In vitro developmental competence of alpaca (Vicugna pacos) and llama (Lama glama) oocytes after parthenogenetic activation

The study was designed to compare the cleavage and blastocysts rate of in vitro matured alpaca and llama oocytes after chemical activation. Alpaca (n = 90) and llama (n = 85) ovaries were collected at a local slaughterhouse and transported within 2-3 h to the laboratory. Cumulus oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter and classified according to the number of cumulus cell layers and cytoplasm morphology. A total of 350 and 400COCs were collected from the alpaca and llama abattoir-derived ovaries, respectively (average, 3.8 vs. 4.7COCs per ovary, respectively). Only category 1 and 2COCs collected from alpaca (n = 280) and llama (n = 340) were in vitro matured for 26-28 h in medium TCM 199 at 39 °C in an atmosphere of 5% CO₂ in humidified air. After in vitro maturation, oocytes were denuded of cumulus cells by vortex agitation, for 2 min in mSOF-HEPES solution at 0.1% hyaluronidase. Mature (MII) alpaca (n = 224) and llama (n = 240) oocytes were activated using 5 μM Ionomycin in SOF-HEPES supplemented with 1 mg/ml BSA at room temperature for 4 min followed by incubation in mSOF-IVC supplemented with 3 mg/ml BSA, 2 mM 6-dimethylaminopurine (6-DMAP) and 12.5 μM cytochalasin B for 3 h at 39 °C in an atmosphere of 5% O₂, 5% CO₂ and 90% N₂ in humidified air. Then, oocytes were transferred to 40 μl drops of mSOF-IVC supplemented with 3 mg/ml BSA and cultured for 8 days at 39 °C in an atmosphere of 5% O₂, 5% CO₂ and 90% N₂ in humidified air. A greater proportion of category 3COCs was collected from alpaca than llama ovaries; however, there were not significant differences in the remaining COCs categories between species. A total of 224 and 240 alpaca and llama matured oocytes were chemically activated, respectively. Cleavage (62.5 ± 2.7 vs. 66.6 ± 5.2), morula (47.0 ± 2.0 vs. 45.8 ± 1.4) and blastocyst (22.5 ± 1.3 vs. 18.7 ± 1.0) development rate did not differ between groups. In conclusion, alpaca and llama oocytes can be effectively activated after a sequential incubation with 5 μM Ionomycin and 2 mM 6-DMAP/12.5 μM cytochalasin B resulting in consistent in vitro embryo development rates that could be used to assess oocyte viability/functionality after in vitro maturation or cryopreservation.