31 INTRAFOLLICULAR TRANSFER OF FRESH AND VITRIFIED IMMATURE BOVINE OOCYTES: AN OPTION FOR EMBRYO PRODUCTION

Abstract

The association of a technique that guarantees the embryo quality of in vivo blastocyst and otherwise allows the increment of embryo production, such as the in vitro model, would result in a healthier and cheaper embryo. Immature oocytes intrafollicular transfer (IOIFT) is a technique in which immature oocytes obtained by ovum pickup are injected into a dominant follicle of a synchronized recipient. We hypothesised that IOIFT could support embryo development even after oocyte vitrification. We aimed to compare IOIFT or traditional in vitro embryo system using fresh and vitrified immature oocytes. Cumulus-oocyte complexes (COC) were obtained from slaughterhouse ovaries; after selection, half of COC were vitrified by cryotop method. Vitrified and fresh oocytes were either cultured in vitro or transferred to a follicle on the recipient ovary. Four groups were used: (1) fresh immature oocytes (VitroF); (2) vitrified/warmed immature oocyte (VitroV), both (1) and (2) were in vitro matured, fertilized, and cultured; (3) fresh immature oocytes submitted to IOIFT (VivoF), and (4) vitrified/warmed immature oocytes submitted to IOIFT (VivoV). Recipients heifers (n = 12) were synchronized with the following protocol: on Day –10 a progesterone device (P₄, Primer) was inserted together with the administration of 2 mL of oestradiol benzoate (RIC-BE); at Day –8 the devices were removed simultaneously to the administration of 2 mL of prostaglandin (Veteglan); Day –1, 1 mL of oestradiol benzoate was administered. The COC from VivoF or VivoV groups were injected into dominant follicle (>10 mm), 58 h after P₄ removal. The intrafollicular injections were guided by a 7.5-MHz ultrasound vaginal probe (Aloka) using a modified aspiration system. For the injection, 25 COC were placed into a needle (27 G), with 80 μL of follicular fluid. An insulin syringe served to perform the injections. A single dose of semen was used for AI, soon after IOIFT, and embryos were recovered by uterine flushing 7 days later. The results of embryo development and total cell number and apoptotic cells (TUNEL) are present in Table 1. The results obtained for fresh oocytes suggest that IFIOT technique may be an option for bovine embryo production. Despite, it does not improve embryo development or increase total cell number when vitrified and warmed immature oocytes are used.

Table 1.  Cleaved and blastocyst rates, total cell number, and apoptotic cell counting of expanded blastocyst of fresh (F) and vitrified (V) cumulus-oocyte complexes that were in vitro (Vitro) or immature oocytes intrafollicular transfer (Vivo) produced