78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20 h after IVF, groups of presumptive zygotes were cultured in 30 μL of SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 205) or 5 μg mL⁻¹ of carnosine (n = 209) under humidified air with 5% CO₂, 5% O₂, and 88% N₂. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n = 25 control and n = 27 carnosine) were frozen in 1.5 M ethylene glycol + 0.1 M sucrose. Embryos were thawed and then cultured for 72 h in SOF-BSAaa + 1% oestrus cow serum for re-expansion and hatching rate assessments at +24 h, +48 h, and +72 h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 48) or 5 μg mL⁻¹ of carnosine (n = 48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15 min for up to 168 h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student’s t-test for Experiment 2 were used, and differences were considered significant at P < 0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0 ± 4.2 v.85.2 ± 3.8, P = 0.7; 46.9 ± 7.1 v. 45.0 ± 7.5, P = 0.7; 24.1 ± 2.0 v. 24.0 ± 6.5, P = 0.6; respectively). After thawing, the re-expansion at +24 h was not different between groups (74.1 v. 48.0% for carnosine and control groups, respectively; P = 0.06). However, at +48 h and +72 h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4 ± 4.5% v. 40.0 ± 3.8% and 59.3 ± 3.8% v. 24.0 ± 3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1 ± 3.9 v. 33.8 ± 6.1; P = 0.3), C2 (8.2 ± 8.9 v. 8.9 ± 0.9; P = 0.07), and B4 (147.0 ± 9.5 v. 145.4 ± 11.6; P = 0.6), whereas C3 was significantly different within groups: 59.9 ± 9.6 v. 51.8 ± 6.7 (P = 0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5 μg mL⁻¹ carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality.

This work was supported by FECUND European project (grant agreement number 312097).