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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.

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