MALE REPRODUCTIVE SYSTEM

The male reproductive tract developed from the

wolffian duct and body or mesonephric tubules.
 The male reproductive organs consist of two testes which are present in the scrotum ,
ducts, accessory sex glands , penis and prepuce.
 The testis produces spermatozoa and the male sex hormone (androgens).
 The scrotum helps in maintaining optimum temperature for the spermatozoa
production.
 The other structures help in transport of semen from site of production to the site of
deposition ie. Vagina
 It comprises of three components, they are,
o The primary sex organ / gonads – the testes.
o The accessory sex organ; the epididymis, ductus deferens, vesicular glands,
prostate gland and bulbourethral glands.
o The copulatory organ – the penis

Basic components of male reproductive system includes

 Scrotum
 Spermatic cord
 Testis
 Epididymis
 Vas deferens or ductus deferens
 Pelvic urethra
 Accessory sex glands
o Seminal vesicle or vesicular gland
o Prostate gland
o Cowper’s gland or Bulbo-urethral gland
 Penile urethra or extrapelvic urethra
 Penis
 Prepuce

EMBRYOLOGY OF MALE REPRODUCTIVE SYSTEM

 The urogenital system is formed mainly from mesodermal tissue that in early
embryonic life forms the nephric and genital regions.
 In the cranial portion of the nephric region, segmental tubules initially form the
pronephros each with a pronephric duct that runs to the primitive cloaca.

 Later other segmental tubules form caudal to the pronephros and unite with the
pronephric ducts to form another temporary excretory organ,
 the mesonephros andmesonephric ducts or Wolffian body and ducts and the
pronephros degenerates.
 Later a third and more permanent excretory organ forms more caudally from an out
growth of the mesonephric duct to become the metanephros or true kidney with its
ureter and bladder.
 The mesonephros then degenerates but its duct system is utilized in the male to
transport spermatozoa from the testes to the pelvic urethra.
 Remnants of the paramesonephric or mesonephric ducts may persist in the adult
male as the uterus masculinus and appendix testis.
 In the fetus most of the urine from the fetal kidneys passes into the bladder and
through the urachus into the allantoic cavity.
 The testes form initially as undifferentiated gonads late in the embryonic period in
the genital region or gonadal or genital ridge between the dorsal mesentery and the
mesonephros.
 Although the sex of the embryo is determined at the time of fertilization, sexual
differentiation doesn’t occur until early fetal period after the primordial germ cells
have migrated to the gonadal ridge from the wall of the yolk sac in the region of the
hind gut and structural changes occur in the gonad.
 Testis cords, rete testis cords and the tunica albugeniea form early in the fetal period.
 Primary medullary cords are characteristic of the early male gonad.

Primary medullary cords are characteristic of the

early male gonad.
 These testis cords of primitive germ cells and supportive sertoli cells remain solid
until just before puberty.When the seminiferous tubules are formed and they join the
rete testis by means of tubuli recti.
 The rete testis in turn enters the efferent tubules, epididymis and ductus deferens that
are formed from the mesonephric tubules and duct.
 Certain tubules of the mesonephros that do not form the testicular duct system may
remain as small remnants called the ductuli aberrants and appendix testis near the
head of t epididymis and the paradidymis near the body or tail of the epididymis
 During the middle trimester of gestation, the interstitial or Leydig cells are numerous
especially in the equine fetus.
 In the very early fetal period, the external genitalia in the cloacae region are also in
the indifferent stage characterized by a genital tubercle which elongates to form
the phallus or penis with two genital folds that form the urethra and the two genital
swellings that form the scrotum.
 Development of the external genitalia in males is hormonally controlled by androgens
presumably from the fetal gonad.

Undifferentiated genital structures in the embryo and their adult male

counterparts
 EMBRYONIC STRUCTURE ADULT
Gonad Testis
Mesentry Mesorchium
Gubernaculum Ligamentum testis
Paramesonephric duct or Appendix testis and uterus
mullerian duct masculinus
Mesonephric duct or wolffian Efferent tubules, epididymis, vas
duct and body deferens , ampulla
Genital tubercle Penis
Genital folds Penile urethra
Genital swelling Scrotum
TESTICULAR DESCENT
 Testis during the foetal life is located in the abdominal cavity, they pass through the
abdominal inguinal canal, and inside the scrotum
o Enlargement of the gubernaculum and its disappearance play an important role
in testicular descent
o Intra-abdominal pressure in the foetus also determines testis descent
o Androgen from foetal testis favours testis descent

Androgen from foetal testis favours testis

descent

Time of testicular descent

o Stallion - 9 -11 months of gestation

o Cattle -3 ½ - 4 months of gestation
o Sheep and goat -2 ½-3 months of gestation
o Swine -3 months of gestation
o Dogs- 5 days after birth
 Failure of testis to descent into the scrotum is called as cryptorchidism.
 The retained testis is called as cryptorchid testis.

Failure of testis to descent into the scrotum is called as

cryptorchidism.
SCROTUM
Scrotum

 The scrotum is a bi-lobed sac or pouch (derived from the skin and fascia) and is the
external part of male reproductive system which encloses the testis.
 It is located between the thighs except in case of boar and tom cat.
 In boar and tom cat, it is located caudal to the thighs, caudal and ventral to the
ischiatic arch.
 Scrotum externally composed of skin which is relatively devoid of hair except in ram
(male sheep) and buck (male goat) and tom cat (male cat).

 Beneath the skin is the dartos muscle, which consists of fibroelastic tissue and
unstripped muscle.
 Dartos muscle divides the scrotum in to two halves.
 It is closely attached with the tunica vaginalis and scrotal ligament which is a remnant
of gubernaculum. Scrotal ligament is absent in bulls.
 Testis and epididymis are fixed in scrotum by means of scrotal ligament which is
attached near the tail of the epididymis and mesorchium. (Click here to view
animation)

LOCATION OF SCROTUM IN DIFFERENT SPECIES

Scrotum in bull located Scrotum in stallion located
between thighs between thighs

Scrotum in buck located Scrotum in ram located

between thighs between thighs

Scrotum in dog located Scrotum in boar is located

between thighs caudal to the thighs,
caudal and ventral to the
ischiatic arch

Scrotum in tom cat is located caudal to the thighs,

caudal and ventral to the ischiatic arch
Scrotum has the following 8 layers from externally

 Epidermis –
outermost layer
 Dermis
 Dartos muscle
 Connective tissue
 Parietal layer of
vaginal process
(PVP)
 Cavity of vaginal
process (CV)
 Visceral layer of
vaginal process
(VVP)
 Tunica albuginea

 Tunica albuginea penetrate the testicular parenchyma to join at mediastinum.

 These fibrous septa divide the testicular parenchyma into lobules.
 The lobules are having the highly coiled seminiferous tubules.
 About 75% of the testicular mass is composed of seminiferous tubules.

Scrotum, cremaster muscle and pampiniform plexus maintains the

testicular temperature
Functions

 It encloses the male gonad

 It provides suspensory attachment of testes with remaining animal body.
 It protects the testes from adverse stress conditions.
 It serves as a thermic chest of male gonad.
 It maintains gonadal temperature through thermo regulatory mechanism for
optimum spermatogenesis.

The scrotum becomes flaccid and elongated due to relaxation of

the dartos and cremaster muscle, under hot climate. It becomes
contracted and wrinkled during cold climate
Blood supply
 External pudental artery and also Internal pudental artery in boar and cat
Nerve supply

 Genital nerve which is a branch of genito-femoral nerve arising from second to fourth
lumbar nerve and perineal nerve.
 Smooth muscle of the scrotum is supplied by the spermatic plexus from pelvic plexus.

TESTIS

Synonyms: Orchium, male gonad

 Testis originated from Greek word- Orchis

 Testis is the primary andrological organ which produces spermatozoa and male sex
hormone (testosterone).
 Morphologically oval shaped, paired glands. Right and left testicles are separated by
muscular septum which is formed by dartos muscle.
 Located outside the abdominal cavity. In most of the species, located between the
thighs within the scrotum.
 Testes are intra-abdominal in elephant, whales, seal, dolphin, birds, rhinoceros
(testchondas)
 The testis is supported in one of the two scrotal pouches where it is held by its tunics
and by the spermatic cord.

The Spermatic cord is composed of

 Spermatic artery
 Spermatic vein
 Spermatic nerve
 Internal cremaster muscle
 Lymphatic vessels
 Vas deferens
 Tunica vaginalis propria

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Embryonic Development

 Origin of testes is initiated from gonadal / genital ridge. It occurs in 4 phases.

o First Phase
 Migration of the germ cells at the gonadal ridge.
o Second Phase
  Formation and proliferation of the blastema for genesis of indifferent
gonad.
o Third Phase
 Migration of mesonephric cells into gonad.
o Fourth Phase
 Sexual differentiation and its development into testis

The testis consists of the

 Testicular capsule
 Parenchyma
 Mediastinum
 Rete tubules

The testicular parenchyma consists of

 Seminiferous tubules
 Interstitial cells of leydig
 Capillaries
 Lymphatic vessels
 Connective tissue

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Testis proper is covered by 2 capsules

 Tunica vaginalis propria

 Tunica albuginea
 Tunica vaginalis propria is composed of
o Parietal layer – faces scrotum

Visceral layer – faces testis

Internal features

(Press the refresh button to view picture)

 The distal end of the testis is attached to the
scrotum by the scrotal ligament
 The testis proper is closely covered by a thin
serous membrane - tunica vaginalis
propria. Beneath this structure is a dense,
thick connective capsule – tunica albuginea,
from which septa radiate to the mediastinum
testes except in stallion to form the lobules of
the testis.
 Within this lobules are the seminiferous
tubules, which are lined by germinal
epithelium that produces spermatozoa and
sertoli cells which are located in between the
developing germ cells.
 The sertoli cells are otherwise called as Nurse
cells or Pillar like cells or Sustantacular cells.
 The interstitial cells (Leydig), which lie
between the seminiferous tubules, secrete
male hormones.
 The seminiferous tubules converge at the apex
of the lobule at the receptacle to join the
straight tubules.
 The straight tubules are also called as tubuli
recti that enter the rete testis, a structure of
anastomosing spaces located in the
mediastinum testis.
 There is no mediastinum testis in the stallion
as is present in other animals.
 The collecting tubules join the efferent
tubules.
 From the rete testis, 6-24 efferent ducts arise;
these ducts form a single duct called as caput
epididymis.
 On examination of the testes the torturous
configuration of blood vessels is most readily
noted in the tunica albuginea in the bull.
 This is a further provision assisting the heat
regulatory mechanism of the testis.
  The consistency of the testis is usually turgid.
 The parenchyma is yellow to reddish brown in
colour and bulges on section.

Approximate length of seminiferous tubule

Swine 6000 meters

Bull 5000 meters
Ram 4000 meters
Dog 150 meters
Cat 25 meters
Cross section of seminiferous tubules contains
three layers

 Outer capsule
 Basement membrane
 Testicular cells

Testicular cells is composed of

 Germinal cells
 Parenchymal cells or testicular somatic
cells
 Sertoli cells

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Physiological functions of testis

 It produces male sex hormone(androgen).

 It secretes testicular fluid.
 It nourishes the newly born male gamete.
 It helps in the transportation of spermatozoa.
 It provides blood-testis barrier.
 It secretes estrogen in limited amount.

Associated structures
  Testicular ligament
 Testicular mesentry
 Testicular appendix

Testicular ligament

 Fetal ligament which is a derivative of gubernaculum and present during the descent
of the testis in to the scrotum. Later on, it gets atrophied.

Testicular mesentry

 It is a part of primitive mesentry which encloses fetal testis and is present during the
descent of testis in the scrotum.
 It continues in the form of peritoneal fold between testes and epididymis during post-
natal life of livestock.

Testicular appendix

 It is non-functional residual part of embryonal hood during post-natal life of

livestock.

Blood supply
 The testis is richly supplied with blood by the spermatic artery/ testicular artery, a
branch of the abdominal aorta.
 The veins on leaving the testicles form a network, the pampiniform plexus around the
artery in spermatic cord.
 The spermatic vein which issues from this plexus, usually joins with the posterior
venacava on the right side, the left renal vein on left side.

Nerve Supply

 The nerves derived from the renal and posterior mesenteric plexus form the
spermatic plexus around the vessels to which they are chiefly distributed.

SPECIES DIFFERENCES IN TESTICULAR CHARACTERS

TESTICLES' MEASUREMENTS IN FARM
AND PET ANIMALS
Character Bull Stallion Ram Boar Dog Cat
Shape Oval Oval Oval Oval Round Round
to oval to oval
 Length 10 - 15 7.5 - 12.5 7.5 - 10 - 15 2-4 1.2 - 2
(cm) 11.5
Diameter 5 - 8.5 4-7 3.8 - 5-9 1 - 2.5 0.7 -
(cm) 6.8 1.5
Weight 200 - 200 - 300 200 - 600 7 - 15 0.7 -
(gm) 500 400 1.5
Plane Vertical Horizontal Vertical Vetrical Oblique Verical

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SPECIES DIFFERENCES IN TESTICLES OF FARM AND PET ANIMALS

Bull

 Shape: Oval
 Length: 10-15 cm
 Diameter: 5-8 cm
 Weight: 200-500 gm
 Long axis: Vertical
 Head of epididymis: Lies dorsal to the testis
 Body of epididymis: Lies caudal and medial to the testis
 Tail of epididymis: Lies ventral to the testis
 Vas deferens: Lies parallel to the body of the epididymis and medial and cranial to it
 Location: Anterior prepubic region
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Stallion

 Shape: Oval but compressed from side to side

 Length: 7.5-12.5 cm
 Thickness : About 5 cm
 Weight: 200-300 gm
 Long axis: Horizontal
 Head of epididymis: Lies cranial to the testis
 Body of epididymis: Lies dorsal to the testis
 Tail of epididymis: Lies caudal to the testis
 Vas deferens: Lies dorsal and medial to the body of the epididymis and testis
 Location: Intermediate to the prepubic and perineal region

Appendix testis
Remnant of the paramesonephric duct, present as a
small protruding structure on the cranial pole of the
 testis. It is also seen occasionally in ram.

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Ram and Buck

 Shape: Oval
 Length: 7.5-11.5 cm
 Diameter: 3.8-6.8 cm
 Weight: 200-400 gm
 Long axis: Vertical
 Head of epididymis: Lies dorsal to the testis
 Body of epididymis: Lies medial to the testis
 Tail of epididymis: Lies caudal to the testis
 Location: Anterior prepubic region

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Boar

 Shape: Oval
 Length: 10-15 cm
 Diameter: 5-9 cm
 Weight: 500-800 gm
 Long axis: Vertical
 Head of epididymis: Lies ventral to the testis
 Body of epididymis: Lies cranial to the testis
  Tail of epididymis: Lies dorsal to the testis
 Vas deferens: Lies medial and cranial to the testis
 Spermatic cord is long
 Location: Caudal to thighs, caudal and ventral to the ischiatic arch (perineal region)

 Click here to view animation

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Dog

 Shape: Oval
 Length: 2-4 cm
 Diameter: 1.2-2.5 cm
 Weight: 7-15 gm
 Long axis: Horizontal and
oblique
 Head of epididymis: Lies
cranial to the testis
 Body of epididymis: Lies
dorsal to the testis
 Tail of epididymis: Lies
caudal to the testis
  Vas deferens: Lies dorsal
and medial to the body of
the epididymis
 Location: Intermediate to
the prepubic and perineal
region

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Tom cat

 Shape: Oval
 Length: 1.2-2 cm
 Diameter: 0.7-1.5 cm
 Location: Caudal to thighs, caudal and ventral to the ischiatic arch (perineal region)
 THERMOREGULATION OF TESTIS

 For effective functioning, the testes have to be kept at a temperature of 4-

6° C lower than the body temperature.

Scrotal skin and sweat glands

 It has got temperature receptors. When there is a elevated environmental

temperature, these receptors elicit the response by producing panting and
sweating and lower the body temperature.
 Scrotal skin is devoid of subcutaneous fat.
 It is enriched with large adrenergic sweat glands.
 Sweating allows scrotum to be cooled by evaporative heat transfer.

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Dartos muscle

 It is an open mesh like smooth muscle layer which lies beneath the scrotal
skin.

By contracting in cold weather to hold the testes against the

body and by relaxing in warm weather, it is the principal
thermoregulator of the testis.
  The contractile characteristics of dartos are androgen dependant and its
ability to contract in cold climates is lost in castrated males.
 Action of dartos muscle in stallion is enhanced by presence of smooth
muscle in the spermatic cord and tunica albuginea.

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External cremaster muscle

 It raises the testis, thereby playing a role in the thermoregulation of testis.

 It contracts and relaxes, creating a ‘pumping action’ on the pampiniform
plexus, thus facilitating blood flow and enhancing cooling efficiency.

Pampiniform plexus

 In the proximal end of testis, testicular artery is coiled and is surrounded

by network of spermatic vein. This arrangement is called as pampiniform
plexus.
 Blood present in vein cools the incoming blood of artery.

Tunica albuginea

 It is richly supplied with network of blood vessels and plays role in

regulation of testicular temperature
 In human beings, the difference between body temperature and testicular
temperature is 2oC.
o In bulls : 4oC
o In ram : 5-7oC

The anatomical arrangement of testis and features of testicular blood vessels

maintains the testicular temperature
BLOOD TESTIS BARRIERS
Seminiferous tubules are not penetrated by blood and lymph
vessels

 The peritubular cells (Myoid cells) surrounding the seminiferous tubules and the
sertoli cell junctional complexes form the blood testis barrier.
 The primary function is to prevent autoimmune reactions from destroying the
developing germ cells.
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Myoid cells (View the picture)

 They are the incomplete or partial barriers located in the basement membrane of
seminiferous tubules.
 This barrier is poorly developed in bull, ram and boar.
 It is not important in farm animals.

Sertoli cell junction

 The tight junctions formed between two adjacent sertoli cells divide the germ cells in
two compartments as basal compartments and adluminal compartments.
 They are the true blood testis barrier.

The chemical changes which occur in blood cannot occur within

the seminiferous tubules.

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 EPIDIDYMIS

It is composed of a single, torturous, coiled tubule starting from the proximal portion of the
testis and is responsible for nutrition, maturation, transport and storage of spermatozoa.

Epididymis consists of three parts

 Head of epididymis (or) caput

o The proximal or spermatic cord end of the epididymis is more or less flat, broad
and U-shaped.
o It is a fimbriated tubular mass.
o It is hidden due to its enclosure with a fat pad.
 Body of epididymis (or) corpora (or) corpus
o It is the intermediate narrow part.
o It is a cylindrical tubular mass.
o It is apparently visible.
 Tail (or) cauda
o It is the distal enlarged end.
o It is an inverted triangular tubular mass.
o It is apparently visible.
 o It is the storage site for spermatozoa.

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 Epididymis is covered by a smooth muscle coat which transports the sperms by

peristaltic movement.
 It has more secretory and absorptive activity.
 The epididymal duct provides the environment for gaining of fertility by spermatozoa.
 Spermatozoal removal from the epididymis is caused by periodic contractions of the
epididymis and ductus deferens, resulting in a gradual trickle of spermatozoa out of
the tail, through the ductus deferens, in to the pelvic urethra where they are flushed
out of the tract during urination. This allows removal of sperm from the epididymis
on a continual basis.
 Grossly, the epididymis appears as an approximately cylindrical organ.
 Spermatozoa stored in the epididymis retain fertilizing capacity for several weeks.
 The cauda epididymis contains about 75% of total epididymal spermatozoa.

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The tail of the epididymis acts as a reservoir of fully mature

sperm and becomes turgid with stored sperm in sexually
active animals.

 The length of the epididymis varies with the species. It is about 20 metres in stallion,
30 metres in bull and 50 metres in boar.
 The epididymis is closely attached by fibrous tissue to the surface of the testis proper.
 This is continuous with the vas deferens.

Functions of the epididymis

 Absorption
 Secretion
 Maturation of fluid
 Transportation and
 Storage of spermatozoa
INGUINAL CANAL
Inguinal canal is a slit like space between the internal and
external inguinal ring.
  Internal inguinal ring is formed by internal oblique abdominal muscle and external
inguinal ring is formed by the tendons of external oblique abdominal muscle.
 In case of dog, bull and boar, internal inguinal ring is larger but they have short
inguinal canal.
 In stallion, inguinal canal is longer but it has smaller internal inguinal ring.

VAS DEFERENS OR DUCTUS DEFERENS

 Vas deferens extends from tail of epididymis to colliculus seminalis of the

pelvic urethra.

Click on the refresh button to view animation

 It is a siphon shaped structure for the exit of spermatozoa.

 Vas deferens passes parallel to the testis into the spermatic cord through
the inguinal canal and dorsomedially to the neck of the urinary bladder.
 It has uniform thickness up to the urinary bladder.
 Thickness is 3 mm in bulls and 6 mm in stallion.
 Blood supply: Internal pudic artery and spermatic artery.
 Nerve supply: Pelvic plexus.

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Ampulla
Terminal fusiform enlarged glandular portion of vas deferens is called as
ampulla of the vas deferens

 Bull: Length – 10 -15 cm, Diameter – 1-1.5 cm

 Stallion: Length – 15-20 cm, Diameter – 2-2.5 cm

Ampulla is absent in dog and cat.

 It is smaller in boars.
 Ampulla lies dorsal to the urinary bladder and passes beneath the body of the prostate
and opens in the round prominence into the pelvic urethra called as colliculus
seminalis.

ACCESSORY SEX GLANDS OF MALE

 The secondary andrological organs, which are monitoring the associated function for
production of seminal fluid (seminal plasma) are termed as andrological accessory
glands .
 The glandular apparatus consist of
o Seminal vesicle or vesicular gland
o Prostate gland
o Cowper’s gland

SEMINAL VESICLE
 Paired accessory sex gland located in the floor of the
pelvis, dorsal and lateral to the ampulla or neck of
the bladder.
 In ruminants and boar, it is a lobulatory gland in
which lobulation have small central dilatation.

Name of the seminal vesicle in all domestic

animals is vesicular gland except in stallion.

 In stallion - large central dilatation is present.

 In stallion, structure of seminal vesicle is more
bladder like and glands are located on the walls.
 Secretion of the seminal vesicle adds nutrients,
volume and buffer to the semen.
 They open in the pelvic urethra in close proximity
with the opening of vas
 deferens (Colliculus seminalis )

Seminal vesicle in all domestic animals do not

store spermatozoa
Function

 The salient physiological functions of seminal vesicle are

o To produce vital biochemical secretion.The seminal vesicle is the
site for production of fructose and citric acid. The fructose is the
main source of nutrition /energy for the spermatozoan motility.
The remaining other biochemicals are also essential for
spermatozoal survivability and maintenance.
o To secrete vesicular fluid. The seminal vesicle is the main source
for the production of liquid part of ejaculate. The vesicular fluid in
the liquid state is the main source of flow-current and gives
impetus for floating vesicular motion due to surface tension of its
liquid state, which helps the male gamete during its swimming in
the liquid seminal plasma by propelling of spermatazoal
locomotory apparatus through dynamic energy.
o The liquid secretion of seminal vesicle helps in lubrication of
urethra for movement of spermatozoa.
o Final volume of semen is related to the quantity of secretion of
seminal vesicle.

Blood supply

 Both middle rectal artery and the inferior vesicle artery are the arterial
source of seminal vesicle which is the branch of internal iliac artery.

Nerve supply

 The muscular wall of the seminal vesicle is provided with a plexus of

nerve fibres and contain small sympathetic ganglion.

Species wise anatomy (Click on the images for detailed pictures)

Bull Bio
me
 Vesicular glands are lobulated, 10-15 cm, try
long and 2-4 cm, in diameter . of
Se
Stallion
mi
 Seminal vesicles are smooth, 15-20 cm, long nal
and about 2.5-5 cm, in diameter. Ve
 A small portion of seminal vesicle is covered sicl
with peritoneum in case of bull and stallion. e
in
Boar Dif
fer
 Vesicular glands are proportionately very ent
large, 12-15 cm, long, 5-8 cm wide and 5-8 Sp
cm, thick. eci
 They cover the caudal portion of the bladder
es
and extend in to the abdominal cavity.
 The volume of ejaculated semen is maximum
in porcine species which is probably due to
comparatively heavier and enlarged size of
seminal vesicle in boar.
 It might be a characteristic features of
species.

Ram and Buck

 Resemble those of the bull .

Dog and Cat

Seminal vesicle absent in dog and cat

Species Length (cm) Breadth (cm) Thickness (cm) Weight (gm)
Bull 10 - 15 2-4 2 75
Stallion 15 - 20 2. - 5 5 -
Ram 12 - 15 5-8 4 200
 Boar 4-5 2 1.5 5
PROSTATE GLAND

 Prostate gland has various forms in domestic male farm animals.

 It is located in the floor of the pelvis on or around the neck of the urinary
bladder or cranial portion of the pelvic urethra or caudal to the neck of the
bladder.
 It adds its secretion at the time of ejaculation by means of many ducts
opening on the pelvic urethra.
 The secretions are released in the pelvic urethra near the colliculus
seminalis.

Functions

 The salient physiological functions of prostate gland are

o to secrete prostatic fluid.
o to secrete more quantity of serous and less quantity of mucous
secretion.
o to stimulate the spermatozoal motility.
o to help in the formation of vaginal-plug in opposite sex.
o to secrete little quantity of certain vital bio-chemicals like fructose
and citric acid for additional supply of nutrition/energy to
spermatozoa during its nourishment.
o to provide passage for onward-transit of glandular secretion-from the
gland to its outlet (i.e.pelvic urethra).
o To provide liquid medium for the transport of sperm in female
reproductive tract.

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Blood supply

 The blood supply of prostate is by internal pudendal artery.

Nerve supply

 The prostate is supplied by pelvic plexus.

Species difference
 SPECIES PARS PROPRIA (cm) PARS DISSEMINATA (cm)
Bull 3x1x1 12 x 1.5 x 1.0
Boar 3x3x1 17 x 1.0 x 1.0

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Bull

 Body – Wide 2.5 – 4 cm, Length – 1-1.5 cm. Thickness 1-1.5 cm.
 It can be felt as a small protuberance in the cranial end of pelvic urethra by
rectal examination. Pars disseminata surrounds the pelvic urethra.

Prostate gland in bull has two parts (disseminated portion)

 pars disseminata
 body of prostrate (Pars propria).

Ram

 It is diffused over a large portion of the pelvic urethra.

Ram has no body of prostate gland and it has only pars

disseminata

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Stallion

 It is situated over the neck of the bladder and cranial portion of the
urethra.

Boar

Prostate gland in stallion has two lateral lobes in the cranial end
of pelvic urethra and are connected by a structure
called isthumus

 Prostate gland is covered by the seminal vesicle.

 Pars disseminata is quite extensive in boars as in case of bull and
ram.

 Body is located dorsal to the urinary bladder.

Dog

Prostate gland in dogs opens by two excretory

ducts

 Prostate gland is larger in size, surrounds the neck of the bladder, located
in the cranial border of pubis, the size varies with the age.
 Enlarged in older dogs.

BULBO-URETHRAL GLAND OR COWPER’S GLAND

 They are paired glands located on either side of the pelvic urethra near the ischiatic
arch.
 Ovoid in shape or walnut shaped
 The salient physiological functions of cowper’s gland are
o To provide pre-ejaculatory secretion,
o To clean the urethral passage through flushing the urethra from urine and
micro-organism, debris and crystal, for maintaining the hygienic condition of
urethral orifice.
o To supplement nutrition or energy to spermatozoa.
o To lubricate the urethral passage.
o To lubricate the vagina of opposite sex partner during mating.
o To maintain optimum pH level of urethral passage which provides favourable
condition for spermatozoa.

Species Diameter (cm) Length (cm)

Bull 1.5 - 3.0 -
Stallion 2.5 - 5.0 -
Ram 0.5 - 1.0 -
Boar 2.5 - 3.0 12.0

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Bull

 The size of the cowper’s gland is smaller in bull when compared to stallion.
 Single duct from each gland is present.

Stallion

 Each gland in stallion has got 6- 8 excretory ducts opening into the urethra.

Boars

 It is large, dense, thick and cylindrical in shape. Single duct from each gland is
present.

Dog

Cowper’s gland is absent in dogs

Tom Cat

Cowper’s gland in tom cat is as large as in case of prostate in cats

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Blood supply

 The internal pudic artery

Nerve supply

 The pelvic plexus

URETHRAL GLANDS
 Present in human beings but absent in bull, stallion, dog, cat and boar.

In bull, boar and ram, pelvic urethra contains disseminate part of

theprostate gland and should not be called as urethral glands
UTERUS MASCULINUS
It is a rudimentary structure and remnant of the
paramesonephric duct in male and is located between
 ampullae and seminal vesicles.

 It is situated on the caudal dorsal surface of the bladder and cranial to the prostate.
 50 -70 % of bulls have uterus masculinus.

URETHRA
The urethra in males is the common passage for the excretion of
urine as well as for the transportation of semen

 It is about 115 cm in length in bulls.

 The urethra has three distinct parts namely pelvic part, bulb of urethra and the penile
part.
 In bull, the pelvic part of urethra is about 20 cm in length and is situated on pelvic
floor.
 The pelvic urethra is enclosed by heavy urethral muscle or wilson’s muscle which
helps in ejaculation and micturition by its forcible contraction.
 The bulb of urethra is extra pelvic part situated at ischial arch and is bending, ventral
to the pelvis.
 The penile urethra runs inside the penis proper.

PENIS
Penis is a copulatory organ in
male

 Almost cylindrical in shape.

 It extends from ischiatic arch to the umbilical region except in tom cat.
 Penis is supported by penile fascia and skin.
 Prescrotally, it is situated in the sheath or prepuce.

Terminal portion of penis which is freely moving within the

sheath is called as glans penis

Penis has got three parts

 Root or base or phallus root

 Body or phallus body
 Tip of penis or glans penis or phallus tip
 Phallus root (Otherwise called as, Root of penis or Penile root or Radix penis )
o It is originating part of penis, which is attached to ischial bone an ischio-
cavernosus muscle.
 Phallus body (Otherwise called as, Body of penis or Penile body or Corpus penis )
 o It is middle part of penis which is formed by fusion of two crura, corpus
cavernosum and corpus spongiosum. It is the major part of penis.
 Phallus tip (Otherwise called as, Tip of penis or Penile tip or Glans penis )
o It is the exterior part of penis, which is attached to phallus body. It is a conical
tapering end of almond shape.

Besides above classification, the penis may be classified

into two parts (viz., pelvic internal and preputial external
parts) on the basis of location.
Further, the penis can also be divided into two parts (viz.,
protruded and non-protruded parts) on the basis of its
protrusions.

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Type of penis
 The type of penis varies according to the ratio of connective tissue and erectile tissue.
 The penis of male livestock can be grouped into two different types, depending on its
internal micro-architect.
o Fibrous type
o Vascular type .
 Fibrous type (Musculo-fibrous/ or Fibro-elastic type)
o The fibrous penis contains large ratio of connective tissue.

The well developed fibrous tissue of penis, is a characteristic

feature of bovine, porcine, ovine and caprines.

 Vascular type (Musculo-cavernous)

The well developed vascular sinuses and elastic fibres of penis is

a characteristic feature of equine, canine and feline

o The vascular type of penis contains large ratio of erectile tissue.

 The salient physiological functions of penis are

o To provide stiffness through engorgement of numerous labyrinth of haemo-
sinuses with inflow of blood on proper sexual stimulation.
o To locate the vulva of opposite sex during coitus.
o To perform intromission.
o To ejaculate semen into female genitalia.
o To urinate.
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Muscles of Penis
 Body of penis consists of corpus cavernosum
penis muscle which is surrounded by a thick
connective tissue capsule called as tunica
albuginea.
 Beneath the corpus cavernosum penis is
the corpus cavernosum urethrae which
surrounds the urethral orifice. These two muscles
are spongy in nature and divided into many
spaces. Erection of the penis is caused by
distention of theses spaces with blood.
 Root of penis is formed by two crura that fasten
the penis on either side of the ischiatic arch.

 The ischio-cavernosus muscle or erector penis muscle is a short

paired muscle from tuber-ischii and sacro-sciatic ligament that is
inserted on the crura and body of the penis. This muscle causes erection
of penis by its compressing and pumping action on the bulbous portion
of the corpus cavernosum penis .
 Retractor penis muscle is a paired smooth muscle originates from
1st and 2nd coccygeal vertebra, divides and meets again below the anus.
 Retractor penis muscle is passing along the caudal ventral surface of the
penis and attaches to the tunica albuginea of the penis. It draws the penis
back into the sheath after ejaculation.
 The muscles of pelvic urethra include Urethral muscle which is a
circular muscle consisting of dorsal and ventral layers of transverse
fibres. It aids in ejaculation and micturition by its forcible contraction.
 The Bulbo-cavernous muscle extends from ischiatic arch to glans
penis. It is a continuation of extra-pelvic urethra. It is thickest at the root
of the penis. It empties the extra pelvic urethra.

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Associated structures of penis

 The penis has some specific associated structures.
Sigmoid flexure

 The sigmoid flexure is a ‘S’ shaped bent of penis which is characteristically found
in cattle-bull, buffalo-bull, boar, ram, buck and some wild animals like giraffe.

Usually sigmoid flexure is post-scrotal in most of species except boar

where it is pre-scrotal.

 Its posterior portion is attached to the retractor penis muscle.

 During copulation the flaccid sigmoid flexure becomes straightened due to relaxation
of retractor penis muscle.
 After copulation the sigmoid flexure takes its original “S” shape, due to contraction of
retractor penis muscle.

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Os-penis (Os priapi or Baculum )

The os-penis is characteristically found in male dog, ferrets and mink.

It is occasionally present in tom-cat

 It is absent in other species. The size of os-penis is grown up as the age advances.
 It may be utilized as age-indicator.
 The os-penis of each species has a characteristic shape which serves as diagnostic
taxonomic structures in certain livestock.

Cork screw penis

The cork screw penis is the modified structure of external

extremity of penis which is a characteristic feature of boar

 This helps in intromission of boar penis in the genitalia of sow ( Cervical canal in sow
is cork screw like).

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Phallus

 The phallus is the external extremity of male genitalia in place of penis which is
a characteristic feature of boar. This helps is ejaculation.

Phalli
  The phalli are the external extremity of male genitalia in place of penis which is a
characteristic feature of ducks and geese. This helps in intromission.

Penile papillae

 The penile papillae are located at anterior portion of penis which is a characteristic
feature of tom-cat, hamster, house-mice and rat. This probably helps in ovulation.

Urethral sinus

 The urethral sinus is located around the end of penis which is characteristic feature
of stallion. This contains urethral sinus diverticulum.

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Urethral sinus diverticulum

 The urethral sinus diverticulum is located at dorsal aspect of urethral sinus which is
characteristic feature of stallion. This helps in accumulation of debris and formation
ofsmegma, called as BEAN which is the main source of pheromones.

Urethral process

 The urethral process is a filliform appendage extending form the anterior portion of
glans penis which is a characteristic feature in ram, buck and giraffe.

Blood supply

 Internal pudendal artery to the root of penis

 Obturator artery to the body of the penis
 External pudendal artery that gives rise to dorsal artery of penis – after passing
through the inguinal canal.

Nerve supply

 Autonomic nerves of the pelvic plexus and haemorroidal and pudendal nerves. The
sensory nerve fibres to the glans penis come from the dorsal nerve of the penis, The
glans penis is plentifully supplied with nerves and nerve endings.

PENIS - SPECIES WISE DIFFERENCES

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Bull

 Characterized by “S” shaped curve or sigmoid flexure (Post-Scrotal)

Sigmoid flexure is post-scrotal in bulls

  Penis in adult bull – 36 inches long from root to tip
 The erectile tissue is small in amount as compared to that of the stallion.
 The diameter of the penis is less than 4-5 cm even in erectile state.
 The glans penis is 7.5 to 12.5 cm long and is rather pointed.

Retractor penis muscle is well developed

 The thickened dorsal portion of the fibrous sheath in the penis is known as
dorsal apical ligament of the penis.
 At the time of erection and service the penis is protruded, 10-24 inches out
of sheath.

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Stallion

Large amount of erectile tissue is present in the penis

of stallion

 Length about 50 cm ; diameter 2.5 to 6 cm ; 15-20 cm lies, free in the

prepuce.
 At the time of erection the penis will double its length and thickness, with
the glans penis gets enlarged three or more times than its normal size
 Dorsal to the urethral process of the glans penis, the urethral sinus or
diverticulum is located which is sometimes filled with smegma which is
called as BEAN.
 Length : 45-55 cm ; at the time of copulation 20-35 cm, may protrude
beyond the preputial opening. The retractor penis muscle is not as strong
as in the bull.

Sigmoid flexure is absent in stallion

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Ram and Buck

Penis of ram is characterized by the presence of urethral process

extending 4-5 cm beyond glans penis

 Length about 30 cm ; Diameter 1.5 -2 cm ; glans penis 5-7.5 cm long

 Well developed sigmoid flexure is present.

Boar

The sigmoid flexure is present but it is

prescrotal

 The cranial portion of the penis has no glans but is spirally twisted
counterclock-wise.

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Dog
 The os-penis is characteristically found in
male dog.

 The penis of dog in its caudal part has two distinct corpora cavernosa in its
caudal part separated by a median septum.
 Length about 6.5 – 25 cm .
 In the cranial free portion, there is a bone called os penis which varies from
5 to 10 cm in length, depending upon the size of the dog. Ventrally, this
bone is grooved for the urethra.
 Penile bone also present in foxes, raccoons and hedgehogs.
 Glans penis has two parts, the pars longa glandis and the bulbus glandis.
 Bulbus glandis expands greatly at the time of erection and prevents
withdrawal during ejaculation.

Bulbus glandis expands greatly at the time of erection and prevents

withdrawal during ejaculation. Vagina also contracts over the penis.
This results in coital lock or copulatory tie

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Cat

 Short
 Directed caudally and downward
 Urethra located dorsally
 Os penis is often lacking but when present , it is short 3-4 mm long.
 No glans penis.

In the cat the bulbus glandis is absent

Penis has terminal cap about 1 cm that contains numerous papillae or spines pointing
towards the base of the penis. These may cause the female to cry out of intromission
PREPUCE
The secondary andrological organ, which is comprised of
cutaneous fold for enveloping the penis, is known as prepuce
(cutaneous sheath)
  It is the double invagination of skin which contains and covers the free portion of
penis, when it is non-erect.
 It covers the body of the penis behind the glans, when the penis is erect.
 Preputial orifice is the external opening of the prepuce.
 Preputial lining is a freely movable membrane or modified skin which is attached
firmly only at glans penis and preputial orifice.

Blood supply: External pudendal artery

Nerve supply: Innervation from pudic, ilio-hypogastric, ilio-inguinal nerves

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Functions

The salient functions of prepuce are:

o To enclose non erected penis within preputial cavity.

o To protect non erected penis from adverse conditions (viz extreme cold or heat,
injury).
o To hold penis external to animal body.
o To maintain penile movement through providing sufficient passage by opening
of preputial sphincter.

Bull

 Prepuce is long and narrow (35-40 cm length).

 Preputial orifice is surrounded by a tuft of long preputial hairs.
 There are usually 2 pairs of cranial and caudal preputial muscles, protractors
and retractors that draw the preputial opening forward or backward.
 The fornix of the prepuce is the point at which the prepuce reflects upon the
penis just caudal to the glans.

Stallion
  Has preputial cavity of 15-20 cm deep and then a second reflection of prepuce to
form the prepuce proper of the penis. The opening between these two cavities is
called as the “preputial ring”.
 The engaging and disengaging of the glans penis in the preputial ring causes the
sucking noise frequently heard when the gelding or stallion trots.

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Ram

 Similar to bull .

Boar

 Prepuce has a small orifice.

 Caudal part of the prepuce is narrow and the cranial part wide.

Dog

 No out-standing feature

Intra preputial urination

Swine and ruminants urinate inside the

prepuce

Extra preputial urination

Horses, dogs and cats extend the penis beyond the sheath and urinate.
INTRODUCTION
Puberty may be defined as the age or time at which the generative
organs become functional and reproduction may occur.

Puberty in male

 Puberty in male is characterized by appearance of secondary sexual characteristics,

sexual desire and presence of viable spermatozoa in semen.
 Pre-pubertal period includes age of infant to puberty. During this period, all these
above characters are absent.
 Puberty does not indicate full reproductive capacity. In bulls, spermatocytes appear in
the seminiferous tubules by 4-6 months of life, spermatids at 6-7 months of age,
 spermatozoa at 7-9 months of age. Seminal secretions are released by 5-7 months of
age.
 The separation of prepuce starts from one month of age and completed by 8 months
of age.

PUBERTY Vs SEXUAL MATURITY

Development of puberty

 Primary spermatocytes appear in the seminiferous tubules by 4 to 6 months and

spermatozoa by 7-9 months of age in bulls.
 Seminal secretion from the accessory glands appears by 5-6 months.
 Separation of the penis from the sheath in bulls proceeds caudally beginning at one
month of age and ending with complete separation by 8 months of age.
 The period from 6 to 10 months of age in bulls is characterized by the accelerated
growth of male genital system, increase in GnRH secretion in the hypothalamus,
increase in concentration of LH in plasma, external manifestations of puberty and the
rapid onset of spermatogenesis.

Sexual maturity

Attainment of puberty does not signify the full reproductive

capacity in males

 A highly significant increase in ejaculate volume, output of motile spermatozoa and

concentration of spermatozoa in bulls for 6-9 months after the onset of puberty.
Hence, there is gap between the attainment of puberty and full reproductive capacity.
 Males attains full potential of reproduction at the time of attainment of sexual
maturity. If puberty is delayed, sexual maturity also gets delayed.
  Because of the lack of maturity after the onset of puberty immature males should be
sparingly used for breeding purposes for ½ to 1 year or more after reaching the
puberty.

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Signs of puberty

 Pubertal period indicate accelerated growth rate of genital system – increased LH

releasing factor in hypothalamus and plasma LH.

Onset of puberty in male is characterized by

 Secondary sexual signs

 Sex desire
 Ability to copulate and presence of viable spermatozoa in
the ejaculate
 First appearance of seminal secretion from accessory sex
glands.

Mechanism for onset of puberty/sex desire

 Onset of puberty in the male domestic animals occurs at approximately the same time
after birth as puberty in females of the same species.
 It is brought about by the release of gonadotrophic hormone from the anterior
pituitary resulting in the secretion of steroid hormones from the gonads that cause
growth of genital organs and secondary sex characteristics.

FACTORS AFFECTING PUBERTY AND SEXUAL MATURITY

 Onset of puberty and sexual maturity in the male are the gradual processes which are
influenced by plane of nutrition and management, cross breeding, chronic diseases,
individual differences and other factors.

Nutrition and management

 Poor somatic growth and emaciation which ultimately delays the onset of puberty due
to the deficient feeding of protein, iodine, phosphorus, copper, iron, cobalt.

The improved nutrition and good management starting at

 earlier age enhances the onset of puberty

 Lack of TDN in the feed or animal get starvated leads to prevent the secretion of
gonadotropic hormones by the anterior pituitary- result in failure of early puberty.

Body size and weight

 Body weight plays more important role than the age for attaining puberty and sexual
maturity.
 If the individual animal gain less weight according to age factor, the puberty and
ultimately sexual maturity will be delayed.

Gonadal growth

 The scrotal circumference is directly proportional to the intensity of sex desire and
spermatozoa production.
 Sex desire: Depending on the available testicular surface area containing number of
leydig cells - site of androgen secretion.
 Spermatozoal production: Depending on the available testicular surface area
containing number of seminiferous tubule - site for spermatogenesis.
 At the age of puberty the diameter of seminiferous tubules is less than the diameter of
the seminiferous tubules at the age of sexual maturity .

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Genetic factors

 The genetic components of young bulls affect the onset of puberty and maturity.
 The larger breeds of cattle and horses have a late onset of puberty than the smaller
breeds.
 The buffalo male calves appear to attain sexual maturity later than the cattle-bull
calves even with good nutrition and management; probably due to genetic difference
(testicular surface area of buffalo is lesser than cattle).
 The cross breeding causes early puberty and maturity.

Species

 Onset of puberty varies with species.

Species Age at puberty

Bull 9 to 12 Months ( range 6-18 months)
 Stallion 18 Months ( range 12 to 24 months)
Boar 5 to 7 Months ( range 4 to 8 months)
Ram and Buck 7 to 8 Months ( range 4 to 12 months)
Dog 7 to 10 months ( range 5 to 12 months)
Cat 8 to 10 months ( range 6 to 15 months)
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Geographical location

 The geographical location for rearing of young animals affects the onset of puberty
and maturity.
 If individual animal of one geographical area raised in another geographical area, the
puberty will be affected adversely.
 Animals located in tropical regions are late in attaining puberty and sexual maturity.

Season

 There is a close relationship among season of birth, body weight and onset of puberty.
 Winter is favourable for sexual maturity in young bulls, used in AI.
 Seasonal influences are there in sheep and buffaloes - hot season delays the onset of
puberty.

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Hormones

 If hormone (FSH and ICSH) release occurs at earlier age, the puberty and sexual
maturity comes earlier.
 If delay the release of hormones leads to delay in the puberty and sexual maturity.

Chronic disease and debility

 Any disease which causes emaciation of individual, delay the onset of puberty and
sexual maturity. Eg. FMD, Johne’s diseases, TB, mange.
 Chronic diseases indirectly due to elevation of body temperature, disturbances of
basal metabolism, thermal stress, anorexia, indigestion, stunted growth, emaciation,
debility, weakness and endocrinological dysfunction affects the onset of puberty.

Sexual stimulation
 The stimulation of sensory apparatus through CNS by hearing, seeing, smelling of
opposite sex, causes early puberty.
 If there is a lack of these stimulation leads to delay the onset of puberty.
 If the male and female are kept together they mature earlier.

ENDOCRINOLOGY OF MALE REPRODUCTION

 Primordial germ cells migrate to the gonads, differentiate into spermatogonia located
in the centre of the primitive testes.
 Development of medullary cords occurs earlier in males than in females.
 The primitive testes produce or secrete an androgen or androgen like
substance which causes development of mesonephric duct system and male external
genitalia and at the same time degeneration and atrophy of the paramesonephric
ducts.
 Excessive and prolonged dosages of androgens given early in foetal life may cause
masculinization of the external genitalia organs of the female fetuses.
 Prior to the onset of puberty releasing hormones are secreted by the hypothalamus
which is carried via the hypothalamo-hypophyseal portal system into the anterior
pituitary and causes the release of FSH and LH.
 FSH acts on the seminiferous tubules of the testes and is responsible for
spermatogenesis i.e., from the initial division of the spermatogonia through the
formation of secondary spermatocytes. Thereafter the testosterone is responsible for
sperm cell development.
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 LH stimulates the growth and development of interstitial cells or Leydig cells in

between the seminiferous tubules of the testes and the secretion of the testosterone
and androgens from these cells. These androgens are secreted into the blood stream
where they cause the development of the secondary sexual characteristics in the male
and development and maintenance of male reproductive tract. The androgens
suppress GnRH, LH, FSH secretion by a negative feedback on the pituitary and
hypothalamus.
  FSH also interacts with receptors on sertoli cells to cause production of androgen
binding protein (ABP), conversion of testosterone to dihydrotestosterone and
estrogen completion of sperm release (spermiation) and secretion of inhibin.
 The ABP forms a complex with androgens and is carried along with the spermatozoa
into the epididymis.
 The epithelial cells of the epididymis require relatively high levels of androgen for
normal function.
 Although much of the testosterone secreted into the seminiferous tubules is converted
into dihydrotestosterone (DHT) by the enzyme 5 alpha reductase, some of the
testosterone gets converted to estrogens by the enzyme aromatase.
 High levels of testosterone, estrogen or progesterone maintained by continuous
injections, feeding or steroid producing tumors of the testes and adrenal gland causes
and atrophy of the seminiferous epithelium and interstitial cells. This process can be
reversed by withdrawal of the exogenous source of steroids resulting in testicular
rebound phenomenon.
 High levels of estrogens found in stallion urine are mainly due to estrogen produced
by interstitial cells and or sertoli cells of the testes.
 Sertoli cell tumor of canine testes is common and they produce high levels of
estrogens which cause feminization and testicular atrophy in males.

The inhibin secreted into the blood stream has a negative

feedback effect on FSH but not on LH secretion.


Androgen binding protein (ABP)

 ABP is produced by sertoli cells under the influence of FSH and forms a complex with
androgen.
 It is carried along with spermatozoa into the epididymis.
 The epithelial cells of the epididymis require high levels of androgen for normal
function- Epididymal transport, sperm maturation and storage.

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TESTOSTERONE
 Testosterone is a steroid hormone soluble in oil and alcohol but not in water.
 Acetate is converted to cholesterol which in turn is converted to progesterone –
androstenedione – testosterone (16 times more active than androstenedione in bulls).

Functions of Testosterone
 Sexual differentiation of external male genetalia and descent of the testes into
scrotum in fetuses or neonates.
 Keratinization of the preputial epithelium, separation of glans penis from prepuce,
growth of the penis and prepuce at puberty.
 Growth and maintenance of accessory sex glands.
 Sexual desire or libido, ability for normal erection and ejaculation.
 Secondary sexual characteristics like hair or horn growth, male attitudes, voice,
increased bone thickness, increased muscle tissue with different distribution of fat
from female.
 Maintenance of secretory and absorptive activities and structure of efferent ducts,
epididymis and ductus deferens including ampulla.
 Spermiogenesis – development and maturation of spermatids and spermatozoa.

In stallion the androstenedione is the major androgen.

BIOSYNTHESIS
 Acetate → Cholesterol → Pregnenalone → Progesterone → Androstenedione →
Testosterone.
 Testosterone is the major androgen produced by bull, dog and human testes.
Androstenedione is the major androgen from the stallion’s testes.

In bull, testosterone is sixteen times more potent than

androstenedione.

 Testosterone secreted from the Leydig cells is carried in the blood stream by an α –
globulin designated as steroid binding globulin.
 99% of the circulatory testosterone is in bound form and 1% free testosterone from
circulation enters the target cell.
 In the cytoplasm of target cells, it is converted by 5- α reductase into
dihydrotestosterone which is biologically active form.

INTRODUCTION
Spermatogenesis is a complex process of cell division and
differentiation resulting in the formation of spermatozoa.

 Spermatozoa are formed in the seminiferous tubules of the testes, by a

series of cell divisions followed by a metamorphosis, produces a highly
differentiated and potentially motile cell, the spermatozoa.
 Spermatogenesis can be divided into two phases
o Spermatocytogenesis
o Spermiogenesis.
 Spermatocytogenesis is the proliferative stage in which primitive germ
cells are multiplied by a series of mitotic divisions followed by the meiotic
divisions which produce the haploid stage spermatid.
 Spermiogenesis is the differentiative phase in which the nucleus and
cytoplasm of spermatid undergo morphologic changes to form the sperm
cells. Metamorphosis of spermatid into spermatozoa, is called as
spermiogenesis.
Last modified: Tuesday, 12 June 2012, 09:56 AM
SPERMATOCYTOGENESIS

 Spermatocytogenesis begins with the mitotic division of spermatogonia on the

basement membrane of seminiferous tubules and proceed towards the lumen.
 Spermatogonia are activated to form active type A spermatogonia. There may be
several generations of type A spermatogonia.
 Most of type A spermatogonia divide to form intermediate spermatogonia.
 Some of the type A are retained as resting type A spermatogonia. In this way, the type
A cells provide daughter cells for the formation of spermatozoa but are not depleted
in the process.
 Intermediate spermatogonia divide to form type B spermatogonia, which undergo
the last of the mitotic divisions to form primary spermatocytes. Spermatocytogenesis
is concluded by the meiotic divisions, which produce secondary spermatocytes, then
spermatids.

The formation of spermatids marks the end of spermatocytogenesis

and the beginning of spermiogenesis.

 The entire divisional process of spermatocytogenesis, from spermatogonia to

spermatid takes approximately 45 days in bulls.

SPERMIOGENESIS
Spermiogenesis consists of 3 major changes

 Condensation of nuclear chromatin

 Formation of tail or flagellum
 Formation of acrosomal cap or membrane.

 Spermiogenesis consists of four phases namely

o Golgi phase
o Cap phase
o Acrosomal phase
 o Maturational phase

GOLGI PHASE

 The newly formed spermatid (A) has a well-developed golgi apparatus.

 Small vesicles of the golgi fuse, giving rise to larger secretory granules
called proacrosomic granules (B)
 Vesicle fusion continues until a large acrosomic vesicle is formed that contains a
dense acrosomic granule (C)
 During the last half of the golgi phase the centrioles migrate to a position opposite to
the acrosomic vesicle
 Proximal centriole (PC) gives rise to the attachment point for the tail
 Distal centriole (DC) will give rise to the developing axoneme inside the cytoplasm of
the spermatid.

The axoneme is the central portion of a flagellum

CAP PHASE
 The acrosomic vesicle flattens and begins to form a distinct cap (B) consisting of an
outer acrosomal membrane (OAM) and inner acrosomal membrane (IAM).
 The golgi (A) migrates towards the caudal part of the cell (B).
 Distal centriole (DC) form the axoneme (AX) or flagellum which projects away from
the nucleus toward the lumen of the seminiferous tubule.

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ACROSOMAL PHASE

 Spermatid nucleus begins to elongate (A).

 Acrosome covers the majority of the anterior nucleus (B).
 A unique system of microtubules known as manchette extends from posterior
nucleus. Portions of manchette attach to the region of the nucleus just posterior to the
acrosome.
 Some of the microtubules of the manchette will become the post nuclear cap.
 During the acrosome phase, spermatid become deeply embedded in sertoli cells with
their tails protruding towards the lumen of the seminiferous tubule .

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MATURATION PHASE

 Manchette migrate towards the tail and begin to disappear.

 Mitochondria are assembled around flagellum to form the middle piece.
  Dense outer fibres form around the flagellum.
 Post-nuclear cap is formed from manchette microtubules.
 Annulus forms the juncture between the middle piece and principal piece.
 During later stages of spermiogenesis, sertoli cells forms the cytoplasm remaining
after elongation of spermatid into a spheroidal lobule called residual body.
 Lobule of cytoplasm is connected to the elongated spermatid by a slender thread
cytoplasm and is inter connected with other residual bodies by intercellular bridges.

Formation of residual bodies completes final maturation and

spermatids are ready for release.

 Sertoli cells also remove degenerating germ cells.

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DURATION OF SPERMATOGENESIS

Entire spermatogenesis takes about 50-60 days in boars, 60-70 days in bull
and ram.

 Spermatogenesis comprised of four to five cycles of seminiferous epithelium.

 Cycle of seminiferous epithelium is defined as series of stages/changes between two
similar cellular associations in the given area of seminiferous tubule.

The time necessary to complete a cycle of the seminiferous epithelium is called

duration of spermatogenesis and varies among domestic animals

DURATION OF THE CYCLE IN DIFFERENT SPECIES

Species Days
Boar 9
Ram 10
Horse 12
Bull 14

Spermatogenic wave

The distance between two similar cellular associations in the seminiferous epithelium
is called spermatogenic wave.
 SPERMIATION
Release of formed germ cells into lumen of the seminiferous tubules is known as
spermiation

 Elongated spermatids are gradually extruded into the lumen of the tubule
 Extrusion continues until only a slender stalk of cytoplasm connects the neck of the
spermatid to the residual body
 Breakage of the stalk results in formation of cytoplasmic droplet in the neck region of
the released spermatozoa (proximal droplet) and retention of interconnected residual
bodies
 Residual body are disposed by sertoli cells

SPERMATOZOA

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The head of spermatozoa has a shape characteristic for each

species.

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 Chromatin is compact.
 Anterior 2/3rd of the nucleus is covered by acrosome which is a membrane bound
lysosome that contains hydrolytic enzymes such as acrosin, hyaluronidase, zonalysin,
esterases and acid hydrolases which are required for penetration of cellular
investments and zona pellucida.
 The tail composed of capitulum, middle piece, the principal piece and the terminal
piece
 The capitulum fits into the implantation socket, a depression in the posterior nucleus
 Anterior portion of the tail consists of laminated columns – gives neck region
flexibility
 Axonemal components of tail originate from the distal centriole consists of 9 pairs of
microtubules arranged radically around 2 central filaments. Surrounding this 9+9+2
arrangement are 9 dense fibres that are unique to the flagellum of spermatozoa.
 The mitochondrial sheath is arranged in a helical pattern around the outer dense
fibre of the tail and contributes to the middle piece
 The middle piece terminates at annulus which demarcates junction between it and
principal piece
 Principal piece makes up majority of tail
 Continues and connects the terminal piece.
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Biometry of spermatozoa in different species

Species Head (µ) Mid piece (µ) Tail (µ) Total length
(µ)
Length Width Length Width Length Width
Cattle 9.05 4.25 14.84 0.67 45.50 0.51 69.59
Buffalo 7.50 4.70 11.95 0.62 42.50 0.50 61.95
Ram 8.20 4.25 14.00 0.80 40.45 0.50 62.65
Buck 8.14 4.30 12.52 0.72 42.77 - 63.43
Boar 8.05 4.18 11.55 0.55 32.94 - 52.54
Stallion 7.00 3.91 9.83 - 42.30 - 59.13
Poultry 12.5 - 4.00 - 80.00 - 96.50
Dog 5.5-7.0 2.0- 7.0- 1.0 40.0-
4.0 13.0 45.0

 The nucleus is oval and flattened and is surrounded by nuclear membrane.

FACTORS AFFECTING SPERMATOGENESIS

 Physical factors
 Chemical factors
 Nutritional factors
 Hormonal factors
 Genetic factors
 Pathological factors
 Age factors

I: PHYSICAL FACTORS
Irradiation

 Irradiation produces interference with spermatogenesis by injuring spermatogonia,

spermatocytes and spermatids.

The spermatocytes are most sensitive to irradiation while leydig and

sertoli cells are quit resistant.

 Steroidogenesis is not affected much by the X-rays. The testosterone level is

maintained as such.

Hyperthermia

High ambient temperature will cause testicular degeneration which

affects the spermatogenesis

 The causes for the elevation of testicular temperature are cryptorchid and ectopic
testes, inguinal hernia, scrotal dermatitis due to irritants, chorioptic mange, myiasis
in sheep and localized skin infections or wounds, contusions and haematomas of the
scrotum and testes, prolonged body temperature as in certain infectious diseases and
in prolonged high environmental temperature particularly associated with high
humidity.
 Males that are unable to rise, often develops testicular degeneration and atrophy due
to the prolonged elevation of testicular temperature from the testes being held close
to the body.
 The temperature more than 410C has detrimental effect on spermatogenesis.
 The cells undergoing meiosis are more sensitive than resting sperm but there is no
change in steroidogenesis.
 Late pachytene primary spermatocytes and early round spermatids are more
sensitive.

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Hypothermia

 Spermatogenesis seems to be more resistant to cooling than to heat and the tunica
dartos and cremaster muscle contract to protect the testes from the effects of cold.
 Damage to spermatogenic function is notices at the temperature below -250 F.
 In hypothermia the stagnation of blood and resultant hypoxia is probably more
damaging than to decrease in temperature.

Light
  The influence of light on control of spermatogenesis is brought through the capacity
of it to control the pituitary gonadotrophin.
 The pineal body operated as a neuroendocrine transducer mediating light effects on
the testes.
 The shortened photoperiod (below 12 hrs) decrease the capacity of the pituitary to
release of gonadotrophin, possibly by reducing sensitivity to gonadal steroid hormone
feedback. Thereby it brings about effect on spermatogenesis.

Low oxygen tension

 Spermatogenesis is severely impaired when adopted males are subjected to low

oxygen tension at high attitude or in experimental chambers.

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II: CHEMICAL FACTORS AFFECTING SPERMATOGENESIS

Antispermatogenic drugs

 Cadmium: Cadmium salts, in small doses produce marked necrosis in the testis which
probably results from a marked increase in testicular blood flow and increase in
permeability of the blood vessels by increasing the intercellular cleft and the blood –
testis barrier. It affects both the spermatogenic and androgenic functions of the testis
but after about one month, the damaged testis gradually regains full androgenic
activity.
 Alkylating agents: Eg. Busulfan. These drugs destroy spermatogonia and in later
stages the germinal epithelium are removed by ‘maturation depletion’.
 Diamines: these drugs affect spermatocytes followed by ‘maturation depletion’.
 Nitrogen-containing compounds: Administration of these drugs cause arrest of
spermatogenesis at primary spermatocyte stage with histological castration changes
in the pituitary.
 Drugs affecting cell division: Drugs like hydroxyurea, interferes with DNA synthesis
and thereby it affects the spermatogenesis

Effects of Environment Agents

 Recent evidence suggests that the pesticides used in agricultural might alter the male
reproductive function.

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Organochlorine derivatives
  DDT (Dichlorodiphenyl Trichloro Ethane): It causes spermatogenic cell degeneration
and spermatogenic activity appeared to be decreased. It causes progressive
histological deterioration followed by loss of germinal cells, karyopyknotic nuclei,
cytoplasmic vascuolization, abnormal spermatids and multinucleated spermatocytes.
 Cyclodines: Dieldrin, aldrin and some indane derivatives may cause germ cell
damage, lowered plasma testosterone levels and decreased prostatic secretion and
these alterations may lead to decrease in spermatogenic activity.
 Benzene hexachloride: It decreases the number of mature sperms and the testis show
degenerative changes, necrosis and cellular proliferation. The seminiferous tubules
are severely damaged and multinucleated giant cells are commonly found.
 Miscellaneous Organochlorine compounds:
o Kepone: (used to control a wide range of inset pests) It produces atropic or
greatly enlarged testes associated with seminiferous histopathology and thereby
interfere with spermatogenesis
o Polychlorpinene: (Insecticide) It causes degeneration of seminiferous tubules
with lack of mature spermatozoa, spermatids and spermatocytes. However
sertoli cells and spermatogonia are present.
 Organophosphates: eg.Dichlorovos and carbamates
o Dichlorovos: It causes degeneration of seminiferous tubules but sertoli cells and
Leydig cells appear normal.
o Carbamates: Carbaryl at various doses increased testosterone hydroxynation
and carbaryl stimulate the conversion of testosterone to dihydrotestosterone.

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Food additives and contaminants

 Diethyl stilbesterol: Diethyl stilbesterol produces atrophy seminiferous tubule

histopathology and atrophic Leydig cells.
 Food colourings: Metanil yellow which is an commonly used dye in India produces
vascular damage and irreversible damage to seminiferous epithelium and thereby
produce spermatogenic cell changes in testes.
 Alcohol: Most important cause of male feminization. The effect of alcohol is
characterized by atrophy and pathology of testes, prostate and seminal vesicles,
lowered plasma testosterone levels, germ cell immaturity and seminiferous tubule
pathology. The changes occur in the absence of liver disease suggesting that alcohol is
a direct testicular toxin. Ethanol inhibits testicular retinal alcohol dehydrogenase
activity. Vitamin A plays an important of spermatogenesis. Alcohol inhibits the rate
of formation of retinol from retinal liver and retina. If this inhibition occurs in the
testes, steroidogenesis might be reduced followed by alteration in spermatogenesis
and accessory gland morphology and function.

Atmospheric pollutants
  Increased atmospheric pressure of carbondioxide reduces spermatid concentrations
in seminiferous tubules and prematurely releases spermatozoa, presumably by
carbondioxide effects on sertoli cells.

III: NUTRITIONAL FACTORS AFFECTING SPERMATOGENESIS

 Low plane of nutrition: the low plane of nutrition or malnutrition cause a greater
stress on pre-pubertal animals than in post- pubertal animals. It causes hypoplasia of
the testes and accessory sex glands and delays puberty. Adversely affect
gonadotropins secretion i.e FSH and LH and thereby reduces the spermatogenic
activity.
 High plane of nutrition: high plane of nutrition is frequently cited as a cause of
infertility especially in fatty, overfed and obese animals. It indicated that excessively
fatty bulls may have enough fat around the testes in the scrotum, especially the dorsal
part to insulate the testes and possibly affect spermatogenesis.

Vitamins deficiencies

 Hypovitaminosis A: germinal and leydig cells are affected by hypovitaminosis A

resulting in poor semen quality, testicular atrophy, hypoplasia of accessory sex glands
and delayed puberty. Vitamin A is epitheliotrophic and the severe deficiency produce
degeneration of seminiferous tubule and interfere with spermatogeneis.
 Hypovitaminosis E: causes testicular damage in rats but in domestic animals the role
of vitamin E on spermatogenesis is insignificant.

IV: HORMONAL FACTORS AFFECTING SPERMATOGENESIS

Exogenous steroids

 Exogenous steroids affect testicular function by altering the secretion of

gonadotropins.

Small doses of testosterone may impair spermatogenesis to a greater

degree than larger doses due to ability of the larger dose to maintain
the seminiferous epithelium.

 In some species steroid suppression of testis has been followed by a rebound

phenomenon after steroid withdrawal.

Pituitary factors

 Reduced spermatogenesis with testicular atrophy may be caused by failure of FSH

and LH.
  Many stress factors affect LH and FSH release, the most important being inanition
due to low plane of nutrition.
 Tumors of the pituitary gland also cause atrophy of the pituitary gland and failure of
gonadotropic production.

V: GENETIC FACTORS AFFECTING SPERMATOGENESIS

 Heredity sperm production is grossly affected by genetic abnormality like testicular
hypoplasia and congenital aplasia of wolffian duct. Testicular hypoplasia in cattle is
due to single recessive autosomal gene with incomplete penetrance. Hereditary
defects in sperm production or maturation cause abnormality of the acrosome and
separation of the head and tail.
 Inbreeding: it increases the abnormal seminiferous tubules and hypoplasia which
will affect the spermatogenesis. This may be due to autosomal recessive gene.
 Cytogenetic disturbances: This causes two abnormalities in the primary
spermatocytes including
o Stickness of chromosome in which they failed to separate at anaphase
o a pyknotic nucleus and multiple spindle formation due to a dysfunction of the
mechanism of cell division due to extra centrosome divisions resulting in the
formation of giant cells with a number of nucleus.
 Hybridization
o Eg. Donkey(Jack) X Horse (mare) = Mule
(62) (64) (63)
 The germ cells of hybrids proceed through mitosis, but there is a block to meiosis
since ‘pairing’ of homologous chromosomes is impossible due to ‘uneven
chromosome number’ having come from parents with different chromosome
numbers.
 Freemartinism: In this condition there will be non inflammatory degeneration of
testes which will affect the spermatogenesis.

VI: PATHOLOGICAL FACTORS AFFECTING SPERMATOGENESIS

 The pathological factors affecting spermatogenesis are divided into two namely
congenital factors and acquired factors.

Congenital factors

 Testicular hypoplasia: It is congenital or hereditary in origin caused by single

recessive autosomal gene with incomplete penetrance leading to lack of meiotic
division and block in spermatogenesis.
 Cryptorchidism: Spermatogenesis is inhibited by elevation of temperature of the
affected testis. The affected testis is usually small in size, soft, flaccid and do not
produce any spermatozoa.
  Imperfect descent of the testes: thermoregulatory system is affected and hence
spermatogenesis is impaired.
 Scrotal or inguinal hernia: this interferes with the normal thermoregulatory
mechanism and thereby affect the spermatogenesis.

Acquired factors

 Testicular degeneration: Several factors like heat, cold, trauma, excessive physical
strain, unilateral castration, irradiation, fever, toxaemia, hot environment, insect
bites on the scrotum, diseases like foot and mouth and other viral diseases,
vaccination against rinderpest and FMD, Vitamin A deficiency, injection of
autologous or homologous testicular material can cause testicular degeneration. Here,
spermatogenesis is affected and there will be presence of spermatocytes in the semen.
The seminiferous tubules are reduced in size.
 Testicular fibrosis: A marked degenerative change in the germinal epithelium
together with increased interstitial tissue is noticed. Semen is usually watery
containing few or no sperms.

VII: AGE FACTOR AFFECTING SPERMATOGENESIS

 Degenerative changes in seminiferous tubules of mammals have been

noticed in aging.
 Focal degeneration and calcification of tubules has been noticed.
 A decline in germinal tissue, an arrest of the later stages of
spermatogenesis and increase in connective tissue are associated with
aging.

Last modified: Monday, 4 June 2012, 02:50 PM

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INTRODUCTION

 The reproductive behaviour in animals is innate in nature. It is an obligatory

component of reproductive process.

The purpose of reproductive behaviour is to promote the opportunity

of copulation and thereby pregnancy will occur and the birth of young
one.

 As the bulls mature the manifestation of sexual behaviour become evident.

 The pituitary gland secretes the gonadotrophic hormones follicle stimulating
hormone (FSH) – maintains seminiferous tubules and initiates spermatogenesis and
luteinizing hormone (LH) - produces androgenic hormone – testosterone which is
responsible for sexual behaviour and secondary sexual characters.
 The male animal exhibits the sexual activity occurs at any time whereas in females it
is restricted only during the time of estrum .
 This behaviour involves series of endocrine and neural events. When the males attain
puberty their behaviour becomes more insistent towards opposite sex.

STAGES OF REPRODUCTIVE BEHAVIOUR

Stages of Reproductive consists of

 Precopulatory behaviour
 Copulatory behavoiour
 Postcopulatory behaviour

PRECOPULATORY BEHAVIOUR
 Each stage of the reproductive behaviour becomes the stimulant for the next stage.

Searching of sexual partner

 The identification of sexual partner involves the various senses like olfactory, optic,
auditory and tactile senses.
 The male animals will search its partner by seeking and identifying the estrual signs
of the female animals.
 Secretions from the female reproductive tract serve as sexual attractants and sexually
stimulate and attract the male to female.
 Pheromone is a volatile substance secreted or released outside the body and is
perceived by the olfactory system of other individuals of the same species.
 Males also produce sex pheromones that attract and
stimulate females eg. Boars.

 Males also produce sex pheromones that attract and stimulate females eg. Boars.
 Boars produce 2 attractants – from preputial pouch and in saliva secreted from
submaxillary salivary gland.
 The active components of the saliva are 3 alpha androstenol and 5 alpha
androstenone. Both the compounds have musk like odour.
 The presence of the male will stimulate the females to intensify its sexual responses.

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 The searching behaviours in are as follows:

Species Male Female

Bovine Approaching the female and Bellowing, restlessness,
testing for lordosis ,Chin increased locomotion,
resting twitching and elevation of
tail
Equine Visual search Increased locomotion and
flagging of tail
Caprine & Neck outstretched and head Restlessness
ovine held horizontally Sniffing
and licking the ano-genital
region,

Nudging and exhibiting

Flehmen’s reflex
Canine Roaming in its territory Roaming
Swine Moving with sows to identify Mild Restlessness
the receptive animal
Feline Prowling Vocalization
o Nudging – touching or pushing gently
o Prowling – move restlessly
Courtship/Sexual display

 The identification of the

sexual partner leads to the
initiation of courtship
behaviour with sniffing of the
vulva by the male, urination
in the presence of male,
flehmen reflex, chin resting,
circling and increased
phonation.
 The common courtship
behaviours in different
species are as follows
o Some pheromones are
less volatile and need to
be detected by
the vomeronasal
organ in the bull, ram,
stallion and to some
extent, the boar.
o The vomeronasal organ
is an accessory olfactory
organ and is connected
to two small openings in
the anterior roof of the
mouth just behind the
upper lip.
o Many species such as
the bull, ram and bucks
smell the genetalia as
well as the urine and
exhibit the flehmen
response which is
characterized by
elevation of head and
curling of the upper lip.
o Curling of the upper lip
 closes the nostrils and
allows a suckling
response to occur in the
nasopalatine duct.
o Less volatile substances
are aspirated into
the nasopalatine
organ where they are
evaluated by sensory
neurons.
o As in males, females also
exhibit flehmen
response to placenta,
newborn animals and
volatile substance.

The Flehmen’s reflex

is noticed in all
animals except swines.
Species Male Female
Bovine Flehmen, nuzzling and licking the Increased grooming and
perineal region mounting of other females
Equine Flehmen and High degree of Urinating in the presence of
excitement stallion
Caprine Flehmen, sniffing and licking of Urinating in the presence of
& ovine vulva, nudging the ewe ram
Canine Sniffing and licking the vulva Immobile stance
Swine Nuzzling, grinding of teeth, foamy Immobile stance
saliva
Feline Biting the queen at neck Crouching, head rubbing and
rolling

 Nuzzling – rub or push against with nose

 Sniffing - smelling
 Grooming - cleaning
  Crouching – bending the knee and brining the body forward and down

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Sexual arousal

 Among the all stimuli the visual stimuli is important for the sexual arousal.
 The courtship behaviours will end in lordosis/immobile stand/willingness to mate,
which stimulates the significant sexual arousal in male animals.
 Once the male identified the female is displaying the lordosis, it is intensively
stimulated.
 The following are the characteristics of sexual arousal in farm animals.

Species Male Female

Bovine Penile protrusion with Homosexual mounting and
dribbling of seminal fluid standing to be mounted
with few spermatozoa,
erection and attempted
mounts
Equine Penile protrusion with no Presents hind quarters to
pre-ejaculatory expulsion male, Clitoreal exposure by
of seminal fluid labial eversion (winking of
clitoris), pulsatile
contraction of labia
Ovine Repeated dorsal elevation Immobile stance
and of scrotum, Penile
caprine protrusion with no
dribbling of seminal fluid
Boar Penile protrusion, shallow Immobile stance
pelvic thrusts, attempted
mounting
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Erection
  The penile erection requires a series of neural and vasomotor reactions.

Erection is characterized by the marked increase in rigidity of

the penis.

 Erection is characterized by the marked increase in rigidity of the penis.

 The rigidity is due to elevated arterial blood flow, dilatation of corporal sinusoids,
restricted venous outflow, elevated intra penile pressure and relaxation of the
retractor penis muscle.
 The penis of the bull, ram and boar is fibroelastic in nature and is having sigmoid
flexure. Therefore during erection in these species the diameter will not increase
much.
 In stallions, the penis is vascular in nature and it is not having sigmoid flexure. So
there is significant enlargement in size of the penis occurs in this species.
 The erection is predominantly under the control of parasympathetic nervous system.
 The reflex stimulation of the testis, urethra, prostate and glans penis cause erection.

Penile protrusion

 The erection will leads to the separation of the glans penis from the prepuce.
 During this period, the dribbling of the secretions of Cowper’s gland is noticed in
bulls.
 The male will keep its chin on the receptive female and the female will stand quietly to
allow the male to mount.

COPULATORY BEHAVIOUR

Mounting

 The sexually stimulated active male mounts the female. Few initial
mountings may not be successful with dribbling from the penis.
 During this process of mounting the movement of hind limbs and
contractions of rectus abdominis muscle will align the penis horizontally
and vertically to seek the vulva for intromission

The male will fix its fore limbs around the females body
and will perform rhythmic pelvic thrusts.

Intromission

Successful entry of the penis into the vagina is called

as intromission.
  The thrusting movement of the pelvis will help intromission.
 The vulvar moisture and heat is identified by to and pro movement of the
penis and the sensory nerves of the penis is stimulated. This is major factor
for intromission.
 Stallion oscillates the pelvis many times, engorgement of penis occurs and
finally the intromission occurs.

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Ejaculation

 The time of ejaculation varies with the species.

 The animals like bull and ram will ejaculate in short period of time (within
one or two seconds) and are called as short copulators whereas the boar
and dog will take longer duration up to 30 minutes for ejaculation and are
called as sustained copulators.
 The stallion is intermediate copulator ( 20 seconds to 1 minute) with
regard to duration of copulation.
 In bull and ram the ejaculation is stimulated by warm temperature of the
vagina. But in stallions and boar, the vaginal pressure is important when
compared to temperature.

Species Volume of Site of deposition Duration of

ejaculate copulation
Bovine 3-5 ml Anterior vagina, external 1-3 sec
os of the cervix
Equine 75-120 ml Cervix and uterus 20-60 sec
Caprine/ovine 0.8-2 ml Fornix vagina 1-2 sec
Swine 200-250 ml uterus 5-30 min
Dog 1-5 ml Uterus 20-30 min
POSTCOPULATORY BEHAVIOUR

Dismounting

 Immediately after ejaculation the dismounting takes place and the penis is
withdrawn into the prepuce.
  Both males and females often display post coital behaviours like vocal
emissions, genital grooming, changing postural relationship, licking and
nuzzling. Post coital play is rare in farm animals like cattle, swine and
horse.
 The male goat licks the penis after ejaculation. The ram stretches its head
and neck .

Refractory period

 The refractory period in which either male, female or both will not engage
in copulatory behaviour.
 This period depends on degree of sexual rest prior to copulation, age of the
male, degree of female novelty and number of previous copulations.
 The refractory period is sometimes erroneously referred as Sexual
exhaustion.

This period should be considered as part of

satiation rather than exhaustion

 Restimulation may occur after the refractory period while no further sexual
behaviour can be induced even if sufficient stimuli are present.
 Most males will not show interest towards female immediately after
ejaculation.
 The period of refractoriness will vary between individual males.
 The period of refractoriness can be modified by environment and new
stimuli
 The boar and stallion reach exhaustion after few ejaculations than bull and
ram.
 The Coolidge effect can be defined as the restoration of mating behaviour
in males that have reached sexual satiation when the original female is
replaced a novel female. In other words, a sexually satiated male can be
restimulated if exposed to a novel female.

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Maximum number of ejaculations for exhaustion (per day)

Animal Average number of Maximum number of

ejaculations ejaculations
 for satisfaction to exhaustion
Bull 20 60-80
Stallion 3 20
Ram and 10 30-40
buck
Boar 3 8

Memory

 Memory is important in both positive and negative way.

Positive experiences during copulation will promote the

reproductive behaviour where as the negative inhibit the
reproductive behaviour.

 Positive experiences will encourage the both the sexes.

FACTORS AFFECTING SEX DRIVE

 The male sexual behaviour (sex drive) is affected by the certain factors
o Individual.
o Sexual experience.
o Presence of opposite sex partner.
o Metrological attributes.
o Hormone.
o Pheromone.
o Management.
o Nutrition.
o Growth.
o Genetic.

Individual animal

 The sex is an inherent instinct of individual.

 The animal having pendulous sheath shows longer reaction time. Whereas, the old
individual has poor-worst sex libido.
 The temperament of individual animals plays a vital role in sexual behaviour.

The individual with strong temperament exhibits vigorous

 response with excellent sex drive. Whereas, the individual with
weak temperament exhibits dull response with poor sex drive.

 Aged animals and young animals show a marked difference in the sex libido.

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Previous sexual experience

 During the first contact with the receptive females the inexperienced young male
animals will show hesitation, spend a long time in exploring the genitalia , mount
with erection descend and try to mount again.
 The efficiency of copulatory capability in males increased by experience.

Opposite sex

 The presence of opposite sex of the same species greatly affects the grading of sex
libido, due to her look, vocal sound, physical touch, body/body fluid smell.
 The males raised with females before puberty had a greater mating frequency than
males raised without females.
 The total number of copulations and the sum of all the courting behaviour activities
are greater for the boars reared in all and mixed sex groups (social free) than for those
raised in social restriction.

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Seasonal influence

 The change of season affects the sex libido of the male.

 The comfortable season indicates excellent sex drive while adverse season indicates
poor sex libido.
 It is observed due to the direct effect of season on the sensibility of the nervous
system.
 In India, the spring season (Feb-April) and autumn season (Oct-Nov) are conducive
for excellent sex drive by indigenous bovine.
 The winter season is pleasant for expression of excellent sex drive by exotic Holstein
bulls in India, due to cold climate.
 The seasonal variations in sex-drive of ram, buck and stallion are seasonality of
pituitary function controlling the secretion of gonadotrophins. The sex libido is
reduced in summer season, due to adverse hot climate.
 The rainy season is conducive for expression of sex libido in full intensity among dog.
 The sex libido is adversely affected with higher ambient temperature.
  The sex libido during early morning hour is fully expressed due to lower ambient
temperature which is forming a conducive environment for expressing sex libido.

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Hormone

 The sex hormones activate the central nervous system. The level of testosterone and
sensitivity of the brain chiefly affect the grading of sex libido in male.

Pheromone

 The pheromone plays a major role in the sexual behaviour of animals.

 The characteristic odour of vaginal discharge, urine and body of female partner infuse
sexual motivation among males.

Management

 The managerial factor is vital for efficiency of sex libido.

 The proper management of male livestock depends on its feeding, housing, watering,
exercising, washing, bedding, health care, prophylaxis, semen collection technique,
semen collection schedule, semen frequency, semen collection time, artificial vaginal
preparation and its temperature, pressure and lubrication.
 The rearing of breeding male in individual pan is better than the rearing of breeding
male in loose field.
 With the increased frequency of copulation, the sex libido either may decrease or even
stop.
 The sex drive is improved in the boar when they are allowed exercise.
 The change of site for semen collection or the change of animal attendant affects sex
libido of the male sex libido.

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Nutrition

 The balanced ration plane is always preferably advised for optimum sex drive of male.
 Either the low ration plane or the high ration plane show negative trend on sex drive
of animal.

Growth

 Usually the proper body growth is expressing a good sex libido

  While, the overweight male becomes lazy, dull, experiences pain during copulation
and exhibits poor sex libido.
 The general body growth also affects indirectly the growth of testes.
 The bull having well developed testis show better sexual behaviour. Whereas the bull
having relatively ill-developed testes shows poor sexual behaviour.
 The testes volume is positively correlated with daily gain and all testis measurements
are closely correlated with testosterone concentration.
 The growth rate of male livestock during initial age is comparatively more important
than during later age of animal life.

Genetic

 The genetic component of the male livestock affects the sex libido.
 The breed and strains affect the libido.
 The dairy breed are more sexually active than beef breed of the cattle-bull.
 The Yorkshire breed of boar have better libido than Duroc breed of boars.
 The cross breeding with exotic animals improves the sex drive of the native animals.

INTRODUCTION

Infertility –Temporary loss of fertility in male characterized by reduced

number of viable spermatozoa in the ejaculate

Nils Lagerlof divided the forms of infertility in males into 3

general categories

 Reduced to complete lack of sexual desire and ability

to copulate (Impotentia Coeundi)
 Inability or reduced ability to fertilize the ovum due to
pathology of testis, mesonephric duct and accessory
sex glands (Impotentia Generandi)
 Miscellaneous forms affecting reproductive organs.

 These above conditions are present in males of all species.

 The degree of each condition present in males varies considerably between species,
breeds, families and individuals. There are many degrees, from mild to severe in each
category of the various forms of infertility.
 Sometimes reduced sexual desire and ability to copulate may be associated with
reduced fertility and poor quality semen but in most cases of infertility in males the
two conditions are not related.
  In male animals semen collection is usually possible so that direct measurement of
semen quality and other test may be applied to diagnose male infertility.
 In examining males for infertility or sterility,
o Accurate breeding and health records on the male and the herd should be
obtained and examined.
o Secondly, there should be a careful, painstaking physical examination of the
male including the observation of his mating behavior.
o Thirdly, one or more thorough semen examination by a trained veterinarian or
qualified laboratory is necessary.
 To evaluate the nature of a male’s infertility and sterility so that proper
recommendations for therapy, treatment or disposal of the animal may be made.
 The prognosis in nearly all forms of infertility or sterility in male animals should be
guarded.

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Potency
Potency is the physical capability of the entire body to coordinate
and performs the male’s normal role at coitus including erection,
mounting, intromission and ejaculation.

 A lack of potency is observed in certain males in all species and is characterized by

symptoms ranging from a complete lack of sexual interest and inability to copulate to
a slight slowness or delay in the exhibition of libido, mounting and copulating.
 Libido is defined as the willingness and eagerness of a bull to attempt to mount and
service .
 In the female animal reproductive behavior is relatively simple requiring only a
willingness to stand to be mounted or an attitude of acceptance. This attitude is
primarily under the control or influence of oestrogen which may
be enhanced by progesterone.
 In the male animal reproductive behavior is more complex requiring identification,
seeking out, teasing and then the performance of the complicated act of
copulation. This is under the control or influence of testosterone and other central
nervous system mechanisms.
 The components of the act of copulation consist of sexual excitement, courtship,
erection, mounting, intromission, ejaculation, dismounting and refractoriness.
 There is a great variation in the duration of these components between species and
individual males within a species. Mating behavior depends to a varying degree
on visual, olfactory, auditory and tactile cues. In all species visual and tactile
cues are probably most important in the actual act of copulation but even in blind
males may copulate if experienced.
 Height, width and color of the female, attitudes of acceptance, such as her stance as
the male approaches, reaction of the female to pressure of the bulls head on her rump
or back, pressure of the shoulder on the mare’s rump or biting of the skin of the
 mare’s rear parts by the stallion and by the boar’s snout lifting of the sow’s rear
quarters, odour of the rear parts in horses and sheep and vocalization by the stallion
and boar are all factors in the precopulatory and copulatory acts in domestic animals.
 Sex drive, libido or sexual desire of the male is largely determined genetically but that
environmental influences play an important role in modifying it. It is well known that
males differ widely in their ability to copulate frequently.
 The differences between animals were due to the reactivity of their tissues, such as
the brain, rather than to the differences in the amount of hormone secreted or
present in the body.
 Thus variations in endocrine function may be linked with changed reactivity in the
target organs, especially the central nervous system, as well as with the hormone
producing organ. Many of the complex components of copulatory behavior are
determined by hormone action during various stages of development and may be
persist after castration.
 It is extremely difficult to evaluate the libido and mounting ability of Brahman, Zebu
or Santa Gerturdis bulls since these breeds copulate mainly at night and rapidly so the
copulatory act can only rarely be observed.
 Innate virility is reflected in the number and frequency of copulations that occur in a
period of time and these vary widely between males.
 Although sex drive, mating behavior or libido is largely genetic nature it is subject to
great modification by many environmental or physical factors.

Males with strong sex drive require more severe and prolonged environmental and
physical insults to significantly affect their mating behavior than do males with weak
sex drive.
FACTORS CAUSING IMPOTENTIA COEUNDI

 Environmental causes
 Joint,Muscle,Bone, Tendon and nerve injuries
 Diseases of the penis ans prepuce
 Miscellaneous causes

ENVIRONMENTAL FACTORS

Nutrition

 Thin emaciated semi starved males or those suffering from deficiencies of TDN,
vitamin A, protein and certain minerals such as phosphorus and cobalt may have a
definitely reduced sex desire
 Inanition is severe enough a complete lack of libido results
 Low level of energy diet in growing males delays the puberty and onset of libido.
  Overfed animals tend to become obese and lazy, often suffer from joint and foot
troubles which may lead to lack of sexual desire. .
 Excessive roughage fed to the bulls and rams may cause great enlargement of the
rumen and abdomen interfering with normal , easy copulation and contributing to a
lack of sexual desire.

Systemic disease

 Any chronic or acute, severe debilitating diseases resulting in rapid or prolonged loss
of weight, or in anorexia, depression and weakness will cause a varying degree of loss
of sexual desire.
 These diseases may include
o Pneumonia,
o Enteritis,
o Tuberculosis,
o Para tuberculosis,
o Severe mange and pediculosis,
o Actinomycosis,
o Lymphocytoma,
o Progressive fat necrosis,
o Severe internal parasitosis,
o Advanced metastatic tumors,
o Alveolar periostitis,
o Traumatic gastritis,
o Severe chronic peritonitis and others.

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Age

 Too young and too old animals usually exhibit a reduced to complete lack of sexual
desire.
 In older animals this may be due to decline in testosterone level, senility, loss of
condition, overuse or to arthritis.
 Inexperience in young male should not be confused with lack of sex desire.

Management

 The management practice plays a major role which affects the sexual desire of male
livestock.
 Libido will vary with the animals depending upon their inherent sex drive and the
way they are trained, handled and managed.
  Young males should be carefully, patiently, quietly trained and handled, especially if
they are to have their semen collected in artificial vagina.
 If a male associates “sex” with pain or punishment, he may decide to give up “sex”.
 Slow breeders can be easily discouraged and made slower by the insertion of a nose
ring in a bull, harsh or abusive handling of the male by attendants, improper restraint
of the mount animal, improper footing, mount animals that are not in estrum or are
too tall or wide, use of an artificial vagina that is too cold or hot, improper preparation
of the male for mounting, breeding large males in a confined area with a low ceiling,
unskilled persons using an artificial vagina and excessive use of a male.
 Males lacking libido if continuously used on the same female for semen collection
frequently develop sexual indifference or satiation.
 Frequent changing of stimulus or mount animal and the collection and breeding site
are indicated in bulls inclined to show a lack of libido.
 The presence of other males near the mount animal or in sight of the breeding male
provides further stimulus.
 Frequent changing of the mount animals, allowing several ‘false’ mounts where
copulation is not allowed and moving the male to several different sites for teasing or
stewing
 Some stallions and jacks with low desire will fail to copulate with mares that are well-
scrubbed and the tails bandaged. Others so vigorously bite the withers and neck of the
mare after mounting that erection is lost. Muzzling if these males is indicated.
 Stallions will perform intromission and make thrusting movements but fail to
ejaculate. This is common in stallions with reduced libido that are being used too
frequently for their reproductive capacity.

The failure to ejaculate can be noted by careful observation

of the stallion at copulation by the

 absence of the usual flagging of the tail,

 absence of the pulsations of the penile urethra,
 the failure of the stallion on dismounting to be content
and relaxed
 the lack of spermatozoa in the tail end sample of
ejaculate collected from the penis as the stallion
dismounts.

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Psychic factors

 Males with a genetically lowered sexual desire are much apt to develop this apparent
psychic refusal to breed.
  Some bulls with apparent psychic impotency may actually be affected with lesion of
spinal columns.
 These bulls could apparently have good sexual desire, mount rapidly, have lordosis in
the lumbar region and the penis could be directed to escutcheon well below the vulva.

 Rapid pelvic thrusts and seeking motions for vulva would occur but penile exposure
was greatly reduced.
 A prolonged period of sexual rest, a change in the site of copulation and careful
preparation may be necessary to encourage the psychic males to again start to breed.
 Shyness or slowness also noticed in boars.

Hypothyroidism, Hypogonadism or Pituitary deficiency

Based on the results of hormonal therapy, it is understood that a lack of hormones is

not a common cause of lack of sexual desire in male animals.
PROGNOSIS AND TREATMENT

Prognosis

 Guarded to poor depending upon the cause and the degree of the inherited or
acquired lack of libido.
 For bulls affected with a lack of libido is often requires 3 to 6 months of proper
handling for sexual desire to improve after adverse environmental influences have
been corrected.

Treatment

Treatment should be given after

 Careful collection of the male’s breeding

history
 Thorough physical examination of the
male and
 Repeated observations of the male
during coitus.

o Proper amounts of good quality grain and roughage should be provided to

reduce obesity (if present) or to increases the body weight (if the male is too
thin)
o Sufficient exercise should be provided.
o Chronic disease status especially parasitism in young males should be corrected
if possible.
o The virility of the male should be assessed and the frequency of services should
be reduced.
o A period of several months sexual rest is desirable in males that have been
excessively over used.
o Changing the site of semen collection.
o Good footing and proper ceiling height in the semen collection yard.
o Proper restraining of the female or teasers while service or semen collection.
o Care, consideration and patience in the handling of the slow breeding male.
o If Artificial Vagina (AV) is used, skilled operator is essential.
o The AV should neither too warm nor too cold and adequate pressure should be
applied to the penis.
o Frequent changes of teasers and a longer period of teasing is often helpful.
o Libido can be restored by allowing the male to stay with one or more quiet non-
pregnant females.
o As a last resort when the condition fails to respond semen may be collected
by using electro ejaculator in bulls, rams and boars and in dogs by using digital
manipulator.
o Drugs and hormone treatments are of questionable value in most males lacking
sex desire.

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o Testosterone may be used but it is not the specific treatment for improving the
libido in males. Prolonged high level of testosterone therapy may cause
testicular degeneration and atrophy of testis.
 Dose: Bull : 100-500mg
 Stallion : 100-500mg
 Ram : 50-100 mg
 Boar : 50-100 mg
 Dogs : 10-50 mg
 Repeated every 5-10 days for several times.
o One or more injection of human Chorionic Gonadotrophin (hCG) at 4-7 days
intervals in doses of 3000-4500 IU for large animals and 100-500 IU for dogs
may help to stimulate testosterone production. This is the specific treatment in
males to improve the libido.
o Pure LH preparation can also be used.
o Males that are obese and lazy possibly due to a hypothyroid condition may
benefit from feeding Iodinated Casein with 3 per cent thyroxine potency at a
rate of 1gm per 100 lb body weight daily.
o Glucocorticoids are also used with questionable success.
 o Injections of vitamins and feeding of trace minerals; protein and iodine have
little value in in most of the slow breeding males.

JOINT, MUSCLE, NERVE, BONE AND TENDON INJURIES AND

PATHOLOGY

 Lesions affecting these structures particularly in hind quarters including hind limbs
may cause reduction in sexual desire.

Coxitis

 Inflammation of hip joint

 Common in dogs and boars
 Characterized by a shot stride and adduction of the limb.
 Rupture of the round ligament may be observed in bulls with degenerative coxitis.
 Hip dislocation in one or both limbs may cause inability to copulate.

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Gonitis

 Common in bulls
 Characterized by a short, stiff gait, distension and enlargement of joint capsule of the
stifle.
 Rupture of the anterior cruciate ligament of the stifle inhibits mounting in bulls and
dogs.

Tarsitis or Degenerative joint lesions in the fetlock or phalangeal joints or ring bone may
result in pain and reluctance to copulate.

 Other conditions causing similar signs of reduced libido include

o Over-grown claws or hooves
o Suppurative podo dermatitis
o Quittors or interdigital granulomas
o Foot rot or interdigital necrosis
o Tendonitis
o Suppurative arthritis of the coffin joint
o Traumatic peroneal nerve paralysis
o Rupture of gastrocnemius muscle or gluteal or croup muscle.
o Laminitis
o Poly arthritis
o Fracture of pelvis (Rare)
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Spinal Diseases

 In bulls and dogs, symptoms of impotency may be related to spinal disease.

 Characterized by stiffness and soreness in the gait, spinal rigidity and pain over the
vertebrae.
 If the spinal cord is compressed then a slight to marked paralysis may be present with
a swaying, unsteady gait, a slightly flaccid tail and dragging of the hind limbs.
 Bulls with spondyl-arthroses especially of the lumbo-sacral joint may develop loss of
sex libido.
 Bulls with spondylosis often involving most intervertebral spaces and discs, exhibited
“Kyposis” a stiff back, stiff hind leg movements or goose stepping and loss of
liveliness and mobility. It is common in bulls over 10 years of age.
 In dogs, especially brachcephalic breeds such as the Dachshund, prolapse of
intervertebral discs causing compression of the spinal cord results in inability to
copulate.
 Tumors also cause compression of spinal cord lead to impotency.

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Spastic syndrome

In Spastic syndrome or Crampiness or Stretches in bulls, may

interfere with or prevent copulation due to prolonged spasms of
the skeletal muscles of the hind limbs and back.

 All breeds are affected. But common in Holstein Friesian and Guernsey breeds.
 It is probably inherited as a single recessive factor with incomplete penetrance.
 Bulls over 3 years of age are affected.
 Spastic signs are not observed when the bull is lying down but become evident or
severe on standing.
 PROGNOSIS AND TREATMENT

Prognosis

 Depends upon the nature and severity of the condition and the species, age and value
of the animla.
 In fractures of the spine, dislocation of the hips and compression of the spinal cord
due to arthritic lesions and tumors in large animals, the prognosis is poor to hopeless.

Treatment

 Sexual rest and close confinement with good footing

 Animals recovered from arthritis should be handbred when the footing is good and
where the rear parts or the entire female animal are placed in a pit so that the male
can easily copulate or bred on AV
 Electro ejaculator can be used in males that are unable to copulate
 Some males can be trained to ejaculate in the AV with all four feet on the ground i.e
without mounting and by only on standing position.
 Surgery may be indicated in intervertebral disc prolapse in dogs.
 Quittors or interdigital granulomas should be removed surgically
 In suppurative arthritis of the coffin joint, prolonged conservative therapy is indicated
 Bovine spastic syndrome is treated with antispasmodic drugs and tranquilizers.
 In arthritis-Glucocorticoids may be given
 Regular hoof trimming in large male animals is indicated.

DISEASES OF THE PENIS AND PREPUCE INCLUDES

 Inability to protrude the penis
 Deviation of the penis or phallocampsis
 Adhesions of the penis and prepuce
 Tumors of the penis and prepuce
 Phimosis or stenosis of the preputial orifice
 Paraphimosis
 Balanoposthitis
 Defects in the penis

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 INABILITY TO PROTRUDE THE PENIS

A congenital anomaly in the development of penis and prepuce with or without

hypospasdias.

 Observed in all species but is seen most commonly in Boston Terriers breed of dog.

Congenitally short penis

 Occurs in bulls, bucks, boars and horses.

 It may be hereditary
 Retractor penis muscle is normal in the bulls.

Bulls with a short penis the sigmoid flexure does not form a sharp
S-curve in the resting state

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A Congenital Short retractor penis muscle

 Described as a recessive character in Friesians

 Myectomy of this paired muscle under epidural or local anesthesia at the midway
between the anus and the base of the scrotum resulted in improvement.
 Affected animals are culled due to hereditary reason.

Psychic Impotency Caused by

 Injury or lesions of the lumbar or scrotal region

 Phimosis
 Stenosis of the preputial orifice
 Tumors of the penis and prepuce
 If a bull is unable to protrude his penis, normally, a careful physical examination of
the penis and prepuce should be made.
 Use of tranquilizers, pudendal nerve blocks or electro ejaculator may be helpful in
arriving an accurate diagnosis.

Bulls with short penis should be slaughtered and not used for breeding purposes
because of the possible hereditary nature of the defect
DEVIATION OF THE PENIS OR PHALLOCAMPSIS
Spiral or corkscrew type of deviation of the penis

 The condition may be inherited

 Affected bulls successfully bred the cow for one or
more years.
 When complete erection occurs the corpus
cavernosum in the free end of the penis rolls inside
the fibrous tunic and the penis slips and pushes
laterally under the dorsal apical ligament and the
glans penis spirals counterclockwise ventrally and
to the tight around the line of penile raphae.
 May be favoured by early maturation of the
supporting structure of the penis and late
maturation and growth of the penis under the
continued influence of testosterone.

“Cork screwing” of the penis occurs at

the peak of erection when the layers
covering the free end the penis is
stretched. If the erection is partial it
does not occur.

 In affected bulls premature full erection occurs

prior to intromission resulting in corkscrewing that
prevents completion of coitus.
 Other less common types of the penis are the
ventral or rainbow and the mild ‘S’ shaped
curvatures and Double penis or diphallus.

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  Persistent frenulum is a band of tissue that extends
from near the ventral tip of the glans penis to the
prepuce.
 Epithelial separation and rupture of frenulum occur
normally a puberty.
 When a persistence of the frenulum occurs there is
usually a blood vessel present in the center of the
tissue band comprising the frenulum.
 There is evidence that this defect is hereditary

Treatment

 The corkscrew or ventral or mild ‘S’ shaped defects of the penis are corrected by
surgical attempts.
 Cutting of the connective tissue band with or without ligation is a simple procedure
and is uniformly successful for the correction of persistent frenulum

Since deviations of the penis are mostly hereditary especially cork screwing, it
is best to cull the affected animals.
ADHESIONS OF THE PENIS AND PREPUCE

Adhesions on the region of the sigmoid flexure

 In bulls and rams due to trauma from horn injuries

 In bulls, local anesthesia to block the dorsal nerve of the penis rarely cause
adhesion
 These adhesions prevent the obliteration or extension of sigmoid flexure at
the time of erection.

Treatment to relive these adhesions of

penis often results in severe adhesions

 An adhesion in the region of fornix of the prepuce to the abdominal wall or to the skin
produces more severe phimosis.
 These adhesions may be secondary to the laceration of the prepuce especially in
young bulls where the prepuce has not completely separated from the glans.
  In older bulls vigorous thrusts may tear the prepuce away from its attachment to the
glans lead to adhesion.
 Infection that follows this injury may produce abscess and/or adhesions to the
surrounding structures results in prevention of free movement of the penis and
prepuce at erection.
 Treatment: Antibiotic therapy both locally and parentally for 7-14 days.
 Sexual rest for 6-12 weeks.

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A ruptured, fractured or broken penis

 Observed in bulls with secondary haematoma

 Broken penis occurs in bulls with a strong sex drive and breeding the cow naturally.
The condition is rare in bulls are hand bred.

 The injury occurs at coitus when the cow goes down under the weight of the bull when
bull mounts or due to a sudden ventral bending of the erect penis against the
escutcheon (between thighs) at the movement the bull thrusts.

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Symptoms, Prognosis and Treament

o Symptoms include shortening of the stride, stiffness, penis and a slight arching
of back.
 o A swelling rapidly develops just cranial to the scrotum that varies in size
depending upon the amount of hemorrhage from the ruptured penis.
o Hemorrhage is profuse
o There is usually no difficulty in urination
o At first swelling is soft and fluctuating, later it becomes firm and hard
o The bull show definite reluctance and inability to copulate
o The blood clot may become infected and produce abscess
o Adhesions may occur between penis, prepuce, abdominal wall and skin
rendering the bull useless for future service
o Differential diagnosis from other conditions such as tumors, chronic fibrous
adhesions and rupture of urethra should be made

Adhesions of the deeper or caudal portion of the prepuce

Prognosis is guarded even following

surgical operation is performed

o Secondary haematoma that become infected rarely respond to treatment and

surgery.
o The surgical operation is usually performed between 4 and 10 days after the
rupture has occurred. Until then the bull should be given parenteral antibiotics.
o Rupture of penis occur in stallion due to the mare’s kicking the erect penis when
the stallion mounts.
o In dog traumatic injury may cause fracture of the penile bone

TUMORS OF THE PENIS AND PREPUCE

Tumors of penis may cause phimosis or
paraphimosis and prevent normal
intromission.

Bulls

 The only significant tumor is the transmissible fibropapilloma caused by virus.

  They are single or multiple, firm, cauliflower like growths.
 Young bulls (9-18 months age) are commonly affected
 Young bulls grouped together frequently mount on each other and may injure
prepuce affording an invasion site for lesions.
 Injury to the penis occurs when the young bulls breeds before the separation of penis
and prepuce.
 Haemorrhage from the sheath after service and refusal to copulate are frequently
noted in bulls with penile tumors.

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Treatment

 Spontaneous recovery from infectious fibropapillomas occurs within 4 Months

 If the tumors are multiple on the penis- treatment is difficult.
 Wart vaccine can be tried. But it has questionable value in affected bulls.
 Semen from affected bulls not to be used because of the danger of transmitting the
virus to cows.
 Surgical removal under pudendal nerve block.

Stallion

 Uncommon
 When present usually squamous cell carcinoma of low malignancy
 Should be differentiated from granulomas caused by habronema larvae
Treatment

 The occasional squamous cell carcinoma can be removed by a liberal incision or if

necessary by amputation of the penis

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Dogs

The transmissible venereal tumor(TVT) is the

most common tumor on the penis and prepuce

 TVT- spread by coitus

 Licking of the vulva or preputial discharge rarely transmits the disease
 Intact cells must be transplanted to transmit the tumor
 Incubation period 5-6 weeks
  All breeds of dogs are susceptible
 It is characterized by a discharge of bloody, fetid exudates from the prepuce of the
male.
 When penis is exposed, grayish-red nodular growths are observed on the penis and
prepuce
 The tumor masses are friable and bleed when handled.
 In advanced cases the tumor may also be observed involving the inguinal lymph gland
 The tumor ulcerates easily
 The transmissible venereal tumor has been reported widespread throughout the
world but most commonly in tropical countries.
 Papillomas, squamous cell carcinomas, sarcomas and other tumors may occur in
sheath or on the penis of dogs.

Treatment

 In the dog surgery may be indicated. The earlier surgery is undertaken, the better the
success of operation.
 In severe cases where it is difficult or impossible to remove all of the involved tissue,
radiation therapy may be indicated.
 Medically treated with Vincristine at the dose rate of 0.025 mg per kg b.w. slow i/v
diluted in saline or distilled water and repeated at 7 days interval until complete
regression.

PHIMOSIS OR STENOSIS OF THE PREPUTIAL ORIFICE

Inability to protrude the penis outside the
sheath or prepuce is called phimosis.

 Stenosis of the preputial orifice is usually acquired due to injuries, wounds and
infections.
 In cattle with pendulous sheaths, the preputial orifice may be stepped on causing
severe contusion and swelling.
 Congenital stenosis in dogs may be corrected by a dorsal incision of the external
preputial orifice.
 In bull, dog or ram, the usual procedure to correct a simple stenosis of the preputial
orifice caused by cicatricle tissue is to remove a triangular portion of the skin from the
ventral portion of the sheath or prepuce.
 The base of the triangle is at the preputial orifice.
 After the skin is removed, an incision is made through the midline of the prepuce to
the apex of the triangle.
 After the careful hemostasis the preputial membrane is suture to the skin by
interrupted catgut sutures.
 Chronic prolapse of the prepuce is a very common cause of posthitis and phimosis. It
is common in Bos indicus cattle.
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 Pendulous sheath, a large preputial orifice, relaxed preputial membranes are

inherited traits.
 In the affected breeds, the prolapse of the prepuce may occur and it becomes dry,
traumatized, swollen and fibrotic.
 Mild cases-the affected bull may be confined.
 The prolapsed organ should be carefully washed, cleaned and dried.
 Oily antibiotics or bland antiseptic preparations are applied and the prolapsed organ
is replaced and held in position by purse-string suture through preputial orifice.
 More severe cases where replacement is not possible, circumcision or even
amputation of the prepuce is necessary.
Prevention

 It is undesirable to operate an affected bull since the prolapse of prepuce is genetically

predisposed.
 Males should be selected for lighter, less pendulous sheaths with smaller preputial
orifice and stronger retractor penis muscle.
 Phimosis may be congenital in young dogs, cats and horses

PARAPHIMOSIS
The inability to withdraw the penis
into the prepuce called as
paraphimosis

 It results in edema, swelling and balanoposthitis.

 It may occur following erection of the penis through a stenotic preputial ring or orifice
caused by a congenital or acquired stricture or by tumors.

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 Paralysis of the penis and paraphimosis may be due to spinal diseases or trauma.

Paralysis of the penis is seen in bulls with

rabies and in horses in the late stages of
dourine.
  It is possible that the posterior paresis reported in a few cases of rhinopneumonitis,
due to equine herpes virus I, might be cause of occasional unexplained cases of penile
paralysis in stallions.
 Paraphimosis secondary to edema and swelling that occurs postoperatively may
follow castration of stallions.
 Resection of the retractor penis muscle in bulls with pendulant sheaths may cause
paraphimosis. It is not observed in the dairy breeds with a sheath that is closely
attached to the abdominal wall.
 The prognosis in paraphimosis in males is guarded and depends upon the
promptness of treatment and the degree of trauma or necrosis present. Support of the
prolapsed penis and sheath is essential to minimize gravitational edema.
 Cases of paraphimosis in horses following castration and the use of tranquilizers
should have the penis supported or held within sheath within an hour or two of its
occurrence to prevent a chronic prolapse of the organ often necessitating amputation.
 Cold packs and pressure may reduce the swollen penis and allow easy replacement.
 In many cases in dogs and occasionally in bulls, enlargement of the preputial
opening is necessary in order to replace the penis and prevent a recurrence of the
condition.
 For necrosis of the penis secondary to acute paraphimosis and for chronic paralysis
and prolapse of the penis in dogs, amputation is recommended.
 In bulls, the cleaning of the penis, the removal of necrotic tissue and the liberal
application of ointment. Gauze should be wrapped around the penis to protect it. The
penis with its gauze dressing should be placed inside the sheath as soon as possible.
 Petroleum jelly should be packed into the sheath to prevent adhesions. Frequent
withdrawal of the penis and applications of ointment are indicated.

BALANOPOSTHITIS

Dog

 Balonoposthitis is common in the bull , ram and dog and uncommon in the the boar
and cat and rare in stallion.

Bull

 The preputial cavity in the bull contains a variety of bacteria, molds, protozoa and
viruses including;
o Vibriosis
o E.coli
o Streptococcus sp.
o Staphylococcs sp.
o Pseudomonas aeruginosa
o C.pyogenes
 o Proteus sp.
o Actinomycetes necrophorus
o Actinobacilli
o Aspergillus, mucor, Absidia (molds)
o Mycoplasma
o Trichomonas fetus.
o IBR-IPV virus

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 No obvious Balanoposthitis generally accompanies the presence of the above

organism with a possible exception of M.tuberculosis and acute form of IBR-IPV.

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 Tuberculosis of the penis and prepuce may be characterized by enlarged,

granulomatous bleeding lesions of the glans penis, adhesions of the penis and
prepuce and sigmoid flexure with secondary phimosis.
 Actinomycosis also affects the sheaths and the glans penis of bulls of the lesions
resembled those of tuberculosis.
 Trauma, abrasions, lacerations of the prepuce or glans penis usually result in the
introduction of the above wound infection organisms into the deeper tissues which
lead to Balanoposthitis.
 Injuries from artificial vagina including loss of rubber band from vagina into the glans
penis.
 Homosexuality among males leads to balanoposthitis.
 IBR-IPV can produce an acute inflammation and ulceration of the penis and prepuce.
Most bulls recover within a one or two weeks. Because of the infectious nature of the
 disease bulls should not be used for services until 6 to 8 weeks from the onset of
attack.

Ram

Balanoposthitis in rams is called “Pizzle rot” or

"Sheath rot" .

 The most common infectious form of Balanoposthitis is due to virus that

causes ulcerative dermatosis, ulceration of the tip and leg and necrotic venereal
disease.
 The lesion on the preputial orifice, prepuce and glans penis are ulcerative and covered
by scab. Removal of the scab reveals a shallow, raw, bleeding crater containing
creamy odorless pus.
 The prepucial lesions may cause phimosis or paraphimosis and an extensive penile
lesion may render the ram useless as a breeder.
 Lesions may also be present on the lip, nostril, feet or vulva.
 The disease should be distinguished from contagious ecthyma and common non
specific lesions occasionally seen on the preputial orifice of rams.
 Ulcerative dermatosis is spread by ventral contact and other means.
 In non-infectious forms, two degrees are recognized,
o an ulceration of the prepuce near the prepucial orifice and a
o stenosis of the orifice with a secondary chronic ulceration of the deeper portions
of the prepuce accompanied by the accumulation of pus and necrotic material.
 This occurs in wethers or rams of 2 to 4 years of age.
 It is often associated with a wet spring and pasturing sheep on lush grass,rye, clover
or Lucerne that are high in protein.
 Changing of the diet to dry feed a lower protein intake, or fasting results in an
improvement in the balanoposthitis.
  In sever cases the sheath may have to be surgically opened ventrally to permit
urination and the saving the life of the animal.
 Extending the preputial incision too far caudally may render a ram useless for service.
Superficial external lesions may be treated by local therapy.

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Horse

 Balanoposthitis in the horse may occur in gelding or stallions.

 Early symptom of dourine, a reportable venereal disease of horses transmitted by
coitus, are swelling, edema and reddening of the penis and prepuce. This disease is
caused byTrypanosoma equiperdum
 In equine venereal balanitis and vulvitis formerly called genital horse pox or coital
exanthema caused by a herpes virus.
 There was no effect on fertility but affected stallions refused or were hesitant to
copulate.
 Cutaneous Habronemiasis, summer sores or genital bursatti may affect the equine
penis and prepuce. The habronema larvae may produce fungoid granulomatous
growths of 1 to 3cm in diameter that may contain firm necrotic irregular shaped
masses or “kunkers”. These lesions bleed readily on manipulation. These lesions may
produce intense pruritis.

Boars

 Balanoposthitis seldom occurs in boars.

 The preputial diverticulum in boar often fills with foul smelling urine and smegma
and externallay may resemble an umbilical hernia.
 The boar’s sheath may become infested with screw worms.
 In pigs with hog cholera, ulcers of the prepuce as a result of infarction.
 Ulceration of the porcine penis due to other causes may be observed. These
conditions in boars are usually easily diagnosed and treated.

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Dog

 In the dog, Balanoposthitis is occasionally observed but rarely it is a cause of failure of

copulation.

 It is characterized by a discharge of pus from the prepuce and it usually responds well
to mild antiseptic douches followed by bland antibiotic ointments or solutions.

Balanoposthitis in the dog should be differentiated

from the prostatitis as both are characterized by pus
appearing at the preputial opening.

 Suppurative ulcerative and follicular balanoposthitis or preputial catarrh is common

in the dog.
 Balanoposthitis in the dog, as in other animals , is due to mixed infections, trauma,
foreign bodies, other general diseases and debility.
 In the dog acquired phimosis is less frequent than is congenital phimosis.

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Prognosis

 It depends upon the severity of the trauma or the infection.

 In mild cases the prognosis is good.
 In severe chronic cases with adhesions between the penis and prepuce or between the
prepuce and adjacent tissues, the prognosis is guarded to poor.
 In cases of prolapse of the prepuce requiring amputation, the prognosis is usually fair
to good if the amount of prepuce is removed is not excessive.
 Some males may be slow to regain their libido following a painful Balanoposthitis.

Treatment

 Treatment of mild cases of balanoposthitis may consist of douching the prepuce with
aqueous or oily antiseptics or antibiotic preparations such as saline, 50 to 200 ppm of
chlorine solutions, 1:2000 acriflavine or potassium permanganate solutions, 1%
hydrogen peroxide solution etc.,
 Caustic or irritating antiseptics should
be avoided.

 Treatments may be repeated at daily to weekly intervals.

 Regular gentle protrusion of the penis may be desirable to prevent possible adhesions.
 Tranquilizers, anesthetics, pudental nerve block and adequate restraint are helpful in
applying treatment for balanoposthitis.
 Sexual rest is essential during and for sometime after the treatment of acute
balanoposthitis to promote recovery and to prevent a loss of libido associated with
painful condition.
 It may be infectious or noninfectious

MISCELLANEOUS CAUSES FOR LOSS OF LIBIDO OR INABILITY

TO COPULATE
 Hernias
 Premature erection
 Loss of sensory innervation of the glans
penis
 Urinary calculi
 Other causes

Hernias

 Umbilical and ventral hernias as well as deep pendulous abdomen may interfere or
prevent normal copulation by affecting the entry of the glans penis into the vagina at a
natural mating.
 Umbilical and small ventral hernias may respond to surgery but since umbilical
hernia may be hereditary the affected male should not be used as a sire.

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Premature erection

 Premature erection may occur in the dog, stallion and certain bulls and interfere with
normal intromission.
 Premature erection in the dog as an obstacle to coitus and a cause of impotency.
Artificial insemination as the quickest and easiest solution for this problem.
 Stallions the glans penis occasionally becomes too large to readily enter the vulva of a
small mare or a mare whose vulva has been sutured to prevent pneumovagina.
  This cause of inability to copulate can usually be overcome by helping to direct the
penis into the vulva before it fully erect, by lubricating the vulva or by incising the
sutured vulvar lips dorsally.
 The mare may be artificially inseminated or may be bred to a stallion of suitable size.
 Bulls with a strong sex drive and with a narrow penis develop corkscrewing or coiling
of the free end of the penis at premature full erection that prevents intromission.

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Loss of sensory innervation of the glans penis

 Prevents natural intromission and the “thrust” reflex necessary for ejaculation and
leads to pronounced decline in sex drive.
 Lack of sensation of the glans penis may be caused by injury to the dorsal nerve of the
penis secondary to rupture and hemorrhage of the cavernosum penis.
 By the improper technique of the injecting local anaesthesia on the dorsal nerve at the
sigmoid flexure of the penis.
 Rarely by an operation to correct the spiraling of the bovine penis or by a rubber band
from an artificial vagina or other source that is placed around the penis and not
removed for several days.
 Lack of sensation of the glans penis may also be due to severe necrosis of the mucosa
of the glans penis due to severe balanitis of an infectious etiology or due to trauma
resulting in severe scarring.
 Amputation of the glans penis due to rubber band or following extensive surgery for
tumor produces the same effect.

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Urinary Calculi

 Urinary calculi lodging in the urethra may occasionally be a acute pain, obstruction
and rupture of the urethra in male domestic animals and cause reluctance to copulate
and inability to copulate.
 In the ram calculi may lodge in the sigmoid flexure area as in bulls but are more
commonly found in the urethral process.
 Calculi are common in the male cat and uncommon in the dog.
 In order to preserve the breeding potential of these animals conservative therapy such
as massage and flushing in the removal of the urethral calculi and aftercare is
indicated, surgery should be used only as a last resort.

Other causes
  Pain caused by infection of the genital organs or peritoneum. It may be a cause of
impotency or refusal to copulate.
 Bulls with acute semino-vesiculitis were slow or refused mount.
 Boars with brucellar orchitis refused to copulate.
 Acute prostatitis in dogs might similarly affect the copulation.
 Severe congenital or acquired cardiac diseases often result in dyspnea and inability to
perform coitus by male animals.

INTRODUCTION
Incapacity or reduced capacity to fertilize
in males is called as Impotentia Generandi.
 Fertility in male is the normal functioning of the testes, accessory sex glands and
ducts to deliver sperm of normal quality and quantity.
 Fertility and potency are correlated or related and may differ in the same animal.
 Infertility or sterility in males is usually characterized by normal sexual desire and the
ability to copulate and ejaculate but the female animals bred by these males may
suffer from infertility due to failure of fertilization or early embryonic death.

Impotentia generandi may be of 2 categories

 Impotentia generandi associated with apparently normal semen

production.
 Impotentia generandi associated with abnormal semen production.
IMPOTENTIA GENERANDI ASSOCIATED WITH NORMAL SEMEN
PRODUCTION
 Bulls infected with brucellosis, vibriosis, trichomoniasis, IBR-IPV virus, mycoplasma
may produce normal semen but service by these bulls causes failure of fertilization,
early embryonic death and repeat breeding in females.
 Active brucellosis in a herd of cattle is associated with symptoms of infertility.
 Intrauterine insemination of brucella infected semen usually results in infertility.
 Abnormal acrosomes eg. knobbed spermatozoa related to the defective
spermatogenesis involving gogli apparatus is the cause of infertility in bulls, boars
and dogs.
 Some strains used for preparing the spermatozoa for morphological examination do
not reveal this defect.
 But Nigrosin-Eosin, India ink or Giemsa show a refractive unstained or lightly stained
area in the anterior pole or acrosome of the head of the spermatozoa.
 The defective acrosome causes the sperm cell incapable of penetrating and fertilizing
the ovum.

The acrosomal defects in spermiogenesis is due to an autosomal

recessive sex linked defect in Friesian bulls.

 Cytogenetic studies of sperms indicated that gene or chromosomal defects may occur
at the time of meiosis and result in infertility.
 Further many infertile bulls had lower DNA content of the spermatozoan nucleus
than fertile bulls.

IMPOTENTIA GENERANDI ASSOCIATED WITH ABNORMAL

SEMEN PRODUCTION
 In this condition sufficient number of healthy fertile sperm cells are not deposited
properly at the time of coitus to cause the fertilization of ovum and the normal
development of the embryo.
 The acrosomal defects in spermiogenesis is due to an autosomal recessive sex linked
defect in Friesian bulls.

Impotentia generandi is due to the pathology of the

 Testis
 Epididymis
 Vas deferens
 Accessory sex glands
 Urethra

PATHOLOGY OF TESTIS
The two most common changes occurring in the testes
causing disturbed spermatogenesis are

 Testicular hypoplasia which is congenital or

hereditary
 Testicular degeneration which is usually acquired

 Testicular pathology due to congenital or heredotory causes includes testicular

hypoplasia, certain defects in the seminiferous tubules and sperm cells including
cytogenetic and chromosomal defects, cryptochidism and inguinal hernias

TESTICULAR HYPOPLASIA

 It is a congenital/hereditary defect
 It may be bilateral or unilateral
 It is due to autosomal recessive genes with incomplete penetration
 Testicular hypoplasia is common in left
side testis than the right side testis

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INCIDENCE, PATHOGENESIS AND ETIOLOGY

 Testicular hypoplasia may be partial or total due to a failure of germinal cells to
develop in the yolk sac, failure to migrate to the gonad, failure to multiply in the
gonad or extensive degeneration of early germinal cells after they have entered the
gonad.
 Testicular hypoplasia is congenital hereditary in origin is caused by a marked lack
of or reduction in spermatogonia in the gonads during fetal life.
 It is observed as a unilateral or bilateral condition at the time of the puberty or later
in all of our domestic animals.
 Most commonly seen in bulls, rams, boars and stallions.

Swedish Highland breed with white coat colour is

commonly affected. Cattle with black ears were not
affected

 In these cattle hypoplasia of left testis occurred about 25 per cent of all bulls, in the
right testis 1 per cent and in both testes 4-5 per cent.
 This condition in cattle is due to a single autosomal recessive gene with incomplete
penetrance.

SYMPTOMS
In most cases - sexual desire is excellent and coitus is prompt.
Owners may not suspect the infertility for sometime.

 The degree of either unilateral or bilateral testicular hypoplasia varies from

complete hypoplasia causing sterility to only slight softer unsuspected
hypoplasia.
 Lowered conception rates are often evident in bilaterally affected males.
 Severe testicular hypoplasia is usually observed in young bulls of 1-2 years
of age.
 In sterile bull with bilateral hypoplasia, the semen is usually clear, and
watery with few or no spermatozoa.
 Centrifugation of the ejaculate and staining of the sediment revealed the
presence of giant cells and medusa cells or ciliated cells from the efferent
tubules.
 Giant cells or multinucleated cells with 6-8 nuclei apparently result from
incomplete maturation division of the primary spermatocytes. These cells
are seen in testicular hypoplasia and severe degeneration.
 Sexual organ develop normally except the affected testis.
 The consistency of the testes was soft, flabby or hard, indurated and
fibrotic.
 Males which are severally affected are nearly sterile, but an occasional
conception may occur in females bred to them.
  The semen picture shows a lower concentration of spermatozoa usually
less than 70,000 per cmm, low motility, many abnormal spermatozoa and
the possible presence of a few giant cells.
 Histologically the seminiferous tubules are very underdeveloped with
usually only the basal layer of cells being present. Varying degrees of
spermatogenesis may be present from spermatogonia, spermatocytes,
spermatids to abnormal or normal mature spermatozoa.
 In some bulls with a moderate to slight amount of testicular hypoplasia the
conception rate is low, about 20-40%. The testicles may appear nearly
normal in size and consistency. The spermatozoa concentration is from
1,00,000 to 5,00,000 per cmm. The motility is usually low and the number
of pathological or abnormal spermatozoa is high about 30% or greater.

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Epididymis

 Depending upon the degree of hypoplasia affected bulls will have a small
firm epididymis especially in the tail region indicating reduced
spermatogenesis and low gonadal sperm reserves.
 Spermatic cords of hypoplasia testicles are shorter as the testes are less
heavy and the scrotum smaller than in normal males.
 Slight or mild cases of testicular hypoplasia may predispose to testicular
degeneration.
DIAGNOSIS, PROGNOSIS AND TREATMENT

Diagnosis

 Based on the above described symptoms.

 The diagnosis of testicular hypoplasia should not be made before 2 years of age in the
bull and horse or before one year of age in the boar, ram ,dog or cat unless the
hypoplasia is marked and the male is well grown.
 The testicular hypoplasia may be erroneously diagnosed in young immature males
that are underdeveloped or are retarded in growth at the generally accepted time of
puberty for the species.

Prognosis

 Prognosis is poor.
 Affected animals should not be used for breeding because the condition may be
hereditary.
 Severally affected animals are sterile or highly infertile.
 Mildly to moderately affected animals may have only a lowered fertility but are more
prone to early testicular degeneration.

Treatment
 The germinal epithelium of the affected animals apparently cannot respond to
gonadotropic therapy because spermatogonia are reduced in numbers or lacking in
hypoplasia of testis

Treatment of testicular hypoplasia in animals has

been unsuccessful
HEREDITARY DEFECTS OF SPERMATOZOA
Hereditary defects of spermatozoa are

 Dag defects
 Returned tails and narrow heads
 Knobbed acrosome defects.
 Cork screw defects
 Diadem defects
 The pseudodroplet defects
 The decapitated sperm defects

DAG DEFECT
Upto 80 percent of the spermatozoa will
have strongly coiled, folded or disrupted or split
tails.

 The fibres of the axial filament were normal in the testis but abnormal when the cells
reached the cauda epididymis.
 The defect is hereditary and recovery has not been reported.
 Fertility is lowered/sterile.
 Confirmative diagnosis is made by electron microscopy.
 This defect is recorded in Danish Jersey bulls
 RETURNED TAILS AND NARROW HEADS
It is a genetic defect reported in Jersey bulls with coiled or returned tails with
a lowered motility rate.
KNOBBED ACROSOME DEFECT
 The acrosomal cap is enlarged 6-8 times.

Examination of Indian ink smear reveals the presence of highly

refractive cyst formation in the anterior part of sperm head.

 The motility of the affected sperm is usually not affected.

 Affected bulls are sterile.
 This hereditary defect is seen in bulls, boars, dogs. But mainly seen in Friesian bulls
and is caused by an autosomal recessive gene.
 CORKSCREW DEFECT
 The mid piece of the affected sperm looks like a cork screw as compared to normal
cylindrical appearance.

It is due to an irregular distribution of the mitochondrial sheath

together with a high incidence of persistent proximal droplets in
 the affected sperms

 Commonly seen in aged bulls in several exotic breeds.

 The defect is due to progressive testicular degeneration resulting in decline in sperm
count.
 Recovery is rare.

DIADEM DEFECT
The affected sperm show a number of small red colored spots
located along the nuclear ring, just like a row of pearls or necklace
or diadem.

 This can be distinctly seen with eosin-nigrosin staining.

 This defect indicates disturbed spermiogenesis and recovery is common.
 The motility of the affected sperm is not impaired.
 Affected bulls will have lowered motility.
 PSEUDODROPLET DEFECT
The defect is located in the centre of the mid-piece and
appears as rounded or elongated or irregularly thickened
areas.

 The mid-piece is often bent at the site of the defect.

 This defect is mistaken for a protoplasmic droplet.
 Some of the defective sperms are alive.
 The fertility of the affected bull is lowered.
 It is common is Friesian bulls.

THE DECAPITATED SPERM DEFECT

 More than 50 percent of sperms have separation of loose
heads and tails.
 Detached tails are bent around a distal droplet and most of the tails exhibit active
motility.
 The defect is hereditary in nature and affected bulls are sterile.
 This defect is mostly seen in Guernsey breed.

INBREEDING, HYBRIDIZATION, FREEMARTINISM AND

INTERSEXES

Inbreeding

 Generally results in reduced fertility accompanied by an increase in the number of

abnormal seminiferous tubules, lowered semen quality and hypoplasia of testis.

Cytogenetic disturbances in spermatogenesis caused abnormalities

in the primary spermatocytes including

 Stickness of chromosomes in which they failed to separate at

anaphase and a pyknotic nucleus and multiple spindle
formation due to a dysfunction of the mechanism of cell
division due to extra centrosome divisions resulting in the
 formation of giant cells.
 In both condition the semen was thin and watery.

The male tortoise-shell cat

 It is rare and it may have an abnormal sex chromosome constitution of XXY

resembling the more common Klinefelter’s syndrome in man.
 These male cats have small testes and are sterile due to failure of the seminiferous
tubules to develop.

Infertility in hybrids

 Usually occurs when there are major difference in the chromosomes of the parents.
 Minor gene differences may be overcome so fertility is possible. A common example
of the hybrid infertility is the horse and ass cross producing the hybrid mule with
chromosome numbers of 64, 62 and 63.

Freemartinism

 Sometimes noninflammatory degenerative changes in the testis of the males born as a

cotwin with the freemartin females are noticed.

Male intesexes

 They are invariably sterile.

 Male pseudohermaphroditism is found in the caprine, porcine and occasionally in
bovine, equine and other species.
 True hermaphroditism is not common in domestic animals.

Intersexes in pigs are common but not

as in goats.
 They also are of the male pseudohermaphrodite type.
 The cause is probably a recessive gene.
 The affected animals are genetic females, the testes are invariably intra-abdominal
and externally a characteristic “fish hook” vulva with a prominent “clitoris” or phallus
is observed in cattle, horses and dogs.

CRYPTORCHIDISM
 In animals, if bilateral cryptorchidism, results in sterility.

In horses cryptorchids may be spoken of as “ridglings,”

“rigs” or “originals”.
 Unilateral cryptorchidism is more common and usually results in near normal
fertility because of normal sperm production from the testis located in the scrotum.
 The term “monorchidism” is incorrect.
 Cryptorchidism or incomplete descent of the testis or retention of the testis occurs in
all domestic species. But seen most commonly stallion, boars, dogs less commonly in
rams and bucks, uncommonly in cattle, and rarely in cats.
 The undescended testis may be located anywhere from just caudal to the kidney to
within the inguinal canal.
 Many abdominal testes are located close to the internal inguinal ring.
 Occasionally testes may be located ectopically under the skin of the ventral caudal
abdomen, alongside the penis, or rarely in the femoral canal or the perineal region.
This is more commonly the case in male pseudohermaphrodites where no scrotum is
present.
 In some cases where the testis is retained in the inguinal canal, it will descend
spontaneously into the scrotum in a few months to a year after birth.
 In a few cases in colts, testes may be descended into the scrotum at birth and later
become cryptorchid and located in the inguinal canal.
 All retained or cryptorchid testes are small, soft and flaccid weighing in the horse 25
to 131 gm. Normal descended testes usually weigh 170 to 325 gm.
 No spermatozoa are produced by the retained testis but well-developed or degenerate
seminiferous tubules may be present.
 Spermatogenesis is completely inhibited by the elevation of the temperature of the
affected testis.
 The interstitial or Leydig cells are not affected so sexual activity is normal or even
exaggerated in bilateral cryptorchids.
 Cryptorchid animals should never be used for breeding.

Cryptorchidism in the horse is inherited in a dominant manner

while in the other species it is a recessive trait.
 Unilateral cryptorchidism more often affects the right testes especially in dogs,
possibly because the right testis develops in the embryo a greater distance from the
scrotum.
 In dogs cryptorchidism is seen most commonly in the brachycephalic breeds
including: Boxers, Pomeranians, Dachshunds, Sealyhams, Cairn Terriers, and also in
Whippets, Chows, Cocker Spaniels, Poodles, and others.
 In most dogs, the testes are in the inguinal canal at birth and descend into the
scrotum during the first week after birth.
 In some dogs the testes don’t descend till near puberty.
 Possibly because dogs are maintained intact for many years, retained testes are
predisposed to neoplasms.
 Sertoli cell tumors and seminomas in undescended testes tend to be more malignant.
 These tumors affected the retained or abdominal right testes more frequently than
the retained left testes.
 Tumors of undescended testes in man occurred 50 times more often than tumors of
scrotal testes. Hence, early removal of undescended testes is recommended.
 Cryptorchidism in swine is a monogenic sex-limited recessive. To eliminate this
condition in swine or others species all parents of cryptorchid animals should be
discarded as breeders.
 The incidence in swine reach 1 to 2 percent in some herds.
 Treatment of cryptorchid animals, especially those where breeding is to be
considered, should be discouraged.
 Orchiopexy has proven to be a failure in producing satisfactory spermatogenesis.

IMPERFECT DESCENT OF THE TESTIS

 In cattle not associated with cryptorchidism has resulted in testes located horizontally
and fairly high in the scrotum.
 This may be due to the attachment of the cremaster muscle to the caudal aspect of the
testis, fixation of the distal end of the scrotum to the perineal region, or lower than
normal attachment of the gubernaculum.
 This imperfect descent of the testes may result in impaired fertility with degeneration
and atrophy due to difficulty of the thermoregulatory mechanism of the testes to
operate properly.
 This condition might be genetic.

TORSION OF TESTIS
 On a horizontal axis is observed most commonly in the stallion, especially trotting
standardbreds.
 The affected testis is freely moveable within the scrotum.
 Often the tail of the epididymis is lateral instead of caudal.
 On assuming a fast trotting gait, pain is evident by abduction or “hiking” of the leg on
the side of the affected testis.
 Some affected stallions appear unable to draw the testis out of the scrotum and into
the inguinal canal.
 Suspensories are of limited value and unilateral or bilateral castration is indicated.
 Torsion of the retained abdominal or inguinal testis may rarely occur.
 This is usually accompanied by severe pain, swelling and congestion of the involved
organ.
 Rotation or torsion of the testis is rarely reported in bulls.
It is reported most commonly in the dog and stallion and rarely in the pig.
SCROTAL AND INGUINAL HERNIA

 If large, may seriously depress fertility by markedly interfering with the normal
thermoregulatory function of the scrotum and testes.
 If the inguinal ring and hernia is small, there is a greater chance for strangulation of
the intestine.
 Inguinal hernia is considered as a common hereditary defect in horses and pigs.
 It is less common in bulls, dogs and rams, and rare in the cat.

Animals with inguinal hernias should

be castrated and not used for breeding.
 Reduction of the hernia and closure of the hernial ring can be performed through the
inguinal canal, through an incision in the abdominal wall just cranial and medial to
the ring or by a flank incision above the ring.

ACQUIRED TESTICULAR PATHOLOGY

 Testicular pathology due to acquired causes is much more common than congenital or
hereditary causes.
 It includes testicular degeneration, orchitis, fibrosis and calcification.
75 to 80 percent of testicular pathology is related to testicular degeneration
including fibrosis and orchitis.
TESTICULAR DEGENERATION
 Testicular degeneration may be mild or severe and is usually bilateral as it is most
commonly due to generalized disease processes.
 Unilateral degeneration can occur secondary to local testicular lesions such as
tumors.

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Testicular degeneration may develop very rapidly within a few

days or hours; while testicular regeneration proceeds slowly over
weeks and months.

 If the basal layers of the germinal epithelium including the spermatogonia and sertoli
cells are destroyed regeneration of the germinal epithelium is not possible and the
animals is sterile.
 The pattern and signs of testicular degeneration are about the same despite the
species or the etiologic factors involved.
 In all cases, degenerative changes noticed in the mechanism of cell division or
centrosomes in the primary spermatocytes are responsible for reduced motility and
increased numbers of pathologic spermatozoa with an abnormal morphology.
 Sperm cell numbers and concentration are decreased depending on the degree of
degeneration of the seminiferous tubules.
 In severe disturbances of spermatogenesis, spermatocytes with restitution nuclei
with double the normal number of chromosomes as well as pyknotic nuclei are
present in the ejaculate.
 Testes with degeneration of the seminiferous tubules are usually atrophic and softer
and smaller than normal testes.
 In chronic cases the testicle may be firm due to fibrosis even calcium may be
deposited especially in the areas just peripheral to the rete testis.
 Histologically it may be difficult to distinguish between slight degrees of testicular
degeneration and hypoplasia.

CAUSES OF TESTICULAR DEGENERATION

 Thermal causes
 Vascular disturbances
 Irradiation
 Hormonal
 Age
 Acute or subacute trauma, stress or disease
 Acute or chronic localized or systemic diseases
 Nutritional causes
 Infectcious diseases
 Poisons or toxins
 Autoimmunization
 Neoplasms of the testis
 Genetic or hereditary factors

THERMAL CAUSES

 Elevation of the testicular temperature in conditions such as cryptorchid and ectopic

testes; inguinal hernias; scrotal dermatitis due to, irritants, choriopic mange, myiasis
in sheep, and localized skin infections or wounds; contusions and haematomas of the
scrotum and testes; prolonged elevated body temperature as in certain infectious
diseases and in prolonged high environmental temperatures, particularly associated
with high humidity.
 Testicular degeneration is most common in tropical climates and involving breeds
originating in the temperate zones.
 Bulls exposed to heat stress for 8 hours a day for 7 days resulted in a deleterious effect
on semen quality reaching its peak at 2 to 3 weeks after the stress with recovery by 9
weeks.
 Rams maintained at ambient temperatures of 900 F or above develop a marked drop
in semen quality with about 10 percent motility and 70 percent abnormal sperm cells
within a few weeks. Recovery was not complete until 2 to 3 months after normal
temperatures were restored.
 High ambient temperatures will also cause lowered fertility due to testicular
degeneration in boars.

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Heat primarily affects the spermatids, the spermatozoa and the

spermatocytes but usually does not affect the spermatogonia.

 Males that lie down for long periods of time such as bulls with bovine spastic
syndrome, or males that are unable to rise, often develop testicular degeneration and
atrophy due to the prolonged elevation of testicular temperature.
 When the scrotal temperature of bulls was raised to 38.40C or 0.30C below body
temperature, the motility and percent of live spermatozoa in the semen decreased to
zero by the second week.
 Damage to spermatogenic function occurred in beef bulls by low temperatures down
to -250F associated with winds of 60 miles per hour causing frostbite, necrosis of skin,
scrotal dermatitis, heat, swelling, testicular degeneration and adhesions.

Heat has no effect on the Leydig cells.

VASCULAR DISTURBANCES

 Interference with the circulation and infarction of the testis can be produced by
manual torsion of the testis or by the emasculatome used for castration of lambs or

calves .
 Congestion and pain of the scrotal testis due to naturally- occurring torsion or
contusion has been reported in racing stallions. The testes would be swollen,
congested and painful for the next 3 to 4 days.
 Dogs with inguinal or abdominal cryptorchid testes may occasionally develop torsion
of the testis with a sudden onset of pain and associated symptoms.
 Hemorrhagic infarction of the testis may follow torsion.
 Tumors of the abdominal testes may also predispose to torsion.

Testicular biopsies, particularly in bulls, often result in focal areas

of testicular necrosis because of vessel damage when a desirable
size of testicular material is obtained
 Effect of faulty testicular biopsy which is leading to tesicular degeration is shown in

picture
 Inflammation of the testicular artery in the horse may be caused by Strongyle larvae,
the equine arteritis virus and other unknown agents. This may produce areas of
testicular degeneration.

 Strongyle larvae can produce adhesions between the testis and its tunics.
 Age- associated vascular lesions such as hyaline degeneration in older bulls, rams and
dogs causing degenerative changes in the seminiferous tubules.
 Varicocele may effect both the circulation of blood and the heat regulatory
mechanism of the pampiniform plexus. Varicoceles may be rarely palpated in rams.
 IRRADIATION
 It produces interference with spermatogenesis by injuring spermatogonia,
spermatocytes and spermatids.

The spermatocytes are most sensitive to irradiation while Leydig

and Sertoli cells are quite resistant.

 The amount of irradiation and length of treatment are highly important in the degree
of effects produced and the rate of recovery

HORMONAL FACTORS

 Testicular degeneration and atrophy of the testes occurs in the dog and rarely in other
animals due to tumors of the anterior pituitary gland or hypothalamus interfering
with the production of gonadotropic hormones. This is called Dystrophia
Adiposagenitalis Syndrome in dogs.
 Excessive estrogenic hormone produced by sertoli cell tumors and testosterone, and
rarely estrogens, produced by Leydig cell tumors may suppress FSH production and
cause testicular degeneration

AGE
Permanent and progressive testicular degeneration occurs fairly
frequently in all species without any indication of its pathogenesis.
 Senile atrophy of the testes is common in dogs over 10 years of age and in cats over 12
years of age.
 Many disease, genetic, and management factors influence this age effect.

ACUTE OR SUB-ACUTE TRAUMA, STESS OR DISEASE

 It may cause rapid or progressive testicular degeneration in males.
 Those that have been associated with reduced fertility and a decline in semen quality
are: shipping under adverse conditions of heat and cold, severe fatigue, excessive
physical work, traumatic gastritis in bulls, liver or abdominal abscesses, multiple
severe contusions with fractured ribs, severe screw-worm infestations, moderate
quittors in bulls, acute laminitis in rams, horses and other species, kickt wounds and
resulting haematomas of the testes and scrotum in stallions and extensive infected
wounds.

ACUTE OR CHRONIC, LOCALIZED OR SYSTEMIC INFECTIOUS

DISEASES

 Common causes of testicular degeneration with moderately to severely reduced

fertility in males.

Infections producing orchitis or epididymitis have a direct effect on

the testes due to the inflammatory reaction causing heat, edema,
congestion, circulatory interference, ischemia and even infarction
due to the thick firm tunica albuginea that restricts normal
swelling of the testicular parenchyma.
 In bacterial diseases localizing in the testes, abscessation may occur
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 Infectious agents resulting in orchitis are: Brucella abortus, both field strains and
strain 19 in bulls, Brucella suis in boars miliary or chronic tubercular infections with
Mycobacterium tuberculosis of the testes in bulls and boars, Corynebacterium
pyogenes, in bulls and rams, Actinomyces bovis in bulls, Malleomyces mallei in
horses; Salmonella abortus equi and “epizootic cellulitis” due to the arteritis and the
influenza viruses in horses; Cornynebacterium ovis and Pasteurella
pseudotuberculosis in rams; lumpy skin disease in cattle; sheep pox virus in rams,
IBR-IPV virus, that markedly affected the spermatocytes and caused arrested
spermatogenesis in bulls.
 Brucella canis in dogs caused scrotal swelling, epididymitis and unilateral or bilateral
testicular degeneration, fibrosis and sterility.
 “EPIVAG” also causes an orchitis with testicular degeneration and atrophy.
 Orchitis in rams from which a P.L.T., agent (chlamydia) was recovered.
 Sporadic infections of the testis with staphylococci, streptococci, E.coli, Proteus and
Pseudomonas organisms have been reported as a cause of orchitis in dogs and other
male domestic animals.
 NUTRITIONAL FACTORS
Diets for males sufficient for growth and maintenance are
adequate for fertility.

 Testicular degeneration is caused by under-feeding or starvation.

 Chronic diseases associated with inanition and debility and testicular atrophy may
include, chronic severe parasitisms, either external or internal such as: lice, mange,
ticks, roundworms, flukes, and etc., senile changes due to worn or diseased teeth,
chronic arthritis, tumors, fat necrosis around the large intestine in bulls, severe
spastic syndrome and other possible diseases interfering with the intake and passage
of food through the gut.
 Malnutrition may complicate these other chronic diseases.
 Improper care and management of bulls accustomed to special care and feeding may
cause a marked loss in weight resulting in impotency and testicular degeneration and
atrophy.
 Severe vitamin A deficiency characterized by night blindness, lacrimation and
opthalmia usually precedes testicular degeneration and poor semen quality.
 Malnutrition, debility and cachexia produce degeneration and atrophy of the
seminiferous epithelium by causing a suppression of the release of gonadotropic
hormones from the pituitary.
 A high level of feeding and the accompanying obesity has no effect on semen quality
in a normal male, but does effect their libido and willingness to breed.

INFECTIOUS DISEASES
 Pneumonia and shipping fever, I.B.R.-I.P.V., equine infectious anemia,
actinobacillosis, strangles, blue-tongue virus vaccination, tick-borne fever infection in
rams.
 Those infectious diseases causing gradual debilitation, loss of weight and testicular
degeneration with atrophy include: foot and mouth disease, actinomycosis, Johne’s
disease, pyelonephritis, chronic peritonitis, and lymphomatosis.

POISONS OR TOXINS

 It may adversely affect the germinal epithelium.

 Testicular degeneration in bulls and rams with hyperkeratosis caused by
the chlorinated napthalenes.
 Antimony compounds injected for the treatment of heartworms in dogs
have been reported to cause temporary sterility.
 Chemicals, metals and earth salts produce degeneration of the
seminiferous epithelium in a variety of species. eg. alkylating agents,
cadmium chloride and amphotericin B.
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AUTO-IMMUNIZATION

 It may play a role in a few cases of testicular degeneration.

 Degeneration has been produced experimentally in rams, bulls and laboratory
animals by the subcutaneous injection of autologous testicular material, together with
Freund’s adjuvant. Degeneration may occur with or without inflammation.

No natural cases of testicular degeneration due to auto-immunization have

been reported.
TESTICULAR NEOPLASMS
Testicular tumors are unusual in most domestic animals except the
dog and possibly old bulls.
 Primary testicular tumors are usually of three main types and originate from
the interstitial cells, the sertoli cells and the germinal epithelium.
 This may be due to a genetic predisposition in dogs and to the maintenances of intact
male dogs until they become senile.
 Large tumors result in testicular degeneration probably due to the compressing effect
of the tumor on the adjacent seminiferous tubules.
 The degeneration may also be due to the excess steroids produced by the interstitial,
or sertoli cell tumors.
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Bulls

 Testicular tumors are seldom observed except in old animals .

 Interstitial cell tumors of a benign type are occasionally observed in bulls over 7 to 10
years of age.
 These may be multiple and in some cases they are large enough to be palpable as
small, round, masses, liver-like in consistency and causing the testes to feel lumpy on
palpation.

Stallions

 Testicular tumors have been described only occasionally.

 Rare dermoid cysts or teratomas of the testicle of the horse are often found in
cryptorchid testes.

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Rams

 Testicular tumors are rare.

Boars

 Testicular tumors are very unusual.

Cats

 Testicular tumors apparently are rare.

Testicular Tumors in Dogs

 Testicular tumors are observed at any age but most commonly in dogs over five years
old.

There was a higher incidence of testicular tumors in dogs than in

humans.
 The three principal testicular tumors in dogs are, seminomas, serteoli cell tumors
(tubular adenomas), and interstitial cell tumors.

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Seminomas

 They are noted in old dogs, over 7 years of age and arise from the germinal epithelium
of the seminiferous tubules.
 These tumors originate in atrophic seminiferous epithelium and that the tumor is
intratubular before becoming diffuse.
 These tumors grow slowly for months but may develop rapidly at any time.
 They are rather soft in consistency and hemorrhage or necrosis may be present.
 Affected dogs may exhibit lameness and pain by crouching and hunching.
 In some cases seminomas and interstitial cell tumors are present together in the
testis.

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Sertoli cell tumors or tubular adenomas

 They are the least common of the principal tumors of the canine testis.
 This tumor arises from the sertoli or nurse cell of the seminiferous tubules and is
usually noted in older dogs.

The tumor is often characterized by marked feminization of the

male by the estrogens secreted by the tumor cells.
 The estrogenic substances produced by the tumor and causing feminization of the
male were not the usual estrogens found in animals.
 The dog may attract other males.
 The penile sheath is swollen.
 There is a definite loss of hair, especially on the abdomen and lower parts of the body,
and a loss of sexual desire.
 Mammary hypertrophy or gynecomastia, and enlargement of the nipples and in some
cases development of mammary tumors may occur.
 Atrophy of the testicular tissues and penis develops
 Pigmentation of the abdominal skin and scrotum, and female distribution of body fat
are exhibited.
 The prostate may undergo marked enlargement due to squamous metaplasis and
cysts; or it may become markedly involuted due to atrophy of the epithelial elements
and collapse of the acini.
 These tumors are rather large in size, firm, nodular, and fibrous on palpation.
 On cross section, sertoli cell tumors are pale yellow or grey in color and may contain
necrotic, cystic, or hemorrhagic foci.
 If no metastases have occurred recovery from the above dramatic symptoms takes
place promptly after the affected testis is removed.

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Intestitial cell tumors or adenomas, or Leydig-cell tumors

They are the most common tumors in

old dogs.
 Although they are present in many old dogs, they are of little clinical significance.
 Most of them are found only at autopsy.
 They are often overlooked because of their small size; 1 mm to 2 cm is their usual size.
 Nodular hyperplasia of the interstitial cell is seen commonly in older dogs with senile
testicular atrophy.
 Nodular hyperplasia is probably a preneoplastic change but the division between
nodular hyperplasia and interstitial cell tumor is arbitrary.
  These tumors probably produce androgens because the prostate in affected dogs is
normal or hypertrophied.
 The tumors consist of single or multiple well-circumscribed nodules, brownish-
orange in color and soft in consistency.
 Large tumors may occasionally by palpated in the testes of old dogs.
 Canine tumors should be carefully differentiated from orchitis or epididymitis,
traumatic swellings or hydrocele
 The hydrocele condition may be either congenital or secondary to orchitis.
 Canine perianal gland tumors are probably androgen dependent tumors that can be
arrested by castration; about 85 per cent occur in older male dogs with a definite
breed incidence in Cocker Spaniels.

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GENETIC OR HEREDITARY FACTORS

Acquired testicular degeneration have been reported in identical
twin bulls.

 Genetic constitution appeared to influence the onset of generalized

arthritis, fat necrosis, traumatic gastritis and associated testicular
degeneration in identical twin bulls.
 Some bulls can more resistant to testicular generation than other bulls
when affected by similar etiologic agents.
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SIGNS OF TESTICULAR DEGENERATION
They are similar in all species of animals and range in degree from
mild to severe depending upon the cause and duration of the
degeneration.

The fertility

 In the male with testicular degeneration may vary from slightly reduced conception
rates to moderate to severe infertility to complete sterility.

The size of the testes

 It is usually reduced from normal to about one-half to two-thirds of its normal size
depending on the duration and degree of atrophy of the seminiferous tubular
epithelium.
  In acute orchitis, inflammatory conditions, obstruction of the efferent ducts, or
testicular tumors, an increase in testicular size is usually noted.

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The consistency of the testes

 It is soft and flabby in acute moderate to severe testicular degeneration.

 In mild testicular degeneration consistency differs only slightly from an elastic, tonic,
slightly tense, fluctuating normal consistency.

The tonometer is useful for the objective measurement of testicular

consistency.
 The tonometer test correlated closely with other tests of semen quality.
 Bulls with soft testes tended to produce fewer sperm cells.
 Acute orchitis is characterized by a tense swelling and enlargement of the testis
accompanied by pain and heat. This should be differentiated from hydrocele or
hemaotcele.
 Chronic degeneration and atrophy of the testes especially in older males is
characterized by fibrosis of the testes often with calcification. Fibrotic testes are hard,
firm and often lack tonicity or elasticity.
 Ultrasonics can be of value in the detection of connective tissue in fibrosis of the
testes.
 Testes containing tumors may be enlarged and have an irregular shape and
consistency on palpation.

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The libido or sex drive

 It is usually not related or associated with testicular degeneration except in acute

orchitis or other painful testicular diseases or in testicular atrophy associated with
severe debility or inanition due to a variety of causes
 Leydig cells are much more resistant to stress factors than the cells of the germinal
epithelium

The semen picture

Points to be cosidered

 It is very helpful in diagnosing testicular degeneration.

  Semen may be collected by the artificial vagina, by electroejaculation or other
methods.

Since it requires about 60 days or more for sperm cells to develop

from spermatogonia until they are ejaculated, it may be desirable
in diagnosing testicular degeneration to take several samples at
weekly or greater intervals.
 The semen production and quality in young immature bulls continues to improve in
motility, concentration and morphology for months after puberty is reached so if an
initial semen sample is poor, subsequent samples are indicated.
 The first semen samples taken after a long period of sexual rest may also be
misleading as these are often of poor quality with many dead spermatozoa.
 In males where testicular degeneration is severe, semen quality is usually poor.

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Sperm cell concentration

 May be reduced to one-third to one-half of normal values or show severe oligospermia

or azoospermia.
 Watery translucent semen is common in affected bulls and rams.

Sperm cell motility

 It is reduced one-third to one-half or more due to an increase in abnormal cells, dead

cells, necrospermia, or poorly viable cells.
 The duration of motility after ejaculation is shortened.

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Sperm cell morphology

 After staining it reveals an increase in abnormal heads, tails and middle pieces.
 The degree of change in the morphology of cells may vary from normal semen which
usually contains from 5 to 15 per cent abnormal cells, to a 15 to 20 per cent increase in
abnormal cells, to a 15 to 20 per cent increases in abnormal cells in mild cases of
testicular degeneration, to a 20 to 30 per cent increase in moderate testicular
degeneration, and to a 35 to 60 per cent or greater increase in severe testicular
degeneration.
 The occurrence of large numbers of primary abnormalities that arise during
spermatocytogenesis or spermatogenesis and early spermiogenesis are more
indicative of a severe testicular degeneration than the presence of secondary
 abnormalities that occur late in spermiogenesis or during storage of spermatozoa in
the epididymis.

The appearance of primitive cells such as giant cells and

spermatocytes with restitution or pyknotic nuclei together with a
marked reduction in sperm cell concentration and motility are
signs of severe degeneration of the testes.
 Testicular biopsies may be of value in certain species such as the dog and horse but
are impractical for technical reasons in the bull.
 Furthermore the above signs and additional tests of sequential semen samples will
accurately reveal the relative degree of testicular degeneration.

PROGNOSIS AND TREATMENT

Prognosis

 The prognosis in testicular degeneration is variable depending upon the causative

factors, the duration and degree of the degeneration and the age and value of the
male.
 Although testicular degeneration can occur rapidly within a few hours, days or a week
or more, recovery is very slow usually requiring 3 to 6 months or more in moderate
to severe cases.
 The prognosis in slight or mild case of testicular degeneration in young or middle
aged bulls due to transient and correctable causes is fair to good.
 Controlled breeding during the recovery period may result in fair to good fertility in
some males.
 The prognosis is guarded to fair in young males with only moderate evidence of
testicular degeneration, in older males suddenly developing infertility due to a
transient disease where advanced testicular changes do not occur; in mild cases of
trauma to the testes.
 The prognosis is poor in progressive senile testicular degeneration, in acute severe
orchitis especially if it is bilateral and associated with abscessation, in advanced
fibrosis and atrophy of the testes, in degeneration associated with hypoplasia in
young to middle-aged males, and in older males with bilateral tumors of the testes.

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Treatment

The treatment of testicular degeneration requires the correction or

the alleviation of the causative factors
 Sexual rest is usually advised, since in most case, fertility is reduced to a point where
the use of the male is questionable.
 Males with mild degrees of degeneration not severely influencing fertility should be
used sparingly so as to maintain as large number of normal, motile spermatozoa in
each ejaculate.
 A balanced ration high in vitamin A and possibly a good quality and variety of protein
are indicated.
 Good quality roughage or pasture is highly desirable.
 Some exercise is usually recommended for the larger farm animals.
 If excessive heat and humidity is the cause of the temporary infertility in bulls, air
conditioning or cooling is indicated.
 Hormones are of no proven value: Testosterone, various FSH products, and
thyroxine have been tried but none have any demonstrated effect in the therapy of
testicular degeneration or hypoplasis.
 In acute orchitis, sexual rest is imperative.
 Physical rest is usually advisable and can be provided by close confinement of the
male.
 In the early acute stages heavy parenteral broad-range antibiotic therapy is indicated.
 Glucocorticoid agents along with the antibiotics might be helpful in reducing the
inflammatory reaction.
 Ice packs should be applied to the testes by a suitable sling or bag fixed between the
legs and tied over the back.This therapy should be continued until the acute, severe
symptoms have subsided.
 When the orchitis is unilateral, removal of the affected testis may hasten recovery or
save the breeding life of a valuable male.The testis should be examined carefully and
cultured after removal to detect the responsible agent so that suitable precautions can
then be taken for the future use of the sire if his fertility returns.

The Brucella-infected bull or boar should not be used either

artificially or naturally.

 Heat or a mild counter-irritant ointment to the inflamed testis and swollen scrotum
should never be used.
 The preferred treatment for testicular tumors is prompt castration or removal of the
affected testis. If metastases have occurred, symptoms caused by the secondary
tumors will usually develop within a few months.
 Cryptorchid testes should be removed to prevent the occurrence of tumors in later
life.

PATOLOGY OF EPIDIDYMIS - EPIDIDYMITIS

 It is the inflammation of the epididymis.

 It is occasionally observed as an acquired lesion in all species of animals and is caused
by or may be secondary to the same factors causing orchitis.
 Infectious causes of epididymitis include: Brucella abortus in bulls, Brucella suis in
boars, Brucella canis in dogs, Brucella ovis (Ram Epididymitis Organism - REO) in
rams.Streptococcus zooepidemicus in a stallion, C. pyogenes and other miscellaneous
organisms such as Streptococci, Staphylococci, Proteus, E. coli, C.
pseudotuberculosis,Actinobacillus seminis and Pseudomonas aeruginosa.
 Infections may be caused by organisms, such as mycoplasma, invading the epididymis
through the vas deferens or by way of the blood or lymph vessels.
 Traumatic injuries may also produce an epididymitis.
 Inflammation of the epididymis may be caused by accidental passage of either normal
or infected urine to the epididymis through the vas deferens.
 Canine distemper virus in sexually mature dogs causes an epididymitis.

A Specific Bovine Venereal Epididymitis and Vaginitis

(“EPIVAG”) a viral disease is usually characterized by a marked
bilateral hardening and swelling of the tail of the epididymis in the
bull. There is no cure for affected males but artificial insemination
has been an effective control measure to prevent the spread of this
venereal diseases .

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 Besnoitiosis due to Besnoitia besnoiti produces elephantiasis of the scrotal skin and
cysts in the scrotum, inguinal canal, testes and epididymitis .These cysts may persist
and calcify, causing sterility.
 A psittacoid , Chlamydia or PLT agent in bulls causes epididymitis, orchitis and
seminal vesiculitis.
 Brucella ovis (ram epidyimitis organisdm ) is a common cause of ram epidymitis and
infertility.
 Ewes bred to infected rams had a greatly increased number of services per
conception and fewer lambs per ewe than ewes bred to non infected control rams.
 The infective organism is spread mainly by venereal contact to susceptible rams
breeding a ewe recently bred by an infected ram, or by rams penned together
mounting each other and having rectal copulation or sodomy between rams.
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 Ewes play a minor role in the spread of the infection and are relatively resistant to the
infection; but occasional abortions due to Brucella ovis are reported.
 Rams are readily infected by the conjunctival or rectal instillation of the organism.
 Rams may spread the organism for 3 to 4 years while ewes may spread infection for a
much shorter period, especially for several days after an occasional abortion.
 Following infections the antibody levels rise by the third week and persist for more
than a year after the bacteremic stage of the disease.
 The organisms localize in the epididymis, seminal vesicles, ampullae, liver, and
kidneys.
 The organisms cause perivascular lesions with edema and fibrosis in the epididymis
resulting in obstruction of the lumen and stasis of the epididymal contents and
finally extravasation of semen that because of its high lipid and mycolic acid content
results in the formation of the spermatic granulomas resembling tubercular
granulomas.
 Over 90 per cent of these lesions affect the tail of the epididymis but occasionally the
body and head of the epididymis may be involved.
 If the lesions are unilateral, fertility is normal or only slightly impaired; if they are
bilateral sterility is usually present.
 It takes 2 to 4 months from the onset of infection to the development of the lesions in
the tail of the epididymis that causes it to become 3 to 5 times normal size and
fibrotic. Some infected rams fail to develop gross palpable lesions.

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Diagnosis

 It is based on clinical palpating of the epididymis to detect induration, spermatic

granulomas and enlargements, especially involving the tail of the epididymis.
 The combination of clinical palpation of the testes and the complement fixation test
on serum was most effective in locating shedders of Brucella ovis.
 A precipitin test for the detection for REO is proved to be simpler to run and more
accurate than the C.F. test.
 Fluorescent antibody techniques and the indirect hemagglutination test are also
promising diagnostic laboratory techniques.

Control

 Controlling ram epididymitis in a flock is highly important to maintaining good

fertility in the flock.
 Before the breeding season all rams that show clinical or serologic evidence of
epididymitis should be eliminated.
 Various vaccination procedures have been used successfully to protect rams against
Brucella ovis.
 The combination of the vaccine with other control practices was highly effective in
infected flocks of sheep.
 Although B. ovis reported to be sensitive to some antibiotics, the treatment of chronic
lesions is of doubtful value.

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Canine Brucellosis

 It is due to Brucella canis causes abortion in the females and swelling of the scrotum,
firm enlargement of the epididymis, especially in the tail, and degeneration and
atrophy of the testis with sterility.
 The disease is spread by direct contact with aborted fetuses, placental tissues and the
infective vaginal discharges of bitches for several weeks after abortion.
 Venereal transmission to females by the semen from chronically infected males
appears probable.
 Following exposure a bacteremia develops within one to three weeks with a
generalized lymphadenitis.
 Antibodies develop that can be detected by the agglutination, test; 1:100 is considered
positive.
 The organisms can often be recovered from the lymph glands, spleen, liver, prostate,
testes and epididymides.
 Clinical lesions need not be present.
 The prognosis is poor in clinically affected male dogs.
 Any possible effective antibiotic therapeutic regimen must be heroic and sustained.
 If the genital organs are seriously damaged or if the lesions are bilateral, recovery is
unlikely.
 Prevention and control of canine brucellosis presently is based on monthly serologic
tests of all dogs in the kennel, and those entering together with segregation and
destruction of positive animals. Brucella canis has caused human infection.
 The prognosis in severe or moderate epididymitis is poor, as obstructions usually
occur preventing the discharge of spermatozoa from that testis.
 There is no practical cure in animals once the epididymis is obstructed.
 Bilateral epididymitis the prognosis is hopeless.
 In a valuable animals if the epididymitis is unilateral and the accessory glands and the
vas deferens have no lesions or infections caused by the same organisms, unilateral
castration may be indicated followed by a long recovery period for the normal testis
during which repeated tests and cultures of the semen may be performed to be certain
the genital infection has been eliminated.

ANOMALIES OF THE EPIDIDYMIS

 May be either congenital or hereditary and consist of
o Spermiostasis due to distention of aberrant efferent or epididymal tubules with
the secondary formation of spermatic granulomas in the region of the head of
the epididymis
o Segmental aplasia of the mesonephiric or Wolffian duct.

o Mesonephric or paramesonephric duct cysts or remnants near the epididymis

or vas deferens.
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Spermiostasis

 Resulting in spermatoceles and spermatic granulomas, resembling tubercular

granulomas, may be caused by blind rudimentary mesonephiric tubles, ductuli
aberrantes.
 These tubules most often are defective efferent tubules.
 They may be attached to the rete or epididymis and therefore produce lesions
involving the head of the epididymis and only rarely other portions of the epididymis.

 The condition is common in bucks and possibly rams, less common in bulls and rare
in the other domestic animals.
 In rams it has been confused with ram epididymitis which usually causes spermatic
granulomas in the tail of the epididymis.
 In bucks the condition is usually bilateral resulting in complete sterility.

Bovine spermiostasis in the mesonephiric ducts is genetically

determined by some recessive hereditary factors.

 Spermiostasis with spermatocele and spermatic granulomas develop slowly thus

young, even bilaterally affected, males may be fertile for a year or more until the
fibrous tissue of the granulomas completely obstructs the mesonephric ducts.
 Spermatozoa and fluid are continued to be produced in the seminiferous tubules and
absorbed in the efferent ducts and head of the epididymis if the obstruction is located
at the distal portion of the head.
 If the obstruction is located at the proximal portion of the head or in the efferent
tubules the back pressure may produce testicular degeneration but the size and
consistency of the testicle may remain nearly normal due to the accumulation of
testicular secretion.

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Segmental aplasia of the mesonephric or Wolffian duct

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 It may occur congenitally in all species but has been well-described in bulls.
 The majority of cases are unilateral and the body, tail, entire epididymis and even a
part or all of the vas deferens may be missing.
 In unilateral cases the bull is fertile.
 In older bulls spermatoceles and/or granulomas may develop just proximal to the
missing segment.
 In unilaterally affected bulls the sperm cell concentration is about one-half normal as
is the total number of spermatozoa per ejaculate.
 Careful clinical palpation of the testes and epididymides, and a rectal examination of
the ampullae and seminal vesicles in the larger species will usually detect the extent of
the segmental aplasia.
 A small epididymis accompanies hypoplasia of the testis.
 Palpation of a very small or missing tail of the epididymis is highly indicative of
segmental aplasia.
 The head of the epididymis, if present, is often enlarged due to distention with sperm.
 If the segmental aplasia is located in the vas deferens, the tail of the epididymis may
be enlarged.
 In rare cases the missing segment may be located in the vas deferens near the urethra
causing distention of the ampulla.
  There is no treatment for this condition, other than possible surgery to reunite the
unaffected portions of the mesonephric duct. Because of the possible hereditary
nature of this anomaly, affected males should be culled.
 Mesonephric or Wolffian, or Paramesonephric or Mullerian Duct Cysts or
Remnants and miscellaneous anomalies may be found in males and are of little
consequence. They may be large and are confused with defects or diseases of the
epididymis on palpation.
 Occasional mesonephric duct cysts may be found in bulls near the head and tail of the
epididymis and some may be lined by ciliated cells, “medusa cysts”.

Paramesonephric cysts or Uterus masculinus

 Seen commonly in 24 to 44 percent of bulls on the dorsal surface of the urogenital

fold between the ampullae and ductus deferens or rarely in or near the epididymis.

TUMORS
Primary tumors of the epididymis are rare in
all animals.

 Testicular tumors in dogs or other animals may invade the epididymis or spread
through the epididymis to the vas deferens and cord.
 Occasionally metastatic tumors may develop in the epididymis.

PATHOLOGY OF THE VAS DEFERENS AND AMPULLITIS

 Infection and inflammation of the vas deferens is usually associated with an orchitis,
epididymitis, or seminal vesiculitis.
 Infection of the vas deferens apparently occurs less commonly in animals in which the
ampullae or dilated proximal portions of the vas deferens are absent.
 In the stallion and bull, infections with organisms such
as B.abortus, Streptococci, C.pyogenes, tubercle bacillus, P. aeurginosa, and others
including viruses, have been observed.
 Segmental aplasia may occasionally be present in the vas deferens, usually
unilaterally.

Careful rectal examination will usually reveal a thickened, firm

and possibly painful enlargement when ampullae are diseased.

 Semen examination may reveal leukocytes and the infective organisms.

 In some cases the semen contains clots of pus and the motility of the spermatozoa is
poor.
 If the motility is good immediately after ejaculation, the spermatozoa often lose their
motility rapidly on storage.
 In cases of segmental aplasia where the missing segment is near the urethra, the
ampullae may become greatly enlarged and distended with spermatozoa but no
inflammatory reaction is present.
 Often the seminal vesicle on the same side of the missing segment is also hypoplastic
or absent.
 Treatment of infected or diseased ampullae is similar to treatment for seminal
vesiculitis.

DISEASES OF ACCESSORY SEX GLANDS

It includes

 Seminal vesiculitis
 Prostatitis
 Prostatic hyperplasia
 Adenocarcinoma of the prostate

SEMINAL VESICULITIS

 Seminal vesiculitis is the most common condition affecting the gland of

bulls, stallions and boars. It is rare in bucks and rams.
 Gross inflammatory lesions are found most commonly in the vesicular
gland of all the accessory sex glands, and the ampullae.
 Seminal vesiculitis may be caused by a variety of pathogenic organisms.
The most common cause is C.pyogenes. This organism may localize in the
seminal vesicle from other primary, pyogenic, infective foci such as
rumenitis, liver abscesses, traumatic gastritis, lung abscess or from a navel
infection in young calf.
 It may possibly enter as an ascending or descending infection from the
prepuce or from an ampulitis, epididymitis or orchitis.
 Infection may be spread in groups of young bulls by homosexual activities
and contact of the penis and prepuce to the rear part of a bull that another
had recently mounted.
 It is possible that certain organisms as C.pyogenes may invade the seminal
vesicle secondary to a temporary infection with a virus or other agent.
 B.abortus is the most common cause for a seminal
vesiculitis. B.abortus causes seminal vesiculitis in bulls.
 B.suis commonly localizes in the seminal vesicles in boars.
 Other organism found in infected seminal vesicles
include: streptococci and staphylococci in bulls and boars. Pseudomonas
 aeruginosa, Mycoplasma bovigenitalium , E.coli ,Mycobacterium
tuberculosis, epivag virus, IBR-IPV, P.L.T.agent or Chlamydia.

The antibiotics used in extended semen had no effect on

Mycoplasma bovigenitalium.

 Seminal vesiculitis affects males of all ages. In bulls it has been reported as
early as 10 months to 1 and 1.5 years of age.

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 Usually there are no external signs of the disease.

 However some males will show signs of mild peritonitis with an arched
back, reduced appetite, pain on defecation or on rectal examination and
hesitation in mounting and thrusting.
 These occasional signs are observed usually in bulls having abscesses of the
seminal vesicle caused by C.pyogenes infection adjacent to the peritoneum.

On rectal examination seminal vesiculitis, especially bovine cases

due to the C.pyogenes are characterized by irregular enlargement
of the gland, fibrosis, peritoneal adhesions, loss of lobulations ,
fluctuation, and abscessation.
  In rare cases, fistulas occur due to rupture of an abscess into the rectum.

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Types of seminal vesiculitis (Galloway’s classification)

 The first type of bovine seminal vesiculitis was usually unilateral and due
to chronic purulent inflammatory lesions with chronic interstitial changes
and was commonly caused by C.pyogenes. Large clots or flocculi were
commonly observed in the semen.
 The second type of seminal vesiculitis was usually bilateral and
characterized by degenerative changes in the epithelium and inflammatory
changes were variable. Culture of these vesicular glands were frequently
negative for bacteria. Large amounts of feulgen positive chromatin masses
were found in the lumen of affected glands and in the semen of the latter
degenerative type of seminal vesiculitis. Semen may be viscid or “ropy ” .
 Leucocytes in the semen may also come from other portions of the
urogenital tract including the prepuce so their presence is not
diagnostic of seminal vesiculitis.

 Semen quality will vary between affected bulls with a lowered motility of the sperm
cells, an elevated pH, a higher catalase activity and a lowered fructose content.
 Although lowered fertility has been associated with seminal vesiculitis, many affected
bulls breeding cows naturally have a good conception rate.
 Frequently bull with a seminal vesiculitis will have another focus of infection in the
testes, epididymitis or ampulla.

DIAGNOSIS, PROGNOSIS AND TREATMENT

Diagnosis

 It is based on the clinical signs noted above.

 Culture of the semen is usually an unsatisfactory method for diagnosing the causative
bacterial agent because of the contamination from the sheath.
 A new technique based on Galloway’s procedure, was developed to collect non-
contaminated urethral samples from the bulls.
 A tranquilizer was administered to quite the bull and allow withdrawal of the penis.
 Rectal massage aided the protrusion of the penis from the sheath.
 The penis was washed with an antiseptic solution and the urethra was irrigated with
sterile saline.
 A 25 cm sterile silastic tube was inserted up the urethra leaving about 4 cm
protruding.
 Rectal massage of the seminal vesicles , prostate and ampullae resulted in the
collection of their secretions in to sterile vials for cultural purposes.

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Prognosis

 Prognosis in seminal vesiculitis is fair to poor depending upon the causative agent ,
the presence of other foci of infection in the reproductive tract, the duration and
severity of the infection and value of the male.
 Males with brucella infections, tuberculosis or mycoplasmosis of the seminal
vesicles, or those with secondary lesions of the testes, epididymides, ampullae or
prostate should be slaughtered.
 Many young bulls with the seminal vesiculitis syndrome with catarrhal or
degenerative seminal vesiculitis overcome the infections spontaneously in a few
months.
 During this period their use for breeding purposes is questionable.
  Bulls with active acute lesions of seminal vesiculitis with a discharge of pus in the
semen should not be used for artificial insemination as only rarely will the antibiotics
used in extended semen destroy the organisms present.
 Many bulls with seminal vesiculitis caused by organisms other than brucella or
mycobacterium may be used naturally or even artificially with quite satisfactory
conception rates especially if a large amount of mucopurulent material is not present
in the ejaculate.
 In C.pyogenes infection, the gland is usually left severely indurated and largely
destroyed.
 Acute cases of seminal vesiculitis tend to become chronic and chronic cases, if
abscessation does not occur, tend to become fibrotic and indurated similar to the
mammary gland following a severe infection.
 In long-standing chronic cases, pus or high leucocytes numbers are seldom observed
in the semen.

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Treatment

Treatment with high doses of broad spectrum antibiotics, or

antibiotics to which the causative agent is sensitive, for two weeks
or longer together with mild massage of the vesicular gland to
remove its contents may result in recovery or elimination of the
infection in some males after 2 to 6 months.

 In recent years surgical removal of the affected vesicular gland has been
recommended for selected bulls in artificial insemination studs. Following surgery
heavy prolonged antibiotic therapy was recommended.
 Regular and frequent examination of the genital tract and semen for a year or more
should be followed after treatment.

PROSTITIS

 Disease of the prostate gland is not common in all animals except the dog in which
prostatitis and hyperplasia of the prostate are common.
 Tumors of the prostate are uncommon. Senile atrophy of the prostate in intact male
dog is rare.

Prostatitis in the dog is often associated with hyperplasia

of the gland.

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 It is probably due to an ascending infection through the urethra but may occur
secondary to hematogenous infection or even descending infections.
 A wide variety of organisms have been isolated from infected glands
including B.canis, E.coli, Streptococci and Proteus.
 Acute prostatitis in the dog is a diffuse or local suppurative inflammatory reaction
with a tendency for abscess formation.
 Bacteria, leukocytes, and blood are frequently found in the urine or observed at the
preputial orifice.
 This should be differentiated from a balanoposthitis.
 Acute prostatitis may be painful and characterized by constipation, an arched back,
elevated temperature and pulse rate, occasionally anorexia and vomiting, and
possibly a leucocytosis. Palpation of the infected gland per rectum causes pain and
“splinting” of the abdomen.
 Chronic prostatitis is occasionally observed.
 The treatment of prostatitis is frequently successful.
 Parenteral injections of broad range antibiotics are indicated over a prolonged period.
 If the ordinary antibiotics do not control the infection, cultures of the prostatic
secretions may be made and the antibiotic sensitivity of the causative organism
determined.
 If prostatitis is due to B.canis the prognosis is poor. Low doses of X- radiation may be
of value in reducing the inflammation in some cases.
 Clinical prostatitis in the bull and boar is rare and may be caused by B.abortus or
suis.

PROSTATIC HYPERPLASIA
It is present in most dogs over 5 years of age that have not been
castrated.
 The condition is probably due to an endocrine imbalance with an excess of
testosterone being secreted which causes an enlargement and hyperplasia of the
gland.

 The moderate to greatly enlarged gland may be smooth or nodular and may contain
small cysts or occasionally excessively large cysts that extend forward into the
abdominal cavity.
 The cyst walls may be calcified.
 In rare cases prostatic calculi have been reported.
 When cysts are not present the preputial discharge from an infected hyperplasia is
usually purulent.
 When cysts are present the preputial discharge from an infected hyperplasia is
watery-grey or bloody.
 The cystic fluid may be voided with the urine producing albuminuria.
 Dogs with marked hyperplasia are usually constipated.

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Rectal impaction and straining predispose to rectal dilation and

the development of a perineal hernia.
 Rectal impaction and straining predispose to rectal dilation and the development of
a perineal hernia.
 Rarely cystitis and hydronephrosis may develop.
 Usually little discomfort is shown by the dog with a hyperplastic prostate unless
constipation or bladder strangulation occurs.
 Obstruction of the urethra does not occur in dogs as in man because of the bilobed
nature of the gland in the dog in contrast to the trilobed structure of the gland in
man.
 Digital examination of the prostate bimanually per rectum and by abdominal
palpation may reveal a nodular or smooth, enlarged prostate.
 It is often helpful to exert pressure through the abdominal wall at the pelvic brim to
push the prostate caudally so that it can be palpated.
 The consistency will vary with the nature of the hyperplasia from firm and fibrous,
to soft, or even fluctuating if cysts are present.
 Normal prostate glands are 2.5 to 3 cm in diameter while hyperplastic glands may be
twice as large or even larger if cysts are present.

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 Radiography may also be helpful in the diagnosis of prostatic enlargement.

 A perineal punch biopsy of the prostate gland is conclusive evidence of the nature of
the enlargement.
 Rectal massage of the gland may give temporary relief by reducing the size of the
cysts.
 Estrogenic therapy, such as injections of 0.5 to 1 mg of stilbestrol per pound of body
weight daily and then gradually reducing the dose during the next few weeks, may
give relief but continued dosing is required.
 Estrogens act by suppressing the pituitary gonadotropins and this causes an atrophy
of the leydig cell and suppression of the testosterone production. the estrogens do not
antagonize the androgens.

The most satisfactory method of reducing the size of the prostrate

is castration, as this removes the source of androgen causing
hyperplasia. Within 2 to 3 weeks after castration the prostate
gland begins to noticeably involute and by 6 to 8 weeks it is
relatively small in size and atrophied.
 However castration does not reduce the large cysts or abscesses in infected glands or
affect caliculi that may rarely be present. In complicated cases, surgery on the gland
may be indicated.
 A chelating agent, sodium diethyldithio-carbamate is used for removing zinc which is
in very high concentration in the prostate gland. This causes a probable cessation of
much of the glandular enzymal activity and atrophy of the gland.
 PROSTATIC HYPERPLASIA
It is present in most dogs over 5 years of age that have not been
castrated.
 The condition is probably due to an endocrine imbalance with an excess of
testosterone being secreted which causes an enlargement and hyperplasia of the
gland.

 The moderate to greatly enlarged gland may be smooth or nodular and may contain
small cysts or occasionally excessively large cysts that extend forward into the
abdominal cavity.
 The cyst walls may be calcified.
 In rare cases prostatic calculi have been reported.
 When cysts are not present the preputial discharge from an infected hyperplasia is
usually purulent.
 When cysts are present the preputial discharge from an infected hyperplasia is
watery-grey or bloody.
 The cystic fluid may be voided with the urine producing albuminuria.
 Dogs with marked hyperplasia are usually constipated.

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Rectal impaction and straining predispose to rectal dilation and

the development of a perineal hernia.
 Rectal impaction and straining predispose to rectal dilation and the development of
a perineal hernia.
 Rarely cystitis and hydronephrosis may develop.
 Usually little discomfort is shown by the dog with a hyperplastic prostate unless
constipation or bladder strangulation occurs.
 Obstruction of the urethra does not occur in dogs as in man because of the bilobed
nature of the gland in the dog in contrast to the trilobed structure of the gland in
man.
 Digital examination of the prostate bimanually per rectum and by abdominal
palpation may reveal a nodular or smooth, enlarged prostate.
 It is often helpful to exert pressure through the abdominal wall at the pelvic brim to
push the prostate caudally so that it can be palpated.
 The consistency will vary with the nature of the hyperplasia from firm and fibrous,
to soft, or even fluctuating if cysts are present.
 Normal prostate glands are 2.5 to 3 cm in diameter while hyperplastic glands may be
twice as large or even larger if cysts are present.

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 Radiography may also be helpful in the diagnosis of prostatic enlargement.

 A perineal punch biopsy of the prostate gland is conclusive evidence of the nature of
the enlargement.
 Rectal massage of the gland may give temporary relief by reducing the size of the
cysts.
 Estrogenic therapy, such as injections of 0.5 to 1 mg of stilbestrol per pound of body
weight daily and then gradually reducing the dose during the next few weeks, may
give relief but continued dosing is required.
 Estrogens act by suppressing the pituitary gonadotropins and this causes an atrophy
of the leydig cell and suppression of the testosterone production. the estrogens do not
antagonize the androgens.

The most satisfactory method of reducing the size of the prostrate

is castration, as this removes the source of androgen causing
hyperplasia. Within 2 to 3 weeks after castration the prostate
gland begins to noticeably involute and by 6 to 8 weeks it is
relatively small in size and atrophied.

 However castration does not reduce the large cysts or abscesses in infected glands or
affect caliculi that may rarely be present. In complicated cases, surgery on the gland
may be indicated.
 A chelating agent, sodium diethyldithio-carbamate is used for removing zinc which is
in very high concentration in the prostate gland. This causes a probable cessation of
much of the glandular enzymal activity and atrophy of the gland.

ADENOCARCINOMA OF THE PROSTATE IN THE DOG

  It is observed only rarely in dogs over 10 years of age.
 Persistent straining at the time of defecation was characteristic of all cases.
 Pain was commonly observed symptom.

Adenocarcinoma of the prostate results in a nodular asymmetrical

enlargement of the gland.
 The tumor may vary in consistency from very hard to soft and is slow to metastasize.
 Castration, estrogen therapy and radiation therapy are of definite value in man in
delaying the growth of the tumor even though metastasis has occurred.
 Prostatectomy would be indicated if the carcinoma had not metastasized. However
the prognosis is poor.
 Other tumors of the prostate are rare.

MISCELLANEOUS FORMS OF INFERTILITY

MISCELLANEOUS MALE FACTORS RESULTING IN FAILURE OF

FERTILIZATION OR CONCEPTION AND REDUCED FERTILITY.

Failure of proper delivery of semen into the vagina at the time of ejaculation may result in
infertility or sterility.

 This may be secondary to lacerations of the ventral caudal portions of the glans penis
with a fistulous opening of the urethra ventrally so semen is deposited in the middle
portion of the vagina and not sprayed into the cranial portion or over the cervix at
ejaculation.
 A similar condition may occur in rams following necrosis of most of the urethral
process secondary to urinary calculi lodging in the process or due to lacerations or
injury to the urethral process.

The inheritance of normal fertility or subfertility

 In males it is difficult to evaluate The inheritance of normal fertility or subfertility

because of the wide variation in fertility between males and the difficulty in
eliminating environment factors except in bulls used artificially on many cows.
 No studies have been made on the transmission of fertility.

The character of high fertility may be transmitted genetically; sons of highly

fertile artificial sires are usually more fertile in artificial service.
COITAL INJURIES IN MALE
It includes
  Balanitis and posthitis
 Injury or trauma to penis
 Hemorrhage from the prepuce and penis
 Herpes coital exanthema
 Fracture of the penis
 Injury of the scrotum and testes
 Miscellaneous injuries

BALANITIS AND POSTHITIS

 Balanitis and posthitis of a severe nature frequently leading to adhesion

and inability to protrude the penis are occasionally observed in bull
especially in young bull with great sexual desire first few weeks on pasture
with cow or heifer.
 Trauma and infection incurred by frequent service and contamination of
sheath or by exposure to the IBR-IPV virus in cow's genital tract result in
severe necrotic & pyogenic infection of penis and prepuce.
 Young ram may be similarly affected
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INJURY OR TRAUMA TO PENIS
 In rare instances in stallion this may be due to mare kicking at the stallion and
striking erect penis at the time of service.
 This may cause haematoma, paraphymosis, laceration or rupture of penis. it is easily
avoided by the proper supervision of breeding and by being certain that mare is
definitely in the estrum.
 If necessary breeding hobble should be used in nervous & excitable mare.
 In the bull occasionally penis catches on the vulval lips in a hymenal remnant or
beneath the vulva. If bull thrusts and penis is bent sharply at right angle. When the
cow suddenly collapsing, a rupture of corpus cavernosum and tunica albuginea,
usually, on the dorsal surface of penis would occur. A haemotoma is thus produced .
This is often spoken of as a ‘’fractured’’ ruptured or broken penis.
 HEMORRHAGE FROM THE PREPUCE AND PENIS
 Following service, it may be due to tumors of the penis or to laceration of the penis or
prepuce.
 In rare instance bleeding form the urethra may be observed in stallions and bulls.
 Several affected boars that had a small vascular outgrowth or polyp in the urethra that
caused blood to be mixed with semen at ejaculation.
 In several bulls irregular-shaped calculi have lodged in the urethra and caused
bleeding at the time of service but for a while only slight or moderate symptoms of
difficult urination were observed .
 In young bulls there may occasionally be a small fistula in the glans penis extending
into the corpus cavernosum.
 On erection a fine stream of blood comes through the fistula.
 Some veterinarians have described the presence on the glans penis of blue-red raised
areas or ulcers that rupture and bleed.
 In artificial breeding, rubber bands from the artificial vagina may slip over the penis
at the time the bull thrusts.
 These usually cause deep lacerations or even amputation of all or part of the glans
penis if they are not removed promptly.
 Such and occurrence may be prevented by not using rubber bands on the artificial
vagina, or by tying them with cord so they cannot slip off.
 Occasionally persons trimming preputial hairs on bulls will snip off the tip of the
penis if vare is not taken to hold the penis caudal to the preputial orifice.
 Bloom reported that bleeding form the urethra in dogs is frequently a symptom of
fracture of the os penis.
 Tumors should be removed. Sexual rest should be give most animals with
hemorrhage from the penis and prepuce.
 In case of urinary calculi the prognosis is often hopeless for future breeding.
 When bleeding occurs from the corpus through fine fistula, sexual rest or surgery to
close the defect are indicated.

HERPES COITAL EXANTHEMA

 This is a vesicular, then ulcerative veneral disease that causes genital discomfort and
reluctance to breed in both sexes.
 The following picture shows the lesions in mare.

 Secondary infection may complicate the condition occasionally but it usually heals.
 Spontaneously within 7-10days.

Coital exanthema is caused by equine herpes virus-1.

FRACTURE OF THE PENIS

 Excessive bending can fracture an erect penis.

 Such bending may occur during vigorous sexual intercourse if the penis is pressed
against the partner pelvic bone.
 The fracture is actually a tear in one of the tube like structure in the penis (corpus
cavernosum ) that hold the extra blood flow that maintain erection.

 The animal has immediate pain and swelling, and the penis appears deformed.
 The injury often damages the structure that controlled erection and after the injury
heals the animal may have difficulty with intercourse, urination or both.

Emergency surgery is usually necessary to repair such fracture to prevent

abnormal curvature of the penis or permanent erectile dysfunction.
INJURY OF THE SCROTUM AND TESTES

 The location of the scrotum makes it susceptible to injury.

 Blunt forces (for example, a kick or crushing below) cause most injuries.

MISCELLANEOUS INJURIES

 At the time of coitus in the stallion may include kick injuries resulting in a
ventral hernia, fracture of the hind limbs, or severe orchitis.
 Breeding hobbles, tying up a front leg and a twitch applied to the mare may
be indicated to prevent kicking.
 In all breeds of animals, especially the larger ones, the footing of the male
should be good and the female restrained properly to prevent the male’s
 slipping and falling, possibly causing gonitis, seen most commonly in the
bull; dislocation of the hip; fractures of the limb or pelvis; fractures of the
spine; muscle or tendon strains or ruptures; as well as the harmful
psychological effect on the male from falling or injuring himself during
coitus.

Large females should not be bred naturally to small males unless

restrained with their rear limbs or all four limbs in a hole or pit.
 Occasionally inguinal hernia with strangulation of the intestine may follow service in
stallions and cause severe colic within 1 to 3 hours.

INTRODUCTION
 Artificial Insemination and Embryo transfer as a means of livestock improvement are
now accepted and utilized worldwide. The increased use of outstanding proven sires
to enhance production potentials, control genital diseases transmitted through
natural service which aid in animal improvement results from the expanding use of
Artificial Insemination and other bio – techniques.

To breed livestock, Artificial Insemination has been accepted as

an established method in all countries.

Terminology

 The term “Artificial Insemination,” commonly called “AI” implies the deposition
of semen into the female reproductive tract by the use of Artificial means
(instruments) rather than by natural service involving the male.
 Although, in American livestock circles, the term “artificial breeding” is often used to
mean the same as Artificial Insemination, the former is not technically correct. Thus,
Artificial Insemination or AI is preferred.
 Artificial Insemination means the deposition of the semen from a male into the
female genitalia during oestrus by mechanical means rather than by the direct service
of the respective male.
 In natural mating, the male ejaculates semen directly into the vagina or near the os
uteri of the female. With the technique of Artificial Insemination semen is collected
into an artificial vagina exteriorly. It is evaluated for its qualities and is extended and
preserved with suitable media prior to use. The processed semen is inseminated into
the reproductive tract of receptive females.

HISTORY AND DEVELOPMENT

 Artificial Insemination was practiced long before the method of selecting superior
males for production traits on the basis of genetic principles were known.

384 - Aristotle Human embryo were developed from

322 menstrual blood.
B.C
1322 Arab Horse Inseminated Mares with
A.D. Breeders Stallion’s semen.
1672 Regnier De Graaf Described the Ovarian follicle
1677 Antoni van Demonstrated motile cells in semen
Leeuwenhock and referred it as “Animalcules.”
and Johann
Hamm
1784 Lazzaro Artificial Insemination in
Spallanzani amphibians and in a bitch, he also
proved that the fertilizing power of
semen resided in the spermatozoa
carried by spermatic fluid.
1786 John Hunter Narrated semen as mawkish and
unpleasant and in taste at first it is
insipid & later pungent and the first
discharge is bluish white in colour &
creamy in consistency & the
subsequent discharge is similar to
that of the common mucus of nose
and less viscid
1799 John Hunter Artificial Insemination in Human
beings.
1890 Repiquet Artificial Insemination in Horses and
advised it as a means of overcoming
sterility.
1890 Sand and Stribolt Obtained 4 successful conceptions
after Artificial Insemination in 8
mares.
1909 Ivanovich Ivanoff First man who successfully did A.I in
(Russian) cattle, sheep and birds
1914 Professor Designed artificial vagina for dogs
Amantea and cattle

(Human
Physiologist)
1933 Walton Described the handling of semen.
1934 Miller and Evans Tried ampullary massage technique
in bulls.
1935 Gunn Devised electroejaculator for rams.
1936 Edward Sorensen Formed the first co-operative
and Jens Gylling artificial breeding association in
Holm Denmark.
1938 E.J.Perry Formed the first co-operative
artificial breeding association at New
Jersey in U.S.A.
1938 Milovanov Devised artificial vagina for bull,
stallion and ram and extenders for
diluting the semen.
1938 Laplaud, Devised electroejaculator for bulls
Thiabault and
Cassou
1949 Polge, Smith and Long time preservation of semen at -
Parkes 79 ° C.
1951 Stewart Reported the birth of first calf born
from frozen semen
1952 Smith and Polge Introduced glycerol as a
cryoprotective agent and freezing of
semen at -196 ° C in liquid nitrogen
1954 Waterloo cattle First to operate 100% A.I programme
breeding using frozen semen
 association
1957 American Practiced the use of long
Breeders Service distance transport of semen in dry
of Madison, ice or in liquid nitrogen.
Wisconsin
Indian Scenario
1939 J. D. First man who did A.I in cattle at the
Sampathkumaran palace dairy herd of Maharaja of
Mysore. Inseminated Hallikar cows
with semen collected from Friesian
bulls.
1939 P. Bhattacharya Established A.I centre at IVRI,
Izatnagar
1942 Reported the birth of first buffalo calf born through A.I.
at Agricultural Institute, Allahabad.
1944 Four regional centres were established throughout India
to implement A.I on large scale at Patna, Bangalore,
Calcutta and Montgomery (Pakistan).
1948 Dr. Veeramani Iyer first did A.I in Tamil Nadu at
Madras Veterinary College, Chennai
1961 Frozen semen technology was first introduced in India
at NDRI, Bangalore.

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 During the First five year plan (April, 1951 to March, 1956) a master project the key
village scheme was launched, which provides for the all round improvement of cattle
and buffaloes in the country
 To bring about rapid genetic improvement in the stock, artificial insemination was
accepted as a major activity of the scheme.
 Under the scheme 600 key villages and 150 artificial insemination centres were
established during the period 1952 to 1956.
 One centre was attached to group of four villages. Each key village had 500 cows
and/or she buffaloes, so that one artificial insemination centre was responsible for
2000 animals.
 Under the second Five-year plan (April, 1956 to March, 1961) the scope of work has
been further extended and by 1957, 400 artificial insemination centre’s were
operating.
 Besides the artificial insemination centre’s in the key village scheme almost all the
states had additional artificial insemination units working in areas outside the key
villages units.
 Some private agencies or co-operative organizations dealing with livestock have also
adopted artificial insemination for breeding work.
 A semen bank has been established at the National Dairy Research Institute of
Bangalore with a view to supplying semen from Jersey bulls for cross-breeding work
& also from bulls of superior Indian Dairy breeds for selective breeding or upgrading
work. Semen from this bank is being flown to different parts of the country.
 The use of AI is other species have generally lagged behind its use in cattle largely
because of the problem of the satisfactory storage of semen.
 According to present status AI is currently used in dairy & beef, cattle, goats, buffalo,
sheep, swine, horses, turkeys, bees, dogs, red fox, fish, mink, humans ?& many other
species in many countries of the world.

Artificial insemination was first attempted in India in 1939 by

Dr. Sampath Kumaran at the palace Dairy farm, Mysore, &
some healthy calves were obtained
ADVANTAGES

 Minimizes the number of sires to be maintained.

 Careful and scientific selection of bulls improves the breeding efficiency.
 Since the transmitting ability of the bulls can be determined quickly and efficiently,
more sires can be evaluated in shorter period, thus helpful in progeny testing.
 Minimizes the occurrence of sexually transmitted diseases such as Brucellosis,
Leptospirosis, Trichomoniasis, Tuberculosis, Listeriosis, Vibriosis and Johne’s
disease.
 Increases sire’s life and thereby provides insurance against death of the individual.
 Eliminates danger and exposure of keeping inferior bulls.
 Periodical evaluation of semen and removal of inferior bulls aid in better fertility
control.
 Provides better identification of animals for improved record keeping and thereby
helpful in selection, disease control and breeding efficiency in the herd.
 Reduces injury and possible mating of unequal sized animals.
 Complete utilization of semen without any wastage due to ageing.
 Reduces sire cost in well established farms.
 Transport of semen to longer distances without being hindered by geographical
boundaries.
 Semen from superior quality bulls which for some reasons unable to copulate can be
collected and used.
 The changes in the reproductive tract of female can be examined and timely
treatment is possible.
 Semen can be stored indefinitely in liquid nitrogen without loosing its fertilizing
potential.

LIMITATIONS
 Trained personnel are required for semen collection and processing.
 If selection and screening of bulls are not properly carried out genetic inferiority,
abnormality and diseases will be transmitted from herd to herd / country to country
very quickly.
 Trained personnel are required for detection of oestrus and performing A. I.
Otherwise conception will be lowered.
 Risk of inbreeding on large-scale introduction of A.I in a small population. However,
this can be overcome by replacing the bulls periodically. Reduces the cost of sires.
 Initial investment on implementation of A.I programme is high.
 Intrauterine insemination of a pregnant female may result in abortion.
 Uncontrolled or unscrupulous operator or owners could substitute sperm of less
valuable animals unless blood typing is routinely employed.
 Requires more time than the natural service.
 Necessitates the knowledge of structure and function of reproductive organs by the
operator.
 Improper cleaning of instruments and unsanitary conditions during handling may
lead to lower fertility.

INTRODUCTION

 One of the most important aspects of artificial insemination is the proper and clean
collection of semen. This involves the proper scheduling of bulls and sexual
preparation as well as techniques of semen collection.
 Collecting a microbe free semen having spermatozoa with vigour, resistance and
livability should be the aim of collection.

Although initial quality of the semen collected depends on the

individual bull, the quality can further be lowered by improper
collection methods.
 Several methods have been developed for the collection of semen.
 These methods of collecting semen have undergone several changes towards
improving quality and quantity of the frozen semen

SEMEN COLLECTION IN BULLS

 Different semen collection methods in
bulls are

 Collection from vagina

 Massage technique
 Artificial vagina method
 Electroejaculation method

Vaginal method ( Spoon or Sponge technique)

 The simplest and earliest method of semen collection was from vagina.
 In this method, the bull is allowed to mount the cow in or out of oestrus naturally and
the semen is collected from vagina by means of a spoon or Sponge or syringe with a
long nozzle.
 This technique is unsatisfactory because selectively small volume of semen is mixed
with large volume of vaginal mucus.To avoid this female out of oestrus can be used.
 Later, Breeder’s bag and urethral fistular methods of semen collection were used.
 All these methods were crude and danger of contamination and disease transmission
was considerable.
 Keeping quality of the semen will be very poor.

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Ampullary Massage technique

 Here semen is collected by massaging the ampullae and seminal vesicle through the
rectum.
 This method was reported as early as 1925 by Case and late well described by Miller
and Evans (1934).

Procedure

 Before starting semen collection by massage method ensure that the bull has not
ejaculated either by natural service or by some other method for two to three days to
ensure that ampulla is full of spermatozoa.
 Secure the bull in a suitable stanchion
 The preputial hair should be clipped
 By tapping the preputial sheath stimulate the urination and empty the bladder
 Properly wash the preputial sheath with warm water and carefully dry it with a clean
towel
 The operator should trimmed his nails quite closely can pass his hand into rectum
after wearing a clean rubber sleeve on his arm and remove all dung from the rectum
  The operator can carefully massage the seminal vesicle, prostate, cowper’s gland to
stimulate some secretion of seminal fluid to rinse the bull’s urethra.
 The seminal vesicles are gently massaged for few times with fingers by backward and
downward strokes towards urethra and a cloudy fluid is expelled.
 Then the ampullae is massaged one by one by gentle, slow and rhythmic manner.
 The ampullae is squeezed/stripped by pressing over floor of the pelvis and the pelvic
urethra may be massaged.
 The massage can be done for 5 minutes. After the massage of the ampullae the S-
curve of the penis should be straightened to allow the escape of semen.
 An assistant holding a glass funnel fitted to a collection vial directly beneath the bull’s
sheath can collect the semen during massaging.
 The quantity of fluid collected from seminal vesicle ranged from .5 to 21 ml and
ampullae ranged from .5 to 23 ml.

Advantages

 Semen can be collected from bulls, which are physically incapable of mounting due to
less libido, lameness or fracture .
 To collect semen from bulls that are refusing to artificial vagina.
 To collect from bulls those are incapable of erection.

Disadvantages

 Chances of contamination by urine or sand.

 Seminal vesicles secretion leads to unbalanced semen components than ejaculated
semen.
 Poor concentration and poor keeping quality.
 Requires skill on the past of operator or training on the past of bull.
 Excessive massage will cause inflammation of the organs.

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Electroejaculator method

 Electrical stimulation of ampullae and seminal vesicle is also a method of collecting

semen from bulls.
 Many instruments has been devised the collection of semen through electro
ejaculation.
 This method was used by Gunn in 1936 for rams. Later it was modified by Rowson
and Murdock, Marden, Dzuik for collection of semen in bulls.
 Thibault et al. (1948) successfully used this for bulls.
  Electro ejaculator consists of
o The instrument consists of a rectal probe with paired rings of copper.
o There are 30 rings which are insulated from each other with ebony.
o Sixty cycles alternate current is used. Voltage is increased rhythmically and
returned back to zero in 3-5 seconds interval.
o The next increase will be higher than the previous one. A bipolar electrode was
devised by Dzuik et al. (1954) for collecting semen.
o At one end it contained 6 metal rings spaced at 4.5 cm apart. These metal rings
work alternate as positive and negative electrodes.

Procedure

 The animal is restrained in a trevis/chute

 The rectum of the animal is flushed with normal saline
 The preputial hairs may be clipped, washed and dried
 Teasing or sexually stimulating the bull before applying electro ejaculate may help to
inform the quality and quantity of semen
 After cleaning the rectum the glove hand with the electrodes is inserted and passed
forward upto 30 cm and the electrodes can be pressed down on the floor of the
rectum directly over the two ampullae between the two diverting seminal vesicles
 An alternate current is passed starting at 5 voltage and return back to zero every 5 to
10 seconds.
 Subsequent stimulations should go high and again come back to zero.
 This is gradually increased up to 15 volts. Generally the erection and ejaculation
occurs at 10-15 volts when 0.5 to 1 ampere current is flowing.
 The semen is collected in a clean sterile glass with a glass funnel.
  The first portion generally consists of merely the watery secretion of the accessory
glands and can be discarded.
 The portion is usually containing semen is collected and used.

Advantages

 Semen can be collected from males that refuse to donate semen in artificial vagina,
lowered sex libido or when injuries or deformities make this impossible.

Helps to collect the semen from bulls that are not

able to mount
 Bulls that are recumbent but having good pedigree can be collected by this method

Disadvantages

 The technique may be painful to bulls. It can not be practiced frequently, since
frequent use of this method may affect the health status of the bulls.
 The quality of semen collected by electro ejaculation cannot be compared with semen
obtained by AV method because the non accessory sex gland secretion.
 The semen collected by this method is usually more in volume and less in sperm
concentration. The contamination of semen will also be more.

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Artificial vagina (AV) method

 The AV method of semen collection has replaced all the old, cumbersome and
unreliable procedures.
 The AV provides the most satisfactory method of collecting semen from bulls.

This is the most widely used and preferred method for routine
semen collection.
 This method has several advantages over other methods and a normal, clear ejaculate
can be collected.
 Artificial vagina is very convenient and is more near to the natural service i.e. it
contributes the threshold of warmth, pressure and friction simulating the normal
characters of female reproductive passage.

Limitations
  Requires trained personnel for collection of semen.

History and type of artificial vagina

 Russians (Kumorov and Nagev, 1932) designed the first artificial vagina. Various
workers made different types of AV.
 The Cornell University workers developed Cornell Model and in US “Danish model”
was developed.
 In tropical countries the “short model” was developed
 Russian model of Artificial Vagina
o Made of rigid rubber cylinder
o 60 cm long and 5.5 cm inner diameter.
o The inner side of rubber cylinder was covered by a thin walled rubber liner, the
ends was turned back over the outer cylinder to form water tight jacket
o The jacket was filled with hot water enough to bring the inside of AV to also
body temperature.
 Danish model of artificial Vagina
o Danish model is commonly used for semen collection in united states.
o Length – 40.70 cm
o Outer diameter – 6.25 cm
o Inner diameter – 5.7 cm
o The above mentioned dimensions may be lowered for smaller bulls in order to
collect the ejaculate near the collecting receptacle

Disadvantage

 Semen receptacle is exposed to light and ambient temperature

 Damage of cold shock to spermatozoa during collection in cold weather.
 Cornell model of Artificial Vagina
 Developed by scientists of Cornell university
 Length – 71.20 cm
 Outer diameter – 6.25 cm
 Inner diameter – 5.7 cm
 This model overcome the advantage of Danish model by covering the collecting
receptacle and exposing to outside environment

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Parts of Artificial Vagina

 Rubber hose pipe or cylinder

 Inner liner
 Directors cone
  Collection vial
 Insulation bag

 The parts AV should be sterilized before it is used for semen collection to avoid the
contamination and disease transmission.
 After sterilization all parts should be dried and stored in a dust free cabinet or
incubator.
 The inner liner should be inserted into the AV and both the ends are turned back over
the ends of the cylinder
 Rubber bands may be attached on both the ends so as to form a jacket between the
liner and rubber cylinder.
 The director cone is fastened over one end of the AV and the glass semen collecting
vial is attached to the smaller end of the cone.
 After assembling AV, half the jacket is filled with warm water of about 65-70 ⁰ C is
poured to get the final temperature of 42.5 to 45 ⁰ C inside the AV.
 The remaining half of the jacket is filled with air. The temperature of AV may vary
depending upon the season, time semen collection and air temperature. (Click here to
view picture).
 The insulation bag is used in colder or hotter environment to avoid cold/heat shock to
the spermatozoa.
 The insulation bag is applied to cover the director cone and collection vial. (Click here
to view picture)
 After assembling the AV, sterile lubricating jelly is applied for 3 to 5 inches of the
interior of liner.
 The temperature of the AV should be checked by the semen collector before taking it
for semen collection. (Click here to view icture)
 Excessive lubrication will cause the contamination over semen samples by carrying
the jelly through the AV over the bull’s pelvis.

Lubricants may be

 K.Y. Jelly
 Sterile white vaseline jelly
 White mineral oil
 Tragacanth gum (3 gm Tragacanth, 5 ml glycerine and 50 ml distilled water)

Preparation of bull

 Semen collection is done in the early morning. The animals will be active and the
stomach will be empty in the morning hours which help in better semen collection.
 The scheduled bulls will be carried from their paddocks to washing area.
 The animals should be washed to remove all the dirt.
 They should be tied surrounding the collection shed and should be dried.
  The bulls should be tied with bull apron and the prepuce should be cleaned with
normal saline
 The bulls are allowed to watch the other bulls mounting which will stimulate the bull.
The forward and backward movement of dummy will also stimulate the bull.
 The protrusion of penis from prepuce will indicate the bull is ready to mount.

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Semen collection

 The teaser should be restrained in a chute.

 The dummy also used occasionally for semen collection .

 The bull is allowed form behind the teaser and 2 to 3 false mountings are given (Click
here to view picture).
 The right handed operator can approach the bull from right side by holding the AV on
his right hand
 When the bull mounts and ready to make the thrust, the operator using the left hand
drawn the penis from sheath and directs it into the AV which is held at an angle that it
is in have with the testis.
 During the entire process the testis itself should not be touched as it will leads to
refusal to service.
 The AV should be directed in such a way that the semen should be ejaculated in
director cone or directly on collection vial
 On completion of the thrust the collection tube is immediately doubled back on the
cone so as to exclude any semen that might has been deposited on testis should not
enter inside the collection vial (as it carries bacteria from penis and sometimes
lubricating jelly)
 After collecting the semen the collection vial is disconnected immediately, closed with
clean stopper / sterile aluminum, plastered with details of bulls, placed in a beaker
containing water of about 34 ⁰ C and examined as soon as possible.

Species Artificial vagina Artificial vagina Artificial vagina

hose liner cone
Length Diameter Length Diameter Length Diameter
Cattle 35 cm 6.5 cm 54 cm 5.5 cm 24cm Upper: 5.0 cm
Lower: 1.5-
2.0cm
Buffalo 30 cm 6.5 cm 54 cm 5.5 cm
 Stallion 54 cm 13 cm *
Sheep 20 cm 4.5cm 30 cm 3.0 cm
Goat 15 cm 4.5cm 30 cm 3.0 cm

* The diameter at the distal end is only 8.5cm.

Hygiene and precautions at collection

 The bull and the location of semen collection should be clean, dry and free from dust
and mud.
 Collection yard should not be overcrowded with bulls and new persons.
 The semen collector should wear protective clothing and gum boots
 The hind portion of the dummy should be as clean as possible.
 During collection the penis should be prevented from coming in contact with the
dummies hind quarter. For this the bull apron can be used. (Click here to view
picture)
 The preputial hairs should be clipped at regular interval and the prepuce should be
washed with normal saline weekly twice (Click here to view picture).
 More than two ejaculates cannot be collected from a bull in one day.
 Don’t grasp the unsheathed penis at the time of collection

The semen collector should not enter into the lab. The semen should
be given through the pass box.

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Preparation of Artificial Vagina

SEMEN COLLECTION IN STALLION

Different semen collection methods in
Stallion are

 Vaginal method
 Collection of tail end sample
 Condom or Breeder's bag
method
 Artificial vagina method

Vaginal method
  Described by Frank
 Semen is collected either from vagina or uterus after natural service
 The mare should be healthy, free from genital diseases and it should not be in foal
heat
 The stallion is allowed to mount, a rubber tube of 2 to 21/2 inch and 3/16 inch
diameter is passed in to vagina and suction by mouth or syringe the semen is collected
in a sterile glass bottle.

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Collection of tail end sample

 After copulation in a stallion to a mare the last portion dripping from glans penis,
urethra of male and vulva is collected in a glass tube
 This last portion or dismount portion will be and 10-30 ml and has lower
concentration.
 This portion is usually contaminated with dirt.

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Condom or Breeder’s bag method

 Used in stallion’s by Mac Leod of Mc Gee.

 Breeder’s bag is a heavy rubber material. It has to be washed, rinsed with distilled
water finally with normal saline. For reuse it should be sterilized properly.
 The stallion’s penis should be washed smegma or dirt removed and dried.
 The bag is applied over the erect penis , squeeze out the air and applied rubber bands.
 The stallion is allowed to mount, the ejaculate is collected in a pre warmed sterile
tube. bottle and kept in a water bath of 35o c for further processing.

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Artificial vagina method

 This method is better than other methods of semen collection due to more
satisfactory results and less contamination.
 There are different types of AV like Missoueri model, Mississipppi model and
Nishikawa model, Colorado model, Hannover model, Krakow – Polish etc.

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Preparation of AV
  Most stallions want a temperature of 45-48o C (depends on stallion's preference).
 Adjust the pressure to stallion's preference. This keeps a lot of water in the AV and
helps prevent cooling.
 Use about a tablespoon of non-spermicidal lube when you are adjusting the pressure.
 Only lubricate about the first third of the AV. Lubricate just before collection to avoid
drying out the lube and to check for foreign objects (thermometers) in the AV.

Semen Collection

It is best to collect a stallion when he is at his Daily

Sperm Output (DSO).
 The DSO means the number of cells the testicular parenchyma can produce daily.
 This is when all the cells he is ejaculating equals the cells he is producing every day.
 It may take 7-10 days in some cases to get to the DSO.
 Normally the sperm output will stabilize at the daily sperm output.
 The mean of last 3 daily collections is DSO.
 You can estimate the DSO by taking 27.5% of second ejaculate (taken 1 hr apart).
From testicular measurements:
o DSO=-3.36 X 109 + (0.066 X 109)(SW in mm)
o DSO=(0.024)(TV)-0.76 . (TV=(4/3)(height X width X length/2).
 For a stallion that has not trained to a dummy, an oestrus mare is needed.
 Select a mare that is calm and does not like to kick.
 If a mare in oestrus is not available , an anestrus mare given 1-2 mg estradiol
cypionate (ECP) will show signs of heat can be used.
 An ovariectomized mare given 2 mg ECP 2-3 days in advance is also ideal
 A dummy or phantom is ideal if the stallion is trained for it. Dummies do not move or
kick. The phantom may also be used for semen collecion in stallion.
 Wrap the mare's tail and tie it off to the side.
 Make sure the restraint is adequate for both mare and stallion.
 Prepare the stallion
 Tease the stallion to erection.
 Wash the penis with warm water only
 All the handlers should be on left side and everyone is advised to pull to the left if
there is a problem.
 The entire collection is a collective effort by everyone involved in getting a semen
sample and keeping everyone safe.
 Approach the mare at an angle and allow the stallion to mount the mare.
 Let the stallion thrust and guide or allow the stallion to insert his penis into the AV.
 Gently touch the ventral penis and feel the urethra for the ejaculatory pulses. Others
can watch for the flagging of the tail that indicates ejaculation.
 After the sample is collected, keep open of the AV upright to avoid spilling the semen
out.
 Immediately drain the water out to prevent the continued exposure of the sperm cells
to hot water.
 The water jacket also forms a 'dam' that will prevent the semen from draining into the
collection bottle.
 Protect the semen from light and cold shock and then begin to analyze it as soon as
possible.
 The stallion will take 20-30 seconds for ejaculation.
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SEMEN COLLECTION IN RAM AND BUCKS

The semen collection in Ram and Bucks
is done by

 Vaginal method
 Artificial vagina method
 Electro ejaculation

Vaginal method

 Here the male is allowed to mount the female in or out of oestrus and the semen is
withdrawn from the vagina.
 Generally female out of oestrus is preferred to avoid the mixing of vaginal secretion
with semen
 It is better to rinse the vagina thoroughly with normal saline or a suitable sterile clean
semen dilutor before semen collection
 After ejaculation the semen is withdrawn from the anterior part of vagina by a glass
pipetted or plexi glass.
 For adopting this method, both male and female should be free from diseases.

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Artificial vagina method

 The method is similar to bulls

 The rubber cylinder will have 20 cm length and 5 cm wide and the inner liner will be
25 cm long and 3.75 cm wide. The AV is prepared as bull AV.
  The inner temperature of AV should be round 45-50 ⁰ C. Half of the AV is filled with
hot water and the remaining is filled with air.
 The liner can be lubricated with non-spermicidal jelly.
 The ram can be easily trained with oestrual ewe. It can be trained with dummy also.
 The bucks prefer oestrual doe only. The non-oestrual doe will move laterally while
mounting.
 Mounting and ejaculation is rapid in ram and buck. So the operator should be alert
while collecting the semen.

The penis should not be touched with the hand. The sheath of
the penis should be grasped and should be directed towards the
AV.

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Electro ejaculation method

 This method was first described Gunn in rams during the year 1936.
 This method can be satisfactorily used in ram and

buck

Procedure

 Restrain the animal

 Empty the rectum by using warm saline enema.
 Pass the electrode into the rectum of the animal after applying K-Y jelly
 Pass the electrical current 2, 5 and 8 volt peaks in a series of 5 seconds on and 5
seconds off by increasing the voltage. The voltage should be increased slowly.
 Hold the penis with gauze and direct the urethral process into the collection vial.
 SEMEN COLLECTION IN BOARS
The semen can be collected by

 Gloved hand method

 Artificial Vagina method
 Electro ejaculation method

Gloved hand method

 The boar is allowed to mount the estrual female or phantom.

 The operator stands on the right side of the dummy
 As the boar mounts the penis will protrude
 Catch the twisted glans penis in gloved hand and exerts pressure on it.
 Relaxation of the pressure will lead to interruption of ejaculation.
 Once the ejaculation is started the pressure should be maintained until the
ejaculation completed.
 The semen is collected in a sterile vial.

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Artificial vagina method

 The boars penis is spirally twisted counter clock wise.

The Artificial Vagina should be able to adopt this spiral type of

penis to get complete ejaculation.

 The length of boar AV- 12.5 cm, Diameter - 4.5 cm

 Inner liner diameter - 40 cm, diameter 3-3.5 cm
 Inside the AV is filled with a heavy rubber casing of 12.5 cm of 4.5 cm diameter of a
rubber sponge.
 The temperature of AV is about 45-50oC. This rubber casing adopts the cork screw
penis and exerts pressure over the penis.
 The AV is passed over the penis and several back and forth movements was done to
lock the penis in the casing.
 The ejaculation commences and continues for about 15 minute with the sperm rich
fraction being emitted on 3rd or 4th minute. frequently these may be a series of sperm
rich fractions interspersed between periods of this sperm fractions and gelatinous
tapioca like secretions.
 Nishikawa and Maule designed a AV in corporating a heavy spiral ring with in the
vagina to stimulate the fold’s of the sow’s genital tract.
  Dummy also used for semen collection in boars.

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Electro ejaculation method

 Electro ejaculation in boar was described by Dzuik and his coworkers and Adams et
al. (1969).
 The rectum is made free of feaces and the probe is inserted. Electric current of 12-20
volts at 5 to 10 second interval is passed to obtain ejaculation.
 Boar must be forcibly restrained and collection is a noisy drawn out affair, resulting in
a heavily contaminated ejaculate.
 Some author described about collection of semen in anaesthetized boars.
 Here the genital organ may be thoroughly cleaned and the penis was removed from
the sheath, held by the hand and electro ejaculator is applied so the semen would flow
in to collection cup.
 But the disadvantage of collecting semen from the anesthetic, pigs were inability to
observe boar’s libido this actions at coitus non physiologic ejaculate of largely free of
accessory gland secretion.
 The electro ejaculation method of semen collection is not satisfactory in boars as in
buck and rams.

SEMEN COLLECTION IN DOGS

 Semen is collected from dogs mainly for breeding soundness examination and for
artificial insemination.
 Semen collected for insemination can be used fresh or cooled and shipped to another
location.
 It is also frozen and can be used at any time. Another indication for collecting semen
is to obtain prostatic fluid for culture or cytology in cases of suspected prostatic
disease.
 In dogs the semen can be collected without the need for a teaser bitch, particularly if
the male has had the experience of semen collection previously.
 However, use of a bitch will almost certainly expedite the procedure and allow more
sperm to be harvested.
 Ideally the teaser should be in estrus, but considering the length of the canine cycle,
that is often difficult to arrange, and a friendly, non-estrus bitch will often serve the
purpose.
 If a bitch is used, it should be controlled with her rear quarters facing the male.

Canine semen is collected using

 Digital pressure and massage

  AV method

Digital pressure and massage

 Restrain the animal. Grasp the prepuce , push it back and expose the tip glans penis .
 Lock your fingers in a ring around the penis, essentially holding the bulbus glandis
inside your fist.
 Apply pressure with forward and backward movement; in most cases, the male will
begin to thrust back and forth.
 Slide the semen collection cup over the protruding penis and slide it over the penis,
pushing the prepuce back over of the bulbus glandis.
 Watch for semen to flow in the collection tube. Most dogs stop thrusting as they begin
to ejaculate.
 The ejaculation will come in three fractions. The first fraction is a clear fluid and has
only few sperms.
 The second fraction will have the actual semen. The third fraction the will have only
prostatic fluid. The different fractions have to be collected in different collection cups.
 Most failures arise because the male is shy or otherwise intimidated.
 It helps to perform the collection on a non-slip surface such as a carpet.
 If the male appears nervous or this is his first time, a teaser bitch may help
considerably.

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Artificial vagina method

 The semen can be collected in artificial vagina also.

 The length of canine AV is 19-20 cm and diameter is 5 cm. The temperature of AV will
be around 40-42 ⁰ C and lubrication is not needed.
 As for as possible do not allow the dogs to urinate and mark the area.
 This act of dog would result in urine in the ejaculate.
 In case the dog urinates and marks the area, a few drops of semen may be discarded
on the ground in order to clean the passage of urethra of the urine residue.
 The dogs penis is stimulated by through the preputial sheath by manipulation.
 When the penis is erected out AV is slipped over it.

In long haired breeds it should be ensured that no hair comes in

between the penis and artificial vagina.
 After semen collection, the artificial vagina is gently removed because the dog’s penis
remains engorged.
  If the AV is not removed in a gentle way there may be damage to the superficial cells,
which may be painful and even cause infection and lack of subsequent libido.

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FACTORS AFFECTING QUALITY AND QUANTITY OF SEMEN

The various factors affecting quality and quantity of semen are

Sexual maturitity

 The process of spermatogenesis is initiated just before puberty the spermatogenesis is

at it’s peak during sexual maturity.

Senile changes

 The age of the male livestock affects the semen production such as spermatozoal
concentration and seminal volume.

Season

 The seasonal variation is observed on the process of spermatogenesis as well as

plasmogenesis .in male live stock via., buffalo –bull, stallion ,ram.
 The quantity of semen ejaculate is greatly declined due to adverse summer season in
buffalo bull which is probably due to less activity of accessory gland (seminal vesicle).

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Climate

 The environmental temperature, relative humidity and wind flow rate have a great
effect on the process of spermatogenesis.
 The extreme hot or extreme cold has adverse effect on spermatogenesis and semen
quality.
 The day length also affects the spermatogenesis among ram in cold countries

Nutrition

 The under nourished male livestock especially in terms of protein ,A,D and E vitamins
and certain trace elements (iron ,cobalt, copper, phosphorus)have adversely affect the
semen quality.

Endocrine

 The testicle and pituitary gland play an important role in spermatogenesis through
production of testosterone and ABP from male gonad and LH/ICSH and FSH from
hypophysis.

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Hormone

 The androgen is the principle hormone for initial impetus for spermatogenesis.
 The androgen is secreted by leydig cells under the control of LH/FSH .the FSH also
helps in the spermatogenesis through influencing the production of ABP (Androgen
Binding Protein) by sertoli cell . Hence the level of above hormone is vital.

Pshycological factors

 The pshycho –neuric and pshyco-somatic factors also affect semen production.

Sexual stimulant

 If the male livestock is placed near it’s female sexual partner then it will stimulates
the process of semen production through pheromone and various extraneous
stimulies.

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Semen collection technology

  The output of good quantity and quality of semen can be improved through proper
semen collection techniques via., sexual preparation and sexual stimulation prior to
ejaculation.

Two false mounts will improve the semen

quality.
 The proper temperature, lubrication and pressure of artificial vagina, site of semen
collection , dummy and attendant affects the semen production.
 The frequency of semen collection is an important factor for semen quality.
 The method of semen collection techniques ( AV, electroejaculation ,massage method
) also affect the semen production .

Management factors

 The care and management of male livestock directly affects the quantity and quality of
semen ejaculate .The exercise of animal also affects it.

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Health

 The general and reproductive health conditions of male livestock affects the semen
production.

Genetics

 The species , subspecies and breed affect the quantity and quality of semen ejaculate .

Individual variation

 The production of semen chiefly depends on the individual male livestock.

FACTORS AFFECTING QUALITY OF SEMEN AFTER COLLECTION

Semen which may be of good quality when collected may
subsequently deteriorate. Semen of all species are sensitive to
environmental and other changes and extreme care should be
taken to avoid damage when handling semen

Temperature
  The temperature of semen in most of the domestic animals except for on ejaculation
is about that of the body. (37.50 C)
 The exposure of semen to temperature above this increases the metabolic rate,
exhausts the energy reserves and decreases the life span of spermatozoa .
 Temperatures above 450 C will kills the spermatozoa
 Reduction temperature will reduce the metabolism of spermatozoa but a sudden drop
in temperature , particularly to below 10*c will cause irreversible loss of their viability
. motility is not regained on rewarming and fertilizing ability of the spermatozoa is
lost. This is called cold shock and can occur through careless over exposure to cold air
or by use of cold collecting glass or microscope slide.

Sunlight

 Direct sunlight is detrimental to semen. Short exposure to sunlight may reduce the
viability of spermatozoa and 30-40 minutes exposure will kill them. Consequently it is
best to avoid exposure of semen to direct sunlight at all times, and all collection and
handlig procedures should be carried out indoors and away from shunny windows .
 It’s also wise to avoid prolonged exposure to strong fluorescent lights or UV radiation.

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Contact with metal

 Contact with metal of any kind is harmful to spermatozoa. For this reason only glass
utensils or equipment made of inert synthetic materials should be used for collection ,
dilution and storage of semen and for insemination.

Contact with water

 Water reduces the osmotic pressure of seminal plasma and may there by kill the
spermatozoa. Thus water is a powerful spermicidal agent and semen should never be
allowed to come into contact with it .
 All equipments should be thoroughly dried before use, including the AV and
collecting glasses.

Impurities and bacteria

 Bacteria, dust, hairs, urine and other such contaminants that may get into semen will
reduce the viability or kill the spermatozoa .
 Contamination of semen occurs most frequently during collection and can best be
avoided by thoroughly cleaning the prepuce of the male before hand.
 After collection the semen should be protected from airborne contaminants by
keeping it covered with aluminium foil or a watch glass .
  Aerosol fly – sprays or antiseptics spray should not be used as these too are very
harmful to spermatozoa and can persist in the atmosphere for sometime after use
 Proliferation of microbes in the semen can be controlled by addition of antibiotics to
the diluents.

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Disinfectants

 Disinfectants and antiseptics are harmful to spermatozoa and their use should be
avoided .for sterilization of equipment 70% alchocol in water is suitable but all
equipment should be dried before use.

Long exposure to air

 Oxygen in the air increases the metabolic activity of spermatozoa and lactic acid
accumulates in the semen .lactic acid may reduce the pH of the semen below the
optimum of 7 and the viability of the spermatozoa will be reduced accordingly. Semen
shoulde be therefore used for insemination or storage as soon as possible after
collection .

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Buffering capacity of dilute

 The media used for dilution of semen should have the capacity to maintain the
optimum ph in diluted semen. Shifts in pH either above 7 (alkaline) or below 7 (acid)
reduce the viability of spermatozoa.
 The recommended semen diluents contain buffers that is substances able to maintain
the medium surrounding the spermatozoa at optimum pH.

Osmotic pressure of diluent

 The components dissolved in the medium surrounding the spermatozoa may exert a
pressure on the cell membrane this is known as osmotic pressure, and it increases
with the concentration of solute in the medium.
 Media in which concentration of solute is equivalent to that within the cell membrane
are said to be isotonic .media with lower solute concentration are hypotonic and those
with higher concentration are hypertonic .
 Both hypotonic and hypertonic media are harmful to spermatozoa , there is only a
narrow range of tonicity over which a diluent can be altered with out affecting the
viability of spermatozoa .spermatozoa remain motile longest when suspended in
isotonic diluent .
 INTRODUCTION
 Semen evaluation alone should not be interpreted as the sole indicator of the fertility
of the bull.
 Suitability of semen for artificial insemination depends on semen characteristics.

Evaluation of semen provides information on the complex of sexual

functions and therefore must not be neglected

 It has not been possible so far to evaluate semen on the basis of a single trait.
 The results of these tests are essential to judge the efficiency of semen prior to use. As
soon as the semen sample is received, it is kept in the laboratory at 35 degree Celsius
till examination is over.
 The evaluation of semen involves handling of living cells and therefore care should be
taken to avoid creation of artifacts and damage to the cells between collection and
completion of tests.
 There are standard tests carried out for the evaluation of semen quality in artificial
insemination techniques and the bulls are rated accordingly.
 The semen is the normal discharge of the male at time of mating. It is a suspension of
spermatozoa in a fluid medium called seminal plasma.
 Spermatozoa originates from the testes, primary sex glands and stored in the
epididymis constitute 10% of the total volume, while the seminal plasma is a mixture
of secretions from seminal vesicles, cowper’s gland, prostate gland, ampullae and
epididymis.
 Secretions of seminal vesicles form nearly 55% of the total volume of the bull semen.
Seminal plasma with all its favorable biochemical composition nourishes the sperm
cells at the time of ejaculation.
 It takes about 60 days before spermatozoa appear in the ejaculated semen.
 The factors that affect the characteristics of semen include breed, frequency of
collection, age of the bull, condition of the bull, season, degree of sexual excitement
prior to collection and skill of the operator.
 The changes in the spermatozoa are due to aging, cold shock, dilution and
preservation.
 Generally life of the spermatozoa is limited by source of energy it contains.
 The semen examination is of great diagnostic value in determining the cause, severity
and degree of pathological conditions of the testes and other genital organs.
 The quality of the semen can be useful in assessing the fertility of a male animal.
 The quality of the semen will vary only in narrow limits between different ejaculates
of a bull.
 During adverse conditions the semen quality will be affected at a very faster rate but
the recovery takes a longer time.
 The semen is a highly fragile material. The semen quality should be assessed
immediately after collection.
  Once the sample is received inside the laboratory it should be kept in a water bath of
370C and evaluation should be started.
 There are several parameters are available to assess the semen quality.
 The good quality semen will have better fertility but the poor quality semen will have
poor fertility.
 There are combination of tests are used to assess the semen quality.

There is no any single test available to certify a semen sample.

PRECAUTION TO BE TAKEN TO MAINTAIN SEMEN QUALITY

 The AV or containers used for semen collection should be clean and free from
contaminants that might injure sperms such as alcohol, excessive petroleum jelly,
powder present on new liners and antiseptics and chemicals of any kind.
 At the time of collection excessive dirt and debris should be kept out of A.V. water and
urine adversely affects sperms. Excessive blood and serum also affect the semen
quality.
 Immediately after collection the semen vial should be placed in a water bath at 37°C.
 Overheating and rapid chilling of semen should be avoided as it affects the semen
quality.
 Too much agitation and shaking of semen should be avoided.

Exposure of semen to sunlight should be avoided

DIFFERENT TESTS TO ASSESS THE SPERM QUALITY
 There are different test are available to assess the semen quality and classified under
following headings.

I. Macroscopic examination

 Volume
 Colour
 Consistency/density
 Presence of foreign materials
 Gross motility

II Microscopic examination

 Mass activity
 Individual motility
 Concentration estimation
 Live and dead sperm estimation
 Abnormal sperm estimation
 Acrosome integrity evaluation
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III. Biochemical tests

 pH estimation
 Methylene blue reduction test (MBRT)
 Hypoosmotic swelling test (HOST)
 Fructolytic index
 Oxygen uptake/ utilization test
 Pyruvate utilisation test
 Glutamic oxaloacetic transaminase (GOT) activity
 Hyaluronidase activity
 Resazurin test
 Buffering capacity test
 Alkaline & acid phosphatase test
 Millovanov’s resistance test

IV.Resistance to environment

 Low temperature storage / resistance to cold shock

 High temperature viability test
 Resistance to sodium chloride / millovanov’s resistance test (R-test)

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V. Physical evaluation

 Objective measurement of motility

 Impedance change frequency
 Filteration through sephadex

VI.Chemical Evaluation

 DNA estimation
 Ascorbic acid content
 Potassium & sodium estimation
 Fructose level in semen
 Citric acid

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VII.Post- thaw evaluation of semen

  Post – thaw motility
 Post – thaw acrosomal integrity
 Post – thaw acrosomal enzyme release
 Post – thaw incubation survival or viability at 37 ° c

VIII. Biological Test

 Zona free hamster egg penetration test (HEPT)

 Zona binding assay (ZBA)

Even though various tests are available only few important tests are
commonly followed as routine tests. Other tests are followed only during
specific conditions.
MACROSCOPIC SEMEN EVALUATION

 Volume
 Colour
 Consistency/density
 Presence of foreign materials
 Gross motility

VOLUME

 Read from the graduated semen collection vial immediately after collection.
 Volume remains fairly constant for each species.
 Volume varies among individuals and between ejaculates within the same individual.
 Volume increases with age and body size of animal and changes with general
reproductive health and vigour and frequency of service.
 Teasing and stewing of bulls are practiced to increase the volume of semen.

Semen volume increases up to 6-8 years

of age.

Decreased volume is noticed in following conditions

 Young males
 Males used excessively
 Incomplete ejaculation or failure of ejaculation
 Bilateral seminal vesiculitis

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 Volume of semen in difference species

Species Volume of Semen

Average (ml) Range (ml)
Bull 4 1-15
Stallion 70 30-250
Ram and Buck 1 0.7-3
Boar 250 1.25-400
Dog 10 1.25
Cat 0.04 0.01-0.12
Fowl 0.75 0.25-2.0
Man - 2-6
Elephants - 50-100

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Nomenclatures for different evaluation criteria

Explanation Terminology
No sperm Aspermia
Reduced Hypospermia
Increased Hyperspermia
COLOUR

 Bull and buck semen is highly concentrated and hence the colour of their
semen may be milky white, creamy or opaque.
 Buffalo semen is whitish when compared to bull semen.
  In stallion, boar and dog the colour of semen is pearly white to grey and
translucent.
 Any deviation from the normal color should be suspected for pathological
conditions.

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Based on color bull semen is graded as

o Creamy
o Thick milky
o Milky
o Thin milky
o Watery

Yellow colour of semen is normal in some bulls due to riboflavin

content, which is secreted, from ampulla or seminal vesicles
 The colour of semen depends upon the concentration of spermatozoa.

 The highly concentrated semen will be creamy in colour and if there is only very few
or no sperms then the colour will be watery.
 Certain abnormal colours encountered are
 Colour Abnormal due to
Brownish Orchitis (Blood pigments)
Dark red to pink Hemorrhage in male reproductive tract
blood
Yellow green Pseudomonas aerogenosa infection - pus
This colour appears on keeping semen
some time after collection
Light brown Contamination with dung
Dull and dirty white Increased number of spermatogenic cells
Yellow Presence of urine
Chunk clots/Curdy Infection
appearance
VISCOSITY AND DENSITY
 Semen may have various consistencies like creamy, miky or watery. This largely
depend on sperm concentration.

 Viscosity is assessed by using semen delivery pipettes. Viscosity increases with sperm
concentration.

Semen with high concentration are more viscous (creamy)

whereas semen with less concentration are less viscous
(watery).

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 The specific gravity of bull semen is 1.0361 There is a positive correlation between
specific gravity and sperm cell concentration.
 Density gives a rapid assessment of concentration. The score used to express density
is ‘D’.

Colour Density Grade

Creamy DDDD
Milky DDD
Thin milky DD
Translucent & cloudy D
Watery O

 Certain pathological conditions of testis and accessory sex glands may affect the
consistency of semen. Some examples are

Pathological condition Consistency of

semen
Epididymitis Less milky semen
Catarrhal conditions of accessory sex Thick viscous semen
glands
Seminal vesiculitis Purulent flocculi
PRESENCE OF FOREIGN MATERIALS

 The foreign materials may enter into the semen from the animal, environment or
from AV and will affect the quality of semen.
  The following are the materials may be present inside the semen
o Dung, pus, urine, hair, dust – from animals
o Sand, bedding materials, dry dung, insects – environment
o Water, lubricant jelly, dusting powder - AV

GROSS MOTILITY

 A drop of semen is placed on a warm glass slide and is observed in naked eye.
 A semen sample with good motility will show wavy motion when it is observed.
 Sperms with poor quality will not show wave motion.
 Based on the wavy motion the semen sample is graded.

Wavy/swirling motion of sperms is assessed in gross motility by naked eye.

Swirling pattern definitely indicates the sperms are live. Since the semen is not
examined under microscope, assessment of individual sperms cannot be made
in gross motility.
MICROSCOPIC SEMEN EVALUATION

II Microscopic examination

 Mass activity
 Individual motility
 Concentration estimation
 Live and dead sperm estimation
 Abnormal sperm estimation
 Acrosome integrity evaluation

Last modified: Monday, 4 June 2012, 03:33 PM

MASS ACTIVITY
The collective movement of sperms or their wave motion is
called as mass activity

S.No. Findings Description Grade

1 Very vigorous forward Excellent 5
motion, extremely rapid
waves and eddies, about
90-100 % active sperms.
2 Vigorous, progressive Good 4
movement with rapid
and abruptly forming
waves and eddies, about
70-80% sperms are
motile.
3 Progressive rapid Fair 3
movement of sperm,
slow moving waves and
eddies, 50-60% sperms
are motile.
4 Oscillatory or rotary Poor 2
movement, no waves
and eddies, 30-40%
progressively motile
sperms.
5 Stationary bunting or Very poor 1
weak rotary movements,
10-20% scattered
progressive sperms.
 6 Immotile sperms. All dead 0

 The evaluation depends upon the experience of the operator.

 5 and 4th grade samples are acceptable. Others should be discarded.
 The collective movement of sperms or their wave motion is called as mass activity.
 It is estimated by keeping a semen drop on a warm glass slide and examining under
low power microscope with out putting coverslip.
 Mass activity may be classified into five grade scale depending upon the wave motion
and rate of sperm activity.

INDIVIDUAL MOTILITY

 Motility is the most common and extensively used tool for estimating the semen
quality.
 The movement of individual sperm is taken into consideration while estimating the
motility.

All the fertile sperms are motile. But all the motile
sperms are not fertile.

 To assess the individual motility diluted semen (diluted with physiological saline or
3% sodium citrate or Tris buffer or Tris-egg yolk extender) is kept on a warm slide
and covered with cover slip.
 The slide is kept on the stage warmer of the phase contrast microscope and examined
under high power. The various types of motility observed may be
o Progressive movement - sperms with very rapid straight forward direction

o Initial motilty 10-20%,

o Initial motilty 30-40%,
o Initial motilty 60-70%,
o Initial motilty 70-80%
o Initial motilty 80%
 Circular movement - sperms with movement in circular path
 Reverse movement - sperms moving in reverse manner
 Oscillatory movement - sperms with jerky movement
 The progressively motile sperms are only taken into account, while estimating the
initial motility.
 The progressive sperms will cover a distance of 100-120 µ in a second.
 Based on progressive motility, the semen samples are graded as follows.
S.No. Progressive motility Grade
1 90-100% Excellent
2 70-80% Good
3 50-60% Fair
4 30-40% Poor
5 0-20% Very poor
The semen collected after long period of time will have more dead sperms.

 The sperm with circular motility indicates cold shock to the sperms.
 The heat/thermal shock also affects the sperm motility.
 A good semen sample should have an initial motility of 70%.

ESTIMATION OF SPERM CONCENTRATION

 Accurate determination of the number of sperm and volume of the ejaculate

determines how many females can be inseminated.

In fresh semen samples the concentration estimation helps to

determine the dilution rate and in frozen semen samples it is done
to ensure sufficient concentration is packed in the straws

Concentration estimation can be done by

 Visual examination
 Cell volume method
 Colorimeter
 Photometer
 Opacity tubes
 Computer assisted semen analyzer (CASA)
 Haemocytometer method

Visual examination

 Here the semen sample is seen visually by naked eye and the concentration is
estimated approximately.
 The estimation depends on the experience of the person.
 This method is giving satisfactory result in samples with high concentration in bulls
and rams.
 But it will not be useful in samples with medium or low concentration.
  This method is not useful in stallion, boar and dogs.

Sperm concentration and semen color in bulls and rams

S.No. Bull Ram Color

(sperms/cmm) (sperms/cmm)
1 - 2,500,000 Thick creamy
2 2,000,000 2,000,000 Creamy
3 1,000,000 1,000,000 Thick milky
4 500,000 500,000 Milky
5 100,000 100,000 Cloudy, watery,
translucent
6 Less than 50,000 Less than 50,000 Clear, transparent,
watery

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Cell volume method

 The semen is centrifuged immediately after collection.

 The sperms will deposit at bottom and the seminal plasma will come to top.
 The packed volume of sperms is measured and based on this the concentration is
estimated.
 This is not an accurate method because the materials other than the sperms will
interfere with the result.

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Calorimeter

 Here the optical density of the semen sample is measured and from which the
concentration of the sample is arrived.
 The colorimeters are designed to measure the percentage of light transmitted through
a light absorbing media can be used.
 The percentage of transmission is a function of the concentration of the light
absorbing agents in the medium.
 This principle is applied to estimate the sperm concentration in a semen sample.
 The percentage of transmission recorded has to be converted as sperm concentration.
  To standardize this colorimeter initially the percentage transmission has to be
recorded for a large number of semen samples of various concentrations.
 The actual sperm concentration of the same samples has to be estimated by
haemocytometer method.
 Finally the correlation between the sperm concentration and light transmission has to
be found.
 Based on this a working chart has to be prepared and it can be used for routine
concentration.
 The instrument has to be calibrated once in a month.

Advantages

 Faster than other methods.

 Results are reliable
Disadvantage

 Initial standardization requires time

 Operation requires time and skill.
 The media used for suspending the semen should dust free otherwise the results will
be wrong.

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Photometer

 It is the advanced form of colorimeter. Here the principle of the equipment is same as
colorimeter.
 But the instrument itself will display the final concentration, dilution rate and
number of doses can be made. It is faster than the colorimeter.
 The instrument has to be calibrated once in two weeks with haemocytometer to
ensure better working condition.
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Opacity tubes

 Brown’s opacity tubes have been used for estimating sperm concentration.
 It is a faster method and can be used in field conditions.
 Opacity increases with increase in concentration of semen.
 Here 0.1 ml of semen is diluted with 9.9 ml of formal saline (dilution rate 1:100). This
is matched with the Brown’s opacity tubes.
 Concentration per cmm = Opacity tube number x dilution rate x 100 x 5
 Semen samples with more epithelial cells, casts, dusts and lubricants will give poor
result. However this method is not used nowadays.

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Computer Assisted Semen Analyzer (CASA)

 It is the most advanced method. Here a special instrument CASA is used to assess the
sperm concentration.
 But the process is tedious and the instrument is costly. So it is not in regular
practice.
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Haemocytometer

 It is a very old method used to assess the sperm concentration but is most accurate
method.
 The procedure is as same as RBC estimation.

Advantages

 Accurate method
 It is used when few bulls requires estimation

Disadvantages

 Requires skill
 The procedure is time consuming.
 So it cannot be used in places where large samples are processed.

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CONCENTRATION ESTIMATION BY HAEMOCYTOMETER

Materials required

 Semen sample (fresh/frozen)

 Haemocytometer set
 Phase contrast microscope
 Watch glass
 Dilution fluid (0.1% formal saline or distilled water or 3% chlorazene solution)
 Eosin powder
 Blotting paper

Procedure

 Mix the semen sample gently to get uniform distribution of sperms.

 Place the semen in a sterile watch glass.
 Put a speck of eosin powder and mix it with semen.
 Aspirate the semen from watch glass into a RBC pipette upto 0.5 mark.
 Clean the tip of the pipette with blotting paper.
 Draw the dilution fluid in the same RBC pipette upto 101 mark.
 Roll the pipette between palm of the hands for 2 minutes to ensure through mixing of
the fluid and semen.
 Discard first few drops.
  Charge the haemocytometer by releasing the fluid below the coverslip which is placed
over the haemocytometer.
 While charging overflowing and air bubble formation should be avoided.
 Wait for 1-2 minutes for the sperms to settle.
 Examine the charged haemocytometer for under low power and then in high
power. (Click here to view picture).
 The sperm counting is done in RBC chamber. (Click here to view picture).
 Count the number of sperms in left top, right top, right bottom, left bottom and center
squares of RBC chamber and calculate the concentration. Counting can be done in
other directions also but it should be in unbiased manner(Click here to view picture).
 While counting sperms in individual chamber it should be done in such a way that
biasness should not be there. (Click here to view picture)

Calculation

Length of one small square = 1/20 mm

Breadth of one small square = 1/20 mm
Depth of one small square = 1/10 mm
Volume of one small square = length x breadth x
depth

= 1/20 x 1/20 x 1/10

= 1/4000 mm3
Total number of small squares counted = 16 x 5 = 80
Volume of semen in 80 squares = 1/4000 x 80

= 1/50 mm3
Consider the total number of spermatozoa counted N
is
The dilution rate is 1:200
Number of sperms are occupying 1/50 mm3 space =N
So sperms occupied in 1 mm3 space = N x 50 x 200 mm3

= N x 10000 mm3
Number of sperms in 1 ml of semen = N x 10000 x 1000

= N x 107 millions

Normal sperm concentration in different species

S.No. Species Concentration

1 Cattle bull 1200 (800-1400 millions/ml)
2 Buffalo bull 800 (600-1200 millions/ml)
3 Stallion 250 (200-600 millions/ml)
4 Ram and buck 3000 (2000-4000 millions/ml)
5 Boar 250 (200-500 millions/ml)
6 Dog 250 (125-500 millions/ml)
7 Man 150 (100-200 millions/ml)

Nomenclature

S.No. Terminology Explaination

1 Normozoospermia Normal sperm concentration
2 Oligozoospermia Reduced sperm concentraion
3 Polyzoospermia Increased sperm concentration
4 Azoospermia Zero sperm concentration
LIVE AND DEAD SPERM ESTIMATION

Introduction

 The estimation of live and dead sperm is an important quality control test in all
semen banks.
 The live sperm concentration is directly related with the fertility.
 The live sperm percentage is also directly related with motility.
 More the number of motile sperms higher will be the live sperms.

But the live sperm percentage will not give indication about
progressively motile sperms.
  Sperms with other motilities like circular, oscillatory and bunting movements also
will be live.
 Semen samples with lower motility will also have normal live sperm percentage.
 So the results should always be correlated with motility and the interpretation should
be done critically.

Principle

 The spermatozoa will have a plasma membrane as its outer covering.

 This plasma membrane is intact in live sperms and its integrity is lost in dead
sperms.
 The live and dead sperm is assessed by a vital stain - eosin-nigrosin which was found
by Hancock during 1951.
 The eosin is a vital stain which can only pass through the loosely integrated plasma
membrane of dead sperm and stains it as pink color.
 The stain will not pass through the live sperm and it appears as white in color. The
nigrosin is a negative stain which gives background color.
 Initially the live and dead sperm was assessed by Mayer et al. (1947) by using eosin
(vital stain) and opal blue (background stain).
 Later Hancock (1951) used eosin (vital stain) and nigrosin (background stain) and it is
most commonly followed.

Procedure and result interpretation

Materials required

 Semen sample (fresh/frozen)

 Glass slides
 Eosin stain (5%)
 Nigrosin stain (10%)
 Immersion oil

Phase contrast microscope

Preparation of 5% eosin stain

Chemical Quantity
Eosin yellow powder 5 gm
2.9% sodium citrate 100 ml

 Weigh the eosin powder, put in pestle and mortar.

  Prepare 2.9% sodium citrate solution, boil it.
 Add the boiling solution to stain and grind it well. Finally filter and store it at 4 ⁰ C.

Preparation of 10% nigrosin stain

Chemical Quantity
Nigrosin water soluble powder 10 gm
2.9% sodium citrate 100 ml

 The preparation of the stain is as per the above method. After filtration the stain is
stored in an air tight bottle at 4° C.

Staining procedure

 A drop of eosin, four drops of nigrosin and a small drop of semen are placed on a
clean, grease free slide.
 Mix the semen first with eosin and then immediately with nigrosin stain.
 The mixture is taken on the edge of a slide and pulled across the top of another slide
leaving a smear
 Allow it to dry in air.
 200 spermatozoa are counted under oil immersion at a magnification of 1000X in
different areas of smear and the live sperm percent is calculated.
 Calculation

Interpretation

 Unstained sperms are considered as LIVE

 Sperms stained pink by eosin are considered as DEAD
 Partially stained sperms are considered as DEAD
 The fresh semen sample should have 80% live sperms
 The frozen semen samples should have 50% live sperms .
 If a semen sample will have more dead sperms it is called as necrozoospermia
 ESTIMATION OF SPERM ABNORMALITY

Introduction

 Any deviation from normal morphological structure of spermatozoa is called as

abnormal sperm.
 Every ejaculate will have some abnormal sperms in it.
 An increased prevalence of abnormal sperm is associated with a decrease in fertility.
 The appearance of an increased number of abnormal sperm in the ejaculate is a
reflection of lesions of the testes and/or the excurrent duct system and provides a
convenient clinical diagnostic aid.
 Overall length of a bull spermatozoon : 68 – 74 m
 Head - length : 8 – 10 m : width: 4 – 5 m
 Neck : 0.3 – 1.5 m
 Mid-piece : 8 – 10 m
 Tail - principle piece : 45 – 50 m : end piece : 2 – 4 m

60 per cent of the anterior head is covered by acrosome or galea capitis.

Sperm abnormality are produced by

 Inheritance
 Disease
 Adverse environmental effects
 Improper handling of semen.

The sperm abnormality increases with

 Extremes of temperature
 Reproductive infection
 Congenital testicular hypoplasia
 Acquired testicular degeneration
 Epididymal dysfunction
 Ephemeral fever
 To a subtle extent with advancing age

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Classification of Sperm Abnormality

 There are different types of classifications available. They are
o Based on source of abnormality
o Based on effect on fertility
 o Based on the location of abnormality

Based on source of abnormality

Primary abnormality

 They are appearing at the time of formation of spermatozoa.

 This is mainly caused due to aberration in spermatogenesis.
 These abnormalities are arising due to developmental defects in seminiferous
tubules of diseased, degenerative or hypoplastic or inherited sterile conditions.
 Primary abnormalities are dangerous and they can be transmitted to their
young ones also.

Secondary abnormality

 These abnormalities are acquired at the time of transport of spermatozoa

through epididymis, accessory sex glands and urethra.

Tertiary abnormality

 Caused by the semen handler after collection. It is mainly due mishandling.


 Based on the effect on fertility
 Major abnormalities – Abnormalities which are having more effect on fertility
is called as major abnormalities.

 Minor abnormalities - Some abnormalities will have lesser effect and is called as
minor abnormalities.

 Based on the location of the abnormality

 This depends upon the region where the abnormality is located. It is divided in to
head, mid piece and tail abnormalities.

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Methods of Assessing Sperm Morphology

 Sperm morphology is assessed by
o Eosin-Nigrosin stain
o Rose Bengal stain
o Indian ink stain
o Wright’s stain
o Casaretts’s stain
 o Buffered formal saline method (Wet smear method)
 An eosin-nigrosin stain is commonly used as a morphology stain.
 The eosin-nigrosin sperm smears prepared for live and dead count is satisfactory for
assessing abnormal spermatozoa.

Eosin-nigrosi staining method

Materials required

 Semen sample
 Glass slides
 Eosin stain (5%)
 Nigrosin stain (10%)
 Immersion oil
 Phase contrast microscope

Preparation of 5% eosin stain

Chemical Quantity
Eosin powder 5 gm
2.9% sodium citrate 100 ml

 Weigh the eosin powder, put in pestle and mortar.

 Prepare 2.9% sodium citrate solution, boil it.
 Add the boiling solution to stain and grind it well. Finally filter and store it at 4 ° C.

Preparation of 10% nigrosin stain

Chemical Quantity
Nigrosin powder 10 gm
2.9% sodium citrate 100 ml

 The preparation of the stain is as per the above method.

 After filtration the stain is stored in an air tight bottle at 4 ° C.

Staining procedure (Click here to view video demonstration)

 A drop of eosin, four drops of nigrosine and a small drop of semen are placed on a
clean, grease free slide.
 Mix the semen first with eosin and then immediately with nigrosin stain.
 The mixture is taken on the edge of a slide and pulled across the top of another slide
leaving a smear .
 Allow it to dry in air.
 200 spermatozoa are counted under oil immersion at a magnification of 1000X in
different areas of smear and classify them as normal, head abnormal, mid piece
abnormal and tail abnormal sperms.
Calculation

Precautions

 Avoid artefacts at the time of preparation of the smear on the slide.

 Avoid cold shock and use of hypotonic solution if diluted semen is used.

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Rose bengal stain method

Preparation of Rose Bengal stain

 Rose Bengal - 3 gm
 Formalin (37-41%) - 1 ml
 Distilled water - 100 ml.
 The stain is prepared by adding and grinding the contents in pestle and mortar.

Staining method

 Prepare a thin smear of semen in a clean grease free slide and dry it
 Place slide in a jar containing Rose Bengal stain for 20 minutes
 Wash the slide in running tap water
 Dry it and examine under oil immersion microscope
 Count 200 sperms and express the results

Interpretation of result
 Classification of sperm abnormalities

Head Neck Mid piece Tail

abnormalities abnormalities abnormalities abnormalities
Microcephalic Swollen neck Coiled mid Coiled tail
head piece
Macrocephalic Broken neck Kinked/Bent Terminally
head mid piece coiled tail
Pear shaped Broken mid Broken tail
head piece
Tapering head Bowed mid Dag defect
piece
Round short Abaxial mid Stump tail
head piece
Lost acrosome Double mid Double tail
piece
Loose head Immature
(normal & Sperms
abnormal)
Knobbed Proximal
acrosome droplet
Diadem defect Distal droplet
Double head Teratoid

Inference

 A maximum of 20 per cent sperm abnormalities are allowed in bull semen (major
7.5% and minor 12.5%)
 Hereditary defects should not exceed 5 %
 Specific minor abnormalities should not exceed 10%

Interpretation of spermiogram

 An increased prevalence of sperm with morphologic abnormalities such as head

abnormalities and retained proximal droplets may be evidence of either
 sexual immaturity or degenerative changes in the seminiferous epithelium of the
testes.
 Abnormal sperm will usually disappear from the ejaculated as the bull ages and, in
some cases, as the testes become larger.
 A bull that does not exhibit a normal spermiogram by 18 month age is a poor risk as a
future breeding animal.
 In case of matured bull, it has to be differentiated whether the degenerative changes
are transient or permanent which is very difficult and is complicated by the fact that
49 days are required for sperm to sperm to complete the spermatogenic cycle plus an
additional two weeks are required for passage of the sperm through the epididymis.
 The presence of more that 15% major abnormalities or more than 30% total
abnormalities, especially when coupled with other findings such as palpable testicular
or epididymal lesions or inadequate scrotal circumference, is sufficient reason to
classify a bull as questionable or, depending upon severity, as an unsatisfactory
potential breeder.
 However, interpretation of a spermiogram requires information concerning the bull’s
breeding history and the results obtained from a through clinical examination of the
bull.

ACROSOME INTEGRITY

Introduction

 Acrosome a cap like structure on the head of the spermatozoa covers 60% of the
anterior portion of the sperm head.
 The morphology of the acrosome should be maintained for the sperm to
undergo capacitation and acrosome reaction in the female reproductive tract for
attaining fertilizing ability.
 Appreciable modifications to the structure of the plasma membrane and the outer
acrosomal membrane follow after capacitation has run its course, in the form of
acrosome reaction.
 Acrosome reaction consists of fusion at multiple points between the two membranes
and formation of vesicles made up of fragments of two membranes.
 The sperm must be able to undergo these changes in the female reproductive tract to
attain fertilizing ability by release of specific enzymes.
 The subcellular enzymes present in the acrosome facilitates disolution and
penetration of the zona by the spermatozoa which will leads to the union of male and
female nucleus.
 For this to happen the acrosome should be intact. Maintenance of optimum fertility
depends on the acrosome being structurally and biochemically intact.
 Acrosome can be detached from sperm under the influence of different physical and
chemical factors.
 Freezing and thawing can also bring about damage to the acrosome.
  Hence the acrosomal cap has received considerable attention in sperm morphology
due to its importance during fertilization.
 Any damage or loss of the acrosome leads to infertility or sterility problem. Hence the
evaluation of the acrosomal status gets importance.

The acrosome is evaluated by the

Giemsa stain.

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Materials required

 Stock Giemsa stain

 Semen (fresh/frozen)
 Glass slides
 5% formaldehyde

Preparation of stock Giemsa Stain

Chemical Quantity
Giemsa powder 1 gm
Glycerol 60 ml
Methanol 66 ml

 Weigh the Giemsa powder, put in a pestle and mortar.

 The glycerol is added slowly and grind it well. Then add methanol slowly and grind it.
 Finally the solution is filtered and stored in an amber colored bottle.

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Preparation of working Giemsa solution Preparation of Sorenson’s

Buffer Preparation of 5% formaldehyde

 It is prepared immediately prior to use

Chemical Quantity
Stock Giemsa stain 3 ml
Sorenson’s buffer 2 ml
 Distilled water 45 ml
Chemical Quantity
Na2HPo4.2H2O (21.682 gm/500 ml) 200 ml
KHPo4H2O (22.254 gm/500 ml) 80 ml
Stock buffer 280 ml
Chemical Quantity
Formalin (37-41% W/V) 12.5 ml
Distilled water 87.5 ml

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Staining procedure and result interpretaion

Procedure ( Video demonstration: Giemsa staing procedure Part 1 & Giemsa

staing procedure Part 2)

 A drop of diluted semen (1:5 to 1:10 in 2.9 per cent sodium citrate) is kept on a clean,
grease free, pre-warmed glass slide and is dried in air. The frozen thawed semen
needs no dilution.
 The slide is immersed in 5% formaldehyde f or 30 minutes for fixing semen smear.
 The slide is washed in running tap water and dries it.
 The slide is immersed in working Giemsa solution for 3 hours at 37 ⁰ C.
 Finally wash the slide in running tap water and dry it.
 Examine the slide under oil immersion of phase contrast microscope and count 200
sperms.
 The acrosome based on the morphology is classified as follows

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S.No. Defect Picture

1 Normal acrosome
2 Partially lost acrosome
3 Completely lost acrosome
 4 Ruffled/wrinkled acrosome
5 Knobbed acrosome
6 Diadem defect
7 Detached galea capitis
8 Apical notch/ridge

 The fresh semen should have 80 per cent and above acrosome integrity and the frozen
semen should have minimum of 65 per cent intact acrosome.
 The hereditary defects like knobbed acrosome and diadem defect should not exceed 5
per cent.

Various acrosomal defects are given below.

BIOCHEMICAL TESTS

 pH estimation
 Methylene blue reduction test (MBRT)
 Hypoosmotic swelling test (HOST)
 Fructolytic index
 Oxygen uptake/ utilization test
 Pyruvate utilisation test
 Glutamic oxaloacetic transaminase (GOT) activity
 Hyaluronidase activity
 Resazurin test
 Buffering capacity test
 Alkaline & acid phosphatase test
 Millovanov’s resistance test
 HYDROGEN ION CONCENTRATION OR pH OF THE SEMEN
 The pH of the domestic animals at the time of collection is having practical value in
accepting or rejecting a semen sample.
 Depending upon the concentration and activity of the spermatozoa the pH of the
semen varies.
 Since the sperm concentration and activity can be measured directly there is no better
information gained with pH.
 The pH of the semen can best be measured with pH meter. It can be measured with
pH paper or by using dyes also.
 The use of narrow range indicators is more convenient in the routine work of artificial
insemination.

pH paper

 It is more convenient for routine work.

 Tear the paper strip from the indicator book (touch the paper only at one end).
 Tip the torn end of the paper in a drop of semen.
 Look for the first change in color.
 Find the matching color on the cover book and note the corresponding pH.

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Indicator dyes (Bromothymol blue or Bromo cresole purple or Phenol red)

 Take two capillary tubes which are marked for equal volume
 Take the semen in on capillary and dye in the other capillary up to the marked
volume.
 Add the semen and dye in a watch glass and mix it well.
 Take the mixed solution in a clean capillary tube and the color is compared with the
comparator set (which has set of capillary tube according to the pH).
 pH of the semen is assessed by reading the matching capillary from the comparator
set.
 The ranges of pH that can be assessed by using different dyes are as follows.
o Bromothymol blue - 5.2-6.8
o Bromo cresole purple – 6.0-7.6
o Phenol red – 6.8 – 8.4.

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pH of semen of different species

Species pH
 Bull 6.8
Stallion 7.4
Ram/Buck 6.8
Boar 6.8
Dog 6.7
Cat 7.4

 pH of the semen affected during

o Inflammatory conditions affecting the accessory sex glands
o When bulls are used excessively – Alkaline pH
o Incomplete ejaculation – Alkaline pH

METHYLENE BLUE DYE REDUCTION TEST

Principle

Methylene blue dye reduction test (MBRT) is a very simple test

used to measure the metabolic activity of the spermatozoa.

 This test is based on the principle that the hydrogen ions are liberated during sperm
metabolism which will reduce the blue colored methylene blue into colorless
leucomethylene blue.
 The hydrogen ions are released due to the dehydrogenase enzyme present in active
sperm.
 The time taken to change the color is directly related with the motility and
concentration of the spermatozoa.
 More the motility and concentration will lead to more the hydrogen ion release which
will cause less time taken by the methylene blue to colourless leucomethylene blue.

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Procedure and interpretaion of reuslt

Materials required

 Fresh semen
 Egg yolk citrate diluent
 5 ml test tube
 Incubator
  Methylene blue solution (50 mg of methylene blue in 100 ml of 2.9 per cent sodium
citrate buffer)
 Liquid paraffin
 Water bath

Procedure

 Take 0.2 ml of fresh semen and 0.8 ml of egg yolk citrate diluent in a sterile 5 ml test
tube
 Add 0.1 ml methylene blue solution and mix the contents
 Place 1 cm layer of liquid paraffin
 Keep the test tube in a water bath of 46.5° C
 Observe the time taken to change in color from blue to colorless

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Result

Based on the time taken to change the color, the semen sample is classified as follows.
S.No. Time interval Classification
1 3 - 5 min Good
2 9 min Average
3 > 9 min Poor
HYPO OSMOTIC SWELLING TEST (HOST)

Introduction and Principle of the test

 The integrity of plasma membrane is a pre-requisite for maintaining fertility.

 The spermatozoa undergo important events in the female reproductive tract like
capacitation and acrosome reaction to attain fertilizing ability.
 For these events to take place the spermatozoa must maintain the functional integrity
of cell membrane.
 Further the process of fertilization involves complex biochemical and physiological
events that are not measured by the gross physical indicators used in routine semen
evaluation.
 Hence there is a need for a test that is inexpensive, technically simple and reasonably
accurate that could be used as an adjunct to the standard semen evaluation methods.
 Fluid transport occurs in an intact cell membrane under hypo osmotic conditions
until equilibration is reached between inside and outside the cell.
 Due to influx of fluid there will be bulging or bending of the tail fibre occurs.
  This phenomenon is known as "tail curling" or the sperm with curled tail is known as
"swollen sperm".
 Spermatozoa with chemically and physically intact membrane will show tail curling
under hypo osmotic conditions whereas spermatozoa with an inactive membrane will
not.
 During cryopreservation, spermatozoa are subjected to stress that can alter
membrane integrity.

Hypo osmotic swelling test (HOST) is found to be useful as a

measure of cryosurvival of spermatozoa.

 Various media are used to provide hypo osmotic conditions to spermatozoa to test the
functional integrity of cell membrane.
 Distilled water can be used as hypo osmotic medium in HOST.
 The results of the studies on HOST and fertility of frozen semen in bulls indicate that
with higher HOS spermatozoa give higher fertility rate.
 HOS also has positive correlation with freezability of semen.

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Pre requisites

Materials required

 Fresh/frozen semen
 Sugar tube
 Distilled water/ HOS media
 Water bath
 Pipette and tips
 Preparation of HOS media

For 75 mosm media

 Sodium citrate - 0.367 gm

 Fructose - 0.675 gm
 Distilled water - 100 ml

The osmolality of water is '0'. This can be used as hypo osmotic

media. Otherwise hypo osmotic media (with different osmolalities
like 75, 100, 125, 150 & 250 mOsm) can be prepared with sodium
citrate and fructose in certain combination.
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Procedure and interpretion of result

Procedure

 Take 0.9 ml of HOS media /distilled water in a sugar tube

 Keep the tube in 37 ° C water bath for 5 minutes to bring the temperature of media to
37 ° C
 Then add 0.1 ml of semen
 Incubate the mixture at 37 ° C water bath for 30 minutes
 Place a drop of semen in a clean, grease free glass slide and put a cover slip over it.

Examine under phase contrast microscope to see the tail curling

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If the clarity is not there, do the following procedure

 Make a thin smear of the incubated mixture in a clean slide and dry it.
 Stain the slide with 3% Rose Bengal stain for 20 minutes
 Wash the slide under running tap water and dry it
 Examine the slide under oil immersion microscope for tail curling
 Count at least 200 spermatozoa
o Calculate the percentage of spermatozoa with showing tail curling.
o Samples showing higher percentage of sperms with tail curling indicate good
samples

Calculation
 The fresh semen samples should have a minimum of 70% and above HOS
reacted sperms and the frozen semen samples should have a minimum of 50%
reacted sperm.
FRUCTOLYSIS INDEX

Introduction

 Fructose, a glycolysable sugar, present in the semen, which is produced by the

accessory sex glands, is utilized as a source of energy by active spermatozoa.
 Its level in semen is regulated by the male sex hormone testosterone.

Fructolytic index is defined as the amount of fructose utilized by

109 spermatozoa in one hour at 370C.

 Greater the metabolic activity of spermatozoa more will be the amount of fructose
metabolized in any semen sample.
 The quality of the semen can be assessed by measuring the rate of utilization of
fructose.
 Fructolysis in the semen is assessed by measuring the disappearance of sugars and
accumulation of lactic acid by a constant number of spermatozoa in a specific time
and under specified conditions.
 Gassner and Hill (1952), Bonadonna and Pozzi (1954), Probine et al. (1958) reported
a relationship between fructolysis and fertility.
 A highly significant relationship was found between fructolysis and concentration of
live sperm.
 In buffalo semen fructose level is low as compared to cattle semen. Fructolysis was
lowest during summer and superior during spring season.
 Mann (1948) for the first time proposed fructolysis as an index for evaluating the
activity of semen.

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Materials required and procedure for estiation of fructose

Materials required

 Phosphate buffered saline (0.25 M) with pH 7.4. (Prepare the phosphate buffer by
mixing 9.6 ml of 3.4% potassium dihydrogen phosphate and 40.4 ml of 3.55% sodium
hydrogen phosphate solutions)
o Zinc sulphate (0.2%)
o Sodium hydroxide solution (N/10)
o Resorsinol (0.1%) in ethanol
 o Hydrochloric acid (30%)
o Electric calorimeter

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Procedure for estimation of fructose

 Modified method on Mann (1964) for fructose estimation in semen sample is as

follows.
 Take 0.4 ml of fresh semen in a tube containing 0.6 ml of 0.25 M phosphate buffer
(pH 7.4).
 0.1 ml of above mixture semen buffer is added to 1.9 ml of distilled water.
 To the above mixture add 1 ml of 2% zinc sulphate solution and 1 ml of N/10 NaOH
solution for deproteinisation (zero hour sample).
 0.9 ml of semen buffer mixture (from Sr.No. a) is incubated at 370C for one hour.
 After incubation deproteinise it as in step (b) & (c) [one hour sample].
 Heat both the deproteinised samples in boiling water for one minute.
 Cool and filter the samples.
 Take 0.5 ml of above filterate in two separate tubes and make the volume to 2 ml with
distilled water.
 Add 2 ml of 0.1% resorcinol in ethanol and add 6 ml of 30% HCl to each sample.
 Mix the contents properly and maintain the sample in water bath at 80oC for 10
minutes.
 Immediately cool the contents to room temperature in running water. Reddish brown
colour develops in the solution.
 Compare the intensity of colour with the help of a visual electric calorimeter (Dubosco
Hellige) with a standard fructose solution of equal volume run as a blank.
 Standard fructose solution is prepared by dissolving 400 mg of fructose in 100 ml of
saturated benzoic acid. 0.02% and 0.1% fructose solutions are prepared out of 0.4%
stock solution for use of comparison with one hour and zero hour samples
respectively.
 Prepare the blank solution by adding 0.5 ml of 2% zinc sulphate and 0.5 ml sodium
hydroxide solutions to 1.0 ml of standard fructose solution.
 Then follow the steps described from step No. 10 onwards.

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Calculation
 A standard curve is prepared by taking 0.02 – 0.1% standard fructose solution in
satrated benzoic acid.
 The amount of fructose is calculated from this curve.
 Blank is prepared by adding all the reagents and in place of semen water is added.
 The amount of fructose present in semen immediately after collection and present
after one hour’s incubation are worked out.
 The difference between the two concentrations of fructose is on account of fructolysis
in one hour.
 Fructolysis may also be worked out in respect of 109 live spermatozoa.
 This method is useful as compared to 109 sperm concentration and provides a true
comparison of metabolic activities of semen samples.

OXYGEN UPTAKE OR UTILIZATION TEST

 If the sperms are very active they take up higher volume of oxygen per unit of time.
 The respiratory quotient (ZO2) is calculated as volume of CO2 produced by the
spermatozoa per unit of time divided by the volume of O2 consumed in the same unit
of time.
 The ZO2 value of bull semen is to be 21. This test indicates activity and live percentage
of spermatozoa.

PYRUVATE UTILISATION TEST

 Found by Melrose during 1952.

 The quality of semen samples could be graded according to the oxygen consumption
after the addition of pyruvate and pyruvate plus 2:4 dinitrophenol. Flouride was
added to the sample to reduce the exogenous metaboloism to a low level.
 When added with pyruvate the oxygen consumption in high fertile bull semen and
low fertile bull semen is increased but on addition of 2:4 dinitrophenol the oxygen
uptake is increasing two fold in high fertile bulls but not in low fertile bulls.
 The oxygen uptake was measured with the help of monometric equipment.

GLUTAMIC OXALOACETIC TRANSAMINASE (GOT) ACTIVITY

 To record the extent of damage occurred to spermatozoa, there is no satisfactory
method except to depend upon biochemical tests to estimate the presence of enzyme
(GOT) in seminal plasma after freezing and thawing.
 Normally it is not found in seminal plasma. The presence of this enzyme indicates the
damage to spermatozoa

HYALURONIDASE ACTIVITY
 This enzyme is present in acrosomal system of spermatozoa.
 Integrity of acrosome is directly involved in fertilizing capacity of spermatozoa.
 Presence of hyaluranidase activity in extracellular fluid will indicate acrosomal
damage.

RESAZURIN TEST
It is used as an indicator of metabolic
activity of semen.
 On account of dehydrogenase activity of semen blue colour of resazurine changes to
pink at first and finally to colourless state.
 Good semen sample reduces resazurine to pink colour in one minute and requires
four minutes to become colourless

BUFFERING CAPACITY TEST

 It is the chemical ability of a fluid to absorb acid or alkali with little change in pH.
 Bull semen
o highly buffered at pH below 5.5 and above 9.0
o moderate well buffered at pH 5.5 to 6.5 and 8.0 to 9.0
o lacks buffering capacity at pH 6.5 to 8.0

 Its presence reflects the functional state of accessory sex glands and metabolic activity
of spermatozoa.
 Increase in conception rate in cows following inseminations with semen containing
increasing order of alkaline phosphatase.

MILLOVANOV’S RESISTANCE TEST

 This test assesses the ability of spermatozoa to withstand the action of 1 per cent
Sodium chloride solution.
 The resistance is denoted as the millilitre of 1per cent Sodium chloride solution
required to stop the progressive motility of all spermatozoa in 0.02 ml of semen.
Procedure

 Pipette out 0.02 ml of semen into a 200 ml capacity flask

 Add 1% sodium chloride solution volumes each of 10 ml from a pipette
 Assess the progressive motility of semen after each addition 10 ml of sodium chloride.
 Sodium chloride is added at intervals till the progressive motility of spermatozoa is
ceased
 Good quality semen shows ‘R’ value to be not less than 5000.

RESISTANCE TO ENVIRONMENT

 Low temperature storage / resistance to cold shock

 High temperature viability test
 Resistance to sodium chloride / millovanov’s resistance test (R-test)

HIGH TEMPERATURE VIABILITY TEST

 There is high correlation between the time of incubation at 100° F required for all
spermatozoa to lose their motility and conception rate.

COLD SHOCK RESISTANCE TEST

Introduction

 This test is done to assess the fertilizability/preservability (5°C) of the semen of a

particular bull.
 The spermatozoa are subjected to unfavourable condition such as cold (0°C).
 The percentage of resistant live sperm is counted after cold shock.
 There is a good correlation between percentage of sperms which withstand cold shock
and motility after 5 days of preservation under chilled condition (5°C).

Materials required

 Test tube
 Semen sample
 Microscope
 Crushed ice
 Eosin and Nigrosin stain
Procedure

 Examine the live sperm percentage of the fresh semen immediately after collection.
 Take one ml of freshly collected in a test tube and place it in a beaker containing
crushed ice (0⁰C) for 10 minutes.
 Determine the percentage of live sperm and motility after cold shock.
 Compare the percentage of live sperm in fresh semen sample and semen after expose
to crushed ice.
 Then estimate the storage ability and freezability of spermatozoa.

RESISTANCE TO SODIUM CHLORIDE

 The amount of 1 per cent sodium chloride solution required to cease the
motility reflects the resistance of spermatozoa.
Last modified: Monday, 4 June 2012, 03:38 PM
PHYSICAL METHODS OF EVALUATION OF SEMEN

Objective method of measurement of motility

 Computer assisted semen analysis system ( phase contrast microscope, cameras and
control monitor with personal computer with 640 kb CPU co-processor and digitizer
with 256 grey valves terminal printer) will yield exact data regarding motility,
concentration, velocity of cells, ratio of live cells, quantity of required diluent and
straws.
 It has the capacity of storing entire production information.

Impedance change frequency

 This technique involves the passage of electric current with very low voltage in the
freshly collected semen.
 This impedance caused by motile spermatozoa is recorded in the meter.
 Semen charged with electric current during impedance change frequency test is safe
and can be used subsequently for Artificial Insemination.

Filtration through Sephadex

PHYSICAL METHODS OF EVALUATION OF SEMEN

Objective method of measurement of motility

 Computer assisted semen analysis system ( phase contrast microscope, cameras and
control monitor with personal computer with 640 kb CPU co-processor and digitizer
 with 256 grey valves terminal printer) will yield exact data regarding motility,
concentration, velocity of cells, ratio of live cells, quantity of required diluent and
straws.
 It has the capacity of storing entire production information.

Impedance change frequency

 This technique involves the passage of electric current with very low voltage in the
freshly collected semen.
 This impedance caused by motile spermatozoa is recorded in the meter.
 Semen charged with electric current during impedance change frequency test is safe
and can be used subsequently for Artificial Insemination.

Filtration through Sephadex

CHEMICAL TESTS TO EVALUATE SEMEN

Chemical tests available are

 DNA estimation
 Ascorbic acid content

DNA estimation

 The available literature shows that the quantity and quality of DNA content
of sperm alters during storage, seasonal variation and aging of the male.
 This alteration of DNA content may be co-related with fertility.
 Gledhill (1966) reported that in infertile bulls the amount of DNA content
may not vary but the quality of basic protein alters.

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Ascorbic acid content

 The ascorbic acid content in semen may be co-related with the fertility of
semen (Philips et al., 1940 and Reid et al., 1947).
 Hence evaluation of semen for ascorbic acid content may be desirable.

Procedure

 This method was described by Roy et al., 1950.

 Take 0.2 ml of fresh semen in a sterile test tube.
  Add 0.8 ml trichlor acetic acid.
 Mix the contents well.
 Filter the mixture through Whatman filter paper No. 1.
 Add 0.05 ml of 2,6 dichlorophenol indophenol solution in a centrifuge
tube.
 Titer it against protein free filtrate of semen in a micropipette.
 Check the end point where the pink color disappears.
 A blank solution of 0.2 ml of standard ascorbic acid plus 0.8 ml of 10%
trichlor acetic acid is titrated against 0.05 ml of 2, 6 dichlorophenol
indophenol dye.

Titration reading of standard

Ascorbic acid concentration = ------------------------------------ x 1.2

Titration reading of unknown

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Preparation of dye

 Extract 100 mg of 2, 6 dichlorophenol indophenol through a filter paper

with 25 ml of boiling distilled water. It can be kept as stock solution. This
can be stored in refrigerated temperature. On the day of estimation 5 ml of
stock solution is made up to 100 ml by adding freshly boiled and cooled
distilled water.

Standard ascorbic acid

 Stock solution can be prepared by dissolving 60 mg of ascorbic acid in 5%

acetic acid and the volume 100 ml. For routine use 1 ml of the the stock
solution is diluted with 5% actetic acid to 100 ml. Thus 1.2 gm of ascorbic
acid are present in 100 ml of solution.

POST THAW EVALUATION OF SEMEN

 Post – thaw motility

 Post – thaw acrosomal integrity
 Post – thaw acrosomal enzyme release
  Post – thaw incubation survival or viability at 37 ° c

POST-THAW MOTILITY
 Post thaw motility is examined 24 hours after freezing the semen.
 Thaw the frozen semen straw at a water bath of 370C for 30 seconds.
 Wipe the straw and cut the ends of the straw and collect the semen in a small test
tube.
 Keep a small drop on a clean, grease free glass slide.
 Cover it with cover slip and examine under the phase contrast microscope.
 Motility based on progressive motile sperms is evaluated on the nearest 5 percentage.
 If motility differs by 10 per cent or more between two straws of the same batch a third
straw is examined.

The minimum post thaw motility of 50 percentage

is acceptable.

 In routine practice only post thaw motility is performed and it is the most reliable test
when carried out by an experienced person.

Click the links below to view video:

o PTM -10% (Buffalo semen)

o PTM -50-60% (Buffalo semen)
o PTM -60-70% (Buffalo semen)
o PTM-Jerky and circular (Buffalo semen)
o PTM -50-60% (Bull semen)
o PTM -60-70% (Bull semen)

Note: The following three tests are done only as added tests when it is suspected that
semen may be of lowered fertility but meets minimum standards for fertility.

POST-THAW ACROSOMAL INTEGRITY

 The acrosome covers 60 per cent of the anterior portion of the nucleus.
 Acrosome can be detached from sperm under the influence of different physical and
chemical factors.
 The enzymes localized in the acrosome determine the sperm penetrating and
fertilizing capacity.

Optimum fertility depends on the acrosome being structurally and

 biochemically intact.
POST-THAW RELEASE OF ACROSOMAL ENZYMES

 Leakage of enzymes (hyaluronidase and acrosin) from the acrosome to the seminal
plasma can be used as an indicator of acrosomal changes.
 This test involves special chemicals, equipments and it is time consuming.

POST THAW INCUBATION SURVIVAL OF SPERMATOZOA AT 370C

 Thaw the frozen semen straw at a water bath of 370C for 30 seconds
 Wipe the straw and cut the ends of the straw and collect the semen in a small test
tube.
 Keep a small drop on a clean, grease free glass slide.
 Cover it with cover slip and examine under the phase contrast microscope and
estimate the post thaw motility.
 Incubate the semen in the test tube in the same water bath at 370C.
 Estimate the progressive motility at every half an hour interval till the motility
completely ceases.

Longer the survival, greater the chance for the sperm to survive in
the female genitalia on insemination resulting in better fertility.

 Individual variations among bulls have been observed for post thaw survival of
spermatozoa.

BIOLOGICAL TEST

In vitro Fertility Assessment of sperms

 The widespread use of AI in livestock breeding, initiated scientists to persistently

develop laboratory tests that would accurately predict the fertility of sires.
 The commonly employed semen evaluation tests have been effective in controlling the
quality of bovine frozen semen used for A.I. but cannot be relied upon to accurately
predict fertility.
 Hence, there is a need for evolving some methods to assess the fertility of
spermatozoa in vitro before it is used for AI.
 Zona-free hamster ova penetration bioassay is a recently developed technique that
could be successfully employed for in vitro fertility assessment of spermatozoa.
 Zona-free ova of most mammalian species retain very strong or fairly strong species
specificity, rejecting entry of spermatozoa of most other species.
 The Golden hamster is exceptional and permits sperm entry of wide variety of other
species provided the sperm have completed capacitation.
 The hamster egg penetration is a heterologous sperm penetration
test. The ova of any species will not allow the other species sperms
to enter inside even after the removal of the zona pellucida. But the
hamster egg will allow the other species sperms after the zona
removal by enzymatic digestion.

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 Sperm penetration bioassay (SPB) has been evolved as a reliable test to assess the
fertility of spermatozoa.
 Various reports also indicate that there is a positive correlation between bioassay
measures and fertility.
 SPB could also be used in screening and selection of donors with high fertility for in
vitro fertilization and embryo transfer studies.
 The logistics of SPB is some what cumbersome, because a colony of hamsters is to be
maintained, require scheduled hormonal injections, the animal has to be sacrificed
and the oocytes are to be recovered at the optimum time.
 Further, the spermatozoa to be tested should also be available at a fixed time.
 To eliminate these problems, the use of cryopreserved hamster oocytes has been
attempted.
 In addition, cryopreservation of hamster oocytes has practical advantages such as
long-term storage, ready supply and easy transportation of viable oocytes.
 Ready availability of hamster oocytes is essential for infertility clinics and semen
banks where maintenance of hamsters is difficult.
 Now, by research in this Semen Bank, it is established that cryopreserved hamster
oocytes can also be successfully utilised for SPB.

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SELECTION AND SUPEROVULATION OF HAMSTER AND

COLLECTION AND PREPARATION OF OOCYTE

Experimental animals

 Healthy, sexually matured young female golden hamsters (Mesocricetus auratus) of

eight to 12 weeks of age weighing between 60 and 120 g should be used for carrying
out this test.

Superovulation and recovery of oocytes

  The number of oocytes ovulated in the normal cycle is increased by superovulating
the hamsters with Pregnant Mares' Serum Gonadotrophin (PMSG) and human
Chorionic Gonadotrophin (hCG).

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PMSG

 50 I.U. of PMSG (Folligon) should be injected intraperitoneally using a sterile single

use syringe for stimulating the follicular growth.

hCG

 After 78 to 82 hours of PMSG injection, 75 I.U. of hCG (Chorulon) should be

administered intraperitoneally using a sterile disposable syringe for superovulation.

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Medium for handling oocytes

 For handling oocytes, the Biggers, Whitten and Whittingham (BWW) medium
(Biggers et al. 1971) with minor modifications should be used.
 The tonicity of the medium should be adjusted to 290-300 mosmol /kg and the pH of
the medium should be adjusted to 7.2 to 7.3.
 The media used for the study, should be prepared as freshmedium, and sterilized by
filtration using sterile single use syringe filter unit.
 The triple glass distilled water for the preparation of media should also be autoclaved
before use.

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Recovery of the oviduct

 After 16 to 17 hours of hCG injection, the hamster should be sacrificed by exposing to

toxic dose of chloroform anesthesia in a closed chamber.
 Then, the hamster should be fixed on a dissection board and under aseptic
precautions and the abdomen is opened by midventral incision.
 By using curved scissors, the oviducts are removed after severing the uterine and
ovarian ends.
 The oviducts are then transferred to a drop of handling medium placed in a sterile
single use 35 x 10 mm petridish.

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Recovery of cumulus mass

 The petridish containing oviducts are viewed under the stereozoom microscope and
observed for the contractions and relaxations of the ampullar portion of the oviducts.
 The cumulus mass are then released by puncturing the ampullar portion with pointed
forceps.

Digestion of cumulus mass

 The released cumulus mass is then transferred to a drop of freshly prepared 0.1%
solution of hyaluronidase in mBWW medium and allowed for five minutes for the
digestion to take place.

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Washing of oocytes

 After digestion, the oocytes are released from cumulus cells.

 The freed oocytes are then aspirated and washed three to four times by successive
transfer of the oocytes to drops of medium to remove the cell debris.

Evaluation of oocytes

 The washed oocytes should be evaluated under stereozoom microscope for their
morphology.
 Bright, shiny oocytes with uniform, spherical, regular cytoplasm and an intact zona
pellucida are considered as normal.

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Removal of zona pellucida

 Zona pellucida is removed by treating the oocytes with freshly prepared 0.1% trypsin
in mBWW medium for 30 to 60 seconds.
 During digestion, the oocytes should be constantly monitored through the stereozoom
microscope to avoid excessive digestion.
 After the dissolution of zona, the zona-free oocytes are washed three or four times in
drops of fresh handling medium and kept ready for coincubation with capacitated bull
spermatozoa.

PREPARATION OF SEMEN

Semen samples
  The frozen semen samples of bulls maintained at the Semen Banks can be used for
carrying out this test.
 On the day of use, the semen straws should be thawed at 37oC for 30 seconds in a
water bath and used for capacitation.

Medium for sperm handling

 The mBWW medium used for handling of oocytes can be used as medium for
handling spermatozoa.
 The capacitation medium is prepared by adding bovine serum albumin at a
concentration of 35 mg/ml of mBWW medium.

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Swim-up technique

 Swim-up technique should be adopted to improve the quality of semen to be used for
sperm penetration bioassay.
 Four mini straws (0.25 ml) are thawed, to which 5 ml of mBWW medium was added
to remove the egg yolk and seminal plasma by centrifugation.
 The supernatant is discarded and the pellet is resuspended in 0.5 ml of medium.
 100 to 200 ml of sperm suspension is placed in three centrifuge tubes, to which two
ml of fresh medium is carefully layered.
 The tubes are then placed in an air incubator at 37oC for one hour in slanting position
for the motile spermatozoa to swim-up.
 At the end of one hour, the supernatant is collected and centrifuged.
 After centrifugation, the supernatant is discarded and the sperm pellet is again
resuspended in fresh medium.

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Capacitation of spermatozoa

 After swim-up, the sperm suspension is capacitated in 0.5 mM concentration of

calcium ionophore A23158 (CaI) for one minute and centrifuged to remove CaI.
 The supernatant is discarded and the pellet is resuspended in capacitation medium.
 The sperm concentration is adjusted to 107 million spermatozoa per ml and kept in an
air incubator at 37oC for one hour.
 The individual sperm motility is estimated and observing head to head association of
two to three spermatozoa can confirm capacitation.

Sperm - Oocyte Co-incubation

  After one hour of capacitation, 100 ml of capacitated sperm drops is placed in a sterile
disposable petridish to which 15-20 zona-free oocytes are added.
 Mineral oil should be layered over the sperm drop to avoid possible loss by
evaporation.
 The petridishes are then incubated in air incubator at 37oC for three hours to allow
sperm-oocyte coincubation.
 At the end of coincubation, the oocyte-sperm complexes are washed several times
with handling medium to remove loosely attached spermatozoa.
 The oocytes are then subjected to either fluorescent staining or aceto-orcein staining
to assess sperm penetration.
 Spermatozoa with decondensed heads and associated tails are considered to have
penetrated the oocyte

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Scoring

 The fertilizing ability of frozen spermatozoa of different bulls is assessed by

fertilization percentage and fertilization index.

INTRODUCTION
Preparation of semen diluent is a vital step in any semen
processing laboratory.

 The function of semen dilution is not only to increase the viability of sperm in
vitro for providing nutrients and buffer salts.
  The purpose of diluting semen is to extend its volume so that many females can be
bred from a single ejaculate.
 Fluids used for diluting also prolong the life of sperm, provide energy to the sperm
and one of the ingredients acts as a so called ‘buffer’.
 The purpose of the buffer is to keep the fluid at the degree of alkalinity necessary to
preserve the life of the sperm.
 Such extenders are used to preserve its fertilizing capacities throughout the various
procedural stages necessary prior to its proper introduction into the female
reproductive tracts at estrus.
 Dilution and preservation of semen is carried out with suitable media, which are non-
toxic and provide nutrients for the maintenance of spermatozoa along with egg yolk.

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A satisfactory diluent must have

 Similar osmotic pressure - isotonic to blood, semen and milk (around 275-300
mOsm) and should be capable of maintaining the entire storage period.
 Proper balance of mineral elements which are essential to the life of sperm cells.
 All sperm cell nutrients for both aerobic and anaerobic metabolic processes.
 Lipoproteins and/or lecithin to protect sperm cell against cold shock.
 Chemical means for buffering toxic end products of sperm cell metabolism.
 Antibiotics to inhibit bacterial multiplication.
 Source material to protect from sulphydryl containing enzymes.
 Materials to protect from freezing injuries.
 These are some of the properties of good diluent.

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 Hence, precise weighing of chemicals is absolutely necessary to ensure correct

composition. The pH is a good indicator and it should be assessed frequently to
ascertain the quality of semen.
 The sperm concentration is determined and the dilution rate is calculated.
 Dilution should be done within 10 minutes after collection. Delaying the dilution
adversely affects the semen quality.
 Both the semen and diluent should be at the same temperature (30 ° C) at the time of
dilution.
 Semen is diluted with equal quantity of diluent immediately after collection, poured
slowly and gently along the sides of the collection tube and then the whole content is
emptied into the pre-measured remaining diluent with caution

The death of spermatozoa outside the animal body is due to one of

the three following causes
  Destruction of the spermatozoa due to senescence.
 Expenditure of nutritive material
 Autointoxication of metabolic products.

Storage of spermatozoa outside the animal body therefore involves

 Delaying the senescence.

 Slowing down the metabolic processes
 Supply of nutritive materials
 Provision of substances which prevents autointoxication of metabolic products.

EXTENDERS FOR FREEZING OF SEMEN

Successful freezing of semen will depend upon prevention of
damage caused by

 Internal ice crystal formation that affects the structure of the

spermatozoa.
 The increase in solute concentration as water is withdrawn
from the suspension medium.
 By interaction of these two physical factors.

 These above damages can be prevented by suspending the sperm in ideal extender,
which could afford cryoprotection during freezing.

A satisfactory diluent must have

 Similar osmotic pressure - isotonic to blood, semen and milk (around 275-
300 mOsm) and should be capable of maintaining the entire storage
period.
 Proper balance of mineral elements which are essential to the life of
sperm cells.
 All sperm cell nutrients for both aerobic and anaerobic metabolic
processes.
 Lipoproteins and/or lecithin to protect sperm cell against cold shock.
 Chemical means for buffering toxic end products of sperm cell
metabolism.
 Antibiotics to inhibit bacterial multiplication.
  Source material to protect from sulphydryl containing enzymes.
 Materials to protect from freezing injuries.
 These are some of the properties of good diluent.

THE BASIC COMPONENTS OF EXTENDERS

The basic components of extenders for freezing of bovine spermatozoa are

 Buffer
 Fructose
 Glycerol
 Egg yolk
 Antibiotics

BUFFER

 A variety of organic and inorganic buffers have been used for deep freezing of semen.
 But an ideal buffer should prolong the life if spermatozoa at room temperature
penetrate cells and acts intracellular buffer, less toxic in the critical temperature
during freezing and better clarity under microscope.
 The most commonly used diluent in most of the laboratories for deep freezing of
semen is the inorganic buffer called Tris buffer.

Tris is commonly called as universal diluents which prolongs the

life of sperm at room temperature, penetrate sperm and acts as
intra cellular buffer, less toxic at the critical low freezing
temperature.

Preparation of buffer

 Weigh the required chemicals accurately in an analytical balance.

 Mix the chemicals in double distilled water thoroughly in a conical flask.
 Adjust the pH of buffer to 6.5 – 7.0
 Boil the contents for few minutes in a water bath.
 Cool and add antibiotics.
 Store the buffer in a refrigerator

FRUCTOSE
Addition of fructose provides glycolysable substrate for sperm,
prevent agglutination, maintain required osmotic tension and
electronic balance and gives better cryoprotection during deep
 freezing.
 Fructose added in the diluent is the major energy source for the sperm

GLYCEROL
Glycerol is the most widely and commonly used cryoprotective
agent for bull spermatozoa.
 It was the spectacular discovery of Polge in 1949 that showed that the death of
spermatozoa could be avoided if the cells were suspended in a medium containing
glycerol.
 The possible modes of action of glycerol are:
o Modifies the size and shape of ice crystals formed.
o Binds water and decrease freezing point of solution and less ice is formed.
o Acts through salt buffering mechanism.
o Reduces solute concentration.
o Prevents denaturation of proteins and rupture of plasma membrane.

EGG YOLK

 When spermatozoa is cooled to 5 ° C they are subjected to cold shock, which causes
leakage of intracellular enzymes and other materials present in the spermatozoa.
 This damage can be prevented by providing addition of lecithin, protein, lipoprotein
and similar compounds present in the egg yolk.
 In addition the glucose, proteins and vitamins present in the egg are utilized by the
spermatozoa and also protect the enzymes and antiagglutinic factors present seminal
plasma.
 The value of the yolk of the hen’s egg as a diluting medium in semen preservation was
found by Philips during 1939.
 It helped in rapid growth in recent years of artificial insemination throughout the
world.

Egg yolk protects lipoprotein sheath of sperm against

cold shock.
 The active principle in yolk responsible for this is now thought to be lecithin or a
similar phospholipid occurring either free or in combination with protein.
 It contains glucose, proteins, vitamins and required viscosity index which may be of
advantage to the sperm cells.
 The hydrogen peroxide formed, which is toxic to the sperm, may be destroyed by
additions of the enzyme catalase to the yolk diluents, under anaerobic conditions the
problem does not occur.
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Preparation of Egg Yolk

 Egg yolk is commonly added with buffer at the ratio of 1: 4 (20%) to

prepare the extender for dilution.
 Unfertilized egg is used for this purpose.
 The egg should be purchase from disease free, known flock.
 Fresh egg immediately after laying is purchased
 The egg immediately after receiving is wiped with dry cotton and stored at
refrigerator at 4-5 ° C
 Maximum it can be stored for 3 days at refrigerator temperature of 4-5 ° C
 Immediately before use it is cleaned with swab of 70% alcohol
 It is opened at the narrow end with the help of a sterilized foreceps.
 The white of an egg (albumin) is drained off with the help of an egg yolk separator
and yolk is placed on a sterile filter paper.
 If required yolk may be transferred to another filter paper to clear traces of albumin.
 Then the vitelline membrane is punctured with a sterile glass rod and yolk is allowed
to fall in a sterilized graduated cylinder directly at the bottom without touching the
walls.
 The required quantity is prepared and added with autoclaved buffer (added with
glycerol) and mixed with magnetic stirrer.

ANTIBIOTICS
 Many of the organisms present in the semen are non-pathogenic.
 The organisms which are contagious and spread through the semen are Brucella
abortus, Vibrio fetus, Trichomonos fetus, Leptospira pomona, Mycobacterium
tuberculosis,Mycobacterium paratuberculosis and viral agents like Infectious Bovine
Rhinotracheitis and FMD.
 Refrigeration greatly suppresses the multiplication of organisms but does not
necessarily stop it.
 Some of the organisms like Pseudomonos aeroginosa, Trichomonas fetus, Leptospira
pomona, FMD and IBR-IPV survive after freezing also.
 Penicillin and Streptomycin are two antibiotics widely used since 1950 and are still in
use. They are relatively harmless to sperm cells and inhibits broad spectrum
microorganisms.
 Penicillin G – 500-1000 IU/ml and Dihydrostreptomycin 500-1000 µg/ml is
adequate for routine use.
 Antibiotics will not completely eleminate Brucella abortus, Trichomonos
fetus, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, viral agents
like Infectious Bovine Rhinotracheitis and Foot and Mouth virus and Rickettsiae and
Fungi.
 The addition of antibiotic is not an extra precaution and cannot be taken as
effective tool to permit use of semen from infected bulls or unhygienic
proceedings.
PRECAUTIONS IN DILUENT PREPARATION
 Only double glass distilled water, fresh eggs and pure chemicals are used for
preparing diluents.
 Care is taken not to rupture the yolk membrane before it becomes free of white.
 The yolk membrane should be retained with filter paper and discarded.
 Enough buffer solution is prepared in bulk, sterilised and kept in refrigerator.
 Diluents are prepared on the previous day itself of collection and kept in a refrigerator
until used before dilution, the temperature of the diluent should be 30 ° C and this is
achieved by keeping it in a water bath for 15-20 minutes before dilution.
 The semen is always kept at 30 ° C during evaluation.
 Dilution should be done within 10 minutes after collection.
 Glycerol when added should be warmed to 40 ° C. After addition, the glycerol should
be mixed well with the diluents by stirring gently with a sterile glass rod for 20
minutes.
 Always use sterilised glassware during the entire operation.

DILUENT PREPARATION
The diluents preparation involves two
steps

 Preparation of buffer
 Preparation of diluents

Points to be considered before preparation of diluent

 The chemicals should be purchased from reliable source.

 The compounds used should be chemically pure and weighing should be accurate.
 The triple glass distilled water or Millipore water should be used for dissolving the
salts.
 Completely dissolve one salt before adding another salt.
 The buffer solution should be sterilized by autoclaving before use.

PREPARATION OF BUFFER

Materials required

 Triple ditillation water/Millipore water

  Chemicals (Tris, Citric acid monohydrate, Fructose)
 Glycerol
 Weigh the chemicals as per the requirement given above for preparation of 1 litre
diluent

Buffer preparation (for 1 litre diluent)

 Take the triple distilled water in a sterile beaker according to the requirement
 Weigh the chemicals accurately
 Add the chemicals one by one and dissolve it by magnetic stirrer by required speed
 Finally add the glycerol at 7% level ie. 70 ml
 Make the volume upto 800 ml by adding distilled water
 Completely mix the glycerol by magnetic stirrer for at least 20 minutes
 Care should be taken while stirring that air bubbles should not be formed by over
speed of the stirrer

The pH of buffer should be 6.8-

6.9 Glycerol 7%

Sterilization of buffer

 The prepared buffer is sterilized by autoclaving at 100 ⁰ C for 15 min at 5 psi.

 Cool the buffer to room temperature
 Generally the buffer is prepared on previous day evening, sterilized and kept under
laminar air flow over night.
 The diluent is prepared by adding egg yolk in the morning immediately before semen
collection.

PREPARATION OF DILUENT
 The egg yolk is prepared as per the standard procedure

 For 1 litre of diluent, 200 ml of egg yolk is required

 The egg yolk is added and mixed by using magnetic stirrer
 The antibiotics are also added while stirring with egg yolk
 Care should be taken while stirring that air bubbles should not be formed by over
speed of the stirrer
 The pH of the diluents should be 6.7-6.8.

Egg yolk is added @ 20%.

CALCULATION OF DILUTION RATE
The dilution rate is calculated according to the number of sperms
 to be packed in a straw. In both French mini and medium straws
20 million sperms are packed.

For French mini straw

 The French mini straw is having 20 million sperms in 0.25 ml.

 Let us consider the

 The total diluted volume is 62.5 ml which includes the semen volume also. So to the 5
ml semen 57.5 ml diluents is added to make the final volume of 62.5 ml.

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For French medium straw

 The French medium straw is having 20 million sperms in 0.5 ml.

 Let us consider the
  The total diluted volume is 125 ml which includes the semen volume also. So to the 5
ml semen 120 ml diluents is added to make the final volume of 125 ml.

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Dilution of semen

 Immediately after semen collection the volume, concentration and initial motility are
estimated.
 A minimum of 2.5 ml volume, 500 million concentration and 70% initial motility is
required to process a semen sample.
 The semen is diluted initially with 1:1 dilution for assessing the initial motility.
 Then the dilution rate is calculated as per the above method (depends upon the
packing volume) and the final dilution is made in a sterile beaker.
 While diluting the semen the diluents should not be poured directly over the semen.
 It should be poured in the side walls of the beaker without air bubble formation.
 DILUENTS FOR LIQUID SEMEN

Sodium citrate egg yolk diluent

Composition

 Tri-Sodium citrate dihydrate : 9.375g

 Glycine : 12.5g
 Glucose anhydrous : 18.75g
 Distilled water : 1 litre
 Penicillin G sodium : 1 lakh I.U. / vial
 Dihydrostreptomycin : 1g / vial
 Add 20 ml of egg yolk with 80 ml of buffer to prepare the diluent.

Tris buffer

Composition

 Tris (hydroxy methyl aminomethane) – 0.3M: 30.4812g

 Citric acid monohydrate : 17.0g
 Fructose : 12.5g
 Distilled water : up to 850 ml
 Penicillin G sodium or ] : 10 lakhs / vial
 Benzyl Penicillin ]
 Dihydrostreptomycin : 1g / vial
 The pH should be adjusted to 6.75

Reconstituted skim milk diluent

 For dilution of semen either whole milk or skim milk or reconstituted skim milk can
be used. These were heated to 92 ° C to 95 ° C for 8 to 10 minutes in a water bath.
 Too much heating is harmful because of its effect on the lactose. Ultra-heat-treated
long-life milk could also be used wherein sterilisation and heating is not necessary.
 A small volume of egg yolk is added to milk, which improves the sperm survival, and
inactivates the spermicide in milk.

Composition

 Powdered skim milk : 9 – 10 g

 Distilled water : 100 ml
 Egg yolk : 5 – 10 ml
 Glucose : 0.9g
 DILUENTS FOR FROZEN SEMEN

Sodium citrate egg yolk diluent

Composition

 Diluent for frozen semen : 420 ml

 Glycerol : 80 ml
 Glycerol is warmed to about 40 ° C before addition and mix gently for 20 minutes.

Tris buffer

Composition

 Tris (hydroxy methyl amino methane)- 0.3M : 30.4812g

 Citric acid monohydrate : 17.0g
 Fructose : 12.5g
 Glass distilled water : 850 ml
 Benzyl Penicillin : 1 lakh I.U. / vial
 Dihydrostreptomycin : 1g / vial
 From the basic diluent take : 336 ml
 Glycerol : 64 ml
 Egg yolk : 100 ml
 This should be mixed gently for 20 minutes.

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Reconstituted skim milk diluent

Composition

 No difference in fertility was found between semen from the same source extended
with heated skim milk to which 5 – 10 per cent glycerol can be added.
 It is better maintained for several days at 5 ° C with milk glycerol extender.
 The semen can be added directly to glycerolized milk yolk extender.

Note: Even though so much diluents are present for buffalo semen, the sodium citrate egg
yolk diluent is a better one.

INTRODUCTION

Methods of semen preservation

There are several extenders are available to preserve cattle and buffalo bull spermatozoa at
different temperatures for certain of period time.

Based on the temperature of storage, there are three different methods are
available to store the semen namely

 Preservation at room temperature (18 – 25oC)

 Preservation at refrigerator temperature (4 – 6oC)
 Preservation at ultra low temperature (- 79oC to – 196oC)

PRESERVATION AT ROOM TEMPERATURE

 This is a very old method of semen preservation.

In areas where refrigeration facility is poor, semen may be stored

at ambient temperature.
 The keeping quality of semen is poor at room temperature due to the faster utilisation
of nutrients by the spermatozoa and also due to bacterial multiplication.
 So obviously it is mandate to add sufficient antibiotic to control the multiplication of
bacteria.
 Various workers have evolved different extenders for the preservation of semen at
room temperature (18 – 25oC). Some of them are as follows.

ILLINI VARIABLE TEMPERATURE (IVT) DILUENT

IVT diluent can be used to store the semen for 3-6 days at room
temperature.
 Van Demark and Sharma has evolved this diluent during 1957 at the University of
Illinois, USA.
 They could preserve semen at room temperature with the help of carbon dioxide gas.
 Co2 temporarily immobilizes spermatozoa and thus reduces their metabolism.
 The composition of IVT dilutor

S.No. Composition Quantity

1 Sodium citrate dehydrate 2.000 gm
2 Sodium bicarbonate 0.210 gm
3 Glucose 0.300 gm
 4 Potassium chloride 0.040 gm
5 Sulphanilamide 0.300 gm
6 Distilled water To make the volume up to 100 ml

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 The above contents are dissolved in boiling double/triple glass distilled water.
 It is cooled to room temperature and is saturated with carbon dioxide by bubbling the
gas through it for ten minutes or until the pH reaches to 6.3.
 Egg yolk is mixed at 10 per cent level and then antibiotics are added. Then the semen
is ampouled and sealed.
 Before placing the ampoules must be flushed with with Co2.
 Semen ampoules are maintained best at room temperature of 20 to 25oC and can be
stored upto 6 days with 76 per cent fertility on non return basis. Ulaganathan (1970)
reported that ascorbic acid supplementation (20 mg/100 ml) to the IVT diluent
significantly improved the keeping quality of buffalo sperm stored at 21-31oC.
 Use of CO2 for semen preservation is more useful at places where semen is collected
daily, so that semen may be dispatched without refrigeration.
 For longer storage of semen this method does not appear satisfactory.

CORNELL UNIVERSITY EXTENDER (CUE)

 Foote et al. (1958) at Cornell University developed the following dilutor for the
preservation of semen at room temperature (20-24oC).

S.No. Components Quantity

1 Sodium bicarbonate 0.210 per cent
2 Sodium citrate 1.450 per cent
3 Potassium chloride 0.040 per cent
4 Glucose 0.300 per cent
5 Glycine 0.909 per cent

 To this citric acid 0.12% and 2 mg of catalase per 100 ml of dilutor was added.
Antibiotics are also added to control the bacteria.
 Reaction of citric acid and bicarbonate releases CO2 which helps in prolonging sperm
life.
 Small quantity of CO2 is also produced by spermatozoa from the breakdown of glycine
via glyoxylate mechanism, and glyoxylate itself is an effective inhibitor of respiration.
  Thus, in medium to which glycine and citric acid are added in addition to bicarbonate,
there is no need to infuse extra CO2.
 The gas generated in the medium is sufficient to curtail respiration of spermatozoa
and immobilize them temporarily.
 Addition of catalase at room temperature preservation was necessary to act on
hydrogen peroxide produced by spermatozoa from amino acids.
 Semen can be preserved for 3-6 days with 70-80% fertility on non return basis.

MILOVANOV'S METHOD/CARBONATE-PHOSPHATE METHOD

 It is also called as “carbonate phosphate method”.

 It was developed by Milovanov (USSR) during 1959.
 Composition of this media is as follows

Part A

S.No. Components Quantity

1 Potassium dihydrogen phosphate 72 mg
2 Distilled water 10 ml

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Part B

S.No. Components Quantity

1 Sodium citrate 2.025 gm
2 Glucose 0.570 gm
3 Sodium bicarbonate 0.125 gm
4 Penicillin G sodium 60,000 mg
5 Dihydro streptomycin sulphate 0.100 gm
6 Sulphanilamide 0.300 gm
7 Distilled water 90,000 ml

Mix Part A and Part B in the ratio of 1:9.

Part C
  Add 11 ml of egg yolk to the above solution (AB).
 The antibiotics are added as per the schedule.
 One ml of the diluted semen are sealed off in glass ampoules and stored at 20-25oC.

COCONUT MILK EXTENDER (CME)

 Charl's Norman et al. (1958) identified that the cocont milk can be used for semen
preservation.
 This media was later improved by adding catalase, polymixine and mycoststin.
 The composition is as follows.

Part A

S.No. Components Quantity

1 Sodium citrate dehydrate 2.2 gm
2 Penicillin G Sodium 60 mgm
3 Dihydrostreptomycin sulphate 135 mgm
4 Sulphanilamide 300 mgm
5 Polymixine B Sulphate 10 mgm
6 Aqueous solution of catalase 15,000 units
7 Mycoststin (dissolve 10 mgm in 50 ml of distilled water) 1,000 unit
8 Glass distilled water 70 ml

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Part B

 Coconut milk (water) - 15 ml

 Boil the coconut milk for 10 minutes, filter and add to Part A

Part C

 Egg yolk - 7 ml
 Add this egg yolk to AB. Bring the volume to 100 ml using distilled water. Adjust the
pH to 7.4
 The vials are filled without air space.
o Wrap it with aluminium foil.
 o Store at dark place
o During tansport use insulated bag to avoid advese effects
 Norman (1968) used this dilutor for buffalo semen and could preserve buffalo semen
for seven days at room temperature.

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 Coconut milk has the following chemical composition

S.No. Components Quantity (per litre)

1 Calcium 9.0 mg
2 Potassium 53.7 mg
3 Magnesium 19.0 mg
4 Chloride 57.6 mg
5 Sodium 4.2 mg
6 Phosphorus 2.4 mg
7 Protein 0.156 mg
8 Glucose 1.8 mg
9 Fructose 3.8 mg
10 Specific gravity 1020
11 pH 4.9

 This extender is not commercially successful due to the nonavailability of the good
quality coconut through out the year.

CAPROGEN
 It was developed in New Zealand. The initial development of this extender for 5 ⁰ C
storage was modified by adding 2-5 per cent egg yolk and catalase to decompose the
H2O2 that is formed as a product of sperm metabolism.
 The optimum temperature range for caprogen is considered to be 18-24 ⁰ C.

MG32 EXTENDER
 The composition of the diluent is given below
 S.No. Components Quantity
1 Sodium bicarbonate 200 mg
2 Sodium chloride 680 mg
3 Sodium dihydrogen phosphate 150 mg
4 Potassium Chloride 40 mg
5 Calcium chloride 20 mg
6 Magnesium chloride 20 mg
7 Distilled water 100 ml

 It was developed by Ramamurthy during 1973.

 Used successfully in buffalo semen.

STORING SEMEN AT REFRIGERATION (4 - 60C) TEMPERATURE

 Here the semen is preserved under refrigeration temperature.

Until the invention of frozen semen technology this

was the most common method used to preserve the
semen.

 Various semen diluents have been evolved by different workers.

 The semen is preserved in these diluents at 4-6 ⁰ C which can be used for
insemination upto 72 hours. Some of the diluents are described.

EGG YOLK PHOSPHATE EXTENDER (EYP)

 Philips and Lardy (1940) develope this dilutor.

Composition

S.No. Components Quantity

1 Disodium hydrogen phosphate 2.0 gm
2 Potassium dihydrogen phosphate 0.2 gm
3 Distilled water 100 ml
 Equal parts of this buffer solution and egg yolk are mixed together to constitute the
dilutor.
 This dilutor preserves bull semen for 72 to 96 hours and buffalo semen for an average
of 48-72 hours.
 In this dilutor, visibility of sperm motility was not very clear when examined under
microscope.
 In field tests this extender provided good fertility.

EGG YOLK CITRATE EXTENDER (EYC)

 Salisbury et al. (1941) evolved egg yolk citrate dilutor which has become popular for
diluting and preservation of bull and buffalo bull semen.
 The citrate available in this diluents is chealating agent and it disperses the fat
globules of the egg yolk and improves visibilty when examined under microscope.

S.No. Components Quantity

1 Sodium citrate dihydrate 2.9 gm
2 Distilled water 100 ml

 Equal parts of this buffer and egg yolk are mixed.

 Swanson (1949) observed good results by mixing 3 parts of this buffer and one part of
egg yolk.
 Subsequent works identified that 20 per cent egg yolk is satisfactory.
 EYC dilutor present very clear film of semen under microscope.
 This dilutor is widely used for the preservation of bull and buffalo bull semen upto 72
hours.
 This is the basic extender from which various new extenders developed.

CAPROGEN

 The citrate-buffered yolk diluents was improved by gassing with nitrogen and adding
caproic acid.
 This led to an extender called “caprogen”. The composition is as follows

S.No. Components Quantity

1 Sodium citrate dihydrate 2.0 per cent
2 Glucose 0.3 per cent
3 Glycine 1.0 per cent
 4 Glycerol 1.25 per cent
5 Caproic acid 0.0325 per cent
6 Sulphacetamide 0.01 per cent
7 Egg yolk 20 per cent

 This was used routinely in New Zealand for extending sperm stored at 5o.
 Later this was modified for ambient temperature use by reducing the egg yolk
content.

KAMPSCHMIDT OR EGG YOLK GLUCOSE BICARBONATE

EXTENDER (EYGB)

 It was developed by Kampschmidt et al. (1951).

 These workers identified that the reduction of electrolyte contents in the medium and
its replacement with metabolisable sugar will increase the viability of spermatozoa at
5oC.
 The workers evolved a dilutor with the following composition

S.No. Components Solution Quantity

1 Dextrose 5% solution 4 part
2 Sodium bicarbonate 1.3 % solution 1 part
3 Sulphamethazine 2 % solution 4 parts
4 Egg yolk 1 part
5 Penicillin G sodium 1 lac unit
6 Dihydrostreptomycin 100 mg
7 Distilled water make up to 100 ml

 This medium has been reported superior to egg yolk citrate for buffalo semen.

D2 DILUTOR

 Singh and Tomar (1959) modified the above dilutor and named it as D2 dilutor. The
composition is as follows.

S.No. Components Solution Quantity

 1 Sodium bicarbonate 1.3 % solution 10 parts
2 Glucose anhydrous 5 % solution 40 parts
3 Fructose 5 % solution 25 parts
4 Egg yolk 25 parts

 Tomar and Desai (1961) claimed it to be efficient medium for buffalo semen. Tomar et
al.
 (1968) used the above D2 medium in routine practice for buffalo and Zebu semen.
 Buffalo semen was maintained fit for use for 4 days while Zebu semen was preserved
for 5-6 days.
 Fertility ranged around 45% in Zebu and 40% in buffaloes.

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D5 DILUTOR
 Another bicarbonate dilutor named as D5 was reported by Tomar and Desai (1961) for
buffalo semen.
 The medium was observed to be better than D5. It consists of the following ingredients

S.No. Components Quantity

1 Potassium bicarbonate 0.25 gm
2 Sodium citrate dihydrate 11.222 gm
3 Glucose anhydrous 18.750 gm
4 Fructose 5.125 gm

 Distilled water added to make upto one litre.

 80 ml of the buffer solution is mixed with 20 ml of egg yolk

GLYCINE DILUTOR

 Knoop and Krano (1944) evolved a dilutor for bull semen with the following
composition

S.No. Components Quantity

1 Glycine 1.09%
 2 Sodium phosphate 0.20%
3 Potassium phosphate 0.08%

 Equal volumes of buffer and egg yolk are mixed.

 Roy and Bishop (1954) used equal parts of egg yolk and 4% glycine solution. This
dilutor is inferior for buffalo semen preservation.
 This dilutor is not used even for the bull semen for the reason that it affects the
morphology of spermatozoa particularly the acrosome is damaged and the fertility is
poor (Strom, 1956; Mukherjee and Dott, 1960).

MILK DILUTOR
 Thacker and Almquist (1951) have shown that boiled homogenized milk and boiled
pasteurized skim milk are excellent dilutors for bull semen and presented the first of a
series of papers on their successful use of heated milk as a semen diluents.
 Boiled, filtered milk gave highly satisfactory results at extension ratios upto 1:25.
 Thacker and Almquist (1953) showed that spermicidal action of unheated milk is
associated with albumin containing fraction of milk.
 Flipse et al. (1954) indicated that “Lactenin” an antistreptococcal agent is normally
present in milk in its albumin fraction which is toxic to sperm.
 This can be inactivated by heating to 92⁰C for 10 minutes only. Use of whole or skim
milk in semen extenders also protects sperm against cold shock.

The milk protein, casein, has been established as the agent

responsible for prevention of cold shock.

 Salisbury (1957) has reviewed the use of liquid whole milk, skim milk and powdered
milk as bull semen extenders.
 Milk combined with sugar, glycine, glycerol and egg yolk were reviewed.

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 Milk dilutor is prepared as follows:

o Obtain fresh, homogenized, pasteurized cow’s milk or fresh pasteurized skim
milk from the reliable dairy.
o Using an accurate thermometer, heat the milk to 92 to 95⁰C and hold at this
temperature exactly for 10 minutes in the top portion of a covered glass double
boiler or in a covered vessel placed directly on a hot plate.
o Cool the milk to room temperature and remove the fat layer; or milk may be
drawn with pipette from its deeper layer by piercing through fat layer.
 Add the following to the above milk
 Suphanilamide - 0.3 gm/ml
 Penicillin G sodium - 1000 units/ml
 Dihydrostreptomycin sulphate - 1000 µg/ml
 The protective fraction of the milk is composed of phospholipids. The toxic fraction of
milk (lactenin) is removed with the removal of albumin from milk.
 Michajilov apparently considered that heating process released glucose from the
disaccharide, lactose, in the milk, thus providing available carbohydrate substrate for
the cells.
 The release of sulpha-hydral (SH) groups from milk by heating process produces an
intensely reduced condition, which protect the SH containing enzymes of the
spermatozoa and by limiting the available oxygen in storage tubes, forces the cells
into anaerobic metabolic activity, which is one important form metabolic control.
 Egg yolk which contains sulpha-hydryl group containing, compounds improves the
quality of heated milk as a semen extender.
 A level of 10 to 20% of egg yolk greatly improves the keeping quality of semen. Joshi
et al (1967) used heated cow milk for preservation of buffalo semen upto 4 days.
 The use of skim milk dilutors for preserving buffalo semen was reported by Tomar
and Desai (1961) and the significant improvement in the efficiency of skim milk
medium was observed when egg yolk and glycine or fructose were incorporated in it.
 Kale (1963) also reported successful preservation of buffalo semen by incorporating
egg yolk and glucose in boiled milk.
 In skim milk dilutor visibility of sperm was better in comparison to whole milk.
 Jesichen (1965) reported the use of powedered milk extender (Laiciphos) with 10%
egg yolk and observed good fertility.
 “Spermasol” is another commercial preparation which has been used successfully.

CITRIC ACID WHEY

 Ganguli et al (1973) and Bhosrekar and Ganguli (1974) developed a skim milk based
diluents known as citric acid whey [CAW] at NDRI, Karnal and claimed to be superior
to some conventional extenders.
 However, reports from elsewhere have not confirmed such claim of superiority for the
CAW extender (Fluckiger et al., 1976, Singh 1977, Sall et al., 1980 and Sidhu and
Guraya 1979).

TRIS-YOLK EXTENDER
 Davis et al (1963) and Good et al (1966) studied certain organic compounds and
found excellent buffering capacity when used as semen extenders.
 Tris (hydroxymethyl amino methane (C4H11NO3) is an organic compound which in
combination with glucose or fructose proved an ideal semen dilutor.
 Yassen (1970) reported better preservation of buffalo semen with tris extender.
 Recently, Tris dilutor is being extensively used for bull and buffalo bull semen
preservation by almost all the advanced countries.
  Tris dilutor may be prepared as follows.

S.No. Contents Quantity

1 Tris 3.028 gm
2 Citric acid 1.675 gm
3 Fructose 1.250 gm
4 Glycerol 7 ml
5 Penicillin G sodium 1 lac unit
6 Dihydrostreptomycin 10 mg
7 Distilled water (make upto) 80 ml

 20% egg yolk is added and mixed properly.

 This dilutor was found to be superior for both Zebu and buffalo bull semen.
 The preservability and the fertility were found to be better those other dilutors.

 Tris-egg yolk diluent can best be used for cattle abd buffalo bull semen.
 It contains 7% glycerol and 20% egg yolk.

STORING SEMEN AT ULTRA LOW TEMPERATURE

 The advances in semen preservation techniques leads to the storing at ultra low
temperature.
 The commonly used dilutor for the preservation of semen at subzero temperature are
as follows

TRIS-EGG YOLK-GLYCEROL DILUENT/UNIVERSAL DILUENT

Composition

S.No. Components Quantity

1 Tris 3.028 gm
2 Citric acid 1.675 gm
3 Fructose 1.250 gm
4 Glycerol 7 ml
 5 Penicillin G sodium 1 lac unit
6 Dihydrostreptomycin 100 mg
7 Distilled water Make upto 80 ml
8 Egg yolk 20 ml

 Inclusion of glycerol at room temperature was found as satisfactory as 5⁰C.

 Tris dilutor allows filling and sealing of poly vinyl straws at room temperature while
freezing the semen.

SODIUM CITRATE EXTENDER

 Egg yolk citrate is prepared as per the procedure given in refrigerated storage of
semen (except sulphanilamide- because sulphanilamide crystallizes during freezing
and hence it is not used in dilutors used for deep freezing).
 Sodium citrate dihydrate - 2.9 gm
 Penicillin G sodium - 1 lac unit
 Dihydrostreptomycin sulphate - 100 mg
 Distilled water - make upto 100 ml
 To the above solution 20% egg yolk and 10% glycerol.

MILK EXTENDER

 The preparation is explained as milk diluents for refrigerated storage.

 The sulphanilamide is not added but the glyceol is added at the level of 10%.

LACTOSE EGG YOLK EXTENDER

 The sugars like glucose, lactose, raffinose and sucrose have been used with semen for
freezing bull spermatozoa.
 The results indicated that sperm survival rate increased as the molecular weight of the
sugar is increased.

PACKING SYSTEMS OF FROZEN SEMEN

 Ampoule method
 Pellet method
 Straw technique
 Tupol method
 o Other packing techniques such as pipettes, bulk, sausages, gelatin capsules, etc.,
are not economically feasible.

AMPOULE METHOD

Method of freezing

 This method was developed in USA by Macpherson (1954), Vandemark and Kinney
(1954).
 Immediately after equilibration the semen of o.5-1.0 ml is placed in previously
marked glass ampoules
 These ampoules were sealed over flame leaving 0.5 ml air space
 The ampoules are placed in ethyl alcohol or acetone bath at 5⁰C
 Small quantity of solid carbondioxide the temperature was brought from 5⁰C to -15⁰C
(at the rate of 1-2⁰C/min).
 Now more solid carbondioxide is added to bring the temperature to -79⁰C (at the rate
of 4-5⁰C/min).
 Six to eight ampoules are arranged in clips or metal cane and each cane is marked
with identification of individual bull.
 Then the ampoules are stored in solid carbondioxide at -79⁰C or in liquid nitrogen at-
196⁰C.

Semen is placed in glass ampoules, sealed, frozen and preserved in

liquid Nitrogen.

 For insemination, the ampoule is thawed in warm water, ampoule is cut, semen is
drawn into glass catheter and is used as liquid semen.

Merits

 Since the semen is sealed inside ampoule, contamination during storage is avoided.
 Identification of sample is possible as the bull number etc. can be marked on the
ampoule.

Demerits

 As the semen is frozen in a large volume, the freezability and fertility are less.
 It occupies more space in frozen semen containers.
 While thawing and insemination, about 8 – 10 per cent semen is lost.
 Use of glass catheters for A.I. has several drawbacks.

PELLET METHOD
Pellet method was developed by Nagase and Niwa (1963) in
 Japan.
 Here the semen is rapidly frozen as small pellets on solid carbondioxide following
dilution with media containing carbohydrate and glycerol.

Freezing method

The volume of semen pellet is 0.1-0.2 ml. The freezing is done with
the help of solid carbondioxide.
 The media used for pellet method is as follow

S.No. Components Quantity(ml)

1 Lactose 75.30
2 Glycerol 4.7
3 Egg yolk 20

 Antibiotics as per the schedule

 The semen is diluted with the above diluents in a glass tube.
 It is placed in 250 ml beaker having water at 30⁰C.
 The beaker along with the semen is placed in a refrigerator/cold handling cabinet at
5⁰C for 5 hours.
 0.1-0.2 ml of semen is taken in a calibrated pipette and is dropped on solid
carbondioxide.
 To facilitate this, the solid carbondioxide was smoothened to a flat surface with an
electric sander and small depressions were made by the use of metal die.
 The depressions were approximately 2mm in diameter and 1 mm in deep.
 The semen is allowed for 10 minutes for freezing on the solid carbondioxide.
 Then they are collected by using pre cooled forceps in to a goblet and stored in liquid
nitrogen.

Thawing

 The pellet is thawed by placing it in 1 ml 3% sodium citrate solution containing 1.5%

fructose at 20⁰C.
 The dilute semen is taken in glass catheter and inseminated.

Advantages

 Economical method
 Requires less storage space
Disadvantage

 As the pellets are stored directly identification is difficult

 As the pellet is placed directly in to liquid nitrogen the chance of contamination with
organisms in liquid nitrogen is more
 When the pellets are taken with forceps for storing and during thawing they may
break and gets attached to forceps or may fall anywhere. This leads to loss of sperms.
 The freezability of sperms by this method is moderate.
 The thawing is a tedious process when compared to other methods.
 The insemination is done with glass catheters which is having disadvantages

STRAW TECHNIQUE
 The straw method was introduced by scientist of Denmark during 1940 for packing of
liquid semen.
 Adler (1960) first frozen the semen packed in straws by using liquid nitrogen vapour.
 This technique was later modified by Cassou in 1965 which was called as “medium
straws”.
 This was in use for a period of time. Later Cassou in 1968 brought further
improvement by reducing diameter for better freezing which is called as “mini
straws”.
 Most of the straws recently used were called as “French straws” - made of poly vinyl
chloride.

The semen straws are made of poly vinyl

chloride.

 Later in German, Simmet (1972) introduced a new straw called “mini tube” or
“German straws” or “Landshut system”.
 Both the ends of this straw was sealed with either steel, glass or plastic beads.
 For insemination, special sheath is required by which the ball and minitube can be
retained in the sheath allowing only the semen to enter the uterus.
 Another straw known as “Continental straws” made of polypropylene was developed
in United States.

The dimensions of French and German straws are as follows

S.No. Type of straw Length Diameter Volume
1 French medium 135 mm 2.8 mm 0.5 ml
2 French mini 135 mm 2.0 mm 0.25 ml
3 German straw 65 mm 2.8 mm 0.25 ml
  The French straws have two ends – factory seal end and laboratory seal end. In this
the factory seal end was formed in the manufacturers themselves.
 The laboratory seal end will be open when it is purchased. The laboratory seal has to
be created in the semen processing laboratory after filling the straw with diluted
semen before freezing.
 For creating laboratory seal earlier the poly vinyl alcohol was used. Recently by using
filling and sealing machine, after filling by pressing the straw mechanically the
laboratory seal is created.

 Factory seal has two cotton plugs with PVA powder in-
between them. This seal is formed by while manufacuring
semen straw at factory.
 Laboratory seal - formed with PVA powder or sealing
machine after filling of semen. This seal is formed at semen
processing laboratoty.

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Advantages of straw method

 Semen is processed as thin film with its greater surface area to volume ratio. Here
there is rapid exchange and better revival rate.
 Reduced volume of the semen is better tolerated by uterus
 The diameter of the artificial insemination gun is small so it is easy to pass it through
cervix when it is in heat.
 Complete delivery of semen into uterus is ensured.
 Identification is easy by using different color straws, different color poly vinyl alcohol
powder for different breeds
 Details like Bull name/No., Breed, Date of collection/batch No. Name of semen bank,
first or second ejaculate and other details if required can be printed on the straw.
 Use of steel gun is safety when compared to glass catheter.
 The disposable plastic sheath used to cover the AI gun can prevent the disease
transmission.
 Straws occupies less storage space when compared to ampoules
 Since the straws is handled with automatic machines the hygiene is ensured
 TUPOL METHOD
Developed by Roy (1974). It is made of
polythene.

 Roy (1974) used polythene tubes named “Tupol” for freezing semen at -196⁰C.
 One end of the tube is immersed in the diluted semen and the other end is attached
with the syringe and the semen is sucked into the tube.
 Then 3-4 inches pieces are cut with electrically heated copper with which seals the cut
ends.
 Insemination is done by cutting one end of the tube and taking the semen in the
syringe or glass catheter as per the liquid semen insemination.
 There is no measure to keep the length of the tube uniform and the sealing is not very
satisfactory.
 More over sealing is done by means of heat, so certain numbers of sperms are
damaged at both ends.
 Therefore volume of semen and number of sperm per dose of insemination is
affected.
 However this method is used in places where other facilities are not available.

METHODOLOGY OF DEEP FREEZING OF SEMEN

Introducion

 The introduction of freezing of semen led to the better possibility of international

exchange of semen.
 It is possible to use any kind of superior bull semen at any point of time.
 The technique of freezing semen helped in the great development of artificial
insemination industry.

The advantages of frozen semen are

 It permits maximum utilization of semen collected from a sire.

 Long term preservation of semen for any time period is possible.
 Semen can be used even after death of the bull.
  Transport of semen is easy even to international level.
 Transport cost is frozen semen is less when compared to liquid semen.
 Selective breeding is possible as per the wish of the owner.
 It eliminates the maintenance of breeding bull in a herd thereby economic benefit to
the owner is ensured.
 Number of bulls required for breeding purpose can be minimized.
 Aids in progeny testing of bulls.
 Round the clock facility is possible even at villages.
 Door step insemination even at remote places is possible.

PREPARATION OF DILUENT
 It is better to prepare the buffer in the previous day evening and diluent in the
morning or one hour before semen collection.

For deep freezing of semen the Tris-egg yolk-glycerol diluent is

used.
 The preparation of the diluent is carried out as per the standard procedure.

The buffer is prepared on previous day evening and autoclaved. It

is kept outside at room temperature under laminar air flow to
cool. On the day of semen collection the diluent is prepared by
adding the egg yolk and antibiotic to the autoclved buffer.

The diluent preparation involves two steps

 Preparation of buffer
 Preparation of diluents

Points to be considered before preparation of diluent

 The chemicals should be purchased from reliable source.

The chemicals of only highest purity (GR or AR grade) should be

purchased to prepare buffer and diluent.

 The compounds used should be chemically pure and weighing should be accurate.
  The triple glass distilled water or Millipore water should be used for dissolving the
salts.
 Completely dissolve one salt before adding another salt.
 The buffer solution should be sterilized by autoclaving before use.

Preparation of buffer

Materials required

 Triple distillation water/Millipore water

 Chemicals (Tris, Citric acid monohydrate, Fructose)
 Glycerol
 Weigh the chemicals as per the requirement given above for preparation of 1 liter
diluent

Buffer preparation (for 1 litre diluent)

o Take the triple distilled water in a sterile beaker according to the requirement
o Weigh the chemicals accurately
o Add the chemicals one by one and dissolve it by magnetic stirrer by required
speed
o Finally add the glycerol at 7% level ie. 70 ml
o Make the volume up to 800 ml by adding distilled water
o Completely mix the glycerol by magnetic stirrer for at least 20 minutes
o Care should be taken while stirring that air bubbles should not be formed by
over speed of the stirrer
o The pH of buffer should be 6.8-6.9.

o Glycerol is added @ 7%.

o pH of buffer - 6.8-6.9.

Sterilization of buffer

o The prepared buffer is sterilized by autoclaving at 100⁰C for 15 min at 5 psi.

o Cool the buffer to room temperature
o Generally the buffer is prepared on previous day evening, sterilized and kept
under laminar air flow over night.
o The diluent is prepared by adding egg yolk in the morning immediately before
semen collection.

Preparation of diluent

o The egg yolk is prepared as per the standard procedure

 o For 1 liter of diluent, 200 ml of egg yolk is required
o The egg yolk is added and mixed by using magnetic stirrer
o The antibiotics are also added while stirring with egg yolk
o Care should be taken while stirring that air bubbles should not be formed by
over speed of the stirrer
o The pH of the diluents should be 6.7-6.8.

o Egg yolk is added @ 20%.

o pH of buffer - 6.7-6.8.

COLLECTION OF SEMEN
 The semen collection is done in morning hours.
 The bulls will be active and the stomach will be empty in the morning hours which
help in better semen collection.
 The scheduled bulls will be carried from their paddocks to washing area.
 The animals should be washed to remove all the dirt.
 They should be tied surrounding the collection shed and should be dried.
 The bulls should be tied with bull apron and the prepuce should be cleaned with
normal saline.
 The dummies are placed in trevis and restrained.
 The bull will be brought behind the dummy and 2-3 false mountings will be allowed.
When it is mounting with complete thrust the semen is collected with artificial vagina.

Note: After collection the animals are given 20 minutes refractory period for
second collection. But this time varies from bull to bull.
EVALUATION OF SEMEN
 Immediately after collection the collection vial is taken out of AV and the rims are
covered with aluminium foil.
 The details of the collection are pasted on the vial (bull No., ejaculate number etc).
 It is placed in a 34 ⁰ C water bath in the passbox (The lab is tightly sealed at the time
of processing to maintain hygiene. So the passbox helps to transport of semen from
collection yard to lab. But the passbox should not be opened at both the sides
simultaneously).
 Inside the lab the semen is evaluated for volume, color, consistency and presence of
foreign matter and then placed in a water bath of 34 ⁰ C.
 This temperature has to be maintained till the final dilution of semen.
 One drop of semen is placed on glass slide and mass activity recorded.
 Samples showing more than +3 are taken for estimating concentration of
spermatozoa.
 The concentration is estimated by using calorimeter or photometer.
 The semen is diluted with diluent in 1:1 ratio.
  The initial motility is assessed by placing a drop of 1:1 diluted semen on a warm slide
and placing a cover slip on it.
 The initial motility is estimated by using phase contrast microscope.
 A sample with 70% and above initial motility is selected for further processing.
 The entire process of above mentioned semen evaluation has to be completed within
5-7 minutes.

The minimum requirement for fresh semen to be processed are as follows

 Volume - 2.5 ml
 Concentration - 500 million and
above
 Initial motility - 70%

DILUTION OF SEMEN

 The French mini/medium straw is packed with 20 million sperms.

 The final dilution is calculated based on volume, concentration and packing volume.
 Till the final dilution rate is decided, 1:1 diluted semen has to be kept in water bath of
34 ⁰ C.
 This time period will also help to adjust and stabilize the semen with the diluents.
 Then it is placed outside the water bath at room temperature (20-22 ⁰ C).
 Here it is placed for 10 minutes to bring the 1:1 diluted semen temperature to 20 ⁰ C.
 The final dilution is done at room temperature of 20-22 ⁰ C.

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Example

Calculation of dilution rate

 For French mini straw

o The French mini straw is having 20 million sperms in 0.25 ml.
o Let us consider the

 The total diluted volume is 62.5 ml which includes the semen volume also.
 So to the 5 ml semen 57.5 ml diluents is added to make the final volume of 62.5 ml.
 The semen is already diluted with 1:1 dilution rate ie to 5 ml of semen 5 ml diluent
was already added.
 So the final volume available is 10 ml. So 52.5 ml of diluent has to be added as per the
dilution rate.
 In a sterile beaker (of 100 ml) add 40 ml of diluent. To this add the 1:1 diluted semen
ie 10 ml.
 The remaining 7.5 ml diluent is added to the collection vial and it is poured into the
beaker.
  When the diluents or semen is poured into the beaker it has to be passed via the side
walls of the beaker.
 It should not be poured directly into the beaker. The air bubble formation should be
avoided while dilution is done.

FILLING AND SEALING OF SEMEN STRAWS

Filling of straws (Manual method)

 The manual method of filling requires vacuum pump, filling comb, rubber tube, straw
clips and polyvinyl alchol powder (PVA).
 The straws (15 medium or 20 mini) are clipped together by using straw clips. The
clipped straws are cooled to +5°C in the cold handling cabinet.
 The straws in the clamps are fitted on the filling comb (factory seal near the nozzle),
which in turn is fixed to vacuum pump through the rubber tube.
 Semen is taken in the bath of the bubbler and filling is done by operating the vacuum
pump and slowly dipping the open end of the straw.
 Due to negative pressure semen is drawn into the straws. Appearance of two distinct
bands in the factory seal indicate complete filling of straws.
 A uniform air Space is created in all the filled straws by pushing the open ends of the
straw on to the teeth of the bubbler.
 The bubbler and the bath are discarded after use. This small amount of air space is
required in order to allow for expansion of the semen during freezing.
 In it’s a absence the sealing will be pushed out by the frozen semen.
 Further this air space also prevents contact of scissors with semen at the time of
cutting of straw before insemination, thus preventing possible contamination.

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Sealing of straws (manual method)

The sealing powder PVA is available in 10

different colours.
 The French straws are available in 16 different colours.
 The combination of both assures 160 positive identifications.
 The sealing powder is spread on a clean glass dish to provide a thickness of 4-5 m.m.
 The open ends of filled straws are dipped into the powder and when the powder
penetrates 4-5 m.m. into the straw a satisfactory seal is made.
 This is known as ‘laboratory seal’ which will appear as a single band.
 Immediately after sealing the sealed end of the straws are placed in water bath at
20°C for 10 minutes for firm sealing.
 Then the straws are taken out and wiped of water and sent for printing of the straws.
  At the same time this allows the sealing to become firmer and the excess powder on
the ends falls to the bottom of water bath.
 The entire operation of filling and sealing has to be done at + 5 ° C within the clod
handling cabinet.

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Filling and sealing by machine

 Automatic filling and sealing machine is also available for filling and sealing of semen.
 Both operations are done within a shorter time and with least handling of semen.
 This will be the ideal way of filling and sealing where large scale freezing is
undertaken daily.
 The machine MRS – 1, MRS-3, of IMV, France have capacity to fill and seal 3600 and
12960 as the sealing is done by compressing the end of the straw.
 Recently MRS 5 has been launched which is faster than above.
 PRINTING (IDENTIFICATION) OF STRAWS

 The filled and sealed straws are sent for the printing.
 The automatic straw printing machine manufactured by IMV, France has got a
capacity to print 11,160 straws per hour.

The details like bull No., breed, semen bank, batch No./date of
collection, ejaculate No. etc can be printed on the straw for easy
identification
 RACKING

 The straws are arranged rapidly on the freezing rack by the use of use of a racking
platform.
 The entire operation from dilution to racking is carried out in room temperature of 20
⁰ C.
 EQUILIBRATION OF SEMEN
This is the pre-freeze storage period of spermatozoa in the diluent
at +5°C.

 This equilibration period is given for the glycerol to bring about its
beneficial action on the spermatozoa.
 The equilibration is done in cold handling cabinet (CHC) box.
 The straws are placed over freezing racks and kept inside the CHC box.

Normally equilibration period of 4 hours for cattle bull semen and 3

hours for buffalo bull semen is sufficient

 At the end of equilibration period the pre-freeze motility is recorded.

 Samples showing more than 60-70 per cent motility are taken, for ‘Test
freezing’.
 Test freezing is done for 2-3 straws for each sample, frozen, in liquid
nitrogen vapour and plunged in liquid nitrogen.
 After few minutes the revival rate is determined and satisfactory samples
are taken up for filling and further processing.

FREEZING OF SEMEN

Freezing the straws in liquid nitrogen vapour (manual freezing)

 Freezing is carried out in a wide mouthed container (LR 320, LR 250) especially
designed for freezing of straws.
 The actual freezing is done by holding the equilibrated straws at 4.5 cm form the level
of liquids nitrogen thereby exposing to the vapours of liquid nitrogen.

When the temperature of semen reaches – 130°C to – 150°C they

are transferred to goblets and submerged in liquid nitrogen for
storage at – 196 ° C.
 The main advantage of horizontal freezing is the efficiency and simplicity of the
method.
 The racks hold the straws at a constant level and the straws are frozen at even rate all
along the length of the straw.
 Ensure that the level of liquids nitrogen is 1-2 mm above the safety freezing net.
  After that the vapour temperature is adjusted by wafting and the racks with straws are
kept on the freezing net so that the straws are about 4-5 c.m. form the level of liquid
nitrogen.
 The recommended temperature for freezing semen is LR 320 unit is
 Medium straws: - 150 ° C to - 160 ° C for 7 minutes.
 Mini straws : - 120 ° C to - 130 ° C for 9 minutes
 These containers are used for freezing as well as storage of semen.
 The maximum storage capacity of LR 250 will be about 70, 000 French medium
straws can be stored in 3 layers.
 With LR 320 one lakh French medium straws stored in 4 layer.
 The height of canisters to be used for LR 250 and LR 320 containers should be 25 cm
and 40 cm respectively.
 Normally semen banks should plan and store a minimum of 4 months of its supply to
the field in advance.

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Freezing in bio freezer

 Where large scale freezing is carried out the freezing is done by using bio freezer.
 Here the freezing programme is already fed in a computer.
 It can be altered whenever required. The bio freezer is connected with liquid nitrogen
tank and the chamber temperature is brought to +5 ⁰ C.
 Transfer of freezing rack from cold handling cabinet to freezing unit should be done
in the shortest possible time to avoid any increase of semen temperature above +5°C.
 To achieve this cold handling cabinet and freezing unit is kept in the same lab.
 The freezing racks are placed inside the bio freezer and it is allowed for 5 minutes to
get stabilized at +5 ⁰ C.
 Then freezing is started. The entire process will take 9 minutes where the temperature
will reach from +5 ⁰ C to -140 ⁰ C.

PRELIMINARY STORAGE AND POST FREEZE EXAMINATION

Preliminary storing

 After primary freezing the straws are plunged into the plastic goblet filled with liquid
nitrogen.
 When the boil – off ceases the goblets is transferred to canister of frozen semen
container for storage at – 196 ° C.

Post-freezing examination

 Post thaw motility of samples is assessed after 24 hours of storage.

  Samples having less than 50 percent progressive motility (under phase contrast
microscope) are discarded.
 Good sample are transferred to permanent storage containers for preservation.

 STORAGE OF SEMEN

The aim of storage of semen is to prolong the fertilizing capacity of

spermatozoa by reducing or arresting their motility and metabolic reactions
SHORT-TERM LIQUID STORAGE OF SEMEN
Fertile spermatozoa can be preserved for a short period of time
without freezing.
 The gametes of many species can be stored in liquid media which is maintained either
at ambient temperature or in a refrigerator at 4-5˚c.
 There are definite advantages to store gametes at ambient temperature, especially
where refrigeration facilities may not be readily available.
 The daily fluctuations encountered in storage temperature can present serious
problems in maintaining fertile spermatozoa.
 Ambient temperature storage requires prevention of microbial contamination and
effective prevention of microbial proliferation without impairment of gametes
viability.
 For successful short term liquid storage of semen it is necessary neither to extend the
time that spermatozoa are able to maintain high metabolic rates or to slow the
process down.
 The first effective ambient temperature storage medium was based on the fact that
carbon dioxide markedly reduced the rate of sperm metabolism.
 This storage, medium which was termed Ilini Variable Temperature IVT) extender,
was saturated with carbon dioxide by bubbling the pure gar through an egg yolk –
citrate extender.
 Other ambient temperature extenders, such as CUE (Cornell University Extenders),
utilize similar metabolic inhibition principles.
 Another ambient temperature storage medium called Caprogen extender was
extensively field tested in New Zealand and adopted for routine use at a storage
temperature of 18-24 C.
 The Caprogen extenders is a modified citrate buffered egg yolk medium containing
Caproic acid, Glucose, Glycine, sulfacetamide, penicillin, and streptomycin and is
saturated with nitrogen gas.
 The fertility of bovine spermatozoa can be maintained for about 3 days using the
common ambient-temperature storage media.

LONG TERM STORAGE (CRYOPRESERVATION TECHNIQUE)

  The obvious limitations of fluid storage of semen, whether it may be at ambient or
refrigeration temperature, have been over-come by cryopreservation.
 The discovery by Polge and colleagues (Polge et al., 1949) of the cryoprotectant
properties of glycerol emphasized the advantages of preserving semen at ultra-low
temperatures.

Spermatozoa of many species can be stored at liquid nitrogen temperatures (-

196˚C) for indefinite periods and, after thawing, retain relatively high rates of
fertility.
STORAGE OF FROZEN SEMEN

 The frozen semen straws should always be kept submerged in liquid nitrogen in liquid
nitrogen container.

For this the level of liquid nitrogen should be maintained a

minimum of 15 cm and above.

 The semen containers should be periodically topped with liquid nitrogen.

 The semen straws should be kept in a plastic goblet which in turn should be kept in
canister.
 The goblet should not be tightly packed with semen straws.
 There should be space for liquid nitrogen to go inside to maintain the temperature at
lower level.
 Always keep identification slip or colored plastic sticks in the goblet.
 The slip or stick should contain the information about the semen straws for easy and
quick identification.
 Don’t keep different breeds’ semen straws in a goblet. The semen straws of same
breed but from different bulls should be stored with proper partition.
 The goblets used must be slightly shorter than straws to enable quick removal of
straws. The commonly used goblets are 12 cm in height.
 A 35 mm diameter goblet holds 85 medium straws and 65 mm goblet holds 300
medium straws.
 The frozen semen should never be touched with hands. The straws should always be
removed with pre-cooled stainless steel forceps.
 Frozen semen is exposed to elevated temperature when the semen is transferred or
taken from a storage container.
 The increase of temperature is determined by length of time exposed, ambient
temperature, air circulation, solar radiation, level of liquid nitrogen in container and
height to which the canister is raised above the neck.
 The technician must be efficient enough to pick desired straw within 10 seconds.
 This will minimize the fluctuation in temperature of straws while handling.
 If semen is to be transferred from one canister to other, keep both the canisters
submerged in liquid nitrogen kept in a thermocool box and carry out the transfer
quickly.
 During storage and handling of frozen semen, any rise in temperature above -130 ⁰ C
should be avoided.
 The key factor enabling the successful long term storage of frozen semen is low
temperature.

HANDLING DURING AND AFTER THAWING OF FROZEN SEMEN

 The semen straw should be taken with pre-cooled forceps.

 The straw should be given a jerk to remove all the liquid nitrogen attached over the
surface.

Thaw the semen straw at 37 ⁰ C water bath either horizontally or

vertically for 30 seconds.

 Don’t thaw more than 2-3 straws at a time.

 After thawing the semen should be used immediately.
 The straw should be wiped thoroughly to remove all the water.
 The semen straw should be cut at laboratory seal end where the air space is available.
 The AI gun should be loaded correctly after pulling the plunger down.
 The sheath should be applied over the AI gun and the button should be placed on the
sheath.
 The semen should be deposited by pushing the plunger smoothly
 Do's and Don't in handling of liquid nitrogen

SHIPMENT OF SEMEN

 Role of artificial insemination is much more valued when semen of bulls is

transported to distant and wide spread areas.
 With the transport of semen usefulness of bull is exploited, and there is less cost
incurred on the programme of artificial insemination.
 With the convenience of transport, semen collection stations are opened at important
places from where semen is dispatched to all insemination centres.
 Fertility of bull semen transported through bus service and cycle to long distance was
not deteriorated on first day of use. But there may be minor losses in fertility as a
result of transportation of semen.
 However, such disadvantage may be insignificant, while considering the manifold
facilities and conveniences together with economic advantages met with centralized
scheme of semen collection and more use of A1 bulls.

SHIPMENT OF LIQUID SEMEN

  Semen can be transported with all types of transport facilities.
 Best type of transport is that which takes less time to reach the insemination centre
and gives least jerks and jolts during transport.
 A time interval of more than 24 hours fail to maintain requisite temperature in semen
container.

Normally, semen should reach all insemination centres within 12

hours from its collection.

 When semen is sent through rail or bus, proper instructions should be issued not to
expose the container to sun.
 Loose handling and dumping of semen containers in godown should be prohibited.
 The container may better be marked “ Living Biological Product” and “Gentle
Handling Not To Be Exposed To Sun.”
 Transport of semen with cycle is not advisable for more than 10 km during hot
season. Motor cycle is not a good transport for semen.

Usefulness of semen transport depends to a great deal on the

method of packing . If semen is subjected to jolts and variable
temperature, sperm motility is not maintained during transport.

TOP

Some of the precautions observed to minimize transport shocks, are as follows

 Semen vials should be filled up completely and minimum air space should be allowed
in them.
 This method not only safeguards the spermatozoa against frequent shocks, but it also
minimizes the chances of hydrogen peroxide production in semen.
 Semen vials should be maintained in jackets (glass or plastic) which are properly
secured and capped. With such precaution spermatozoa do not get cold shock, and
semen is not polluted with ice or ice water during transport.
 Semen vials when properly corked, should be wrapped one by one in cotton wool
paddings or in foam packings. These packages are then placed in a water-proof
container, so that semen vials do not come in direct contact with water during
transport.
 Sufficient crushed ice should be filled in thermos flask to maintain a temperature
below 10˚C during transport. Thermos flasks of 4 to 8 pints capacity should be used
for transport, as flasks with less capacity do not keep ice longer.
 A point may arise if it is necessary to lower down the temperature of semen to 5˚C to
7˚C
 In the case of semen preserved at room temperature (20˚C to 25˚C), transport is very
convenient. But in India, the environment is not so favourable as in the temperature
zones.
 The room temperature in India and similar tropical countries varies from about 15˚C
in winter moths to 40˚Cin summers. During most of the months. viz, from 35˚C to
45˚C. Consequently, the transport of semen preserved at room temperature cannot be
materialized without some cooling device.
 Ordinary semen shipper may be prepared by fixing thermos flask in a strong light
metal box with perfect insulation.
 Iron or wooden crates may be used to protect thermos flasks and shippers from
damage during rail or road transport. When semen is carried by a messenger, no such
precautions are necessary.

TOP

SHIPMENT OF FROZEN SEMEN

 The two principal refrigerants used in experimental and routine shipping of frozen
semen in recent years dry ice (Co2) and liquid nitrogen.
 Dry ice functions at -110˚ F, (-79˚C.) and liquid nitrogen at -320˚F, (-196˚C), and
hence the latter has a longer safety factor.

DRY ICE

 Ampoules of frozen semen can be shipped in much the same manner as liquid semen
when the refrigerant is dry ice.
 First of all, the container should be one well known for its insulating properties.
Suppliers now have on hand equipment that is both strong and light in weight, made
of such material as expanded styrene.
 One of the good shippers is round in shape, has a capacity of 75 ampoules and a
loaded weight of 20 lb. Its safety period is three days.
 Another model especially designed for international transport has a loaded weight of
70 lb., holds up to 500 ampoules, is 18 by 18 by 24 inches in size, and has a safety
period of six days. Both models are guaranteed to hold the temperature down to -
75˚C, or lower when properly packaged.
 As many as a dozen ampoules of frozen semen have also been shipped in cake of dry
ice about 5 by 5 by 9 inches in size.

LIQUID NITROGEN
 Importing frozen semen with the aid of liquid nitrogen as the refrigerant is being
favored in areas where dry ice is not obtainable, where the nitrogen is available at a
reasonable price, and where the electric current is not dependable.
 The major advantage of frozen semen is its capacity to remain uninfluenced over long
distance transport.
 Another most important advantage is its storage potential extending over several
years and the semen may be available even after the death of the bull.
 For storage and transportation of frozen semen, various sizes of liquid nitrogen
containers are available. The frozen semen can be transported through road, rail or
air.

It is advantageous if it takes less time to reach the place with

minimum jerks during transport.

 This depends upon distance and availability of transport facility.

 While transporting frozen semen the temperature should be maintained -196 ⁰ C.
 The liquid nitrogen holding capacity and evaporation rate depends on the size and
type of container.
 The liquid nitrogen should be topped up during the transport of straws.
 The wooden crate or similar protective device like hard board or card board may be
used during transport of semen to avoid shock to the container.

CARE AND MAINTENANCE OF LN2 CONTAINERS

 The cryogenic containers are double walled vessels with annular space evacuated and
sealed. In addition several types of insulation
o Vacuum alone
o Expanded foam
o Gas filled powder and fibrous materials
o Evacuated powder
o Evacuated superinsulation is used as thermal insulators.
 The outer walls of the containers are made up of stainless steel, carbon steel or
aluminum alloys.
 Extreme care should be exercised while handling the containers.
 The cryogenic equipment is specific and they are meant to store only the particular
liquid for which they are made.
 Welding or piercing the container wall is dangerous.
 Keep the container in upright position.
 Protect the container against shock and rough handling. During transport support the
container with soft padding.
 Protect against direct sunlight or hot blowing winds.
 Charging of warm container should be slow.
 Avoid frequent cooling and warming of the containers. Thermal stress may cause so
much strain within it, that the inner wall of the container may crack.

Appearance of moisture on the outer wall of the container is a sign

 of damaged container.
 Do not dry the container meant for regular use.
 Find out the evaporation rate for each container, so that the periodicity of topping
could be organized.
 When vacuum disappears, the insulating capacity is lost. The manufacturers alone
should do repairing of damaged containers.
 Hence, containers should be handled very gently particularly during transport.

ASSESSMENT OF LIQUID NITROGEN LEVEL

 The evaporation rate of liquid nitrogen varies

o From container to container
o Temperature of the room in which stored
o Number of times the container is opened.
 Hence periodic checking of level of liquid nitrogen is essential. The
minimum level of liquid nitrogen should keep the straws completely
submerged in the liquid nitrogen.
o To measure the level of liquid nitrogen, a dipstick (slender stick made of wood
or metal) should be used. The stick is gently lower into the container and rests it
at the bottom.
o Hollow tube should not be used. After 5-10 seconds take it out and wave in air.
o The atmospheric air condenses as a frost on the dipstick to the level of liquid
nitrogen.
o Read the level one c.m below the end of frost line giving allowance for boiling of
liquid nitrogen when dipstick is inserted.
o By using calibration chart the volume of nitrogen can be estimated. Frequent
measuring leads to unnecessary evaporation of liquid nitrogen.
 Weighment method
o Specific gravity of liquid nitrogen is 0.82. (one litre of LN2 = 0.82kg or one kg of
LN2 = 1.22 litres by volume).
o Based on known weight of empty container and weight after filling liquid
nitrogen, the assessment can be made.

INTRODUCTION - TECHNIQUE OF ARTIFICIAL INSEMINATION

Of all the reproductive organs, the cervix play a key role in AI, as it
is the external entrance to the uterus, which must be located and
penetrated with the inseminating instrument.
 The cervix is normally tightly closed, except during the periods of heat and
parturition. Semen is deposited in the vagina in natural service, but AI requires the
deposition of semen in the uterus in cattle.
  The object of insemination technique is to place good quality semen in the accurate
place which will give best chance of conception.
 There are different types of insemination techniques are available.
 These insemination techniques depend upon the size of the female animals of
different species and difference in their anatomy

Points to be considered before inseminating the animals

 The animal should have appropriate body weight to carry the pregnancy – minimum
of 250 kg in cattle heifers and 300 kg in buffalo heifers.
 Animal should be in estrum.
 The reproductive tract should be free of infection.
 Discharge should be clear.
 Congenital or hereditary anatomical defects of reproductive tract should not be there.
 Animal should not be pregnant. Before performing AI rule out pregnancy.
 If it is post partum animal, there should be gap of 60 days from the time of
parturition.
 If there was abnormal calving in previous parturition, the time gap of 90 days is
preferable.
 The type of semen – breed, bull no should be decided well in advance.
 Insemination while standing posture is recommended.
 Stress should be avoided.

CATTLE
Technique of AI in cattle includes

 Vaginal Insemination
 Cervical Insemination Using Speculum method
 Recto-vaginal method

VAGINAL INSEMINATION
Here a pipette is inserted in to vagina and the semen is deposited in
anterior vagina or near to cervix.

 The major disadvantages of this method are

o The vaginal environment is not conducive for the longer life of the sperm
o Deposition of more number of sperms is necessary to get moderate result

CERVICAL INSEMINATION

Using speculum method

  Older method- done with the help of vaginal speculum after cleaning the external
genitalia of the cow.

With the aid of light, a long insemination pipette containing 0.1-

0.2ml of diluted semen is introduced and deposited into the
external os of the cervix.

Disadvantages

 It is fairly satisfactory but requires frequent cleaning and disinfection of the speculum
or use of a different speculum on each cow.
 When large number of cows are inseminated the sterilization of speculum sometimes
will become problem.
 Chances of injury and infection of vagina and cervix will lower the fertility.
 In some cows it is difficult to introduce the semen into cervical lumen and it is
deposited in external os which will lower the conception rate.

RECTO-VAGINAL METHOD

 It is more effective and widely used.

 This rectovaginal method is performed in animals in standing position.
 The animal should be restrained in a service crate.
 The perineal region of the animal should be washed with running tap water
to remove the dung and dirt. The water should be wiped off.
 The inseminators hand is covered with disposable rubber sleeve.

The lubrication is done with non-irritant soap and the hand is

passed into the rectum.

 The excessive dung inside the rectum is removed. But complete removal is
not advisable because a minimum level of dung is essential for better
operation.
 Frequent removal of hand from the rectum while removing dung also
should be avoided because it will lead to ballooning of the rectum.
 Vulva and vulval lips are carefully wiped or if necessary, washed and then
wiped dry with cotton or a paper towel.
 The cervix is grasped with gloved hand and the external os is located.
 If the external os is hard to locate, the cervix may be hold by the fingers
and the thumb is placed over the external os.
  The vulval lips are widened apart by an attendant and the insemination
gun is inserted through the vulva and vagina and into the external os of the
cervix
 If folds of vaginal wall interfere, the cervix is pulled or pushed forwarded to
straighten the lumen of the vagina.
 By manipulation of the cervix with the hand in the rectum, the
insemination catheter is passed just through the annular rings of the
cervix.
 The semen is deposited through the cervix into the body of the uterus and
the remainder in the internal os of the cervix, while the catheter is
withdrawn.
 Semen is expelled slowly to avoid sperm losses in the catheter.
 A common error is to penetrate the catheter beyond the body of the uterus
into the uterine horns.

Advantages of recto-vaginal technique of insemination

Deposition of semen at proper site is ensured without loss of semen

 Chance of infection are minimized and fertility rate is high compared to speculum
method.
 Injury/bleeding caused while using speculum is avoided in this method
 Estrum is confirmed while examining the animal per rectum
 Inseminating pregnant animal is also avoided because the rectal palpation of genital
organs rules out pregnancy.

CONSTRAINTS OF FROZEN SEMEN

 The frozen semen is inseminated invariably by the rectovaginal method only.

 Because the frozen thawed semen will have only 10 million live sperms and it will
have more abnormalities and less acrosomal integrity when compared to chilled
semen.
 So the maximum chance should be given for the sperms to reach the site of
fertilization by depositing the semen at the internal os or in body of the uterus by
rectovaginal method.
 The conception rate for artificial insemination with frozen semen is low.
 There was a temporary decline in fertility rate when frozen semen was used in the
field.
 The reason was investigated and traced to improper handling or deposition of semen
by the inseminator.
 Thawing and insemination are the two important phases to be
handled by the inseminator with utmost care.
 With chilled semen, deposition of semen either in cervix or uterus results in same
conception.
 While with frozen semen, if semen is deposited in the vagina conception is almost
excluded, if deposited in the cervix the conception rate is lowered and optimum
conception occurs with deposition in the body of uterus.
 The service of skilled inseminator is essential in maintaining optimum fertility in the
field.
 It has been observed that inseminators who had been inseminating for many years
were only 25% accurate in semen deposition.
 Another study is revealed that most inseminators had a tendency for too deep
deposition, most frequently in to the right horn.
 This could have a marked effect on conception in animals ovulating from the left
ovary.

TYPES OF SEMEN STRAWS

The frozen semen are stored in different types of straws. They are

o French medium straw (0.5 ml)

o French mini straw (o.25 ml)
o German mini tube (0.25 ml)

THAWING FRENCH SEMEN STRAW

 Lift the neck plug of the LN2 container

 Precool the forceps in the liquid nitrogen vapour for 5-10 seconds
 Lift the canister from the neck and move it to its opposite edge. Now the body of the
canister will come to centre
 Lift the canister to the level of neck
 Identify and lift the straw by forceps
 The whole operation of lifting of canister and removal of straw should be done as fast
as possible ie. with in 5-10 seconds. Otherwise immerse the canister into the liquid
nitrogen.
 Immediately after removing the semen straw give a jerk to remove the liquid
nitrogen on the straw and also to avoid popping of the seal.
 If the straw is having cracks or broken or defective seals the straw should be
discarded.

Put the semen straw in a water bath of 37 degree celsius for 30

seconds .
 While thawing the semen straw should be completely submerged in water.
 The thawing water should be fresh and clean. The container used to hold the thawing
water should also be clean.
 Thaw the semen straw either in horizontal or vertical manner
 After the thawing, remove the straw and wipe with cotton to remove all the water on
the surface of the straw.

TOP

LOADING OF AI GUN (FRENCH STRAW)

 Use appropriate insemination gun for respective semen straws or use universal gun to
load the straws.

 By gently tapping the straws bring the air space near to laboratory seal
 Place the straw in the chamber of the insemination gun. While placing the factory
seal should go down and the laboratory seal should come up.
 Cut the air space area of the straw by keeping the straw in upright position.
 While cutting it should not be cut too close to the semen or should not be cut at angles
which will lead to back flow of the semen or wastage of the semen at the time of
insemination.
 Fit the sterile sheath over the straw and the chamber of the inseminating gun.
 Obtain perfect fit between extremity of the straw and the cone of the sheath .
 The sheath should be fixed with the button of the inseminating gun.
 In Universal AI gun auto lock is available which will hold the sheath firmly.
 After placing the sheath, do not touch the tip of the sheath. Handle only the base of
the sheath.
 The post thaw survival of spermatozoa is poor.

For maximum reproductive efficiency thawed semen should be used

immediately. So do not thaw more than one straw at one time.
 SITE OF SEMEN DEPOSITION
Frozen thawed semen is deposited in internal os of cervix or body
of uterus. Avoid depositing in horn of the uterus.
 The post thaw survival of spermatozoa is poor.
 For maximum reproductive efficiency thawed semen should be used immediatedly.
 So donot thaw morethan one straw at one time.

Frozen thawed semen is deposited in internal os of cervix or body

of uterus.

INSEMINATING WITH GERMAN MINITUBE STRAW

 For German minitube straws the gun and sheath are different than French gun and
sheath.
 The gun is made of stainless steel rod of 18 inch long and the sheath has special
arrangement at its tip and is called as plug tipped sheath.
 The minitube after cutting at one end is inserted in the sheath (cut end downward)
and then gun is fitted.
 At the time of insemination, the semen is deposited by pressing the gun and the
sealing ball is retained at the tip of the sheath.
 After insemination it is taken out and the sheath along with the empty minitube are
discarded.
 By this method with German gun medium French straw can also be inseminated but
with the French gun minitube cannot be inseminated.

THAWING SEMEN FROM AMPOULES

 The ampoule is taken out from the liquid nitrogen container carefully.

Thaw the ampoules in iced water for 6-7

minutes.

 Wipe the ampoule with absorbent cotton.

 Cut and draw the semen in a sterilized catheter fitted to a plastic/glass syringe with
rubber connector.
 Inseminate the animal using speculum.

TECHNIQUE OF AI IN SHEEP AND GOAT

There are three methods of artificially inseminating small ruminants

 Cervical AI
 Trans cervical AI
 Laparoscopic AI

CERVICAL AI

 After identifying the ewe/doe in heat, the animal is restrained by a second person who
straddles the doe’s neck and elevates the hindquarters to a vertical position while
holding the hind limbs tightly flexed.
 The operator holds the speculum in one hand and pipette or inseminating gun in the
other hand.
 After holding the female in position, the vulva is cleaned.
 Insert lubricated speculum in a slow and gentle manner and locate the cervix.
 Centre the end of the speculum over the os uteri.
 Cervix should be of a red- purple colouration with a whitish mucous present, if the
female is truly in heat.
 Insert pipette or inseminating gun into the speculum to the cervix.
 Gently manipulate the instrument through the cervical canal and deposit semen near
the uterine end of the cervix or just inside the uterus.
 Do not enter too far into the uterus as the semen will then tend to be dumped into one
born or the other.
 Deposit semen slowly, tanking at least five seconds.
 Withdraw the instrument slowly and then carefully remove the speculum.
 TRANS CERVICAL AI
 Non- surgical technique
 It can be done with the female standing or in a breeding cradle.
 The first step is to insert a lubricated speculum into the vagina.
 A light source is fixed to the speculum so that the operator can visualize the opening
of the cervix,
 Surgical forceps are used to tie off the tissue at the opening of the cervix.
 The endoscope is inserted into the opening of the cervix.
 The bent tip of the instrument helps to maneuver through the first ring.
 The operator looks through the eyepieces to navigate the scope through the rings of
the cervix.
 Once the final cervical ring is penetrated, semen is deposited in the body of uterus.

Higher doses of sperm (atleast 100 million sperm cells) are required
for trans-cervical AI.

LAPAROSCOPIC AI

 It bypasses the cervix and deposits semen directly into the uterine horns.
 It is minimally invasive and a minor surgical procedure.
 The ewe/doe’s abdomen is sheared and scrubbed and a local anesthetic is injected
under the skin.
 Two small incisions are made with a surgical blade,
 Trocars and trocar sleeves are inserted through the incisions and pushed through the
body wall into the peritoneum.
 The trocars are replaced with a laparoscope and manipulating probe.
 The operator looks through the laparoscope to locate the female’s reproductive tract.
 The body cavity is inflated with CO2 to allow the uterus to be observed.
 Once the tract is manipulated, the probe is replaced with an insemination pipette and
semen is injected into the lumen of each uterine horn.
 After insemination, the equipment is removed and the female is replaced and allowed
to walk to a recovery pen.
 If there is no bleeding, it is not necessary to close the incision sites.
  The ewe is given an antibiotic injection to prevent possible infection.
 It takes less than 5 minutes per animal when performed by a skilled operator and
pregnancy rates of 70-85% have been reported.

Disadvantages

 It requires specialized equipments and drugs.

 It can be performed only by a skilled and trained professional.
 It is a costly procedure.
HORSES

 The mare is restrained by hobbles, backed against baled hay or a board wall or put in
a breeding chute to protect the inseminator.
 The mare should be suitably restrained after bandaging the mare’s tail with sterile
gauze or a bandage.
 The buttocks, perineal region and external genitalia are thoroughly scrubbed with
soap and water and then wiped and rinsed with clear water.
 The operator inserts his left hand, which is encased in a plastic sleeve-lightly
lubricated with water –soluble, non-spermicidal lubricating jelly into the vagina and
the index finger is inserted into the cervix.
 Inseminating catheter is guided into the uterus to deposit 20-50ml of raw (or)
extended semen.
 Possible entry of air into vagina should be prevented on withdrawal of the arm.

PIGS

 Insemination can be done without restraining the sow because with some rubbing
and pressure on back the sow stands calmly during AI.
 The insemination tube is guided into the cervix because the vagina tapers directly into
the cervix which itself tapers.
 It is not possible to pass the catheter into the uterus but with inflated cuff, most of 50
ml is forced into the uterus.

Large semen volume and high sperm numbers are

required in sows.
 DOGS

 AI in the canine generally is based on the use of fresh, unextended semen.

 Generally, the entire sperm rich fraction is deposited into the anterior
vagina of the estrus female.
 A plastic pipette is passed dorso-cranially into the vagina of the bitch to the
external cervical os.
 The semen is slowly injected, followed by elevation of the bitch’s
hindquarters for 5 minutes. Elevation helps prevent the premature loss of
semen from the vaginal tract.
 Intracervical or intrauterine insemination via the vagina is nearly
impossible.
 Vaginal stimulation with a gloved finger may aid in the transport of semen into the
uterus via genital contractions and altering vaginal pressures.
 The use of transvaginal insemination with frozen semen has produced extremely
erratic results in conception rate and litter size.

 The most consistent results are obtained through surgical intrauterine insemination
of frozen semen containing 100×106 spermatozoa.

CATS

 The queen is inseminated vaginally with a minimum of 5×106 spermatozoa.

 The insemination pipette is passed through the vagina to the area of the cervix, and
the insemination dose is expelled.
 The time of breeding coincides with the maximum hypertrophy of the vaginal
epithelial cells.
 Consequently, a vaginal wash is performed to check that the queen is in estrus prior to
insemination.

After insemination, the queen must be given LH or human

chorionic gonadotrophin to induce ovulation.