Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws.
Abstract: Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.
Publication Date: 2000-09-16 PubMed ID: 10990353DOI: 10.1016/s0093-691x(00)00330-7Google Scholar: Lookup
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- Journal Article
Summary
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The research paper reports on the success of vitrifying (a freezing process) both mature and immature horse and bovine egg cells through the use of ethylene glycol, in a type of cryopreservation technique.
Experimental Design
- The study was conducted to compare the viability of sperm cells of horses and cows, which were made to vitrify or freeze using ethylene glycol and Ficoll.
- The egg cells were immediately vitrified after being collected from the slaughterhouse ovaries or after a period of maturation, 36 to 40 hours for horse egg cells and 22 to 24 hours for cow egg cells.
- The immature egg cells were matured after thawing. A special cryoprotectant mix (TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose or EFS) was used to prepare the egg cells before vitrification.
- The egg cells were stored in straws in small amounts of cryoprotectant and submerged in liquid nitrogen. The process of warming straws and dilution of the cryoprotectant was done at a temperature of 37 degrees Celsius using a particular mix.
Results
- The maturation of egg cells to metaphase II after thawing did not significantly vary among the groups within the same species.
- It was observed that all horse treatment groups had lower maturation rates than the cow groups. The live/dead status of the egg cells did not differ among treatments within species.
- There were no significant differences in the percentage of egg cells that survived and reached metaphase II among the treatment groups of each species. Also, there were no significant differences in the rate of mature cortical granule distribution within species.
- However, more bvine egg cells had mature cortical granule distribution and nuclear maturation than horse egg cells. Comparing nuclear and cytoplasmic maturation, only 30% equine oocytes had both while 70% of bovine oocytes did, indicating a significant difference.
Conclusion
- In conclusion, vitrification was successful in cryopreserving both immature and mature horse and bovine egg cells.
- However, the survival rate of egg cell vitrification was found to be lower in horses than cows.
Cite This Article
APA
Hurtt AE, Landim-Alvarenga F, Seidel GE, Squires EL.
(2000).
Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws.
Theriogenology, 54(1), 119-128.
https://doi.org/10.1016/s0093-691x(00)00330-7 Publication
Researcher Affiliations
- Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Animals
- Cattle / physiology
- Cell Survival
- Ethylene Glycol
- Female
- Ficoll
- Horses / physiology
- Oocytes
- Solutions
- Sucrose
- Tissue Preservation / methods
- Tissue Preservation / veterinary
Citations
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- Tharasanit T, Thuwanut P. Oocyte Cryopreservation in Domestic Animals and Humans: Principles, Techniques and Updated Outcomes. Animals (Basel) 2021 Oct 13;11(10).
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- Nikseresht M, Toori MA, Rasti T, Kashani IR, Mahmoudi R. The Nuclear Maturation and Embryo Development of Mice Germinal Vesicle Oocytes with and without Cumulus Cell after Vitrification. J Clin Diagn Res 2015 Jan;9(1):AF01-4.
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- Turathum B, Saikhun K, Sangsuwan P, Kitiyanant Y. Effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. Reprod Biol Endocrinol 2010 Jun 22;8:70.
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