MORPHOMETRIC AND MOLECULAR STUDIES OF THE CORRELATION BETWEEN CHRONIC MASTITIS AND INFERTILITY IN DAIRY CATTLE

Dairy cow fertility has declined over the past decades even within the recent developments in functional genomics and genetic selections. There is overwhelming evidence that increasing genetic merit for milk yield considerably reduces fertility and the strength of negative associations between yield and fertility is obviously towards a rising trend. The deterioration of resistance or increased susceptibility to mastitis as well as reduced fertility in today’s cow, are the examples of undesirable side-effects of the high emphasis on production traits in selection decision. Although, recent studies suggested an association between reproductive failure and mastitis in lactating dairy cows, our understanding of how mastitis affects reproduction is still limited. In this context, we investigated the effects of naturally occurring chronic mastitis on fertility particularly at the level of bovine ovarian stroma with matrix fibers, follicular growth factors, characteristic micro-architecture of stromal vasculature, as well as growth and development of follicles by macroscopic and microscopic morphological as well as immuno-detection based molecular analysis of the ovaries collected at slaughter. Ovaries and milk samples were collected from 74 cows at slaughter. Milk samples from each quarter, were analyzed following National Mastitis Council procedures. Following macroscopic assessment, ovaries of 63 animals were scored as healthy, intermediate and pathological. Based on the presence of major pathogens and somatic cell count results, animals were sorted in 9 groups but only the two extremes were considered for further analysis: uninfected (n = 8) and affected by chronic mastitis (n = 9). Primordial, primary and secondary follicles were counted and scored on similar surface area of ovary sections for each animal (mean ± SD= 5.65 ± 0.25 cm2). They were analyzed with Fisher’s exact test and the association between health status and follicle number was estimated by odds ratios ± confidence limits. Stromal cells with matrix collagen fibres were visualized with Masson trichrome staining. Vasculature area in the ovarian cortex of healthy and mastitic animals was identified using Bandeiraea simplicifolia-I (BSL-I) Lectin. Results were quantified with the dedicated software MacBiophotonics image J, NIH, USA and subsequently analyzed with t-test for statistical significance. Follicles were further characterized by immunostaining with a GDF-9 specific antibody. GDF positive follicular area was measured as well as using a relative scale its fluorescence was semi-quantified. Our results indicate no noticeable macroscopic alteration amongst various macroscopic parameters. Similarly, no (P>0.05) difference was observed between the numbers of primordial and primary follicles in healthy and affected animals. In contrast, the number of secondary follicles was significantly lower in sick animals (Odds ratio 10.50*; p<0.05), indicating a 10 times higher risk for a mastitis animal to have less than 2 secondary follicles per square centimeter. In the healthy and mastitic animals the stromal collagen were (1.61±0.90% vs. 6.046±1.85% P<0.001), indicating mastitis is linked with stromal alterations. Ovarian stromal vasculature represented the cortical area of 6.38±0.66 % in healthy vs. 4.24±0.37%; (P<0.001) in affected cows. Likewise, reduced GDF-9 expressed cortical area, healthy vs. sick (1.97±0.83% vs. 1.04±0.11%; P<0.05), with less immuno-fluorescence was observed in mastitic animals. Our results show that chronic mastitis is associated with considerable alterations in follicle growth and differentiation with a decreased ability of primary follicles to develop into the secondary state in affected animals. This is accompanied by a significant increase of stromal collagen with reduced vasculature and the down regulation of the follicle differentiation factor GDF-9. All together, these findings substantiate the hypothesis that mastitis can reduce fertility by exerting a negative effect on ovarian function.