56 Antifreeze protein type I increases mitochondrial activity and reduces reactive oxygen species levels in frozen-thawed in vivo-derived sheep embryos

Antifreeze proteins (AFPs) have been used as cryoprotective agents due to their properties for cell protection at negative temperatures, which can increase the success of embryo cryopreservation. This study assessed the effects of different concentrations of AFP type I (0.1 or 0.5 µg/mL) added to the slow-freezing solution for in vivo-derived sheep embryo cryopreservation. Good quality (Grade I and II) embryos (n = 135) were collected transcervically from 37 superovulated ewes and allocated into a slow-freezing solution: AFP-free (CONT; n = 39); 0.1 µg/mL of AFP I (AFP0.1; n = 53); or 0.5 µg/mL of AFP I (AFP0.5; n = 43). Freezing followed cooling from 20°C until −6°C (3°C min⁻¹); stabilisation during 15 min and seeding in −6°C after 5 min; cooling to −32°C (−0.5°C min⁻¹), before being plunged into liquid N₂. After thawing, embryos were in vitro cultured (IVC) in synthetic oviducal fluid medium at 38.5°C, 5% CO_2, and 5% O₂ for 48 h. The survival rate was assessed at 24 and 48 h, and the blastocyst hatching rate at 48 h; analysed by Pearson’s chi-squared test. At 24 and 48 h of IVC, viability, mitochondrial activity, intracellular reactive oxygen species (ROS), and glutathione (GSH) levels were evaluated in groups of five viable embryos per treatment and compared by one-way ANOVA followed by Tukey test (normal distribution), or by Kruskal-Wallis followed by Dunn’s test (non-normal distribution). The embryos evaluated at 24 h of IVC were submitted to RT-qPCR for metabolism (CDX2, OCT4, PRDX1) and quality genes (HSP70, BAX, BCL2, CDH1, AQP3). The expression of each gene was normalised using GAPDH values and compared by Ct method. All analyses were performed in IBM SPSS software, and P < 0.05 was considered significant and 0.05 < P < 0.10 as a tendency. In vitro survival rate was similar (P > 0.05) in embryos among groups in 24 h (CONT: 48.7%; AFP0.1: 52.8%; and AFP0.5: 44.2%) and 48 h (CONT: 56.4%; AFP0.1: 60.4%; and AFP0.5: 53.5%). A tendency (P = 0.09) for a higher blastocyst hatching rate at 48 h was noted in AFP0.1 (61.8%; 21/34) compared with AFP0.5 (33.3%; 9/27), and both groups were similar to CONT (50.0%; 11/22). An increased (P < 0.05) mitochondrial activity (fluorescence arbitrary unit × 10⁻³/µm²) at 24 h was observed in re-expanded blastocysts of AFP0.1 (5.0 ± 1.6) and AFP0.5 (5.2 ± 2.4), compared with CONT (1.1 ± 0.6). A reduction in intracellular ROS levels (P < 0.05) was observed at 24 h in AFP0.1 (5.9 ± 0.7) compared with CONT (10.8 ± 1.6), and both were similar to AFP0.5 (7.4 ± 1.4). No differences (P > 0.05) were noted in these parameters in re-expanded blastocysts at 48 h of IVC. For GSH levels and viability, no differences were observed among groups at any time point (P > 0.05). Regarding gene expression, no difference (P > 0.05) was observed, but there was a tendency for downregulation in AQP3 for AFP0.5 compared with CONT (P = 0.08), and upregulation for BCL2 in AFP0.5 compared to AFP0.1 (P = 0.09). In conclusion, the addition of AFP I can provide some benefits to frozen-thawed in vivo-derived sheep embryos without affecting embryonic survival and gene expression.

This research was supported by CAPES, CNPq, and FAPERJ.