37 In vitro maturation and fertilization in white-tailed deer (Odocoileus virginianus) oocytes vitrified with trehalose or sucrose

Abstract

White-tailed deer (WTD) invitro embryo production is not well documented, due to the seasonality of the wildlife species and available hunting permits. The objective of the present study was to conduct IVM and IVF using WTD oocytes vitrified with trehalose (TH) or sucrose (SC). A total of 121 immature oocytes were obtained from ovaries (n=18) using the slicing technique from hunter-slaughtered deer, not more than 2h after death. The WTD oocytes were vitrified using a solid surface vitrification technique in two different groups: 1) TH (n=60) and 2) SC (n=61). Oocyte warming (OW) was done using four concentrations (OW1 1 M, OW2 0.5 M, OW3 0.25 M, and OW4 0 M) in the oocytes after thawing. Then, a sample was used to evaluate viability for TH (n=5) and SC (n=5) (MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.5mgmL−1) and nuclear status (NS) for TH (n=4) and SC (n=5) with 4′,6-diamidino-2-phenylindole (1μgmL−1), defining the stage as germinal vesicle (GV), MI, and unable to evaluate (NE). The remaining oocytes (n=88) were used for IVM for 36h in tissue culture medium-199 supplemented with human menopausal hormone (75 UImL−1) and epidermal growth factor (10ngmL−1). The evaluation of NS was defined (GV, MI considered immature and MII matured). The IVF (n=15) took place in Tyrode's albumin lactate pyruvate media supplemented with heparin (10μgmL−1), penicillamine (0.075mgmL−1), hypotaurine (10M), epinephrine (1μM), and bovine serum albumin fraction V (0.4%), with a final semen concentration of 3×106 spermmL−1. Frozen straws of conventional WTD semen were used; the straws were split into 3 parts, with one fraction thawed and capacitated using “swim-up” technique. After 24h of incubation at 38.5°C with 5% CO2, and humidified air, nuclear evaluation was made as fertilized (F), not fertilized (NF), or NE. A Fisher exact test was used (SAS, 9.0 version for Windows), α=0.05. After warming, for the TH group (n=5), viability was 60% and nuclear status (n=4) was 50% GV, 50% MI, and 0% NE. For the SC group (n=5), viability was 40% and nuclear status (n=5) was 60% GV, 20% MI, and 20% NE. After 36h IVM, NS evaluation in the TH group (n=38) was 66% GV, 24% MI, 0% MII, and 10% NE; for the SC group (n=50), 84% GV, 10% MI, 2% MII, and 4% NE, not a statistically significant difference (P>0.05). For IVF after 24h, the NS evaluation in the TH treatment (n=10) was 0% F, 60% NF, and 40% NE versus the SC treatment (n=5) with 20% F, 40% NF, and 40% NE, not a statistically significant difference (P>0.05). A statistically significant difference (P>0.05) was not found between the TH and SC groups with regard to post-thaw viability, IVM, and IVF. Future research with larger numbers of immature or mature oocytes is suggested for further evaluation of both TH and SC for WTD oocyte vitrification for use with invitro-production techniques as a model for other endangered cervid species.