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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 programs4. Vitrification by ultra-rapid cooling and warming of oocytes is currently considered a standard approach for livestock oocytes cryopreservation5. In ruminants, before vitrification, oocytes are usually matured in vitro, after retrieval from follicles obtained from abattoir-derived ovaries2. Adult, and especially prepubertal ovaries4,6, can indeed supply a virtually unlimited number of oocytes to be cryopreserved.
In cattle, after oocyte vitrification and warming, blastocyst yields at >10% have been commonly reported by several laboratories during the last decade3. However, in small ruminants oocyte vitrification is still considered relatively new for both juvenile and adult oocytes, and a standard method for sheep oocyte vitrification remains to be established2,5. Despite recent advancements, the vitrified and warmed oocyte indeed presents several functional and structural alterations that limit their developmental potential7,8,9. Thus, few articles have reported blastocyst development at 10% or more in vitrified/warmed sheep oocytes2. Several approaches have been investigated to reduce the above-mentioned alterations: optimizing the composition of the vitrification and thawing solutions10,11; experimenting with the use of different cryo-devices8,12,13; and applying specific treatments during in vitro maturation (IVM)4,14,15 and/or during the recovery time after warming6.
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 culture period.
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
2. Oocyte collection and maturation
3. Semen collection, freezing and thawing procedures
4. In vitro fertilization and embryo culture
5. Oocyte vitrification and warming
NOTE: Perform vitrification following the method of minimum essential volume (MEV) using device cryotops17.
6. Assessment of oocyte quality post-warming
7. Oocyte survival assessment
8. Evaluation of mitochondrial activity and ROS intracellular levels by confocal laser scanning microscopy
9. Statistical Analyses
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
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.
The authors declare they have no competing financial interests.
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.
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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