19 In vitro embryonic development from oocytes collected by ovum pickup of superstimulated females and nonstimulated slaughterhouse ovaries of alpaca (Vicugna pacos)

Abstract

The objective of this study was to evaluate the embryonic development of alpaca oocytes collected by ovum pickup from superstimulated females (OPU, Group 1) and from slaughterhouse ovaries of 8 non-superstimulated females (SHO, Group 2) using a conventional aspiration technique (20 G needle and a 3-mL syringe). A total of 8 nonpregnant alpacas, 3 to 4 years old, were superstimulated with a single dose of 200 IU of equine chorionic gonadotrophin (eCG, Day = 0). Three days later, alpacas were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. A total of 101 follicles were aspirated, recovering 67 oocytes (66.3%) by OPU using an endocavity transducer attached to a 21 G needle adapted for alpacas. The follicular fluid was aspirated using a regulated vacuum pump (40 mmHg) into a tube containing 5 mL of phosphate-buffered saline (PBS), 0.2% bovine serum albumin (BSA), and 10 IU mL⁻¹ heparin, at 37°C. In the SHO group we used 16 ovaries maintained at 28°C. The recovery of oocytes was carried out within 3 h of ovary collection. We aspirated 155 follicles from SHO and recovered 117 oocytes (75.5%). After collection, all oocytes recovered were morphologically classified into categories (I and II) and cultured for 26 h in an incubator (5% CO₂ in air at 38.5°C), in TCM-199 supplemented with 0.2 mmol sodium pyruvate, 50 µg mL⁻¹ gentamicin sulphate, 0.02 IU mL⁻¹ FSH, 1 µg mL⁻¹ oestradiol 17β, and 10% fetal calf serum (FCS). After maturation, oocytes were in vitro fertilized with epididymal spermatozoa recovered from postmortem males and co-cultured for 18 to 20 h. After this period, all cleaved oocytes were incubated (5% CO₂ in air, 38.5°C) for 6 days in synthetic oviductal fluid-serum medium. Number and morphological quality of oocytes collected, in vitro cleaved, and embryos ratea were registered and compared between groups. Statistical significance was determined using Kruskal–Wallis test. The mean and standard error were calculated from average of the percentages obtained in each repetition. Results indicated that the mean number of oocytes collected per ovary was higher (P < 0.05) using SHO (7.8 ± 2.4) than OPU (4.5 ± 3.0). Also, the number of oocytes classified as category I, was higher in the SHO compared with OPU group (56% vs. 30% respectively; P < 0.05); however, category II oocytes were the same (16% vs. 15%, respectively). There was no difference in early development (cleavage) rate between OPU (57 ± 2.0) and SHO (49 ± 1.5) groups. However, there was difference in the rate of development (P < 0.05) between OPU and SHO groups to reach the morula stage (56 ± 2.0 vs. 42 ± 1.7, respectively) and early blastocyst stage (55 ± 2.0 vs. 34 ± 1.4, respectively). In conclusion, oocyte quality could be affected by hormonal stimulation or by the quality of follicles aspirated by OPU. In contrast, oocytes recovered from live animals by OPU have greater capability of embryonic development in vitro than oocytes recovered from slaughterhouse ovaries.