66 Effect of the co-culture system and the culture medium on invitro embryo development in alpacas (Vicugna pacos)

Abstract

The aim was to evaluate 4 co-culture systems and 4 culture medium types to produce alpaca embryos by IVF. Gametes were obtained from ovaries and testes collected from a slaughterhouse. Oocytes were recovered by follicle aspiration using a 5-mL syringe. Oocytes with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.02IU mL−1 FSH, 1µg mL−1 oestradiol 17β, 0.2mM sodium pyruvate, and 50µg mL−1 gentamicin sulphate. After maturation, oocytes were placed in FERT-TALP (fertilization- Tyrode’s medium with albumin, lactate, and pyruvate) solution for 30min before IVF with epididymal sperm. Motile sperm were selected by swim-up method. Oocytes were co-cultured with 3×106 spermatozoa mL−1 for 18 to 20h under the same atmospheric conditions mentioned above. In Experiment 1, we evaluated 4 co-culture systems: ear fibroblasts (T1, n=224), fetal fibroblasts (T2, n=154), granulosa cells (T3, n=225), oviduct cells (T4, n=169) and synthetic oviductal fluid (SOF) invitro culture (IVC) (T5, n=116). As culture base, we used 0.5mL of SOF IVC medium supplemented with 10% FCS in all treatments. In Experiment 2, we evaluated 4 culture media: TCM-199+10% FCS (T1, n=137), CR1aa+3mg of bovine serum albumin (BSA) mL−1 (T2, n=85), KSOM medium+3mg of BSA mL−1 (T3, n=110), and SOF IVC+10% FCS (T4, n=66). The volume of culture medium used was 0.5mL in 4-well Nunc plates for each treatment. The time (8 days) and culture (38.5°C, 5% CO2 in air and high humidity) conditions, and change of medium (each 24h) were the same in both experiments. Statistical significance was determined using ANOVA. The mean and s.d. were calculated from the average of the percentages obtained in each repetition. In experiment 1, the cleavage rates were higher (P<0.05) in co-culture with fetal fibroblasts (46%±0.17), oviduct cells (50%±0.09), and ear fibroblasts (58%±0.17) than with granulosa cells (28%±0.12) and SOF IVC (30%±0.18). Also, the morula rates were higher (P<0.05) in co-culture with fetal fibroblasts (35%±0.16), oviduct cells (31%±0.01), and ear fibroblasts (35%±0.14) than with granulosa cells (11%±0.01) and SOF IVC (27%±0.17). In contrast, there were no differences in blastocyst rates between co-culture with granulosa cells (10%±0.04), SOF IVC (12%±0.09), and fetal fibroblasts (24%±0.14). However, there were differences between co-culture with oviduct cells (19%±0.06) and ear fibroblasts (32%±0.14). In Experiment 2, there were no differences in cleavage rates between TCM-199+FCS (28%±0.12), CR1aa+BSA (44%±0.24), KSOM+BSA (38%±0.19), and SOF-IVC+FCS (50%±0.20). However, there were differences in morula rates between CR1aa+BSA (7%±0.09) and SOF IVC+FCS (35%±0.21), TCM-199+FCS (23%±0.09), and KSOM+BSA (27%±0.15). We obtained a higher blastocyst rates in SOF IVC+BSA (24±0.12) and KSOM+BSA than with CR1aa+BSA (3±0.06) and TCM-199+FCS (9±0.03). In conclusion, KSOM and SOF-IVC were the best media for culture, and oviduct cells and ear and fetal fibroblasts were the best cells to produce alpaca embryos by IVF.