314 Effect of macromolecule supplementation during in vitro maturation on the developmental competence of goat oocytes

Abstract

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20-22�h (6.0% CO2 in air, 38.7�C) in modified SOF medium (1.5�mM glucose, 3.0�mM L-lactate, 0.1�mM pyruvate, 1.0�mM glutamine, 0.1�mM taurine) supplemented with 1�_�MEM nonessential amino acids, 0.5�_�MEM essential amino acids, 1�_�MEM vitamins, 0.1�mM cysteamine, 5�_g mL-1 insulin, 5�_g�mL-1 transferrin, 5�ng mL-1 selenium, 50�ng�mL-1 EGF, 0.01�U mL-1 LH and FSH, and 50�_g�mL-1 gentamicin. Treatments were: (1) 1�mg�mL-1 PVA (protein-free, defined); (2) 4�mg�mL-1 BSA (semi-defined); (3) 0.5�mM citrate and 0.5�mg�mL-1 hylauronate (C�+�H, defined); and (4) 0.5�mM citrate and 0.5�mg�mL-1 hylauronate with 4�mg�mL-1 BSA (C�+�H�+�BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant's medium with 7.7�mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50�_L) containing COC at a final concentration of 14-15�_�106 sperm�mL-1. Gametes were coincubated in the presence of heparin (25�_g�mL-1) for 22-24�h in 7% CO2 in air at 38.7�C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7�C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented �SEM and probability values P�<�0.05 were considered significant. The use of BSA did not improve (P�>�0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P�>�0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions.