Maturação e vitrificação de oócitos eqüinos incubados em meio contendo hormônio do crescimento e fator de crescimento semelhante à insulina-I

Abstract

The objective of this study was to investigate the effects of equine growth hormone (eGH) and its relationship with insulin-like growth factor-1 (IGF-1) on the in vitro maturation (IVM) and development of equine oocytes. Additionally, we also evaluated the morphological and structural integrity of all equine oocytes subjected to IVM and cryopreservation. Complex cumulus oocytes (COCs) were cultured in TCM199 supplemented with 0.1% BSA and antibiotics. COC’s (n=122) were placed in a four-well dish supplemented according to four treatments, as follows: a) control, no additives (n=34); b) 400 ng/mL of eGH (n=31); c) 200 ng/mL of IGF-1 (n=35); and d) 400 ng/mL of eGH + 200 ng/mL of IGF-1 (n=22). After maturation, oocytes were fixed (n=37) or subjected to the vitrification protocol (n=85). Cryopreserved oocytes were exposed to 1,4 M dimethyl sulfoxide (DMSO) + 1,8 M ethylene glycol (EG) + 1% PVA copolymer (20%vinyl acetate and 80% vinyl alcohol) for 3 min, and then transfer to 2,8 M DMSO + 3,6 M EG + 0,6 M sucrose + 1% PVA copolymer for 30 sec. Next, the oocytes were loaded to the open pulled straw and transferred to liquid nitrogen vapor, then held in vapor for 3 sec before being plunged into liquid nitrogen. After cryopreservation, the oocytes were thawed and transferred into a PBS containing a decreasing sucrose concentration, for 5 min of each concentration: 0.5M, 0.25M, 0.125M and PBS with 0.4% BSA. Nuclear and cytoplasmic maturation status were assessed by laser scanning confocal microscopy. Maturation rates of oocytes were 26.1% control, 23.5% eGH, 31.8% IGF-I and 17.6% eGH + IGF-I, weren’t significant difference among the treatment groups (p>0,05). However, the results suggest that the eGH + IGF-1 group can develop the assessment resumption of meiosis (MI+MII=86,7%). There weren’t morphological abnormalities after vitrification/thawing. In conclusion, the addition of eGH and/or IGF-I to our IVM protocol didn’t increase rates of nuclear and cytoplasmic maturation. Oocytes subject to vitrification with copolymer PVA had an intact plasma membrane and didn’t show any sign of vacuolization or degeneration. This is the first report showing the vitrification with PVA in equine oocytes.