126 EFFECT OF REDUCING GLUCOSE CONCENTRATION DURING IN VITRO EMBRYO CULTURE IN BUFFALO (BUBALUS BUBALIS)

The current knowledge on metabolism and glucose utilisation of preimplantation bovine and ovine embryos suggest the reduction of glucose concentration during early culture. On the contrary, it has been demonstrated that glucose is absolutely required for in vitro culture of buffalo embryos, as indicated by the poor efficiency recorded in the absence of this substrate during early embryonic development (Monaco et al. 2006 Reprod. Domest. Anim. 41, 332). However, complete removal of glucose from culture medium throughout pre-elongation development is unlikely to benefit the embryo because glucose plays other roles including ribose and NADPH production through the pentose-phosphate pathway. Therefore, the aim of this study was to investigate the effect of reducing glucose concentration up to 0.15 mM (1/10 compared to the standard concentration in SOF) on embryo development in buffalo. In order to evaluate the role of this substrate during development, glucose was reduced at different stages of embryo culture. Cumulus–oocyte complexes (n = 573, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287). On day 1 (Day 0 = IVF), zygotes were cultured in SOF with group A) 1.5 mM glucose (standard concentration in SOF) throughout culture (control); group B) 1.5 mM glucose for early culture (Day 1 to Day 4) and 0.15 mM glucose for late culture (Day 4 to Day 7); group C) 0.15 mM glucose throughout culture; and group D) 0.15 mM glucose for early culture and 1.5 mM glucose for subsequent culture. In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 4, and blastocyst yield, in relation to cleaved embryos, was recorded on Day 7. Differences among groups in blastocyst rate were analysed by chi-square test. The reduction of glucose concentration did not affect cleavage rate (73.7 v. 65.1%, respectively, for Groups A-B and C-D). Nevertheless, blastocyst rates significantly decreased when glucose was reduced throughout culture (Group C: 10.1%; P < 0.01) and to a limited degree during early culture (Group D: 17.2%; P < 0.05) compared with the control (Group A: 38.3%). On the contrary, a decreased glucose concentration during late culture did not reduce embryo development (Group B: 35.18%). This finding indicates that energy requirements of buffalo embryos during IVC are different from those of sheep and cattle, which show a significant rise in glucose uptake just around compaction, i.e. during late culture (Thompson et al. 1991 Reprod. Fertil. Dev. 3, 571–576; Thompson et al. 1996 J. Reprod. Fertil. 106, 299–306). In conclusion, in buffalo, unlike sheep and cattle, glucose is more critical for early embryo development than for post-compaction development, suggesting the importance of developing other strategies for optimizing in vitro embryo production efficiency.