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Cryopreservation of equine oocytes by 2-step freezing.
Abstract: Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.
Publication Date: 1994-01-01 PubMed ID: 16727612DOI: 10.1016/0093-691x(94)90856-7Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research presents findings on the effectiveness of cryopreservation methods for immature equine oocytes. Cryoprotectants used for freezing were ethylene glycol (EG), 1,2-propanediol (PD), or glycerol (GL). The subsequently thawed oocytes’ maturity was evaluated, revealing the most successful cryoprotectant to be ethylene glycol.
Research Methodology
- The study collected immature equine oocytes from slaughterhouse ovaries for cryopreservation.
- The oocytes were frozen in a solution containing a cryoprotectant (EG, PD, or GL) and sucrose. This solution was placed in straws which were then cooled to a specific temperature before being frozen in liquid nitrogen.
- The straws were rapidly thawed and the thawed oocytes were in vitro cultured for 32 hours to determine the rate of maturation.
- The researchers also tested the capability of the cryopreserved oocytes to be successfully fertilized by stallion spermatozoa.
- The spermatozoa were processed through a series of actions, including preincubation with caffeine, ionophore treatment, and insemination for a set period.
- Two groups of oocytes – those matured in vitro post-thawing and the other matured immediately after thawing – were exposed to this processed spermatozoa for fertilization tests.
Main Findings
- Of the three cryoprotectants, ethylene glycol (EG) had the best results, with 15.8% of frozen-thawed oocytes reaching the Metaphase II (MII) stage. 1,2-Propanediol (PD) had a maturation rate of 5.8%. Glycerol (GL) was unsuccessful, with none of the oocytes maturing.
- In comparison, 63.3% of nonfrozen control oocytes reached the MII stage when matured in vitro, showing the relative effectiveness of the non-cryopreserved control method.
- In terms of fertilization after freezing in EG, both matured and immature oocytes showed significant capability to be penetrated by stallion spermatozoa.
- The count of spermatozoa per penetrated oocyte was similar in both the cryopreserved oocytes and the nonfrozen controls.
- The overall results showed that immature equine oocytes can reach the MII stage in vitro after cryopreservation in EG or PD, but not GL. Furthermore, they can be successfully penetrated and fertilized by stallion spermatozoa.
Conclusion
The study provides insight into the potential use and optimization of cryopreservation techniques for equine oocytes. Utilizing EG or PD as cryoprotectants can lead to successful freezing, thawing, and subsequent in vitro maturation of the oocytes. Moreover, these cryopreserved oocytes exhibited a significant potential for successful fertilization.
Cite This Article
APA
Hochi S, Fujimoto T, Choi YH, Braun J, Oguri N.
(1994).
Cryopreservation of equine oocytes by 2-step freezing.
Theriogenology, 42(7), 1085-1094.
https://doi.org/10.1016/0093-691x(94)90856-7 Publication
Researcher Affiliations
- Laboratory of Horse Production Obihiro University of Agriculture and Veterinary Medicine Obihiro, Hokkaido 080, Japan.
Citations
This article has been cited 2 times.- Prentice JR, Anzar M. Cryopreservation of Mammalian oocyte for conservation of animal genetics. Vet Med Int 2010 Sep 21;2011.
- Rimayanti R, Khairullah AR, Madyawati SP, Rantam FA, Mustofa I, Widjiati W, Srianto P, Akintunde AO, Pratama BP, Ahmad RZ, Wardhani BWK, Khalisa AT, Wibowo S. The role of heat shock protein 70 on oocyte apoptosis during vitrification. Open Vet J 2025;15(10):4847-4864.
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