86 Comparison of in vivo and in vitro embryo production in Bonsmara donors

Abstract

A retrospective analysis of embryo production records from 2013 to 2017 was carried out to evaluate the in vivo and in vitro production (IVP) of embryos in donors of the Bonsmara breed (i.e. tropically adapted Bos taurus). Only donors with production records of both in vivo and in vitro embryos during the same period were used. A total of 127 superovulations and ova/embryo collections of 19 donors were evaluated. The donors were superstimulated with the following protocol: on Day 0 they received a device with 1 g of progesterone (DIB, Zoetis, Argentina), 50 mg of rogesterone (Progestar, Zoetis), and 5 mg of oestradiol-17β (17ßOestradiol, Rio de Janeiro, Argentina) or 2 mg of oestradiol benzoate (Gonadiol, Zoetis) intramuscularly (IM) at the same time. Superstimulatory treatments were initiated on the morning of Day 4 with Folltropin-V (Vetoquinol, France; total dose = 240 to 340 mg IM) in twice-daily decreasing doses over 4 days. All donors received 2 IM injections of 500 µg of cloprostenol (Ciclase DL, Zoetis) on the morning and afternoon of Day 6 and; the intravaginal devices were removed on the morning of Day 7 and 100 µg of Gonadorelin (gonadotropin-releasing hormone, Gonasyn gdr, Zoetis) was given on the morning of Day 8. Donors were inseminated using semen from 9 Bonsmara bulls, 12 and 24 h after gonadotropin-releasing hormone. On Day 15, ova/embryos were collected and classified according to IETS standards. A total of 89 follicular aspirations (ovum pickup) of 19 donors for IVP were evaluated. The ovum pickups were performed at random stages of oestrous cycle, without superstimulation or other hormone treatments. A total of 1109 viable oocytes (12.5 ± 0.9 per ovum pickup) were collected and matured for 24 h in 100-µL drops of maturation medium (TCM-199, supplemented with hormones) under mineral oil and incubated at 38.5°C in 5.5% CO₂ and humidity at saturation. Fertilization was performed using 3 Bonsmara bulls that were also used for in vivo embryo production. Viable sperm were obtained using the percoll gradient technique (45-90%). The sperm pellet was dissolved in TL-Sperm, centrifuged, and then diluted to a final concentration of 1.5 × 10⁶ sperm/mL. Zygotes were stripped and placed in drops of 100 µL of SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO₂, 7% O₂, and humidity at saturation for 7 days. The culture medium was renewed on Days 3 and 5. The data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), a P-value <0.05 was considered significant. The mean (± standard error of the means) number of CL, ova/embryos collected, fertilized ova, and transferable embryos were 12.9 ± 0.6, 8.8 ± 0.6, 6.6 ± 0.5, and 4.7 ± 0.4, respectively. A total of 662 oocytes (66.3 ± 2.4%) cleaved 48 h post-IVF. On Day 7, an average of 4.4 ± 0.3 embryos were produced. No differences were detected in the number of transferable embryos produced in vivo v. those produced in vitro. Furthermore, no significant differences were found between the techniques or bulls on the proportion of embryos produced in relation to the ova/embryos or oocytes obtained (in vivo 51.5 ± 3.2% v. in vitro 42.9 ± 2.5%). In conclusion, the in vivo and in vitro production of embryos are both effective alternatives to increase the number of offspring from valuable Bonsmara donors.