Bioenergetic/oxidative balance in feline morulae and blastocysts produced in vitro after temporary oocyte holding at room temperature versus cold ovary storage

A major problem in in vitro embryo production (IVEP) is the availability of methods for ovary or oocyte preservation and transport. The suitability of a given method can be assessed by analizing the bioenergetic/oxidative balance of obtained embryos, indicating oocyte/embryo health status during the whole IVEP chain. Unlike other species, cat oocytes have the unique ability to mature in vitro after cold ovary storage (COS; 1), which provides opportunities to rescue oocytes after ovariohysterectomy or sudden death. An alternative strategy is oocyte holding at room temperature (r.t.) in a medium without meiotic inhibitors, namely EH medium, as established in the mare (EH, 2). The aim of this study was to evaluate the effects of temporary (6h) oocyte holding at r.t. in EH medium versus COS on the development and bioenergetic/oxidative balance of feline late stage embryos, morulae (M) and blastocysts (Bl), obtained after in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVEC). The study was conducted in autumn 2016 in 7 replicates. Ovaries were collected from 17 anestrous queens by routine ovariohysterectomy at the Veterinary Hospital of University of Bari. For each queen, one ovary underwent immediate slicing and retrieved cumulus-oocyte complexes (COCs) were placed at r.t. (22-27°C) in the dark for 6h in EH medium (40% Earle’s, 40% Hank’s salts-buffered M199 and 20% FCS; 2), and the other ovary was placed in PBS solution and stored for 6h at 4°C (1) until slicing. Only COCs with uniform, darkly pigmented ooplasm and intact cumulus (1) underwent 24h IVM in a TCM199-based medium at 38.5°C under 5% CO2 in air (3). Fresh sperm cells were collected by flushing cauda epididymis of male cats, following routine orchiectomy. COCs and sperm cells (2x106/ml) were coincubated for 18-22h for IVF under 5% CO2 in air. IVEC lasted for 7 days under 5% CO2, 5% O2, 90% N2. IVF and IVEC were performed in SOF media (3). Embryo development was followed up to M and Bl stage and confirmed by nuclear chromatin evaluation. The bioenergetic/oxidative status of M and Bl was assessed by confocal microscopy after mitochondria and reactive oxygen species (ROS) labeling (4). Data were analysed by Chi-square and Student’s t Test (Significance when P≤0.05). A total of 359 oocytes were used, 185 after EH and 174 after COS. No differences were observed between the two groups for embryo cleavage (22/185, 12% vs 21/174, 12%; P>0.05), M/cleaved (4/22, 18% vs 8/21, 38%; P>0.05), Bl/cleaved (2/22, 9% vs 5/21, 24%; P>0.05) and number of nuclei (M: 39.5±30.4 vs 40±0; P>0.05; Bl: 100±14.1 vs 107.6±21.9; P>0.05). Compared with the EH group, COS-embryos showed mitochondria over-activity (arbitrary densitometry units, ADU: 38.2±15.0 vs 19.9±12.2; P<0.0001), increased ROS levels (ADU: 17.8±7.9 vs 10.9±4.1; P<0.0001) and significantly lower mitochondria/ROS colocalization (Pearson coefficient: 0.25±0.1 vs 0.34±0.2; P<0.001), overall indicating worse health conditions. We conclude that EH could be a valid alternative to COS for feline oocyte hortterm preservation, as it allowed to produce similar rates of M+Bl and these embryos showed improved bioenergetic/oxidative balance. [1]Wolfe & Wildt, J Reprod Fertil 1996;106:135-41. [2]Choi et al, Theriogenology 2006;66:955-63. [3]Martino et al, Reprod Toxicol 2016;65:204–11. [4]Martino et al, Reprod Biol Endocrinol 2013;11:27. [5]Gonzalez et al, Reprod Dom Anim 2012;47:118-20