The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

Embryo production by intrafollicular oocyte transfer (IFOT) represents an alternative for production of a large number of embryos without requiring any hormones and only basic laboratory handling. We aimed to (1) evaluate the efficiency of IFOT using immature oocytes (IFIOT) and (2) compare embryo development after IFIOT using fresh or vitrified immature oocytes. First, six IFIOTs were performed using immature oocytes obtained by ovum pickup. After insemination and uterine flush for embryo recovery, 21.3% of total transferred structures were recovered excluding the recipient's own oocyte or embryo, and of those, 26% (5.5% of transferred cumulus-oocyte complexes [COCs]) were morula or blastocyst. In the second study, we compared fresh and vitrified-warmed immature COCs. Four groups were used: (1) fresh immature COCs (Fresh-Vitro); (2) vitrified immature COCs (Vit-Vitro), with both groups 1 and 2 being matured, fertilized, and cultured in vitro; (3) fresh immature COCs submitted to IFIOT (Fresh-IFIOT); and (4) vitrified immature COCs submitted to IFIOT (Vit-IFIOT). Cumulus-oocyte complexes (n = 25) from Fresh-IFIOT or Vit-IFIOT groups were injected into dominant follicles (>10 mm) of synchronized heifers. After excluding one structure or blastocyst, the recovery rates per transferred oocyte were higher (P < 0.05) for Fresh-IFIOT (47.6%) than for Vit-IFIOT (12.0%). Blastocyst yield per initial oocyte was higher (P < 0.05) for Fresh-Vitro (42.1%) than for Fresh-IFIOT (12.9%). Vit-Vitro presented higher (P < 0.05) embryo development (6.3%), compared to Vit-IFIOT, which did not result in any extra embryo. Although IFOT did not improve developmental competence of vitrified oocytes, we achieved viable blastocysts and pregnancies produced after IFIOT of fresh bovine immature oocytes. Further work on this technique is warranted as an option both for research studies and for clinical bovine embryo production in the absence of laboratory facilities for IVF.

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