133 L-CARNITINE DURING IN VITRO CULTURE ENHANCES THE CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS

Abstract

In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237–256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632–1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25 mM for the experiment. Cumulus–oocytes complexes (n = 288, 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 supplemented with 8 mg mL^–1 BSA, in the absence (control, n = 143) or presence of 0.25 mM L-carnitine (n = 145). In vitro culture was carried out at 38.5°C under 5% CO₂, 7% O₂, and 88% N₂. Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5 M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747–750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24 h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24 h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P < 0.01). In conclusion, these results demonstrated that L-carnitine supplementation of culture medium improves the resistance to cryopreservation of in vitro produced buffalo embryos. We speculate that the increased cryotolerance observed in the presence of L-carnitine may be due to a better utilization of the endogenous lipid stores, resulting in improved embryo quality.