35 Ethanolic extracts of Cerrado plants in cryotolerance of in vitro-produced bovine embryos

Abstract

In vitro embryo culture induces excessive production of reactive oxygen species (ROS) that impair the quality of produced blastocysts, making them less cryotolerant. Supplementation of culture media with antioxidants would be an alternative to minimise these effects. The present study evaluated the effect of ethanolic extracts obtained from Brazilian Cerrado plants on the cryotolerance of in vitro-produced embryos. Bovine ovaries from a slaughterhouse were used to obtain grade I and II oocytes, which were submitted to maturation, fertilization (Day 0), and in vitro culture. After fertilization, zygotes were distributed into five groups: one fresh control group (GCont), cultured in high O₂ tension (~21%), and four groups cryopreserved at Day 7: a control group subject to high O₂ tension without extract supplementation (GDT); a group (G5%) cultured in low O₂ tension (5%) without extract supplementation, and two groups cultured under high O₂ tension, one supplemented with 0.01 mg mL⁻¹ of the ethanolic cagaita extract (Eugenia dysenterica; GCag) and another with 0.01 mg mL⁻¹ of murici extract (Byrsonima crassifolia; GMur). Only the expanded blastocysts (EB) of each group were submitted to the direct-transfer cryopreservation system, with slow freezing with ethylene glycol (Sanches et al. 2016 Theriogenology 85, 1147-1151). A total of 163 thawed embryos were evaluated 12, 24, and 36 h post-thawing for re-expansion, hatching, and embryonic degeneration rate. In addition, after 12 h of post-thaw culture, only the EB embryos were subjected to ROS measurement, quantified in confocal microscopy using 2′,7′-dichlorodihydrofluorescein diacetate, and the rate of apoptosis was obtained by the terminal deoxynucleotidyl transferase dUTP nick end labelling method. Data were analysed by analysis of variance, and the means were compared by TUKEY. A total of 133 EB were used (GCont: 20; GDT: 22; GCag: 35; GMur: 36; G5%: 20). Results are expressed as means ± standard deviation. No differences were observed on re-expansion among treatments and time of culture. However, 24 h post-thaw, hatching and degeneration rates differed between groups. The group GCont had a higher (P < 0.05) hatching rate (47.5 ± 10.3) than the GDT (14.4 ± 6.7) and G5% (10.5 ± 5.8) groups but was similar (P > 0.05) to groups supplemented with extracts GCag (22.2 ± 17.1) and GMur (17.5 ± 17.5). Degeneration rate was higher (P < 0.05) at 12 h for the direct-transfer group (30.2 ± 11.3) and similar among the others (GCont: 0; GCag: 16.8 ± 12; GMur: 10.8 ± 10.8; G5%: 11 ± 11). Nevertheless, GCag had lower embryonic degeneration rates (20 ± 16.7) than the others cryopreserved groups 24 h after thawing (GDT: 37.7 ± 5.5; GMur: 38.9 ± 8.2; G5%: 41.3 ± 14.4). Although ROS levels were similar among all groups (P > 0.05), apoptosis rate was higher (P < 0.05) in the GDT group (23.4 ± 1.9) than in the GCont groups (10.6 ± 1.3): GCag (10.8 ± 1.1), GMur (10.1 ± 1.1), and G5% (7.8 ± 1.2). Therefore, supplementation of ethanolic extracts (0.01 mg mL⁻¹) of cagaita and murici improved the embryo quality by reducing the degeneration and apoptosis rates of cryopreserved embryos when compared with the group without supplementation (GDT). Such extracts may be an alternative to increase the cryotolerance of in vitro-produced bovine embryos.