45 Frozen–thawed embryo transfer: effects of embryo thawing protocol on pregnancy per embryo transfer in Nelore (Bos indicus) cattle recipients

In recent years, the expansion of the use of cryopreserved embryos in cattle has received special attention; however, bovine embryos are still very sensitive to cryopreservation. The main aim of the present research was to study the effects of different embryo thawing protocols on pregnancy per embryo transfer (P/ET) in Nelore cattle recipients. A total of 2740 Nelore breed cows (age: ~48 ± 12 months.; body weight: ~400 ± 50 kg; body condition score: 3.0–3.5) were used as embryo transfer recipients. Four experimental groups of thawed embryos were used: Directly thawed embryos (D; n = 1507), ethylene glycol + sucrose-thawed embryos (EGS; n = 375), sucrose-thawed embryos (S; n = 410), and holding solution-thawed embryos (HS; n = 448). All embryos (morula, early blastocyst, and blastocyst stage) were cryopreserved following the same standard protocol (10% ethylene glycol solution) and stored until used. Immediately before embryo transfer, different embryo thawing protocols were carried out: D: thawing 10 s (air) + 30 s (30°C); EGS: thawing 10 s (air) + 30 s (30°C) + 6 min. 30 s (0.75 EG + 0.5 S) + 6 min, 30 s (0.5 S) + holding solution (4 washes/15 s per wash); S: thawing 10 s (air) + 30 s (30°C) + 6 min, 30 s (0.5 S) + holding solution (4 washes/15 s per wash); HS: thawing 10 s (air) + 30 s (30°C) + holding solution (4 washes/15 s per wash). The pregnancy test was performed by ultrasonography on Day 30 after ET. Chi-squared test, Spearman correlation, and GLMM were performed using SPSS^® v.25. A significant positive correlation was observed between embryo developmental stage (morula and early blastocyst) and P/ET irrespective of the thawing protocol (r = 0.42; P < 0.05). Moreover, there was a significant interaction between embryo thawing protocol and embryo stage on P/ET (P < 0.05). No differences were observed in P/ET among embryo thawing protocols regarding morula and early blastocyst stage (P > 0.05). However, significant differences were observed in P/ET between the D embryo thawing protocol and the rest of the protocols when blastocyst stage embryos were transferred (D: 117/412 (28%) vs EGS: 38/76 (50%) vs S: 34/79 (43%) vs HS: 66/158 (42%); P < 0.05). No differences were observed in P/ET among embryo thawing protocols when all embryos were considered irrespective of the developmental stage (P > 0.05). In conclusion, the embryo thawing protocol affected the P/ET observed when blastocyst-stage embryos were transferred. Despite the P/ET remaining similar among embryo thawing protocols regarding morulae and early blastocysts, the P/ET was slightly improved using morula-stage embryos. Finally, the interaction between the thawing protocol and embryo developmental stage must be considered to improve P/ET in Nelore cattle.

This research was supported by DIRGI-CP2022–005.