99 Short-term storage of equine embryos at 5 or 20°C does not cause lipid peroxidation

Abstract

Maintaining the integrity of equine embryos during storage for transportation is essential for successful conception after transfer. During storage, reactive oxygen species may originate from embryo metabolism, causing lipid peroxidation and increasing its end product malondialdehyde (MDA); MDA is one of the important biomarkers for oxidative stress. This study aimed to evaluate the temperature curve, pH and lipid peroxidation of equine embryos stored in holding medium after 24 h at 5 or 20°C. Embryos (n = 33) were collected on Day 7 (n = 21, 7 embryos per group) or Day 8 (n = 12) after ovulation and assigned to four groups: Day 7 control (D7, fresh); Day 7, 24h at 5°C (E5C); Day 7, 24h at 20°C (E20C); and Day 8 control (D8, fresh 24h time control). After collection, embryos were washed and kept in holding medium (Minitube) for morphological classification and measurements. For pH and lipid peroxidation measurement, embryos were kept in a fixed volume of holding medium (150 µL) within a microtube (200 µL); the microtube was kept within a falcon tube (50 mL) inside of an Equitainer (Hamilton Biovet). The temperature was recorded by a data logger (Testo 175, Testo) every 10 min for 24 h. The pH was assessed by a pH meter using a microelectrode (InLab Ultra-Micro-ISM, Mettler Toledo) for small sample volumes. Lipid peroxidation was assessed using the MDA assay kit (catalog number MAK085, Sigma-Aldrich) according to the manufacturer's instructions. Statistical analyses were performed using the Kruskal-Wallis nonparametric test and Mann-Whitney U to compare differences among groups. Embryo size differed (P < 0.05) between D7 (383 ± 41 µm) and D8 (1044 ± 131 µm). Storage temperature (E5C or E20C) did not affect embryo size (382 ± 47 and 553 ± 99 µm, respectively; P > 0.05). The temperature curve was similar (P > 0.05) among embryos within the treatment groups during the storage period. The pH (7.22 ± 0.07 and 7.22 ± 0.09) did not differ (P > 0.05) between E5C and E20C. Lipid peroxidation levels were below the limit of quantification (0.04 nmol) in all groups. The present findings suggest that holding temperature does not affect the size, pH, or lipid peroxidation of equine embryos stored for 24 h in holding medium. However, our previous studies (Gastal et al. 2018 J. Equine Vet. Sci. 66, 185) have shown that holding temperature influences the expression of genes involved in equine embryo development. In conclusion, variations regarding embryo development and conception rate after transfer of embryos stored at different temperatures might be related to factors other than lipid peroxidation.