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

Resumen:
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 4000005 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 degrees 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 (MB) alpaca (n=224) and llama (n=240) oocytes were activated using 5 mu M lonomycin 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 mu M cytochalasin B for 3 h at 39 degrees C in an atmosphere of 5% O-2,5% CO2 and 90% N-2 in humidified air. Then, oocytes were transferred to 40 mu l drops of mSOF-IVC supplemented with 3 mg/ml BSA and cultured for 8 days at 39 degrees C in an atmosphere of 5% O-2, 5% CO2 and 90% N-2 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 +/- 13 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 mu M lonomycin and 2 mM 6-DMAP/12.5 mu M 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. (C) 2015 Elsevier B.V. All rights reserved.

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