Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

Summary

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

Abstract

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart.

Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

Introduction

Long-term storage of the female gametes can offer a wide range of applications, such as improving domestic animal breeding by genetic selection programs, contributing to preserve biodiversity through the ex-situ wildlife species conservation program, and boosting in vitro biotechnology research and applications thanks to the availability of stored oocytes to be incorporated in in vitro embryo production or nuclear transplantation programs^1,2,3. Juvenile oocyte vitrification would also increase genetic gain by shortening the generation interval in breeding programs

Protocol

The animal protocol and the implemented procedures described below are in accordance with the ethical guidelines in force at the University of Sassari, in compliance with the European Union Directive 86/609/EC and the recommendation of the Commission of the European Communities 2007/526/EC.

1. Preparation of media for oocyte manipulation

1. Prepare the medium for transport of collected ovaries by supplementing Dulbecco's phosphate buffered saline with 0.1 g/L penicillin and 0.1 g/.......

Discussion

Oocyte cryopreservation in domestic animals can allow not only the long-term conservation of female genetic resources, but also advance the development of embryonic biotechnologies. Thus, the development of a standard method for oocyte vitrification would advantage both the livestock and the research sector. In this protocol, a complete method for adult sheep oocyte vitrification is presented and could represent a solid starting point for the development of an efficient vitrification system for juvenile oocyte.

Materials

Name	Company	Catalog Number	Comments
2′,7′-Dichlorofluorescin diacetate	Sigma-Aldrich	D-6883
Albumin bovine fraction V, protease free	Sigma-Aldrich	A3059
Bisbenzimide H 33342 trihydrochloride (Hoechst 33342)	Sigma-Aldrich	14533
Calcium chloride (CaCl2 2H20)	Sigma-Aldrich	C8106
Citric acid	Sigma-Aldrich	C2404
Confocal laser scanning microscope	Leica Microsystems GmbH,Wetzlar	TCS SP5 DMI 6000CS
Cryotop Kitazato	Medical Biological Technologies
Cysteamine	Sigma-Aldrich	M9768
D- (-) Fructose	Sigma-Aldrich	F0127
D(+)Trehalose dehydrate	Sigma-Aldrich	T0167
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2438
Dulbecco Phosphate Buffered Saline	Sigma-Aldrich	D8537
Egg yolk	Sigma-Aldrich	P3556
Ethylene glycol (EG)	Sigma-Aldrich	324558
FSH	Sigma-Aldrich	F4021
Glutamic Acid	Sigma-Aldrich	G5638
Glutaraldehyde	Sigma-Aldrich	G5882
Glycerol	Sigma-Aldrich	G5516
Glycine	Sigma-Aldrich	G8790
Heparin	Sigma-Aldrich	H4149
HEPES	Sigma-Aldrich	H4034
Hypoutarine	Sigma-Aldrich	H1384
Inverted microscope	Diaphot, Nikon
L-Alanine	Sigma-Aldrich	A3534
L-Arginine	Sigma-Aldrich	A3784
L-Asparagine	Sigma-Aldrich	A4284
L-Aspartic Acid	Sigma-Aldrich	A4534
L-Cysteine	Sigma-Aldrich	C7352
L-Cystine	Sigma-Aldrich	C8786
L-Glutamine	Sigma-Aldrich	G3126
LH	Sigma-Aldrich	L6420
L-Histidine	Sigma-Aldrich	H9511
L-Isoleucine	Sigma-Aldrich	I7383
L-Leucine	Sigma-Aldrich	L1512
L-Lysine	Sigma-Aldrich	L1137
L-Methionine	Sigma-Aldrich	M2893
L-Ornithine	Sigma-Aldrich	O6503
L-Phenylalanine	Sigma-Aldrich	P5030
L-Proline	Sigma-Aldrich	P4655
L-Serine	Sigma-Aldrich	S5511
L-Tyrosine	Sigma-Aldrich	T1020
L-Valine	Sigma-Aldrich	V6504
Magnesium chloride heptahydrate (MgSO4.7H2O)	Sigma-Aldrich	M2393
Makler Counting Chamber	Sefi-Medical Instruments ltd.Biosigma S.r.l.
Medium 199	Sigma-Aldrich	M5017
Mineral oil	Sigma-Aldrich	M8410
MitoTracker Red CM-H2XRos	ThermoFisher	M7512
New born calf serum heat inactivated (FCS)	Sigma-Aldrich	N4762
Penicillin G sodium salt	Sigma-Aldrich	P3032
Phenol Red	Sigma-Aldrich	P3532
Polyvinyl alcohol (87-90% hydrolyzed, average mol wt 30,000-70,000)	Sigma-Aldrich	P8136
Potassium Chloride (KCl)	Sigma-Aldrich	P5405
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655
Propidium iodide	Sigma-Aldrich	P4170
Sheep serum	Sigma-Aldrich	S2263
Sodium azide	Sigma-Aldrich	S2202
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S5761
Sodium chloride (NaCl)	Sigma-Aldrich	S9888
Sodium dl-lactate solution syrup	Sigma-Aldrich	L4263
Sodium pyruvate	Sigma-Aldrich	P2256
Sperm Class Analyzer	Microptic S.L.	S.C.A. v 3.2.0
Statistical software Minitab 18.1	2017 Minitab
Stereo microscope	Olimpus	SZ61
Streptomycin sulfate	Sigma-Aldrich	S9137
Taurine	Sigma-Aldrich	T7146
TRIS	Sigma-Aldrich	15,456-3