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Abstract

Introduction

Protocol

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Acknowledgements

Materials

References

Developmental Biology

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

Published: July 10th, 2021

DOI:

10.3791/62272

1Department of Veterinary Medicine, University of Sassari

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.

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.

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 programs1,2,3. Juvenile oocyte vitrification would also increase genetic gain by shortening the generation interval in breeding programs.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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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/.......

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The cryotolerance of oocyte from juvenile donors is lower compared to adult ones. The first effect observed is a lower post-warming survival rate compared to adult oocytes (Figure 1A; χ2 test P<0.001). Juvenile oocytes showed a lower membrane integrity after warming (Figure 1B). The use of trehalose in the maturation medium was intended to verify whether this sugar could reduce cryoinjuries in juvenile oocytes. The data have demonstrated

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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.

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The authors received no specific funding for this work. Professor Maria Grazia Cappai and Dr. Valeria Pasciu are gratefully acknowledged for the video voiceover and for setting up the lab during the video making.

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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

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