Minimum Volume Vitrification of Immature Feline Oocytes

Summary

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

Abstract

In wild animals’ conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator’s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

Introduction

Cryopreservation has become a key step of assisted reproduction techniques (ARTs). In humans, it allows preservation of fertility or postponement of parenthood for medical or personal reasons. In animals, it is necessary to overcome distance and time in planned matings, especially in farm animals and pets, or to preserve genetic material of valuable subjects in conservation programs, particularly in wild endangered species. Gamete cryopreservation is the best choice when the individuals to be bred have not been chosen yet or in order to avoid ethical issues associated with embryo freezing, especially in human medicine¹. Spermatozoa are relative....

Protocol

The procedures hereby depicted did not undergo ethical approval since cat ovaries were collected at veterinary clinics as byproducts from owner-requested routine ovariectomy or ovariohysterectomy.

1. Oocyte collection

1. Before starting the experiments, prepare solutions for ovary and oocyte collection. Prepare ovary collection solution with phosphate buffered saline (PBS) with a mixture of antibiotics and antimycotics (100 IU/mL of penicillin G sodium, 0.1 mg/mL of streptomycin sulph.......

Discussion

Oocyte cryopreservation is a crucial germplasm conservation technique, especially in taxa where many species are endangered, such as Felidae family. In this manuscript, a simple and field-friendly protocol for the vitrification of immature cat oocytes was presented. Laboratory-made media, minimum volume vitrification supports and trained personnel are the key factors for the success of this method, which allows obtaining viable oocytes consistently and repeatedly, as shown by the representative results hereby reported.

Materials

Name	Company	Catalog Number	Comments
Amphotericin B	Sigma-Aldrich	A2942	/
Automatic pipettes & tips	/	/	Needed to pipette 20, 100, 240 and 300 µL
Surgical scalpels	/	/	Size 10 is usually ok
Bunsen beak	/	/	/
Clamps	/	/	Some small (Mosquito clamp) for ovary isolation, some bigger (Klemmer clamp) to work in liquid nitrogen
Cryotop	Kitazato (distributor: MBT - Medical Biological Technologies)	01.CR	Distributors and catalog number may change in different countries
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650	/
Ethylene glycol (EG)	Sigma-Aldrich	E9129	/
Fetal bovine serum (FBS)	Sigma-Aldrich	F9665	/
Glass Pasteur pipettes	/	/	Advised lenght 230 mm (100+130)
Gobelets	/	/	According to the canisters of the storage tank
Heating stage	/	/	All heating stages are ok, as long as they can keep 38ºC
Liquid nitrogen	/	/	/
Medium 199	Sigma-Aldrich	M4530	/
Phosphate-buffered saline (PBS)	Sigma-Aldrich	D8662	/
Penicillin G sodium	Sigma-Aldrich	P3032	/
Polyvinyl alcohol (PVA)	Sigma-Aldrich	P8136	/
Repro plate	Kitazato (distributor: MBT - Medical Biological Technologies)	01.K-2	Distributors and catalog number may change in different countries
Stereomicroscope	/	/	As long as the operator can select the oocytes, other stereomicroscopes are ok
Storage tank	/	/	Any regularly filled tank is ok
Streptomycin sulphate	Sigma-Aldrich	S9137	/
Styrofoam/nitrogen resistant box	/	/	All boxes which can contain liquid nitrogen are ok, as long as the operator is comfortable. Kitazato box is called "Cooling Rack"
Sucrose	Sigma-Aldrich	S1888	/
Timers	/	/	All timers which can be set on times until 9 minutes are ok