efficacy of antibiotics
o Stimulation of uterine contractility
 o Recruitment of fresh neutrophils through mechanical irritation of the
endometrium
 The saline is infused by gravity flow 1 litre at a time, and the washings are
inspected to provide immediate information concerning the nature of the uterine
contents. The lavage should be repeated until the fluid that is recovered is clear.
In most cases, the fluid is evenly distributed in both horns, making transrectal
massage of the uterus unnecessary. If a rectal examination is performed whilst
the catheter is in the uterus care must be taken to avoid contaminating the
catheter. The fluid should be recovered in the same container from which it was
infused, thereby preventing air being aspirated into the uterus via the catheter.
Measurement of the recovered fluid and ultrasonographic examination of the
uterus should be performed after flushing to ensure that all the fluid has been
recovered. This is necessary because you are dealing with a mare with an
impaired ability to drain the uterus spontaneously
 For this reason the process is usually combined with oxytocin injection. Ideally
these mares will be bred only once, but if repeated matings are necessary, uterine
lavage should be performed after each mating
 Large-volume lavage is beneficial in many cases, particularly the mare with a
relatively large (above 2 cm depth) accumulation of fluid after breeding. The
process is time-consuming and there is the possibility of further contamination of
the uterus by passage of a drainage tube

Oxytocin

 The ideal method of treatment will be the use of a non-invasive technique with
early and complete elimination of any intrauterine fluid
 Oxytocin stimulates uterine contractions in the cyclical, pregnant and
postpartum mare and hence can be used as a method to promote uterine
drainage in mares with defective uterine clearance. However, its use was
discouraged because of the worry that it would cause severe colic

Prostaglandin Analogues

 Prostaglandin analogue cloprostenol given at a dose rate of 500 µg IM increased

myometrial activity and assisted in uterine clearance. Of the prostaglandins
administered (PGF2 α, cloprostenol and fenprostalene) cloprostenol produced
the most consistent response. Cloprostenol would seem to be indicated in mares
that have lymphatic stasis as shown by excessive fluid within the endometrium or
large lymphatic cysts. Cloprostenol should not be given more than 24 hours after
ovulation in case of inducing premature luteal regression

Intrauterine Plasma Infusions

 Intrauterine plasma has been used in the susceptible mares and had an
enhancing effect on phagocytosis by uterine neutorphils. However, its use may
only apply to mares without a mechanical clearance problem and thereby
repeatedly fail to become pregnant, but have no history of fluid accumulation
 MANAGEMENT PROTOCOL USEFUL IN THE HIGHLY
SUSCEPTIBLE MARE

Overall management of mares known to produce a large amount (several centimeters

depth) of luminal fluid after mating should be excellent prior to breeding.

 Good hygiene at foaling is essential and all mares should be thoroughly examined
postpartum for the presence of trauma that might compromise the physical
barriers to uterine contamination
 Gynaecological examinations, particularly of the vagina, should be performed as
aseptically as possible
 Thorough digital examination of the cervix can identify fibrosis, lacerations or
adhesions that may need treatment before breeding
 Since, air in the vagina can cause irritation of the mucosa it should be expelled by
applying downward pressure with the hand through the rectal wall
 Attention should be paid to hygiene at mating by using a tail bandage and
washing the mare’s vulva and perineal area with clean water (ideally from a spray
nozzle which avoids the need for buckets)
 Breeding should occur at the optimal time, and the number of breedings should
be minimized. This means that these mares need very close monitoring of the
estrus period by rectal palpation and ultrasonography
 The use of hCG is strongly recommended in such mares in an attempt to ensure
they are bred only once. Prediction of ovulation can also be made easier by not
breeding these mares too early in the year, i.e., before they have begun to cycle
regularly
 If feasible, the use of artificial insemination can be helpful to reduce (but not
eliminate) the inevitable post-breeding endometritis.
 A single breeding must be arranged 1-2 (or even 3) days before the anticipated
time of ovulation
 Ultrasound examination of the uterus 3-12 hours after mating is performed to
assess the amount and echogenicity of any intrauterine fluid
 After 20 minutes the mare should be re-examined and any fluid pooling in the
vagina removed. This is followed by infusion of low volume (30 ml) of water-
soluble, broad-spectrum antibiotics such as already described into the uterus via
a sterile irrigation catheter
 2 x 25 IU of oxytocin should be given by the stud farm personnel that evening and
again in the morning, by the intramuscular route
 In mares with lymphatic stasis, the slower release of prostaglandin (cloprostenol
500 µg im.) may be useful. In addition cloprostenol should be given 6-8 hours
after the first oxytocin injection
 The mare is re-examined the following day and oxytocin treatment repeated if
fluid is still present. Only rarely will a second infusion of antibiotics or lavage
procedure be performed due to the risk of uterine contamination
MODULE-12
INFERTILITY IN FARM AND COMPANION ANIMALS - PART III
Normal fertility requires ovulation of normal ova into a patent, healthy reproductive tract,
insemination with normal semen near the time of ovulation, and maintenance of pregnancy for
approximately 2 months. Infertility is often assumed when a bitch has been bred repeatedly but
does not become pregnant or deliver a litter.
The investigation of infertility in the bitch and queen is complicated by the fact that failure to
conceive does not result in an immediate return to estrus as occurs in polyestrous species.

As with other species, infertility in the bitch and queen may be categorized according to whether
the cause is structural (including congenital,acquired and neoplastic diseases), functional
(including endocrinological abnormalities), infectious or managemental.

INFERTILITY IN THE BITCH

Developing a Problem List

 Infertility or apparent infrertility problems in the bitch are common. Veterinary

advice is often sought after a bitch fails to conceive. If she fails to exhibit
“normal” breeding behavior, when her cycles appear to be unusual, or for myriad
other distrubances.
 “Infertility”therefore, is a huge category comprising a long list of anatomic,
physiologic, and behavioral problems as well as a number of apparent husbandry
misunderstandings.
 Further, a championship or other important title may be earned by the bitch,
ensuring demand for and value of any puppies before any attempt has been made
at breeding.

ASSESSMENT OF THE MALE

 Before embarking on an investigation into the potential causes of infertility in a

bitch, the male should be assessed. Males are so much easier to study than
females as the male is continuously fertile while a female is usually fertile only 1
to 3 weeks per year.
 Male fertility can be established by reviewing the males previous breeding
history. Any male siring a litter or litters within the preceding 1 to 4 months can
usually be assumed to be fertile. It is also helpful to know if the male sired any
litters at the time the bitch in question was in heat and bred. However, even if the
responses to these enquiries are affirmative, the fertility of the male should be
demonstrated with a complete semen analysis.

 All active stud dogs should be tested for brucellosis every 6 months. Less active studs
should be checked yearly and immediately prior to use. A male that has not sired a litter
or has sired litters in the past but not in the preceding 6 to 12 months must be viewed
with suspicion.
 Whenever the male’s fertility is questionable, the owner of the bitch has three main
alternatives:

 have a semen analysis and brucella titer performed on the male,

 utilized an alternative, proven sire on the next heat, and
 evaluate the bitch, realizing she may not be at fault.

 A normal semen analysis is a major step toward ensuring that the male is not at fault.
 Abnormal semen, or an inability to obtain an ejaculate, leaves some suspicion directed
at the male.
EXAMINATION FOR PREGNANCY

History

OBTAINING A “COMPLETE” HISTORY. Before the bitch is examined by the

veterinarian, the various potential causes for infertility must be reduced to a workable
number. In other words, the differential diagnosis for most infertility disorders is
established by obtaining a thorough history from the owner. The initial history should
include information such as

 How well the owners know the bitch?

 Does she live indoors with them or 200 miles away at a hunt club?
 Is she housed alone, with another bitch that recently completed ovarian cycles,
with ovariohysterectomized bitch or with males?
 Is she normal in height and weight for her breed and for her line?
 Is she receiving any medication and is she well or ill?

Items can always be forgotten in reviewing a cases history during a busy workday, and
the question sheet helps to avoid this problem.

AGE AND BREED

 Small dogs reach sexual maturity at a younger age than large dogs.
 Onset of the pubertal estrus in the bitch has been reported to occur at ages
ranging from 6.3 to 23 months, with mean ages of 9.6 to 13.9 months.
 Almost all healthy bitches begin cycling by 24 to 30 months of age.
 The first and second cycles may be irregular, unusual, short, or long.
 Infertility evaluations are delayed in most dogs until they are 24 to 30
months of age.
 Toy poodles may benefit from evaluation earlier in life than Bull Mastiffs.
 Each breed does have distinct average interestrus intervals, but the interestrus
interval varies within a breed.
 As a general rule, almost all breeds cycle once every 4.5 to 10 months. The African
breeds cycle once yearly.
 PHYSICAL EXAMINATION

EXAMINE THE PROBLEM AREA LAST. As with any serious problem, the area of
concern should be the last to be evaluated on physical examaination. This approach
ensures that each bitch receives a complete physical examination prior to an evaluation
of the reproductive tract.

Vulva

 Examination of the reproductive tract usually begins with an external inspection

of the vulva, checking the size and conformation and for presence of any
discharge.
o The small immature vulva or one that is recessed under a fold of tissue
owing to body type or obesity may present impediments to normal
breeding.
o The obese bitch is prone to perivulvar dermatitis.
o A swollen, turgid vulva is suggestive of proestrus
o A swollen and flaccid vulva can be consistent with estrus or approaching
parturition.

Vaginal Discharges

 The bitch in anestrus or diestrus usually has no vaginal discharge.

 A bloody discharge is most suggestive of proestrus, estrus, separation of the
placental sites, or severe vaginitis.
 Greenish black or dark bloody vaginal discharges are associated with placental
separation as well as postpartum “lochia”.

Reddish brown yellowish, or grayish, thick, creamy, malodorous vaginal discharges are often
seen in open-cervix pyometra, metritis, or severe vaginitis.

 Straw-colored vaginal discharges are sometimes seen when bitches are in estrus.
 Clear mucus can precede parturition and is rarely worrisome.

A vaginal cytology specimen should be an integral part of any reproductive evaluation

because it is easy to obtain the sample, inexpensive, and can be extremely informative.
Vaginal cytology should be performed in any bitch with a vaginal discharge.

Digital Examination of the Vestibule and Vagina

 A digital examination of the vaginal vault should be performed routinely on any

bitch examined for breeding soundness.
 If a culture or cytology is needed, it should be obtained prior to the digital
examination.Most bitches are easy to examine.
 The gloved and lubricated index finger should pass easily into the vaginal vault,
allowing assessment of the lumen, the urethral opening, and clitoral size and
 shape. Masses, foreign bodies, strictures, painful vaginitis, or abnormal tissue
bands all prevent easy and painless examination.
 If the digital examination is abnormal but inconclusive, vaginoscopy provides a
more thorough evaluation.
 The use of an otoscope or a vaginal speculum provides an extremely limited view
of the vaginal vault and is of little value in most clinical situations.
 Pediatric proctoscopes are easy to use for vaginoscopy and are relatively
inexpensive, and can be used in all but the smallest of miniature breeds. A more
expensive but smaller diameter alternative is use of an endoscope which provides
far better visualization of the area than an otoscope.

Mammary Glands

 The mammary gland should be palpated in the bitch examined for breeding
soundness.The primary concern is the presence of mammary tumors.
 The glands can also be checked for evidence of lactation, mastitis, inverted teats,
or benign nodules.
 The ventral midline can also be checked for evidence of a previous surgical
incision, which might be a clue suggesting that the bitch has undergone
ovariohysterectomy.

Rectal Examination

 A rectal examination ensures that the pelvic canal has been assessed for previous
fractures or other unsuspected abnormalities.
 Compression of the pelvic canal is a potential cause of dystocia.
 One can also attempt to palpate the vagina ventrally, although the vagina would
have to be extremely abnormal to reveal anything suspicious on palpation.

Abdominal Palpation

 The abdomen should be palpated in an effort to identify and characterize the

uterus. However, except in pregnancy and pyometra, the uterus almost never can
be evaluated with confidence on abdominal palpation.

General Health of the Bitch

 In the clinical evaluation of the infertile bitch, one underlying question is her
overall health status.
 Complete blood counts, chemistry panels, urinalysis, thyroid function, and
adrenocortical function studies can be carried out as an initial step in evaluating
the potentially infertile bitch. However, such extensive diagnostic evaluations are
not required unless the history and/or physical examination dictates that
aggressive diagnostic testing is warranted.
 The bitch that appears healthy to an owner, appears healthy on physical
examination, and has normal ovarian cycles does not have thyroid failure or
adrenocortical disease and rarely has other significant organ disease. Therefore,
 obtaining a complete blood count, urinalysis, and blood urea nitrogen provides a
sufficient data base. However, this approach depends on completing a thorough
history and a competent physical examination. If abnormalities are identified on
history or physical examination, appropriate testing can then be completed which
may clarify the nature of the problem or specifically demonstrate the cause of
infertility.

OWNER MANAGEMENT PRACTICES

 Improper management practices are the cause for a large majority of apparent
infertility problems. A bitch that is bred or attempted to be bred at incorrect
times may be totally normal. She may fail to conceive as a result of being brought
to the male when she is not fertile.
 The common errors in breeding management have already been discussed in
detail under the module on "Breeding and Artificial Insemination in Dogs". These
and similar parctices do not consistently result in conception.
 They may work in a majority of bitches, but some normal bitches fail to conceive
if bred according to such criteria.

INFERTILITY IN A BRUCELLA NEGATIVE BITCHWITH

NORMAL OVARIAN CYCLES, NORMAL INTERESTROUS PERIOD
AND ALLOWS BREEDING

 Management problems are the most common cause of apparent infertility in the
bitch with a normal cycle. The entire question regarding proper management for
an individual bitch can be answered through obtaining a thorough history with
corrections made as needed in past practices, behavior observation, vaginal
cytology review, and monitoring plasma progesterone concentrations.
 This approach answers the following question:
o How is the owner managing this bitch?
o When does standing heat begin?
o How long does standing heat persist?
o What is the first day of true diestrus?
o When is the bitch truly fertile?
o What are her ideal breeding dates?
o Does she ovulate?
o When does she ovulate?
o Does she have the luteal function necessary to support pregnancy?

MANAGEMENT PROBLEMS

 Adoption of a reliable breeding schedule while simultaneously studying follicular

function and the time of ovulation which may help to correct management
related problems. If the problem is physiologic, it may be identified and treated
 appropriately. A thorough review of breeding practices have already been dealt in
the module on “Breeding Management and Artificial Insemination in Dogs”.
 The ovarian function during proestrus and estrus can be monitored using Vaginal
cytology and Progesterone estimations. Vaginal smears can be used to identify
day of onset of estrus and one can count back six days from Day 1 of diestrus to
predict the day of ovulation. Obtaining vaginal smears, staining and examining
them.
o The evaluation of the bitch can be enhanced by serial monitoring of serum
progesterone levels to accurately predict the progression of the bitch
through proestrus and estrus and to predict the time of ovulation.
o The ovarian function during diestrus to determine whether ovulations
have occurred can be monitored by identifying day 1 of diestrus based on
vaginal exfoliative cytology coupled with plasma progesterone
concentrations obtained between 10 and 20th day of diestrus.
 Shipping or transport practices should be reviewed. It is not known wheter
transporting or stress affects ovulation or conception in the bitch, hower, it is
better to avoid during one cycle to see whether the infertility problem can be
resolved. the bitch can be bred locally to avoid transportation.
 All previously or currently used medications must be reviewed. Previous use of
gonadotropins may have long-term deleterious effects on pituitary function while
previous progesterone or estrogen administration may result in subclinical cystic
endometrial hyperplasia with infertility being the only outward effect seen by the
owner or veterinarian.
 Hypothyroidism is often described as “common” with signs such as
o persistent anestrus,
o prolonged interestrus interval, and
o prolonged proestrus,
o some bitches demonstrate normal reproductive activity, pregnancy, and
parturition. The diagnosis of hypothyroidism should always be viewed
with suspicion, not because the disease does not exist, but simply because
most dogs treated for the disease are not so afflicted.

INFECTIONS

Brucella Infection

 Brucella canis classically causes abortion late in gestation, resorption in early

gestation, birth of still born puppies, and infertility. All bitches included in
breeding programs, especially those with an infertility problem, should be
evaluated for canine brucellosis. The rapid slide agglutination test is an
excellent screening test. False negative test results are unlikely, and a negative
result can be trusted. Bitches that are seropositive should be retested using the
tube agglutination method, because false-positive results do occur.

Other Infections
  Bacterial infections have been implicated as a cause of infertility in the bitch.
These infections are thought to be subclinical in the infertile bitch, only
occasionally resulting in obvious vaginitis, metritis, pyometra, or systemic
infection.
 Most normal bitches have bacterial flora present in the anterior vagina, and
similar types of aerobic bacteria are present in the vaginal vaults of infertile
bitches. Hence, it is difficult to establish the role of bacterial infections in canine
infertility.
 Treatment with vaginal douches for 2 to 3 weeks, with or without systemic
antibiotics, may be beneficial but such therapies should be reserved for bitches
with obvious clinical signs of infection, such as purulent vaginal discharge.

Mycoplasma and Ureaplasma

 A syndrome of poor conception, early embryonic death, embryonal or fetal

resorption, abortion, stillborn pups, weak newborns, and neonatal death has
been suggested to be caused by mycoplasma and ureaplasma. However, these
organisms are present in the vaginal tract of the normal bitch and hence cannot
always be considered as a cause for infertility. If large numbers of these
organisms are identified in pure or nearly pure growth from the vaginal vault of a
breeding bitch with an infertility problem, these microorganisms may be at fault.
Management includes isolation of the animal and tetracycline or
chloramphenicol therapy for 10 to 14 days.

Viral Infections

 Viral infections, specifically herpes virus have been isolated in dogs – that had
abortions and stillbirths. However, viral infections as a cause of infertility are not
well documented.

CHRONIC ENDOMETRITIS-CYSTIC ENDOMETRIAL

HYPERPLASIA

 The bitch with chronic endometrial disease is likely to be infertile. These dogs
could experience normal ovarian cycles, ovulate, and have fertilized eggs, but fail
to support pregnancy because of the abnormal uterine environment that prevents
implantation or that would result in fetal resorption.
 Chronic endometritis or CEH can be extremely difficult to confirm. The diagnosis
is suspected if the non-pregnant uterus is thickened or abnormally large in
anestrus or diestrus. Although a thickened uterine wall is a potentially palpable
abnormality, it is difficult to be certain that one is palpating the uterus.
 Visualizing the non-pregnant uterus using abdominal ultrasonography is a
potential method for documenting the presence of a thickened endometrium or
of intraluminal fluid. Uterine biopsy is the only method of confirming a
diagnosis, a procedure usually requiring laparotomy.
 EARLY FETAL RESORPTION

 Early fetal resorption usually appears to both owner and veterinarian as primary
infertility because early pregnancy is so difficult to confirm. Pregnancy cannot be
recognized by palpation until after 21 days of gestation, and then the diagnosis is
subjective.
 Radiographically, pregnancy cannot be confirmed until 42 to 45 days of
gestation.
 The earliest that pregnancy can be identified is approximately 16 days after first
breeding, using ultrasonography. This tool has been helpful in recognizing early
fetal resorption.
 Early fetal resorption suggests an endometrial disorder failure of corpora lutea to
support pregnancy infectious disease such as brucellosis, fetal defects or some
less common disorder.


HYPOLUTEOIDISM

 Plasma progesterone concentrations begin to rise prior to the onset of standing

heat and decline to basal levels immediately prior to parturition. The first 6 to 7
weeks of diestrus are usually associated with progesterone concentrations of 50
ng/ml. Any bitch diagnosed as having an infertility problem should be evaluated
with a plasma progesterone concentration 10 to 20 days after termination of
standing heat and then once or twice weekly thereafter. These studies should be
completed in conjunction with evaluation by abdominal ultrasonography.
 If the progesterone concentration is below 1.0 ng/ml, either the bitch never
ovulated or the corpora lutea have failed to synthesize and /or secrete
progesterone. Serum progesterone concentrations above 2-4 ng/ml should be
sufficient to maintain pregnancy. If the progesterone concentration is less than
2 ng/ml, the amount of progesterone secreted may be insufficient to maintain
pregnancy and abortion or fetal resorption may result. If fetuses are observed on
abdominal ultrasonography early in gestation, abortion or fetal resorption should
become demonstrable with repeated ultrasound examinations. Progesterone
therapy can be given but should be recommended only with great caution.

OCCLUSION OF THE UTERUS OR OVIDUCTS

 Bilateral segmental aplasia or other causes of obstruction of the uterine horns, or

occlusion of both oviducts, could result in a bitch that cycles, ovulates, and breeds
normally, but fails to conceive. Bilateral occlusion prevents the sperm from ever
reaching the egg.
 Diagnosis can be made by
o Hysterosalpingography which involves passing radio opaque dye from the
vagina through the cervix and uterus into both oviducts. It is an excellent
theoretic tool but is difficult to employ on a practical basis.
 o An alternative to the radiographic study is direct visualization.
Laparoscopy is not a good tool because the oviducts are not visible with a
laparoscope. Laparotomy is the only realistic remaining tool. Surgery also
allows uterine biopsies and cultures to be obtained.
 Little can be done if a bitch is diagnosed as having bilateral uterine or oviductal
occlusion. Unilateral occlusion does not result in infertility. Opening an occluded
uterine horn or oviduct has not been described, and such bitches are permanently
infertile.

MISCELLANEOUS CAUSES OF INFERTILITY

 Among the recognized causes of infertility in species other than the dog, when the
female has normal cycles and the male is fertile,are
o antisperm antibodies produced by the female or spermicidal substances
within secretions of the cervix.
o antiegg zona pellucida antibodies have been developed in bitches through
immunization procedures. Such antibodies do result in infertility.

THE BITCH WITH SHORTENED INTERESTRUS INTERVAL

Idiopathic shortened Ovarian Cycles

 Bitches normally have an interestrus interval of 5 to 11 months. The German

Shepherd dog and the Rottweiler are breeds that often have fertile cycles every
4.5 months.
 Apparent infertility occurs when a bitch enters proestrus prior to completion of
the uterine repair. Infertility could be the result of implantation failure caused by
an abnormal endometrium that has not recovered from the previous effects of
progesterone.
 Prior to making a diagnosis or instituting therapy, a complete history of the bitch
must be obtained and studied. The bitch that cycles at less than 4 month intervals
is typically normal in all respects and is infertile only as a result of incomplete
uterine involution.
 Young bitches often have irregular, frequent, or silent ovarian cycles. By the age
of 2 to 3 years, ovarian cycles should be regular. Thus, it is recommended not
to treat any bitch for frequent cycles until she is at least 2.5 to 3 years
of age
 Treatment for the bitch older than 3 years of age that cycles too frequently is to
medically induce a normal anestrus period. This can usually be accomplished by
treating the bitch with mibolerone drops for a period of 6 months. Medication is
started 6 to 8 weeks after the end of the previous standing heat. One must
ascertain that the bitch is not pregnant prior to beginning mibolerone therapy
because this potent synthetic androgen causes urogenital defects in female
fetuses and the bitch also may undergo some virilization, but these signs are
reversible and the drug is not thought to alter future reproductive performance.
The bitch should be bred during the first estrus that follows therapy. This estrus
 can begin immediately or as long as 6 to 9 months after discontinuation of
therapy.

Follicular Cysts

 Ovarian follicular cysts have been implicated as a cause for shortened interestrus
intervals in the bitch. Follicular cysts are well recognized in association with
prolongation of proestrus and/or estrus. Abdominal ultrasonography is the only
practical means of diagnosing an ovarian cyst. Treatment includes surgical
removal of the cyst or the cyst and ovary.

Uterine Disease

 Uterine disease has been suggested as a cause for shortened inter estrous
intervals and a diagnosis of this condition requires histologic evaluation of
uterine tissue obtained by uterine biopsy.

Split Heats

 Split heats are observed in young pubertal bitches but can occur at any time in
life. In split heat, follicles develop, produce estrogen and the bitch exhibits all
signs of proestrus. However, ovulations do not occur. Four to 10 weeks later, the
bitch once again enters into estrus. The second half of split heat is always an
ovulatory heat. Diagnosis is by vaginal cytology and serum progesterone
estimations.

Ovulation Failure

 Failure to ovulate may result in failure to form corpora lutea and failure to
synthesize progesterone. The entire diestrus phase of the ovarian cycle is skipped,
and, therefore, the phase of uterine involution is also brief. Diagnosis is based on
serial serum progesterone determinations. It is not known how this diagnosis
differs from that of split heats. In the bitch less than 3 years of age, no treatment
is recommended. In the bitch older than 3 years of age, an attempt to stimulate
ovulation can be undertaken with luteinizing hormone or human chorionic
gonadotrophin administered the day before or the day after first breeding

THE BITCH WITH PROLONGED INTERESTRUS INTERVAL

Idiopathic Prolongation of the Interestrous Interval

 A thorough history should be obtained to make sure that, outside of the

reproductive tract, the bitch is healthy. Interestrous intervals increase as the
bitch becomes older. An interval of 10 to 13 months for a bitch older than 6 to 8
years of age is not worrisome. However, such prolonged intervals are not typical
of the 2 to 6 year-old bitch.
  It will help to know how closely an owner watches his dog and detects proestrus
and estrus. Sometimes, a heat may simply be missed by the owner if no males are
present and the owner does not specifically examine the vulva once or twice each
week. Further evaluations involve
o Physical Examination
o Breed
o In-House Examination
o General Approach
o Hypothyroidism

Physical Examination

 Thorough physical examination is should follow only after obtaining a thorough

history. A physical examination need not be too elaborate, but neither should it
be omitted. The finding of a serious heart murmur, unsuspected organomegaly, a
mass that was not expected, or a variety of other problems must be investigated
in order to better treat the patient and to understand any potential cause for
delay in an ovarian cycle.

Breed

 Certain breeds like Basenji and the wolf-hybrid cycle on a yearly basis. The bitch
that cycles less often than every 10 months and appears infertile is of greatest
concern.

In-Hospital Evaluation

 Increase in length between ovarian cycles in the bitch can occur secondary to
o An underlying illness
o Any major medical disorder has the potential for delaying the onset of an
ovarian cycle.
 Hypothyroidism is a disorder most often associated with long
interestrous periods is hypothyroidism.
 Ovarian Cysts or Neoplasm
 Silent Heat

THE BITCH WITH PRIMARY OR SECONDARY ANESTRUS

 Primary anestrus is referred to a condition of a bitch that has never had an

ovarian cycle. Secondary anestrus is that of a bitch that has had one or more
ovarian cycles but subsequently fails to cycle.
 Current history such as the dog’s age, breed, past history, and physical
examination should be assessed before any major tests are undertaken. Failure to
cycle, is a problem that is usually not pursued until the bitch is older than 2 to 3
years of age. Evaluation of dogs with secondary anestrus should include all
suggested approaches in the previous section on prolonged interestrous intervals.
  Secondary anestrus can occur after the onset of thyroid, other endocrine or
nonendocrine disease. These bitches should be thoroughly evaluated with history,
physical examination, and laboratory testing and if all testing is normal, it is wise
to wait at least 16 to 20 months from the previous cycle, in case one or two heat
cycles were silent and, therefore, missed. The dog should be closely monitored
during this time by the owner, and the veterinarian can recommend serial testing.

Previous Ovariohysterectomy

 If the past history of a bitch is not known, one cause for failure to cycle is
previous ovariohysterectomy. Examination of the ventral midline for an incision
scar provides initial evidence for an earlier spay. One may need to clip hair away
from this area to be certain. The condition can be confirmed by plasma
estimations for LH and FSH determinations. The ovario-hysterectomized female
has persistent elevation in LH and FSH concentrations.

Silent Heat

 Silent heat can be difficult to detect. Bitches in this condition may not have vulvar
enlargement or a sanguineous vaginal discharge, or may not attract or allow
breeding by males. Silent heats should be considered a possible cause for primary
anestrus, especially if the owners of a bitch have little or no experience with an
intact female, if the bitch is housed separately from any contact with a male dog,
or if the bitch is not closely observed.
 Diagnosis of silent heat can be done by
o Trying to bring the bitch into contact with a male once weekly to help
recognize estrus.
o Close visual examination of the vulva once or twice weekly as it is an
excellent method for detecting silent heat.
o Close observation which allows the owner to develop some experience with
the anestrus appearance of the vulva. Mild enlargement of the vulva or a
slight bloody discharge is easier to see, and the owner is more comfortable
identifying signs of early proestrus.
o Adopting more aggressive methods of evaluating bitches suspected of
having silent heats that include weekly reading of vaginal cytology smears
or monthly serum progesterone assessments.

Drug-Induced Anestrus

 Anestrus may be induced by drugs specifically marketed for that purpose and by
drugs that result in anestrus as a side effect. Marketed drugs include androgens,
which might be used by an owner interested in increasing the strength and/or
endurance of his or her pet, without realizing effects on the hypothalamic
pituitary-ovarian axis. Progestagens are used in the treatment of a variety of
maladies, with prolongation of anestrus as a side effect. Glucocorticoids can have
negative feedback effects on the pituitary, suppressing gonadotrophin activity
and preventing ovarian cycles.
Underlying Disease

 Any illness, mild as well as severe, can interfere with ovarian cycle activity in the
bitch. Obtaining a thorough history as well as performing a complete and
competent physical examination is important. Abnormalities identified in these
areas must be pursued as potential explanations for the infertility problem and to
avoid the mistake of separating the reproductive tract from the rest of the animal.
 When silent heats, previous ovariohysterectomy, and owner error are considered
unlikely explanations for apparent failure to cycles, blood and urine testing is
advisable. It is recommended that a CBC, serum chemistry profile, urinalysis, and
serum thyroid concentration be obtained and reviewed. Another integral
component of screening a bitch for unsuspected problems is abdominal
ultrasonography. This is a noninvasive means of evaluating abdominal
structures, including the uterus for thickening and/or fluid and the ovaries for
masses or cysts.

Hypothyroidism

 Hypothyroidism has become a “popular” diagnosis in explaining the cause for a

bitch failing to cycle. Although the potential exists for a hypothyroid bitch to
exhibit primary or secondary anestrus, these dogs should have signs of
hypothyroidism. The alert, active, vibrant bitch is rarely hypothyroid. As
previously stated, hypothyroidism is over diagnosed by the profession and by
breeders / trainers.
 Hypothyroid dogs rapidly respond to thyroid replacement by becoming more
alert, active, and responsive within days of initiating replacement therapy. Their
appetite quickly improves and weight loss follows. Improvement in hair coat may
take weeks. These dogs typically begin ovarian cycles within 3 to 6 months of
initiating therapy. If these responses are not observed, the thyroid hormone
replacement dose is inadequate, a second medical problem exists, or the
diagnosis is not correct.

Glucocorticoid Excess

 Glucocorticoids are used in the treatment of numerous small animal problems.

Glucocorticoids have negative feedback effects on pituitary adrenocorticotropin
secreting and similar effects on suppressing secretion of both FSH and LH. A
bitch receiving glucocorticoid therapy may not exhibit ovarian cycles unless the
steroid dosages are kept to a minimum or administration is discontinued.
Naturally occurring hyperadrenocorticism is not usually a major consideration in
the noncycling female because most bitches with Cushing’s syndrome are older
than 8 years of age. Therefore, their failure to cycle is not recognized as a problem
by the owners, whereas the other major signs of Cushing’s syndrome are more
obvious, and worrisome.

Premature Ovarian Failure/Ovarian Aplasia

  A bitch that has never exhibited ovarian cycles may not have ovaries. Other
causes of the aplasia may exist. Secondary hypoplasia simply suggests that one or
more ovarian cycles preceded premature ovarian failure.
 The diagnosis of ovarian failure may be suspected when all other differential
diagnoses are excluded and attempts to induce estrus fail. One method of
confirming such a diagnosis is random evaluation of plasma FSH and LH
concentrations. Premature ovarian failure is associated with extremely increased
plasma FSH and LH concentrations due to absence of negative feedback to the
pituitary and hypothalamus Alternatively, exploratory surgery can be undertaken
to inspect the reproductive tract and to biopsy the uterus and ovaries for
confirmation of abnormalities.

Progesterone-Secreting Ovarian Cyst

 This is a well –recognized but uncommon syndrome resulting in prolongation of

diestrus. To the owner, the condition usually appears to be a prolongation of
anestrus. By definition such a bitch must have had an ovarian cycle and would be
classified as having secondary anestrus. However, if this follows a silent heat, the
prolonged anestrus may appear to be primary. In either situation, abdominal
ultrasonography, together with assessment of the plasma progesterone
concentration, is diagnostic in most bitches. Treatment with prostaglandins to
lyse these cysts is not usually successful, and their surgical removal is
recommended.
 Induction of estrus in anestrus dogs have been dealt with in detail in the module
on Induction of estrus.

PERSISTANT PROESTRUS AND/OR ESTRUS

 Persistent estrus is defined as a bitch willing to breed for longer than 21 to 28

consecutive days in any one ovarian cycle. Alternatively, and less directly,
persistent estrus is defined as more than 21 to 28 consecutive days of greater than
80 to 90% superficial cells observed on vaginal cytology. Persistent presence of a
large percentage of superficial ells on vaginal cytology is strong evidence for
continued increases in serum estrogen concentration.
 Bitches with persistent proestrus have an enlarged vulva and persistent vaginal
bleeding, attract males, and demonstrate estrogen effects on the reproductive
tract. Although the causes for persistent proestrus/estrus may vary among
bitches, the final common denominator is continued exposure to increases in
serum estrogen concentration.

Exogenous estrogen excess

 The use of parenteral estrogen to prevent pregnancy and in some bitches to

prevent urinary incontinence may lead to this condition. If estrogens were
administered, the type and dosage should be ascertained. In addition to
 associations between exogenous estrogen and development of pyometra or bone
marrow aplasia, ovarian cysts are recognized sequel to such medication.

Endogenous estrogen excess

 Rarely does the young bitch in her first or second ovarian cycle fails to ovulate
and may exhibit prolonged proestrus or estrus activity due to continued follicular
estrogen secretion. This would probably due to inadequate amounts of estrogen
to induce the LH surge or a failure in LH to induce ovulation leading to
development of follicular cysts.

DISORDERS OF SEXUAL DEVELOPMENT

Normal Sexual Development

 The sex chromosome constitution of the sperm determines the sex of mammals
at fertilization. The embryo develops as a male if the fertilizing sperm has a Y
chromosome. If the sperm contains an X chromosome, the embryo develops as a
female. The genital system of early developing embryos is neither male nor
female. Eventually, the embryo without a Y chromosome develops an ovary from
the undifferentiated gonad, and the Mullerian system persists as the fallopian
tubes, uterus, and cranial vagina. The urogenital sinus and external genitalia
develop in a female pattern.
 In the presence of a Y chromosome the indifferent gonad develops into a testis,
which produces both testosterone and Mullerian inhibiting substance (MIS),
secreted by sertoli cells, causes regression of the Mullerian duct system.
Testosterone secreted by Leydig cells stimulates formation of the epididymis and
vas deferens from the Wolffian duct system as well as the male urethra, penis.,
prostate, and scrotum.
 Normal sexual development occurs in three steps
o Step 1: Establishment of chromosomal sex
o Step 2: Development of gonadal sex
o Step 3: Development of phenotypic sex.
 An error in any one of these steps can result in a disorder of sexual development
which may be occult or obvious to the owner and/or veterinarian. Normal dogs
have a total chromosome number of 78. These 78 chromosomes include 38 pairs
of non-sex chromosomes and 2 sex chromosomes. The sex chromosome
constitution of females is XX, whereas that of males is XY.

Abnormal Sexual Development

 Dogs with obvious abnormalities in their external genitalia may be quickly

recognized, but abnormalities may occur at any step in development. Intersex
conditions include congenital malformations of the genital system such that the
gender of the individual is ambiguous. “Interesx” is a generic term including
numerous disorders. True hermaphrodites are individuals with both testicular
 and ovarian tissue, either combined in one gonad or existing as separate gonads.
Pseudo-hermaphrodites have the gonads of one gender but have reproductive
organs having characteristics of the opposite gender. Male
pseudohermaphrodites have testes but have some female features, such as the
presence of a uterus or external genitalia that are primarily female. Female
pseudohermaphrodites have ovaries but are masculinized to some degree.
 Gonadal gender is best determined by histology of the gonads. Phenotypic sex
can be established following review of a thorough description of both internal and
external genitalia. It is necessary to determine (1) whether the vulva or prepuce is
appropriate in form and position; (2) whether a clitoris or penis is present; (3)
what is the location of the urethral opening; and (4) whether the dog has a
prostate or caudal vagina.

Abnormalities of Chromosomal Sex

 Dogs and cats with abnormalities of chromosomal sex are normal-appearing

males or females that have underdeveloped rather than ambiguous genitalia.
 Animals that are chimeras or mosaics may be exceptions to this rule. Most
animals with chromosomal sex abnormalities are sterile, with no treatment
advised. Abnormalities of chromosal sex include
o The XXY Syndrome
o The XO Syndrome
o The XXX Syndrome
o Chimeras and Mosaics
o True Hermaphrodites

Abnormalities of Phenotypic Sex

 Abnormalities of Phenotypic Sex include

o Pseudohermaphrodites-Female and male pseudohermaphrodites have
some degree of sexual ambiguity in the genitalia. Their chromosomal sex
and gonadal sex agree, but their external appearance is reversed; that is,
they are phenotypically abnormal.
 Female Pseudohermaphrodites
 Male Pseudohermaphrodites

Hypospadias

 This condition was defined in the previous section the XX male. Hypospadias
may occur from a variety of causes and the result is an incomplete fusion of the
urethral folds leading to the formation of the male urethra.
 The Boston Terrier may have a familial predisposition to hypospadias.
Cryptorchidism is the most common defect associated with hypospadias.

FAILURE TO PERMIT BREEDING

Mismanagement

 The most common cause for bitches to refuse attempts at mounting by a male is
an owner choosing incorrect breeding dates. Another potential cause of failing to
permit breeding, however, is vaginal defects.

Behavior

 Bitches may be managed properly but still consistently refuse to breed with a
particular male. Mate preference appears to be one potential cause for this
problem. Therefore, if no other cause is evident the owner should attempt to
breed the bitch to another, more dominant male before investigating unusual
problems.


INFERTILITY IN THE QUEEN

Introduction

 Infertility in the queen is a nonspecific, historical complaint that relies on historical

reproductive information for consideration of appropriate causes and appropriate
diagnostic plans. Infertile queens can be assigned to one of the four groups
o Failure to cycle.
o Those with prolonged interestrus intervals.
o Those with prolonged sexual receptivity.
o Cycling queens that refuse copulation with the male.

FAILURE TO CYCLE

Previous Ovariohysterectomy

 If a queen displays no estrus activity whatsoever, one should check to see if she
has previously undergone an OVH. Usually, checking for a “spay” incision is all
that is needed

General Health

The cat’s general health must be thoroughly evaluated. This usually involves a good
history, complete physical examination, and a routine blood and urine data base.
Estrous cycles can be interrupted or can cease in an animal under the stress of

 a poor diet
 compromising illness
 overcrowding
 exposure to extremes in temperature
 inadequate exposure to light
  the stress of a show circuit
 traveling
 drug therapy, especially progestagens and glucocorticoids
 variety of ovarian and uterine neoplasias

Silent Heat

 Perhaps the best example of silent heat is the cat housed with a number of other
cats. If a cat is low on the “pecking order” or if overcrowding exists, its cycles may
be completely undetectable, that is silent, to humans and apparently to other
cats.
 Diagnostic methods used to diagnose this condition is to teach an owner how to
obtain vaginal smears from the cat.
o Follicular phases are reflected as an increase in the percentage of
superficial cells present.
o Alternatively, once-or twice-weekly plasma samples can be assayed for
estrogen concentration.
 If either study suggests normal follicular function, the cat should be completely
isolated and maintained on 14 hours of light and 10 hours of darkness. Usually,
her estrus activity is more apparent on this regimen and removal from the other
cats. One could also attempt to induce estrus medically.

Premature Ovarian Failure

 The functional longevity of the ovaries in queens is not known, although many
queens do not continue estrous cycle activity beyond 11 to 13 years of age.
 Queens beyond 8 years of age are not usually used in breeding programs. The
ovaries, abnormally, may cease functioning earlier. This results in a permanent
condition interpreted as prolonged anestrus by the owner.
 Premature ovarian failure is suspected when all other differentials are excluded
from the list of potential diagnoses and induction procedures fail. One could
assay LH and FSH concentrations in the plasma to confirm diagnosis. Persistent
elevation of these hormones is consistent with nonfunctioning ovaries.

Disorders of Sexual Development

 Phenotypically normal females may not have functional ovaries secondary to

chromosomal abnormalities. Several such cats have been reported.
 Karyotyping can be performed to recognize these disorders. Exploratory surgery
can be performed to examine the reproductive tract and to do biopsy or remove
any abnormal tissue. One can also attempt to induce estrus medically to rule out
any likelihood of a disorder in sexual development.

Induction of Estrus

 Once the veterinarian is certain that no anatomic defect, organic illness, or

medication would explain failure to cycle, an attempt can be made to induce a
 cycle medically. This procedure can be used in queens that have never cycled,
queens with prolonged acquired anestrus, or queens with highly irregular cycles.
Because the queen is an induced ovulator, one must rely on the male to induce
ovulation once estrus is induced, or an attempt can be made to induce ovulation
medically.

PROLONGED INTERESTROUS INTERVALS

Pseudopregnancy

 A queen that enters estrus every 30 to 60 days may be ovulating and experiencing
repeated pseudopregnancies. This has been observed in queens that have never
been bred. Ovulation in some queens can be induced by petting, obtaining
vaginal cytology smears, or less obvious factors. These queens are typically
healthy and are fertile if breed. The diagnosis of pseudopregnancy can be
confirmed by demonstrating an elevation in the plasma progesterone
concentration 1 to 3 weeks after estrus.
 Queens must receive adequate food, housing, light, and general care if they are to
cycle normally. Owner observation is also valuable to be certain that some cycles
are not missed. A general health examination and laboratory evaluation are also
worthwhile because an underlying illness may interrupt cyclic ovarian activity.

Cystic Follicles

 Cystic follicles could result in either prolonged estrus behavior or a prolonged

interestrous interval. Although difficult to diagnose, functioning follicles create
persistent estrus via production of estrogen, which can be assayed. The most
reliable and easiest method of diagnosing cystic follicles is abdominal
ultrasonography. Cystic structures are usually easily identified and remain
relatively static in size and shape. If the plasma estrogen concentration is
persistently increased the diagnosis is further supported.

PROLONGED SEXUAL RECEPTIVITY - NYMPHOMANIA

 Normal queens may exhibit prolonged sexual receptivity despite having normal
waves of follicular function. In other words, their estrus behavior overlaps
interestrous intervals and persistent estrus results. In most cats this is considered
a normal phenomenon not requiring treatment.The ideal therapy, if any, is to
induce ovulation via breeding to a normal or vasectomized tomcat. Artificial
vaginal stimulation could also be used to induce ovulation and cause an end to
persistent behavioral estrus.
 Follicular cysts often produce signs of persistent estrus. A persistent follicle
becomes a persistent source of estrogen and any queen under a constant
influence of estrogen displays continuous estrus behavior. The diagnosis is made
by demonstrating increased plasma estrogen concentrations for more than 3
 weeks without evidence of normal cyclicity in a queen with a cyst associated with
one ovary on abdominal ultrasonography.
 Persistent estrus in cats older than 5 years is consistent with the presence of
granulose cell tumors. This is the most common ovarian neoplasm in cats. Such
tumors are more likely to be malignant in cats than in other species.
 Treatment could consist of attempts at breeding to ovulate the cyst. One could
attempt to induce rupture of the follicle(s) by administering 250IU of hCG IM
once daily for 2 days. The recommended treatment is surgical removal of the cyst,
with or without the associated ovary. Usually it is difficult to remove the cyst
without the ovary. These cats remain fertile with one ovary.

FAILURE TO PERMIT BREEDING

 A similar group of differential diagnoses exist in the queen, including

management problems, behavior disorders, vaginal or vulvar defects, and
miscellancous obstructions.
 Vulvar and vaginal and vaginal atresias have been diagnosed in our practices.
Vaginal strictures are rare but must be considered in a queen that fails to permit
breeding.

MODULE-13
ANESTRUS

 Anestrus meaning “without cyclicity” is a condition when the female does not
exhibit regular estrous cycles due to insufficient GnRH release from the
hypothalamus to stimulate and maintain gonadotrophin secretion. The ovaries
are relatively inactive and neither ovulatory follicles nor corpus luteum are
present.
 Anestrus is observed more commonly either after parturition as postpartum or
pre service anestrus and following service as post service anestrus when
conception does not occur.
 There are two categories:
o Class I or False anestrus - with functional CL.
o Class II or True anestrus - with no functional CL.

FALSE ANESTRUS

 False Anestrus may be classified as

o Anestrus due to pregnancy.
o Anestrus due to persistent corpus luteum (CL) – Conditions associated
with uterine pathology such as pyometra, mummified fetus, fetal
maceration, other disease states, mucometra and hydrometra.
 o Anestrus associated with CL of pregnancy that terminated early and not
recognized.
o Subestrus, weak or silent estrus and unobserved estrus.
 Normal cyclical changes in the genital organs but the signs of heat
are not exhibited or not observed.Most common especially in
buffalo cows. Common during post partum period.
o Anestrus following fetal maceration wherein most of the fetal parts appear
to have been expelled.

Note: Persistent CL does not occur in the presence of a normal non-pregnant uterus.
Many veterinarians tend to call wrongly a cyclic CL as persistent CL.

CAUSES

 Physiological basis is not known, but it may be due to a lack of estrogen and a
potentiating action of progesterone and is seen associated with
o Advanced age
o Arthritis
o Poor nutrition
o Seasonal stress
o Suckling
 Unobserved estrum may be due to managerial deficiencies and short period of
estrus.

TRUE ANESTRUS

 Small inactive ovaries - no functional

CL.
 May be due to an insufficient release
of gonadotropins or failure of ovaries
to respond.
 PREPUBERTAL ANESTRUS

 Pubertal estrus represents the initiation of the reproductive cycle, and this first
estrus generally occurs by a certain age relative to the animal’s weight. Heifers
must attain approximately two thirds of their adult size before they will reach
puberty. With good nutritional management, most Bos Taurus heifers attain their
 pubertal weight between 8 and 13 months of age. Failure of estrus expression
past this time is prepubertal anestrus.
 Clinically, heifers generally fall into one of two categories:
o The acyclic heifer of the same age as the rest of a cycling group
 Related to an abnormal reproductive tract. Freemartins,
hermaphrodites and aplasia are readily diagnosed by palpation
 Cyclicity of herd mates indicates that the problem does not affect
the entire group. Debilitating disease such as chronic pneumonia
can delay puberty by decreasing rate of gain, and this appears to be
a functional dietary problem
o Several acyclic heifers in a group of the same age or a group of mixed ages.
 Management practices play a vital role in the second category,
which comprises acyclic heifers of similar or diverse ages. Since the
onset of puberty is influenced by the level of available nutrition,
heifers of similar ages that are fed a suboptimal energy diet will
show a prolonged prepubertal anestrus period.
 Similarly, groups of heifers of diverse ages that are housed together
and given a balanced ration may contain several acyclic animals.
Larger or more aggressive herd mates consume a greater portion of
the available nutrition and tend to cycle first. Puberty, however, is
not postponed indefinitely, and eventually the entire population
cycle. There is an inherent danger in breeding these late heifers
before they have developed adequate body size as they tend to have
more dystocia problems and are prone to very long postpartum
anestrous periods.
o Certain infectious diseases can also produce anestrus in heifers. Blue
tongue and bovine diarrhea virus are capable of causing an acute ovaritis,
which leads to varying degrees of ovarian atrophy. Animals with complete
atrophy are anestrus unless stimulated with exogenous hormones. They
promptly return to the anestrus state when hormone therapy is
withdrawn.
o Growth –stimulating implants must be used with caution in prepubertal
heifers that will be used as breeding animals. Synovex-H and Zeranol
(Ralgro) can delay pubertal estrus and may affect future fertility.

POST PARTUM ANESTRUS

 A period of anestrus following parturition is a normal physiological event, and

ovarian cyclicity resumes as the uterus involutes. The anestrous period becomes
abnormal when its duration extends past the accepted average. The duration of
the average anestrus interval is influenced by age, breed, environmental factors
and genetic background.
 The normal postpartum cow undergoes surges of Follicle Stimulating Hormone
(FSH) which promote follicular growth that may be detected as early as 9 to 15
days postpartum in the dairy cow.
 The estradiol surges interact with the neuroendocrine centers, which results in
increased sensitivity to gonadotropin-releasing hormone (GnRH).
 Concurrently, the plasma luteinizing hormone (LH) level rise, and the number
and magnitude of episodic LH peaks tend to increase during the first 2 weeks
postpartum. This correlates with the fact that the quantity of LH at the pituitary
level increases after parturition and that GnRH sensitivity returns at about 8 to
10 days postpartum.
 At approximately 2 to 3 weeks postpartum the LH levels are able to induce
ovulation of one of the ovarian follicles.
 The corpus luteum of this first ovulation has a lower progesterone content and
may not be as responsive to LH, which results in a shortest lifespan. Also, due to
the absence of progesterone prior to this ovulation the estrus in generally silent.
Normal behavioral estrus generally develops at successive heats.
 Factors that modify this course of events can be divided into three categories:
lactational effects, nutritional effects and organic disease. Anestrus may be due to
a combination of the above factors.
 A clinical examination of a true anestrus animal typically reveals ovaries that
range from small and firm with no palpable structures to those that have multiple
medium size follicles (5 to 10 mm). Although most cases of anestrus will
eventually resolve themselves, this leads to a prolonged calving interval, which is
economically unsound.
 Lactation has been shown to cause an extension of the anestrus period and that
suckling decreased the average plasma LH concentrations and lengthened the
interval to the development of episodic ovulation and blocked the return to
cyclicity.
 Extension of the normal anestrus period can be caused by a deficient diet,
particularly one low in energy. The low producers would still cycle first; however,
the length of anestrus would become protracted.
 Organic dysfunction in the postpartum cow that causes anestrus may be related
to a primary uterine disease of a secondary systematic problem.
 Placental retention, metritis, chronic debilitating diseases, such as leg injuries,
displaced abomasums are associated with the anestrus state. The etiologic agent
may be chronic stress, or it may be related to the reduction in nutrient intake due
to a decrease in the animal’s desire and ability to eat properly. This is further
supported by a concurrent decrease in milk production, indicating the inability to
support lactation.

POST SERVICE ANESTRUS

 Post service anestrus is a normal event following insemination if the animal has
conceived. Approximately 5 per cent of the pregnant cows or heifers may exhibit
behavioural signs of estrus early in the gestation period.
 Following breeding, the animals are closely observed for estrus activity, which
should occur 18 to 23 days after breeding if they failed to conceive or to maintain
the embryo past day 12 aft5er ovulation. If the animal remains anestrus she is
presented for a pregnancy examination at 35 to 40 days after breeding. If the cow
 or heifer is nongravid at this time, the next estrus is expected in a few days, and
reinsemination is advised.
 Cases of anestrus other than pregnancy most frequently are due to estrus
detection failure, cystic follicular degeneration, pyometra, early embryonic death
and uterus unicornis, rarely granulos cell tumors, or leimyomas.
 True cases of postservice anestrus, in which the ovaries are nonfunctional or only
have multiple small follicles, are uncommon and reflect a severe nutiritional
deficienty and/or systemic disease. These animals tend to remain in the anestrus
state until the underlying illness is resolved.

DIAGNOSIS

Based on Rectal Examination

 The ovaries appear small and smooth. In buffaloes the ovaries appear spindle
like.
 Should be confirmed by repeated examinations at 10 days interval.
 Ultrasound examinations at regular intervals can be done to diagnose and
confirm anestrus.
 Progesterone estimations at intervals of 10 days would aid in confirmation. Low
progesterone levels at both times indicate true anestrus.

CLINICAL TREATMENT

 Therapy for the induction of cyclicity in the anestrus animal has been attempted
with a variety of exogenous hormones and management practices. For economic
consideration it is important to have heifers calve at 2 years of age and cows calve
every 12 months.
 Many hormone treatments have been utilized to hasten the onset of puberty or to
decrease the interval from calving to conception. Unfortunately, due to the
number of variables, including age, weight, diet and management hormones do
not always give consistent results.
 The overriding considerations for correcting an anestrus problem are that the
animal be healthy, have palpable follicular development and have access to good
feed.
 Induction of a pubertal estrus in heifers depends largely on the weight of the
animal after she reaches 13 months of age. Optimum results will be obtained if
the heifer is near the average weight at puberty for her breed. If a heifer is very
light she may not continue to cycle after an attempt at puberty induction.
o The treatment Syncro-Mate B, which combines a 6 mg Norgestomet ear
implant with an injection of 3 mg Norgestomet plus 5 mg estradiol
valerate, has given the best results. The implant is removed 9 days later,
and estrus ensues in 50 to 94 per cent of the animals within 120 hours.
Pregnancy rates following insemination during this induced estrus have
been reported to be as high as 50 per cent. The mechanism behind this
therapy attempts to mimic the short luteal phase associated with the
 “silent” pubertal estrus. The estradiol valerate causes luteal regression if a
functional corpus luteum is present.
o The injectable Norgestomet prevents luteinization of additional follicles by
the progesterone negative feedback mechanism. The 9-day period of
implantation is important in promoting estrus expression by causing
progestogen priming necessary for a psychic estrus. Also the implant helps
decrease the incidence of induced corpora lutea having a reduced lifespan
by promoting a normal LH release pattern following implant removal.
o The use of hormonal therapy in the anestrus suckling beef or lactating
dairy cow are usually done 45 to 90 days postpartum and as with heifers,
success depends on their nutritional status and body condition. Hormone
therapy is the same as that described for acyclic heifers; however, best
results are obtained by removing the nursing calves for 48 hours at the
time of implant removal (Shang treatment). Estrus occurs 24 to 48 hours
later, and first service conception rates have been reported to range from
40 to 70 per cent under ideal conditions. As with heifers, the progestogen
implants decrease the incidence of short luteal lifespan following the
induced estrus, although the implants do not completely eliminate this
phenomenon.
o Weaning without prior hormone therapy results in a higher expression of
estrus when compared with nonweaned cows.
 In general, the protocol involved involves the following steps:
o Improve Nutrition
 Extra feeding of a concentrate mixture or grains like maize, cholam,
kambu, etc., and at least small amount of green fodder along with
other roughages.
o Supplement Minerals
 Specific patent preparations which contain important minerals.
 Standard mineral mixture.
o Improve Managerial Practice
 Eradication of internal and external parasitism.
 Proper housing.
 Elimination of stressful factors.
o Specific
 GnRH 0.5 mg IM. to induce estrus. May be repeated after 10 days
 GnRH analogue Buserelin 0.02 mg IM.
 PMSG or FSH is not advisable as they can cause superovulation
 Short term progestogens- CIDR, PRID or Ear implant
induces heat even in anestrus animals.
 Progesterone injection followed by hCG or combination of
progesterone + PMSG + estrogen.
 Clomiphene citrate. 300 mg. daily for 5 days after drenching with
CuSO4 solution.
 MODULE-14
OVULATORY DEFECTS

Among the Ovulatory defects viz. Cystic Ovary, Delayed ovulation and Anovulation, the
economic losses due to cystic ovarian degeneration is the highest and is due to a
prolonged calving interval, increased culling rates and costs of veterinary intervention.

The traditional definition of a follicular cyst has been a structure of at least 2.5 cm
diameter that persists for ten days or more in the absence of a corpus luteum. However,
recent findings suggest that follicular cysts may be smaller than 2.5 cm, especially when
several cystic structures are present. Luteal cysts are typically 3 cm or more in diameter
and persist for at least 14 days.

CLINICAL SIGNS

 Affected cows may exhibit nymphomania or anestrus

 Due to aggressive sexual behaviour, they are called Buller’s
 Relaxation of sacrosciatic ligaments and upward displacement of the coccygeal
bones or elevation of the tail head called as sterility hump develop in the long
standing cases
 Cows with sterility hump are more prone for hip dislocation and pelvic fracture
as well as endometritis.
 Other signs include tendency for vaginal prolapse, pneumovagina, hydrometra
and mucometra. They gain a steer- like appearance.
 Microscopically the cystic dilation of the endometrial glands and hyperplasia of
the uterine mucosa take place and in the follicle the thecal layer and/ or
granulosa layer may be affected

DIFFERENCE BETWEEN FOLLICULAR AND LUTEAL CYSTS


Features Follicular cyst Luteal cyst
Nature of cyst Anovulatory Anovulatory
Persistence in ovary > 10 days Prolonged period
Diameter >2.5 cm >2.5 cm
Presence in ovary Multiple in both ovaries Often single
Symptoms Nymphomania – frequent, Anestrus – no sign of estrus, if
irregular, prolonged or continuous untreated for a prolonged period some
oestrus, prolonged period accept become virilized develop masculine
riding of another cow, frequent conformation, attempt to mount other
attempts to mount on other cows– cows, and may not allow mounting by
sexually aggressive– “ Bullers ”, others.
relaxation of the sacrosciatic
ligaments -upward displacement
of coccyx - “ Sterility hump ”.

CLINICAL DIAGNOSIS

 Based on clinical signs

 Based on rectal examination

Follicular Cyst Luteal Cyst

Both the ovaries greatly enlarged. One of the ovaries greatly enlarged.
Multiple cysts in both ovaries. Single cyst in one of the ovaries
Follicle wall thin, fluid filled, fluctuate Cystic wall thickened, fluid filled,
and smooth surface. fluctuate and smooth surface.
Readily rupture. Difficult to rupture.
Vagina, clitoris and vulva: swollen. No change.
Cervix: Large and dilated. Closed.
Uterus: Thickened, large, tonic and Flaccid.
oedematous.
Accurate diagnosis of cystic conditions is possible with single examination. If
doubtful, repeat examination after 10 days.

 Milk or plasma progesterone estimation

o Milk or Plasma Progesterone estimation may aid in diagnosis.
o Plasma or milk progesterone levels vary according to type of cyst.
o In milk, follicular cysts typically show levels less than 5 ng/ml, with luteal
cysts showing levels above 5 ng/ml.
 Ultrasonography
o Most accurate and practical diagnostic technique to identify and
differentiate between follicular and luteal cysts
o Wall thickness is much less in follicular cysts (average of 2.5 mm, range 1-
6 mm) than in luteal cysts (5.3 mm, range 3-9 mm).
o With follicular cysts, there appears to be a correlation between
ultrasonographic wall thickness and hormone concentrations within that
cyst, with thicker-walled cysts (average 2.5 mm) showing high
 progesterone and low estradiol concentrations, and vice versa in thinner-
walled cysts.

PROGNOSIS

 Good in early cases.

 Poor in long standing cases, where severe cystic degeneration of the
endometrium and atrophy of the uterine wall has taken place.

Treatment

 If diagnosed as cystic irrespective of the type any one of the following treatment
line may be attempted.
 LH: 2500-5000 IU I/V – optimum and economic
 GnRH:
o 100-250 µg I/M to luteinize
o 0.5 to 1.5 mg for ovulation
 Synthetic analogue, Buserelin: 0.02 mg I/M
 Following LH or GnRH treatment, the cysts undergo luteinization and most of
the cows re-establish ovarian cycle and exhibit estrus in 18-23 days.
 Following LH or GnRH, PGF2 α- 25 mg may be administered after 9-12 days to
cut short the cycle length
 GnRH or PGF 2 α may be preferred for luteal cyst which is however difficult to
differentiate from follicular cyst, in which PGF2α alone is ineffective
 Progestogens: CIDR, PRID or Ear implant are also effective.
 Progesterone: 100mg intramuscular for 14 days.
 Corticosteroids: 10-40 mg Betamethasone or 10-20 mg Dexamethasone.Found to
be as effective as LH or GnRH. Repeated if necessary (average 1.9 injections).
Suppresses the release of ACTH and also LH and upon the release of exogenous
block, LH is released in bulk.
 Potassium iodide: 30 Gm–divided into 6 doses. Daily oral administration
reported to be successful.
 Other lines of treatment tried include: clomiphene citrate, oxytocin, testosterone,
estrogen, etc.

Reasons for Reduced Recovery Rate

 Inability of the cystic structures to respond to GnRH induced LH release because

of fibrosis, degeneration of the theca and granulosa cells in the cyst.
 Insufficiency of the LH receptors
 Decreased sensitivity to LH
 Low pituitary responsiveness to GnRH or low activity of the secreted LH

DELAYED OVULATION
  Ovulation in the cow is atypical since it occurs 10-12 h after the end of estrus and
18-26 h after the ovulatory LH peak.
 Ovulatory defects may be due to endocrine deficiency or imbalance and
mechanical factors.
 Incidence: 2 to 18%
 Delayed ovulation is generally assumed to be one of the causes of failure of
conception.
 Certain cows have prolonged estrus. However, this is opined to be re lated to a
delay in corpus luteum (CL) assuming normal steroidogenesis rather than to the
delayed ovulation.
 Conception rate is reduced in cows that ovulated by the second day after oestrus.

Diagnosis

 Diagnosis is difficult and requires sequential rectal palpation of the ovaries.

Treatment

 Repeated AI at 24 h interval two or three times.

 GnRH: Encouraging results.Dose: Natural GnRH, 100 µg i/m. Buserelin, 5-10 µg
i/m.
 Luteinizing hormone ( LH ): Dose- 1000-1500 IU. i/m or i/v.

ANOVULATION

 Associated with those conditions which can predispose to true anestrus.

 May occur when the cow goes into anestrus or during the first cycle after
parturition.
 Diagnosis of anovulation can only be made retrospectively by noting on rectal
palpation that a follicle persists longer than one would have suspected.
 Anovulatory follicle undergoes luteinization and regresses like a normal CL after
17-18 days.

MODULE-15
REPEAT BREEDING

A repeat breeder cow is defined as one that,

 Has experienced three or more unsuccessful services.

 Have normal estrous cycles with approximately 21 days intervals.
 Is free from palpable abnormalities.
 Shows no abnormal vaginal discharges.
 Has calved at least once before.
 Is less than 10 years old.
However, this definition is quite restrictive and may not fit in all cases. All the major
causes can be grouped into two categories of those causing

 Fertilization Failure
 Early Embryonic Death

FAILURE OF FERTILIZATION

 Fertilization failure may result from death of the egg before sperm entry,
structural and functional abnormality in the egg or sperm, physical barriers in
the female genital tract preventing gamete transport to the site of fertilization, or
ovulatory failure.

Causes

Abnormal eggs

 Several types of morphologic and functional abnormalities have been observed in

unfertilized eggs, e.g., giant egg, oval shaped egg, lentil-shaped egg, and ruptured
zone pellucida.
 Failure to undergo fertilization and normal embryonic development may be due
to inherent abnormalities of the egg or to environmental factors.
 In sheep, some of the conception failures at the beginning of the breeding season
are associated with a high incidence of abnormal ova.

Abnormal Sperms

 The physiologic significance of abnormal sperm in relation to fertilization failure

has not been studied in animals other than cattle. Certain forms of male infertility
 are related to structural defects of the DNA protein complex. Sperm aging and
injury may cause
o Alterations in the acrosomal cap that may prevent defective spermatozoa
from fertilizing the egg. In bull, ram, and boar, a good correlation exists
between fertility and acrosomal integrity.
o Leakage of vital intracellular constituents such as cyclic AMP or the
formation of lipid peroxides from sperm plasmalogen when sperm are
stored under anaerobic conditions.
o A gradual decrease in the fertilizing capacity of aging of spermatozoa in
the female genital tract.

Structural Barriers to Fertilization

 Congenital or acquired defects of the female genital tract interfere with transport
of the sperm and/or the ovum to the site of fertilization
o Congenital defects are the result of arrested development of the different
segments of the Mullerian ducts or of an incomplete fusion of these ducts
caudally. A classic congenital anomaly associated with the gene for white
coat color is “white heifer disease” in cattle, in which the prenatal,
development of the Mullerian ducts is arrested, and the vaginal canal is
obstructed by the presence of an abnormally developed hymen. It can be
differentiated from the freemartin syndrome by the presence of normal
ovaries, vulva, and labia.
o Common anatomic abnormalities are adhesions of the infundibulum to the
ovary or uterine horns; this interferes with the pick-up of the egg or causes
a mechanical obstruction of one part of the reproductive duct system.
Bilateral or unilateral missing segments of the reproductive tract also
cause anatomic sterility.

Phytoestrogens

 Reproductive failure occurs more in sheep than in cattle grazing on plants that
contain compounds with estrogenic activity, e.g., subterranean clover and red
clover.
 The estrogenic activity is due to plant isoflavones and related substance with
hydroxyl groups. Cows and ewes fed estrogenic forage may suffer impaired
ovarian function, often accompanied by reduced conception rates and increased
embryonic loss. In cows, clinical signs resemble those associated with cystic
ovaries.
 The infertility is temporary, normally resolving within one month after removal
from the estrogenic feed. Ewes grazed on estrogenic pastures around the time of
joining, shed fewer ova and have a reduced chance of conception.
 Fertility is improved within 3 weeks, after the ewes are moved into nonestrogenic
pastures. The pathologic changes in temporary infertility are due to actions of
estrogen on the hypophyseal – ovarian axis and on sperm transport.
  Ewes grazed for several seasons on estrogenic pastures mate and ovulate, but
fertilization rate is depressed as a result of failure of sperm transport caused by
severe changes occurring in the cervix.

EARLY EMBRYONIC MORTALITY

 Embryonic mortality denotes the death of fertilized ova and embryos up to the
end of implantation. About 25 to 40% of embryos are normally lost in farm
species. It is also noted in large litters of swine and during multiple pregnancies
in cattle and sheep.
 Mortality is more common during the early than the late embryonic period. Early
embryonic mortality should be regarded as a normal process of eliminating unfit
genotypes in each generation, particularly in large litters of swine and multiple
pregnancies in cattle and sheep.
 In the past it was believed that the bovine conceptus was resorbed but transrectal
ultrasound examination has demonstrated that that the conceptus and its
breakdown products apparently are eliminated by expulsion through the cervix,
which either goes unnoticed or appears as a vulval discharge of clear mucus.
 Embryonic mortality after natural breeding or artificial insemination accounts for
the majority of reproductive failures in the cattle, with a mortality rate of up to
40% of all fertilized eggs. In cattle, most embryonic deaths occur between days 8
and 16 during hatching of the blastocyst and implantation without affecting cycle
lengths. Since, most embryos die between days 9 and 15 infertile ewes may
experience normal as well as prolonged cycles.

Cattle

 Most of the embryonic loss in cattle occurs between days 8 and 16 after
insemination.
 The timing of insemination is important as insemination too late in the estrous
period leads to ovum ageing and embryonic death. Artificial insemination during
pregnancy will induce loss, either through mechanical trauma to fetal membranes
or the introduction of infection.
 Nutritional causes such as B-carotene, selenium, phosphorus and copper
deficiencies have all been implicated in embryonic loss, but unequivocal data are
not available. High intakes of crude protein, in particular rumen – degradable
protein have been associated with reduced fertility. This is said to be due to the
toxic effects of blood urea or ammonia on the embryo.
 Stress, e.g. heat stress, has also been shown to result in embryonic loss. A high
rate of increase in milk yield and high milk yield per se in early lactation are
negatively correlated with fertility and this could be considered a metabolic
stress.

Horses
  The commonest cause of embryonic loss in mares is twin conceptions as
competition for placental space usually results in one fetus growing more slowly
than the other and the smaller fetus, with a smaller placenta, dies. Death of one
fetus often results in the loss of the second.
 Other intrinsic factors which are thought to be related to embryonic loss in the
mare include oviductal secretions, embryonic vesicle mobility and uterine
environment. Since, the mare’s embryo is at a more advanced stage whilst still in
the uterine tube, the environment may be relatively more important in this
species than others.
 In addition the embryonic vesicle remains free in the lumen of the uterine horn
much longer in the mare than in other species and the degree of mobility of this
vesicle is thought to be important in maternal recognition of pregnancy. Greater
mobility enhances the suppression of luteolysis and results in higher levels of
progesterone. As regards the uterine environment, recurrent endometritis and
post-service infection lead to perivascular fibrosis, and this is a common cause of
embryonic and fetal death between 40 and 90 days of gestation. Increased
maternal age has also been associated with increased embryonic loss, but this
may merely reflect increased chronic uterine pathology.
 Other factors such as lactation and service at the foal heat also result in higher
embryonic death rates, although the latter may be due to lactational stress.
Stress, due to transportation, is thought to cause embryonic losses in the mare.
However, recent studies failed to confirm this even though transport did result in
raised plasma ascorbic acid levels, which have been associated with prolonged
stress. Nutritional stress, in the form of restricted energy intake, does increase
embryonic loss.

Sheep

 Nutrition, specifically energy level, is known to affect embryonic survival in sheep

in a complex manner. Low body condition at mating is detrimental to embryo
survival, irrespective of post mating nutrition. However, in ewes that lose weight
post – service, embryo mortality is increased. Prolonged, moderate under
nutrition has more effect on ewe lambs than adult ewes.
 Nutritional energy may exert its effect via peripheral blood progesterone between
food intake and progesterone levels. Other nutrients important in embryo
survival are vitamin E and selenium. Certain plants, such as kale and Veratrum
californicum, will cause embryonic death.
 The latter is also a teratogenic agent. The effects of nutrition may be exaggerated
or confounded by differences in ovulation rate since losses have often been
reported to be disproportionately high in twin ovulations. In breeds with very
high ovulation rates the embryonic death rate rises proportionately, but this is
probably due to limitations of uterine space.
 High environmental temperature, particularly in the first week after mating, has
been shown experimentally to increase the embryonic death rate dramatically.
This could be important in climatic heat waves. However, if there is diurnal
variation, as would occur naturally, the loss is much lower.
  Physiological stress, such as that produced by overcrowding or handling of sheep,
also increases embryonic loss. This may be due to excess secretion of
progesterone by the adrenals and/or raised corticosteroid levels. The age of the
ewe is also important, since there is some evidence that ewe lambs have a higher
incidence of embryonic loss than mature ewes.

Goats

 Goats are particularly susceptible to non-infectious fetal loss, and this is

particularly true of the Angora breed. Losses are also common in poorly fed
animals of any breed.
 Another reported cause of fetal loss is dosing with anthelmintics such as carbon
tetrachloride and phenothiazine.

Pigs

 Ovulation rate is not usually a limiting factor in productivity in the pig but, in
general, as ovulation rates increase, the embryo survival rate decreases. This can
be demonstrated in gilts, where the ovulation rate can be artificially increased but
embryo survival rate decreases. Even if early embryonic death does not occur
with high ovulation rates, a problem may arise later in pregnancy with
competition for uterine space. It has been suggested that a higher fetal death rate
exists when there are more than five fetuses per horn, with those embryos in the
middle of the horn being smaller.
 Apart from the above intrinsic factors, extrinsic factors such as nutrition and
stress play an important part in embryonic loss in the pig. For example, it is well
documented that high energy levels after service result in reduced embryo
survival. Stress, associated with extremes of temperature, or certain management
systems such as sow stalls or tethers, is also known to result in increased embryo
mortality. Other husbandry policies such as lactation length, also affect
embryonic death rates, and lactation lengths of less than 3 weeks produce a
marked rise in embryonic mortality presumably due to a poor uterine
environment.

Sequel to Embryonic or Fetal Death

 Following early embryonic death the embryonic tissue are usually resorbed, and
the animal returns to estrus if there is no other conceptus in the uterus. If death
occurs before there has been maternal recognition of pregnancy the estrous cycle
is not prolonged. If it occurs after recognition has taken place, the estrous cycle
will be prolonged.
 If death of the embryo is due to an infection then, even though the embryonic
material may be absorbed, a pyometra may follow. In cattle this condition is
characterized by persistence of the corpus luteum, closed cervix and pus
accumulation in the uterine body and horns. It is a particular characteristic of
infection with Tritrichomonas fetus. If fetal death occurs after ossification of the
 bones has begun, complete resorption of fetal material cannot take place, instead,
fetal mummification occurs.

CAUSES OF EMBRYONIC MORTALITY

Causes for early embryonic death

 Embryonic mortality can be due to maternal factors, embryonic factors, or to

embryonic-maternal interactions. Maternal failure tends to affect an entire litter,
resulting in complete loss of pregnancy.
 In contrast, embryonic failure affects embryos individually, often leaving others
in litter unharmed. In other cases the maternal environment may be insufficient,
allowing the support of only a few strong embryos.

Endocrine Factors
  Accelerated or delayed transport of the egg, as a result of estrogen – progesterone
imbalance, leads to preimplantation death. An abnormally undersized conceptus
might not be able to counteract the uterine luteolytic effect, with consequent
regression of the CL and termination of pregnancy. In swine, as stated previously,
at least four living blastocysts are needed by day 10 of pregnancy to counteract
the uterine luteolytic effects.
 A critical period of embryonic survival is the late blastocyst stage. Normally, the
developing CL secretes progesterone, which acts on the female tract in close
synchrony with the development of the embryos. The cause and effect
relationship between luteolysis and embryonic deaths is controversial.
Apparently, embryonic mortality in cattle is not caused by a progesterone
deficiency during the luteal phase of the cycle; luteal regression follows rather
than precedes embryonic mortality. However, a diminished response to
circulating luteotrophic hormones may contribute to embryo mortality in
subfertile cows.

Lactation

 Embryonic mortality occurs during lactation in cattle, sheep, and horses and is
characterized by prolonged estrous cycles after breeding. Mating of mares at foal
heat leads to early embryonic mortality, which has been attributed to reduce
effectiveness of uterine defense mechanisms, stress of lactation, and incomplete
regeneration of the endometrium.
 Sows bred after weaning at 7 days of lactation suffer high embryonic losses
between days 9 and 20 of pregnancy.

Nutrition of the Dam

 Caloric intake and specific nutritional deficiencies affect ovulation rate and
fertilization rate, as well as cause embryonic death. Also extremes in the level of
feeding are detrimental to embryo survival, so too are extremes in the supply of
specific dietary nutrients.
 In dairy cows, high intakes of rumen degradable protein may lead to embryonic
mortality. This effect may be mediated through a reduction in the pH of the
uterine environment during the luteal phases of the cycle in which the embryo
must grow.
 In swine, high caloric intake or continuous unlimited feeding increases ovulation
rate, thereby increasing the incidence of embryonic mortality before
implantation. However, following implantation, unlimited feeding decreases fetal
death.
 In sheep, full feeding before breeding also increases ovulation rate as well as
embryonic mortality. Poor body condition of ewes at mating increases the
incidence of embryonic mortality, whereas moderate feed restriction from day 20
to 100 of pregnancy is less likely to reduce lambing percentages. Under nutrition
affects twin ovulators more than single ovulators because both embryos are lost
in the former, while a single embryo survives in the latter. Thus, more twin than
single ovulating ewes are barren.
  In the mare, the critical period for embryonic resorption is between 25 and days
after ovulation. No resorption occurs if mares are maintained on an adequate
plane of nutrition until 35 days after service.

Age of the Dam

 A higher incidence of embryonic mortality is observed in gilts and in sows after

the fifth gestation. In the ewe, the incidence of late embryonic loss is higher in
ewe lambs and ewes over 6 years than it is in mature ewes, which is due to factors
associated with the embryo rather than the uterine environment.

Overcrowding in Utero

 Because the degree of placental development is primarily influenced by the

availability of space and vascular supply within the uterus, increasing the number
of implantations decreases the vascular supply to each site and restricts placental
development. This results in a high embryonic and fetal mortality rate and
probably explains the higher incidence of embryonic mortality in cattle and sheep
following twin rather than single ovulation. It should be noted, however, that
uterine capacity does not limit the ability of the cow and ewe to carry twins,
provided they are located in separate uterine horns. In cattle, embryo transfer
experiments have shown a higher embryonic mortality rate in recipients which
received two embryos in a single uterine horn. This loss may be due to
overcrowding and intrauterine competition for nutrients.
 In cattle and sheep with multiple ovulations, the number of embryos surviving is
reduced to a fairly constant number within the first 3 or 4 weeks of pregnancy,
which implies that embryonic loss increases as the number of eggs shed
increases. Mortality does not seem to be due to a deficiency of progesterone. In
prolific breeds of sheep, late embryonic deaths occur in ewes with more than five
ovulations.
 Transuterine migration of embryos is of importance for equal distribution of
embryos in the two horns of the uterus in polytocous species such as swine. In its
absence, there is a high incidence of embryonic mortality in swine.

Thermal Stress

 Embryonic mortality increases in a number of species following exposure of the

mother to elevated ambient temperatures, especially in tropical areas the effects
of thermal stress on the early embryo are not apparent until the later stage of its
development. Fertilized eggs of sheep and cattle, when subjected to high
temperatures either in vitro or in vivo are damaged but continue to develop, only
to die during the critical stages of implantation.
 Reduced fertility of summer heat-stressed dairy cows may result from decreased
viability and developmental capacity of 6 day –old to 8 day-old embryos and may
account for the well-documented seasonal reduction in the efficiency of artificial
insemination during summer. Heat stress between days 8 and 17 of pregnancy
may also alter the uterine environment as well as growth and secretory activity of
 the conceptus. Apparently heat stress antagonizes the inhibitory effects of the
embryo on the uterine secretion of PGF2a.
 Several studies have demonstrated that the pig embryo is most susceptible to
heat stress before day 18 of pregnancy particularly during implantation. A greater
incidence of embryonic deaths was noted among gilts exposed to high
temperatures 8 to 16 days post breeding than among those exposed during 0 to 8
days post breeding.

Semen

 A portion of all embryonic mortality is attributable to the male and the mating
system. Genetic factors that are transmitted by the male to the embryo may be
inherited, may arise from testicular tissue, or may occur in spermatozoa after
they are released from the testis.
 Infertile matings by highly fertile bulls are primarily due to embryonic mortality,
while those of bulls with low fertility are due to fertilization failure and
embryonic deaths. In swine, semen stored for 3 days before insemination
produced zygotes much more susceptible to early embryonic death, presumably
owing to the reduced DNA content in aged spermatozoa.

Incompatibility

 The inherited genotype of the male may include a variety of genetic factors that
lead to incompatibility and early embryonic loss. There may be incompatibility
between spermatozoa and mother, between spermatozoa and egg, or between
zygote and mother.
 Immunologic incompatibilities may block fertilization or cause embryonic, fetal,
or neonatal mortality. In cattle, homozygosity for certain blood groups and
certain substances related to transferring and J-antigen in sera are associated
with increased embryonic loss as well as decreased fertilization rate.

DIAGNOSIS AND TREATMENT

Diagnosis

Based on reproductive history and clinical examination

Treatment

Specific treatments for conditions like delayed ovulation, endometritis may be carried
out. Since most of the cases do not reveal any specific condition the following guidelines
may be adopted.

 Bring the animal into positive energy balance.

 Do AI twice at each estrus preferably at 12 or 24 h interval.
  Check the semen quality - use only high quality semen - (This is often taken for
granted and ignored).
 Clitorial stimulation for at least three seconds at AI.
 Administration of 100 µg of GnRH or 1000-1500 IU of LH at the time of AI.
 Administration of 500 mg of depot progesterone on the 5th day of AI.
 Skipping of AI, administration of PGF 2 alpha after 9-10 days and fixed time AI
twice at 72 and 96 h.
 Intrauterine infusion of 1 million units of procaine penicillin diluted in saline
three times at the onset of estrus, 8 h after AI and 24 h later.
 Skipping of AI and intrauterine infusion of 1 to 1.5 million units of procaine
penicillin in 20 ml of sterile saline daily for 3-4 days.
 Skipping of AI and intrauterine infusion of 2 ml of Lugol’s solution diluted in 8
ml of sterile saline.
 Flushing of the uterus with normal saline - under moderate pressure as being
done in embryo transfer (to remove cellular debris and also mild block in the
uterine tubes).
 Administration of different hormones and antibiotics may preferably be tried at
separate estrus.

INTRAPERITONEAL INSEMINATION

 Mammalian sperm interactions with the female reproductive tract determine the
drastic reduction in the number of spermatozoa that occurs between the site at
which the ejaculate is deposited and the site at which fertilization takes place.
Physiological and pathological events may occur in the tubular genital organs,
which could act unfavourably on the mechanisms of sperm transport and result
in infertility. Under normal conditions, sperm transport is of an ascending nature
from the site of semen deposition to the fallopian tubes.
 It is generally accepted that some spermatozoa (perhaps in low numbers) pass
through the oviduct in to the peritoneal cavity after natural mating or
insemination. The fate of these spermatozoa is not known, but on their earlier
travel through the genital tract, they become separated from the rest of the
components of the seminal plasma, thus reducing the chances of an immune
response. However, spermatozoa can also approach the site of fertilization from
the peritoneal cavity.

Procedure

Intraperitoneal insemination procedure as outlined by Lopez-Gatius (1995).

 A sterile 19 gauge needle 4 cm long and a glass tube 44 cm long and 10 mm in

diameter and with a conic tip are used as additional accessories to standard AI
equipment.
 Immediately before insemination, the vulva and the perineal region of the heifer
and the glass tube are washed with disinfectant solution.
  For insemination, the glass tube is carefully inserted into the vagina, guided by
the hand, and the conic tip positioned to the dorsal fornix.
 The AI catheter with the needle affixed to the end of the plastic sheath is then
inserted in to the glass tube, and the vaginal wall is pierced in cranial direction
through the fornix by the needle.
 Next, semen is slowly injected in to the peritoneal cavity.
 Frozen semen in 0.25 ml French straws is diluted in Triladyl (Minitub) so that
each unit of semen contains at least 45 million spermatozoa.

Though intraperitoneal insemination appears to a useful technique for the treatment of

a wide scope of infertility problems in human medicine, only 2 previous experiments
have been performed in cattle using this technique. Lopez-Gatius (1995) recorded 9
pregnancies following intraperitoneal insemination of 62 repeat breeder cows. The
pregnancy rate was not different for intraperitoneal or uterine inseminations. Further,
the results suggest that intraperitoneal insemination could be an alternative procedure
to the usual deposition of semen in to the uterus in repeat breeder cows.

MODULE-16
SPECIFIC INFECTIOUS DISEASES CAUSING INFERTILITY-I

 Infectious diseases of cattle adversely affect reproductive performance, either

directly upon the reproductive system or indirectly upon the general state of
health of affected animals.
 Infectious diseases can affect the reproductive system in the following main ways:
o Impaired sperm survival or transport in the female tract, leading to
reduced fertilization rate.
o Direct effects upon the embryo. This includes infections that result in early
embryonic death, and those that infect the more advanced fetus or its
placenta, resulting in abortion, stillbirths or the birth of weak calves.
o Indirect effects upon embryo survival. This includes infections that
adversely affect uterine function and infect the maternal component of the
placenta leading to embryonic death and fetal stillbirth.
o Systemic illness causing fetal losses or a direct impairment of reproductive
cyclicity.

MODULE-21
NON SPECIFIC INFECTIONS OF GENITAL ORGANS

Non specific infections of the genital tract causing infertility in female farm animals include
endometritis, cervicitis and vaginitis. A significant proportion of failures of conception can be
ascribed to varying degrees of chronic endometritis.

ENDOMETRITIS IN COWS
  Endometritis is a localized inflammation of the uterine lining, associated with
chronic postpartum infection of the uterus with pathogenic bacteria
Arcanobacterium pyogenes (Bondurant, 1999).

Etiology

 The causal organisms usually reach the uterus from the vagina at coitus,
insemination, parturition or postpartum, although it is possible in some
circumstances for infection to arrive by the circulation. The great majority of
cows suffer from bacterial contamination of the uterus after calving, but under
normal circumstance this flora is rapidly eliminated. In cows that develop
endometritis, the bacterial flora is not eliminated from the uterus, causing the
endometrium to become inflamed.

Factors Associated with the Development of Endometritis

o Retained fetal membranes.

o Abortion.
o Induced calving.
o Multiple births.
o Dystocia.
o Management factors – state of nutrition, hypocalcaemia, season.
o Return of ovarian cyclicity.
o Bacterial loading.
 The endometritis is almost invariably a sequel to invasion with A. pyogenes.
There is good evidence that there is synergism between A. pyogenes and
Fusobacterium necrophorum, the latter organism producing a leucocidal
endotoxin which interferes with the host’s ability to eliminate A.pyogenes.
Similarly Bacteroides sp. also produces substances that interfere with the
phagocytosis and killing of bacteria.

CLINICAL SIGNS

 The presence of a white or whitish-yellow mucopurulent vaginal discharge in the

post partum cow.
 The volume of discharge is variable, but frequently increases at the time of estrus
when the cervix dilates and there is copious vaginal mucus.
 The cows rarely show any signs of systemic illness, although in a few cases milk
yield and appetite may be slightly reduced.
 Rectal palpation frequently shows a poorly involved uterus which has a doughy
feel.

DIAGNOSIS

 Clinical signs
 Rectal examination
  Vaginal examination
 Whiteside test
 Uterine biopsy
 Bacterial culture

RECTAL EXAMINATION

 Transrectal palpation of the uterus. However, this method is subjective and often
fails to account for normal events and variability in uterine involution or to have
any association with reproductive performance.
 By rectal examination cervical diameter, location of the uterus, symmetry of the
uterine horns, diameter of the uterine horns, texture of the uterine wall, palpable
uterine lumen are noted.

VAGINAL EXAMINATION

 At examination, cows are first inspected for the presence of fresh discharge on
the vulva, perineum, or tail. If discharge is not visible externally cows are
examined using vaginal speculum.
 The speculum is inserted into the vagina up to the level of the external os of the
cervix.
 Inspection of the cervix and vagina is performed with illumination from a
penlight.
 The nature of the discharge may be clear mucus with flakes of pus,
mucopurulent, purulent but not foul smelling.

WHITE SIDE TEST

This test is used to detect sub-clinical endometritis in repeat breeding cows.

Procedure

 The uterine discharges (cervical mucus) is collected aseptically with sterile sheath
and syringe.
 Mixed with equal volume of 5% NaoH in a test tube.
 The mixture is heated up to the boiling point, and
 The intensity of colour changes is graded.

Color Degree
Turbid Normal
Light yellow Mild
Yellow Moderate
Dark yellow Severe
 ENDOMETRIAL BIOPSY

 A relatively easy and safe procedure for the practicing veterinarian to perform.
 Its use in conjunction with a detailed history, rectal and vaginal examinations
and microbial cultures can lead to a more accurate prognosis of difficult breeders
and greater therapeutic efficiency.
 Repeated biopsies do not cause adverse effects on cow’s reproductive capacity.
 Biopsy lesions heal rapidly.
 Hemorrhages are of little or no clinical significance and are quickly resorbed.
 Biopsy specimen should be of sufficient size (4 x 6 mm).
 Specimens should be taken from both the horns and the body of the uterus due to
variability of pathology in each section.

BIOPSY CATHETER

 To obtain in vivo uterine endometrial samples.

 It consists of an outer casing and piston of length 57.5 cm and, diameter of 0.7
cm.
 Distal end of the catheter has a rounded tip to prevent injury to the reproductive
tract and to facilitate the easy entry of the tip through the cervical canal.

BIOPSY TECHNIQUE

 Proper care, disinfection and sterilization of the biopsy instrument are necessary
to prevent microbial contamination. Before taking biopsy, thoroughly scrub and
clean the vulva and surrounding perineal area.
 Evert the vulval lips and introduce the biopsy instrument in closed position
through the vagina and cervix in to the uterus (Step-1).
 Gently push the piston to open the cutting edge. Press a portion of the uterine
wall in to the cavity of the cutting edge (Step-2). Pull the piston caudally to close
the cutting edge so as to remove a piece of the endometrium.
 Withdraw the instrument out of the reproductive tract in closed position (Step-
3).
 Remove the endometrial tissue from the instrument and immediately transfer it
into 10% neutral buffered formalin solution at room temperature.
 Tissues are trimmed, dehydrated, cleared and embedded in paraffin sections and
cut at a thickness of 5-6µ and stained with H&E stain for histological
examination.

INTERPRETATION

 Bovine endometrium is evaluated histologically for:

o periglandular fibrosis.
o cystic glandular changes, and
o cellular infiltration of endometrial stroma.
 Cellular infiltration is the most striking feature of acute endometritis.
 Moderate and severe cases of endometritis are much easier to diagnose on the
basis of the increased number of inflammatory cells spread throughout the
stratum compactum and spongiosum layers compared with few cells seen in mild
endometritis.
 Neutrophils may be present in high numbers during normal estrum –
erroneously suggesting acute endometritis.
 Neutrophils present during the luteal phase – definitely indicative of an acute
endometritis.
 Initial phases of endometritis: diffuse and possibly the periglandular and
perivascular cellular infiltrations are dominated by neutrophils and lymphocytes.

TREATMENT

 Treatment of endometritis is the subject of considerable controversy among veterinary

practitioners, particularly with respect to which therapy to use, and to a lesser extent,
which cows to treat or whether to treat at all (Gilbert, 1992).
 The general therapy of endometritis is to halt and reverse inflammatory changes that
impair fertility practically, treatments aim to reduce the load of pathogenic bacteria and
enhance the processes of uterine defence and repair.
 A wide range of antiseptics antimicrobial agents and hormones have been used as
treatments for endometritis. Objective studies of the effectiveness of these agents have
been difficult because of the multifactorial nature of the disease and many cases of
endometritis are self-limiting and resolve after the resumption of oestrous cyclicity.
o Antibiotic therapy
o Hormones
o Intrauterine infusion of disinfectants
o Immunomodulators

ANTIBIOTIC THERAPY

 Local vs systemic administration - For antimicrobial treatment to be effective, an

effective, concentration of drug must be achieved and maintained of the site of
infection for an adequate period. Several antimicrobial agents are absorbed from
the uterus (sulfonamides, tetracylines, streptomycin, penicillin, ampicillin,
gentamicin and chloromphenicol). The absorption in the immediate postpartum
period is considerably less than that after complete uterine involution. Uterine
pathologic changes (endometritis) result in further decrease of absorption. Poor
absorption results in a high concentration of the drug in the uterine cavity and on
the endometrium on the other hand, adequate concentrations frequently are not
achieved in the subendometrial tissues, vagina, cervix, or ovaries and oviducts.
 Systemic administration usually results in uterine tissue and lumen antibiotic
concentrations equal to blood plasma concentrations. The concentrations are the
same in the normal and the pathologic uterus. The systemic administration gives
a better distribution in the tubular genital tract and to the ovaries. Furthermore,
fetal membranes and abnormal exudate cannot mechanically influence the
distribution. Also, systemic administration eliminates the risk for damage to the
endometrium. Repeated treatment can be carried out relatively simple and
 without introduction of new infections. Because, there are reasons to assume that
a moderate to severe uterine infection seldom is localized only to the superficial
layer of the endometrium, therapeutic strategies would have to consider systemic
treatment.
 In the treatment of chronic endometritis with antimicrobial substances, it is
preferable to administer the substance by the intrauterine route provided an
adequate dose rate is used, this will result in effective minimum inhibitory
concentrations (MICs) reaching the endometrium and being established in the
intraluminal secretions. The latter point is important for the effective treatment
of the disease, since sub therapeutic dose rates are frequently used.
 Several antibiotics are inappropriate for the treatment of uterine infections
Nitrofurazone is an irritant and has an adverse effect on fertility.
Aminoglycosides are not effective in the predominantly anaerobic environment of
the infected uterus. Sulphonamides are ineffective because of the presence of
para-aminobenzoic acid metabolites in the lumen of the infected uterus.
Penicillins are susceptible to degradation by the large numbers of penicillinase
producing bacteria that are present.
 A broad-spectrum antibiotic, such as oxytetracycline, used at a dose rate of up to
22 mg/kg, will provide effective MICs in the lumen and uterine tissues for intra
uterine treatment with oxytetracycline total doses of 0.5 to 5 g may be used. The
lower dose (0.5 to 2 g) is unlikely to yield adequate concentrations in the large
postpartum cavity. Systemic administration of penicillin results in genital tract
tissue and lumen concentrations similar to blood plasma concentrations in the
cows. Other antibiotics such as Penicillins, Metronidazole, Ciprofloxacin and
Cephalosporins are administered systemically as well as intrauterine for the
treatment of uterine infections.

HORMONE:PROSTAGLANDIN F2 ALPHA

 When there is a palpable mature CL on the ovary it is arguable that the best-
method of treating clinical endometritis is with PGF 2α or its synthetic analogues.
When a CL is present, PGF2α causes luteolysis, thereby stimulating the return to
estrus and reducing the high progesterone concentrations.

INTRAUTERINE INFUSION OF DISINFECTANTS

 Intrauterine infusions with various disinfectants such as lugols’ iodine and

povidone iodine are relatively common for treatment of postpartum infections.
Although positive results occasionally have been reported, few controlled
evaluations have been made. Because intrauterine use of disinfectants may
suppress the uterine defense mechanisms eg. phagocytosis, the use of
intrauterine infusions in the postpartum cow is not recommended.

ROLE OF IMMUNOMODULATORS
Lipopolysaccharides of E.coli

 Serotype 026:B6.
 Dissolve 100 μg in 20 ml of PBS (pH 7.4).
 Administer on day 0 (estrum) through intra uterine route.

Oyster glycogen

 PMN migration into the uterine lumen of healthy cows is stimulated after
intrauterine administration of oyster glycogen, up to 90% of all cells identified in
uterine secretions being neutrophils.
 Variable concentrations of oyster glycogen between 0.1-10% all in 60 ml of
vehicle produced identical responses with a peak PMN concentration 12 h after
administration.

Leukotriene B4

 Leukotriene B4 (LTB4) is an effective chemo-attractant, stimulating preferential

migration of PMNs into the lumen of the bovine uterus.
 A single intrauterine treatment of a 30 nmol/L solution increased the
intrauterine leucocyte count 5-10 times within 24 h.

Autologous plasma

 Collect ~300 ml of blood from oestrus animal in JML blood bag. Keep in ice and
transport to the lab. Transfer in to 50 ml of sterile centrifuge plastic vials;
centrifuge at 3000 RPM for 15 min, separate the plasma and stored at -20 °C.
 Administer 50 ml of plasma through intra uterine route on days 1, 2, and 3 (day 0
– estrum).

ENDOMETRITIS IN MARES

 Reduced fertility associated with endometritis, both acute and chronic, has been
recognized for many years in brood mares. This subfertility is due to a hostile
environment for the developing conceptus, and in some cases, the endometritis
causes early regression of the CL.
 The term `endometritis’ refers to the acute or chronic inflammatory process
involving the endometrium. These changes frequently occur as a result of
microbial infection, but they can also be due to non-infectious causes.
 One of the main obstacles to producing the maximum number of live, healthy
foals from mares bred during the previous season is the mare, which is
susceptible to persistent acute endometritis following breeding.

ENDOMETRITIS IN SOWS, EWES AND GOATS

  A form of endometritis characterized by profuse vaginal discharge at the onset of
estrus has been described in Europe and other regions. The causative agent is
usually Staphylococcus hyicus or E coli , and the disease seems to be transmitted
at mating or artificial insemination; signs are seen 15-25 days later during the
subsequent proestrus or estrus. Infection may be of long duration with signs
recurring at each estrus.
 There is some correlation with serum concentration of progesterone and
endometritis. When inseminations are done at the time when progesterone levels
begin to rise after ovulation, the possibility of inducing endometritis is more.
Some sows recover spontaneously, but there does not seem to be any effective
treatment for those that do not. At necropsy, copious quantities of purulent
exudate may be found in the uterus, making this condition more akin to
pyometra. Some virgin gilts also show endometritis. The cause is unclear but may
occur secondary to vulval biting in finishing houses or due to the fact that many
gilts attain puberty prior to transport with infection if the uterus occurring at gilt
finishing houses. Endometritis also occurs following parturition due to dystocia,
traumatic injury, abortion or unhygienic manipulations.
 Endometritis has been seen in sheep and goats.In commercial sheep and goat
flocks, diagnosis is seldom made antemortem, and treatment is generally
impractical. In animals with a persistent uterine discharge, remnants of a
macerated fetus should be considered as a nidus of chronic infection.

MODULE-22
NORMAL TARGETABLE HERD FERTILITY PARAMETERS

Reproductive traits Goal

Calving interval 365-380 days
Less than 40
Avg. days to 1st observed heat
days
% cows in heat by 60 days post calving Greater than 90
Avg. days open to 1st breeding 50-60 days
Avg. days open to conception 85-100 days
Services/conception 1.5-1.7
First service conception rate
A) replacements 65-70%
B) producing females 55-60%
Greater than
% breeding intervals between 18-24 days
85%
% cows open greater than 120 days Less than 10%
Dry period length 45-60 days
Avg. age at 1st calving* 24 months
Avg. age at 1st breeding* 15 months
% cows pregnant less than or equal to 3
90%
Al/services
% cows pregnant on examination 80-85%
Abortion rate Less than 5%
Cull rate for infertility Less than 10%

NON-RETURN RATE TO FIRST INSEMINATION

 This is the percentage of cows or heifers, in a particular group, which have not
been presented for a repeat insemination within a specific period of time.
 The periods are usually 30-60 days or 49 days.
 This is used, particularly in artificial insemination centers, to monitor the fertility
of bulls and the performance of inseminators.

CALVING INTERVAL AND CALVING INDEX

 The calving interval is the interval in days for an individual cow between
successive calvings; the calving index is the mean calving interval of all the cows
in a herd at a specific point in time, calculated retrospectively from their most
recent calving date. These two measurements have been used traditionally as a
measure of fertility, since they indicate how closely the individual cow or herd
approximates to the accepted optimum of 365 days.
 The disadvantages of these measurements are that they are historical in that they
are calculated retrospectively; furthermore, the calving index can give an
overoptimistic assessment of fertility when many of the cows that fail to become
pregnant are culled.

CALVING TO CONCEPTION INTERVAL

 The calving interval (or index CI) is the sum of two components, (a) the interval
from the last calving date to the date of conception and (b) the length of gestation
thus:
o CI = a + b
 Therefore
o CI = 85 days + 280 days = 365 days
 The calving to conception interval (CCI) is calculated by counting the number of
days from calving to the service which resulted in pregnancy (effective service);
this is usually the last recorded service date. The CCI is a useful measurement of
fertility but requires a positive diagnosis of pregnancy to be made. It is influenced
by two factors; how soon after calving the cows are served or inseminated and
how readily they become pregnant when they have been served.
 The CCI can be expressed thus:
o Mean CCI = c +d
 Where ‘c’ is the mean calving to first service interval and ‘d’ is the
mean first service to conception interval, therefore
o Mean CCI = 65 days + 20- days = 85 days.
  The mean CCI is a useful measure of fertility, provided that the interval from
calving to first service is stated, since this probably will have the greatest
influence upon its length.

DAYS OPEN

 This is defined as the interval, in days, from calving to the subsequent effective
service date of those cows that conceive, and from calving to culling or death for
those cows that did not conceive.
 Numerically, it will always be greater than the mean CCI unless all cows that are
served conceive, in which case it would be the same.

Days open (%) = (Total service period in days in all lactations /Total herd life in days)
x 100

CALVING TO FIRST SERVICE INTERVAL

 In the case of a herd that calves all the year round a mean value of 65 days should
result in a mean CCI of 85 days.The factors that influence the calving to first
service interval are:
o Breeding policy of the farm. Although cows will return to estrus after
calving as early as 2-3 weeks, they should not be served before 45 days,
and in the case of first calving, high-yielding cows and those that have had
dystocia and problems during the puerperium slightly longer time should
elapse. Thus, in a seasonal calving herd, those that calve early in the
season will have their first service delayed and, for those that calve late, it
may be necessary to advance the date of first service thereby tightening the
calving pattern
o Delayed return of cyclical activity after calving, i.e. true anestrus
o Failure to detect estrus in those cows that have resumed normal cyclical
activity.
 Factors (2) and (3) can be improved by ensuring that cows have returned to
cyclical activity postpartum. This can be done by regular and routine examination
of those cows, per rectum, that have failed to be seen in estrus by 42 days
postpartum and by the use of milk progesterone assays.
 Detection of estrus depends upon the herdsman knowing the true signs of estrus,
having a regular routine, recording the events and using estrus detection aids.

OVERALL PREGNANCY RATE

 This (originally called the overall conception rate) is the number of services given
to a defined group of cows or heifers, over a specified period of time which result
in a diagnosed pregnancy not less than 42 days after service; the figure is
expressed as a percentage of the total number of all services and should include
culled cows.
 The method of pregnancy diagnosis should be specified. The first service
pregnancy rate is usually calculated separately and obviously refers to first
services only. Thus in a 12 month period, if 100 cows receive 180 services, of
which 90 resulted in a confirmed pregnancy, the overall pregnancy rate would be
50 per cent.
 The pregnancy rate is influenced by:
o The correct timing of artificial insemination, which will be dependent
particularly on the accuracy of estrus detection.
o Correct artificial insemination technique, handling and storage of semen.
o Good fertility of the bull if natural service is used, and the absence of
venereal disease.
o Adequate nutritional status of cows and heifers at the time of service and
afterwards.
o Complete uterine involution and absence of uterine infection.
 The pregnancy rate to first service and overall pregnancy rate are very useful
measures of fertility; the latter is used to calculate the reproductive efficiency of
the herd.
 The rates for the first service are usually slightly higher than those for all services
because the latter group will include those cows that may be sterile and receive
many services before they are culled.
 In order to identify the influence of management changes, particularly nutrition,
it is worthwhile calculating these two parameters on a monthly basis provided
that there is a minimum of 10 services per month.

ESTRUS DETECTION RATE

 Improving the detection of estrus has a much greater influence upon reducing the
calving to conception interval than improving the pregnancy rates; the latter can
only be improved up to a certain level. This enables herdsmen to anticipate the
time of a subsequent estrus and thus improves the detection rate. It also enables
the early detection of acyclic cows.
 It is possible to estimate the estrus detection rate but it is important to stress that
it is an estimate and not an accurate measurement. A number of different
methods are used and they all have some measure of inaccuracy. One method is
to determine the number of supposed missed estrous periods. This an interval of
36-48 days suggests that one estrus has been missed and an interval of 54-72
days suggests that two have been missed, although this latter range is fairly wide
and can lead to errors. The percentage of estrus detection rate (EDR) is calculated
thus:
o Estrus detection rate (%) =(No. of interservice interval recorded /(No.
of interservice interval recorded + No. of missed estrous period) ) x 100
o Efficiency of estrus detection (%) = (No. of estrus detected / Total
No. of estruses ) x 100
o Accuracy of estrus detection (%) =(No. of estrus detected / (No. of
estrus detected + No. of false estrus detection ))x 100
  One simple method of assessing the estrus detection rate at routine sessions of
pregnancy diagnosis will be the number of cows that are assumed by the
herdsman to be pregnant, and thus submitted for examination, but are found to
be non-pregnant. Non-pregnant cows should have returned to estrus since
service or artificial insemination, and hence should have been seen in estrus.
 In many apparently well-managed dairy herds where the calving to first service
interval is on target, there is a failure to detect returns to estrus in non-pregnant
cows. This will result in a large number of inter estrus intervals that are two or
three times the normal interval. Poor estrus detection may be due to:
 Poor accommodation inhibiting cows from exhibiting overt signs of
estrus.
 Poor lighting or identification of animals.
 Failure to record signs of approaching estrus and signs of true
estrus.
 Inadequate regimen for observing cows for signs of estrus, perhaps
due to the herdsman being overworked

MODULE-23
SEXUAL HEALTH CONTROL AND HERD HEALTH
REPRODUCTIVE PROGRAMME

IDENTIFYING THE INFERTILE COW

The responsibility of identifying the infertile cow lies with the farmer, which requires
accurate records of reproductive events. Once identified veterinary help should be
sought. The following cows should be identified for examination:

 Cows not observed in estrus by 42 days after calving or cows not served or
inseminated by 63 days after calving
 Cows that regularly and repeatedly return to estrus after service or AI
 Cows with signs of nymphomania (persistent estrus or repeated, short, irregular
inter estrus intervals)
 Cows with repeated irregular, extended inter-estrus intervals
 Cows with abnormal vulvar discharge

HISTORY

 Before performing a detailed gynecological examination it is important to obtain

a detailed and accurate history, particularly the breeding history, of the cow. The
following should be obtained:
o Age
o Parity (there are certain conditions which can be excluded in nulliparous
as opposed to parous individuals).
o Date of last calving, together with information on the occurrence of
dystocia, retained placenta or puerperal infection.
o Dates of observed estrus since calving, if recorded.
 o Presence of any abnormal discharge.
o Numbers and dates of services or inseminations.
o Dates of observed estrus since service or artificial insemination.
o Previous fertility records, particularly calving conception intervals and
services per conception.
o Details of feeding management and milk yield; in suckler cows the number
of calves suckled.
o Details of health, i.e. signs of milk fever, mastitis or ketosis.
o Details of fertility of other cows or heifers in the group or herd.

DIAGNOSTIC TESTS

 Most diagnostic tests are of limited value, although single blood or milk
progesterone assays are useful to identify the presence of luteal tissue if
concentrations are high (4-6ng/ml in plasma or 12-18ng/ml in milk).
 Bacterial swabbing and culture and endometrial biopsy are of limited value. The
PSP (phenolsulphonphthalein) test for tubal patency can also be used to
demonstrate occluded uterine tubes.

SUMMARY OF SIGNS OF INFERTILITY

 The following summary describes a procedure for investigating an infertile animal on the
basis of the clinical history, signs and examination, with an indication of a possible
diagnosis of the cause and its treatment. Animals can be catagorised into those having
o No Observed Estrus
o Regularly Returning to Estrus
o Short Interestrus Intervals
o Anestrum

ABORTION

 This is defined as the production of one or more calves between 152 and 270 days
of gestation: they are either born dead or survive for less than 24 hours.
 The cow should be isolated, the fetus and fetal membranes should be retained
and the case treated as a suspected Brucella abortion.
 The physical appearance of the fetus and fetal membranes should be noted, the
fetus aged approximately and this confirmed by the service or insemination date
if available.
 Elimination of infection as a cause is made by being unable to demonstrate
organisms in the fetus, fetal membranes vaginal and uterine discharges and/or by
the demonstration of specific antibodies in body fluids.
 Where possible the whole fetus should be submitted to the laboratory for cultural
examination.Possible infectious causes of abortion are:

Infectious Causes Occurance

Brucella abortus 6-9 months

Leptospira spp 6-9 months

Listeria monocytogenes sporadic outbreaks at 6-9 months

Campylobacter fetus (veneralis) 5-7 months

Trichomonas fetus occurs before 5 months

Salmonella spp: especially S. Dublin usually sporadic with no specific time,

although usually about 7 months
Actinomyces (Corynebacterium) usually sporadic and occurs at any stage
pyogenes
Mycobacterium tuberculosis occurs at any stage

Mycotic agents, Aspergillus spp;, Absidia occurs from 4 months to term

spp. Mucoralis group
Bacillus lichenkformis sporadic late abortions

Neospora caninum late abortions

Infectious bovine rhinotracheitis- occurs at 4-7 months

infectious pustular vulvovaginitis (IBR-
IPV) virus
Bovine viral diarrhea (BVD) virus occurs at any stage

INVESTIGATION OF ABORTIONS

 The approach to investigating the cause of abortion will depend upon the
frequency. If sporadic, then a full laboratory investigation is probably
unnecessary because many abortions are not associated with infection.
 However, if it exceeds 3-5 per cent of the herd-and it is important to consider
stillbirths and premature calving (excluding twins) in this calculation-then a
thorough investigation should be implemented.
 MODULE-24
ROLE OF HORMONES IN FEMALE REPRODUCTION

Reproduction in mammals is largely under the control of the endocrine glands and the
hormones they secrete. Since the nervous system also plays an important role in
reproduction we say, reproduction is under neurohumoral control or neuroendocrine
control.

Reproduction is regulated by a remarkable interplay between the nervous system and

the endocrine system. Both work as a team to initiate, co-ordinate and regulate
reproductive functions.

DEFINITIONS

Before learning about tha endocrine glands and the hormones they secrete it is
important that we know the certain definitions.

 Endocrinology
o A science concerned with chemical integration of the body. Integration is a
key word related to the function of the nervous system.
 Endocrine glands
o Endocrine glands are those ductless glands of the body whose secretion
goes directly in to the blood stream.
 Exocrine glands
o Exocrine glands are those glands of the body whose secretion is carried
away by a duct.
 Hormone
o The word hormone originated from a Greek word meaning "I Stir up or
stimulate". A hormone is a chemical substance produced in one part of
the body (restricted area) that diffuses or is transported to another area
where it influences activity and tends to integrate component parts of the
organism.
o Hormones regulate (decrease or increase) the rates of specific processes
but do not contribute energy to the process or initiate metabolic reactions.
Instead, hormones influence an existing reaction which is usually one
involving enzymes.
 Local Hormone or Para Hormones
o In strict sense are not hormones, but are chemical messengers or
regulators. Eg. Prostaglandins, erythropoietin, and histami

HYPOTHALAMUS
The hypothalamus, the key brain center that controlls all our reproductive activities
lies at the base of the brain, bordered anteriorly by the optic chiasma, posteriorly by the
mammillary bodies, dorsally by the thalamus and ventrally by the sphenoid bone.

 Its size is 1/300 of the entire brain.

 Within the hypothalamus are numerous neurons. Clusters or groups of these
neurons are called hypothalamic nuclei, each of which have a specific name.
 The hypothalamic nuclei of importance are:
o The preoptic nucleii(PON), the anterior hypothalamic area (AHA),
suprachiasmatic nucleii(SCN) that make up the surge center
o The ventromedian nucleii(VMN), the arcuate nucleii(ARC) and the median
eminence(ME) that make up the tonic center, and
o paraventricular nucleus(PVN).

 The medial portion of the hypothalamus known as third ventricle of the

brain separates most of the paired nuclei.
 Neurons in the hypothalamus communicate with the anterior lobe of the pituitary
using a special circulatory modification known as the hypothalamo-
hypophyseal portal system.

Pituitary gland

 The pituitary gland lies below the hypothalamus in a bony depression in the
sphenoid bone called the Sella turcica.
 It consists of anterior and posterior lobes

 .

HYPOTHALAMO-HYPOPHYSEAL PORTAL SYSTEM

 Axons from neurons in the surge center and the tonic center extend to the stalk
region where their endings terminate upon blood vessels of the hypothalamo-
hypophyseal portal system.

 The portal system consists of
o The Superior hypophyseal artery(SHA)
o Primary portal plexus(PPP) (where the neurons of the surge center and
tonic center terminate),
o The medial hypophyseal artery (MHA)that supplies part of the anterior
lobe of the pituitary,
o The portal vessels(PV) that transport the blood containing releasing
hormones and,
o The secondary portal plexus(SPP) that delivers blood and releasing
hormones to the cells of the anterior lobe.
  The terminal portion of the hypothalamic neurons release neuropeptides that
enter specialized capillary system at the stalk of the pituitary. Blood enters the
capillary system from the superior hypophyseal artery that divides into small
arterial capillary forming plexus (primary portal plexus). The releasing hormones
are transferred to the secondary portal plexus in the anterior lobe of the pituitary
where the releasing hormones cause pituitary cells to release other hormones.
The hypothalomo-hypophyseal portal system is important as it allows for minute
quantities of releasing hormones to act directly on the cells of the anterior lobe of
the pituitary before GnRH gets diluted by the circulation.
 The posterior lobe of the pituitary does not have a portal system. Neurons from
certain hypothalamic nuclei (PVN) extend directly into the postetrior lobe of the
pituitary where the neurohormone is released into a simple arteriovenous
capillary plexus. For eg. Cell bodies in the Para Ventricular Nucleus (PVN)
synthesize oxytocin that is transported down the axon to the terminals in the
posterior lobe. If the neuron is stimulated, oxytocin is released into the blood.

ENDOCRINE REGULATION OF REPRODUCTION

 In contrast to the neural system, the endocrine system depends on hormones to

cause the responses.
 Hormone in extremely small quantities can bring about dramatic physiologic
responses.
 Hormones are classified as having relatively short half lives.
 Hormonal half life is defined as the time required for one half of the quantity
of the hormone to disappear from the blood or from the body.
 Short half lives are important because once the hormone is secreted and released
into the blood and cause a response, it is rapidly degraded so that further or
unnecessary responses do not occur. However, when hormones such as
progesterone are produced during pregnancy, the action brought about by the
hormone continues as long as the hormone is present.
 Compared to neural control, hormonal control is slower and has durations of
minutes, hours or even days.

MECHANISM CONTROLLING SECRETION

Positive and Negative Feedback are the major Controllers of Reproductive

Hormones.

 Negative feedback: inhibits GnRH neurons. For eg., High progesterone

inhibits GnRH neurons which secrete only basal levels of GnRH. Such basal
levels will allow for some follicular development but only to the extent where
insufficient estrogen production is there. Hence, when progesterone levels are
high, animal does not cycle. The tonic center in both and female is believed to
respond mostly to negative feedback.
  Positive feedback: activates the GnRH neurons in the hypothalamus. When
estradiol reaches threshold level, the surge center will be positively stimulated
and will release large quantities of LH that stimulate ovulation.

CLASSIFICATION OF HORMONES

 Hormones of reproduction are classified based on

o Chemical structure
o Glandular origin
o Mode of action

CLASSIFICATION BASED ON THEIR STRUCTURE

 Based on their structure, the hormones are classified into

o Proteins
o Steroids
o Fatty acids

Proteins or Polypeptide Hormones

 Proteins or polypeptide hormones have molecular weight of 300 to 70,000

Daltons and are easily broken down by enzymes. They must be administered by
injections.
 They contain an α and β subunit. The α subunit for FSH, LH and TSH are
identical but the β subunit is unique to each species. When two α or two β
subunits combine the resulting molecule will have no activity. If α subunit of one
hormone combines with the β subunit of another hormone, the activity of the
molecule will be determined by the β sub unit only.
 The amount of carbohydrate present on the surface of the protein determines the
duration of half life of the hormone.
 Glycoprotein hormones can be degraded easily by proteolytic enzymes in the
digestive tract. There fore, they are not effective when given orally.

Steroids

 Steroid hormones have the common cyclopentano perhydro phenantherene ring

nucleus.
 They have a molecular weight of 300 to 400 Daltons.
 Precursor is cholesterol
 Natural steroids are not effective by oral administration.
 Synthetic and plant steroids can be administered orally and by injection.
 Steroid hormones are sexual promoters and cause profound changes in both male
and female reproductive tract.

Fatty acids
 Have a molecular weight of approximately 400 Daltons, and
 Can be administered by injection.

BASED ON GLANDULAR ORIGIN

 Reproductive hormones originate from:

o Hypothalamus
o Pituitary
o Gonads
o Uterus, and
o Placenta
 Based on their origin, they are classified as
o Hypothalamic hormones eg. GnRH.
o Pituitary Hormones eg. FSH/LH
o Gonadal Hormones eg. Estrogen, Progesterone
o Uterine hormones eg. PGF2 α
o Placental hormones eg. hCG, eCG.

MODE OF ACTION

 Neurohormones : Synthesized by neurons and released directly into the blood

and cause response in a target tissue. eg. Oxytocin. A neurohormone can act on
any number of tissues provided that the tissue has cellular receptors for the
neurohormone.
 Releasing hormones: Synthesized by neurons in hypothalamus and cause
release of hormones from pituitary. eg. GnRH.
 Gonadotrophins: Hormones released by the gonadotroph cells of the anterior
pituitary and stimulate gonads. The suffix `tropin’ means having an affinity for.
eg. FSH and LH.
 Sexual promoters (Steroids): Produced by the gonads of both male and
female to stimulate the reproductive tract, to regulate function of hypothalamus
and anterior pituitary and to regulate reproductive behaviour.
 Pregnancy maintenance hormones: are responsible for maintenance of
pregnancy. (eg. Progesterone) and in some cases, assist the female in her
lactation ability.
 General metabolic hormones: promote metabolic well being. eg. Thyroxin,
adrenal corticoids and somatrophin.
 Luteolytic hormones: cause destruction of the corpus luteum. The suffix
`lytic’ is a derivative of word lysis. eg. PGF 2 α

HYPOTHALAMIC HORMONE - OXYTOCIN

 First hormonal peptide identified in the mammals.

 Synthesized in the supraoptic and paraventricular nuclei of the hypothalamus
and stored and released from posterior pituitary.
  Synthesized along with the carrier proteins called neurophysins.
 Transported in small secretory vesicles enclosed by a membrane. Secretory
vesicles flow down the hypothalamic–hypophyseal nerve axons by axoplasmic
streaming and are stored at nerve endings next to capillary beds in the
neurohypophysis.
 Also produced by the corpus luteum of cow, ewe and human.
 Therefore, oxytocin has two sites of origin:
o the ovary, and
o the hypothalamus.
 Functions of oxytocin
 Oxytocin in Greek means 'rapid birth'

 Causes contraction of oviduct and thus involved in transport of male and female
gametes in oviduct.
 Estrogen enhances responsiveness of smooth muscle to oxytocin.
 Causes milk let down.
 Ovarian oxytocin is involved in luteal function by acting on the endometrium of
the uterus to induce PGF2 alpha release which causes lysis of CL.

HYPOTHALAMIC HORMONE - GONADOTROPHIC RELEASING

HORMONE

 GnRH is a deca peptide containing 10 amino acids.

 Molecular weight is 1183 daltons.
 Causes release of FSH and LH from the anterior pituitary.
 The C- terminal portion of this molecule is necessary for attaching to receptor
while first 3 amino acids are necessary for activating LH and FSH release.
 The hormones of the anterior pituitary, adrenal cortex, thyroid and gonads
feedback to inhibit and in some instances to stimulate the secretion of the
hormone.
 Two types of Analogs to LHRH have been synthesized:
o GnRH antagonists - bind to receptor sites on the pituitary but do not
induce release of LH or FSH and block the action of the natural hormone.
o GnRH agonists - that induce release of more LH and FSH than natural
GnRH. Increased biological activity is due to their ability to stay bound to
pituitary receptors longer than natural hormone and their ability to resist
enzyme degradation.

Functions

 Controls the release of FSH and LH

 PITUITARY GONADOTROPHIC HORMONES

 The anterior pituitary gland secretes three glycoprotein hormones viz.

o Follicle Stimulating Hormone (FSH)
o Leutenizing Hormone (LH)
o Prolactin
 Consist of two non-identical subunits termed a and ß
 The alpha subunit is identical within species for FSH, LH and TSH.
 Molecular weight is 32,000 daltons with each subunit having molecular weight of 16,000
daltons.
 Alpha and beta subunits by themselves have no biological activity.
 Alpha subunit of one hormone (LH alpha) is recombined with beta subunit of another
hormone (FSH beta), the molecule regains FSH biologic activity or activity of the beta
subunit.
 If two alpha subunits or two beta subunits are combined, no biologic activity is noted.

FOLLICLE STIMULATING HORMONE

 Alpha subunit contains 92 amino acids with CHO side chains at aa 52 and 78 :
the beta subunit has between 108 to 118 amino acids with 2 CHO side chains
at aminoacid 7 and 24.
 Combination of alpha and beta subunit are necessary to provide tertiary structure
for recognition by the FSH receptor in the gonad.
 Six different species of FSH in a single animal has been identified.
 Half life of 2.0–2.5 h.

Functions

 Stimulates growth and maturation of the graafian follicle in the ovary.

 FSH along with LH causes estrogen production from the ovary and testes.

LUTEINISING HORMONE

 Also called Luteotrophin or Interstitial Cell Stimulating Hormone (ICSH).

 Glycoprotein composed of alpha and beta subunit with a molecular weight of
30,000 daltons.
 Half life of 30 minutes.

Functions

 Tonic or basal levels of LH act in conjunction with FSH to induce estrogen

secretion from the large graafian follicle.
 Preovulatory LH surge causes rupture of follicle and ovulation.
 LH is the major luteotrophic substance (maintains activity of corpus luteum).
 Stimulates interstitial cells (Leydig cells) in male to produce androgens.
 CONTROL OF LH AND FSH SECRETIONS

Tonic LH and FSH Release

 Serum LH and FSH are released in a tonic or basal fashion in both male and
female.
 Tonic levels are controlled by negative feed back of estrogen and inhibin from
gonads.
 The arcuate nucleus, ventromedian nucleus and the median eminence control the
tonic release of LH and FSH.

Preovulatory LH and FSH Release

 Preovulatory surge of LH and FSH occurs in female prior to ovulation.

 Initiated by increase in estrogen which exerts a positive feed back on the
hypothalamus inducing release of GnRH surge.
 Preoptic and anterior hypothalamic nuclei controls preovulatory surge of LH and
FSH.

PROLACTIN

 Polypeptide hormone containing 198 aa and a molecular weight of 24, 000

Daltons.
 Prolactin molecules are similar in structure to growth hormone and in some sp.
have similar biologic properties.

Functions

 Prolactin initiates and maintains lactation.

 It is considered as a gonadotropic hormone because of its leutrotropic properties
in bitches and rodents. However in domestic animals, LH is the major luteotropic
hormone.
 Prolactin may mediate the seasonal and lactational effects on reproduction in
farm animals.

PROSTAGLANDINS

 The prostaglandins are a group of compounds synthesized in the body from

arachadonic acid and found in many tissues of the body. The prostaglandins have
been arranged into 8 major series according to chemical makeup: A, B, C, D, E, F,
G and H. There may be different prostaglandins within a series.
 Many different physiological and pharmacological actions reportedly affect a
number of body systems:
 o Central nervous
o Cardiovascular
o Urinary
o Gastrointestinal
o Respiratory
o Reproductive
o Certain prostaglandins are involved in the inflammatory reaction and
allied adverse reactions of the body to injury.
 Currently the only prostaglandin available commercially is prostaglandin F2
Alpha (PGF2Alpha).
 Prostaglandin F2 alpha (PGF2α) release is brought about by the activation of
endometrial oxytocin receptors.
 The receptor concentration increased after 10 day exposure to progesterone and
is brought about by estrogen from antral follicles in late luteal phase.
 If the animal becomes pregnant, the early embryo through ectoderm secretes the
protein, bovine trophoblast interferon which prevents PGF 2α release, which is the
signal for maternal recognition of pregnancy.

MECHANISM OF ACTION

 Reduction in arterial blood supply by vasoconstriction leading to luteolysis.

 Binding with specific binding receptor sites developed on the plasma membrane
of luteal cells. Binding interferes or prevents PGF2 α binding.
 PGF2α binding brings about intracellular changes and affects steroidogenesis.

FUNCTIONS

 PGF2α is involved in ovulation in ewes and cows. Ovulation is blocked by

administration of indomethacin, an inhibitor of PG synthesis. LH release is not
affected. Hence action of PGs may be only at the level of ovarian follicle.

PGE

 Stimulates contractions of the uterus,

 Dilatation of blood vessels and has no luteolytic action.

PGF2α

 Stimulates contraction of the uterus,

 Aids in sperm transport in the male and female reproductive tracts,
 Causes constriction of blood vessels,
 Has luteolytic properties by venous constriction,
 Helps in parturition and a parallel rise in the level of PGF 2α along with estrogen
is observed. Primary effect of PGF2α is the myometrial contraction which favours
release of oxytocin.
 GONADAL HORMONES

 Primarily secreted by ovary and testes. The adrenals and placenta are other sources.
 They are of 4 types
o Androgens
o Estrogens
o Progesterone
o Relaxin
 The first three are steroid hormones while relaxin is a protein.
 Steroid hormones have a basic or common nucleus called the
cyclopentanoperhydrophenanantherene nucleus. It consists of a three, six member fully
hydrogenated phenantherene rings designated as A,B,C and D.
o An 18 carbon steroid has estrogen activity
o A 19 carbon steroid has androgen activity
o A 21 carbon steroid has progesterone activity
 The secretory activity of steroid hormones is under endocrine control of anterior
pituitary.


ESTROGEN
(Functions, Applications and Side effects)

 The estrogens are steroidal hormones synthesized from cholesterol and produced
primarily by the ovaries, placenta and corpus luteum.
 A significant source of estrogens is the testes of stallions and boars, while a minor source
is the adrenal gland.
 The estrogens are metabolized by the liver and excreted in the bile, feces, and the urine
(horses and ruminants).
 Progesterone decreases the effects of the estrogen and FSH and LH may be involved in
estrogen secretion.

Functions

Some important physiological actions of estrogens include:

 Maturation growth and development of the reproductive organs.

 Stimulation of normal physiological processes of the tubular reproductive tract.
o growth of the uterine muscle
o development of the endometrial lining of the uterus
o increase the vascularity of the uterus
 Induction of behavioral estrus
 The production of edema in folds of the mucosa at the utero-tubal junction
 Dilation of the cervix
 Protein anabolism in ruminants
 Under the influence of the estrogens the uterus is less susceptible to infection
 They play a role in the normal health and function of the skin
 They produce contractions of the uterus
 They enhance the effects of oxytocin on uterine motility
  The estrogens inhibit the secretion of FSH and LH via a negative feedback
mechanism
 They are required for the development of the secondary sex characteristics of the
female including hair growth, deposition of body fat, mammary gland
development, plumage, etc.
 The estrogens are involved in the regression of the corpus luteum.

TOP

Applications

 They are used to evacuate the uterus in cases of fetal mummification, fetal
maceration and pyometra.
 They are used to induce abortion in all species
 They can be used in the treatment of postpartum metritis and retained fetal
membranes
 Used in the management of misalliance in dogs
 To produce signs of estrus in anestrual animals
 Treating cases of estrogen responsive urinary incontinence
 In virgin heifers and dry cows estrogen can be used to stimulate mammary
development and lactation
 Large doses of estrogen after parturition can be used to inhibit lactation and
relieve congestion of the mammary glands.
 Used as growth promotants in beef cattle
 Used in the management of skin condition in spayed bitches
 In treating male dogs with prostatic hyperplasia
 Can be used to decrease libido in males

TOP

Side effects

 Prolonged use or large doses can produce cystic ovaries or ovarian atrophy
 Because of its effect on the ligaments, the estrogens predispose to prolapse of the
vagina and rectum and dislocations and fractures of the pelvic bones.
 In the dog excessive amounts or prolonged administration of estrogens can
produce a fatal anemia - leukopenia and thrombocytopenia.
 In some species the estrogens in combination with progesterone may increase the
incidence of cystic endometrial hyperplasia - pyometra complex.

PROGESTERONE

 These include the naturally occurring steroid progesterone which is synthesized

from cholesterol and produced by the corpus luteum (main source), placenta
(especially of the ewe and mare after the first 1/3 and 1/2 of gestation
respectively), the adrenal gland and the testes, as well as a number of synthetic
 progestogens which are much more potent and have a longer half-life than
progesterone.

Functions

 Progesterone causes growth of the glandular system of the endometrium of the

uterus, and the secretions from the endometrial glands (uterine milk) for the
nutrition of the ovum and the attachment of the embryo.

 Progesterone plays a role in the maintenance of pregnancy (and

pseudopregnancy) by providing a favorable environment for survival of the
embryo.
 Progesterone causes growth of the alveolar system of the mammary gland.
 Progesterone inhibits the smooth muscle activity of the uterus - renders it less
sensitive to oxytocin.
 Target tissues are relatively insensitive to progesterone unless primed by
estrogen - At low levels progesterone acts with estrogen to stimulate ovulation by
promoting LH release.
 At high levels progesterone inhibits the secretion of FSH and LH via a negative
feedback. However enough FSH is released so that follicles may develop during
the luteal phase of the cycle (diestrual ovulation seen in the mare, and also during
the gestation period in some species - the mare and to some extent the cow).
 Progesterone increases the efficiency of nutrient utilization.

Applications

 Prevent or control habitual abortion due to an actual or possible progesterone

deficiency.
 Estrum deferment or suppression in the ewe, cow, sow, mare and bitch.
 Synchronization of estrus in the mare, ewe, cow and sow.
 In heifers the progestins are used to promote growth - suppresses heat
 To treat post-partum hemorrhage in the bitch.
 In the treatment of cows with cystic ovaries progesterone withdrawal
 In cats progesterone has been used to treat miliary eczema and eosinophilic
granulomas.
 Progesterone has a calming as well as androgenic effect and has been used to
manage antisocial or aggressive behavior in mares, stallions, dogs and cats.

Problems

 If injections are given beyond the recommended length of time, prolongation of

the gestation period and fetal death may occur.

ANDROGENS
  Androgens are 19-carbon steroids with a hydroxyl or oxygen at positions 3 and 17
and a double bond at position 4.
 The androgens are called 17-Ketosteroids when qxygen is found at position 17.
 Testosterone is the principal circulating androgen in the male being produced by
the interstitial cells of the testis.
 Other minor sources include the adrenal cortex and ovaries in females.

Functions

 Testosterone plays a role in

o Maturation, growth and development of the repro- ductive organs and

secondary sex characteristics of the male.
o Erythropoesis
o Protein anabolism
o Maintainance of the secretory responses of the accessory sex organs-
provide the fluid component of semen.
o Suppressing the secretion of the pituitary gonado- trophins through
negative feedback.

Applications

 In general,the use of testosterone in large animal is limited to:

o Developing teaser animals
o Induce libido in geldings
 In general, there is no fertility condition in the male due to a hormonal cause that
uniformly respond to endocrine therapy. The androgens have been used in small
animals to manage several problems.

RELAXIN

 Relaxin is a polypeptide hormone containing alpha and beta subunits that are
connected by two disulphide bonds.
 It has amolecular weight of 5700 daltons. Inhibin and insulins are structurally
similar, but their biological actions are similar.
 Relaxin is primarily secreted by the corpus luteum during pregnancy. In some
species the placenta also secrete relaxin.
 In canines, relaxin is a pregnancy specific hormone.
 The main biological action of relaxin is
o Dilatation of cervix and vagina before parturition.
o It also inhibits uterine contractions
o Causes increased growth of the mammary gland if given in conjunction
with estradiol.
o In the Guinea pig, relaxin causes seperation of the pubic symphysis bone
within 6 hrs after injection. Seperation of pubic symphysis normally
 occurs during parturition in this species. MODULE-25
BREEDING MANAGEMENT IN BITCHES

o The most common problem encountered by veterinarians working in canine

reproduction is the “potentially” infertile bitch or stud. Owners bring these dogs
to their veterinarian with the major concern that the dogs are failing to produce
puppies. It is important to emphasize that a vast majority of these dogs are
healthy fertile animals whose apparent infertility problems are related to a
misunderstanding of proper breeding management.

METHODS OF BREEDING

 Methods of breeding include

o Out-crossing
o In-breeding
o Line breeding
 Out-crossing is the mating of two dogs within the same breed that are less
closely related than the breed average.
 In-breeding occurs when closely related dogs are breed such as parents and
offspring or brother and sister.
 Line breeding (which is a form of in-breeding) occurs with the repeated use of
1-2 dogs (usually males) for breeding to increase a certain trait.
 The problems with in-breeding are that it increases the manifestation of
undesirable traits. Because of this it is advisable to evaluate at least five
generations of pedigree on any bitch bought into the in-breeding program,
especially those bitches with relatives that have demonstrated potentially
inherited diseases, and if the bitch is in any way a likely carrier of a serious
genetic disorder breeding from her should be discouraged. Also where significant
in-breeding is present out-crossing should be seriously considered.

GOAL IN BREEDING PROGRAMME

 The simple goal in any breeding program is to have sufficient sperm numbers
present in the uterus and oviducts to achieve the optimal chance for fertilization
of mature eggs.
 Mature occytes are typically fertilized during the 3 to 8 days following the
luteinizing hormone surge, representing a period beginning 24 to 48 hours after
ovulation of primary immature oocytes.
 Using reliable, clinically practical methods for estimating the day of the LH surge
can be quite valuable. These criteria include
o Behavior observation
o Vaginal cytology
 o Vaginoscopy
o Hormone assays
 When used together they enhance the chances of a bitch being inseminated at the
proper times.
 Further, normal sperm are known to survive and retain the capacity for fertilizing
mature oocytes within the uterus of the bitch for at least 4 to 6 days, and in some
instance for as long as 11 days.
 Using this information, a breeding program can be offered to a client with
reasonable confidence of success.
o

ERRORS IN BREEDING MANAGEMENT

 People who own male dogs allow only one or two breedings per cycle. Since, there
is so much of variation in the duration of proestrus and estrus and in the time of
ovulation, it is almost impossible to fix two breeding dates without using any
breeding management tool.
 Most breeders and petowners breed their dogs on the 9, 11 or 13th day of the cycle
assuming that all dogs enter into estrus on the 9th day. Such predetermined dates
are fine if it works, but what if the perfectly normal, but not average, bitch is in
proestrus for 16 days or proestrus lasts 4 days and estrus lasts 4 days?
 Some breeders begin breedings when the bloody vaginal discharge of proestrus
becomes clear and /or strawcolored. Some normal bitches may have a bloody
vaginal discharge throughout proestrus, estrus and even into diestrus. Others
may discontinue bleeding days before the onset of estrus.
 Most breeders tend to depend on the male dog to choose the breeding dates. Male
response to a bitch is simply unreliable. Some males always want to breed. Other
males never want to breed because they may be submissive to a bitch, again
rendering the male worthless as a guide to breeding.
 People always assume that the male is always fertile. Any male may quickly
become transiently or premanently infertile. Any time the fertility of the male is
questioned a semen analysis is warranted.

PREBREEDING EXAMINATION

GENERAL EXAMINATION, TESTING AND GENETIC SCREENING

Prior to breeding the bitch ideally should undergo

 A full physical examination to ensure she is in a fit condition to carry a litter of

pups
 Verification of her vaccination status also whether she is free from parasites such
as worms, fleas and mites.
 Screening tests for genetic diseases is recommended for certain breeds. For
example breeds that are susceptible to hip dysplasia should be radiographed and
hip scored prior to a breeding occurring (dogs should be over 12 months of age
 before being radiographed for hip dysplasia). Bitches that are determined
positive to hip dysplasia should not be bred with due to the nature of the disease.
 Thyroid testing should be considered in breeds that are over presented for
hypothyroidism or in bitches showing signs of thyroid problems.

EXAMINATION AND TESTING OF THE REPRODUCTIVE

SYSTEM

 All bitches presented for breeding need to be examined for abnormal vulvar
conformation and vulvar discharge. Severe conformational problems can
contribute to an inability to breed, persistent inflammation of part of the
reproductive trait and an inability to whelp naturally.
 It is advisable to perform a digital vaginal examination in maiden bitches. We
look for strictures that may interfere with matings, foreign bodies, tumours or
any other abnormalities that may prevent a normal mating.
 Vaginascope should be performed in bitches that have a history of being non-
receptive to mating, that have an abnormal discharge from the vulva, or that have
signs suggesting inflammation of the caudal reproductive trait, such as rubbing
the vulva on the floor, excessively licking the vulva and abnormal vulva odours.

BREEDING BASED ON SEXUAL BEHAVIOUR

 Behavioral estrus is the factor in determining when breeding of the bitch should
begin. Observation of the bitch's response to a male is an inexpensive, straight
forward, and reliable means of determining when to begin and when to end the
breeding phase.
 On day 5 or 6 of proestrus, the bitch should be brought into contact with a male
dog for approximately 10 to 20 minutes. This should be repeated every second or
third day. Breeding should begin whenever the bitch is willing regardless of the
color of the vaginal discharge, the vaginal exfoliative cytology interpretation, or
the day of the cycle and should continue every other day until she is no longer
willing to breed.
 It is recommended to breed the bitch every 2 to 4 days, beginning with the first
day of acceptance and continuing throughout the acceptance period. Dogs that
are in standing heat for longer than 12 days should be bred no more often than
every third or fourth day. Bitches in standing heat for only 3 or 4 days should be
bred every 48 hours.
 It is of paramount importance to recommend to owners that the male continue
breeding the bitch until the bitch refuses to breed or until the first day of diestrus
is documented with vaginal cytology. Fertilization of eggs is most likely occurring
in the final 4 or 5 days of standing heat, regardless of the length of standing heat,
or 4 to 5 days before the onset of diestrus.

BREEDING BASED ON VAGINAL EXFOLIATIVE CYTOLOGY

  Vaginal cytology is a simple, inexpensive, and reliable means of evaluating the
bitch and provides a good reflection of rising plasma estrogen concentrations.
Vaginal smears should be monitored beginning with the second or third day of
proestrus. The day the smear shows more than 80 % superficial plus cornified
cells put together (cytological estus), breedings should begin.
 Since, Vaginal cytology does not directly predict the time of ovulation it is advised
that once breeding begins, it should be allowed to continue until the bitch refuses
to breed. Recommendations are to breed the dog every second, third or fourth
day of estrus.

BREEDING BASED ON VAGINAL ENDOSCOPY

 Vaginoscopy can be used to aid in timing natural breeding. Vaginal mucosa in

proestrus appears rounded and edematous.
 "Wrinkling" or "Crenulation" of mucosa is associated with LH surge. This is the
time to begin breeding. Breeding should be continued throughout the phase of
maximal mucosal crenulation, seen as angulated folds of vaginal mucosa with
sharp profiles.
 Breeding should be discontinued when the vaginal mucosa again becomes flacid
and smooth, with patchy red and white surface which is observed 6 to 10 days
following LH surge.

BREEDING BASED ON HORMONE ASSAYS

LH Assay

 Measurement of the peripheral plasma concentration of LH is a reliable and

accurate method for determining the optimum time to mate. In most countries
there is no readily available commercial assay for canine serum LH, and at
present measurement requires radioimmunoassay. This method is time-
consuming, expensive and there is frequently a delay in obtaining the results,
because samples are assayed in batches in service laboratories. Should LH
concentration be measured, critical matings or insemination can be planned
between four and six days after the LH surge.
 At least one ELIZA assay kit for measuring LH in canine serum has been
marketed for ovulation timing (Status-LH, Synbiotics). The test is stored at room
temperature and has a relatively short shelf-life. The kit may not be accurate if
used beyond the expiration date, or if the foil packet containing the test device
and pipette is opened well before the test is to be run. The preferred sample is
serum. The sample should not be lipemic or hemolyzed. The test should be run
the same day the serum is collected. If that is not possible, the serum should be
refrigerated, not frozen, until the test can be run.
 Because duration of the LH peak averages about one day in bitches, samples must
be drawn daily, at about the same time of day. Because of the tests short shelf-
life, necessity of daily testing, and the requirement of assessing progesterone to
verify accuracy, this assay is rarely used in practice.
Progesterone Assay

 There is a significant preovulatory luteinization in the bitch during and following

the LH surge as there is in many rodent and primate species. Plasma
progesterone concentration begins to increase rapidly from baseline
approximately 2 days before ovulation, during the LH surge.
 Serial monitoring of plasma progesterone concentrations therefore allows
anticipation of ovulation by about 1 to 2 days, and if continued allows
confirmation of ovulation and detection of the fertilization period.
 Since the initial rise in progesterone is progressive, it is only necessary to collect
blood samples every second or even third day, unlike the daily regime required to
detect the LH surge.Daily assays can determine the day of ovulation within 1 or 2
days in most cases, and should be used for very critical breedings and
inseminations.
 Progesterone may be measured most accurately by radioimmunoassay (RIA),
quantitative enzyme-linked immunosorbent assay (ELISA), or immuno-
chemilluminesce assay. Many veterinary diagnostic laboratories have a turn-
around time of 1 day for these accurate assays. RIA of LH in daily serum samples
can pinpoint the day of the LH surge in the majority of dogs, and be accurate
within 1 day in over 90% of cases.
 In-hospital test kits are available for measuring serum concentrations of
progesterone as <3, 3-10 ng/ml and greater than 10 ng/ml. Serum should be
assessed beginning 3 or 4 days after onset of proestrus and should be continued
every other day. At the time of ovulation the level of blood progesterone rises
dramatically & ovulation takes place when values of progesterone are between 4
and 10 ng/ml. Matings should be done at this time.
 It is important to know that at the time of ovulation, the ova are primary oocytes
and require 48 hours to mature. Hence a second mating should be done 48 hours
later to achieve maximum litter size.

RECOMMENDATIONS FOR A BREEDING PROGRAMME

A set of simple guidelines should be available for the conscientious breeder of an

apparently healthy bitch. These are guidelines that cannot harm the animal and usually
increase success of any breeding program.

 Record the first day of vulvar swelling, bloody vaginal discharge, and when males
become obviously interested in the bitch.
 Begin “teasing” the bitch with a male dog on day 5 or 6 of proestrus, and repeat
this procedure every 2 or 3 days to determine the first day of standing heat. In
cases of previous infertility, begin on the first day that proestrus is observed.
 Allow the bitch to be bred, beginning on her first dayof acceptance of the male,
and continue to breed every 2 to 4 days throughout the acceptance period.
 In cases of infertility, as well as bitches with short or prolonged standing heat,
teach an owner how to obtain vaginal smears. Smears should be obtained once
daily throughout apparent proestrus and estrus as well as several days into
 diestrus. The veterinarian can then stain and review slides as they are brought in,
or the entire series of slides can be reviewed after estrus has apparently ended.
The results of slide interpretation can then be correlated with breeding dates and
conception rate. When possible, a series of serum progesterone measurements
with or without vaginoscopy should be considered.
 Complete records should be kept on the dates of proestrus, breeding, and vaginal
smears. Notes should be made on the presence or absence of ties, the length of
each tie, and the behavior of both the male and female. The success of the male in
siring litters with other bitches should be recorded. Records should also be kept
on whelping dates, litter size, health of puppies, length of parturition, interval
between births, and any other valuable information. This includes the reason for
destroying any puppies.

ARTIFICIAL INSEMINATION IN DOGS

 The manual collection and subsequent deposition of semen into the vaginal vault
of a bitch in castrus is a common procedure used by breeders and veterinarians.
 As a result, artificial insemination is frequently requested by the dog owner or
handler. Fresh undiluted semen, semen mixed with an extender, or frozen semen
is used in dogs.
 To enhance changes of success, veterinarians must have a good understanding of
the estrus cycle, semen collection and AI techniques, and potential pitfalls that
may be encountered.

INDICATIONS FOR ARTIFICIAL INSEMINATION

There are several situations in which AI may be used. They are

 inability of the male and female to breed.

o For the bitch, these problems include
 vaginal strictures
 conformational defects
 rear leg weaknes
 psychological problems
 pain
o for the male
 weakness
 arthritis
 back pain
 premature ejaculation
 conformational defects that prevent intromission or a “tie” .
 AI may also be chosen because of a major size difference between the mates.
 Psychological problems may also result in the need for AI.
 Some owners wish to use AI avoid any possible venereal contact between their
dog and its mate, thereby controlling the spread of potentially infectious
diseases. Inseminating semen into the vagina still provides intimate contact
 between bitch and stud dog and therefore any infectious agent that could be
transmitted from the dog to the bitch during natural mating also has the potential
to be transmitted during AI. However AI does avoid transmission of infectious
agents from the bitch to the stud dog. All breeding dogs and bitches should be
Brucella-free as determined by appropriate tests.
 Some male dogs experience prostatic bleeding and hemospermia following
exposure to a bitch “in heat”. The bleeding may be associated with Von
Willebrand’s disease (VWD) but has also been observed in dogs not afficted with
VWD. Regradless, AI can be performed without any contact between stud dog
and bitch, occasionally avoiding this problem.

Fresh extended and frozen forms of semen are being used with increasing frequency.
Because semen collection is the difficult task, insemination of extended or previously
frozen semen remains a relatively simple procedure. The shipment and use of fresh,
extended or frozen semen helps defray the cost and removes the hazards associated with
shipping the female.

SEMEN COLLECTION

 Semen is usually collected in an artificial vagina or in collection cups. One end of

a sterilized, soft rubber, cone-shaped bovine artificial vagina is cut off and to this
is attached a 12 to 15 ml plastic tube. The wide-mouth end of the cone is folded
over and sealed with rubber cement to make a smooth, nonabrasive edge. A small
amount of nonspermicidal lubricating jelly is applied to the inner surface of the
rubber cone. This is the only surface that make penile contact.
 The most difficult task in AI is stimulating the male to ejaculate. Once this is
accomplished, the balance of the procedure is quite simple. With most stud dogs,
semen can be collected in a clean, quiet room with a nonslippery floor. A bitch is
not often needed. However, for experienced males accustomed to natural
breeding, a bitch in heat makes collecting semen easier. With the owner holding
the stud dog to minimize movement and to protect the collector, the penis and
bulbus glandis are gently massaged within the penile sheath.When the bulbus
glandis begins to enlarge, the sheath is slipped posteriorly and the penis with the
bulbus glandis is exteriorized. Failure to exteriorize both the penis and the bulbus
glandis from the sheath usually results in an incomplete erection and failure to
ejaculate or incomplete ejaculation, presumably due to pain.
 Once the penis and bulbus glandis are extruded from the sheath, the collector
firmly holds onto the the base of the penis, proximal to the bulbus glandis. The
thumb and index finger are used, providing both massaging movements and
downward pressure around the base of the bulbus glandis. During or
immediately after achieving an erection, aggressive pelvic thrusting movements
by the stud dog may make it difficult to place the artificial vagina over the penis.
Fortunately, pelvic thrusting is typically short-lived. The initial phase of the
ejaculate consists primarily of sperm-free prostatic fluid, and the sperm-rich
second fraction of the ejaculate usually begins as the pelvic thrusting begins to
subside. Immediately after pelvic thrusting stops, many males try to”step over”
 the collector’s arm as if dismounting the bitch. The collector should simply allow
this movement by the male, which results in a 180° rotation of the engorged penis
such that it is protruding caudally between the rear legs. Digital pressure should
be maintained on the bulbus glandis and the collection continued until the
ejaculate becomes clear.
 Semen is usually collected for a period of 2 to 5 minutes. This represents the
typical duration of the second phase of ejaculation. The clear plastic tube should
already have been connected to the rubber artificial vagina, and the apparatus
can be held under the collector’s arm during the initial stimulation period to
provide some warmth. Canine semen is relatively resistant to cold shock,
alleviating the need for warm water baths or incubators for holding semen. The
use of a clear plastic collection tube allows visualization of the semen. One hand
is kept over the plastic to avoid excessive light exposure. As long as the ejaculate
is obviously whitish or creamy and cloudy the ejaculate continues to be collected.
When the ejaculate becomes clear one can discontinue collection. If one is not
certain when the male has ceased ejaculating the sperm-rich fraction, stop the
collection after 5 minutes. Continued collection only dilutes the sperm with the
third-phase clear, sperm free prostatic fluid, resulting in cumbersome fluid
volumes.
 The collection of the third fraction of the ejaculate is not necessary for successful
insemination. The semen volume that can be handled easily is 3 to 10 ml. the
collection system containing sperm-rich semen can be exchanged for a clean
system if there is any need to evaluate the prostatic fluid.
 The bitch should be inseminated within 5 to 10 minutes of collecting semen. Prior
to insemination the color and consistency of the semen should be noted and a
small drop placed on a warm glass slide. The semen can be quickly evaluated
microscopically to easure that a normal number of live, progressively motile
sperm are present. During this time, the semen is kept warm by holding the tube
in one’s hand, which also minimizes exposure of semen to potentially harmful
ultraviolet rays. Alternatively, a drop of semen can be evaluated by a technician
while the remainder of the semen is being placed into the insemination device.
The presence of abnormal color or consistency, oligospermia, or dead sperm
should be identified.

FRESH SEMEN ARTIFICIAL INSEMINATION

Insemination Procedure

 Although there are a variety of “tools” used for insemination, commonly used
tools are
o 12 ml syringe
o flexible disposable male urinary catheter or rigid plastic insemination
pipette or the Cassous' AI Sheath
o surgical gloves.
 These items should be sterile.
 After the gloves are put on, the semen sample is drawn into the syringe, the
sterile catheter is attached, and the syringe is then filled with an additional 1 to 3
ml of air.
 A gloved, non lubricated index finger is placed into the vaginal vault, palm up. If
a lubricant is used it must be nonspermicidal.
 The catheter is then slid over the top of the finger and passed into the vaginal
vault,avoiding accidental catheterization of the urethra. Sliding the catheter over
the index finger also aids in avoiding the clitoral fossa.
 The catheter follows the dorsal curvature of the vaginal vault.
 The catheter is inserted until resistance is met. The resistance indicates that the
cranial end of the vagina vault has been reached or the catheter has simply
become trapped within vaginal folds.
 The catheter should be gently advanced as far cranially as possible before the
semen is deposited to ensure deposition of spermatozoa near the cervix and into
the uterus.
 Once the syringe containing the semen has been emptied it should be
disconnected from the catheter, filled with a few more milliliters of air,
reattached to the catheter, and emptied, thereby depositing any semen that may
have remined in the catheter.
 Care should be taken to avoid injecting too much air into the vagina, as this may
result in loss of semen out of the vulva once the procedure is completed.
 Once the semen has been deposited, the catheter should be removed and the
hindquarters of the bitch should remain elevated above her head for a minimum
of 20 minutes, thereby aiding the movement of semen anteriorly in the
reproductive tract and into the uterus. Insertion of a gloved finger and the gentle
stroking of the dorsal wall of the vagina during this time may stimulate muscular
contractions within the reproductive tract, further enhancing the movement of
spermatozoa toward the ovaries.
 After elevation of the hindquarters has been completed, the bitch should be kept
quiet for an hour or so to minimize loss of semen out of the vagina. In
addition,pressure should not be applied to the abdomen.
 The entire insemination procedure is rarely a problem for the bitch. There should
not be any pain or discomfort associated with the procedure. For these reasons,
the procedure is rather simple and not time consuming.

EXTENDED CHILLED SEMEN AI

 Semen that has been properly extended and chilled can be refrigerated for several
days yet still yield fertile sperm when warmed and inseminated.
 The extender helps keep the spermatozoal membranes from being harmed by
changes in temperature or shaking during transport, while also providing
nutrients and stabilizing the pH of the medium.
 Semen once extended, is gradually cooled to 5 to 15°C over a period of 30 to 60
minutes.
 Rapid changes in temperature must be avoided.
  When prepared properly, chilled extended spermatozoa easily remain viable for
24 hours and, depending on the technique, may remain viable for as long as 5
days.
 Sort – term preservation of sperm allows overnight air delivery of freshly
extended semen without the costs of frozen semen or shipment of the bitch.
 The ejaculate is extended, packaged in a small container, and shipped in a
thermos-type container. The ejaculater, should remain cold during shipment and
should be kept refrigerated until use.
 The technique for insemination of the bitch with extended semen is as previously
described for insemination with fresh semen.
 Ideally, multiple inseminations beginning 3 to 4 days after the initial rise in blood
progesterone concentration should be completed to maximize conception rate.

FROZEN SEMEN AI

Long –term preservation of semen utilizing deep freezing techniques has been available
for several years.

 The advantages of using frozen semen include

o wider dispersion of desirable genetic traits,
o disease prevention,
o decreased numbers of breeding males in research colony,
o preservation of semen from dogs with diseases that are models of human
disorders,
o elimination of the need for shipping bitches.
 The steps involved in freezing semen for AI include
o Semen collection
o Dilution in an extender
o Equilibration under refrigeration
o Freezing in convenient volumes
o Storage
o Thawing and insemination of the bitch during the peak of her fertile
period.
 Extenders such as egg yolk, glycerol, lactose, skim milk and antibiotics are
typically added to the semen prior to freezing. Extenders have
o nutrients as an energy source,
o buffer against harmful changes in pH,
o provide a physiologic osmotic pressure and concentration of electrolytes,
o prevent growth of bacteria,
o protect cells from cold shock during the cooling process,
o have cryoprotectants that reduce sperm cell damage during freezing and
subsequent thawing.
 Canine sperm is often frozen in 0.5 ml, plastic “French straws.” Straws provide a
convenient format for handling, labeling, storage and thawing.
 Alternative formats include bulk semen volumes sealed in ampoules and semen
frozen in small spherical pellets on a block of dry ice.
 Frozen semen has been stored for as long as 9 years with little to no post-thaw
decrease in sperm motility. More than 4 years have been reported between semen
collection/storage and thawing/conception. Thawing instructions usually
accompany the semen and should be followed exactly.
 At the time of insemination, small drop of thawed semen should be evaluated
microscopically for sperm viability and motility.

CONCEPTION RATES FOLLOWING AI

 The success of AI depends upon several variables and include

o the use of a fertile stud dog with normal semen
o proper handling of the semen during collection and insemination
o and the deposition of semen into the anterior vaginal vault of the bitch at
the appropriate time of estrus.
 The importance of the latter cannot be overemphasized. Determining when to
perform AI should always be based on a combination of
o behavior of the bitch
o sequential vaginal cytology
o plasma progesterone concentrations
o vaginal endoscopy. Evaluation of serial vaginal cytology and plasma
progesterone concentrations, in conjunction with changes in behavior of
the bitch, are easy, relatively inexpensive, and accurate methods to
determine when the bitch should be inseminated.
 Multiple inseminations, performed daily or every other day until diestrus as
confirmed with vaginal cytology, should be done once the decision is made to
perform AI. Since, there is no way to predict exactlywhen ovulation has occurred,
multiple inseminations increase the change of conception by ensuring the
presence of spermatozoa in the female tract both early and late in estrus.
 Artificial insemination may be associated with lower conception rates and
smaller litter sizes than would be achieved with natural breeding. This is likely a
result, of several factors such as
o During natural breeding, semen is pressure-forced through the cervix into
the uterus and oviducts, whereas in AI, the semen is placed posterior to
the cervix.
o The erect penis virtually fills the vaginal vault, compressing the urethral
opening, and the bulbis glandis prevents backward flow of semen. The
large volume of ejaculate produced during the normally prolonged third
phase is forced through the cervix because there is no other place for
distribution.
o With natural breeding, uterine contractions may aid in semen transport.
This is an unlikely event in most AI situations.
o Also in natural breeding the duration of the tie may contribute to
improving conception rates, because the male ejaculates during that entire
period producing a volume of semen too large for the vaginal vault.

CONCEPTION RATES FOLLOWING FRESH SEMEN AI

  Conception rates of 75 to 80% have been reported when AI is performed
properly. This technique places the semen in the cranial vagina.
 Although deposition of semen in the uterus may improve the conception rate,
catheterizing the cervix of a bitch in estrus is not so easy primarily because of the
anatomic conformation of the canine cervix.

CONCEPTION RATES FOLLOWING EXTENDED CHILLED

SEMEN AI

 Conception rates for AI using fresh extended semen are variable but have been
reported to be 60% or higher.
 In some studies conception rates for AI using fresh extended semen are higher
than with frozen semen.
 With fresh extended semen, no damage to the sperm occurs due to freezing, the
cervix is less of a barrier, larger numbers of sperm are usually inseminated, fresh
sperm live longer in the reproductive tract of the bitch, and timing of
inseminations is not as critical as with frozen semen.

MODULE-26
MEDICAL TERMINATION OF PREGNANCY

Unwanted mating or mismating is a common clinical problem in veterinary practice

which may arise because dog owners are not aware that their pet is “in heat” or the
owner may be aware of estrus but underestimated the will of a stud or bitch that wants
to encounter the opposite sex. Therefore, even the best educated and most careful
owners will end up with mismating problems in their bitches.

Ovariohysterectomy would be the best method to prevent birth of puppies following

mismating, but for bitches whose future breeding potential needs to be maintained,
medical termination of pregnancy becomes imperative.

METHODS OF ABORTION INDUCTION

Abortion induction methods involve

 Modification of estrogen progesterone ratio by administration of

estrogen/synthetic estrogen derivatives / glucocorticoids.
 Induction of functional luteal arrest or luteolysis by substances that act directly
(prostaglandins) or indirectly on the CL by inhibiting luteotrophic support. For
eg. dopamine agonists suppress prolactin secretion whereas GnRH antagonists
deplete LH.
 Blockage of progesterone secretion by inhibiting steroidogenesis (epostane).
 Blockage of progesterone action at the receptor levels (anti progesterone
(mifipristone / algepristone).
 ESTROGEN

 Large doses of estrogens have been used for many years to prevent implantation
following mismating. However, currently estrogens are to be avoided because of
their toxic effects.
 Estrogens exert their action by causing closure of the uterotubal junction, alter
the transport time of zygotes and exert direct embryotoxic effect.

Recommended protocols

 Estradiol benzoate @ 0.5 - 3.0 mg every other day for a total of three injections
beginning 4 - 10 days after misalliance.
 Estradiol valerate @ 3.0 - 7.0 mg administered once 4 to 10 days after mismating.

Side effects and Toxicity of Estrogens

 Estrogens at high doses cause

o bone marrow suppression leading to thrombocytopenia, leukopenia,
severe anaemia and death
o development of pyometra related to abnormal increase in estrogen:
progesterone ratio, cystic hyperplasia of the uterine glands, abnormal
epithelial secretion, relaxation of the cervix and ascending infection
o induction and prolongation of estrus behaviour. As a result bitches may
continue to attract males for 7 to 10 days
o less severe side effects include alopecia, skin hyperpigmentation,
mammary gland and vulval enlargement.

PROSTAGLANDINS

 Prostaglandins when administered to the bitch cause lysis of corpora lutea and
decrease in plasma concentrations of progesterone. Since, corpora lutea of the
bitch are highly resistant to prostaglandins than those of other species, repeated
therapy is necessary to achieve complete regression of the corpus luteum.
Prostaglandins also produce contraction of smooth muscle having an ecbolic
effect that may a part of the mechanism of inducing abortion.

Candidates for Prostaglandin Administration

 The bitches must be healthy and less than 7 years of age.

 Pregnancy must be confirmed using ultrasonography

Treatment Protocols
  The half life of PG is only seconds and it remains in circulation for a few minutes
following an intramuscular injection or perhaps a little longer when administered
by subcutaneous injection. Hence repeated administration several times a
day is a must for absolute efficacy, whether using low, moderate, or
high doses. Treatment must be continued until the efficacy is verified by
ultrasound or palpation. Partial abortion of litters can occur if
treatment is discontinued prematurely. Whatever may be the dose, a
minimum of 5 to 7 or sometimes even 9 or more days may be required to
terminate pregnancies.

Drug Dose Day of Duration

pregnancy
(SC injections)
Single Natural 20 – 30µg/kg BID or After day 30 4-7 days longer
dose PGF2α TID
treatment 100 – 250µg/kg BID After day 30 4-7 days longer
Cloprostinol 2.5µg/kg every 48 hrs. After day 30 4-7 days longer
Multi Natural Day 1 Day 2- Day 4 Between day 7 days or longer
dose PGF2α 3 onward 25 and 35 of
treatment 25 50 50 to 100 pregnancy
µg/kg µg/kg µg/kg or
higher

Note

Ultrasound examination is the only reliable means to evaluate the viability of fetus in
early and mid gestation. Radiography to confirm efficacy based on absence of fetal
skeletons would not be reliable until after day 45 of gestation.

Side Effects

 Since prostaglandins act on all smooth muscles of the body, side effects such as
panting, respiratory distress, excess salivation, vomiting, defecation, stranguria
and/or urination are observed within 3 seconds to 3 minutes after injection and
persisted for 4 to 55 minutes. However, bitches adapt to the PGF 2α , with side
effects diminishing after each subsequent injections. After 6 to 8 injections, side
effects were minimal to absent in all dogs, regardless of the dose protocol.
 Side effects can be reduced by concurrent administration of anticholinergic drugs
like atropine @ 0.5 mg/kg SC or IM, 15 minutes before prostaglandin
administration. Walking the animal for 30 minutes following PGF 2α injections
would help to reduced intensity of side effects. One must bear in mind that
physical side effects of PG though frightening are only transient but not life
threatening.
 DOPAMINE AGONISTS

 Prolactin is the major luteotropic hormone throughout the luteal phase in both
pregnant and non pregnant bitches which means the progesterone production
from corpora lutea is prolactin dependent.
 Dopamine agonists like bromocriptine or cabergoline are ergot alkaloids with
strong dopamine D2 receptor agonist activity and suppress prolactin secretion
and thereby progesterone levels.

BROMOCRIPTINE
 Bromocriptine, at dose of 0.1 mg / kg PO BID for a minimum of 6 days
terminated pregnancy after day 30. Treatment should not be discontinued until
all fetuses are expelled (as confirmed by U/S).
 Bromocriptine has potent dopamine receptor activity and produces side effects
including severe vomiting, anorexia and depression. Peripherally acting emesis
inhibitors other than metaclorpromide (Metaclorpromide and Bromocriptine are
antagonistic) could be given prior to administration to prevent emesis.


CABERGOLINE

 Compared to Bromocriptine, Cabergoline is a more potent dopamine agonist with

milder side effects as it is a more specific D-2 dopamine receptor agonist and is
less able to cross the blood brain barrier and have CNS effects. A dose of 5 µg/kg
PO once daily for 5 days after day 32 of LH surge caused abortion in dogs.
 A combination therapy of oral cabergoline at 5 µg/kg per day and low dose of
cloprostenol (PGF2α analogues) injection of 1 µg/kg SC every other day for upto 9
days terminated pregnancy in dogs when administered starting around day 28 of
pregnancy. The objective of this combination is to reduce CL function and
progesterone release by a double mechanism of action.
o Direct local effect of PG on CL steroidogenesis
o Indirect effect due to withdrawal of pituitary prolactin support.
 Resorptions with some vaginal discharge usually occurred 5 to 8 days after the
beginning of treatment which could be visualized and confirmed sonographically.

ANTIPROGESTIN TREATMENT

 Antiprogestins are synthetic steroids which bind with great affinity to

progesterone receptors thereby preventing progesterone induced changes in DNA
transcription. As a result, the effects of progesterone are not there.
 Mifepristone (RU486) is one such antiprogesterone which induced direct
luteolysis. In pregnant bitches, Mifepristone was found to be effective if
administered after day 32 of gestation @ 2.5 mg/kg orally twice daily for 4.5 days.
Pregnancy was terminated without any side effects within 3 to 6 days after
treatment.
 A combination of low doses of prostaglandin E such as misoprostol and
mifepristone is currently being used to improve the action of mifepristone.
 Another analog of RU-486, Aglepristone (RU-534) can be used to prevent
pregnancy when administered as two injections SC of 10 mg/kg 24 hrs apart. This
drug is found to have an efficacy of 97.3% and does not cause any side effects.

CORTICOSTEROIDS
  Dexamethasone when administered beginning at mid gestation can terminate
pregnancy in dogs by activating endogenous mechanisms similar to those
involved in parturition.
 Injectable Dexamethasone (5 mg twice daily IM for 10 days) and oral
Dexamethasone (0.1 to 0.2 mg twice daily at a decreasing dose for 5 to 10 days).
 Some bitches had brownish vaginal discharge at the time of abortion while no
signs were observed in other bitches.
 Side effects like polydipsia and polyurea were observed shortly after start of
treatment and persisted for a few days after end of treatment.
 Restlessness and milk secretion were also observed in some bitches during period
of abortion or resorption.
 Failure of dexamethasone to terminate pregnancy has resulted in birth of live
normal pups at term; delivery of dead pups near term and partial abortions with
expulsion or resorption of few fetuses and then birth of 1 or 2 dead pups at term.

MISCELLANEOUS PREGNANCY TERMINATION AGENTS

Epostane

 Epostane is a steroid molecule that inhibits steroid synthesis at the level of the
hydroxy steroid – dehydrogenase – isomerase enzyme system, thereby inhibiting
the formation of progesterone from pregnanolone. Epostane terminates
pregnancy when given orally at 2.5 to 5 mg/kg for 7 days starting any time after
diestrus begins. No adverse side effects have been observed upto 10 mg/kg; but at
higher doses sterile abscess may form at the site of injection.

GnRH Antagonists

 GnRH antagonists are effective after day 20 of diestrus and act by decreasing
concentration of circulating gonadotropins causing luteolysis, decline in serum
progesterone concentrations, and subsequent pregnancy loss. But, currently this
preparation is not available in clinical practice.

MODULE-27
CLINICAL PSEUDOPREGNANCY – INTRODUCTION

 In dogs, the clinical condition of overt pseudopregnancy called simply

pseudopregnancy, pseudocyesis, false pregnancy or nervous lactation is a
syndrome observed in non-pregnant bitches about 6 to 12 weeks after estrus and
is characterized by clinical signs such as nesting, weight gain, mammary
enlargement and lactation.
 Since, there is no pregnancy recognition system in dogs; the corpus luteum
continues to remain functional in both pregnant and non pregnant bitches.
 Hence, all non pregnant dogs in diestrus stage of the cycle are referred to as
pseudopregnant (physiological pseudopregnancy).
  However, when extreme behavior or atypical mammary activity are presented as
clinical problems involving changes similar to those seen in late pregnancy or the
early post-partum period, the condition can best be termed "clinical
pseudopregnancy" or "overt pseudopregnancy".

PATHO-PHYSIOLOGY

 The occurrence of pseudopregnancy appears to be related to and dependent on a

previous prolonged, and in most cases a very recent, exposure to elevated levels
of progesterone. Further, it has been suggested, based on indirect evidence, that
pseudopregnancy may occur as a result of increased concentrations of prolactin
or an increased sensitivity to prolactin induced by a more rapid than normal
decline of progesterone levels in the late luteal phase. In fact, spaying or
ovariectomy during the luteal phase (i.e., during metestrus/diestrus) induces
pseudopregnancy in some bitches.

Probable and Proposed Causes of Clinical Pseudopregnancy in Female Dogs

 Idiopathic occurrence of a more extensive increase in prolactin than occurs in

normal diestrus.
 Idiopathic increase in sensitivity to the endocrine changes that normally occur in
late diestrus, including the normal progressive decline in progesterone and
modest elevation in prolactin.
 Pseudo-luteal phase induced by administration of exogenous progestins
 Progesterone withdrawal caused by:
a. ovariectomy during diestrus,
b. termination of long-term or short-term progestin therapy
c. idiopathic or prostaglandin-induced abrupt luteolysis
d. antiprogestin therapy
 Idiopathic hyperprolactinemia potentially associated with pituitary
microadenomas.
 Physchogenic or reflexive hyper-prolactinemia occurring in response to
stimulation by surrogate neonates or other visual, physical or social stimulation.

CLINICAL SIGNS

 Common signs include

o Prepartum and maternal-like behaviors.
o Nesting, digging, over-affection, over-protectiveness, over-defensiveness,
aggression, licking, mothering of inanimate objects.
o Mammary enlargement and distension. Mammary hypertrophy is usually
more evident in the most caudal pair of glands although the entire
mammary chain can be involved.
o Lactation and milk release. Milk production during pseudopregnancy
apparently results from the development of not only intra-acinar but also
 intra-canalicular mammary secretion in predisposed bitches. Lactation is
often encouraged by self-nursing or by adoption of unrelated neonates.
o Weight gain
o Anorexia
o Sometimes abdominal contractions that mimic those of parturition
 Less common clinical signs include
o Emesis
o Abdominal enlargement
o Abdominal contractions
o Diarrhoea
o Polyuria
o Polydipsia
o Polyphagia
 Pseudopregnancy is a self limiting condition and signs normally cease after 2 to 4
weeks.
 A high recurrence rate in successive estrous cycles has been reported in
susceptible bitches.
 Pseudopregnancy and prolactin have been implicated in the pathogenesis of
mammary tumors. An increased risk of mammary neoplasia associated with
pseudopregnancy might be explained by a continuous mechanical distension of,
and the accumulation of carcinogenic products within, the mammary acini
caused by the formation and retention of milk.

DIAGNOSIS

 Diagnosis is based on reported clinical signs. Ultrasound or radiography should

be used to differentially diagnose from pregnancy as unscheduled matings may
be overlooked by owners.
 Other conditions of the luteal phase, such as pyometra or recent pregnancy and
abortion, should be ruled out by abdominal ultrasonography or radiography, a
complete blood cell count and additional ancillary testing, including vulval and
vaginal examination.
 It is also important to keep in mind that pseudopregnancy can co-exist with other
reproductive or non-reproductive clinical problems, sometimes making diagnosis
more difficult.

TREATMENT

 Since, pseudopregnancy is typically a self-limiting state, mild cases usually need no

treatment other than discouraging maternal behavior.
 Treatment for pseudopregnancy may either be
o Conservative Treatment
o Aggressive Treatment

CONSERVATIVE THERAPY
  Placing of Elizabethan collars to prevent licking of the mammary glands is
recommended.
 Avoid licking, milking, or use of compresses as they stimulate lactation.
 Withdrawal of water for 5 to 7 consequent nights promotes fluid conservation
and also helps to terminate lactation (normal renal function should be
documented beforehand).
 When behavioral signs are significant, light tranquilization with non-
phenothiazine drugs can be useful.
 Phenothiazines are not recommended in pseudopregnant bitches as they
stimulate prolactin secretion.
 Considering the possible relationship between pseudopregnancy and subsequent
development of mammary tumors the recent approach is to treat
psuedopregnancy with pharmacological agents.

AGGRESSIVE THERAPY

 Pharmacological approaches to the treatment of moderate to severe

pseudopregnancy have historically included steroids, including estrogens,
progestins and androgens. More recently the preferred method of treatment is
the use of prolactin-suppressing drugs, especially dopamine agonists, whereever
available.

Sex Steroid Therapy

 Steroids such as estrogens, progestins and androgens have been traditionally

used to treat pseudopregnancy but the side effects usually outweigh any benefits
of these medications. High doses appear to exert a negative effect, either by
suppressing pituitary prolactin or decreasing sensitivity to prolactin.
 Estrogens
o Estrogens such as diethylstilbestrol, estradiol benzoate or estradiol
cipionate have been used. They may cause signs of proestrus or estrus,
uterine disease such as pyometra, and anemia due to bone marrow
depression. The use of estrogen is not recommended.
 Androgens
o Androgens including testosterone and synthetic androgens can suppress
lactation. Clitoral hypertrophy, virilization, and epiphora are side effects
noticed. The synthetic androgen mibolerone has been shown to reduce the
duration of pseudopregnancy.
 Progestins
o Progestins such as megestrol acetate and medroxyprogesterone acetate,
administered orally, have been used to suppress the symptoms of overt
pseudopregnancy probably by suppressing prolactin secretion or reducing
the tissue sensitivity to prolactin. However, withdrawal of progestin results
in recurrence of symptoms including lactation. Progestin administration
can cause cystic endometrial hyperplasia-pyometra complex and insulin
 resistance, as well as mammary gland nodules, mammary tumors, and
acromegaly. Administration of progestins is therefore not recommended.

Prolactin-Suppression Therapy

 Dopamine Agonists
o Secretion of prolactin by the pituitary is mainly under tonic inhibitory
control of the hypothalamus, mediated by a direct action of dopamine, the
major prolactin inhibiting factor (PIF).
o The dopamine agonists bromocriptine and cabergoline are the most
common ergot compounds used clinically to inhibit prolactin secretion.
 Bromocriptine
o Oral administration of Bromocriptine at varying doses ranging from 10 to
100 µg/kg/day for 10 to 14 days has been recommended by various
authors. Since, it has a short half-life of + 4 to 6 h and should be
administered at least twice a day for greatest efficacy.
o Unlike cabergoline, bromocriptine also crosses the blood-brain barrier and
can stimulate other brain centers in addition to the hypothalamus. Emetic
effects result from stimulation of the hypothalamic vomiting center.
o The ED50 for emesis is near the commonly used therapeutic doses and
digestive side effects are frequent and proportional to the dose. Common
side effects include vomiting, anorexia, depression, and other behavioral
changes. Side effects tend to decrease during the course of treatment.
o To prevent or reduce the incidence of emesis, bromocriptine can be
administered in low and then increasing doses, or administered with the
food. In addition, vomiting can be managed by administration of
anticholinergic drugs such as atropine.
o Care should be taken, when trying to prevent emesis, not to use central
dopamine blockers of synaptic transmission whose action would oppose
that of bromocriptine.
o Bromocriptine is formulated in 2.5 mg tablets for use in humans, and
fractionation of the tablets is necessary to achieve dosages of 10 to 30
µg/kg typically administered to pseudopregnant bitches. This makes it
difficult to administer exact doses, and may have caused an overestimation
of the drug's side effects.
o Preparation of exact dosages is important. Administration of the 10 to 30
µg/kg dose twice or even 3 times a day is preferable to once a day
administration.
 Cabergoline
o Cabergoline has greater bioactivity, superior D2-receptor specificity, and a
longer duration of action compared to bromocriptine.
o It can be effectively administered once a day. Cabergoline crosses the
blood brain barrier only slightly and consequently has much less central
emetic effects than some other dopamine agonists.
o The ED50 for emesis is 4 times the therapeutic dose and gastrointestinal
signs are rare. Cabergoline is used for treating pseudopregnant bitches at a
dose of 5 µg/kg/day for 5 to 10 days, given orally.
Anti-Serotonergic

 Metergoline
o Metergoline, an anti-serotonergic veterinary drug marketed for the
treatment of pseudopregnancy in bitches in Europe and in some Latin
American countries (Contralac®) is an ergot alkaloid which has a
dopaminergic effect and thus reduces prolactin secretion when
administered at high doses.
o It has a short half-life and hence has to be administered twice a day.
o The recommended dose is 0.1 mg/kg, orally, twice a day, for 8 to 10 days.
o Anxiety, aggressiveness, hyper excitation and whining are the most
frequent side effects of metergoline, which are due to its central anti-
serotonergic effect.

OVARIECTOMY

 Predisposed bitches not intended for breeding should be spayed. Ovariectomy is

the only permanent preventive measure. This should preferably be done during
anestrus.
 Ovariectomy during lactation can lead to an extended pseudopregnancy.
 In bitches with a history of overt pseudopregnancy, spaying during diestrus may
provoke an episode of pseudopregnancy 3 to 7 days after surgery .

MODULE-28
TRANSMISSIBLE VENEREAL TUMOUR IN DOGS
Transmissible venereal tumour (TVT) also known as infections sarcoma, venereal granuloma,
transmissible lymphosarcoma or stickler tumour is a benign reticuloendothelial tumour of the
dog that mainly affects the external genitalia and occasionally the internal genitalia. Since, it is
usually transmitted during coitus, it mainly occurs in young sexually mature animals. The
transmissible venereal tumour cells contain an abnormal number of chromosomes ranging from
57- 64 in contrast to the normal 78 of the species.

ETIOLOGY

 TVT arises from allogenic cellular transplants and the abnormal cells of the
neoplasm are the vectors of transmission. Exfoliation and transplantation of
neoplastic cells due to physical contact during mating or licking of affected area is
responsible for spread onto genital, oral or nasal mucosa.
 The implantation of the tumour is facilitated by the presence of any mucosal
lesion or by the loss of mucosal integrity.
 Growth of tumour occurs 15 to 60 days after implantation.
 Metastasis may occur in less than 5-17% of cases.

GROSS AND MICROSCOPIC CHARACTERISTICS

  Small pink to red, 1 mm to 3 mm diameter nodules can be observed 2 or 3 weeks
after transplantation.
 Initial lesions are superficial dermoepidermal or pedunculated. Multiple nodules
then fuse together forming larger, red, hemorrhagic, cauliflower-like, friable
masses of 5 to 7 cm in diameter which then progress deeper into the mucosa as
multilobular subcutaneous lesions with diameters that can exceed 10 - 15 cm.
 Tumours bleed easily and while becoming larger, normally ulcerate and become
contaminated
 Exfoliative cytology shows discrete cells that are round to oval, with moderately
abundant pale blue cytoplasm, an eccentrically located nucleus, with occasional
binucleation and mitotic figures. Single or multiple nucleoli are often observed.
The most characteristic feature of TVT cells is the presence of numerous discrete
clear cytoplasmic vacuoles.

CLINICAL SIGNS

 Clinical signs vary according to localization of the tumours.

 Discharge may be confused with urethritis or cystitis.
 In bitches the tumours are localized in the vestibule and/or caudal vagina,
protrude from the vulva and frequently cause a deformation of the perineal
region. Rarely, the tumour masses interfere with micturition.
 A considerable hemorrhagic vulvar discharge may occur and can cause anaemia if
it persists.
 The discharge can attract males and the condition of the bitch can be mistaken
for estrus by the owners. Infrequently, TVTs can localize in the uterus.
 In cases with extra genital localization of the TVT, clinical diagnosis is usually
more difficult because TVTs cause a variety of signs depending on the anatomical
localization of the tumour, eg., sneezing, epistaxis, epiphora, halitosis and tooth
loss, exophthalmos, skin bumps, facial or oral deformation along with regional
lymph node .

DIAGNOSIS AND TREATMENT

Diagnosis

 Diagnosis is based on
o Physical examination.
o Cytological findings of TVT in exfoliated cells obtained by swabs, fine
needle aspirations or imprints of the tumours.

Treatment

 Several treatments including surgery, radiotherapy, immunotherapy, biotherapy

and chemotherapy have been applied for TVT.
 Surgery has been used extensively for the treatment of small, localized TVTs,
although the recurrence rate can be as high as 50 - 68% in cases of large invasive
 tumours. Contamination of the surgical site with TVT cells is also a source of
recurrence.
 Transmissible venereal tumours are radiosensitive and orthovoltage as well as
cobalt have been used for this purpose.
 Biotherapy studies have also been reported. The intra tumoural application of
Calmette-Guérin's bacillus (BCG) has been used for three weeks with sporadic
success.
 Recurrences have been described after immunotherapy using Staphylococcus
protein A, BCG or a vaccine made from tumoural cells. Biotherapy has
unfortunately also resulted in a high rate of recurrence.
 Chemotherapy has been shown to be the most effective and practical therapy,
with vincristine sulfate being the most frequently used drug. Vincristine sulfate is
administered weekly at a dose of 0.5 to 0.7 mg/m 2 of the body surface area or
0.025 mg/kg IV. Complete remission takes 2-8 injections and occurs in more
than 90% of the treated cases. Vincristine can cause myelosuppression and
gastrointestinal effects resulting in luekopenia and vomiting. A complete WBC
count is recommended prior to each administration. When the WBC count is
below 4,000 m3, further administration should be delayed 3 to 4 days and its
dose reduced to 25% of the initial dose. Local tissue lesions can be caused by
extravasations of the drug during IV.
 Cyclophosphamide @ 5 mg/kg PO for 10 days as a single drug therapy or in
combination with prednisolone @ 3 mg/kg, for 5 days; vinblastine @ 0.1 mg/kg
IV for 4-6 weeks; methotrexate @ 0.1 mg/kg PO every other day can be given
alone or in combination for treatment of TVT. There is no advantage of
combination therapy over vincristine administration alone.
 In cases that fail to resolve with chemotherapy, electro-cauterization, cryo-
cauterization or radiotherapy has found to yield good results.

MODULE-29
INDUCTION OF ESTRUS
Anestrus denotes a state of complete sexual inactivity with no manifestations of estrus. It is not
a disease but a sign of variety of conditions. Although anestrus is observed during certain
physiological states- eg. before puberty, during pregnancy, lactaion and in seasonal breeders- it
is more often a sign of temporary or permanent depression of ovarian activity (true anestrus)
caused by seasonal changes in the physical environment, nutritional deficiencies, lactation stress
and aging.

HORMONES USED FOR INDUCTION OF ESTRUS AND

OVULATION

TYPE OF METHOD OF BIOLOGICAL ACTIVITY

HORMONE ADMINISTRATION
GONADOTROPINS
ECG or PMSG Single injection Mimics FSH and stimulates follicular
growth, long half-life
FSH Single/multiple injections Stimulates follicular growth; short half-
life
hCG Single injection Mimics LH and induces ovulation
GONATROPIN RELEASING HORMONE AGONIST
GnRH-Buselerin Single injection Induces release of LH and FSH from the
anterior pituitary; recruitment and
selection of new dominant follicle
PROGESTOGENS
Progesterone Multiple injections Inhibits ovulation by suppressing LH
secretion; mimics action of CL
Synthetic Oral, subcutaneous implant, Inhibits ovulation by suppressing LH
Progestogens intravaginal pessary/device secretion, mimics action of CL
ESTROGENS
Estradiol Injection, implant Induces premature regression of CL and
Conjugates enhances response to progestogens
PROSTAGLANDINS
PGF2 alpha or Single intramuscular injection Induces regression of CL during
synthetic responsive phases
analogues

INDUCTION OF ESTRUS IN COWS

Extensive studies have been carried out regarding the use of different hormones in
treatment of anestrus. The major hormones used are as follows,

Follicle Stimulating Hormone (FSH)

 As the name indicates the physiological role of this hormone is to induce

follicular growth.
 Once follicular growth is induced, the estrogen secreted by the developing follicle
induces an endogenous Luteinizing hormone (LH) surge for ovulation.
 But, in several cases of anestrus, administration of FSH alone would not benefit,
as ovulation is not ensured.
 Administration of LH or hCG (human chorionic gonadotrophin) may be
necessary to favor ovulation.
 Generally, LH is administered after 48 – 72 h of FSH administration. Follicular
growth can be induced in anestrus animals by exogenous administration of FSH
or equine chorionic gonadotrophin (eCG) also called as pregnant mare serum
gonadotrophin (PMSG).
  The latter is having both FSH & LH activity and preferred for treatment of
anestrus due to its long half-life.

Dosage

 FSH – 1000 IU intra muscular

 eCG (Folligon, Trophovet) – 500 – 1000 IU intra muscular
 hCG (Chorulon) – 1500 IU intra muscular

Gonadotrophin Releasing Hormone (GnRH)

 This hormone can be used to treat anestrus animals either as single or double
injection; the latter method gives better results.
 In single injection method, it is advised to palpate the ovary for presence of
follicle as this hormone acts better when follicle is present in the ovary.
 In double injection method, administration of GnRH at 10 days apart induces
estrus and improves the ovulation and conception rates.

Dosage

 Gonadorelin (Fertagyl) – 250 – 500 µg intra muscular

 Buserelin acetate (Receptal) – 8 – 20 µg intra muscular

Progesterone

 Administration of progesterone mimics the presence of corpus luteum and

induces follicular growth and ovulation when withdrawn.
 There are several methods of administration of this hormone viz. oral,
intravaginal, injection and ear implants.
 The hormone should be administered for a minimum of 10 days and estrus is
induced with in 3 – 5 days of removal of progesterone.
 Oral feeding of progestational compounds continuously for 14 – 18 days induces
estrus within 3 – 5 days of last day of feeding, however, fertility at this estrus is
not optimum. When administered through injection also it has to be
administered for 10 – 14 days

Oral progesterone

 MGA - 0.5 – 1mg/day/cow

 DHPA – 120 – 150 mg/day/cow
 MPA – 180 mg/day/cow
 CAP – 5 – 10 mg/day/cow

Injectable progesterone Combination of Hormones

 17 alpha hydroxy progesterone caproate(Duraprogen, Proluton)

 50 mg/day/heifer
  100 mg/day/cow

 Several combinations of above said hormones (eg. progesterone releasing

intravaginal device- PRID; Norgestomet ear implants) are also used to augment
fertility in anestrus cattle and buffaloes. Recently, administration of GnRH and
PGF2 alpha has been reported to induce estrus within 3 – 5 days of treatment.

GnRH and PGF2 alpha schedule

 Day 1: GnRH (8 m g intra muscular)

 Day 7: GnRH (8 m g intra muscular)
 Day 17: PGF2 a (25 mg – Dinoprost)

Non-Hormonal Approach

 Supplementation of minerals and some herbal drugs fall in this category.

Different workers have reported different success rates with these therapies.
 Simple utero-ovarian massage is also useful in some anestrus cases. Treatment
with different mineral mixtures, boluses, herbal compounds (Aloes compounds,
Prajana, Janova etc) and were also reported to be effective in treatment of
anestrus cases.
 Treatment with Lugol’s iodine was also proved worthy in treating some anestrus
cattle and buffaloes.
 Swabbing of uterin externum with 5% Lugol’s solution causes local irritation and
hyperemia of uterus and ovaries.
 The ovary stimulates the hypophysis reflexly, resulting in release of
gonadotrophic hormones.
 Besides this, iodine stimulates the thyroid glands directly or indirectly and iodine
deficiency, if any, is corrected. It was also reported that swabbing of Lugol’s
iodine releases uterine PGF2 alpha acting via the utero-ovarian and utero-
pituitary-ovarian pathway.

INDUCTION OF ESTRUS IN MARES

The mare is seasonally polyestrous and has several cycles during a particular season
cycling during periods of long daylight thus ensuring that the mare will have foals at the
time of the year most conducive to foal survival, the spring.

Light Year

 The mare is light responsive in that increasing daylight causes cycles to start by
decreasing melatonin while decreasing light turns the mare off. The important
days to remember in the light year are:
o Summer solstice - ~June 21, which is the longest day of the year and the
peak of natural breeding season;
 o Fall equinox - ~September 21, when there is equal light and dark and the
mares are turning off in fall transition;
o Winter solstice - ~December 21, which is the shortest day of the year and
mares are in deepest anestrus;
o Spring equinox - ~March 21 when there is equal light and dark and the
mares are in spring transition.
 Temperature may also influence the onset of cyclicity as cyclicity may be in part
regulated by a neurotransmitter which is also involved in prolactin secretion.
Therefore, temperature is important in the control of prolactin (low temp, low
prolactin) and may therefore also affect neurotransmitters.
 Reduction of the opioid inhibition of the gonadal axis may also play a role in
triggering the onset of the breeding season.
 Normal cycles occur around the Summer solstice, which is the natural breeding
season.

Anestrus

 Anestrus in caused by the secretion of melatonin, which is secreted in response to

increasing darkness.
 The melatonin inhibits GnRH, so the FSH and LH are low.
 Anestrus occurs around Winter solstice wherin 80% of mares undergo anestrus.
 Mares in anestrus are passive to the stallion advances.
 On rectal palpation the
o ovaries are small, smooth, and inactive
o the cervix and uterus are flaccid.
 Vaginoscopic exam reveals a cervix that is pale and dry, and the cervix may even
be open.
 The hormones are all at very low concentrations.
 If the is nutrition poor, the mare may not cycle back in the spring.

Spring Transition

 Melatonin apparently inhibits the production of GnRH in anestrus.

 As the melatonin decreases, GnRH resumes secretion, and FSH and LH also
increase.
 FSH during anestrus is low and irregular, whereas during the cycling season it
becomes bimodal (through pregnancy).
 LH during anestrus is also at basal levels, but increases to a normal pattern in
cycling animals. LH production however, lags behind FSH production.
 With increased FSH, follicles start to grow.
 Most of these follicles are not steroidogenically competent so they do not produce
estrogen.
 They also do not ovulate. In fact, an average of 3.7 waves of follicular
development occur before the first ovulation.
 After several waves, an estrogen producing follicle finally develops and ovulates.
 During transition mares show irregular periods of sexual receptivity, prolonged
heats of 10-20 days, split heats, and heats without ovulation.
  The uterus may be histologically 1 cycle behind the ovaries. In other words, the
uterus may still look anestrus when the mare starts cycling.
 The ovaries have follicles that grow and regress until one is selected to ovulate.
 This occurs when a follicle becomes steroidogenically competent and starts
secreting estrogen.
 The large follicles may persist on the ovaries because of insufficient LH. The
follicles are not cystic !!!
 There is no treatment for this anovulatory receptivity. LH (or hCG) will not make
a transitional mare ovulate.

Treatment

 Nothing prevents transition.

 Regumate- (altrenogest is a progestogen that has no cross reaction with
progesterone. If given at a dose of 1ml/110 lbs for 14 days orally, it shortens and
eases the transitional signs, but does not eliminate transition !!!! The net result is
increased fertility earlier in season. Regumate may induce LH receptors on the
follicles?????
 Progesterone in oil -100 mg/day for 7 days has similar effects as Regumate.

Fall Transition

 Fall transition mirrors spring transition and is characterised by

o prolonged heats,
o irregular cycles,
o large 'hung' or 'autumn' anovulatory follicles that become atretic and the
mare goes into anestrus.
o This is caused from the low LH release because melatonin is taking its grip
again as the day length decreases.
 There is no treatment for fall transition

Induction of Cycling

 You must start no later than Dec. 1 (about 2 months before you want cycles to
begin)
o 10 foot candles or 1 lux is sufficient. This is equivalent to a 100 watt bulb in
a 12X12 box stall or two 40 watt fluorescent bulbs / box stall.
o You need a minimum 14.5 hr. light to start cycles.
o It is imperative that light be added at the end of the day, not the beginning.
 GnRH supplementation
o 100 mcg /hour for eight days at a constant infusion caused most mares to
start cycling.
o 40 mcg every 12 hours 28 days vs. implants (Buserelin implants released
100mcg/day 28 days) showed that 0/15 of the controls ovulated by 28
days, whereas 7/15 injections and 9 /15 implants did. Although the
implants group retrospectively had greater LH before the study began.
 Dopamine D2-antagonists
 o Dopamine effect on gonadotropin release is unclear .
o Antagonists may act directly on ovary, rather than by hypothalamic
pituitary axis
o GnRH is inhibited by dopaminergic neurons acting directly on GnRH
neurons
o if given during anestrus the effect is not as great as if given during
transition or if mares have been exposed to extended photoperiod.
 Domperidone -0.1 mg.kg PO SID
o Transitional mares ovulated 12-22 days after treatment started
o Deep anestrous mares took longer to ovulate (50-60 days) .
 FSH-12.5–mg of purified eFSH intramuscularly twice daily for a maximum of 15
consecutive days

INDUCTION OF ESTRUS IN DOES

 Two non-hormonal methods for altering the breeding of does include the sudden
introduction of the buck and the use of an artificially altered photoperiod.
 In general the sudden introduction of a male to a group of mature females during
the period of transition from anestrus to estrus can be expressed to advance the
breeding season by a matter of weeks, whereas the use of photoperiod alteration
can allow for the breeding of does during the traditional “deep anestrous” time of
year.
 It is also possible to combine the two techniques in order to achieve both “out of
season” breeding and synchronization of estrus.

Buck Effect

 The introduction of a normal or teaser male induces estrus and ovulation in

mature transition period does within 5 to 7 days. The induced estrus and
ovulation are reasonably synchronized.
 The precise mechanism for this induction is not completely understood, but
seems to be mediated through an induced surge of luteinizing hormone (LH). In
any case, a group of does can be bred earlier (e.g., August in the northern
temperate regions) if they are suddenly introduced to an intact male. If it is
desirable to ascertain that the does are responding prior to breeding, as with in
artificial insemination program, then the introduced male can be a surgically
prepared teaser.
 The use of a marking harness, or simply painting the brisket of the teaser, should
allow for the detection of responding does. Since the cycle induced by the buck is
occasionally of abnormally short length and can be associated with insufficient
luteal function, this practice will preclude the possibility of breeding induced
does at an infertile estrus. In cases studied to date an induced “short cycle” is
usually followed by a cycle of normal length.
 Therefore, once it has been determined that the does are cycling in response to a
male, the teaser can be removed and replaced by a breeding buck or by artificial
insemination.
Photoperiod Alteration

 Many studies have shown that the adult ewe is susceptible to induction of estrus
by manipulation of length of daylight. Few controlled studies are available for
mature goats, but it is known that yearling does can be induced into estrus at
least 60 to 80 days early by providing feeding areas of a barn with 19 hours of
artificial light per day, beginning in mid to late winter.
 An inexpensive timer is set to turn on the lights ( a pair of 8-foot, 40-watt
fluorescent tube for each 36 to 40 square meters of pen space) at approximately
0500 and to turn them off 19 hours later.
 Animals should be fed in the evening, to encourage them to expose themselves to
the augmented light. A protocol for lighting and animal management.
 Apparently, the relative decrease in length of day when the artificial light is
terminated, is the stimulus that induces hypothalamic events leading to estrus
and fertile ovulation.
 The physiological mechanisms that translate "decreased" length of daylight into
estrus and ovulation are under study.
 When photoperiod-primed yearling does are suddenly exposed to bucks, there is
a surge of LH within about 2 weeks. The first surge apparently does not induce
ovulation, but may be responsible for the very slight rise in progesterone that
follows and that precedes estrus by 2 to 4 days. This estrus may be tightly
synchronized.
 It would appear , then, that the relative decrease in length of day may condition
hypothalamic responses necessary to allow the “buck effect” in yearling does.
 The success of this method has not been examined in a controlled fashion for
lactating does, but some producers have reported success rates similar to those
achieved with yearlings.

INDUCTION OF ESTRUS IN GILTS AND SOWS

Prepubertal Gilt

 The gilt is first able to ovulate in response to exogenous gonadotropins at

approximately 100 days of age.
 The most common regimens of 500 to 1000 IU PMSG followed in 48 to 96 hours
by approximately 500 to 750 IU hCG and the injection of 400 IU PMSG in
combination with 200 IU hCG.
 The split-dose and combination treatment appear to induce comparable rates of
ovulation and estrus. When PMSG is given without hCG, ovulation rates and the
per cent of gilts showing estrus are reduced relative to treatments utilizing hCG in
conjunction with PMSG.
 Exogenous GnRH has been used in lieu of hCG at 48 to 60 hours after PMSG
administration to induce synchronized ovulation; however, a more reliable
response appears to occur when hCG is given after PMSG. The time of ovulation
following treatment with the PMSG/hCG combination is approximately 110 to
120 hours.
  When either hCG or GnRH is given following PMSG, ovulation occurs at
approximately 40 to 44 hours following the second injection.
 Although conception rates of 40 to 90 per cent are common following
gonadotropin-induced ovulation, less than 60 per cent of gonadotropin-induced
gilts are typically able to maintain successful pregnancies. It is thought that in
some gilts induced corpora lutea (CL) are not able to retain their viability 20 to
30 days after ovulation.
 If females are mated at the spontaneous estrus that occurs in some gilts at
approximately 21 days following induced ovulation, farrowing rates are markedly
improved. The presence of boars after induced ovulation enhances the ability of
gilts to maintain cyclic, ovarian and estrus activities.

Delayed Puberty

 Gilts reared in confinement commonly show an onset of puberty that is

substantially delayed relative to pen mates. As discussed in other, articles, these
anestrus females may have acyclic, anovulatory or cyclic, ovulatory ovaries.
 Most gilts that are anovulatory and anestrous are able to respond to exogenous
gonadotropins with both estrus and ovulation.
 Prepubertal females that are anestrous and have cyclic ovaries (behaviourally
anestrous) are not able to respond to increased circulating levels of estradiol and
thus cannot respond to gonadotropin therapy with estrus.
 They are, however, able to ovulate and to form accessory CL in addition to their
primary CL.

Lactating Sow

 During lactation, the ovaries of sows show only modest follicular development.
There is gradual but progressive development of follicles in response to
exogenous gonadotorpins as early as 15 days postpartum, but pregnancy rate is
improved when treatment is initiated at 25 days or more following farrowing.
 The procedure found to be most effective for inducing ovulation in the lactating
sow involves the administration of 1500 IU of PMSG followed after 96 hours by
1000 IU of hCG. Since estrus is not consistently observed females should be
artificially inseminated at 24 hours and again at 36 to 42 hours following hCG
administration.
 The separation of the sow from her litter for one to three 12-hour periods prior to
treatment appears to improve the response to gonadotropin.

Postweaning Sow

 Following weaning the majority of sows show signs of estrus within 3 to 7 days.
 Exogenous gonadotropins have been used to decrease the weaning-to-estrus
interval and to improve the synchrony of the post weaning estrus. Gonadotropins
have also been used prophylactically to decrease following weaning.
 PMSG can be used alone or in combination with hCG to promote earlier onset of
the post weaning estrus.
  For maximum effectiveness, PMSG should be administered on the day following
weaning.

Anestrous Sow

 It is not uncommon in some herds for sows to fail to return to estrus for 30 or
more days following weaning.
 As with delayed puberty, anestrous sows may have acyclic or cyclic ovarian
activity.
 Females that have anovulatory ovaries are able to respond to PMSG with a fertile
estrus. The combination of estrogen (1 mg of either estradiol benzoate or
estradiol cypionate) and hCG (1000 IU) has been used to return anestrous sows
to productivity.
 However, additional studies are warranted to establish the efficacy of this
treatment. Because of its luteotrophic effects in the pig, exogenous estrogen may
induce prolonged diestrus in anestrous sows with cyclical ovaries.
 Estrogens should be used with caution in the pig.

INDUCTION OF ESTRUS IN BITCHES

 A number of methods to induce estrus in dogs have been reported. Most are
probably not appropriate for application in healthy, normally cycling bitches,
despite interest in shortening and synchronizing cycles for purposes of
accommodating owners schedules, the availability of stud dogs, or the shipments
of chilled or frozen semen, or for purposes of increasing the number, frequency
or size of litters in such animals.
 All of the methods reported, when assessed in repeated or large studies have a
significant failure rate and involve one or more of the following drawbacks:
smaller than normal litters in a significant percentage of successful attempts;
disruption and possible prolongation of the normal cycle; and, theoretically a
possibly increased risk of reproductive tract disease due to premature and
possibly excessive stimulation of the reproductive tract by the administered
hormones or changes in endogenous hormones provoked by the treatment.
 Nevertheless, interest remains high in the development of methods that may be
safe enough, have fewer drawbacks, and/or have a sufficiently high success rate
as to merit clinical application in the course of breeding management.
 Furthermore, some of the current methods would appear to have significant
merit for application in cases of prolonged anestrus and for enhancing fertility of
research bitches in colonies of dogs maintained as animal-models of heritable or
genetically-based diseases of interest in human or veterinary medicine.

Monitoring the Treated Bitch

 Regardless of the method chosen for estrus induction, vaginal cytology should be
obtained on an alternate-day basis.
  It is recommended that natural breeding or artificial insemination begin when
superficial cells compose 60% or more of the exfoliated vaginal epithelial cells
and/or when plasma progesterone concentrations exceed 1 ng/ml.
 Insemination should continue on an alternate-day basis until diestrus is
confirmed..

Drug Availability and Reliability

 Problems arise regarding availability, quality, consistency, and dependability of

the various hormone preparations utilized in the studies completed. This
accounts, to some degree, for the different results seen with similar protocols
used by separate research groups.
 Many of the agents used are not commercially available and others vary
significantly in potency depending on where they are purchased and how they are
prepared.

Use of PMSG or FSH

 PMSG
o A variety of protocols using pregnant mare serum gonadotropin have been
evaluated in one study, approximately 50% of bitches ovulated following 9
consecutive days of IM or subcutaneous PMSG, injected at 44 IU/kg /day
followed by 500 IU of hCG IM on day 10. The responding animals
exhibited behavioral estrus 10 to 15 days after initiating treatment, but this
included only half of the dogs that ovulated.
o In another study, pregnant mare serum was administered to mature
anestrus bitches for 10 consecutive days at subcutaneous does of 500 IU
/day. 250 IU/day, or 20 IU/kg/day. This was followed by a subcutaneous
injection of 500IU of human chorionic gonadotropin on day 10. Abnormal
ovulations with shortened luteal phases, and toxic side effects attributed to
the excess estrogen. Those side effects included thrombocytopenia, uterine
disease, and termination of pregnancy
o If the PMSG was administered for only 5 days before hCG administration
the serum estrogen concentrations were more physiologic and the protocol
resulted in a 50% conception rate. This latter protocol was evaluated in
another study, resulting in excess concentrations of serum estrogen
Differences in results may be due, in part, to difference in the potency of
the PMSG preparations used.
 FSH with or without Estrogen Priming
o Protocols using FSH (Schering Corporation, Kenilworth), as the sole
stimulus for induction of estrus have not been as successful as those using
PMSG. Pre treating bitches with an “estrogen priming” regimen of
diethylstilbestrol (DES) using 5 mg daily for 7 or more days to produce
signs of proestrus, holds promise. Five days after induction of proestrus 5
mg of LH IM was administered; 10mg of FSH IM was administered 9 and
11 days after observing vaginal bleeding. Each bitch so treated became
pregnant.
 o A successful modification of the estrogen-FSH protocol included
administration of DES daily for 4 to 10 days. Duration of DES
administration continued 3 days beyond the first day of induced proestrus
Counting from the first day that signs of induced proestrus were observed,
10 mg of FSH-P was given IM on days 5,9, and 11. Estrus behavior was
observed in 70% of the bitches 5 to 10 days after the initial dose of DES.
Subsequently, 46% ovulated and 30% became pregnant, carrying litters to
term chard et al.

Gondadotropin-Releasing Hormone or a GnRH Agonist

 Use of GnRH or an agonist of that hormone induces a fertile ovarian cycle only if
the pituitary-ovarian axis is normal. These drugs stimulate the secretion of
pituitary gonadotropins, which should, in turn, stimulate the ovaries. Follicle
development, estrogen secretion, behavioral estrus and pituitary-stimulated
ovulation depend on a normal cascade of physiologic events following
“activation” of the system.
 One protocol used a surgically implanted infusion pump that administered a
small dose of GnRH every 90 minutes for 6 to 12 days. In the bitches treated,
proestrus began in 3 to days and fertile estrus in 7 to 14 days. The protocols were
successful at inducing fertile cycles in 37 to 85% of bitches treated. However, the
cumbersome and impractical nature of expensive implanted infusion pumps that
need to be removed make this protocol interesting but unavailable to most
practitioners:
 Another protocol used a constant infusion of a GnRH agonist for 14 days. This
approach resulted in rapid induction of proestrus and estrus, with fertility rates
of 25% when administered immediately following lactation and 50% when given
to anestrus bitches after a non pregnant cycle. Although the results were
promising, the agonist used is not commercially available Also, the small,
inexpensive osmotic pumps require minor surgery for their subcutaneous
placement and removal.
 A less stringent protocol used subcutaneous injections of a GnRH agonist at a
dose of 1 pg/kg TID for 11 days and then 0.5 pg/mg TID for 3 days. Estrus was
observed within 9 to 11 days of initiating treatment in 80% of the dogs, each of
which became pregnant. Despite the inconvenience of a TID injection protocol,
this method may present the best combination of efficacy and clinical utility
among the various approaches involving GnRH.

Dopamine Agonist Administration

 Several studies have reported the use of a dopamine agonist (DA) administered
orally at doses sufficient to lower plasma prolactin as a means to terminate
anestrus either prematurely in normal bitches or therapeutically in cases of
prolonged or persistent anestrus.
 The efficacy has been anecdotally estimated to be about 70%, and possibly higher
in bitches with prolonged anestrus; the resulting proestrus, when induced, has
occurred after a variable duration of treatment ranging from 8 to 40 days; the
 average appears to be about 20 days; duration appears to be dependent on the
stage of anestrus, with longer treatment required in early anestrus.
 Whether the simultaneous reduction in prolactin is part of the mechanism of
action or if the mechanism involves other or additional dopaminergic effects is
not known.
 However, efficacy appears to depend on a dopamine responsiveness sufficient to
also cause suppression of prolactin; bitches that fail to experience suppression of
prolactin also fail to show a clinical proestrus response.
 Two DA treatments reported to be effective have included bromocriptine at
0.05.or 0.1 mg/kg, p.o., q.d. or bid, and cabergoline at 5 ug/kg, p.o, q.d.;
administration is until an induced proestrus is pronounced for 2 days or until the
onset of estrus.

ROTOCOLS FOR INDUCING ESTRUS AND OVULATION IN

ACYCLIC FARM ANIMALS

SPECIES TREATMENT END OF
TREATMENT OF
ESTRUS
CATTLE AND BUFFALO
Prepubertal or Estrogen on day 1 followed by 7-12 days Within 5 days
Postpartum Suckled Cows of progestogen, eCG given on last day
(optional)

Postpartum Milked Cows GnRH on day 14 postpartum 1 day

2-4 days
GnRH (day 0) and PGF (day 6)

SHEEP AND GOATS

Prepubetral or Seasonal Progestogen for 12-21 days with eCG 2-4 days; eCG required
Anestrus given near the end of progestogen for good response
treatment
SWINE
Prepubertal or eCG alone 3-5 days
Postpartum Anestrus eCG on day 1 with h CG given 48-96 h 3-5 days
later 3-5 days
e CG +h CG given on day 1
HORSES
Seasonal Anestrus Lengthen photoperiod by 4 h per day 4 -6 weeks earlier than
Progestogen for 15 days normal
Within 1 week
MODULE-30
SYNCHRONIZATION OF ESTRUS AND OVULATION

A limiting factor for the dairy herd productivity and profitability is reproductive
efficiency. The calving interval of cows in a farm to be economically efficient should be
12-13 months. To maintain this, cows must be pregnant within 85 to 115 days after
calving and a recommended leaving of 35 to 74 day breeding period. The detection of
estrus and rate of conception are integral components in achieving this calving
interval.Though causes of low conception may be multifactorial, synchronization of
estrus and ovulation will bring about a drastic increase in the conception rate in large
herds. Under the Indian field conditions major use of synchronization agents lies in the
management of infertility rather than estrus synchronization. These synchronizing
agents can be effectively used to address the major causes of infertility viz.

 Poor detection of heat.

 Untimely insemination.
 Subestrus / silent estrus and
 Anestrus.

In addition, these agents can be used in individual animals to induce heat and
inseminate at fixed time to improve fertility.

ESTRUS SYNCHRONIZATION

 It is the method of bringing a group of animals into estrum at a predetermined

time. In farm animals this is achieved by progesterone, PGF 2 alpha or a
combination of both.
 Synchronization of the estrus cycle can only take place in females that are already
cycling.

POTENTIAL ADVANTAGES

Potential Advantages in Large Dairy Herds

 Better control of calving interval.

 Reduction on dependence on heat detection.
 Reduction of errors in heat detection.
 Increased reproduction with the same number of cows.
 Maintain same production with fewer numbers of cows.
 Increased number of calves per cow.
 Increased economic use of AI.
 Schedule entry of heifers into the milking herd.
 Reduce labour.
 HOW DO ESTRUS SYNCHRONIZATION PRODUCTS WORK?

Synchronization products control the estrous cycle by influencing structures that are
present on the ovary.

Prostaglandins (PG)

 PG regulates a female's estrous cycle by causing "luteolysis" or regression of the

CL when it is present on the ovary.
 A synthetic prostaglandin will mimic natural PG release from the uterus and will
cause CL regression. Because the CL produces progesterone, PG eliminates the
"progesterone block" and allows follicles to grow and subsequent ovulation to
occur.
 Females with a CL on their ovary when they receive an injection of PG will
usually exhibit estrus 2 to 5 days later. An injection of PG will only regress a CL
that is 6 to 17 days old in the 21-day estrous cycle.
 After the progesterone block is removed, there is an increase in follicle growth
and therefore, an increase in estrogen production and heat and subsequent
ovulation will occur 2 to 5 days after the PG injection.

Estradiol Benzoate

 Estradiol benzoate, the estrogen used in the Syncro-Mate B protocol, will cause
regression of a CL that is 1 to 5 days old. This is the primary reason for using
estradiol instead of a prostaglandin.

Progesterone, MGA and Norgestomet

 Progesterone, MGA and Norgestomet (in the Syncro-Mate B protocol) "hold" the
female from exhibiting heat. During a normal estrous cycle, the CL on the ovary
produces progesterone.
 Feeding (MGA) or implanting (Syncro-Mate B) progesterone in females will play
the same role as progesterone being produced by the CL.
 Progestins will not allow the follicle to ovulate or allow the female to exhibit heat
until the synthetic progestin is removed.

Gonadotrophin Releasing Hormone (GnRH)

 Gonadotrophin releasing hormone causes the release of luteinizing hormone

(LH) and follicle stimulating hormone (FSH) from the anterior pituitary.
 The target tissue for LH and FSH is the ovary. The ovary needs these hormones to
grow follicles that produce the egg. In a synchronization program, LH and FSH
will grow follicles and also are a part of the process that causes ovulation (release
of the egg from the dominant follicle). Because LH and FSH are involved in
follicle maturation, these two hormones also support estrogen production from
the follicle.
  An elevated blood concentration of estrogen is associated with ovulation and
bringing the animal into standing heat.
 Initially estrus control was attempted with progesterone. Though estrus and
ovulation was controlled, the conception rate in the first service was poor. In the
1960’s, the treatment was mostly oral treatment with progestogens and it was
termed ‘grouping ‘.

EVALUATING ESTRUS RESPONSE

The efficacy of synchronizing treatment is evaluated as two parts:

1. Estrus response, which is the proportion of cattle treated that has come to heat in
the peak 24 h.
2. Reproductive performance, which is the conception rate in the controlled estrus.

CRITERIA FOR SUCCESSFUL CONTROLLED BREEDING

Animal requirement

 Animal must be disease free - especially of reproductive tract.

 Heifer must have reached maturity with proper weight >200-250 kgs.
 Nutrition must be adequate with the animal in positive balance.
 Adequate postpartum interval of 45-60 days should be there.
 For prostaglandins it is important that the animals must be cycling.
 The animal should have a normal non-pregnant reproductive tract.

Management requirements

 Proper timing is essential - once the program is initiated it must be carried

through all steps in proper sequence and at proper times.
 Good semen quality is an essential requirement. Under present condition existing
in the field, this is very important.
 Good AI technique must be followed.

PROSTAGLANDINS (PG) AND THEIR ANALOGUES

 PG are a group of naturally occurring compounds. The specific prostaglandin is

F2 alpha (PGF2 alpha). Synthetic PG function in the same manner as the naturally
occurring PG except they are administered in doses large enough to cause luteal
regression according to the schedule we desire.
 When injected at the recommended dosage, these products act by causing rapid
regression of the corpus luteum (CL) on the ovaries of cycling females in Days 6
to 16 of their estrous cycles. The injection decreases the function of the CL, which
 allows these females to return to estrus within 2 to 5 days and synchronizes their
estrous cycles. Females in Days 17 to 20 will be in estrus normally within 1 to 4
days and also will be synchronized. Females in Days 1 to 5 of the cycle and non-
cycling females that do not have a mature CL will not respond to the injection.
Only about 75% of the cows cycling in a herd can be synchronized with one
injection. If all cycling females are to be synchronized, two injections are needed.
 The PGF2 alpha analogues are more potent than the natural PGF 2 alpha and exert
lesser side effects and have a wider margin of safety.
 When the bovine CL matures there is an increase in the number of prostaglandin
receptors in the CL early in the estrous cycle and twice daily injections would
bring about luteolysis. PG has a short biological half life. Generally fertility is not
affected after PG and fixed time AI results in acceptable conception rates.

Effective Response Time

 PG is not effective in estrus, against developing and regressing CL.

SELECTION OF PROGRAM

 Options given below are suitable for individual as well as group of animals.
Selection of programme depends on the skill to identify CL and economy.
o Program A - requires only a single injection for each animal.
o Program B - requires double injection for all animals.
o Program C - requires one injection in about 2/3 of the animals and
double injection in 1/3 of the animals. ie. as the diestrus period is the
longest, when randomly injected the animal is more likely to be in the
diestrus than non-responsive period.
 Use one of the following options for breeding
 Detect heat and breed (Usually 2 to 5 days after injection).
 OR
 Fixed time AI at 72 h after injection with no heat detection.
 OR
 Fixed time AI at 72 and 96 h with no heat detection (better than single
insemination).
 Animal should be observed for the next estrus 18-22 days after this estrus period
and inseminated.

GENERAL COMMENTS

 Prostaglandins are very effective in their function i.e. luteolysis.

 With proper use nearly 100% induction can be achieved.
 They are not fertility drugs.
 Under ideal conditions conception rate of the induced estrus cannot be better
than the normal oestrus.
 Double injection regimen allows for higher synchrony.
  Since PG can be absorbed through the skin, pregnant women and asthmatics
should especially be careful when handling these products.

COMMERCIAL PREPARATIONS

Prostaglandin Commercial Preparations, Dose and Route of Administration

Commercial Content Presentation Dose and Route of

preparations administration
Lutalyse Dinoprost 10 ml vial 25 mg i/m
5mg/ml
12.5mg IVSM
Prosolvin Luprostiol 7.5 2, 10, 20 ml 15 mg i/m
mg/ml
Estrumate Cloprostenol 263 10 ml 500 m g i/m
mcg/ml
Dinofertin Dinoprost 5 ml 25 mg i/m
prostmate 5mg/ml
Bovilene Fenprostalene 2 ml SC injection
Iliren Tiaprost 10 ml 0.75mg /m, 0.45 mg i/v
0.15mg/ml

Administration

Intra muscular route

 Natural PGF2 alpha - 25 mg.

 Synthetic analog - 0.5 mg.

Intra vulvo sub mucosal route

 Natural PGF2 alpha - 10 mg.

 Synthetic analog - 0.2 mg.

Both routes are effective and have been found to give good conception rate.

PROGESTERONE AND PROGESTOGENS

 The luteal phase of the cycle is artificially prolonged and on withdrawal of

progesterone i.e. after the CL has regressed leads to estrus synchronization.

Estrus Response to Progesterone and Progestogens

 Majority of the animals are found to come to estrum within 24-48 h. Estrus
response to a regimen is lower in cattle compared to sheep probably due to
 endocrine differences which results in a shorter duration of estrus, 18-24 h in
cows compared to sheep (36 h) and so there is difficulty in detecting estrus.
 Exogenous progesterone may lead to development of dominant follicle. It may
not be able to exert the same feed back level on LH as the CL and hence there is
increased level of LH leading to the development of a dominant follicle which
would become atretic due to lack of gonadotrophin and this on ovulation would
lead to decreased fertility.

SYNCHROMATE B

 SMB ear implant is approximately 2 x 18 mm size and contains 3 mg of

norgestomet (17 alpha acetoxy 11 beta methyl 19-Nor-Preg 4 ene-3, 20 dione), a
progesterone-like compound.
 The SMB injection contains 2 ml oily solution of 5 mg estradiol valerate and 3 mg
Norgestomet

ADVANTAGES

 Increases pregnancy rates

 Increases probability of estrous detection and insemination
 Used to control the estrous cycle and mimic the reproductive hormones found within
the normal cow
 Control follicular wave development
 Promote ovulation in anestrous cows
 Regresses the corpus luteum in cyclic cows
 Synchronizing estrus and (or) ovulation

USES

 Syncromate B is used for estrous synchronization.

 The implant is placed in the ear for nine days. At the end of the nine day period
the implant is removed and the injection is given.
 The cows or heifers will start to show signs of heat about twenty-four hours after
implant removal.

ADDITIONAL ADVANTAGES

The advantages of this program include administration at any point within the cow's estrus cycle
will result in a synchronized estrus and accidental administration to pregnant cows won't cause
abortion.

PRINCIPLE

 The norgestomet from the injection immediately blocks the release of hormones
that cause ovulation and prevents females from displaying estrus.
  Release of norgestomet from the implant prevents estrus and ovulation over the
next nine days. The estradiol valerate in the injection causes regression of a
mature CL and any new developing CL.
 Together, the estradiol valerate and norgestomet cause luteolysis and advance all
cows to about day 19 of their estrous cycle and hold them there until the implant
is removed. On day 9, when the norgestomet implant (progesterone block) is
removed, cycling returns with the release of hormones which stimulate follicular
growth and estrogen secretion, and cows generally exhibit estrus within the next
one to four days.
 After the implant removal, cows are observed for standing estrus and bred 12 h
later or time inseminated at 48-54 h after implant removal. SMB also induces
estrous cycles in some anestrus cows.

ADMINISTRATION

 SMB ear implant is placed subcutaneously in the backside of the ear. Before
inserting the implant, it is helpful to clip the hair on the back of the ear, and
disinfect the implant site.
 Synchronization is done by administering an injection and an ear implant on day
0 and the implant removed on day 9.
 At the time the implant is being inserted, the animal is given an intramuscular
injection that contains estradiol valerate and norgestomet.

OPTIONS FOR INSEMINATION

 Three options for insemination using the Syncro-Mate B program,

o All females are mass inseminated at a predetermined time. Females
should be inseminated between 48 and 54 h after implant removal without
regard to time of estrus.
o Animals are inseminated 12 h after first observation of standing estrus.
This result in greater conception rates because the timing for insemination
is more accurate and because non-responding cows are not inseminated.
o A combination of the above two methods. Inseminate females that show
estrus before 48 h by the AM-PM rule and mass inseminate non-
responding females at 48-54 h after implant removal.

ADVANTAGES OF PROGESTOGENS

 CL need not be identified.

 All the animals can be administered at the same time.
 Can be given at any time of the cycle.
 Even if the animal is not cycling they bring the animal into estrus.
 All the other conditions with prostaglandins are also applicable to progestogens
for success
 CONTROLLED INTERNAL DRUG RELEASING DEVICE

 PRODUCT INFORMATION

 The CIDR is designed as a T shaped nylon spine molded with a silicone rubber
skin, which contains 1.9 g progesterone.
 The wings of the CIDR insert have the ability to be folded together in order to
insert intravaginally. Once inserted, the wings return to their original T shape
position and apply pressure to the vaginal walls to hold the insert in place.
 The CIDR insert is removed following the treatment period by pulling the plastic
tail that protrudes from the vulva.
 Current research is looking at modifying a CIDR to reduce the initial and residual
progesterone load, while maintaining intended preformance.
 Progesterone is released from the skin of the insert, causing the animals blood
progesterone concentrations to increase rapidly. Maximum concentrations are
reached within an hour after insertion.
 Progesterone acts according to the same mechanism as a typical steroid
hormone.
 Progesterone diffuses through the cell membrane and the nuclear membrane,
binding to the progesterone receptor in the nucleus, thus causing a change in cell
physiology.

CONTROLLED INTERNAL DRUG RELEASING DEVICE

 ADVANTAGES
  Higher pregnancy rate
  Estrus synchronization
  Improved value from semen, because of a higher success rate of artificial
insemination
  Heifers freshening at a younger age
  Exact breeding and calving dates of cows and heifers
  A more profitable herd or flock
CONTROLLED INTERNAL DRUG RELEASING DEVICE

 HOW IT WORKS
  CIDRs are coated with progesterone. Progesterone is a naturally produced
steroid hormone by the corpus luteum of mammalian ovaries. In vivo,
progestrone functions to maintain pregnancy.
  Progesterone provides a potent suppression of estrus, making it important for
estrus synchronization in herds of animals.
   When the CIDR is removed at the end of a treatment period, a rapid drop in
concentration of systemic progesterone occurs in each animal.
  Thus promoting a synchronized estrus effect within the herd, and allowing for
artificial insemination of the herd to take place

CONTROLLED INTERNAL DRUG RELEASING DEVICE

ADMINISTRATION

 CIDRs are coated with progesterone. Progesterone is a naturally produced steroid

hormone by the corpus luteum of mammalian ovaries. In vivo, progestrone
functions to maintain pregnancy.
 Progesterone provides a potent suppression of estrus, making it important for
estrus synchronization in herds of animals.
 When the CIDR is removed at the end of a treatment period, a rapid drop in
concentration of systemic progesterone occurs in each animal.
 Thus promoting a synchronized estrus effect within the herd, and allowing for
artificial insemination of the herd to take place

CONTROLLED INTERNAL DRUG RELEASING DEVICE

 WARNINGS

Taken from product insert:

 Human Warning: Avoid contact with skin by wearing latex gloves when handling
the inserts. Keep this and all medications out of the reach of children.
 Environmental Warning: Store removed EAZI-BREED CIDR Cattle Inserts in a
plastic bag or other sealable container until they can be properly disposed in
accordance with applicable local, state and Federal regulations.
Residue Warning: Neither a pre-slaughter withdrawal interval nor a milk discard
time is required when this product is used according to label directions.

Do Not Use:

 In beef or dairy heifers of insufficient size or age for breeding or in cattle with
abnormal, immature or infected genital tracts.
 In beef cows that are less than 20 days postpartum or in lactating dairy cows less
than 40 days postpartum. The sponsor has not provided effectiveness and animal
safety data for the use of this product in beef cows that are less than 20 days
postpartum or in lactating dairy cows that are less than 40 days postpartum.
 The EAZI-BREED CIDR Cattle Insert in lactating dairy cows concurrently with
LUTALYSE® Sterile Solution or other prostaglandin products for
 synchronization of the return to estrus. The concurrent use with prostaglandin
products is not approved in lactating dairy cows.
 An insert more than once. To prevent the potential transmission of venereal and
blood borne diseases the EAZI-BREED CIDR Cattle Insert should be disposed
after a single use.

PROGESTERONE RELEASING INTRAVAGINAL DEVICE (PRID)

It is a stainless steel flat coil coated with an inert silicone rubber incorporating 1.55 g of
progesterone and contains a 10 mg of estradiol benzoate capsule.

 The PRID is inserted into the anterior vagina, left for 12 days and then
withdrawn.
 Estrus occurs 2-3 days after withdrawal.
 Fixed time AI at 48 and 72 h or only at 56 h after removal is recommended.

COMMERCIAL PREPARATIONS OF PROGESTERONE AND

PROGESTOGENS

Preparations and Route of Administration Duration of

Treatment (in days)
Oral - MGA 14
Intravaginal Sponges 14
Progesterone Releasing Intra Vaginal Device - 9 -11
PRID
Controlled Internal Drug Releasing Device - 11
CIDR
Ear implants 9

 Crestar
 Synchromate B

Injection in oil or other media 14 -18

TECHNIQUES FOR SYNCHRONIZING ESTRUS IN CYCLIC FARM

ANIMALS

Species Method Treatment Regimen End of

Treatment
to Estrus
Cattle and PGF Two injections (11-12 days 3-5 days;
Buffalo apart) AIDE/TAI
 Cattle GnRH+PGF Inject GnRH (day 0), PGF 2-4 days; TAI
(day6)
GnRH+PGF+GnRH Inject GnRH (day 0), PGF 2-4 days; TAI
(day7), GnRH (day 8 or 9)
Progestogen +estrogen Estrogen injection (day 1), 3-5 days;
CIDR (days 1-9) AIDE/TAI
Sheep Progestogen + PGF Progestogen (days 1-7), PGF 2-3 days;
(day 6) AIDE/TAI
Progestogen (pessary) +e Progestogen (12-14 days),e CG 2 days; AIDE
CG (day of pessary removal) or double AI
PGF Two injections (9 days apart)
Goat Progestogen (pessary) Progestogen (18-21 days), e 2-3 days;
+eCG CG (day of pessary removal) breed at estrus
Two injections (11-12 days or double AI
apart)
Swine Progestogen in feed Altrenogest (14-18 days) 4-7 days;
breed at estrus
Horse Progestogen in feed Altrenogest (15 days) 4-7 days;
breed at estrus
PGF One dose to mares in diestrus 4-7 days;
breed at estrus
PGF +H CG PGF (day 1), hCG (day 7-8), 3-5 days;
PGF (day 15), breed at estrus
hCG (day 21-22) 2-4 days

PROTOCOLS FOR SUPEROVULATION IN FARM ANIMALS

Species Pre Treatment Gonadotropin PGF LH

Goat Fit a Progestogen pessary Inject 20 mg PFSH Inject 125-250 μg None
for 17 days one day before cloprostenol
pessary removal
sheep Fit a Progestogen pessary Inject 20 mg PFSH Inject 1000 μg
for 12 days one day before Cloprostenol
pessary removal
Cattle and Progestogen for 7-8 days Inject 20-30 mg Inject 25 mg PGF Inject (IM) 25
buffalo pFSH (Lutalyse) (day 2) mg porcine LH
(day 5)
Cattle Day-7 Inject 80 mg FSH Inject 25 mg PGF Inject (IM) 25
Progestogen+GnRH (day 0-4) (Lutalyse) (day 2) mg porcine LH
implant (day 5)
Pig Feed Altrenogest for 15 Inject 1500 IU eCG None None
days one day before end
of feeding period

MODULE-31
FOLLICULAR DEVELOPMENT

 In the primordial follicle reserve, formed during fetal life or soon after birth,
follicles are released which continues to grow until ovulation or until the follicle
degenerates, which is the case with majority of the follicle.
 Ovarian follicular development in cows and ewes is a progressive and recurring
process with two or three waves of follicular growth occurring in each cycle. In
cows two waves appear to be more common but three waves are frequent in long
cycles.
 Thus even though the follicular phase comprises only about 20% of the estrous
cycle, the process of follicular growth and degeneration (known as follicular
dynamics) occurs continuously throughout the entire estrous cycle.If the ovaries
are examined at any point during the estrous cycle significant number of antral
follicles of various sizes will be seen. These antral follicles have been classified as
small, medium and large depending on their diameter.

DYNAMICS OF ANTRAL FOLLICLES

 Dynamics of Antral follicles consists of

o Recruitment
o Selection
o Dominance
o Atresia

Recruitment

 Recruitment is the phase of follicular development in which a group of small

antral follicles begin to grow and produce estradiol.
 Some of these recruited follicles undergo atresia.
 Following recruitment, groups of growing follicles, which have not undergone
atresia, are selected.

election

 Selection involves the emergence of dominant follicles from the host of previously
recruited antral follicles.
 Selected follicles may become dominant or they may undergo atresia.

Dominance
  As the selected follicle proceeds towards dominance, they continue to produce
increasing amounts of estrogen as well as inhibin.
 In the cows and mare (monotocus sp) these are several selected follicles but only
one will develop into the dominant follicle.
 Increasing inhibin levels and reduced blood supply to other follicles, the
dominant follicle causes inhibition of other follicles.
 Suppressed FSH concentration in blood along with reduced blood supply results
in atresia.
 Only those follicles receiving a large blood supply and thus higher levels of
gonadotrophin continue to grow.
o Recruitment = High FSH + Low LH + No Inhibin + No Estradiol
o Selection = Low FSH + Moderate LH + Low Inhibin
o Dominance = Low FSH + High LH + High Inhibin
o Atresia = Degeneration of Follicles

ENDOCRINOLOGY OF FOLLICULAR GROWTH AND OVULATION

 FSH plays a major role in antrum formation.

 This gonadotrophin stimulates granulose cell mitosis and follicular fluid
formation.
 FSH also increases the sensitivity of granulosa cells to LH by increasing LH
receptors, which prepares for ovulation. On the other hand theca cells are
stimulated only by LH and LH receptors are present from the beginning of theca
cell formation
 During recruitment FSH and LH begin to increase, thus promoting follicle
development.
 As follicles enter the selection phase, inhibin is produced by the follicles and
inhibits FSH released by the anterior pituitary.
 As follicular dominance phase is entered, the large follicle produces more and
more estrogen and thus causes the preovulatory surge center to release LH surge.
In addition FSH concentration is reduced because of inhibin. This causes antral
follicles to undergo atresia. FSH and LH are also essential for steroidogenesis
(two cell two hormone theory).

MODULE-32
OVULATION

Ovulation is the release of oocyte from mature graafian follicle. Ovulation in

mammalian ovary occurs on any point in the ovarian surface while ovulation in mares is
restricted to ovulatory fossa. In cow, sheep and horses, ovulation occurs at random
irrespective of which ovary contains previous CL. In some animals ovulation
consistently alternates between ovaries and in others (whales) ovulation may
predominate in one ovary.In the rhesus monkey, the CL retards subsequent follicular
growth so that ovulation alternates between ovaries.
TYPES OF OVULATORS

 Spontaneous Ovulators (eg. Cow, mare, sheep goat dog). Ovulation occurs
spontaneously irrespective of whether mating has taken place or not.
 Induced Ovulators or Reflex Ovulators (eg. Cat, mink, ferret). Copulation is
a must for ovulation to take place.

THEORIES OF OVULATION

Follicular Pressure Theory

 As the follicle grows the amount of liquor folliculi also increases. This liquor
folliculi exerts pressure on the follicular wall thereby follicles rupture.
 However, this was not the case with cystic ovaries where there was both an
increase in follicular size and follicular fluid but there was no rupture. This theory
was not acceptable.

High Osmotic Pressure Theory

 Liquor folliculi consists of more amounts of electrolytes particularly Na and K.

Increased osmotic pressure leads to rupture of the follicle.
 In cystic ovaries size of the follicle and amount of follicular fluids along with
electrolyte content also increased but still there was no rupture. Hence, This
theory was also not acceptable.

Ischemic Theory

 Increased follicular fluid exerts pressure against follicular wall.

 At one point due to pressure, ischemia occurs and leads to stigma formation and
ovulation. Theory was partly acceptable.

Follicular Wall Thinning and Rupture

 Nearing ovulation, the blood supply to the follicle increases Thinning of the
follicular wall occurs at one point called as stigma. Ovarian contractions and
follicular rupture occurs. This theory was accepted.
 Recent theory is that ovulation is a combination of physiological, biochemical and
biophysical mechanisms.

OVULATORY PROCESS
 A Surge of LH occurs at the beginning of estrus prior to ovulation when
progesterone is at its minimal levels and estradiol has reached its highest cyclical
values.
 Several tissue layers separate the oocyte from the outside of the follicle. These are
the surface epithelium, the tunica albugenia, theca externa, and interna,
basement membrane and granulosa cells. All these tissue layers have to be
broken down before ovulation can take place.
 An increased blood flow near ovulation occurs to all classes of follicles but the
follicle destined to ovulate receives the largest volume of blood and has capillaries
more permeable than those in other follicle.
 As the follicle enlarges it begins to protrude from the surface of the ovaries, the
vascularity of the follicular surface increases except at its centre, which is devoid
of blood vessels. This avascular area is the future point of rupture.
 Meanwhile there is dissociation of cumulus cells which detaches the oocyte from
the membrana granulosa and now the oocyte is surrounded by the radiata cells.
 Resumption of Meiosis (nuclear maturation) occurs 3 h after LH surge and ends 1
h before ovulation when the first polar body is extruded.
 Cumulus cells actively secrete a viscous mass enclosing the oocyte and its corona.
After follicular rupture the viscous mass spreads at the ovarian surface to
facilitate the pickup of oocytes by the fimbriae.
 The LH surge also causes a temporary shift in steroid secretions by increasing
progesterone secretion. This progesterone stimulates collagenase activity in the
 follicular wall reading to dissociation of bundles of collagen fibers (increase in
plasmin activity causes an increase in follicular wall elasticity).
 The LH surge also causes an increase of PGF2 alpha and PGE2 levels. These
prostaglandins play a basic role in follicular rupture, and inhibition of their
synthesis prevents ovulation.
o PGE2 stimulates production of plasminogen activator thus increasing
plasmin activity which increases in follicular wall elasticity and is involved
in tissues cell migration and thereby causes mixing of theca and granulosa
cells during CL formation.
o PGF2 alpha causes rupture of the epithelial cell lysosomes at the follicular
epithelium. Their hydrolase’s destroy the underlying albugenia cells and
then the theca cells. After lysosomal rupture, epithelial cells scale off. The
wall of the follicular apex becomes thin in a circumscribed area called the
stigma. The PGF2 alpha causes contraction of the smooth muscle cells
that are present in the ovarian stroma and theca externa thus leading to
ovarian contractions and follicular contractions. These ovarian
contractions cause follicular rupture and follicular contractions causes
expulsion of the oocyte.
 At the time of ovulation the ovum, together with surrounding cells in a gelatinous
mass, protrudes at the ovarian surface and is swept into the ostium of the oviduct
by the action of the motile kinocilia of the fimbriae.

OVULATION CHARACTERISTICS IN DOMESTIC ANIMALS

Species Ovulation Duration of estrus Ovulation time

type
Cow Spontaneous 14-18 h 12-18 h after end of estrus
Mare Spontaneous 4-7 days Last 2 days of estrus
Sows Spontaneous 2-3 days Last day of estrus
Ewe Spontaneous 1-2 days Last day of estrus
Bitch Spontaneous 7-9 days First 3 days of estrus
Cat Induced 4 days if copulation 1 day after mating
occurs
no ovulation occurs if copulation
9-10 days without does not take place
copulation
MODULE-33
NON-SURGICAL EMBRYO TRANSFER IN BOVINES

FIRST SUCCESSFUL RECORDS OF EMBRYO TRANSFER

Species Year Authors

Rabbit 1890 Heape
Goat 1932 Warwick & Berry
Rat 1933 Nicholas
Sheep 1933 Warwick et.al.
Mouse 1942 Fekete & Little
Cow 1951 Willet et. al.
Pig 1951 Kvasnickii
Horse 1974 Oguri & Tsutsumi
Human 1978 Steptoe & Edwards

APPLICATIONS OF EMBRYO TRANSFER

 Genetic improvement
o Genetic variation
o Selection of dams
o Selection intensity
o Generation interval
 Genetic screening
 Disease control
 Import and export
 Circumvention of infertility
 Twinning
 Conservation

SELECTION OF DONOR

Two Major Criteria are involved in Selection of Donors

o Genetic merit
o Reproductive performance
 Good body condition and preferably gaining body weight.
 Disease free
 A minimum of 50-60 days post partum, and
 Cycling regularly

Clinical Examination
  Rectal palpation should be performed to rule out any adhesions or other palpable
lesions of the cervix, uterus and ovaries.
 The patency of cervical canal should be checked using a cervical dilator, especially
in heifers. This will help to overcome the frustration that might arise if unable to
negotiate the cervix after superovulation with expensive hormones.
 Vaccinate against prevailing diseases.
 Single embryos or multiple embryos may be collected from naturally ovulating or
superovulated cows, respectively.
 Recommended donor-recipient ratio:
o For every flushing program, at least 2-4 donors should be prepared and
synchronized with their recipients.
o Recommended ratio is 8-10 potential recipients per donor.

SUPEROVULATION

 Superovulation is defined as the increased ovulatory response, above the number

that would be expected to occur naturally, generated in an animal by the
administration of exogenous gonadotrophic hormones.

General Considerations

 In the process of embryo transfer, superovulation is the least predictable step

with great variation in response due to age, breed, lactational status, nutritional
status, season and stage of the cycle at which treatment is initiated.

Superovulation with FSH

o Follicle stimulating hormone (FSH) or equine chorionic gonadotropin

(eCG), formerly called pregnant mare serum gonadotropin (PMSG) are
used.
o Due the short half-life of FSH twice-daily injections over a period of 4-5
days is required.
 Initiation of Treatment
o Treatment should be initiated during the mid-luteal phase (day 8 to 12) of
the donor’s cycle, and with the use of prostaglandins (PGF2alpha) to
synchronize the cycles of the donors and the recipients.
o Alternatively, treatment can be initiated on day 16 or 17 (day 0 = estrus) of
the donor’s natural estrous cycle.
 Schedule
o In four day FSH regimen, prostaglandins (PG) (25-35 mg PGF2α or 500
mcg PG analog IM) are administered at the time of the fifth and sixth FSH
injections. Estrus follows in 2 days and ovulation in 3 days. In
superovulated cows, the interval from PG to the onset of estrus is 12-24 h
shorter than in naturally ovulating cows or heifers. Hence, in recipients PG
should be injected 24 h before the donors.
 Response
 o The response ranges from zero to 20 or more ovulations, with an average
of 8-10.
o No difference in response between a 4-day and a 5-day regimen.
o If a donor fails to respond to an established superovulatory treatment
regime, the second attempt with the same or a similar regimen is also
likely to result in failure.
 Preparations
o Folltropin-V (Bioniche): Twice daily injections in decreasing doses are
recommended.
 Comparable Products
o Ovagen (ICP Ltd; Auckland New Zealand).
o Pluset (Serono; Rome, Italy).
o Stimufol (Rhone Merrieux; Lyon France).

Superovulation with Pregnant Mare Serum Gonadotropin (PMSG)

 PMSG is a foreign protein that is antigenic, may lead to a reduced response after
repeated use.
 Due to its long half-life, a single injection is sufficient.
 The FSH/LH ratio: 1.9 to 95.5 depending on the stage of gestation at which the
serum was collected.
 Administration of gonadotropin releasing hormone or LH to donor cows at the
onset of estrus in an attempt to precipitate or to group ovulations does not offer
any advantage.
 Stage of administration
o On day 16 or 17 of a normal estrous cycle.
o If used in conjunction with prostaglandin (PG), PMSG is administered
between day 8-12 of an estrous cycle followed by PG 48 to 72 h later.
 Dose: 1500-3000 IU.
 Route of administration: SC or IM.

Assessment of Superovulatory Response

 It is difficult to accurately assess the number of ovulations by palpation of the CL

per rectum when the number exceeds 4 to 6 per ovary or when several
anovulatory follicles are also present.

SUPEROVULATION WITH FSH

 Follicle stimulating hormone (FSH) or equine chorionic gonadotropin (eCG),

formerly called pregnant mare serum gonadotropin (PMSG) are used.
 Due the short half-life of FSH twice-daily injections over a period of 4-5 days is
required.

Initiation of Treatment
  Treatment is initiated during the mid-luteal phase (day 8 to 12) of the donor’s
cycle, and with the use of prostaglandins (PGF 2alpha) to synchronize the cycles of
the donors and the recipients.
 Alternatively, treatment is initiated on day 16 or 17 (day 0 = estrus) of the donor’s
natural estrous cycle.

Schedule

 In four day FSH regimen, prostaglandins (PG) (25-35 mg PGF 2α or 500 mcg PG
analog IM) are administered at the time of the fifth and sixth FSH injections.
Estrus follows in 2 days and ovulation in 3 days. In superovulated cows, the
interval from PG to the onset of estrus is 12-24 h shorter than in naturally
ovulating cows or heifers. Hence, in recipients PG should be injected 24 h before
the donors.

Response

 The response ranges from zero to 20 or more ovulations, average 8-10.

 No difference in response between a 4-day and a 5-day regimen.
 If a donor fails to respond to an established superovulatory treatment regime, the
second attempt with the same or a similar regimen is also likely to result in
failure.

Preparations

 Folltropin-V (Bioniche): Twice daily injections in decreasing doses are

recommended.

Comparable Products

 Ovagen (ICP Ltd; Auckland New Zealand).

 Pluset (Serono; Rome, Italy).
 Stimufol (Rhone Merrieux; Lyon France).

SUPEROVULATION WITH PREGNANT MARE SERUM

GONADOTROPIN (PMSG)

 PMSG is a foreign protein that is antigenic, may lead to a reduced response after
repeated use.
 Due to its long half-life, a single injection is sufficient.
 The FSH/LH ratio: 1.9 to 95.5 depending on the stage of gestation at which the
serum was collected.
 Administration of gonadotropin releasing hormone or LH to donor cows at the
onset of estrus in an attempt to precipitate or to group ovulations does not offer
any advantage.
Stage of Administration

 On day 16 or 17 of a normal estrous cycle.

 If used in conjunction with prostaglandin (PG), PMSG is administered between
day 8-12 of an estrous cycle followed by PG 48 to 72 h later.
o Dose: 1500-3000 IU.
o Route of Administration: SC or IM.

ASSESSMENT OF SUPEROVULATORY RESPONSE

 It is difficult to accurately assess the number of ovulations by palpation of the CL

per rectum when the number exceeds 4 to 6 per ovary or when several
anovulatory follicles are also present.

ESTRUS DETECTION

 Accurate estrus detection is necessary for

o Timely insemination of the donor, and
o To assess the degree of synchronization of estrus and ovulation between
the donor and recipients.
 The age of the embryo is calculated from the time of onset of estrus.

Methods of Estrus Detection

 Direct observation of estrus behavior, especially mounting, remains the most

reliable method.

Estrus detection aids are available, including the use of teaser animals, tailhead marking or
painting, heat detector patches, and pedometers.

Monitoring of Estrus and Ovulation

 Frequent rectal palpation of the uterus and ovaries.

 By ultrasonography.
 By frequent milk or plasma progesterone assay.

BREEDING OF DONOR

 Twice AI with a 10-12 h interval beginning 4 -6 h after the onset of estrus, to

cover the range of time over which the ovulations may occur.
 Depending on the quality of the frozen semen, a double inseminating dose may
be used at each insemination especially in cows with a large pendulous uterus.

NON SURGICAL EMBRYO FLUSHING

Optimum Time of Flushing

 Bovine embryos descend into the uterus around day 4.5 (estrus = day 0) and shed
their zona pellucida (“hatch”) between days 8-10.
 Optimum time for nonsurgical flushings is between days 6-8.

TECHNIQUES EMPLOYED

Foley Catheter

 A 2-way round tip with a 30 ml inflatable balloon.

 French size #16 to 24.
 The 2-way catheter has one channel for inflation of the balloon plus a single
channel for alternate inflow and outflow of flushing medium.

Rusch Catheter (Germany)

 Red rubber 68 cm long, 18 French gauge, and balloon catheter.

 It has a self-contained locking stylet and a tip which extends 4.5 cm beyond the
balloon and offers advantage in older cows.
 After the stylet has been withdrawn 3-4 cm the catheter can be directed further
into the horn because the rubber is stiffer than the Foley catheter.
 Advantageous in older animals with long pendulous uteri. The uterine lumen is
flushed by alternating in- and out-flow.

Three-way Collection Device (IMV, France)

 A large and rigid, stainless steel.

 One channel serves to inflate the balloon, a second one, the stainless steel
cannula, to introduce the flushing medium, and the third, a small flexible
catheter which can be advanced into the tip of the horn, to recover the flushing
medium.

FLUSHING AND HOLDING MEDIA

 Phosphate - (Dulbecco’s phosphate buffered saline, DPBS): advantage is that they

maintain the pH during exposure to the air.
 Bicarbonate-buffered solutions: need a gas phase of 5% CO2, which means that
they require a closed system.
 The pH of most body fluids is 7.2-7.4 and osmolarity in the range of 270-300
mOsmol.
 1% heat-treated bovine serum (10 ml) or Bovine serum albumin (BSA) 0.04% is
added to each individual 1-liter bottle of flushing medium which has been
warmed to a temperature between 30-37 °C. Serum may act as a protein source
for embryo growth and membrane stabilization, and renders embryos less sticky.
 For flushing, PBS only needs to contain penicillin and 1-2% serum. 10-20%
serum is added to the flushing medium to make a holding medium which can also
be used for short-term (less than 24 h) culture.
 The holding medium is filter sterilized by filtration through a 0.22 µm or 0.45 µm
millipore filter attached to a large disposable syringe.
 Embryos are stored in the same type of medium that was used for flushing.
Changing embryos from a phosphate- to a bicarbonate- buffered medium is
undesirable because of the possible changes in osmolarity, pH and energy
substrates.
 The holding dishes should be covered to minimize contamination and
evaporation.
 Embryos must be changed to a fresh dish of holding medium from time to time
(every 2 h) to further minimize the effects of contamination and evaporation.

FLUSHING PROCEDURE

 Restrain the donor in a chute.

 Carefully remove the faeces from the rectum avoiding sucking of air, and assess
the number of ovulations (CL).
 Administered (4-6 ml of 2 % lidocaine) epidurally to prevent defecation and
straining.
 Wash the vulva and perineal region thoroughly with plain water and wipe it dry.
 Tie the tail to one side of the animal.
 Use an appropriate cervical dilator properly covered with a sanitary sleeve before
they are introduced into the vagina to expand and straighten the small or
tortuous cervical canal. Perforate this protective cover just before the instrument
enters the external os of the cervix. Exercise extreme caution while using a rigid,
relatively sharp-pointed dilator as it can readily perforate the uterine wall.
 Part the lips of the vulva and the Foley catheter, with the stylet in place, is
inserted into the vagina and on into the lumen of the cervix. Carefully manipulate
into the appropriate horn until the inflatable balloon is situated at the base of the
uterine horn. Alternately, the catheter with the balloon may be fixed just anterior
to the internal os of the cervix, in the body of the uterus so as to flush both horns
simultaneously.
 Slowly inflate the balloon with 15-20 cc of air in adult cows and 10-15 cc of air in
heifers. The endometrium can easily be split by over distension, resulting in
haemorrhage and escape of the flushing solution into the mesometrium from
which it cannot be recovered.
 Position the catheter and remove the stylet.
 Connect the catheter via a Y-junction by sterile tubing to a 1 litre bottle or bag of
flushing medium. Connect the remaining arm of the Y-junction to a free piece of
tubing.
 Quick-release clamps are used to check the flow of medium in both pieces of
tubing. The flushing medium bottle is suspended one metre above the level of the
uterus and when the outlet tubing is clamped, the flushing solution from flows in
to the uterus by gravity flow.
  Extend the horn of the uterus by elevating the tubo-uterine junction and by
carrying it anteriorly. When the inflow stops, clamp the inlet tubing and release
the clamp on the outlet tubing.
 Allow the fluid to directly pass through an embryo filter (75 µm pore size).
 If filters are not used, the effluent may be collected in a 1-liter graduated cylinder.
Allow the embryos to settle for 20-30 min and carefully siphon off the
supernatant. Start the siphoning by aspiration using a small syringe fitted at the
other end of the tubing and gently lower a length of small diameter (e.g. 1 mm)
tubing into the cylinder until the end of the tubing reaches the 75 ml mark.
Examine the remaining 75 ml directly under a stereomicroscope.
 If the returning fluid is blood tinged, the red cells may be washed directly through
the filter by opening both clamps between the bottle of flushing solution and the
filter. Never allow the filter to run completely dry leaving the embryos on the
filter disk exposed to the air. Maintain a minimum layer of 1 cm fluid by
regulation with the clamp on the tubing attached to the bottom of the filter unit.
 During the final collection of the flushing solution, administer 50 IU of oxytocin
IV, which sometimes may aid in the recovery of the residual portion of the
medium from the uterus.
 Repeat the same flushing procedure for the opposite horn using a separate sterile
catheter.

IDENTIFICATION AND HANDLING OF EMBRYOS

Identification

 The embryo is spherical and is composed of cells (blastomeres) surrounded by a

gelatin-like shell, and acellular matrix known as the zona pellucida (ZP).
 The zona is spherical and translucent, thus is clearly distinguishable from cellular
debris.
 Due to its shape the embryo tends to roll on the bottom of the (searching) dish.
 The overall diameter of the bovine embryo is 150-180 μm including a ZP
thickness of 12-15 μm.
 The diameter remains constant until expansion of the blastocele begins.

Important Criteria

 Shape of the embryo

 Presence of a zona pellucida
 Size
 Color, and
 Knowledge of the age of the embryo.

Handling

 As soon as an embryo is identified, it is immediately transfered to a small petri

dish containing fresh, filter sterilized medium.
  The embryos are serially rinsed through at least three different dishes containing
fresh sterile medium using a sterile pipette.

MORPHOLOGICAL CLASSIFICATION OF EMBRYOS

Embryos recovered 5-8 days after estrus are classified morphologically into the
following groups.

Morula

 Blastomeres are round in shape and are not tightly connected to each other.
Individual blastomeres are difficult to discern from one another. The cellular
mass of the embryo occupies most of the perivitelline space.

Compact Morula (Tight Morula)

 The shape of a tight morula is similar to a golf ball, in that the outer edge is
slightly bumpy (scalloped) in appearance because of compaction. Individual
blastomeres are no longer distinguishable. Cells on the surface of the mass are
polygonal in shape. The embryo mass occupies 60 to 70% of the perivitelline
space.

Early Blastocyst

 A tiny transparent (clear) space is visible which contains fluid. This area is the
beginning of the blastocele. The embryo occupies 70 to 80% of the perivitelline
space.

Blastocyst

 The prominent blastocele cavity comprises more than 70% of the volume of the
embryo. Two groups of cells are present and clearly recognizable as the
trophoblastic layer beneath the zona pellucida and the darker inner cell mass
occupying one side of the embryo. The perivitelline space may still be visible but
is very small.

Expanding or Expanded Blastocyst

 There is no perivitelline space between the layer of trophoblastic cells and the
inside of the zona. The zona pellucida becomes thinner as the blastocyst expands.
A small (well compacted) inner cell mass positioned on one side of the embryo is
observed. The color of the embryo is pale to clear because of the large amount of
fluid present inside.

Hatched Blastocyst
  Ultimately the blastocyst expands to the point of rupture and the embryo escapes
from the disrupted zona. Hatched blastocysts may be spherical with a well-
defined blastocele or they may be collapsed, resembling debris. Identification of
embryos at this stage can be difficult for the inexperienced operator. When zona-
free, or hatched, blastocysts are collected, there is a greater risk of damage due to
handling. Furthermore, hatched blastocysts are “sticky” and may adhere to
tubing and glassware. Embryo filters should not be used when there is a
possibility that hatched embryos will be recovered (>day 7.5).
 Excellent, good and fair quality embryos are considered transferable. Excellent
and good quality embryos are freezable.

CODES FOR EMBRYO QUALITY

(Source: International Embryo Transfer Society Manual, Ch 9, p106).

Code 1: Excellent or Good

 Symmetrical and spherical embryo mass with individual blastomeres (cells) that
are uniform in size, color and density.
 Consistent with its expected stage of development.
 Irregularities should be relatively minor and at least 85% of the cellular material
should be an intact, viable embryo mass. This judgement should be based on the
percentage of embryo cells represented by the extruded material in the
perivitelline space.
 The zona pellucida should be smooth and have no concave or flat surfaces that
might cause the embryo to adhere to a petri dish or a straw.

Code 2: Fair

 Moderate irregularities in overall shape of the embryo mass or size, color and
density of the individual cells.
 At least 50% of the cellular material should be an intact, viable embryo mass.

Code 3: Poor

 Major irregularities in shape of the embryo mass, or size, color and density of
individual cells.
 At least 25% of the cellular material should be an intact, viable embryo mass.

Code 4: Dead or Degenerating

 Degenerating embryos, oocytes or 1-cell embryos; non-viable.

EMBRYO SCREENING PROCEDURE

 Embryo searching equipment is prepared before the uterus of the donor is
flushed when a concentrating filter is used.
 One to three 100 x 100 mm square, grid bottom dishes are used to search one
filter.
 Each dish is labeled with the donor’s number and the sequence in which it was
filled from the filter.
 Fresh straight PBS is drawn into a 30 or 35 ml syringe with sterile precautions. A
22 gauge 1 inch needle is then attached to the syringe. This syringe is used to
rinse the filter. The presence of serum in the PBS must be avoided during this
procedure to prevent foaming.
 The medium, left in the filter from the flush, is swirled and poured into first dish.
 The filter is held at an angle and rinsed into the dish. More fluid may be needed if
the filter contains mucus. Steps 4 and 5 are repeated using as many dishes as
necessary, until the filter is completely clear (no mucus and/or tissue debris left
on the filter). Each dish will safely hold 75 ml.
 Five to seven ml of serum or 0.4 percent BSA are added to the searching dish and
stirred in the medium while clearing bubbles from the edge of the dish.
 Dishes are searched under a stereomicroscope (15X). The dish is systematically
moved along the reference lines to ensure the entire dish is searched. When
embryos are identified, they are immediately transferred into a small petri dish
containing sterile, filtered holding medium (PBS + 10-20 % serum, or 0.4 percent
BSA). All dishes should be kept covered between searches to avoid
contamination, and particularly evaporation when placed in an incubator.
 The embryo is drawn from the searching dish into a micropipette attached to a
0.5 ml syringe while observing under the microscope. Then, the embryo is
transferred to the small holding dish. The number of good and/or bad embryos
are tentatively recorded on the lid of the holding dish. The estimated number of
corpora lutea usually serves as a guide to the number of embryos to be searched
for.
 After all the embryos from one donor are accumulated in one small holding dish,
they are transferred three times, each time to a dish containing freshly filtered
PBS plus serum. This procedure serves to rinse the embryos.

LOADING THE STRAW

 Load the embryos individually in sterile 0.25 ml French straws just before
transfer in to suitable recipients.

 The embryos are now graded and prepared for the next procedure; culturing,
transferring, freezing, splitting or sexing.
 Grading is facilitated under higher magnification (100 X) with a regular light
microscope, preferably an inverted microscope with adequate working room on
the stage.
 Alternatively, when a large 500 to 1000 ml graduated cylinder is used, the
embryos are allowed to settle to the bottom of the cylinder for 20 to 30 minutes.
All but the bottom 75 ml of flushing medium are slowly siphoned off with a small
 diameter piece of tubing. The final 75 ml are gently swirled and then poured into
a searching dish. The cylinder is rinsed 2 to 3 times with small volumes of
flushing medium (containing 1 % serum) and emptied into a searching dish. This
is followed by steps 7 through 12 as before.

Aspirate the embryo from the holding dish into the straw with the aid of a 1 ml tuberculin
syringe attached to the plug end of the straw.

 First aspirate a 3 cm column of medium, followed by a 0.5 cm column of air, then

a 3 cm column of medium containing the embryo, followed by another air bubble.
Fill the remainder of the straw with medium until the initial column of medium
wets and solidifies the plug.

SELECTION OF RECIPIENTS

Selection of Cows or Heifers

Select cows and heifers

 with large body frame, disease free and in good body condition.
 with a minimum of 2 normal cycles before inclusion.
 that are not too fat and should preferably gain 0.1 - 0.2 kg / day.
 that are vaccinated against common abortion diseases.

GENERAL CONSIDERATIONS
  An average bovine donor yields 6 to 8 transferable embryos.Therefore, Prepare a
minimum of 8 recipients per donor.
 Screen atleast 12 recipients in order to definitely identify 8 with active corpora
lutea.
 If 8 are injected with prostaglandins, 6 on the average will be suitably
synchronized with the donor.
 Inject recipients with prostaglandins one day earlier than the donor.
 Due to prior gonadotropin treatment, the donor comes into oestrus 48 h after the
prostaglandins, while the recipients which did not receive any gonadotropin
treatment will come into oestrus 72 h after prostaglandin treatment.
 Since all donors will not respond to the superovulatory treatment, for optimal
efficiency, 2-4 donors should be superovulated at the same time to permit sharing
of the prepared recipients and avoid the expensive frustration consequent to
single donor preparation.

PROSTAGLANDIN PROTOCOL FOR ESTRUS

SYNCHRONIZATION OF DONOR AND RECIPIENTS

Recipients can be synchronized to exhibit heat on the same day or just ahead of the
donor with:

Method - 1

 In animals with a palpable CL, inject 25 mg PGF 2α or 0.5 mg PG-analog lM.

 Estrus may be expected in 2-4 days with a peak on the third day.

Method - 2

 Regardless of the presence or absence of a CL, all recipients may be administered

prostaglandins.
 A second PG is administered 11 days later.
 Estrus will peak on the third day after the second injection.
 ostaglandin Protocol

Day What To Do - What To Do -

DONOR RECIPIENTS
0 In heat / No Al PG
10 FSH Nothing
11 FSH PG am
12 FSH
Nothing
PG am/pm
13 FSH Nothing
 14 Estrus +Al Estrus/No Al
21 Collect embryos Transfer

SYNCHROMATE- B PROTOCOL FOR ESTRUS

SYNCHRONIZATION OF DONAR AND RECIPIENTS

 Progestogen containing ear implant.

 Each SMB ear implant contains 6 mg of the synthetic progesterone norgestomet.
 In recipient animals, estrus synchronization is induced by placement of 6 mg
Syncromate-B (SMB) ear implant.
 Under aseptic precautions, the SMB ear implant is inserted on the outer surface
of the ear under the skin, avoiding blood vessels, using SMB applicator.
 At the time of implant insertion 0.5 ml of SMB injection (1.5 mg of norgestomet
and 2.5 mg of estradiol valerate) is administered intramuscularly.
 On day-10 after the insertion of the implant, the animals receive a single non-
superovulatory intramuscular injection of 400 IU of PMSG and 10 mg of PGF 2α.
  The implant is removed on day-11.
  The scab at the point of insertion is gently teased and removed using a sterile
needle.
  By gently squeezing the skin over the implant from the top end, the implant is
removed.
  Apply antiseptic at the site following implant removal.
 Syncromate-B Protocol

Day What To Do - DONOR What To Do -

RECIPIENT
0 SMB Implant +injection SMB implant + injection

7 FSH Nothing

8 FSH PG

9 FSH + PG + implant removal Implant removal

10 FSH Nothing

Oestrus + AI Oestrus
 12 AI Nothing

18 Collect embryos Transfer

NON-SURGICAL EMBRYO TRANSFER

 Performed non-surgically similar to artificial insemination.

 Requires a high degree of dexterity and skill.
 Contamination of the uterus should be minimised because it is more susceptible
to infection during the luteal phase.
 Faeces is evacuated from the rectum and the side of the CL is determined.
 Epidural anesthesia is induced to prevent defecation and to minimize straining.
 The perineal region is washed and the vulva is wiped dry.
 In the laboratory, the embryo is loaded into the 0.25 ml French straw. The straw
is inserted into the transfer or Al gun. The transfer gun is covered with a
sterilized sheath and fixed in place with the 0-ring. The gun is wrapped with a
second, sterile, larger (sanitary) sheath which is closed at the distal end over the
first to serve as a protective cover and permit passage of the gun through the
vagina without coming into contact with the vaginal flora.
  The embryo is gently deposited approximately one-third of the way into the
uterine horn ipsilateral to the CL and the gun is slowly withdrawn.
  It is important to bear in mind that trauma to the delicate endometrium can
result in bleeding, and blood (complement in the serum) is embryocidal.
  In Embryo Placement the tip of the transfer instrument can be seen through
the incision in this slaughterhouse specimen. Deeper placement may be ideal, but
excessive manipulation and trauma to the endometrium must be avoided.
CARE OF DONOR ANIMALS

 Immediately after flushing of donor animals, Prostaglandin F 2 alpha (25 mg; i/m)
is administered to cause rapid reduction in the size of the ovary, and to prevent
the establishment of pregnancy from an unflushed embryo if left in the uterus.
 Administration of Prostaglandin F2 alpha to donor animals will induce estrus
usually within 3-5 days - Remember not to breed the donor on this induced
estrus.
 Any contamination that might have gained entry at the time of the embryo
flushing will be eliminated during this induced estrus.
 Superovulation can be carried out thrice at two months interval without
drastically affecting the response.
 In between the superovulatory treatments, if the animals come in to estrus, they
can be artificially inseminated and single embryo flushing may be performed.
 CARE OF RECIPIENT ANIMALS

 Pregnant recipient cows should be maintained similar to that of other pregnant

cows.
 Presumptive pregnancy diagnosis can be made based on
o Palpation of the functional CL.
o Indirect estimation of progesterone in the milk or plasma on day 21-24 of
their cycle (16-19 days after transfer on day 5), or
o Identification of conceptus by ultrasonography around day 27.
 Definitive pregnancy examinations: at six weeks.
 Confirmative diagnosis: at three months by rectal examination.

SURGICAL EMBRYO TRANSFER IN GOATS

 There are 502 million goats in the world, approximately 56.5% of which are in
Asia. About 6 % of the world’s goats are found in developed countries and 94 % in
developing countries.
 Genetic improvement necessitates substitution of genetically superior animals for
those of little genetic merit.
 Embryo transfer is an advanced, but well established, animal breeding
technology.
 The procedure of superovulation followed by recovery of embryos and transfer to
synchronized recipients has proved to be an effective means of increasing the
contribution of superior females to the gene pool of the population.
 During the last two decades, tremendous progress has been made in the female
germplasm use through multiple ovulation and embryo transfer.
 The first record of successful embryo transfer in goat was reported by Warwick
and Berry (1949). Hunter et. al., (1955) transferred nineteen, 2-16 cell embryos to
18 recipient ewes, eight lambs were born. This procedure remains to be the basis
of surgical embryo transfer in sheep and goats today.

What is Surgical Embryo Transfer?

 Surgical embryo transfer is the process whereby embryos are surgically flushed
from the reproductive tract (either from the oviduct or uterus) of a "donor"
goat and surgically transferred in to suitably synchronized "recipient" in order
to establish a surrogate pregnancy.

SELECTION OF DONORS AND RECIPIENTS

Donors

 Virgin does 6 months and older.

Recipients

 Multiparous does (age approximately 1-1.5 years). The main purpose of having
multiparous does as recipients is to reduce the possible risk of dystocia that may
be seen with primiparous animals.

HORMONES USED FOR SUPEROVULATION

 Generally, the principles of superovulation in goats are similar as in cattle. The

following gonadotrophin preparations are most commonly used for
superovulation in goats
o Pregnant Mare Serum Gonadotrophin (PMSG): Administered as a single
subcutaneous or intramuscular injection given one day prior to the last
synchronization treatment.
o Pituitary Follicle Stimulating Hormone (FSH-P): is given at 12h intervals
in decreasing doses for 3-4 days on days 5-16 of the estrous cycle.
Prostaglandin F2 alpha (PGF2α)
o Horse Anterior Pituitary Extract (HAP)

SUPEROVULATION PROTOCOL

 There are a number of different superovulation protocols that may be used in the
goat. The following protocol produces optimal embryos
o The timing of oestrus in the donors is synchronized on day-0 with the
placement of a subcutaneous synchromate–B (6 mg of norgestomet),
SMB-ear implant.
o At the time of implant insertion 0.5 ml of SMB injection (1.5 mg of
norgestomet and 2.5 mg of estradiol valerate) is administered
intramuscularly.
o Inj. PGF2α (10 mg) is administered intramuscular on day 11.
o Starting from day-9, after insertion of implant a total of 200 mg of FSH-P
is administered intramuscularly over four days in twice daily injections. A
decreasing dose format starting with 32 mg the first day, ending with 16
mg on the fourth day of FSH-P treatment.
o The implant is removed on day-11.
o On day-13 the animals are observed for estrus signs and bred in the
evening to fertile males.

ESTRUS SIGNS

 Standing estrus lasts approximately 24 h in young does but may last 2-3 days in
mature does.
 The doe in estrus walks restlessly along the perimeter of her enclosure, searching
for a way to reach the buck, or remain close to the fence.
 The vulva becomes somewhat swollen and the doe’s tail wags vigorously. This tail
wag can often be observed even in the absence of a buck.
 The doe stands firmly when a buck attempts to mount and may even back up the
buck.
 The vaginal discharge at the beginning of estrus is clear and colorless, becomes
progressively white and more opaque towards the end of standing estrus.
 Ovulation typically occurs near the end of standing estrus and approximately 24
h after a serum peak in Luteinising hormone (LH).

SURGICAL EMBRYO COLLECTION

 Embryos are flushed on the second day following breeding or 72 h following

implant removal.
 Few hours prior to surgical collection embryo flushing medium and culture
droplets are prepared under sterile lab conditions and equilibrated at 38.5-39 °C
in a 5 % CO2 incubator.
 The animals are prepared for surgical collection and transfer by fasting for 24 h
and with holding water for 12 h prior to surgery.
 The animals are subjected to mid ventral laparatomy and anaesthesia induced by
i/m administration of 0.22 mg/kg.b.wt.
 xylazine followed by 11mg /kg.b.wt. ketamine 10 minutes later. This combination
leads to smooth induction with anaesthesia lasting approximately 45 minutes.

SURGICALLY EMBRYO TRANSFER INTO SURROGATE

 Only morphologically normal and 2-4 cell stage embryos are selected for transfer.
 Under the same surgical procedures as for the donors, the fallopian tube is
located and the embryos in minimum medium are transferred into the lumen to a
depth of 2-3 cm using a pipette with a sequencing tip after assessing the ovarian
response.
 Each recipient goat usually receives 2-3 embryos ipsilateral to the ovary
containing one or more corpora lutea.

RECIPIENT MANAGEMENT

 It is advisable that feed intake only be maintained or even reduced for recipients
after transfer, as excess feeding during early pregnancy reduces pregnancy rates.
 Recipient does are first evaluated by ultrasonography approximately day-35 from
the first day of estrus to detect pregnancies.
 A confirmatory ultrasound at day-55 will provide the most reliable indication of
viable pregnancy with fetal number and viability evaluated.
 Pregnant does are monitored daily throughout pregnancy and appropriate pre-
kidding procedures are performed.
 After completion of the gestation period (150 + days), the kids will be born.
 POTENTIAL USE OF EMBRYO TRANSFER IN EQUINES

 From a purely academic point of view, equine embryo transfer can be used for the
following:
o To obtain foals from subfertile mares;
o To better manage older, valuable broodmares;
o To circumvent problems with neonatal isoerythrolysis (jaundice);
o To obtain foals from mares engaged in competition;
o To manage mares that chronically abort twins;
o To further the knowledge of the mechanisms of the maternal recognition
of pregnancy;
o To produce multiple offspring
o To advance genetic progress.
 The first successful equine embryo transfers were reported in England a little
more than 10 years ago. Acceptance of the technique as an approved method for
producing foals by a major American breed association occurred less than 5 years
ago.
 The procedure was initially adopted to produce a single foal per year from barren
mares that could not carry a pregnancy to term; however, more recently, embryo
transfer is also being used to produce pregnancies from maiden fillies that are in
show competition. This is probably most often done in the Arabian breed.
 Irrespective of the reason for performing embryo transfer, the efficacy of the
procedure in horses is confounded by the inability to produce multiple viable
embryos via superovulation, a relatively short in vitro survival of the equine
embryo, difficulty in synchronizing ovulation among donors and recipients and
the high incidence of uterine infections in barren donor mares.
 Moreover, in the United States and in virtually all other countries, the two major
racing breeds, Standardbreds and Thoroughbreds, do not accept foals produced
by embryo transfer in their registries.


PREPARATION OF MEDIA, COLLECTION AND HANDLING OF

EMBRYOS

 Dulbecco’s phosphate-buffered saline (PBS) supplemented with 1 per cent heat-

inactivated fetal bovine serum appears to be most suitable medium for field use.
Dulbecco’s PBS is supplemented with 1 per cent fetal bovine serum and 100 IU
penicillin plus 100 μg streptomucin/ml to prepare the embryo recovery medium.
Embryo culture and transfer medium is prepared by adding 20 per cent heat-
inactivated fetal bovine serum to a volume of recovery medium. This is then
Millipore filtered through a 0.22 μm disposable Falcon filter.

Collection of Embryo

 Embryo collections are performed non surgically on days 6 to 9 after ovulation (

day of ovulation = day 0). The equine embryo does not enter the uterus until day
 6 after ovulation. The diameter of equine embryos over days 6 to 9 after ovulation
will range from 0.1 to 4.5 mm. By day 8 the equine embryo is generally 10-fold
larger than a bovine embryo of the same age and are too large to be transferred
without breakage.
 The nonsurgical collection procedure is a modification of the one developed for
cattle. An extended 30-French Foley or an 18-French Rusch catheter with a 30-
ml inflatable cuff is inserted through the cervix into the uterine body and is
secured in position by inflating the cuff with 1.5 to 30 ml of sterile water or
recovery medium. Once the catheter is properly positioned, both uterine horns
and the uterine body are filled simultaneously with 1 liter of medium by gravity
flow. The medium is then collected by gravity flow into sterile, 1 liter Erlenmeyer
flasks or graduated cylinders. This procedure is repeated three additional times.
Uterine palpation is used to facilitate recovery of medium from the uterus during
the last three flushes.

Embryo Handling

 Embyros are of greater density than the medium and therefore settle to the
bottom of the collection vessel within approximately 20 to 30 minutes following
collection. The upper 850 to 900 ml of medium is removed by pouring or
siphoning into another sterile container. The bottom portion of the medium is
then poured into a gridded sterile plastic petri dish. Attempts are first made to
identify the embryo macroscopically, and then the dish is searched into a 14-
gauge catheter (Soverign) attached to a 1-ml syringe and is then deposited into a
sterile plastic petri dish containing transfer medium. The embryo is then gently
agitated (washed) for approximately 1 to 2 minutes and then placed in a second
Petri dish containing transfer medium. The embryo is stored in the dish until
transfer.
 Results have indicated that equine embryos do not remain viable for more than
approximately 3 hours in Dulbecco’s PBS. Therefore, transport of fresh equine
embryos over long distances would seem impossible. Embryo freezing would be
an obvious solution to this problem.

TRANSFER OF EMBRYOS

Nonsurgical

 Embryos are aspirated into a Luter Flex 22-inch sterile large animal pipette or
similar pipette, which contains 10,000 IU penicillin plus 10,000 μg streptomycin.
The aspiration procedure has the following sequence: 1 ml antibiotics, 0.25 ml
air, 0.5 ml transfer medium, 0.25 air, 0.5 ml transfer medium containing the
embryo, 0.25 air and 0.05 ml transfer medium.
 The perineal area of the recipient is scrubbed with a dilute betadine solution and
water before transfer. The infusion pipette containing the embryo is passed
through the vagina and into the cervix following manual dilation of the external
cervical os.
  The operator’s hand is covered by a plastic palpation sleeve and a sterile surgical
glove. Before passing the pipette into the uterine body, the operator’s hand is
transferred from the vagina into the rectum to grasp the uterus.
 The uterus is elevated, and the pipette is than passed is then passed into the
lumen of the uterine body. Precaution is taken to keep physical trauma to the
endometrium to a minimum. The contents of the pipette are deposited from the
internal cervical and uterine bifurcation.

Surgical

 The most practical method for field use is via a flank in incision. This method is
similar to that used in cattle. Mares are given 250 mg Xylazine IV and 25 mg of
acepromazine IM.
 The paralumbar fossa is prepared for aseptic surgery and the incision is
infiltrated with 30 to 50 ml of lidocaine. A 15- to 20-cm vertical incision is made,
the muscle layers and peritoneum are bluntly dissected.
 The tip of the uterine horn adjacent to the corpus luteum is exteriorized, and a
small puncture is made into the cutting needle. The embryo is loaded into a 14-
gauge large animal Sovereign catheter or glass pipette in a total volume of
approximately 0.5 ml of transfer medium.
 The embryo is usually positioned between two air spaces in the catheter to
stabilize its position.
 The catheter is passed through the uterine puncture, and its contents are
deposited into the uterine lumen. The abdominal wall is then closed in a routine
manner

SPECIFIC USES OF EMBRYO TRANSFER IN SWINES

 The major reason for performing commercial embryo transfer in swine is to

prevent and/or control disease. Although swine producers invariably exploit
superior females, this is seldom the primary reason given for doing embryo
transfer in swine, because even the most valuable donors and embryos are
inexpensive compared with costs incurred when some of the common diseases of
swine are introduced into a susceptible herd.
 Export of swine embryos is likely to become another important reason for doing
embryo transfer once long-term storage of swine embryos becomes possible.

SYNCHRONIZATION OF ESTRUS, SUPEROVULATION AND

INSEMINATION

Synchronization of Estrus

 There are two methods commonly used to synchronize estrus for embryo transfer
purposes. The first method involves weaning a group of sows on the same day,
with estrus occurring 4 to 10 days later. However, if the sows are injected
subcutaneously with 500 to 750 IU of pregnant mare serum gonadotropin
 (PMSG) at weaning, a high proportion of sows will come into estrus 4 to 5 days
later.
 Another method frequently used to synchronize sows is breeding and then
aborting sows when they are between days 16 and 45 of pregnancy. Sows are
aborted with one injection followed 12 hours later by a second injection of
prostaglandin F2α (PGF2α) or one of its analogues.
 A high proportion of sows come into estrus 4 to 7 days after treatment, and
conception rates are high. Better synchrony can be achieved by injecting 500 to
750 IU of PMSG 12 hours after the second injection of PGF 2α. Often, a group of
sows will be synchronized by using both the weaning and the abortion methods.
 Two other methods are sometimes used to synchronize estrus in swine.
Pseudopregnancy may be induced with daily injections of estrogen preparation
on days 11 through 15 of the estrous cycle. The corpora lutea of pseudopregnancy,
which can be maintained for as long as 90 to 120 days, can be induced to regress
with PGF2α.
 Most sows return to estrus 4 to 7 days later. Another synchronization method is
to inject or to feed progestogens for about 14 to 16 days. However, most of these
progestogens induce ovarian cysts and are seldom used.

Superovulation

 Sows are usually superovulated with one injection of 1200 to 1500 IU of PMSG at
weaning or 24 hours after the first injection of PGF 2α in sows that were
synchronized by first being made pregnant or pesudopregnant.
 For gilts and sows in which embryos are collected on more than two consecutive
estrous cycles, the time of estrus is not controlled, and the animals are not usually
superovulated. If these sows are superovulated, PMSG is given on alternate
estrous cycles 4 to 5 days before the expected onset of estrus.
 As with other species, the superovulatory response is quite variable within and
among breeds. However, the average response for small groups of sows to 1200
to 1500 IU of PMSG ranges from 30 to 45 ovulations.
 Ovulation rates of 45 or more are not desirable because of the increase in the
proportion of abnormal embryos and the proportion of unfertilized eggs.
 Human chorionic gonadotropin (hCG), which can be used to control the time of
ovulation, is seldom used by embryo transfer specialists. If hCG is used, 500 IU is
given 3 to 4 days after administration of PMSG. Ovulation occurs about 40 to 42
hours after hCG injection.

Insemination

 For optimum conception and fertilization rate, donors should be mated or

inseminated every 12 hours throughout estrus. If hCG is used to control the time
of ovulation, the most important inseminations of fresh and frozen semen are
those done at 24 and 36 hours, respectively, after hCG injection.
 The volume of the inseminate should be 50 to 100 ml and contain at least 4 to 5
billion live spermatozoa.
 COLLECTION AND HANDLING OF EMBRYOS

Embryo collection

Timing

 Swine embryos are usually collected 4 to 6 days after the onset of estrus.
 Four days after the onset of estrus most embryos are at the four-to-eight –cell
stage, whereas on the sixth day after the onset of estrus, most are in the expanded
unhatched blastocyst stage.
 Most collections of swine embryos are done 4 days after the onset of estrus
because four-to-eight cell embryo are easily identified and evaluated.
 In contrast, morulae and the early blastocysts, which are most frequently
collected on day 5, are more difficult to identify and to distinguish from
unfertilized eggs.
 Collection and transfer are seldom done before day 4, not only because embryos
which are usually located in the oviduct of the donor, must be transferred to the
oviduct of the recipient, but also because it is more difficult to deposit embryos in
the oviduct than in the uterus.
 The collection of embryos on day 7 or later is not usually done because sows that
receive hatched blastocysts may be less likely to farrow than sows that receive
unhatched embryos.

Surgical Collection

 Embryos are collected surgically in a clinic or laboratory. Anesthesia is induced

by injecting a barbiturate into the marginal ear vein and is maintained with
halothane using a closed-circuit anesthesia machine.
 A small mid ventral incision is made to expose one ovary along with the adjacent
oviduct and about 30 cm of the uterine horn. A small incision is made for
insertion of a glass cannula on the antimesometrial side of the uterine horn at
about 20 to 25 cm from the uterotubal junction.
 To avoid contaminating the cannula with blood when it is introduced into the
uterine horn, it is important (1) to squeeze the blood vessels on the mesometrial
side with thumb and forefinger while forcing the blunt end of a scalpel handle
through the wall of the uterus on the opposite side and (2) to insert the cannula
into the uterine lumen as soon as pressure on the blood vessels is removed.
 The glass cannula should be about 12 to 15 cm long and 9 to 11 cm in diameter.
The end that goes into the uterine horn should be cut at a 45°angle and flared.
The opposite end of the cannula should have a bend of about 45° located 1 to 2
cm from the end. The glass cannula is inserted about 2 to 3 cm into the uterine
horn and is held in place with a towel clamp.
 To collect the embryos about 40 to 50 ml of the medium warmed to 37° C is
placed in a syringe fitted with a blunt 12 to 14-gauge needle. The needle is
inserted into the oviduct, and the entire medium is flushed into the oviduct,
through the uterine horn, out the cannula and into a Petri dish. After removing
 the entire flushing medium from the uterine horn, the incision is closed before
repeating the entire procedure in the second uterine horn.
 Following the surgical collection of embryos, swines are especially prone to form
adhesions of the reproductive organs. Therefore, to reduce the possibility of
adhesion forming, it is essential to (1) maintain asepsis throughout the
procedure, (2) handle the reproductive organs gently and only when necessary
and (3) keep the exposed reproductive organs moist at all times with saline or
another physiological solution.

Handling, storage and evaluation of embryos

Searching for and Handling Embryos

 The flushings, are examined for embryo with a stereomicroscope. Searches are
done at 10 to 20x magnification and the evaluation of embryos at 50x or 70x.
Good optics and high magnification are particularly important for distinguishing
morulae and early blastocysts from unfertilized eggs.
 As embryos are located, they are transferred to culture plates or other dishes that
contain fresh medium warmed to 37ºC. After several rinses in fresh medium, the
embryos are stored until transferred to the recipient.
 Tuberculin syringes fitted with a tom cat catheter or a glass pipette are frequently
used to handle embryos.

Short-term Storage

 The medium for the flushing procedure can also be used to store embryos in vitro
for several hours. Some of the media used for flushing and storing embryos
include Brinster’s solution, Ham’s F-10 and TCM-199 with bicarbonate.
 Embryos should be stored in fresh medium at 37°C. Although not recommended,
it is possible to obtain acceptable conception rates with embryos stored at room
temperature for 2 hours.
 Swine embryos have been cultured for 24 hours without a decrease in embryonic
survival rates after transfer to recipients.

Evaluation of Embryos

 Evaluation of the quality of embryos is done by examining the general

morphologic appearance of the embryo at the time of collection. In general, the
cleavage rate of embryos collected from a donor is quite uniform.
 On days 4, 5 or 6 the stage of development for most normally developing embryos
ranges from four to eight cells, eight cells to morulae and blastocysts,
respectively.
 Once embryos pass the eight –cell stage, cells fuse, making it difficult to identify
and to count individual cells. Therefore, considerable experience is required to
distinguish 8- to 16-cell embryos, morulae and early blastocysts from
degenerating unfertilized eggs. Because four- to eight –cell embryos are easily
identified and evaluated, most embryo transfer specialists prefer to collect
 embryos from donors when the embryos are expected to be at the four-to-eight-
cell stage. However, in a recent study it was shown that sows that received
morulae were more likely to farrow than sows that received four-to-eight –cell
embryos.

TRANSFER OF EMBRYOS

 Practical methods for the nonsurgical transfer of swine embryos have not been
developed. Surgical transfers are usually done on the farm rather than in a clinic
or a laboratory to reduce the risk of introducing disease. Anesthesia is induced
and maintained by injecting a barbiturate into the marginal ear vein. The
reproductive tract is reached through a mid ventral incision. Corpora should be
examined for appropriate stage of development before embryos are transferred to
the recipient. The uterine horns should also be examined for abnormalities,
especially if gilts are used as recipients.
 Embryo are transferred to the recipient by one of two methods. In one method a
fine catheter or pipette that contains the embryos is passed through a small
puncture wound into the lumen of the uterus. The embryos are deposited wound
does not require sutures. Inexperienced individuals should be especially careful
not to deposit the embryos into the endometrium or the myometrium. Depositing
embryos into the wall of the uterus is more easily done in swine than in bovine or
in ovine. However, this complication and hemorrhaging of the puncture wound,
which sometimes occur, can be avoided. This is accomplished by introducing a
tom cat catheter or a piece of rubber tubing, which contains the embryos, into the
oviduct. The distal end of the tubing is held firmly in place while the embryos are
flushed into the uterus with a syringe that is attached to the other end of the
tubing.

FACTORS AFFECTING RESULTS

 The day of collection and transfer of embryos may influence results. Best results
are obtained when the donor comes into estrus from 2 days before to 1 day after
the recipient. Higher rates of farrowing may be possible when embryos are
collected and transferred within 6 days after the onset estrus.
 For optimum results, at least 12 embryos of high quality should be transferred to
each recipient. Pregnancy fails to occur if there are too few embryos between days
12 and 17 or if embryos are not distributed throughout most of the uterine horns.
 As in other species, the time and conditions of in vitro storage also influence
results. Until culture methods improve, swine embryos should be transferred to
the recipient as soon as possible after collection. If embryos must be stored for 4
hours or more before transfer, better results may be obtained by collecting
embryos that are past the eight-cell stage.
 Lastly, considerable experience with surgical collection of embryos is required to
minimize the possibility of donors forming adhesions of the reproductive tract,
 which can cause infertility. Sows are more likely than ewes and cows to form
adhesions following the surgical collection of embryos.

MODULE-34
IMMUNOMODULATION FOR ENHANCEMENT OF FECUNDITY

Immunomodulaton in reproductive disorders of livestock is a new area of

immunotherapeutics. It is mainly used to treat inflammatory conditions of the
reproductive disorders such as endometritis, metritis, vaginitis in which the immunity
barrier of the host is breached. Immunomodulaton is also done using plant
preparations. The normal uterine defense mechanism is brought about by

 The anatomical barriers which include vulva, vestibule and cervix,

 The physical barrier which includes the cervical-vaginal mucous.
 The contractions of circular and longitudinal uterine muscles that help to propel the
microbes outside.
 Chemical barrier which consists epithelial and stromal cells which secrete soluble
factors and cause cell to cell adhesions
 Immunological barrier constituted by the cellular immunity comprising of
polymorphonuclear inflammatory cells (PMNs), lymphocytes and humoral immunity by
antibodies.

An ideal immunomodulator should meet the following criteria.

 It should be nontoxic for animals even at high doses.

 It should not have teratogenic, carcinogenic or any other side effects.
 It should have a short withdrawal period with low tissue residues. It should not
be secreted in milk or come in eggs.
 It should stimulate both specific as well as paraspecific immune responses in the
body.
 It should act as adjuvant when given with vaccines.
 The immunomodulator or its breakdown products should be either inactive or
readily biodegradable in the environment.

UTERINE CELLULAR AND HUMORAL IMMUNITY

Uterine Cellular Immunity

 Polymorphonuclear inflammatory cells (PMNs), blood monocytes and tissue

macrophytes are regarded as ‘professional phagocytes’ in the cellular defenses
against pathogenic microorganisms.
  Phagocytosis involves chemotaxis, adherence and attachment of leucocytes to cell
surface antigens presented by the organism before it is ingested by the phagocyte
and finally digested.

Uterine Humoral Immunity

 Immunoglobulin concentrations in uterine secretions reflect both the extent of

the endometrial inflammatory process in the face of microbial challenge.
 Immunoglobulins have been found in bovine uterine secretions and their
protective role against pathogens have been widely reported.

INFLUENCE OF STEROID HORMONES ON UTERINE IMMUNE

RESPONSES

 The cyclical pattern of steroid hormone concentrations, characteristic for

different stages of estrous cycle, regulates potential pathogenicity of micro-
organisms that contaminate the uterus postpartum.
 Steroid hormones influence functional activity of leucocytes migrating into
uterus.
o During estrogen phase of the ovarian cycle there is increased mucus
production, and identified PMN activity;
o During the luteal phase, there is reduced endometrial epithelial
permeability to bacteria that delays leukocyte stimulation, and an absence
of detoxifying agents in uterine secretions.
 Disruptions of these mechanisms allow opportunist pathogens, mostly
microorganisms found in posterior gastrointestinal tract and around perineal
area.
 Inflammation of endometrium can occur following
o coitus,
o artificial insemination (AI),
o more commonly in cattle after parturition,
o in majority of cattle,1-4 weeks after calving,
 Micro-organisms contaminate the uterine lumen but self-cure usually occurs
within 6 weeks postpartum. In those cows unable to eliminate infection,
endometritis may develop subsequently. Endometritis causes a significant delay
in calving to conception intervals.

CAUSATIVE ORGANISM FOR THE INFLAMMATION OF THE

BOVINE GENITALIA

 Various Gram-positive and Gram negative aerobes and anaerobes, have been
isolated from the bovine uterus following coitus, Al, or postpartum.
 The bacterium most frequently isolated is Acranobacter pyogenes (formerly
known as Corynebacterium pyogenes). Other bacteria, such as Streptococci spp.,
 Staphlococci spp. and Escherichia coli have also been cultured and identified with
endometritis of varying severity.
 Anaerobes affecting bovine genitalia are Fusobacterium necrophorum and
Bacteroides melaninogenicus.
 Diagnosis is usually done by rectal palpation,vaginal examination, bacterial
culture, endometrial biopsy, endometrial cytological examination and Ultrasound
examination of the genitalia.
 Intrauterine oxygen reductase potential (Eh) can be used to find the degree of
bacterial infection as more Eh potential indicates infection. Moreover, pH in
bacterial infection as more Eh potential indicates infection. Moreover, pH in
bacterial infection ranges between 6.9-7.3. Peripheral blood haptoglobin, which is
an acute phase protein synthesized in the liver in repose to tissue damage has
been used as a marker for endometritis , its major function is to bind free
haemoglobin and protect the host from the oxidative activity of haemoglobin.
Another circulating acute phase protein, α1-acid glycoprotein is also screened for
detecting endometritis.

USE OF IMMUNOMODULATORS IN TREATMENT

 Plenty of research asociated with immunomodulators of uterine defence

mechanism and their application as treatments for metritis or endometritis has
been carried out in mares.

Hormones

 Hormones commonly used as immunomodulators are

o Prostaglandins
o E.Coli Lipopolysaccharides
o Derivatized Polysaccharides
o Oyster Glycogen
o Leukotriene B4

Other Agents

 Other agents commonly used as immunomodulators are

o Plant Preparations

HORMONES

Prostaglandin

 Prostaglandin F2α is a naturally occurring luteolytic agent in non-pregnant

ruminants. Oxytocin stimulates uterine secretion of PGF2α which in turn,
stimulates oxytocin release from the corpus luteum; this luteal oxytocin positively
stimulates endometrial secretion of PGF2α. As a result, peripheral blood
concentrations of progesterone progressively fall from day 16 of the estrous cycle
 until estrus, when they are undetectable; conversely, estradiol concentrations,
rise to reach a peak when the animal shows standing heat. Hence, there are three
reasons for using PFG2α for treatment of endometritis.
 Exogenous prostaglandin therapy administered to cattle presenting a functional
corpus luteum will induce luteolysis and bring the animal into heat, thereby
removing the suppressive affect of progesterone on the uterine defense
mechanism or, alternatively, stimulate it through estrogen causing myometrial
contraction which expels debris and micro-organisms that contaminate the
uterine lumen after calving. Moreover, PGF2α may have stimulatory effect on the
phagocytic activity of uterine leucocytes. Hence, the luteolytic action of PGF2α
has been used to treat endometritis in cows where a functional corpus luteum is
present.

E.coli Lipopolysaccharides

 E. coli lipopolyaccharides (LPS) are thought to act as a chemo attractant to

PMNs. This increase in numbers of PMNs in the endometrium may help to
resolve endometritis in both cows and mares. A single intrauterine infusion of
100 μg. E. coli LPS at estrus in repeat breeding cows cleared the bacterial
infection from the uterine lumen within one estrous cycle. Following this
treatment, a majority of the repeat breeding cows with turbid vaginal discharge
conceived in a preliminary trial. Similar results have been described following
intrauterine administration of LPS in ewes. When E. coli endotoxin has been
similarly administered to cows 5 days after calving at rates of 5 g/kg body-weight,
the toxin was absorbed with associated transient clinical signs causing
enhancement in immune response.
 The contaminating micro-organisms associated with uterine infection
postpartum may themselves secrete endotoxins detectable in peripheral blood,
and may interfere with ovarian function characterized by short cycles in cows.
Variation between species in endotoxin absorption from uterus postpartum may
be associated with differences in placentation and the extent of trophoblast
invasion of the endometrium. In species, where there is little clinical or
physiological evidence of endotoxin absorption, an intact endometrium may act
as a cytological barrier to totally or partially exclude the toxin.

Derivatized Polysaccharides

 They are generally high molecular weight dextrans.

 Their action is complex possibly by T or B cell mitogenicity e.g. oyster glycogen.

Oyster Glycogen

 Intrauterine administration of oyster glycogen (OG) caused PMN migration, up

to 90% of all cells identified in uterine secretions being neutrophils.
 Measurable immunoglobin (IgG) concentrations were found in uterine secretions
following administration.
Leukotriene B4

 They are generally glycoproteins of molecular weight around 20 kDa.

 They up regulate immune response. It is an effective chemo attractant,
stimulating preferential migration of PMNs into the lumen of the bovine uterus.
 A single intrauterine treatment of a 30 mm01/l solution increased the
intrauterine leukocyte count 5-10 times within 24h.
 Granulocyte-macrophage colony stimulating factor (GM-CSF; lymphokines) are
highly effective chemo attractants in mares, but their activity in cattle has not
been studied.

OTHER AGENTS USED AS IMMUNOMODULATORS

Plant Preparation

 The active principles of plants may be carbohydrates, glycosides, tannins, lipids

and akaloids. WHO has also been recommended the promotion of native,
practices and conservation and cultivation of medicinal plants.
 Despite these facts, little attention has been given to describe and explore the
traditional herbal medicines used by the local and tribal communities in specific
areas for the control and treatment of various reproductive disorders in farm
animals.

Plants Used in Disorders

 Uterine disorders
o A wide variety of medicinal plants and their preparations are found to be
useful in treatment of reproductive ailments.
o They include Abroma augausta, Aristolochia bracteata, Datura alba,
Mytrus communis, Salvadora species, Saraca indica, and ViI species.
 Anestrum
o Anestrum in cows/buffaloes can be overcome by feeding fenugreek
powder. Feeding of bamboo leaves brings cattle and buffaloes into regular
heat. Feeding of leaves of jute plant (about 2-2.5 kg) brings animal into
heat.
o Feeding leaves of Mann tree (approx:15-20 kg) can overcome anestrus
condition.
o A mixture of black pepper (10 grains) and Vanghuchi (20-25 gm) is given
twice a day at interval of 6-8 hrs for 1-2 days for treatment of anestrus.
 Decreased Conception Rate
o Feeding 200 gm germinated Bengal gram (Cicer arietnum) soaked
overnight to the animal continuously for one week.
o Along with this pounded leaves and unopened fruit of Yanai (Pedalium
murex) may be given once a day for three days without adding water.
 Retained Placenta
 o Bamboo leaves and bark are boiled with paddy husk and fed to cows for
expulsion of placenta.
o Ficus bengalensis is used for treatment of retained placenta. Leaves and
twigs of ber (Zyzphus mauritiana) are collected and burnt. The ash is given
to the animal with water to induce the placenta to drop.
o About 250 g of leaves of jingara used for retention of placenta showed 60%
success rate.
o To prevent abortion farmers feed piece of stem of banana (Musa
paradisiacal).After conception, buffalo is fed 10-15 kg; cow is fed 5-10 kg
pieces of stem of banana. It is fed for five times over a period of 2-3 days.
It helps to reduce internal heat and improves health. This practice has
been in use for the last 30-40 years.
 Uterine Infection
o The immunomodulatory property of Aristolochia indica (Ischamur) can be
proved an aid in preventing the uterine infection by augmenting local
immune system. Approximately 100 gm of root or bark of Convolvus
micrphyllus (roots) powdered and mixed with 300 ml of water and boiled.
This concoction is filtered and then cooled. This is given to affected
animals once a day for 3 days.
 Equiman
o This is an immunomodulating phytopreparation having its affect on
bovine immune cells. Equimen is a fixed combination of Echinaces
purpurea, Thuja occidentalis and elemental phosphorous in different
concentration. The preparation reduces the spontaneously generated
reactive oxygen species (ROS) by neutrophils.
o Phytotherapy has been followed in the treatment of animals from
thousand of years since ancient time. Plant based drugs (natural drugs)
may be used directly i.e. they may be collected, dried and used as a
therapeutic agents (crude drugs) or their active principles, separated by
various chemical process which are employed as medicines.
o


DR.KARUN KANT KAMAL

BVN-10023