170 NATURALLY OCCURRING CHRONIC MASTITIS COMPROMISES FOLLICULOGENESIS, AFFECTS VASCULARIZATION, AND INTERACTS WITH DIFFERENTIATION FACTOR GDF-9 IN BOVINE OVARIAN STROMA

Abstract

Recent studies have suggested an association between reproductive failure and mastitis in lactating dairy cows, but our understanding of how mastitis affects reproduction is still limited. In the present study we investigated the effects of naturally occurring chronic mastitis on the population dynamics of ovarian follicles. Ovaries and milk samples were collected from 74 cows at slaughter. Milk samples from each quarter, were analyzed following National Mastitis Council procedures. Based on the presence of major pathogens and somatic cell count results, animals were sorted in 9 groups, but only the 2 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. Vasculature area in the ovarian cortex of healthy and mastitic animals was identified using Bandeiraea simplicifolia-I lectin (BSL-I). 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. The intensity of the staining was semi-quantified using a relative scale: 0, 1, and 2 for no, weak, and strong staining, respectively. Our results indicate no (P > 0.05) difference 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 10 times higher risk for a mastitic animal to have less than 2 secondary follicles per square centimeter. Ovarian stromal vasculature represented the 6.38 ± 0.66% of cortical area in healthy animals v. 4.24 ± 0.37% (P < 0.001) in affected cows. The GDF-9 immunostaining revealed decreased fluorescence intensity 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 decrease of ovarian vasculature and the down-regulation of the follicle differentiation-associated factor GDF. The present findings substantiate the hypothesis that mastitis can reduce fertility by exerting a negative effect on ovarian function.