103 Seasonal effects on follicular metabolome in Italian Mediterranean buffalo

Buffalo is a short-day breeder, and reproductive seasonality is a major factor impairing buffalo farming. An evident effect of season was demonstrated on oocyte developmental competence, known to be affected by follicular environment (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The aim of this work was to evaluate the differences in the follicular metabolome between seasons. Buffalo ovaries were collected at a slaughterhouse in autumn (breeding season, BS), and in mid-winter (non-breeding season, NBS). Follicular fluid (FF) was aspirated from follicles (Ø < 0.8 cm), poured into a dish to remove the oocyte, and centrifuged (300 g × 10 min at 4°C) to separate FF from granulosa cells. The supernatant was centrifuged (2000 g × 10 min and 6500 g × 30 min) and stored at −80°C. After chemical extraction, nuclear magnetic resonance spectroscopy was performed on 4 FF samples (pool of 30/35 follicles) per each season. Orthogonal partial least-squares discriminant analysis (OPLS-DA) and Variable importance in projection (VIP) were carried out. The OPLS-DA showed that samples collected in BS and NBS clustered separately, indicating seasonal differences in the metabolomic composition of FF. Among the 15 metabolites showing the greatest variation between seasons (VIP), most were reduced (lactate, trimethylamine, lysine, serine, cysteine, glutamate, glutathione, glycerophosphocholine, proline, choline, and phosphocholine), with only three increased (β-hydroxybutyrate, leucine, and isoleucine) in NBS. Particularly interesting is the finding of lower levels of glutathione and its precursors (cysteine and glutamate) and of serine (a precursor of cysteine) in the FF of NBS, indicating impaired protection from oxidative stress that may account for decreased oocyte competence. Furthermore, choline and its derivatives, fundamental for biological membranes and markers of human oocyte competence (Wallace et al. 2012 Fertil. Steril. 97, 1078-1084), were reduced in NBS. Among amino acids, particular attention goes to leucine and isoleucine, also known to be decreased in bovine follicles associated with more competent oocytes (Sinclair et al. 2008 Reprod. Biomed. Online 16, 859-868). Finally, β-hydroxybutyrate, a marker of negative energetic balance (NEB), was higher in FF of NBS, in agreement with the observations that buffalo, due to their tropical origins, undergo NEB more easily during colder months, coinciding in Italy with NBS. In conclusion, new potential fertility markers were identified in buffalo FF, laying the basis for corrective strategies to implement IVM media, and improve the developmental competence of oocytes collected during the NBS.