163 EFFECTS OF SERUM AND L-CARNITINE ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

Abstract

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O₂, 5% CO₂, and 90% N₂. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O₂, 5% CO₂, and 90% N₂. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P < 0.05) development to the blastocyst (19.7 ± 1.1% v. 25.3 ± 1.4%) and advanced blastocyst (9.1 ± 0.8% v. 12.4 ± 1.2%) stages. While l-carnitine did not affect blastocyst development, advanced blastocyst development was reduced (P < 0.05) for l-carnitine at 3.03 mM (0 mM: 10.9 ± 1.2%, 0.75 mM: 12.2 ± 1.4%, 1.5 mM: 13.5 ± 1.5%, 3.03 mM: 7.0 ± 1.0%). Serum reduced (P < 0.01) re-expansion (78.1 ± 3.4% v. 65.5 ± 3.1%, 81.0 ± 3.0% v. 68.4 ± 2.7%, 78.4 ± 3.4% v. 65.8 ± 3.1%, for 24, 48, and 72 h, respectively) and hatching (52.0 ± 4.0% v. 39.8 ± 3.6%, 61.2 ± 4.1% v. 45.4 ± 3.8%, 61.2 ± 4.1% v. 45.4 ± 3.8%, for 24, 48, and 72 h, respectively) rates at all time points. In contrast, treatment of embryos with l-carnitine during culture increased (P < 0.05) post-thaw re-expansion rates at 24 and 48 h, regardless of concentration (Table 1). In conclusion, post-thaw viability of bovine embryos PIV can be improved by the addition of l-carnitine during culture. Moreover, the beneficial effects of l-carnitine on cryosurvival are not dependent on serum supplementation.

Table 1.  Effect of addition of l-carnitine during culture on post-thaw re-expansion and hatching rates