Effect of nutritional status of dairy cows on the quality of ovarian follicles: an in vitro study

Abstract

The reproductive efficiency of dairy cattle around the world is declining as greater selection pressure for increased milk production is applied. In addition, nutrition also adversely affects reproductive performance. The purpose of this study was to investigate if the metabolic profile, that is to say, metabolism-associated serum analytes, bore any relation to the in vitro follicle growth and progression of ovarian cortex cultured in the presence of cows sera. Foetal calf serum (FCS) usually at a concentration of 10% in culture medium such as M199, forms the basis of most tissue culture systems including ovarian follicle cultures and has proved suitable for various species. In this study, cow serum (CS), sourced from a large number (17) of differently managed farms and/or animals to provide some variation in metabolic profile was used to replace FCS in the culture media. To date, no studies have been reported using sera other than foetal calf serum or calf serum for the in vitro culture of ovarian tissue. This study has shown that bovine ovarian follicle growth and progression can be supported in vitro by culture medium containing cow serum (CS). Morphologically the ovarian cortex remained viable after being cultured for a 10 day period in medium containing the gonadotropin follicle stimulating hormone (FSH) and supplemented with 10% CS. To assess the degree of follicular growth and progression occuring in vitro, quantitation criteria based on histological classification of follicles as either F0 (primordial follicle- one layer of flattened somatic cells around the oocyte) or F₁ (primary follicle-a single layer of cuboidal granulosa cells around the oocyte) or F₂ (secondary follicle-surrounded by two or more complete layers of cuboidal granulosa cells) and their diameters as measured using image analysis software, were established. Follicles with diameters within diameter limits of 23-32, 41-53 and 55-70µm were classed as F₀, F₁ or F₂ respectively. To confirm if the follicle progression seen morphometrically in vitro was being driven by the genes known to regulate the process in vivo, the expression of Growth Differention Factor-9 (GDF-9) and Bone Morphogenetic Protein-15) (BMP-15) as represented by the relative concentrations of their respective mRNAs was determined by Quantitative Polymerase Chain reaction (qPCR), was assessed. Relative to the reference gene (actin), GDF-9 and BMP-15 were expressed nearly 14x and 6x respectively in cultures demonstrating high follicle growth. In the absence of FSH in the cultures, gene expression levels were not elevated. Follicular growth and progression levels was negatively correlated with plasma concentrations of β-hydroxybutyric acid but not with other metabolic profile analytes or the fertility marker, antimüllerian hormone (AMH). This suggests that in vitro follicular growth in CS is influenced by the physiological/reproductive status of the cow and we conclude that an in vitro culture system is a practical approach for a sensitive follicle growth bioassay to assess the effect of the internal millieu with respect to fertility.