Feline ovarian tissue vitrification: fixation may affect follicle grading

All feline species except the domestic cat are currently classified as endangered. Vitrification of feline ovarian tissue is an innovative field friendly conservation approach suitable to all ages with possibility of retaining both reproductive and endocrine functions of the ovary. Despite several reported protocols of feline ovarian tissue vitrification accompanied by varying degrees of successes, no standardisation has been achieved yet. Thus, we aimed at comparing three vitrification protocols and evaluate the effect of two fixatives on follicular grading. Total of 32 fragments (1x1x1 mm) of ovarian cortex from 4 domestic cats were obtained and divided into four groups. One group (fresh) was fixed as control, while the other three were placed on 30G needles (4 fragments/needle) for vitrification. Three different vitrification protocols (A, B, C) were tested. Protocol A involved equilibration in 7.5% dimethyl sulfoxide (DMSO), 7.5% ethylene glycol (EG) and 20% fetal bovine serum (FBS) for 10 min, then equilibration in 15% DMSO, 15% EG, 20% FBS for 10 min at 4°C [1], and finally vitrification (i.e., direct plunging of the needle with fragments in liquid nitrogen). Protocols B and C, instead, were completely carried out at room temperature and based on a vitrification solution (VS) containing 10% DMSO, 26% EG, 2.5% polyvinylpyrrolidone and 1M sucrose in MEM + 20 mg/ml bovine serum albumin (BSA) [2]. Protocol B involved equilibration for 7 min in VS1 (25% VS in MEM + BSA), 4 min in VS2 (50% VS in MEM + BSA) and 3 min in VS3 (100% VS). Protocol C used the same VS, but timings were halved. Warming was performed in the same way for all protocols. Fragments were warmed in MEM + 1M sucrose for 15 sec, and then washed for 5 minutes in MEM with decreasing concentrations of sucrose (0.5M, 0.25M and 0M sucrose). One fragment from each treatment was fixed in Bouin’s solution and one in 10% neutral buffered formalin. During histological evaluation, follicles were morphologically graded into four grades and classified based on stages of development. Morphologically normal follicles with homogenous oocyte cytoplasm were observed in samples fixed in Bouin’s solution, while formalin-fixed samples appeared with oocyte cytoplasmic vacuoles. There was no significant (P >0.05) variation among the three protocols with regards to the various grades of follicles in samples fixed with Bouin’s solution (A, B, C, respectively, grade 1: 16.5%, 17.8% and 16.8%, grade 2: 50.6%, 54.7%, 47.4%, grade 3: 18.4%, 15.7%, 19.4%, grade 4: 14.6%, 12.4%, 16.8% respectively). On the other hand, irrespective of the treatments significant lower proportion of grade 1 follicles were observed in formalin compared to Bouin-fixed samples (average 0.5% vs. 27.4%, respectively). In conclusion, lower equilibration time presented in protocol C could be used for feline ovarian tissue vitrification and use of formalin in fixing tissue fragments intended for morphological evaluation may lead to erroneous follicle grading. Supported by Polish National Agency for Academic Exchange, Grant PPI/APM/2019/1/00044/U/00001. [1] Mouttham L, Comizzoli, P. The preservation of vital functions in cat ovarian tissues during vitrification depends more on the temperature of the cryoprotectant exposure than on the sucrose supplementation. Cryobiology 2016;73:187-95. [2] Amorim CA, Jacobs S, Devireddy RV, et al. Successful vitrification and autografting of baboon (Papio anubis) ovarian tissue. Hum Reprod 2013;28:2146-56.