Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions

For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 °C and three different media: Dulbecco's phosphate buffered saline (DPBS), TCM199 and Emcare, were tested for two different embryo ages: D4 embryos (collected 144 h after hCG treatment) and D5 embryos (collected 168 h after hCG). After slaughter of the donor gilts, embryos were collected and transported at 25 °C to the lab where morulas and blastocyst were selected (D4 n=222; D5 n=167) and randomly used as controls or distributed over the treatment groups. Developmental stage and embryo diameter were assessed by normal light microscopy, while total number of cells and incidence of apoptosis were assessed using a fluorescent embryo quality staining technique that combines three different dyes: Ethidium Homodimer (EthD-1), TUNEL and Hoechst 33342. Following 24 h storage, D5 embryos had higher rates of hatching (24%) and degeneration (9%) compared to D4 embryos (10 and 4%, respectively; P<0.05). Embryos stored at 38 °C had higher rates of hatching (37%) compared to those ones stored at 25 °C (13%) or 18 °C (0%; P<0.01). More embryos hatched when stored in medium Dulbecco's phosphate buffered saline (DPBS) or in TCM199 compared to those stored in Emcare (P<0.05). A higher percentage of embryos stored at 18 °C degenerated compared to those stored at 25 or 38 °C (P<0.01). No significant increase in apoptosis was observed after storage compared to the rates of apoptosis at 0 h (controls) or between the different storage groups. Based on the results we conclude that D4 porcine embryos produced in vivo, selected under normal light microscopy and stored at 25 °C in a serum free medium for 24 h will have a suitable developmental stage for ET and a high embryo quality.