Repeat breeding in cattle and buffalo: New concepts in diagnosis

and therapy
Prof G N Purohit

Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Science
Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan 334001 India

Repeat breeding (RB) is one of the biggest reproductive problems in cattle. A lot of reasons have been
assigned for such a problem with failure of fertilization and early embryonic deaths being the two
most common reasons. Some workers feel that modern high-producing Holstein cows have reduced
fertility because of intensive selection for high yields. Because of seasonal suppression of fertility
repeat breeding in buffaloes should be limited to the breeding season. The incidence of the condition
varies widely in different studies and being multifactorial the therapeutic options would depend upon
the cause of RB. In this manuscript the advances in diagnostic approaches and therapy are briefly
explained.

Diagnostic Methods

In view of the wide variety of causes that can result in RB, the diagnostic procedures in the present
manuscript have been classified into the following groups each of which is then discussed in points:

1. Record analysis
2. Visual
3. Trans-rectal 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 peri-parturient disease that have resulted in suboptimal fertility. Insights into poor
fertility can be traced in individual cows by record analysis. However, more often, cows/buffaloes
presented to clinicians with RB originate from diverse changing management strategies, with no
records.

Visual Inspection
Visual inspection of cows can throw some light on the probable cause of animals not conceiving.
One should keep in mind the below mentioned points
1) Focus on proper estrus detection
2) Color, consistency and clarity of cervico-vaginal mucus is important
3) Vaginal discharge should be stringy and not thin and voluminous
4) Ejaculatory thrust of males during natural mating should be visually observed
5) Urovagina is an uncommon problem but when present require surgical correction.

Transrectal palpation
Carefully performed transrectal palpation can help clinicians in arriving at certain decisions.
In particular the clinicians must look for the following which can be a cause of RB:
1) Uterine tone- poor uterine tone at insemination warrants careful consideration
2) Deformed cervices- Such anatomic defects pose difficulty in proper insemination
3) Ovulation detection- Delayed ovulation or anovulation/ovarian cysts
4) CL palpation-Improper CL formation can be a reason for RB
5) Enlarged fallopian tubes/ Ovarobursal adhesions

Vaginoscopy
Although vaginoscopy is a good tool however;
1) Absence of abnormal discharge does not guarantee absence of uterine inflammation
2) Single examinations lack accuracy
3) Discharges are absent when cervix is closed
4) Metricheck is an alternative device

Tests to evaluate uterine health

a) Uterine pH
1) Varies from 7.2-7.6 but uterine sample difficult to collect
2) Both low (6.9) and high (8.5) are detrimental
3) Cows on high protein diet have altered pH
4) pH of uterus difficult to detect clinically

b) Uterine Microbiology
1) Uterine sample must be appropriately collected
2) Sub clinical infections are clinically obscure
3) White side test may give poor results with subclinical endometritis
4) Campylobacter a common cause of poor conception is difficult to be detected

c) Uterine biopsy and cytology

Uterine Biopsy require appropriate instrumentation and since the technique is invasive it must
be reserved for specialized cases only
Uterine Cytology
Cytology evaluation of uterine fluid / cells is an appropriate procedure to evaluate uterine
health and has been suggested widely employing the below mentioned procedures. The
number of PMNs in the sample obtained are evaluated and it is validated that endometritis is
existent. However, there is lack of standards for using cytology as a diagnostic tool for sub
clinical endometritis and is difficult to perform under most clinical settings.
1 Low volume flush (2-5ml)
2 Cytobrush

Metabolic Profile
There appears to be a complex mechanism involved in the interplay of various serum
metabolites: the macro (glucose, total protein and lipids) and micro (calcium, phosphorus,
various vitamins and trace minerals) nutrients with the different reproductive events;
however, because there is an indirect neuro-hormonal mechanism, it is difficult to establish
concrete clinical norms that would predict potential fertility.

In vivo imaging techniques

Ultrasonography (USG) is the most important diagnostic modality that has emerged in the
past, is non-invasive and can help in evaluating the cause of RB specially when performed
sequentially. A single examination is less useful. USG can be used for
1) Evaluating the follicle, CL, ovulation, ovarian cysts.
2) Endometrial folding occurs at estrus as seen by USG which can be helpful in estrus
detection.
3) For evaluation of ovulation examination at 12 hours intervals are suggested. Corpus
luteum (CL) development can be evaluated by Day 4 to 5
4) Detection of embryonic deaths

Hormone assays
Assay of plasma progesterone can be helpful in vadiating the CL
1) Higher progesterone on the day of estrus (Suprabasal progesterone) that arise from
adrenals due to stress leads to poor conception
2) Progesterone assay on the day of estrus and 7 days later can be useful

Immunological and in vitro tests

1) Antibodies against sperm can be detected in the serum
2) Such tests are more useful in humans.

Testing tubal patency

Tubal patency testing is suggested in cows/buffaloes with obscure infertility with no uterine
infection or ovarian dysfunction. PSP Dye test is an old test and some new tests have also
been performed.
1) For PSP Dye test fix a foley catheter is one uterine horn and pass PSP dye (10-20ml). Dye
color appears in urine with in 30 minute if tube is patent and after 3-4 hours if tube is
occluded.
2) Contrast sonography by infusing Echovest 400 as contrast medium in uterus followed by
USG was found useful in one recent study (Kauffold et al 2009)
3) Laproscopic chromopertubation hysterosalpingography not yet reported for cows

Hysteroscopy
The direct visualization of uterus using a hysteroscope can be a good approach to evaluate the
uterine health. A problem with the introduction of hysteroscope in cattle is the anatomic
structure of the cervix. Only a few reports depict the use of this modality
1) Flexible scopes are better
2) Bovine cervix offer the maximum difficulty
3) Sacral epidural anesthesia is suggested
4) Air insufflations/ saline introduction is suggested after reaching the uterus.

Therapies for RB cattle and buffalo

The therapy of RB cows/buffaloes is deemed to be instituted only when estrus 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, and the presence of infectious disease such as
Campylobacteriosis or Trichomoniasis must be investigated along with evaluation of the bull
and the nutrition of the herd.

Treatment of individual cows/buffaloes at most situations remains difficult as a proportion of

animals are always present with obscure reasons of poor fertility. Moreover, most diagnostic
modalities described are largely unavailable to the treating clinician. With limited facilities
the therapeutic approach usually must be aimed sequentially at

(i) Combating uterine infection (endometritis),

(ii) Correcting ovulatory disturbances,
(iii) Supplementing 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 buffaloes. The detailed
therapeutic regimens used widely are described below.

i) Endometritis
Most repeat breeder cows/buffaloes have subclinical uterine infections. The therapies for sub-
clinical endometritis include the intra-uterine infusion of antiseptics, antimicrobials and other
alternative therapies that are described below
Antimicrobials
1) Non spermicidal antibiotics 8-12 hours post insemination
2) Antibiotic + imidazole derivatives for 3-5 days followed by insemination in subsequent
estrus.
3) In vitro sensitivity testing is difficult unless the samples are properly collected
4) Antifungal can be considered if endometritis is chronic and prolonged therapy has been
instituted.

Alternative therapies

PG / Uterine lavage Prostaglandins administered on Day 5 of estrus, return cows to estrus

that favors uterine clearance of infection. Uterine lavage with normal saline along with PG
enhances uterine health.

Immunomodulators
Immunomodulators are potent chemotractants that favor the influx of PMNs in uterine lumen
and thus enhance uterine clearance of microbes. Some of the immunomodulators are
mentioned below:
1) 100 mg of E coli LPS in 20 ml of PBS intrauterine once or twice at an interval of 1 week.
2) 500 mg of enzymes oyster glycogen in 60 ml PBS intrauterine once or twic at an interval
of 1 week.
3) 10-20 ml of homologous or hetrologous serum intra-uterine once.
4) 4 mM Taurine +50 mM fructose in PBS
5) Chymotrypsin
6) Acetylcysteine 600 mg in 50 ml of distilled water intra-uterine
Antioxidants
Antioxidants such as vitamin C (Ascorbic acid 20 mL administered intrauterine) alter the
uterine pH and also favor uterine health due to its antioxidant capacity. Similarly, 3-4 weekly
IM injections of vitamin A and E are also known to improve uterine health.

ii) Ovulation induction

Ovulation induction is required if cows/buffaloes evidence delayed ovulation/anovulation.
The usual and the newer therapies are mentioned below
1) hCG- 1500-5000 IU intravenous or 5000-10000 IU intramuscular
2) GnRH
3) 500 ml of 25% dextrose + insulin 5 ml at AI
4) 300 mg Clomiphene citrate for 2-3 days after a 1% copper sulfate drench
5) 2000- 4000 mg meformin orally + clomiphene
6) Bromocryptine 10 mg orally for 2-3 days
Reducing stress to reduce supra bursal progesterone
Therapy of ovarian cysts is required if they are present.

Luteal insufficiency
Luteal inadequacy due to diminished response to the circulating luteotrophic hormones leads to
insufficient progesterone production during the luteal phase after breeding, and could be the cause of
embryonic death. The serum progesterone is known to be low in RB cows and buffaloes. The
therapies are thus oriented towards improving the CL function and include the following:

1) GnRH at the time of insemination or day 11–13 post AI

2) hCG at the time of insemination and/or on Day 4–7, or day 15–16 post AI
3) Progesterone injection 500-750 mg IM on Day 5 post insemination and repeated at weekly
intervals for 2 weeks, or chlormadinone (10 mg) daily oral feeding from Day 14-23 or
progestagen vaginal implant from Day 5 till Day 13 of insemination.
4) Recombinant bovine somatotropin (500 mg SC) at the time of estrus and 10 days later.

Other therapies include administration of drugs/agents that prevent luteolysis and include
5) Feeding of 50-100 gm fish oil from Day 5 till Day 16 of insemination or feeding of 100-
250 gm of linseed oil for similar intervals.
6) Administration of non-steroidal anti-inflammatory drugs like Flunixin meglumine 1.1
mg/Kg IM on Day 14 to 16, or Meloxicam 0.5 mg/Kg or Caprofen 1.4 mg/Kg body weight
SC.
Recently the IM administration of aromatase inhibitor letrozole in oil has shown to improve
ovulation and CL function. Letrozole administered IM (1mg/kg in oil) on Day 3 of ovulation
increased the CL diameter and the subsequent diameters of the dominant follicles. The use of
OvSynch protocol to repeat breeder cows and buffaloes starting from Day 5-12 of the estrous
cycle have been mentioned to improve the conception rates.

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 impacts of nutrition on fertility appear to be complex, and recommendations for
formulating effective dietary strategies to improve conception rates and prevent embryonic
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. Clinical cases suffering from the RB syndrome at many locations suffer from
multiple deficiencies, especially those of glucose, 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.

Improving the Timing and Technique of Insemination

Much improvement can be expected by improving the timing of insemination essentially by

appropriate estrus detection. A sizeable proportion of cows evidence prolonged estrus
periods and such animals pose problems to time insemination. Multiple inseminations or
ovulation induction treatments are suggested in such animals. Heifers frequently evidence
short estrus periods and it is suggested to time insemination slightly earlier. Buffaloes pose a
greater difficulty in estrus detection and sub-estrus is frequent, hence timing inseminations
becomes difficult. A recent report depicts that the conception rates to artificial insemination
improved by 5–27% over many Asian countries when the personnel involved in the AI were
given various levels of training. Importance should also be attached to the use of semen from
high fertility bulls. Also the frozen semen should be evaluated from time to time along with
the LN2 refilling and other procedures.

Avoiding Periparturient Disease

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 during the dry
period and feeding of high-fibre low-energy chopped straw during the dry period. However,
although parturient problems appear to be unavoidable, stress must be attached to parturient
hygiene. Many locations where cattle and buffaloes 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, inadequate nutrition, high
production, social factors and environment on reproduction has been explained in many other
publications. It is nearly impossible to avoid all forms of stress in dairy cows and buffaloes, but
when animals 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.

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; such 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, intra-peritoneal insemination, use of herbal drugs and embryo transfer at 7–8
days of estrus with or without AI at estrus. 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.

Further reading

Purohit GN (2008) Recent developments in the diagnosis and therapy of repeat breeding cows and
buffaloes. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural
Resources 2008 3, No. 062 pages 1-33.

Parmar SC, Dhami AJ, Hadiya KK, Parmar CP (2016) Early embryonic deaths in bovines: An
overview. Raksha Tech Review 6(1):6-12.

Yapura MJ, Zwiefelhofer EM, Pierson RA, Adams GP (2018) Aromatase inhibitors: A new
approach for controlling ovarian function in cattle. Theriogenology 112:18-25.

Paksoy Z, Das H (2013) Nonsteroid Anti-Inflammatory Drugs to Improve Fertility in Cows. In

Tech Open Chapter 5 In: Success in Artificial Insemination - Quality of Semen and Diagnostics
Employed. Page 73-92.

Saraswat CS, Purohit GN (2016) Repeat breeding: Incidence, risk factors and diagnosis in
buffaloes. Asian Pacific Journal of Reproduction. 5(2):87-95.

Mandhwani R, Bhardwaz A, Kumar S, Shivhare M, Aich R (2017). Insights into bovine

endometritis with special reference to phytotherapy. Vet World 10(12):1529-1532.

Purohit GN (2020) Prospects of hysteroscopy in large domestic animals. Journal of Entomology

and Zollogy Studies. 8(1):466-469.

Purohit GN, Ruhil S, Khinchar V (2015). Postpartum endometritis in dairy cows: current status of
diagnosis, therapy and prevention. Theriogenology Insight 5(1):1-23.

Kauffold J, Groeger S, Bergman K, Wehrend A (2009). Use of contrast sonography to test for tubal
patency in dairy cattle. J Reprod Development 55(3):335-338.