|Year : 2020 | Volume
| Issue : 1 | Page : 44-48
Identification of pathogenic microorganisms of repeat breeder dairy cows and a hyperimmune treatment approach
Seyed Morteza Aghamiri1, Mohammad Rahim Ahmadi2, Masoud Haghkhah3, Abdollah Derakhshandeh3
1 Department of Clinical Sciences, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
2 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
3 Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
|Date of Submission||13-May-2019|
|Date of Decision||05-Sep-2019|
|Date of Acceptance||29-Oct-2019|
|Date of Web Publication||21-Jan-2020|
Mohammad Rahim Ahmadi
Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz
Source of Support: None, Conflict of Interest: None
Objective: To investigate the bacterial infections in repeat breeder dairy cows and to evaluate the treatment effects of hyperimmune serum against two main endometritis bacteria, Escherichia (E.) coli and Trueperella pyogenes.
Methods: A total of 29 Holstein multiparous cows with three or more unsuccessful artificial inseminations were carried out and examined to confirm the absence of reproductive tract abnormality and vaginal discharges. Uterine lavage was performed to collect uterine samples for bacterial and fungal cultures. In addition, polymerase chain reaction (PCR) was done to detect Trueperella pyogenes, E. coli, Fusobacterium necrophorum, and Prevotella melaninogenicus. The cytological study was done on cervical mucus. The hyperimmune serums produced against Trueperella pyogenes and E. coli were infused into the uterus of repeat breeder cows and two doses of prostaglandin F2α were administrated intramuscularly within 14 days’ interval.
Results: There were 10 positive samples in the bacterial culture with 19 isolations and no growth of the main causative bacteria of endometritis. In the PCR method, no Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenicus were found. However, 11 positive samples of E. coli were identified by PCR. Cladosporium was detected in one case of repeat breeder cows. The median (interquartile range) of neutrophils in vaginal discharge was 12 (22.5). Eighteen from twenty-nine (62.1%) repeat breeder and eight from eleven (72%) cows with E. coli infections in PCR were made pregnant in the first artificial insemination after treatment by intrauterine infusion of hyperimmune serum and prostaglandin F2 α.
Conclusions: Intrauterine administration of hyperimmune serum could be an alternative to antibiotics for the treatment of repeat breeder cows.
Keywords: Cow; Repeat breeder; Bacterial and fungal culture; Polymerase chain reaction; Hyperimmune serum
|How to cite this article:|
Aghamiri SM, Ahmadi MR, Haghkhah M, Derakhshandeh A. Identification of pathogenic microorganisms of repeat breeder dairy cows and a hyperimmune treatment approach. Asian Pac J Reprod 2020;9:44-8
|How to cite this URL:|
Aghamiri SM, Ahmadi MR, Haghkhah M, Derakhshandeh A. Identification of pathogenic microorganisms of repeat breeder dairy cows and a hyperimmune treatment approach. Asian Pac J Reprod [serial online] 2020 [cited 2021 Sep 29];9:44-8. Available from: http://www.apjr.net/text.asp?2020/9/1/44/275528
| 1. Introduction|| |
The cows with healthy clinical and functional genital tracts that do not get pregnant after three or more artificial inseminations are called repeat breeder cows. About 9%-24% of dairy cows are shown to have this problem,, and cause wastage cost via increasing calving interval, insemination cost, and culling rate,. The causes of repeat breeder syndrome are categorized into three main reasons including ovulation delay, uterine environment and luteal deficiency. Many studies have reported that various factors lead to repeat breeder syndrome such as infectious agents by affecting the sperm or embryo, hormones deficiency and luteal problems, immunologic problem and anti-sperm antibodies, oocytes quality, nutrition, feeding, weather and season,,,,. There are various proposed protocols for treatment of repeat breeder cows such as injection of gonadotropin-releasing hormone or its analogue around artificial insemination,,, post-insemination supplementation with progesterone,,, treatment of human chorionic gonadotropin 5 days after artificial insemination,, intrauterine antibiotics,, and antiseptics, uterine lavage by saline or even acupuncture and moxibustion. Also, the effect of dimethyl sulfoxide was suggested on clearing the uterus from bacterial biofilm as well as improving the fertility of repeat breeder cows after antibiotic treatment.
This study was to detect the presence of the main bacterial causes of endometritis [Escherichia coli (E. coli), Trueperella pyogenes (T. pyogenes), Fusobacterium necrophorum (F. necrophorum), and Prevotella melaninogenicus (P. melaninogenicus)] by polymerase chain reaction (PCR) method in repeat breeder cows and to evaluate of treatment effects of hyperimmune serum against two major pathogen bacteria of endometritis, E. coli and T. pyogenes, on repeat breeder dairy cows.
| 2. Materials and methods|| |
A total of 29 repeat breeder multiparous dairy cows were chosen from a dairy farm in the north of Shiraz, Fars Province, Iran (29° 58′34″N, 52°40′45″E). Cyclic cows with three or more unsuccessful artificial inseminations were classified as repeat breeders. The animals had no detectable abnormality in the reproductive tract and their vaginal discharges were clear during estrus. The average of days in milk of these cows was (346.78 ± 67.18) days. The mean of artificial insemination number was 6.19 ± 2.06. The annual average milk yields of the cows were 10 000 L. The body condition score of all cows were 2.75-3.50 (scale 1-5). The cows were housed in free-stall barns and fed by alfalfa hay, corn silage, and concentrates (a balanced mixture of barley and corn grain, soybean meal, wheat bran, vitamins, and minerals).
2.2. Clinical examination
To confirm the absence of clinical abnormalities, the uterine horns and ovaries of the cows were examined by rectal palpation and 5-MHz linear transducer ultrasonography (Easi-Scan®, BCF, UK). The vaginal discharges of the cows were evaluated by a gloved hand and only the animals with clear mucus were chosen for this study.
2.3. Uterine samples collection
To collect uterine samples for bacterial culture and PCR, uterine lavage was performed on 29 clinically healthy repeat breeder cows by infusion of 60 mL saline solution into the uterus and retraction about 10 mL of it. The collected samples were transferred to the laboratory on ice. All bacterial and fungal cultures were done in less than 6 h from sampling.
2.4. Cervical cytology of samples
The discharges of cervical mucus were collected for cytological study. Gentle collection of the discharge was done by a 50 mL syringe connected to a plastic uterine pipette. By putting a drop of cervical mucus between two slides, thin smears were prepared. The smears were stained with Giemsa and neutrophils were counted in 20 microscopic fields (× 1 000).
2.5. Bacteriological and fungal culture
Aerobic culture on sheep blood agar and MacConkey agar (Merck, Germany) was done at 37 °C for 48 h. Anaerobic culture on sheep blood agar was performed up to seven days. The isolates were identified by standard biochemical tests. Spot inoculation technique was used on Sabouraud Dextrose agar to diagnose fungal infection. The inoculated uterine discharges on Sabouraud Dextrose agar were incubated at 25 °C for 2 weeks. Based on colony characteristics and staining reaction, the identification of fungal agents was performed.
2.6. DNA extraction from uterine fluid
The samples were centrifuged at 5 000 × g for 10 min. After transferring the sediment into 2 mL micro tubes, 200 μL of the suspension was used to extract DNA by AccuPrep® Genomic DNA Extraction Kit (Bioneer, South Korea).
2.7. Detection of E. coli, T. pyogenes, F. necrophorum and P. melaninogenicus by PCR
Standard PCR method was performed to detect E. coli, T. pyogenes, F. necrophorum, and P. melaninogenicus by predesigned primers [Table 1]. The positive controls were E. coli ATCC 35218, P. melaninogenicus ATCC 25845, native T. pyogenes and F. necrophorum. Amplification products were visualized by electrophoresis through 1% (wt/vol) agarose gel with 0.5 μg/mL ethidium bromide. If the amplicons had expected molecular size, they were considered as positive [Figure 1].
|Figure 1: PCR amplification products of genomic DNA of uterine discharges. A: Escherichia coli (340 bp); B: Trueperella pyogenes (270 bp); C: Fusobacterium necrophorum (402 bp); D: Prevotella melaninogenicus (389 bp). M: molecular weight marker; C+: positive control; Lane 1–5: samples from uterine discharges.|
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2.8. Treatment of repeat breeder cows using hyperimmune serum
Yavari et al showed T. pyogenes and E. coli were the most prevalent bacteria in the Iranian dairy cows with clinical endometritis, In this study we used the hyperimmune serums that had been produced before examination of cows based their inoculation. For the preparation of the hyperimmune serums, soy broth cultured bacteria pellets had been sonicated. Then by two-week intervals, each bacterial antigen had been injected subcutaneously to two healthy heifers. Antibodies had been reached peak value after eight injections. The hyperimmune serums had been prepared for uterine infusion by mixing 25 mL of hyperimmune serums separated from heifers’ blood with 25 mL sterile 1 × phosphate buffer saline. Following sampling, hyperimmune serum was infused into uterus and two doses of prostaglandin F2α, 500 mg of cloprostenol sodium (EstroPlan®, IM, UK) were administrated intramuscularly within 14 days’ interval. Subsequently, the cows were inseminated about 12 h after the standing heat by the same person. Pregnancies were diagnosed by ultrasound examination (Easi-Scan®, BCF, UK) about 35-40 days after artificial insemination.
2.9. Statistical analysis
Data were analyzed by SPSS software (SPSS for Windows, version 11.5, SPSS Inc, Chicago, Illinois). Pregnancy among different groups of culture and PCR results were compared by the Chi-square test. Significant difference was considered when P<0.05.
2.10. Ethics approval
All experiments were approved by the State Committee on Animal Ethics, Shiraz University, Shiraz, Iran (ethical approval number: 90GCU1M1599). The recommendations of the European Council Directive (2010/63/EU) of September 22, 2010, were followed by the Ethics Committee.
| 3. Results|| |
3.1. Bacteriological and fungal culture
There were 10 (34.5%) positive samples in the bacterial culture (19 isolations) and the remaining 19 (65.5%) showed no bacterial growth. The isolated bacteria were Actinobacter spp. (21.05%), Micrococcus spp. (10.52%), Pasteurella caballi (10.52%), Staphylococcus saprophyticus (5.26%), Staphylococcus lentus (5.26%), Streptococcus dysgalactiae (5.26%), Bacillus coagulans (5.26%), Bacillus lentus (5.26%), Kurthia spp. (5.26%), Corynebacterium renale (5.26%), Rhodococcus equi (5.26%), Actinomyces spp. (5.26%), Burkholderia spp. (5.26%), Nocardia spp. (5.26%). By fungal culture, Cladosporium was detected in the one case of repeat breeder cows.
3.2. PCR detection
Following the PCR method to diagnose the major pathogens of chronic subclinical endometritis, no T. pyogenes, F. necrophorum, and P. melaninogenicus were found. However, 11 positive samples of E. coli were identified by PCR.
3.3. Cervical cytology
The median of polymorphonuclear neutrophils (PMNs) in the vaginal discharge was 12 (min: 0, max: 50, interquartile range: 23). The median of PMNs in the positive and negative bacterial culture and also, in the positive and negative PCR were shown in [Table 2]. In addition, there were grouping repeat breeder cows by the cut-off point at 8% PMNs. The percentage of PMNs was ≤ 8 in 10 and > 8 in 19 cows. Positive results of bacterial culture and PCR were not caused more cows positioning on > 8% PMNs category [Table 2].
|Table 2: Comparison of PMNs (median and interquartile range) and PMNs categories with culture and PCR detection of bacteria in repeat breeder cows.|
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3.4. Pregnancy results
Eighteen cows from twenty-nine (62.1%) were pregnant in the first artificial insemination after treatment by intrauterine infusion of hyperimmune serum and PGF2α. Pregnancy of positive bacterial culture was higher than negative cows after treatment insignificantly (90% versus 47.4%). Also, more positive PCR cows were pregnant after treatment than negative that it was not significant [Table 3]. The median (interquartile range) of PMNs in pregnant and non-pregnant cows after treatment was 15 (23.5) and 11 (19), respectively. On the PMNs grouping, 50% (5/10) of ≤ 8% category and 68% (13/19) of > 8% category were pregnant after treatment that their difference was not significant.
|Table 3: Comparison of pregnancy with culture and PCR detection of bacteria in repeat breeder cows.|
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| 4. Discussion|| |
Repeat breeder syndrome is one of the main problems in the reproduction of cattle, especially in high producing dairy cows. Infectious agents are one of the multiple causes that were mentioned for repeat breeder syndrome,,,. E. coli is the most common bacteria isolated from the uterus in the first week after parturition but in weeks 2 and 3 T. pyogenes and its related bacteria, F. necrophorum and P. melaninogenicus are common causes of uterine diseases such as endometritis,. These four bacteria are the major pathogens of endometritis. It is suggested that non-specific genital infections are most prevalent in repeat breeder cows and many different bacteria were isolated from their uterus. Bacterial cultures of this study have shown many different infections. But any of the pathogen bacteria were not grown. However, in the PCR method 11 cows were positive for E. coli. Some studies have not found relations between infectious agents and repeat breeder syndrome but maybe using better methods to detect microorganisms could change it. Just like there are some reports on many cases of the negative bacterial culture in the clinical endometritis of cows, and also, bacterial biofilms detected in 60% of the uterus of the repeat breeder cows showing probable inefficiency of the traditional methods to detect bacteria and infectious agents in the uterus. Nowadays, PCR is an accurate and fast method of the culture-independent identification of microorganisms and there are numbers of PCR studies on uterine discharges,,,. These observations could probably show the necessity of more bacterial investigation of the repeat breeder cows with accurate methods.
In this study, one case of Cladosporium was grown from uterine lavage fluids. There are some reports of fungi and yeast in uterine samples of cows with repeat breeder syndrome and endometritis. Some studies have mentioned the subclinical endometritis as a cause of repeat breeder syndrome,,. The median of PMNs of cervical discharges was 12 (22.5). Because of the different techniques for acquiring samples, various PMNs number threshold was used such as 1%, 3% and 5% in uterine discharges. In this study, cut-off points at 8% PMNs have set for cervical discharges and there was no difference in post-treatment pregnancy. In this study, lesser cows with positive PCR (45.5%) compared to negative (77.8%) were positioned on the > 8% PMNs category [Table 2]. Inactive forms of bacteria such as biofilms may not stimulate increasing uterine PMNs but could be detected by PCR methods. Also, some other bacteria may increase PMNs that not found in the present study.
In our study, 18 from 29 cows (62.1%) with repeat breeder syndrome were conceived after treatment with hyperimmune serum. Recently, researches were focused on hormonal and functional causes of repeat breeder syndrome mostly,,,, because of physiological changes of high producing cows and their problems with ovulation time and corpus luteum function. Also, in many studies, infectious agents were not a serious problem in repeat breeder cows but it has been advised that reproductive indexes could be improved by antibiotic treatment. There was no significant difference in treatments of repeat breeder cows between uterine lavage with normal saline with or without cephapirin. It appears that causes of repeat breeder syndrome in dairy cows vary in different countries, even from farm to farm, which is related to the management of transition period, parturition and the postpartum period, genital tract infections, nutrition, genetics, estrus detection, and artificial insemination protocols,,.
Ahmadi et al treated clinical endometritis in dairy cows by the administration of the hyperimmune serum and reported acceptable clinical improvement compared to oxytetracycline and prostaglandin F2α. The limited effects of most of the antibiotics on major pathogenic bacteria in the uterus, inhibition of cellular immune response and withdrawal times for milk consumption,, have reduced the tendency for the intrauterine administration of antibiotics. In this study, the hyperimmune serum against two major uterine pathogens, T. pyogenes, and E. coli were produced. Eighteen out of 29 (62%) repeat breeder cows were conceived by first post- treatment insemination. Also, eight from 11 (72%) cows with E. coli infections in PCR were pregnant after hyperimmune treatment. So, hyperimmune serum could be a specific, effective and harmless replacement of antibiotics in the treatment of repeat breeder cows. Besides the bacterial result, the improvement effects of hyperimmune serum may show bacterial presence and their importance in the occurrence of repeat breeder syndrome. The presence of these bacteria in the uterus could interfere conception by exerting toxic effects on the embryo or endometrium,,. Although the effects of other components of serum such as complement proteins, anti-inflammatory, growth factors should not be ignored. Lange- Consiglio et al reported 70% of repeat breeder cows were conceived by intrauterine administration of platelet concentration 48 h after artificial insemination and it is because of improvement of the uterine environment and counteract subclinical endometritis.
In conclusion, this study proved that PCR is an accurate method of detecting uterine bacterial infections including repeat breeder cow syndrome. The intrauterine administration of hyperimmune serum could be a harmless alternative to antibiotics, reduce the loss brought by repeat breeder syndrome and prevent the culling of the valuable cows.
Conflict of interest statement
All authors declare that there is no conflict of interest.
Seyed Morteza Aghamiri participated in the sampling and laboratory tests such as PCR, bacterial culture and cervical cytology. Mohammad Rahim Ahmadi designed the project, prepared hyperimmune serum and performed sampling and treatments. Masoud Haghkhah and Abdollah Derakhshandeh participated in the PCR test and bacterial and fungal culture.
| References|| |
Ferreira RM, Chiaratti MR, Macabelli CH, Rodrigues CA, Ferraz ML, Watanabe YF, et al
. The infertility of repeat-breeder cows during summer is associated with decreased mitochondrial DNA and increased expression of mitochondrial and apoptotic genes in oocytes. Biol Reprod
Båge R, Gustafsson H, Larsson B, Forsberg M, Rodrıguez-Martınez H. Repeat breeding in dairy heifers: Follicular dynamics and estrous cycle characteristics in relation to sexual hormone patterns. Theriogenology
Yusuf M, Nakao T, Ranasinghe RBK, Gautam G, Long ST, Yoshida C, et al
. Reproductive performance of repeat breeders in dairy herds. Theriogenology
Amiridis G, Tsiligianni T, Dovolou E, Rekkas C, Vouzaras D, Menegatos I. Combined administration of gonadotropin-releasing hormone, progesterone, and meloxicam is an effective treatment for the repeat- breeder cow. Theriogenology
Moss N, Lean I, Reid S, Hodgson D. Risk factors for repeat-breeder syndrome in New South Wales dairy cows. Prev Vet Med
Noakes DE, Parkinson TJ, England GC. Veterinary reproduction and obstetrics
. London: WB Saunders Elsevier, Ltd; 2009.
Sood P, Zachut M, Dekel I, Dube H, Jacoby S, Moallem U. Preovulatory follicle characteristics and oocyte competence in repeat breeder dairy cows. J Dairy Sci
Srivastava S, Shinde S, Singh S, Mehrotra S, Verma M, Singh A, et al
. Antisperm antibodies in repeat-breeding cows: Frequency, detection and validation of threshold levels employing sperm immobilization, sperm agglutination and immunoperoxidase assay. Reprod Domest Anim
Kharche S, Srivastava S. Dose dependent effect of GnRH analogue on pregnancy rate of repeat breeder crossbred cows. Anim Reprod Sci
Peters A. Veterinary clinical application of GnRH—Questions of efficacy. Anim Reprod Sci
Kendall N, Flint A, Mann G. Incidence and treatment of inadequate postovulatory progesterone concentrations in repeat breeder cows. Vet J
Villarroel A, Martino A, BonDurant RH, Dèletang F, Sischo WM. Effect of post-insemination supplementation with PRID on pregnancy in repeat- breeder Holstein cows. Theriogenology
Khoramian B, Farzaneh N, Garoussi MT, Mohri M. Comparison of the effects of gonadotropin-releasing hormone, human chorionic gonadotropin or progesterone on pregnancy per artificial insemination in repeat-breeder dairy cows. Res Vet Sci
Ahmadi MR, Dehghan SA. Evaluation of the treatment of repeat breeder dairy cows with uterine lavage plus PGF2α, with and without cephapirin. Turk J Vet Anim Sci
Ahmadi MR, Derakhshandeh A, Shirian S, Daneshbod Y, Ansari-Lari M, Nazifi S. Detection of bacterial biofilm in uterine of repeat breeder dairy cows. Asian Pac J Reprod
Perez-Marin CC, Moreno LM, Calero GV. Clinical approach to the repeat breeder cow syndrome. In: Perez-Marin CC (ed.). A bird’s-eye view of veterinary medicine
. Rijeka: InTech; 2012.
Dubuc J, Duffield T, Leslie K, Walton J, LeBlanc S. Definitions and diagnosis of postpartum endometritis in dairy cows. J Dairy Sci
Aghamiri S, Haghkhah M, Ahmadi M, Gheisari H. Development of a multiplex PCR for the identification of major pathogenic bacteria of post-partum endometritis in dairy cows. Reprod Domest Anim
Ahmadi MR, Nazifi S, Reza Ghaisari H. Comparative cervical cytology and conception rate in postpartum dairy cows. Vet Arhiv
Markey B, Leonard F, Archambault M, Cullinane A, Maguire D. Clinical veterinary microbiology
. 2 ed. St Louis: Mosby Ltd; 2013.
Derakhshandeh A, Aghamiri SM, Ahmadi MR, Mirzaei A. Prevalence of opportunistic fungi and their possible role in postpartum endometritis in dairy cows. Vet Sci Dev
Yavari M, Haghkhah M, Ahmadi MR. Bacterial study of clinical postpartum endometritis in Holstein dairy cows. J Vet Res
Ahmadi MR, Hosseini A, Gheisari HR, Yavari M. Preliminary trial in treatment of postpartum endometritis with intrauterine application of hyperimmune serum in dairy cows. Asian Pac J Trop Dis
Singh M, Sharma A, Sharma A, Kumar P. Repeat breeding and its treatment in dairy cattle of himachal pradesh (India) - A review. Indian J Anim Reprod
LeBlanc SJ, Osawa T, Dubuc J. Reproductive tract defense and disease in postpartum dairy cows. Theriogenology
Williams EJ, Fischer DP, Pfeiffer DU, England GC, Noakes DE, Dobson H, et al
. Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. Theriogenology
Pothmann H, Prunner I, Wagener K, Jaureguiberry M, de la Sota RL, Erber R, et al
. The prevalence of subclinical endometritis and intrauterine infections in repeat breeder cows. Theriogenology
Westermann S, Drillich M, Kaufmann T, Madoz L, Heuwieser W. A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows. Theriogenology
Guo MY, Wang GQ, Shao G, Tian W, Xu Y, Xie G. Identification of intrauterine Mycoplasma in endometritis of dairy cow isolates by 16S rDNA nested PCR-DGGE. J Anim Vet Adv
Bicalho M, Machado V, Oikonomou G, Gilbert R, Bicalho R. Association between virulence factors of Escherichia coli, Fusobacterium necrophorum
, and Arcanobacterium pyogenes
and uterine diseases of dairy cows. Vet Microbiol
Liu M, Wu C, Liu Y, Zhao J, Yang Y, Shen J. Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes
in bovine endometritis. J Dairy Sci
Sharma S, Singh M. Mycotic endometritis in cows and its therapeutic management. Intas Polivet
Bogado Pascottini O, Hostens M, Opsomer G. Cytological endometritis diagnosed at artificial insemination in repeat breeder dairy cows. Reprod Domest Anim
Lange-Consiglio A, Cazzaniga N, Garlappi R, Spelta C, Pollera C, Perrini C, et al
. Platelet concentrate in bovine reproduction: effects on in vitro
embryo production and after intrauterine administration in repeat breeder cows. Reprod Biol Endocrinol
Salasel B, Mokhtari A. Effect of early postpartum PGF2α treatment on reproductive performance in dairy cows with calving and puerperal traits. Theriogenology
Lucy M. Reproductive loss in high-producing dairy cattle: Where will it end? J Dairy Sci
Jaureguiberry M, Giuliodori M, Mang A, Madoz L, Pothmann H, Drillich M, et al
. Repeat breeder cows with fluid in the uterine lumen had poorer fertility. J Dairy Sci
Katagiri S, Takahashi Y. Potential relationship between normalization of endometrial epidermal growth factor profile and restoration of fertility in repeat breeder cows. Anim Reprod Sci
Hill J, Gilbert R. Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium. Aust Vet J
Kasimanickam R, Kasimanickam V, Kastelic J. Mucin 1 and cytokines mRNA in endometrium of dairy cows with postpartum uterine disease or repeat breeding. Theriogenology
[Table 1], [Table 2], [Table 3]