Genital microbial profile of repeat breeding in a Kangal sheep flock: a survey of ewes and rams under natural mating

Infertility in sheep remains a significant concern in modern livestock production, with non-pregnancy rates during the breeding season increasing considerably in recent decades. The present study was conducted in a Kangal sheep flock with a documented history of reproductive failure, characterised by consecutive estrous cycles without conception. A total of 905 multiparous Kangal ewes (3–6 years old) and 44 Kangal rams (4–6 years old), all with documented fertility records, were enrolled at the beginning of the breeding season. Before mating, all ewes underwent transrectal ultrasonographic examination and evaluation of vaginal discharge. Following this screening, 880 ewes with no ultrasonographic abnormalities and normal vaginal discharge were monitored throughout the breeding season. Of these, 171 ewes that failed to conceive after the first mating underwent a second mating cycle, resulting in 50 additional pregnancies. The remaining 121 non-pregnant ewes underwent a third mating, resulting in 36 additional pregnancies. Blood samples were subsequently collected from the 85 ewes that remained non-pregnant after three consecutive mating attempts. Of these, 21 tested seropositive for Coxiella burnetii and border disease virus (BDV) and were excluded from the study. Vaginal swabs were collected from 25 of the remaining 64 ewes that tested negative for Brucella spp., C. burnetii , and BDV. These animals were classified as repeat breeders or clinically affected animals (Group 2). An equal number of ewes ( n =25) that tested negative for these pathogens and conceived after a single mating cycle were assigned to the healthy control group (Group 1). Preputial swabs were also collected from 15 breeding rams to evaluate the potential for venereal transmission. All swab samples were subjected to microbiological culture and polymerase chain reaction (PCR) analysis following DNA extraction. The targeted pathogens included Brucella spp., Mycoplasma spp., Chlamydophila spp., Campylobacter spp., Coxiella burnetii , Salmonella Abortusovis, Trueperella pyogenes , and Klebsiella pneumoniae . Although Mycoplasma spp., T. pyogenes , and Campylobacter spp. were detected in swab samples from both groups, the prevalence of Mycoplasma spp. ( p _adj <0.001) and Mycoplasma bovigenitalium ( p adj=0.05) was significantly higher in Group 2 than in Group 1. The increased detection of these microorganisms in reproductively compromised ewes suggests a potential role in the aetiology of ovine infertility, either through direct pathogenic effects or by contributing to reproductive tract dysbiosis. However, further controlled studies are warranted to establish a definitive causal relationship between these microorganisms and reproductive failure in sheep.