repeat breeding in cattle and buffaloes

Review

Recent developments in the diagnosis and therapy of repeat breedingcows 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: [email protected]

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

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

modern high-producing Holstein cows have reduced ferti-

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

http://www.cababstractsplus.org/cabreviews

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

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

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, asmore 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


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

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

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

Incidence (%) BreedType ofmanagement Reference

Cows14.4–27.0 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 Jersey�HF VM [474]8.98 Crossbred VM [41]6.8 Crossbred VM [475]19.8 Jersey�Gir FM [476]8.20–9.30 HF�Tharparkar FM [477]7–25.0 Cows, buffaloes FM [478]7.4–18.6 Danish Red, Sahiwal, HF FM [190]25.9 HF�Gir 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 HF�Deoni 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 Sahiwal�Friesian FM [488]7–17.0 Rathi VM [280]12.0 HF crossbred Clinics [489]

Buffaloes7.37 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.


G.N. Purohit 3

[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

Various reproductive tract abnormalities have been

described to be a reason for RB both in cattle [20,

111–114] and buffalo [115, 116]. Only a few of these

described conditions can be discerned clinically, such as

infections of the tubular tract from vagina to fallopian

tubes or ovarian cysts and sometimes ovarobursal adhe-

sions.

Infectious agents present in the genital tract may

hamper fertilization and early embryo development as

well. These organisms when present in pathological num-

bers may produce some toxins or render the uterine

milieu unfavourable for conception. Infections must be

suspected when there is a moderate to mild degree of

endometritis, evident because of flakes of pus or floccu-

lent material in cervico-vaginal mucus discharge at oes-

trus. However, when the infections are subclinical they

remain obscure clinically but still may hamper conception.

A wide variety of bacteria, viruses, fungi and protozoa

have been revealed to hamper conception and have been

widely reviewed elsewhere [30, 117–123], with bacteria

being a particularly common problem.

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

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

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

stressors, e.g. high milk production, postpartum

disorders and negative energy balance, inflammations and

infections, lameness, social factors, transport and heat

stress.

Highmilk 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].

Diagnostic Methods

In view of the wide variety of causes that can result in RB,

the diagnostic procedures in the present review have

been classified into the following groups:

1. Record analysis

2. Visual

3. Recto-genital palpation

4. Vaginoscopy

5. Tests to evaluate uterine health:

(a) Uterine pH

(b) Uterine microbiology

(c) Uterine biopsy and cytology

6. Metabolic profiles

7. In vivo imaging techniques

8. Immunological tests

9. Endoscopy

10. Tubal patency testing

11. Hormone assays

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.

Visual

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



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.

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


G.N. Purohit 5

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 40mm hemisphere of silicon attached

to a 500mm 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

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 (2–5ml) 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.



Metabolic Profile

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

morphological changes otherwise undetected by techni-

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

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

Parameter

Cows Buffaloes

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]


G.N. Purohit 7

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

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

30min; 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.

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 cm�9.5 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 cm�10mm

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


G.N. Purohit 9

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

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 100mg of E. coli LPS

dissolved in 20ml 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 500mg)

dissolved in 60ml of vehicle or 30 nmol/l of leukotriene B4increases 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

(50–100ml 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

that has been tried is lysozyme [346] with a good success

rate. Some medicaments like 4mM taurine and 50mM

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


G.N. Purohit 11

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 100mg 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 500ml of 25% dextrose at oestrus along with

insulin (5ml 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]. Lopez-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

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 300mg 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 (300mg 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 2000–4000mg 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 10mg given orally

12 h before and at the time of AI. In trials on ewes,

0.6–1.2mg 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 200mg injection of proges-

terone in oil [412] or insertion of intravaginal progestagen

implants [413, 414], 100mg 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 (100mg) on day 0,

followed by prostaglandin on day 7, GnRH (100mg) on day9 and AI 16–20 h later. The second dose of GnRH assures

ovulation of the newly formed follicle. Lopez-Gatius and

Lopez-Bejar [387] were of the opinion that a dose of

500mg 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

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 15mm 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].


G.N. Purohit 13

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

(500mg) on day 5 post AI [461], chlormadinone daily

oral feeding (10mg) 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 (500mg 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

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

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


G.N. Purohit 15

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

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

Female

Investigate andadvise

1. Nutrition (preparturient)2. Collect samples for investigation of infectious disease3. Reduce stress4. Metabolic profiles

Naturalmating

Male

Herd

AI

1. Evaluate semen and AI techniques

Repeat breeder cow/buffalo Exclude effects ofseason

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 AI4. 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

1. Infectious disease (i) Trichomonas (ii) Campylobacter2. Semen evaluation3. Age of bull

Figure 1 Diagnostic approaches for repeat breeding



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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