85 Influence of Short-Term Storage on Gene Expression of Equine Embryos

Abstract

Short-term storage for embryo transportation is a routine procedure in equine embryo transfer programs. The success rate after transfer of stored embryos varies among different protocols when embryos are transported overnight. To the best of our knowledge, there is no study evaluating the effect of different temperatures during storage for 24 h on gene expression of equine embryos. Therefore, this study aimed to evaluate the effects of storage of equine embryos for 24 h at 2 temperatures (20°C or 5°C) on the expression of genes related to embryo development (IGF2, H19, POU5F1, SOX2), and regulation of DNA methylation (DNMT1, DNMT3a, DNMT3b). Embryos (n = 24) were collected on Day 7 (n = 18) or Day 8 (n = 6) after ovulation and assigned to 4 groups: Day 7 control (D7, fresh); Day 7, 24 h at 5°C (E5C); Day 7, 24 h at 20°C (E20C); and Day 8 control (D8, fresh 24-h time control). After flushing, embryos were washed and kept in holding medium (Minitube, Tiefenbach, Germany) for morphological classification and measurements. Fresh and stored embryos were treated with pronase (10 mg mL⁻¹), washed with PBS solution and placed in RLT Lysis buffer (Qiagen, Hilden, Germany) for RNA extraction. Total RNA was extracted from each individual embryo using the RNeasy mini kit (Qiagen) following the recommended protocol for animal tissues. After RNA purification, RNA quality was assessed and quantified. Subsequently, cDNA synthesis was performed for RT-qPCR analysis. Two replicates were performed and the relative gene expression was calculated using the 2(–delta delta CT) method, with the target gene expression levels normalized to the geometric mean of PSMB4/SNRPD3. The software SPSS v.24 (IBM/SPSS, Armonk, NY, USA) was used for statistical analyses using the nonparametric tests Kruskal-Wallis and Mann-Whitney U-test to compare differences among groups. Embryos sizes differed (P < 0.05) between D7 (431 ± 48 mm) and D8 (1114 ± 205 mm). Storage temperature did not affect (P > 0.05) embryo size. The mRNA expression of H19 and IGF2 was similar (P > 0.05) among all groups. Expression of POU5F1 and SOX2 was higher (P < 0.05) in D7 and E5C embryos compared with D8 embryos. In addition, E20C had similar (P > 0.05) expression of POU5F1 with D7, E5C, and D8, but lower (P < 0.05) expression of SOX2 when compared with D7. Expression of DNMT1 and DNMT3a were similar (P > 0.05) among D7, D8, and E5C, but lower (P < 0.05) in E20C. Furthermore, expression of DNMT3b was lower (P < 0.05) in D8 and E20C embryos compared with D7 and E5C. In conclusion, temperature during short-term storage seems not to affect the expression of IGF2 and H19 but influences the expression of POU5F1, SOX2, DNMT1, DNMT3a, and DNMT3b. Therefore, these findings suggest that embryos stored at 20°C sustain the pattern of gene expression similar to that of fresh embryos at Day 8, whereas embryos stored at 5°C maintain the gene expression similar to that of fresh embryos at Day 7. Thus, alterations caused by temperature during short-term storage on the expression of genes related to embryo development and DNA methylation may modify the pattern of equine embryonic tissue development, requiring further investigation.