Supplementation of Amino Acids in Serum Free IVM-Media and its Effect on In Vitro Maturation and Fertilization of Buffalo Oocytes

AMINO acids are an important component of culture media and their beneficial role is reported in different species. It was hypothesized that supplementation of amino acids to in vitro maturation (IVM) media may affect IVM and fertilization of buffalo oocytes. The objective was to study the effect of supplementing IVM media with essential amino acids (EAA), non-essential amino acids (NEAA) or both (EAA+NEAA) on in vitro maturation (Experiment I) and fertilization (Experiment II) of buffalo oocytes. In experiment I, cumulus-oocyte complexes aspirated from follicles (2-8 mm in diameter) were matured in IVM media: (1) Maturation medium (MM; TCM-199 enriched with BSA 6mg/mL, 1 mu g/mL estradiol-17 beta, 10 IU/mL LH, 0.5 mu g/mL FSH and 50 ug/mL gentamicin) alone as control; (2) MM supplemented with 2% EAA solution; (3) MM enriched with 1% NEAA solution; and (4) MM added with 2% EAA solution + 1% NEAA solution at 38.5 degrees C in an atmosphere with 5% CO2 and 95% humidity under mineral oil.. The degree of cumulus expansion and degree of nuclear maturation (Aceto orcein staining) was measured after 24 h of incubation. In experiment II, following maturation as in experiment I, the oocytes were co incubated with sperms at 39 degrees C in 5% CO2 in air with maximum humidity. After 18 h, oocytes were stained as described earlier and sperm penetration rate was recorded. Cumulus expansion was higher (P<0.05) in medium supplemented with EAA, NEAA and EAA+NEAA compared to control. Nuclear maturation rate and Sperm penetration rate remained similar in media supplemented with EAA, NEAA, EAA+NEAA and control. Results indicated that supplementation of maturation media with amino acids have beneficial effect on oocyte maturation in terms of cumulus expansion. However, non-significant effect on sperm penetration rate suggested further studies to evaluate the effect of amino acids presence of in vitro maturation media on subsequent culturing of embryos.