Based on our observations, the overall incidence of RBS was 12.9% in the villages of Meerut District. In different places, incidences have been reported to vary from 1 to 30%, under farm and field conditions
(Bhat et al., 2012; Saraswat and Purohit, 2016;
Asaduzzaman et al., 2016). Out of 78 RBS cases presented, the cases that were associated with and without uterine infection were 44.87% (35/78) and 55.13% (43/78), respectively. The infection ranged from low grade to detectable type in sub-clinical and clinical form as 29.49% and 15.38%, respectively.
Thus, the study showed that primarily nearly 40 to 50% of the repeat breeder animals suffered from uterine infection. However, most of the owners remain ignorant towards the infectious secretions and repeatedly complain of poor fertility, without knowing the actual cause. This may be due to the reason that, all the cows that were presented had normal feeding behaviour and usual cyclicity.
Noakes et al., (2001) have also described subclinical uterine infection as the primary cause of RBS in bovines. It may cause direct endometrial damage or may affect conception by producing toxins. It is also known to have an inhibitory effect on growth of the largest follicle
(Heidari et al., 2016) which in turn may lead to impaired corpus luteum development and function
(Pande et al., 2013). Various other reports also suggest that improved uterine health helps in better conception rates and reduces RBS in bovines
(Kumar et al., 2013; Singh et al., 2017).
Cervical mucus characteristics, white-side test and endometrial cytology
The nature and type of mucus and cervical secretions were categorized to produce a clinical score for differentiating the samples (Fig 1).
The mean score of the mucous character, odour, pH and number of PMN cells in RBS cases associated with and without uterine infection, is presented in Table 1. The cases where uterine infection was associated with RBS, the cows had either mucoid discharge with flecks of pus or discharge containing less than 50% white and yellowish white pus, whereas in other RBS cases, the cervico-vaginal mucous was clear and translucent. Similar trend was found with odour score (Table 1). The overall mean pH of infected sample was more than 8 in 25 animals (69.45%) in uterine infection positive RBS cases. The white side test came to be positive in 28 animals out of 35 animals (77.14%).
The final diagnosis was based on endometrial cytology, wherein a higher proportion of samples were diagnosed as positive for infection (35 out of 78 samples, 44.87%). Very few or no PMN cells were observed in other RBS associated cases (55.13%). The number of PMN cells was significantly higher (P<0.001) in the uterine infection positive RBS cases (Table 1, Fig 2).
Uterine infection associated with RBS is difficult to diagnose under field conditions in normal cyclic cows. Examination of cervical mucous may not always lead to a confirmed diagnosis. However, the present study suggests that if a case is presented as a RBS, the owner should be asked to bring the animal for examination only when it is in oestrus. White side test on cervical mucus would be helpful in diagnosis under field conditions, however it may not be full proof. Endometrial cytology will be the best technique to be employed for confirmation. It is easy, quick, inexpensive and gives reliable results. Earlier reports suggest that white side test and endometrial cytology
(Bhat et al., 2014; Dutt et al., 2017) both may aid in diagnosing sub-clinical uterine infection.
Microbial examination of cervical mucous
The bacteriological investigation of the samples revealed the presence of several bacterial species. Gram negative bacilli were the most common isolate found in the uterus (45%). Other isolates were
Trueperella spp.,
Pseudomonas spp.,
Staphylococcus spp. and
Escherichia coli (Fig 3). Mixed infections were found in 27.48% cases. One animal was also found to be infected with yeast (Fig 3).
E.
coli,
Staphylococcus,
Trueperella,
Bacillus spp.
etc. have also been commonly found, identified and recorded in the past studies in repeat breeders
(Gani et al., 2008; Dutt et al., 2017). However, there are scanty reports which have described the role of yeast in bovine infertility
(Saini et al., 2019).
Antibiotic sensitivity test
The antibiogram of all positive animals varied greatly in terms of zone of inhibition. According to result, the response for Tetracyclin (Fig 4) was found to be best in maximum number of infected cases (17/35; 48.57%) followed by Cephalexin (8/35; 22.86%), Chloramphenicol (7/35; 20.0%), Streptomycin (3/35; 8.57%). Clear zone of inhibition was either absent or had irregular boundaries with scanty colonies in the vicinity for Ampicillin, Penicillin G, Sulphatriad, Gentamicin, Cefepime and Co-trimoxazole discs.
The result of antibiogram study of past reports, had documented use of antibiotics like Gentamicin and Ciprofloxacin
(Sharma et al., 2009), Levofloxacin
(Pandey et al., 2018), Ceftriaxone
(Manjhi et al., 2019), Ampicillin and Amoxycillin
(Yadav and Jadhav, 2018) to be most efficacious and rated efficacy of tetracyclines in the range of 20 to 60%.
Dutt et al., (2017) reported that uterine infection associated microbes shows drug resistance against Oxytetracycline.
However, in this study, tetracycline appeared to be the most effective drug. This was in congruous with reports of
Konigsson et al., (2001) and
Ingale et al., (2016). Few other studies have also reported intrauterine infusion of oxytetracycline to be most useful
(Gohar et al., 2018; Manimaran et al., 2019) in treating uterine infections in bovines. The variations in the reports may be due to the involvement of diverse types of infectious agent. A set list of medications, may be difficult to be prescribed in uterine infection and antibiogram may be helpful in detecting and monitoring trends in antimicrobial resistance.
Animals’ response to the treatment
In this study, the antimicrobial susceptibility was assessed individually and the cases were treated likewise, one-to-one. To the single case, where the infection was due to a yeast, the intra-uterine treatment was done by 0.05% povidone-iodine solution.
On examination of cervical mucous in subsequent oestrus, the infection appeared to be considerably controlled in all the 35 animals. They exuded clear mucous (CM score=0), white-side test was negative and the per cent PMNs infiltration had reduced to 0 to 4%. The animals were allowed to bred, and 57.14% (20 out of 35) were found to be pregnant when pregnancy was checked after 2.5 months following insemination. Rest, fifteen animals, which repeated again, were given Buserelin acetate (Receptal®VET, 2.5 mL) shot along with second AI. Out of fifteen, 5 had confirmed pregnancy while ten, remained to be repeat breeders following second AI. Thus, overall uterine infection associated RBS cases were effectively treated in 71.43% cases.
