Supplementation of culture medium with L-carnitine improves the development and cryotolerance of in vitro-produced porcine embryos.

Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic role during development. This study investigated the role of lipid metabolism and the interaction between carbohydrate and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-3 (PZM-3), which contains pyruvate and lactate, 3mM L-carnitine was the only dose that improved cleavage rates compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12mM increased cleavage rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0-3: pyruvate and lactate; Days 4-7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing PZM-3 with 3mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3mM L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo's requirements for carbohydrates are not adequately met.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.