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Recent developments in the diagnosis and therapy of repeat breeding cows

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Article  in  CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources · January 2010
DOI: 10.1079/PAVSNNR20083062

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 CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2008 3, No. 062

Review

Recent developments in the diagnosis and therapy of repeat breeding

cows and buffaloes
G.N. Purohit*

Address: Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Science,
Bikaner 334001, Rajasthan, India.

*Correspondence: Email: gnpvog@yahoo.co.in

Received: 15 May 2008

Accepted: 15 July 2008

doi: 10.1079/PAVSNNR20083062

The electronic version of this article is the definitive one. It is located here: http://www.cababstractsplus.org/cabreviews

g CAB International 2008 (Online ISSN 1749-8848)

Abstract

Repeat breeding (RB) continues to be a big problem for breeders and veterinary clinicians. A brief
mention is made of the common aetiological and risk factors for RB in cattle and buffaloes, and the
possible diagnostic and therapeutic approaches are described in detail. Important diagnostic tools
could include rectogenital palpation, vaginoscopy, uterine cytology and the in vivo imaging tech-
nique of ultrasonography. When considering the most common causes of RB, vaginoscopy and
palpation continue to be the only diagnostic tools available to clinicians at many locations, while by
using ultrasonography, diagnostic accuracy can be increased markedly, especially when dealing with
individual cows or buffaloes. Contrarily, when dealing with herds, metabolic profiles and sampling
to detect infectious disease must be the clinicians’ choice. Of pertinent consideration are the
management regimens and feeding practices. Despite the development of many diagnostic pro-
cedures such as hormone assays, colour Doppler sonography, and hysteroscopy, diagnosing the
cause of pregnancy failure in an individual cow/buffalo continues to be difficult, as a proportion of
animals demonstrate obscure infertility. The choice of a therapeutic regimen depends on the
possible cause of RB. Recent advances in the therapy of endometritis include the use of immu-
nomodulators such as Escherichia coli lipopolysaccharide, use of eicosanoid PGF2a and therapy with
enzymes with or without therapy with antibiotics, the use of which continues to be debatable. The
therapy of ovulation induction in various ovulatory disturbances includes regimens utilizing hCG,
GnRH, prostaglandins and their combinations. It appears that RB animals with aberrations of
oestrus cycle do demonstrate such ovulation asynchronies. Suprabasal progesterone concentra-
tion at oestrus is thought to be an important contributor of RB, but remediation of this is largely
unknown although reducing stress appears to be a probable method. Luteal insufficiency can be
resolved by administration of hCG and GnRH or progestagens. A brief mention is made of ways of
improving management and insemination procedures.

Keywords: Cow, Buffalo, Repeat breeding, Diagnosis, Treatment

Review Methodology: We searched the following databases: CAB Abstracts, Animal Breeding Abstracts, and PubMed (keyword
search terms used: repeat breeding, in vivo imaging, immunoinfertility, endometritis, ovarian cysts, infertility, endoscopy and metabolic
profiles). In addition, we used the references from the articles obtained by this method to check for additional relevant material. We
also spoke to colleagues and checked for any upcoming studies not yet published.

Introduction modern high-producing Holstein cows have reduced ferti-

The repeat breeding (RB) syndrome continues to be a
major problem in cattle and buffalo breeding, leading to
large economic losses to the dairy producers [1, 2]. Some
authors consider RB to be overemphasized and that
lity because of intensive selection for high yields [3–7];
however, others do not concur with this view [8, 9].
Recently, RB cows have been defined as a heterogeneous
group of subfertile cows with no anatomical abnormalities
or infections that exhibit a variety of reproductive

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2 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
disturbances in a consistent pattern over three or more
consecutive heat cycles of normal duration (17–25 days)
[10]. Concurrence does exist for a similar definition in the
water buffalo [11], as cattle and buffalo are an integral part
of the mixed-crop-livestock smallholder farming systems
in the developing countries of the Asia-Pacific region [12].
However, buffaloes are considered tangentially seasonal
animals and their reproductive efficiency is usually nega-
tively affected by increasing the length of daylight, with the
obvious exception of the equatorial regions, where the
reproductive function is mostly dictated by the availability
of feedstuff rather then length of light hours [13–17].
Because of these and other subtle differences between
cattle and buffalo (fertility in buffaloes is considered lower
than in cattle), [18] essentially therefore RB in the buffalo
must be considered only during the breeding season. It
has long been argued that the causes of the RB syndrome
are either failure of fertilization [19] or early embryonic
deaths [20–23], with embryonic deaths accounting for the
major share of the reproductive wastage in dairy cattle
[21, 24–34]. However, such descriptions are few for the
buffalo [35] but the phenomena are usually considered
to be similarly existent [11, 36]. More recently, it has
been considered that the cow, the bull and a range of
environmental and handling factors often overlapping each
other result in RB and it is often difficult to determine
the primary origin [10]. Such problems are much more
difficult to trace when a farmer’s individual cows or
buffaloes from a variety of management practices have to
be investigated instead of investigations on a herd. In
general, the pregnancy rates are higher where natural
service is the method of breeding compared with artificial
insemination [37, 38]. Therefore, RB has also to be
viewed in this perspective. In general, the veterinary
clinicians in many developing countries face the problem
of treating such problem cattle and buffaloes with little to
diagnose and lack of a systematic therapeutic regimen.
This review focuses on the possible diagnostic modalities
and therapeutic regimens in RB cows and buffaloes.
Incidence
For various countries, the incidence of the problem
has been described in cattle and buffaloes to range from 5
to 35% [21, 39–41]. However, such an assessment of
incidence suffers from limited data over a few or many
herds. A seasonal influence on the appearance of the
problem has been depicted, with the hot season being
unfavourable. The incidence of RB in cattle and buffalo in
various reports is presented in Table 1.
Aetiology and Risk Factors
The aetiology of RB has been widely reviewed [1, 22, 23,
42–44] but to correlate diagnostic and therapeutic
http://www.cababstractsplus.org/cabreviews
approaches, a brief mention is made of the possible
aetiological and risk factors.
The aetiology of RB appears to be multifactorial. All
or any of the causes described for pregnancy failures in
cattle and buffaloes are evidenced clinically in the form
of RB. When evaluating females for RB, the causes for
fertilization failures in part or in toto that would result
from male gamete abnormality or hypofunction must be
first eliminated. The possibility of failures caused by semen
abnormality appears to be ruled out when artificial insemi-
nation using semen of high fertility is used. However, it
must be kept in mind that frozen semen has a shorter life
span, < 12 h in the female tract, compared with ejaculated
semen [45] and a lower fertility compared with fresh
semen because of lower viability post thaw and sublethal
dysfunction in a proportion of the surviving subpopulation
[46]. A problem with buffalo semen appears to be the
season during which semen is collected, because semen
collected during hot summer months appears to offer
suboptimal fertility compared with that collected during
the winter months [47–50]. Such seasonal variations,
however, are not known to cause deleterious changes in
sperm quality in swamp Thai buffaloes [51]. A brief
mention is made herewith of the possible aetiological
factors that can contribute to the RB syndrome in cattle
and buffaloes.
Nutritional Inadequacies
Some of the nutritional deficiencies that are known to
result in pregnancy failures include lack of energy [52–60],
excess of dietary protein [61–63], and deficiencies in
micronutrients [64] such as calcium, phosphorus and iodine
[65–69], cobalt, copper, zinc and magnesium [65, 70],
vitamin A [71–74] and selenium and vitamin E [69, 75–77].
Vitamin A and b-carotene [3, 78] deficiency or excess of
body metabolites such as glucose, urea, albumin, globulin,
and non-esterified fatty acids may directly or indirectly
affect follicle growth, conception and embryonic develop-
ment. In buffaloes, only a few descriptions [79, 80] of
inadequate nutrition and RB are available; moreover, the
important aspects of nutritional management of dairy cows
for optimum postpartum fertility [81–84] appear different
from those found in buffaloes, as more important for cows is
the resumption of postpartum oestrus, which occurs 90
days after calving only in 39–49% of buffaloes, the rest
remaining in anoestrus for 150 days [16].
Hormonal Dysfunction
The entire events of pregnancy establishment from
ovulation of a viable competent oocyte to fertilization, to
implantation and subsequent growth of an embryo in utero
are dependent on a complex chain of rhythmic hormone
secretion and binding [85]. A slight deviation in any of
 G.N. Purohit 3

Table 1 Incidence of repeat breeding in cows and buffaloes in various studies

Type of
Incidence (%) Breed management Reference
Cows
14.4–27.0 Hariana FM [469]
10 Swedish breed FM [42]
5.5–33.3 Red Sindhi FM [470]
10–15 HF FM [178]
21.4–28.2 Red Sindhi FM [471]
18.1–24.4 Sahiwal FM [471]
16.5 Tharparkar FM [471]
16.4–18.8 Crossbred FM [471]
5.0 HF FM [472]
16.6–58.8 Fulani Cows VM [473]
8.98 JerseyHF VM [474]
8.98 Crossbred VM [41]
6.8 Crossbred VM [475]
19.8 JerseyGir FM [476]
8.20–9.30 HFTharparkar FM [477]
7–25.0 Cows, buffaloes FM [478]
7.4–18.6 Danish Red, Sahiwal, HF FM [190]
25.9 HFGir FM [479]
5.0 HF FM [480]
24.0 HF FM [1]
8.0 Red Kandhari FM [481]
3.0 Crossbred FM [481]
15.8 Crossbred FM [482]
8.33 Egyptian cows FM [3]
10 HF FM [28]
4.2 HFDeoni FM [483]
9.77 Crossbred FM [484]
17.8 Crossbred FM [485]
10 HF FM [29]
7.3 Crossbred VM [486]
25.1 HF FM [487]
28.4 SahiwalFriesian FM [488]
7–17.0 Rathi VM [280]
12.0 HF crossbred Clinics [489]
Buffaloes
7.37 Jaffarabadi FM [490]
10.76 Murrah, Nilli-Ravi FM [40]
6.0 ND, Murrah VM [41]
5–20% Murrah FM [491]
8.06–28.84 ND, Murrah, Nilli-Ravi Clinics [492, 493]
8.33 Egyptian VM [494]
1.9 ND, Murrah VM [486]
6.1 Mixed VM [37]
7.57 Mixed VM [495]
HF, Holstein Friesian; FM, farm management; VM, village management; ND, Non-descript.

the hormones may change or affect the establishment
of pregnancy. Disturbances of ovulation in part because of
faulty luteinizing hormone (LH) secretion, a prolonged
duration of standing oestrus or improper steroid genesis
appear to be major causes of RB in dairy cattle [86–88].
An important feature of buffalo reproduction appears to
be the levels of plasma prolactin, which are elevated
during summer and known to suppress progesterone
concentrations [7, 89] besides low luteal progesterone
that accounts for 50% of embryonic mortalities in buffa-
loes during summer [35]. Besides the importance of
optimal progesterone concentrations during the luteal
phase, which may depress thyroid function during summer
[90, 91] and culminate into pregnancy failures because of
early embryonic deaths or lack of signal transduction
between the mother and embryo, importance is currently
attached to the suprabasal concentrations of progester-
one (higher basal progesterone at oestrus) at oestrus in
dairy cows [92–98]. Such reports are largely unavailable
for the buffalo. Moreover, in the buffalo the steroid
secretion is inherently low at oestrus [99] and behavioural
oestrus is associated with high progesterone levels [100,
101]. A higher progesterone results in poor conception
rates largely because of ovulation–insemination mismatch.
Ovulatory disturbances commonly encountered in RB
animals include delayed ovulation [86, 102] anovulation

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4 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources

[88, 103–105] and ovarian cysts [106–110]. A manifest-
ation of hormonal dysfunction could be aberrant oestrus
expression in dairy cows [6, 10].
Infectious Agents and Reproductive Tract
Abnormalities
stressors, e.g. high milk production, postpartum
disorders and negative energy balance, inflammations and
infections, lameness, social factors, transport and heat
stress.
High milk yield, high parity and calving in winter were risk
factors for several reproductive disorders, which in turn
delayed insemination and conception in dairy cows [155].

Various reproductive tract abnormalities have been

described to be a reason for RB both in cattle [20, Diagnostic Methods
111–114] and buffalo [115, 116]. Only a few of these
described conditions can be discerned clinically, such as In view of the wide variety of causes that can result in RB,
infections of the tubular tract from vagina to fallopian the diagnostic procedures in the present review have
tubes or ovarian cysts and sometimes ovarobursal adhe- been classified into the following groups:
sions.
Infectious agents present in the genital tract may 1. Record analysis
hamper fertilization and early embryo development as 2. Visual
well. These organisms when present in pathological num- 3. Recto-genital palpation
bers may produce some toxins or render the uterine 4. Vaginoscopy
milieu unfavourable for conception. Infections must be 5. Tests to evaluate uterine health:
suspected when there is a moderate to mild degree of (a) Uterine pH
endometritis, evident because of flakes of pus or floccu- (b) Uterine microbiology
lent material in cervico-vaginal mucus discharge at oes- (c) Uterine biopsy and cytology
trus. However, when the infections are subclinical they 6. Metabolic profiles
remain obscure clinically but still may hamper conception. 7. In vivo imaging techniques
A wide variety of bacteria, viruses, fungi and protozoa 8. Immunological tests
have been revealed to hamper conception and have been 9. Endoscopy
widely reviewed elsewhere [30, 117–123], with bacteria 10. Tubal patency testing
being a particularly common problem. 11. Hormone assays

Genetic Problems and Immunoinfertility

Chromosomal abnormalities are considered by a few
authors as aetiological agents contributing to conception
failures [24, 124–128].
Immunological incompatibility of the sperm and oocyte
because of production of anti-sperm antibodies has been
documented [129–142] in both cattle and buffaloes to be
one reason for fertilization failures.
Miscellaneous
Record Analysis
Analysis of records when traced retrospectively would
provide the number of actual inseminations and previous
periparturient disease that have resulted in suboptimal
fertility. Insights into poor fertility can be traced in indi-
vidual cows by record analysis. However, more often,
cows/buffaloes presented to clinicians with RB originate
from diverse changing management strategies, with no
records.

High environmental temperatures [143, 144], season, size Visual

of herd/type of housing, age [143–148], environmental
pollutants [149], milk yield, lactation and difficult calvings
[21, 150, 151], metabolic disorders [81], postpartum
metritis and ovarian cysts [152] are a few of the other risk
factors that may increase the incidence of RB in cattle and
buffaloes. Stress has been addressed as a cause of
impaired reproductive efficiency [153] and the hormonal
mechanism for effect on fertility is common irrespective
of the stressor involved. In a stressful situation, the
function of the hypothalamus–pituitary–gonadal axis
might be disrupted at each level [154]. Many factors in
modern dairy farming have been identified as potential
The importance of visual observations in diagnosing an
animal that would subsequently repeat to service or
artificial insemination (AI) lies in the fact that human fac-
tors such as improper oestrus detection or insemination
asynchrony may many times contribute to the failure of
pregnancy establishment in an individual animal. Cruz
[156] has stressed that the poor results in insemination
programmes have been largely the result of human factors
such as improper insemination techniques or improper
timing of AI. Visual observations that need the attention
of the inseminators include the colour, consistency and

http://www.cababstractsplus.org/cabreviews

clarity of the cervico-vaginal mucus, vulvar oedema and
vaginal congestion. A cow showing a cervico-vaginal
mucus discharge that is watery, thin or copious should be
viewed as one with suspicious subsequent fertility. In
natural mating programmes, which are more readily
practised in the buffalo, size differences in the male and
female or services without any ejaculatory thrusts may
result in conception failures. The incidence of RB with
individual bulls has been commented on recently [157],
and it must be kept in mind that when more females are
failing to conceive to a particular male, the reason often
lies in that male which must be investigated and it is often
better to replace it. An uncommon condition that is often
missed by insemination personnel is uro-vagina. The urine
pool of vagina contaminates the semen, often reducing its
fertility. A thorough vaginoscopic examination must be
undertaken in animals that have doubtful fertility to
exclude the possibility of any vaginal growths, scars or
adhesions that may impair fertility.
Recto-genital Palpation
Recto-genital palpation of both uterine horns, cervical os
and the ovaries is by far the commonest diagnostic
method used for cows and buffaloes repeating to natural
services/inseminations, yielding little information as to
the cause of pregnancy failures. However, a systematic
approach would definitely give some clue to this multi
factorial problem. Animals with poor uterine tone at
insemination often have poor conception; hence this must
receive attention but not guiding. Moreover, such an
evaluation is subjective and often graded differently by
different clinicians. Pleuriparous cows, especially those
which have had some periparturient problem, have irre-
gularly shaped cervices, creating difficulty in the intro-
duction of the insemination pipettes. Such problems
appear to be rare in buffaloes. The optimum size of the
ovulatory follicles at AI has been commented on else-
where [158]. However, such evaluations are far from
perfect by rectal palpation, and extremely difficult for the
buffalo. An important event during pregnancy establish-
ment is the ovulation of the follicle. For estimation of
ovulation, palpations must be done every 12 h from AI
till the finding of an ovulation depression. Such an eva-
luation again is difficult for the buffalo. Evaluating the early
or late corpus luteum (CL) to rule out luteal insufficiency
is subjective and the predictions are often suboptimal.
Prediction of early foetal deaths by rectal palpation when
it occurs beyond day 45 gestation is often possible, but
then such deaths often result in voiding of foetal fluids,
blood and/or foetus itself and is considered an abortion
rather than an early embryonic death. Moreover, much of
the loss of potential offspring in cattle is concentrated
during the first 42 days after breeding [32] and in parti-
cular 6–20 days after breeding [24, 159–162], when it is
seldom possible by rectal palpation to evaluate pregnancy.
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G.N. Purohit 5
A common belief that pregnancy diagnosis by rectal
palpation between days 35–41 by the foetal membrane
slip can often result in foetal death has been proved
to be wrong when palpations are preformed with care
[163–165].
Rectal palpation can help in diagnosing grossly enlarged
fallopian tubes, but enlargements and constrictions of
minor nature cannot be evaluated by rectal palpation
alone. Likewise, ovarobursal adhesions can be diagnosed
by recto-genital palpation [22, 23].
Vaginoscopy
Examining the vagina and cervix by vaginoscopy to
determine the presence or obsence of small quantities of
infected material inside has been a common clinical
method to estimate uterine infection. Miller et al. [542]
concluded that vaginoscopic examination is a more
accurate method for detecting uterine infections than
palpation per rectum. Cows with abnormal vaginal dis-
charge on vaginoscopic examination have poor repro-
ductive performance [166, 167]. However, vaginoscopy
often fails to identify all cows that are truly at risk of poor
reproductive performance and the absence of discharge at
vaginoscopy does not necessarily indicate absence of
uterine inflammation [168, 169]. The presence of dis-
charge in the vagina and its identification by vaginoscopy
may be influenced by the severity of the infection, myo-
metrial contraction, uterine clearance mechanisms, peri-
neal conformation, body condition, postural changes and
exercise. Discharges may not be detected in cows in
which the cervix is closed, although these cows may
harbour infection. A single vaginoscopic examination
therefore lacks accuracy and may result in undiagnosed
and untreated endometritis [168]. An alternative
approach for sampling of vaginal contents using a novel
device termed ‘Metricheck’ was found to be more sensi-
tive in detecting endometritis compared with vaginoscopy
[170].
Tests to Evaluate Uterine Health
Uterine pH
The pH of the uterine lumen during different stages of the
oestrus cycle varies widely, with the lowest pH occurring
2 days prior to ovulation; however, at oestrus it is known
to be 7.30 [171]. Samples of uterine secretions are diffi-
cult to collect although a few studies point out that this
can serve as a partial indicator of the uterine milieu where
normal gamete transport and development of the embryo
can occur.
A reduction in pH from 7.2 to 6.9–7.1 has been
reported to compromise embryonic development [172,
173]. In contrast, cows/buffaloes with metritis show a pH
6 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
from 8.23 to 8.80 [174, 175]. The pH of vaginal mucus was
8.5+1.16 in repeat breeder cows compared with
7.2+1.10 in normal breeding cows [132].
An important flaw in the estimation of the uterine pH is
the lack of development of special sensing probes that can
directly be placed in the bovine/bubaline uterus. Feeding
diets high in protein alters the uterine environment by
reducing concentrations of magnesium, potassium and
phosphorus in uterine secretions [61] and by reducing
uterine pH [62, 176].
Uterine Microbiology
A wide variety of microbes normally harbour the uterine
lumen and only when their numbers are high are they
evident clinically in the form of purulent mucus flakes of
pus and changes in odour of the genital discharges. It has
been shown in many studies that mostly mixed infections
are present in the uteri of RB cows and buffaloes [177–
180]. In the author’s experience, a subclinical uterine
infection remains clinically obscure as also observed in
some previous studies [181–183] and this appears to be
one of the leading causes of fertilization failure both in
cattle and buffaloes. Diagnostic tests to evaluate such
subclinical uterine infections (endometritis) have been
developed to a limited extent, but suffer from incor-
poration of suboptimal uterine secretion collection
methods and accuracy of diagnosis. One such test, the
‘white side test’, uses cervical mucus of suspected cows
(with metritis/endometritis), which is heated with sodium
hydroxide solution up to boiling point. The reaction is
considered positive if the colour turns yellow. A corre-
lation between the number of leucocytes present in the
mucus and the intensity of yellow colour is the basis of
this test [175, 184].
Other recent developments to evaluate endometritis
include the novel intravaginal device ‘Metricheck’, men-
tioned earlier, which is known to be more sensitive in
detecting endometritis than vaginoscopy [170]. The
device consists of a 40 mm hemisphere of silicon attached
to a 500 mm long stainless steel rod. The device is
inserted through cleaned vulvar lips, advanced to the
cranial extent of the vaginal fornix and then retracted
back. Purulent material may be visualized within the
concave surface, or adherent to the convex surface, of the
device.
Periparturient problems often contribute to some
microbes being harboured in the genital tract, resulting in
deviant fertility of obscure nature. Microbes such as
Campylobacter are difficult to isolate, but their relative
presence is insignificant with the widespread use of AI.
When fertility of a herd is in question, it is often desirable
to collect random samples from the uteri of some
cows using proper techniques in order to find out
the probable aetiological agent. However, such tests are
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time-consuming and often inconclusive in diagnosing the
cause of RB in an individual animal.
Uterine biopsy and cytology
Carefully performed uterine biopsies can often reveal the
changes in the endometrium and the extent of cellular
infiltration and/or cellular morphology changes [185].
However, they can seldom help in formulating therapeutic
measures and therefore their use is, and must be,
reserved for forbidden cases suspected for uterine
growths, enlargements or malfunctions, in which they
supplement as substantial evidence for removal/discard of
such animals. In RB cows the glandular secretions and
supranuclear vacuolations are observed in histological
sections prepared from collected biopsies [186]. Changes
observed in cows with endometritis include denudation of
epithelial lining and infiltration of lymphocyte and neutro-
phils [177, 187–189]. Biopsies of RB buffaloes have also
revealed endometritis of varying degree [190]. The
endometrial EGF concentration is altered in RB cows and
can serve as a potential marker for the identification of
cows that would turn out to be repeat breeder [191]. The
sensitivity and specificity of uterine biopsy for pregnancy
was found to be 92 and 77% [188]. The histological
findings of inflammatory changes and fibrosis were cor-
related with presence of bacteria [192–194].
There is a growing body of evidence on the use of
uterine cytology as a means of evaluating uterine health
[168, 169, 195, 196]. There is an increase in the percen-
tage of polymorphonuclear leucocytes (PMNs) during
clinical and subclinical uterine inflammation. Different
procedures have been described for obtaining the uterine
cells and performing a count, and include flushing the
uterus with small amounts (2–5 ml) of fluid [169] or using
a commercially available cytobrush [197]. When using a
fluid recovery procedure after infusion into the uterus,
the fluid must be centrifuged to concentrate the cells in a
small amount of medium in order to enable effective
cellular concentration and a good slide preparation. There
appears to be lack of standard for identification of uterine
health and labelling of endometritis based on endometrial
cytology. The cell counts vary with respect to days
postpartum. The threshold for defining subclinical endo-
metritis was finding of PMNs >18% at 20–33 days post-
partum, whereas the respective threshold at 39–47 days
postpartum was >10% [168]. Between 40 and 60 days of
parturition the threshold for endometritis was only 5%
[169]. RB cows and buffaloes usually presented to the
clinician belong to a diverse group of animals that had
calved from 60 to 120 days previously. It therefore
remains to be seen how endometrial cytology can help in
defining subclinical endometritis in these animals. More-
over, a simple technique that can be used by most clini-
cians would require simplicity of technique and specificity
and consistency of interpretations.
 G.N. Purohit 7

Table 2 Blood biochemical constituents in normal breeding and repeat breeding cows and buffaloes in various studies

Cows Buffaloes
Parameter Normal RB Reference Normal RB Reference
Glucose (mg%) 47.16–84.54 45.6–97.73 [398, 525–527] 62.5–90.00 52.5–82.5 [537]
Cholesterol (mg%) 83.0–249.22 77.8–182.37 [527–529] 40.23–144.98 52.15–73.01 [537, 538]
Hb (gm%) 9.06–11.74 8.98–9.71 [527, 530, 531] 7.8–9.4 7–8.6 [537]
Ca (mg%) 6.17–10.73 6.60–69.65 [529, 532–534] 9.33–15.00 9–16 [537, 539]
P (mg%) 4.22–8.19 3.37–8.03 [525, 532, 533] 4.5–8.99 5.5–8.0 [537, 540]
Fe (mg/dl) 1.9–2.4 2.47–11.3 [532, 535, 536] 0.03–0.62 – [539]
Mn (mg/ml) 0.46–0.58 0.17–0.19 [526, 532] – – –
Zn (mg/ml) 1.09–3.14 0.65–1.19 [526, 531, 534] 0.17–1.00 0.88 [539, 541]
Cu (mg/ml) 0.65–1.14 0.22–0.99 [531, 534] 0.14–0.88 0.62 [539, 541]
Blood urea (mg%) 18.80 28.88 [526] – – –
Vitamin A (mg/dl) 41.216 37.14 [532] – – –
Co (mg/ml) 2.18–9.67 0.85 [534] 0.02 0.16 [541]
Na (meq/l) 133.7 140 [536] 182–184 – [539]
K (meq/l) 4.44 4.27 [536] 6.53–7.4 – [539]
Cl (meq/l) 96.0 96.1 [536] – – –
Mg (mg%) 3.19–22.57 2.56–9.82 [533, 534] 3.30–3.68 – [539]

Metabolic Profile morphological changes otherwise undetected by techni-

There appears to be a complex mechanism involved in the
interplay of various serum metabolites: the macro (glu-
cose, total protein and lipids) and micro (calcium, phos-
phorus, various vitamins and trace minerals) nutrients
with the different reproductive events; however, because
there is an indirect neuro-hormonal mechanism [52], it is
difficult to establish concrete clinical norms that would
predict potential fertility. The importance of negative
energy balance on reproduction has been stressed else-
where [59] as has been the impact of heat stress [143]. An
important parameter that could have some diagnostic
significance could be circulating blood glucose, as low
levels are known to affect oestrogen production by the
dominant preovulatory follicle [59] and levels of IGF-1.
Table 2 depicts the serum biochemical metabolites
reported for normal breeding and RB cattle and buffalo in
various studies. In one study, the blood metabolites glo-
bulin, albumin, urea and b-hydroxybutyrate did not cor-
relate with reproductive performance in dual purpose
cows in Mexico [198]. However, this is not always true
with RB cattle. A metabolic profile analysis of various
blood parameters, such as blood haematocrit, glucose,
cholesterol and calcium, can diagnose malnutrition and
therefore be useful in high-producing dairy cows [199].
The author partially concurs with this view and suggests a
complete blood biochemistry to be undertaken in RB
animals wherever possible.
In Vivo Imaging Techniques
By far, the most important diagnostic modality for
reproductive diagnosis is ultrasonography (USG). The
diagnostic significance of USG lies in the fact that the
technique is non-invasive, and ovarian and uterine
ques like rectal palpation can be detected and traced
[200]. Follicular growth pattern in RB cows revealed that
such cows more frequently had two follicular waves
corresponding to longer cycles [201]. The CL becomes
visible by USG after 3 days of ovulation [202], and USG is
considered reliable for measuring follicles and detecting
CL [203]. Moreover, the health of the uterus can be
evaluated. At oestrus, there is distinct folding of endo-
metrium, and in uterine inflammation, echogenic snowy
patches are visible sonographically [204]. Ovarian dys-
function is known to be common in RB animals [10]
and includes ovarian cysts, ovulation defects, luteal
dysfunction and a prolonged life span of pre-ovulatory
follicle [98]. Ovulation can be traced by regular scanning
at least at 12 h intervals from AI. This would rule out
ovulation–insemination asynchrony. Cows and buffaloes
not ovulating within 24 h of an insemination must either
be re-inseminated or considered for an ovulation induc-
tion treatment along with AI. A single USG examination,
however, cannot detect ovarian function, and therefore
repeated examinations are necessary. In RB cows the
USG examination therefore must be done at AI to
determine the presence of an ovulatory follicle and then
repeated at 12 h intervals to find out if ovulation has
occurred, and subsequent examinations have to be done
at 4 day intervals to observe CL formation [10]. Although
variable, the optimum size diametric of a follicle at AI
would be 1.5–2.0 cm in dairy cows and 0.9–1.8 cm in
buffalo; however, it has been shown that follicle size has
no effect on fertility when ovulation occurs spontaneously
[158].
USG can delineate cows/buffaloes with subclinical
uterine infection (endometritis). A uterine lumen with a
diameter of 0.2 cm and presence of echogenic content in
the uterus is considered to indicate endometritis and is
known to have a significant negative association with

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8 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
conception rate and proportion of cows pregnant [168,
205]. Other important determinants of pregnancy failures
that can be detected by USG are luteal formation and
embryonic deaths. The morphological characters of CL
on both the bovine and bubaline ovaries can well be
visualized by day 5 of oestrus. However, this does not
precisely predict functionality of this temporary endocrine
gland. It is known that pregnancy can be diagnosed with
around 100% accuracy at day 25 [206] and the embryonic
heart beat can first be visualized at around day 25 of
gestation [207]. It is therefore possible to trace death/
resorption of embryos, which is otherwise not possible
by any other diagnostic modality. The maximum early
embryonic deaths in cattle occur by day 20 [161]. Late
embryonic deaths (day 27–42) account for 10–20%
of embryonic deaths in cattle [208], and these could be
more readily detected by USG. The presence of amniotic
vesicle/foetus at a particular moment by USG and dis-
appearance at a later time clearly suggests embryonic
death.
Improvements in the in vivo imaging technique [209]
include computer-assisted image analysis of USG [210–
214], three-dimensional USG [215], colour Doppler USG
[216–221] and magnetic resonance imaging (MRI) [222–
227]. A better understanding of the picture components
obtained by USG can be obtained by image analysis by
computers. The data obtained from sonographic assess-
ment are standardized and incorporated into computer
analysis software. These modalities widen the under-
standing of the sonograms.
Colour Doppler USG is meant to demonstrate changes
that occur in circulation to the uterus, ovaries or ovarian
structure and hence can provide new information about
physiological changes that occur in the genital organ. The
application of these and other in vivo imaging techniques
such as MRI has widened our understanding of basic
reproductive processes. Prototypes of MRI instruments
for intravaginal or intrarectal use are being developed to
make this technique more user-friendly [209]. However,
due to the high cost of these equipments and the skills
required, the use of these modern in vivo imaging tech-
niques is currently limited and is beyond the scope of
diagnosis in RB animals.
Circulating Hormone Assays
Perturbations of the reproductive hormones, especially
LH and the ovarian steroids oestrogen and progesterone,
can affect pregnancy establishment. A delay/deviation in
the secretion of LH peak surge can affect ovulation and
the development of CL, but such deviant secretions
appear to be multifactorial (e.g. linked to stress, heat,
insufficient oestrogen by the follicle, and high prolactin). In
buffaloes, a consecutive LH peak, usually accompanied by
a double ovulation, has been recorded to be occurring in a
small proportion of animals [228]. Clinical assays of this
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hormone needs repeated blood sampling and costlier
methodology (such as radioimmunoassay (RIA), [229])
that makes such assays impractical under most bovine and
bubaline practice. The levels of progesterone both at
oestrus and during the luteal phase appear to be critical
from most studies in dairy cattle [30, 34, 230–232] and
buffaloes [35, 233]. Higher basal progesterone (the so-
called suprabasal (SB) progesterone) can be considered as
a tool for the identification of repeat breeder heifers
[95, 96, 234, 235] and buffaloes [93], provided that heat
detection and AI timing are optimal. It has been shown
that as the progesterone level at AI rises, conception rate
in cows declines [236]. The season appears to have dis-
tinct effects on buffalo endocrinology, especially the
thyroid and prolactin secretions.
The thyroid function appears to be depressed during
summer and in poorly reproducing buffaloes [91]. Buffa-
loes with low-plasma protein-bound iodine had low pro-
gesterone and a higher number of services per conception
[90]. Similarly, prolactin and progesterone are negatively
correlated during summer [7, 89]. High levels of prolactin
during summer and low LH result in poor reproductive
efficiency in buffaloes during summer. Hormonal profiles
in the course of oestrus cycle are on the whole similar in
cattle and buffalo [17].
In spite of the impact of seasonal influences on buffa-
loes, progesterone production appears to be crucial for
embryo viability in buffaloes [35], similar to cattle, and
both a late post-ovulatory progesterone rise and low
luteal phase concentration are associated with poor
embryo development and production of insufficient in-
terferon IFN to preserve luteal regression [30, 35, 102,
237–241]. It can probably be concluded from a large
number of published studies that whenever possible,
progesterone assays must be done on individual cows/
buffaloes at AI and 7 days later. They can serve as diag-
nostic parameters as to whether the animal would con-
ceive and continue the pregnancy or not. Such inferences,
however, need to be validated experimentally with larger
trials.
Immunological and In Vitro Tests
Immunoinfertility appears to have received more atten-
tion in human species. Antisperm antibodies are known to
be present in serum of both sexes in human studies [242–
245] and they result in sperm-immobilizing activity, lead-
ing to penetration reduction of sperm in cervical mucus
and resultant reduced fertility [245–247].
The detection of these antibodies in cattle is usually
carried out by determination of these antisperm anti-
bodies in serum [137, 146, 147] or cervical mucus [129,
130, 132, 134] and similarly in buffaloes [135, 138, 140].
Farahani et. al. [131], however, found agglutinating (Aggl)
and immunofluorescent (IF) antibodies in serum from
repeat breeder, fertile cows and virgin heifers with no

sperm-immobilizing antibodies. The antibodies were
assumed to be produced naturally with no need of female
exposure to sperm antigens as all virgin heifers also
demonstrated Aggl and IF antibodies in their serum. Their
study pointed out that antibodies against sperm are not
responsible for reduced fertility in RB cows. Contrary to
earlier reports in buffaloes, Kanchev et al. [138] also found
that circulating agglutinating antisperm antibodies are
very rarely detected in the buffalo cows with unexplained
infertility after several inseminations. The role of anti-
sperm antibodies therefore in RB cows/buffaloes remains
suspicious and hence puts a question mark on such
tests. Methods of predicting the in vivo potential fertility
of frozen bull/buffalo bull semen could include the cervical
mucus penetration assay [248, 249] or penetration of
sperm in polyacrylamide gel [139]. These tests and
tests like those performed on oocytes such as the zona
binding assay and hemizona assay [250, 251] and pene-
tration of zona-free hamster oocytes by bull sperm
[252] in combination can provide valuable information
about a semen donor, an insemination dose or a method
for semen preservation [253]. However, a pregnancy
remains the most appropriate test of potential bull
fertility.
Tubal Patency Testing
The fallopian tube is a particularly complex structure and,
as such, an ideal method for its clinical assessment is very
difficult to obtain [254]. The incidence of fallopian tube
lesions is known to be 6.85% (range 2.6–9.0%) with
ovarobursal adhesions being the commonest lesion in
cows [255]. The occlusion of the fallopian tubes can result
in lowered fertility when it is unilateral and sterility when
it is bilateral [22, 23]. The occlusions may not essentially
be because of any lesion inside [256]. An instrument for
diagnosing oviduct patency in cows was developed a long
time ago [257], with many subsequent modifications. The
usual test utilizes infusion of phenol sulpfopthalein (PSP)
dye using a two-way catheter into the uterine horn and
detection of the dye in urine. In animals with non-
occluded fallopian tube, the dye is present in urine within
30 min; however, in cows with non-patent (occluded)
fallopian tube, the dye is not visible in urine for up to 2 h
[258–260]. When the same procedure is to be repeated
in the other horn, a gap of at least 12–24 h must be
provided or the dye must be changed [22, 23]. Patency
testing must be taken up in animals when other diagnostic
procedures have yielded no conclusive information and
the animal continues to evidence obscure infertility.
Improvements in methods of assessment of fallopian tube
functions in human medicine include hysterosalpingo-
graphy and laparoscopic chromopertubation [261]. How-
ever, such techniques need the attention of veterinary
practitioners.
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G.N. Purohit 9
Endoscopy
A potential means of evaluating morphology and func-
tional means of live tissues could be the endoscopic
visualization of the uterus and other genital organs.
Endoscopy has been used to a limited extent for
visualization and surgical intervention in cows and buffa-
loes. The use of a flexible fibre-optic endoscope for
clinical assessment of the uterus and intrauterine therapy
has been described for the mare [262]. However, similar
reports are very few for cattle [263]. Direct hysteroscopy
has been used to evaluate the uterus. A paediatric gastro-
scope (130 cm9.5 cm) and air insufflation allow good
visibility of both uterine cornua [264]. The only difficulty
experienced in cows is traversing the cervical canal [264],
which has in fact prevented the widespread use of hys-
teroscopy in bovine reproductive diagnosis. Endoscopic
techniques using a flank approach and a 60 cm10 mm
rigid endoscope (laparoscope) have been used to view the
ovaries of conscious cattle [265, 266] and buffaloes [267,
268] employing CO2 insufflation and a head-down tilt. In
addition, endoscopy has been used to examine the ovaries
by colpotomy [269] or flank methods, particularly for
follicular aspiration [270–272] or oviductal transfer of
in vitro produced embryos [273]. The general principles of
laparoscopy [274, 275] and laparoscopic surgeries in adult
cattle have been described recently [276]. Laparoscopic
ovariectomy in standing cows has similarly been described
recently with indications for use in research and removal
of tumour-affected ovaries [277].
Therapeutic Regimens
The therapy of RB cows/buffaloes is deemed to be insti-
tuted only when oestrus detection and breeding (natural
or AI) protocols are optimal. Therapies in a herd with
suboptimal fertility constitute corrective measures to
prevent/combat disease and/or deficiency and reducing
stress. Temporary replacement/change of the bull may
take care of infertility because of the bull.
The therapeutic regimens in a herd with reproductive
failures must be aimed at the correction of the most
probable causes.
Treatment of individual cows/buffaloes at most situa-
tions remains difficult as a proportion of animals [22, 23]
are always present with obscure reasons of poor fertility.
Moreover, most diagnostic modalities described are lar-
gely unavailable to the treating clinician. With limited
facilities the therapeutic approach usually must be aimed
sequentially at (i) combating uterine infection (endome-
tritis), (ii) correcting ovulatory disturbances, (iii) supple-
menting for luteal insufficiency and (iv) improving
management. When applied with sufficient caution, one or
all of these approaches would culminate in the successful
establishment of a pregnancy both in dairy cows and
10 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
buffaloes. The detailed therapeutic regimens used widely
are described below.
Endometritis
The clinical forms of bacterial complications of the bovine
uterus have been described recently [123], and accord-
ingly uterine infection that commonly occurs in repeat
breeder cows and buffaloes falls under the definition of
subclinical endometritis which clinically evidences after 8
weeks of parturition and a complete lack of cervical dis-
charge with pathogenomic property [168, 169, 278]. Being
a common cause of pregnancy failures, subclinical endo-
metritis must always be suspected in repeat breeder cows
and buffaloes, once we have assured the male factors
(semen quality, bull). When infection with microbes in the
uterus is suspected, there are a multitude of therapeutic
approaches that have been used widely.
Antimicrobials
Antiseptics such as dilute Lugol’s iodine [279] or povidone
iodine [280, 281] have shown promise, to a limited extent,
in therapy. Likewise, limited data show the success of the
administration of antimicrobials in such cases ([282, 283].
Antimicrobial drugs administered the day after insemin-
ation to rid the uterus of organisms that might be detri-
mental to the survival of the conceptus have been
commonly used in many countries [284]. Alternatively,
antibiotics may be infused in the uterus for 3–5 days
during oestrous and insemination is done in subsequent
oestrus. Conflicting reports depict the limited effect of
treatment [285–288] and a promising benefit of treatment
both in cattle [289–295] and buffalo [296–298]. The limit-
ations of intrauterine therapy are development of drug
resistance, inconsistent results and milk disposal after
treatment that render such treatments uneconomical
[299]. Moreover, the uterus seems to have a considerable
capacity of spontaneous recovery, and a large proportion
of animals probably do not require any therapy at all,
especially under the aspect that some therapies are inef-
fective and might even cause more harm than benefit
[300]. A similar validation [301] suggests that when the
endometritis is severe, intrauterine infusion offers bene-
ficial effects: however, in slight endometritis a similar
treatment had a negative effect on reproductive efficiency.
Ott [543] considers that the result of intrauterine therapy
is generally poor not only in repeat breeders, but also in
animals moderately affected by endometritis.
The in vitro sensitivity patterns of various antibiotics on
the cervico-vaginal mucus collected from repeat breeder
cows [302–306] and buffaloes [307] have been described
in an attempt to formulate the most effective antibiotic for
therapy. However, until the specimens are collected
directly from the uterus using specialized instrumentation
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they do not represent the true picture of uterine infec-
tions as the mucus may be contaminated with microbes
residing in the cervix and vagina and hence they cannot be
recommended widely.
When clinical or subclinical endometritis is suspected,
the authors feel that, because of their low cost, properly
administered antibiotics must be the clinician’s first choice
if flakes of pus are evident in the vagina or cervico-vaginal
mucus as also suggested previously [308], and flushing of
the uterus with normal saline as suggested previously
[309, 310] must be considered when more of pus is evi-
dent clinically or when therapy alone with antibiotics fails.
The route of administration for antibiotics in subclinical
endometritis must be intrauterine as it leads to high
concentrations of the drug in the uterine cavity and
endometrium, and a relatively small amount is absorbed
into the systemic circulation [311]. Systemic antibiotic
administration should therefore be opted in treatment of
more serious cases of metritis [312]. The most traditional
antibiotic of choice has been oxytetracycline; however,
because of its locally irritative character and increase in
the minimal inhibitory concentrations (MIC) during the
last decade, high doses (2–4 g/day, for 3–5 days) are
required [123], which suggests opting for better alter-
natives. The expected in vivo efficacy of other traditional
antibiotics (amoxicillin, and aminoglycosides) is question-
able [282, 283, 313–316]. The efficacy of nitrofurazones
continues to be debatable, and only the clinical efficacy of
fluroquinolones has shown some advantage [317, 318] but
the MIC of quinolones is not known. Likewise, the efficacy
of penicillins given via an intrauterine route is doubtful
[319]. Recently, the new (third and fourth) generation
cephalosporins have shown efficacy against most uterine
pathogens at low MIC values [316] and the first-
generation cephalosporin (cephapirin) is recommended
for intrauterine use [166, 197, 320] as the drug of choice
for subclinical endometritis [123]. It is suggested under
most bovine and bubaline situations to combine an anti-
biotic with an imidazole derivative (metronidazole or
tinidazole) to take care of anaerobic microbes and pro-
tozoa that may inadvertently be present [318].
Addition of antifungal agents as suggested previously
[321–323] must be considered when the endometritis
turns out to be chronic after too much of therapy with
antimicrobials.
Plenty of alternative therapies for treatment of endo-
metritis have appeared in the recent past. Of these, the
most potent and safe approach appears to be the use of
prostglandins (PG) [288, 324, 325]. The uterus has an
increased influx of PMNs, an increased blood supply,
increased mucus production [326] and enhanced uterine
production of leukotriene B4 during the oestrus period
because of increase in proinflamatory cytokines stimu-
lated by PG. The immune functions of the uterus are thus
enhanced [327]. Hence, returning cows and buffaloes to
oestrus at short intervals would lead to endogenous
clearance of microbes and cure from endometritis. This

can usually be achieved by injecting a PG from 5–10 days
of oestrus alone [5, 288, 301] or preceded by a uterine
lavage [310]. In clinical practice it is sometimes recom-
mended to keep the cow/buffalo indoors at 2–3 oestruses
to avoid matings and this works in recovery from endo-
metritis well, but suffers from the longer time interval that
passes by before the animal conceives and it is always
better to use prostaglandins.
Immunomodulators
In the recent past, several therapies alternative to the use
of antibiotics have been suggested for the therapy of
endometritis. The intrauterine infusion of immunomodu-
lators such as E. coli lipopolysaccarides (endotoxin) [195,
328–335], oyster glycogen [209, 283, 336–338], infusion
of serum, plasma or hyperimmune serum [283, 331, 334]
or leukotriene B4 [339] has been reported widely.
These immunomodulators act as a chemoattractant
to the PMNs through stimulation of interleukins [340]
produced by monocytes and macrophages. The PMNs,
blood monocytes and macrophages are regarded as the
professional phagocytes in the cellular defences against
pathogenic micro-organisms [330]. After experimental
intrauterine infection, the PMN population within the
uterine lumen usually increases [341, 342].
A single intrauterine infusion of 100 mg of E. coli LPS
dissolved in 20 ml of phosphate-buffered saline (PBS)
results in increase in the uterine neutrophils (of up to
80%) within 6 h, which remains for 72 h [232, 233]. Like-
wise, 0.1–10% oyster glycogen (OG) (usually 500 mg)
dissolved in 60 ml of vehicle or 30 nmol/l of leukotriene B4
increases the PMN concentration within 12–24 h of
administration [195, 339]. Within 72 h of administration
of either LPS or OG, the denuded epithelium of endo-
metritis-affected crossbred cows was reduced and the
psuedostratification of uterine endometrium was com-
pletely cured [337]. However, LPS was found to cure all
types of endometritis except the chronic type with cystic
dilatation of endometrial glands [337]. Using these treat-
ments the endometritis would usually be cured and cows
can be inseminated at subsequent oestrus. However, LPS
is known to suppress follicular growth, decrease oestra-
diol production and delay the LH surge and ovulation
[343, 344], and thus the subsequent cycle may be delayed.
Addition of a small amount of autologous serum or plasma
(50–100 ml for 2–3 days) to uterine secretions increases
the opsonizing capacity and significantly enhances the
phagocytic ability of PMNs [283].
Besides the use of immunomodulators, some other
therapies suggested for resolving endometritis include the
use of enzymes and antioxidants. Enzymes like trypsin,
chymotrypsin and papain when infused into the uterus
resulted in a cure rate of 59.7% (Revealed by absence of
vaginal discharge at re-examination); however, the con-
ception rates were suboptimal [345]. Another enzyme
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G.N. Purohit 11
that has been tried is lysozyme [346] with a good success
rate. Some medicaments like 4 mM taurine and 50 mM
fructose in PBS [347] and ascorbic acid (vitamin C) have
been used for intrauterine infusion in order to change the
uterine pH prior to insemination, and act as an antioxidant
(G.N. Purohit, unpublished work). Antioxidants such as
vitamins C and E are known to modulate the oxidative
stress and reduce the endometrial damage both at the
biochemical and histological levels [348]. The stressors,
free radicals and b-endorphins were higher in repeat
breeder cows in a recent study [349]. However, whether
the benefit of intrauterine infusion of antioxidants like
vitamin C in repeat breeder cows in our clinical trials was
by resolving endometrial damage or by reducing the
concentrations of b-endorphins and free radicals gener-
ated because of stress remains to be validated. Moreover,
frozen semen might have damaged sperms or sperms with
altered function because of the reactive oxygen species
generated during the freeze thaw process [350] as semen
has little antioxidants to protect them [351]. An infusion
of antioxidants before AI might reduce the uterine luminal
reactive oxygen species and the b-endorphin that might
reduce the functional competence of frozen spermatozoa,
which might not have been offered by parentral admin-
istration of antioxidants. A positive correlation has been
previously observed between lipid peroxidation levels of
plasma and cervical mucus of cows [352].
Correction of Ovarian Dysfunction
Ovulatory disturbances culminating in RB cows and buf-
faloes include anovulation and ovarian cysts. The effects of
both delayed ovulation and anovulation in terms of end
result appear similar: pregnancy failure and hence RB. The
patterns of follicular development during periods of anovu-
lation have been described for cattle [353]. Delayed
ovulation results in poor fertility [102]. The underlying
physiology of anovulation seems to be a lack of a pre-
ovulatory surge in response to the high, oestrual con-
centrations of oestradiol [105], presumably because of
lack of progesterone priming of hypothalamus or a mul-
titude of other factors. Anovulation conditions with
growth of follicles to deviation but not to ovulatory size
may be because of undernutrition, suckling or diseased
condition. Another reason for anovulation may be the
presence of suprabasal progesterone concentrations
during oestrus, which has an inhibitory effect on the
positive feedback of high oestradiol concentrations on the
hypothalamus, resulting in high LH pulse frequency and
effects on follicular growth [354]. Spontaneously occur-
ring delayed ovulation or anovulatory conditions have
been reported in connection with RB in postpartum cows
[103, 104]. Several studies have reported development of
persistently dominant follicles subsequent to pharmaco-
logically induced suprabasal progesterone concentrations
in dairy cows, with inhibitory effects on oestrus signs and
12 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
LH release in a dose-dependent manner [355–358]. In
cases of anovulation, there is some evidence for an absent
or deficient LH surge trigger mechanism: therefore,
GnRH treatment can stimulate ovulation and, in some
cases, result in increased pregnancy rates [359, 360]. It
thus appears that many anovulating events are primarily
the result not of ovarian disorders but rather of deficient
hypothalamic function. Different types of anovulatory
conditions have been described in cattle [105]: however,
clinically, therapies to stimulate ovulation in dairy cows
and buffaloes with delayed ovulation/anovulation essen-
tially remain the same and include administration of either
hCG (1500–5000 IU, intravenous, or 5000–10 000 IU, in-
tramuscularly) [36, 114, 361–364] or 100 mg of GnRH
[359, 360, 365–377] or hMG [378]. These therapies
usually evoke LH release [360, 379] but the CL formed by
hCG injection had a shorter life span [380, 381] and 67%
of the induced short cycles were followed by a return to
acyclicity [382, 383]. Alternatives to these well-known
therapies include administration of glucose and insulin,
prostaglandins, metformin, antiprolactins and clomifene.
The LH surge is known to be complex and affected by
an interplay of various endocrine, neurocrine, metabolic
and cellular events. Low levels of glucose, insulin and
insulin-like growth factors all affect the LH surge [59]. It
has been reported that intramuscular (IM) administration
of 0.2 IU/kg bovine insulin to dairy cows on days 8, 9 and
10 of oestrus resulted in increased concentrations of
progesterone in treated cows and high levels of insulin
and glucose in cows that conceived than those that did
not conceive [384]. The authors have attempted treat-
ment of dairy cows with delayed ovulation by adminis-
tration of 500 ml of 25% dextrose at oestrus along with
insulin (5 ml of bovine insulin). In preliminary trials, 30 of
the 50 cows treated with such a treatment had timely
ovulation and conceived subsequently. However, such
therapies need to be validated further in planned studies
before they can be recommended.
Prostaglandins are known to be involved in the ovul-
ation process as they increase the intrafollicular fluid
pressure and follicle wall thinning [327] and as such can be
used as agents to facilitate ovulation. Moreover, the pre-
sence of luteal tissue at the time of final follicle growth
may hamper ovulation [385]. López-Gatius et al. [386]
noticed that intravenous cloprostenol at AI promoted
ovulation in repeat breeder cows and cows with stress.
Treatment of ovarian cysts with GnRH is also known to
yield a better therapeutic outcome when combined with a
simultaneous prostaglandin [387] administration.
Clomiphene citrate, an antioestrogen, is known to
exert direct antiovulatory and oestrogen antagonistic
actions in rats [388]. Tamoxifen and clomifen are mixed
antagonist–agonists of oestrogen action and belong to the
group of type I antioestrogens [389]. Type I oestrogen
antagonists partially inhibit the action of agonists, but due
to their own inherent weak agonistic properties, they also
induce, to some extent, oestrogenic responses. The
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degree of agonistic or antagonistic activity depends on the
species, organ, tissue or cell type that is being examined
[390]. In women, clomiphene is a well-known pharma-
ceutical agent for ovulation induction in patients with
polycystic ovarian disease [391–393]. It may exert action
directly on pituitary gland to augment oestrogen-induced
LH release [394]. Only sparse reports are available for the
use of this drug in animal studies. A dose of 300 mg of
clomiphene citrate administered to cows after a 1%
copper sulphate drench has been suggested for the
treatment of anoestrus in cows and buffaloes [395–398]
and for the treatment of RB [399] and cystic ovarian
disease [400] in cows. In known cases of ovulatory dis-
turbances in cattle and buffaloes, clomiphene should thus
be started preferably 1 day before oestrous (300 mg orally
after copper sulphate at 12 h intervals) until the onset of
oestrous. The up-regulation of receptors that follows a
down-regulation would facilitate LH release and ovulation.
It has been suggested in human therapy that in clomi-
phene-resistant women, metformin, an insulin sensitizer,
combined with clomiphene, could be a better option for
ovulation induction in patients with polycystic ovarian
syndrome [391, 393, 401] as insulin sensitizers improve
hyperinsulinaemia and hyperandrogenism in treated
women [402]. However, such therapeutic agents need
validation in the ovulation induction programmes for
cattle, and buffaloes as well. The authors have clinically
used 2000–4000 mg of metformin given orally to RB cows,
but the outcome is unknown, at the moment.
Yet another medicament, the antiprolactin bromo-
cryptine have been suggested by some clinicians to help in
ovulation induction in RB cows (P.K. Pareek, personal
communication) with suggestions of 10 mg given orally
12 h before and at the time of AI. In trials on ewes,
0.6–1.2 mg of bromocryptine administered orally for 3–12
days decreased prolactin but did not affect FSH, the mean
time of LH preovulatory surge or LH concentration in LH
surge [403, 404]. The administration of antiprolactins in
ovulation induction therefore remains questionable.
Besides ovulatory failures resulting in failure of con-
ception because of ovulation–insemination asynchrony,
the most common ovulatory disturbance recognized in
dairy cattle is the cystic ovarian disease. The problem is
less known (with an incidence of 0.2–4%) clinically in the
buffalo although reported in many studies [405–409]. The
description of the presence of follicular cysts is pre-
sumptive as signs of nymphomania, mucometra, and fre-
quent oestrous have never been recorded in buffaloes
with cystic ovaries [16].
Cows with single or multiple follicular ovarian cysts and
normal oestrus cycle lengths are often presented to the
clinician with a history of RB. When follicular cysts persist
for prolonged periods in dairy cows, endometrial gland
hypertrophy and pathologies in the uterus many a times
culminate in clinical mucometra with normal oestrus cycle
lengths. The therapeutic management of ovarian cysts in
dairy cattle has been reviewed previously [108, 109, 410,

411]. It has been suggested previously that treatments are
only temporary solutions and it is better to select cows
against cysts [108]. The therapies suggested include
administration of a single IM 200 mg injection of proges-
terone in oil [412] or insertion of intravaginal progestagen
implants [413, 414], 100 mg of GnRH [106, 107, 369,
415–417], hCG [20, 106, 418–420] or prostaglandins
[106, 410, 411], depending on the type of cyst.
More recent literature suggests a combination of these
therapeutic agents [387, 421–423]. Accordingly, GnRH or
hCG treatments are followed by prostaglandin treatments
after 8–10 days. The rationale for this combination
appears to be luteolysis of leutinized tissue formed by
administration of either GnRH, hCG or progesterone.
The complex physiology involved in the formation of cysts
active at the hypothalamus, pituitary, ovary and metabolic
levels and the follicle turnover mechanisms, however,
render the treatments suboptimal with a tendency of
cysts to reform some time after disappearance. The
Ovsynch protocol suggested and used widely [424, 425]
utilizes the administration of GnRH (100 mg) on day 0,
followed by prostaglandin on day 7, GnRH (100 mg) on day
9 and AI 16–20 h later. The second dose of GnRH assures
ovulation of the newly formed follicle. López-Gatius and
López-Béjar [387] were of the opinion that a dose of
500 mg of prostaglandin administered along with GnRH on
the first day of treatment followed by a second dose of
prostaglandin 14 days later resulted in a lower cyst per-
sistence and higher ovulation rate compared with when
GnRH was given alone on the first day of treatment.
Aspiration of follicular fluid from follicular cysts using
transvaginal ultrasound-guided aspiration has been re-
ported to be a new concept in treatment of follicular
ovarian cysts in dairy cows [426]. Whatever the therapy
adopted for treatment of ovarian cysts in dairy cows,
there is a tendency of cysts to reform, and the difficulty in
such a clinical condition lies not in the resolving of the cyst
but in the attainment of a successful pregnancy, which is
extremely difficult when cows develop clinical mucome-
tra. Regimens suggested to resolve mucometra include
oral (3–10 g of potassium iodide for 5–10 days) [106, 419,
427, 428] or injectable administration of elemental iodine
[281] or uterine lavage [309]. However, such therapies
are often unpredictable.
Much has been written regarding suprabasal proges-
terone at oestrus in dairy cows and low conception [92,
95, 96, 235, 236, 429, 430] or ovarian cyst formation
[431–434]. However, little has been suggested on cor-
rection of such a high progesterone concentration at
oestrus. The extra progesterone thought to be of adrenal
origin [435, 436] could not be confirmed to be originating
from the adrenals in studies by Bage et al. [98]. However,
environmental or social stresses were postulated to
originate adrenal stimulation with resultant deviant hor-
mones and RB. The diet and milk production both can
also alter the progesterone [437] and, as such, the
only probable preventive measure to reduce suprabasal
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G.N. Purohit 13
progesterone would be monitoring the diet and reducing
stress. Although deficiency in positive feedback of oes-
trogen to the hypothalamus, leading to a lack of LH surge
with a resultant anovulation, is the widely accepted cause
for ovarian cysts, a more recent postulation for ovarian
cyst formation is the delay (or absence) of the degen-
eration system of the unovulated follicle [438].
Some new concepts in the formation of ovarian cysts in
dairy cattle include a low insulin concentration [432], an
increase in FSH following a reduction in inhibin secretion
[439], a decrease in IGF-1 in follicular fluid [440], altered
oestrogen receptors [441], and alterations in expression
of cytoskeletal proteins in follicles [442]; however, such
insights into the formation of cysts do not necessarily
affect therapeutic regimens.
Luteal Insufficiency
Luteal inadequacy due to diminished response to the
circulating luteotrophic hormones [443] leads to insuffi-
cient progesterone production during the luteal phase
after breeding, and could be the cause of embryonic death
[444]. The serum progesterone is known to be altered in
RB cows [96, 238, 240, 445–448] and buffaloes [78, 239,
443]. Shelton et al. [443] argue that luteal inadequacy,
caused by a diminished response to circulating luteo-
trophic hormones, may contribute to embryo mortality in
subfertile cows. Early in the luteal phase the progesterone
down-regulates the oxytocin receptors (OTRs) for at
least 10 days, thus preventing premature luteolysis [29].
The secretion of antiluteolysin factor IFN-t and bovine
trophoblastic protein-1 (bTP-1) around day 15–16 post
ovulation mainly depends on progesterone concentration
around day 4–5 post ovulation [449]. Moreover, a con-
ceptus has to be around 15 mm long to secrete IFN-t, and
its growth is largely dependent on progesterone levels
[450]. A low progesterone level has been shown to be
significantly related to reduced production of IFN-t by
bovine embryos recovered on day 16 of pregnancy [451].
The most critical period for embryo survival may be
around day 5–6 post insemination when the embryo
descends from the oviduct and enters the uterine lumen.
During this period, progesterone concentrations start
rising and thus any delay in the rise and/or a low luteal
phase progesterone concentration can cause poor
embryo development and hence embryonic death due to
a suboptimal uterine environment on account of low
progesterone [452]. Inadequacies of luteal tissue form-
ation could also arise on account of poor development of
a preovulatory follicle [453], presumably because of low
IGF-1 [454]. Nutritional inadequacies can result in defi-
ciencies of growth hormone and/or insulin with resultant
low IGF-1 secretion [455]. Likewise, low progesterone in
buffaloes is also a result of breeding season, which causes
inadequate functioning of CL [377, 456], possibly because
of high prolactin [7].
14 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
The therapy for luteal insufficiency is based either
on evaluation of blood and milk progesterone on day 5
post insemination, or solely based on presumptions.
Therapeutic regimens include administration of one of the
following:
(i) GnRH at the time of insemination [457] or day 11–13
post AI [373]. The mechanics of GnRH action on
luteal insufficiency prevention are explained else-
where [29, 458].
(ii) hCG on day 4–7 [459] or day 15–16 post AI [460] as
this period coincides with the presence of dominant
follicles in cows having three follicular waves. Peters
[29] suggested that administration of hCG on day 11
and 13 is most beneficial as around this period
maternal recognition of pregnancy occurs. The ben-
efit of hCG or GnRH therapy was postulated to be
because of formation of an accessory corpora lutea
[29, 458]. However, a more recent study showed no
effect of double ovulation (in 35% of animals) on
luteal function or plasma progesterone concentra-
tions [232].
(iii) Progesterone supplemented as a single IM injection
(500 mg) on day 5 post AI [461], chlormadinone daily
oral feeding (10 mg) from day 14–23 [462] or pro-
gestagen vaginal implants from day 5–12 of AI [362,
452, 463–465]. Progesterone supplementation ad-
vances the luteolytic signal and increases embryonic
growth [466] and thus increases pregnancy rates
when given during the first week post AI, the most
critical period of embryo–maternal interactions [4].
(iv) Recombinant bovine somatotropin (500 mg SC) at
the time of oestrus and 10 days later significantly
increases the conception rate because of increase in
circulating progesterone [467].
Besides the above therapies, some other less common
therapies suggested include feeding of linoleic acid, as it is
an inhibitor of prostaglandin synthetase enzyme and thus
delays premature luteolysis and enhances luteal function,
and feeding of fish oil, as it contains docosahexanoic acid
and eicosapentaenoic acid, both of which have antiluteo-
lytic properties [468].
Management Strategies
The overall management of dairy cows and buffaloes is
important as it affects the fertility. Of consideration
are nutrition, timing of insemination and periparturient
disease.
Improving Nutritional Imbalances
The effects of nutrition on fertility in dairy cattle have
been extensively reviewed recently. Poor nutritional
http://www.cababstractsplus.org/cabreviews
management of the dairy cow, particularly before and
after calving, has been considered a key driver of infertility
[81]. Some of the significant reviews appearing recently
include reviews on the effects of macro- and micro-
minerals during the periparturient period [82], the impact
of controlled nutrition during the dry period [83], the
effect of rumen degradable proteins [84], and embryo
survival in dairy cows managed under pastoral conditions
[33]. It appears from all these and other published data
that for today’s high-producing dairy cows, fertility is in
general heading towards a decline [6] although, for the
parous cow, feeding during the dry period and post-
partum appears to be crucial in maintaining high fertility.
Inadequate body condition postpartum has a greater
impact on the probability of conception and embryonic
losses [496]. While poor nutrition during the dry and
early postpartum period results in reduced glucose,
insulin, insulin-like growth factor (IGF-1) and low LH pulse
frequency with concomitant increases in b-hydro-
xybutyrate, non-esterified fatty acids and negative energy
balance all having negative effects on the probability of
conception. Conversely, high nutrition can also increase
the metabolic clearance rate of steroid hormones such as
progesterone and oestradiol, and high rumen degradable
proteins can raise the blood urea nitrogen. All these can
impair conception and embryo survival. However, the
impacts of nutrition on fertility appear to be complex, and
recommendations for formulating effective dietary stra-
tegies to improve conception rates and prevent em-
bryonic losses during the more crucial stages therefore
appear to be difficult. In general however, it has been
recommended that cows must not lose excessive body
condition postpartum, and should not be fed more than
10% of rumen degradable protein. A balanced feed during
the dry period must therefore comprise a low-energy
high-fibre ration containing high levels of chopped straw.
These recommendations, however, do not point out the
possible deficiencies in clinical cases of RB cows and
buffaloes, which may have one or multiple deficiencies or
excesses. It is the author’s presumptive view that clinical
cases suffering from the RB syndrome at many locations
suffer from multiple deficiencies, especially those of glu-
cose, vitamins such as A, E, and C and minerals like
phosphorus, calcium and selenium and, as such, animals
must be supplemented with these nutrients by oral or
injectable supplementation. Some of the published liter-
ature does not concur with the author’s view [497, 498],
while other reports do [494, 499–504], essentially
because most of these trials were performed on well-
managed herds and not on individual cows or buffaloes.
Improving the Timing and Technique of Insemination
Much improvement can be expected by improving the
timing of insemination essentially by appropriate oestrus
detection. A sizeable proportion of cows evidence

prolonged oestrus periods and such animals pose pro-
blems to time insemination [505]. Multiple inseminations
or ovulation induction treatments are suggested in such
animals. Heifers frequently evidence short oestrus periods
and it is suggested to time insemination slightly earlier.
Buffaloes pose a greater difficulty in oestrus detection and
sub-oestrus is frequent, hence timing inseminations
becomes difficult. Vaginal electrical resistance measure-
ments have been suggested for oestrus detection and
timing insemination both in cattle [506] and buffalo [507,
508] with limited success. Likewise, the use of pedo-
meters [544–545] and radio-telemetric devices [546–547]
has been suggested to improve oestrus detection and,
hence, timing of insemination. The usual timeframe sug-
gested for timing inseminations in cattle [509] have been
suggested to be repeated twice at 12 h interval in the
buffalo for optimum conception rates [11]. It has pre-
viously been suggested for cows that if onset of oestrus is
unknown (which is frequent for animals submitted to
inseminators under most situations), inseminations should
be performed within 6 h of initial observation of oestrus
[548] because 24.1% of cows have oestrus of low intensity
and short duration [547]. Fixed-time AI subsequent to an
oestrus synchronization protocol has been shown to
improve fertility of dairy [549] and beef [550–551] cattle.
AI is usually scheduled 60–72 h of a PG injection, and such
protocols significantly improved fertility of cows suffering
from heat stress [549], as heat stress is known to
decrease the intensity and duration of oestrus expression
and increase the incidence of anoestrus and silent ovul-
ation [552]. However, the use of fixed time AI in RB
animals appears to offer little advantage, and other
important aspects mentioned elsewhere in this review
with often overlapping effects are of greater significance.
The usual site of insemination suggested both for cattle
and buffaloes is the mid-cervix; however, some reports
depict benefits of insemination in the uterine horns [510–
513], probably because the functional sperm reservoir
near ovulation is the uterine portion of the oviducts and
not in the cervix [514]. A rigid insemination device, the
‘Ghent’ device was reported to be developed for deep
intrauterine insemination at the uterotubal junction in
dairy cattle [515]. However using the usual AI gun or the
Ghent device or reducing the sperm numbers from 12 to
4 millions had no effect on pregnancy rates with either of
the methods [511, 515]. In trials during summer months
no benefit of depositing semen in the middle of the
uterine horn or uterotubal junction using low or standard
dose of sperms was observed on the pregnancy rates in
cattle [512, 516]. Likewise, Momont et al. [517] concluded
from their trials in cattle that placement of semen in one
horn of the uterus does not appear to be a cause of
decreased or increased pregnancy rates with AI. In buf-
faloes, Zicarelli et al. [518] had contrarily suggested that
inadvertent deposition of semen in the uterine horns of
buffalo due to the small size of the uterine body could be
the reason for low conception rates. Moreover, according
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G.N. Purohit 15
to Vale [49] a pregnancy rate higher than 50% can be
considered good after insemination with frozen thawed
buffalo bull semen. In the author’s view, deposition of
semen in the body of the uterus offers a distinct advantage
in improving the conception rates to AI both in cattle and
buffaloes, compared with when it is deposited in the mid-
cervix. Errors in the preparation of the AI gun or those in
the upkeep of frozen/liquid semen can contribute to
conception failures and hence must be viewed seriously. A
recent report depicts that the conception rates to artifi-
cial insemination improved by 5–27% over many Asian
countries when the personnel involved in the AI were
given various levels of training [12].
Avoiding Periparturient Disease
The role of prevention of problems in the periparturient
period, in particular hypocalcaemia, mastitis and retained
placenta, has been stressed in a recent review [82] as all
are known to have a negative impact on the subsequent
fertility of the cow. Likewise, metabolic diseases during
the postpartum period, such as ketosis and acidosis, or
parturient problems, such as dystocia, predispose cows/
buffaloes to development of postpartum uterine diseases
such as endometritis with the result of more services per
conception [81]. The approaches suggested to reduce the
incidence of these disorders to some extent include the
feeding of anionic salts in combination with adequate
calcium and magnesium [82] during the dry period and
feeding of high-fibre low-energy chopped straw during the
dry period [83]. However, although parturient problems
appear to be unavoidable, stress must be attached to
parturient hygiene. Many locations where cattle and buf-
faloes are raised suffer from extremely poor hygiene.
Moreover, often animals are referred for therapy only
when they have a reduced feed intake/milk production
or develop serious clinical signs. Coupled with this is the
fact that farmers attending calving or manually removing
plancentas often handle animals without any sanitary
measures. These practices are likely to precipitate low
conception levels postpartum presumably because of low-
grade infection or damaged genitalia. It is therefore
important to educate farmers regarding the possible con-
sequences of the poor hygiene at calving and post
partum.
Reducing Stress
Stress appears to play an important role in the modulation
of various biological events including reproduction. The
role of various types of stress because of disease, inade-
quate nutrition, high production, social factors and
environment on reproduction has been explained pre-
viously [154]. It is nearly impossible to avoid all forms of
stress in dairy cows and buffaloes, but when animals
16 Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources
Repeat breeder cow/buffalo
Herd
Female Male
AI
Investigate and 1. Evaluate
advise semen and AI
techniques
1. Nutrition (preparturient)
2. Collect samples for
investigation of Natural
infectious disease mating
3. Reduce stress
4. Metabolic profiles 1. Infectious disease
(i) Trichomonas
(ii) Campylobacter
2. Semen evaluation
3. Age of bull
Figure 1 Diagnostic approaches for repeat breeding
require more number of services per conception,
attempts must be oriented to minimize stress associated
with environment. Cooling of cows/buffaloes during hot
summer months by showering of water is known to
improve fertility. Likewise provision of sufficient shade
and wallowing space essentially improves conception
rates in buffaloes which inherently have a poor thermo-
regulatory mechanism.
Imunoinfertility
It has since long been postulated that sperm, when
deposited in the female tract, can act as an antigen and
evoke production of antibodies leading to immuno-
infertility [242]. Antibodies to sperm may appear both in
the blood and in genital tract secretions of human females
[242]. Both antisperm IgG and IgA have been isolated
from bovine [146] and human subjects [245, 246]. These
antisperm antibodies usually reduce the sperm penetra-
tion of cervical mucus with immobilizing activity of sperms
in cervical mucus of woman [244–246]. Although agglu-
tination of bovine sperm with cervical mucus has been
reported through in vitro studies [129, 130, 146], in vivo
studies have not confirmed their significance [131, 138];
therefore it appears that immunoinfertility is more a
human concern, and in cows and buffaloes the presence of
such a phenomenon continues to be anecdotal. Studies by
Tripathi et al. [147] found that of the 17 sperm-specific
polypeptides detected to be reactive with antisperm
antibodies, only two were reactive with sera from pre-
sumptive immunoinfertile cows. The different methods of
cervical mucus penetration assays used for bovine
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Exclude effects of
season
Individual
1. Investigate for abnormalities of genital
organs like ovaro-bursal adhesions, cystic
ovaries, tumours, stenosis, etc.
2. Investigate for subclinical endometritis.
When no tests possible, treat on
presumptions if there is a history of
periparturient disease.
3. Monitor ovulations/oestrus cycle length
(i) Provide ovulation induction treatments at AI
(ii) Repeat AI/consider I/U AI
4. If animals do not settle, treat for luteal
insufficiency.
5. Supplement with vitamins A,E and C and Ca, P
and Se.
6. PSP dye test – if both fallopian tubes
occluded. Exclude such animals.
7. Cytogenetic-karyotyping
examinations are more suitable to evaluate motility of
sperm than to predict potential fertility [139, 248, 249]
and hence their use to predict infertility of immunogenic
origin similar to that used in human subjects is suboptimal.
Therapies of immunogenic infertility in animal subjects
appear to be simple; changing the semen or intrauterine
insemination would help taking care of any such problem.
Therapies in human subjects are beyond the scope of the
present review and mostly difficult as sperms to which
antibodies are produced continue to travel through the
female genital tract. A few of the approaches include
administration of vitamins C and E and dexamethasone
[247, 519] and intrauterine insemination of vitamin C
(G.N. Purohit, unpublished work) with little success.
Miscellaneous Therapies
Despite all efforts of therapy, a proportion of RB cows
and buffaloes would continue to be infertile for prolonged
periods and they are described to have infertility of
unknown origin [22, 23]; such an infertility should better
be designated as ‘idiopathic’. Therapy of such idiopathic
infertility is seldom possible. Some of the less common
therapies described for cows suffering from the RB
syndrome include acupuncture therapy [520], intraper-
itoneal insemination [521], use of herbal drugs [522, 523]
and embryo transfer at 7–8 days of oestrus with or
without AI at oestrus [524]. Such therapies, however,
have little to offer in improvement of the condition. Cows
that gain excess of body fat are a classic example of
idiopathic infertility. Such cows, when made to lose weight
by severe diet restriction, often conceive. It is difficult to

comment on such infrequent therapeutic approaches as
their results are inconsistent.
Treatment Approaches
The approach to therapeutics in herds or individual ani-
mals is significantly different. When low fertility to either
AI or natural services is a problem in herds, investigations
must be made on the presence of infectious diseases such
as Campylobacteriosis, by random collection of speci-
mens. Moreover, when natural service is being used, the
bull used must be investigated. Specimens of blood should
be collected to evaluate metabolic profiles. Approaches of
feeding suggested during the dry period and the post
parturient period can help to correct some nutritional
inadequacies and those on combating uterine infection can
reduce fertilization failures or embryonic mortalities.
When faced with therapy of individual animals, the
approach should be first to treat uterine infections and
then treat ovulatory disturbances or corpus luteum
inadequacies (Figure 1). In spite of all these therapies, a
small proportion of cows/buffaloes would have infertility
of unknown origin and it is still extremely difficult to
delineate or treat such obscure infertility.
Conclusion
It can be concluded that diagnosis and therapy of RB
continues to be difficult, but when investigating and
treating individual cows/buffaloes, a systematic approach
of combating uterine infection and correcting ovarian
dysfunction or luteal insufficiency would result in a
majority of animals to conceive provided the management
and breeding techniques are optimal. The feeding of high-
producing cows, especially during the periparturient per-
iod, minimizing stress and parturition hygiene are crucial
to obtain high-fertility postpartum, and dairy farmers must
be educated in this regard. While making selection of
cows for high production, stress must now also be laid on
high fertility. Diagnostic approaches such as hysteroscopy
must be stimulated to widen our knowledge of the inner
surfaces of the genital tract.
Acknowledgements
I am grateful to Dr Vijay Kushwaha, Dr Manish Garg
and Dr Arvind Sharma for the endless help they extended
in the preparation of this paper.
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