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Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa.
Abstract: This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.
Publication Date: 2002-03-08 PubMed ID: 11882023 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
- Research Support
- Non-U.S. Gov't
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 investigates the development of horse oocytes (egg cells) after injecting sperm directly into the egg cell using a method called intracytoplasmic sperm injection (ICSI). The study compared the use of fresh and frozen-thawed sperm and was performed both in a laboratory setting (in vitro) and within the body of a female horse (in vivo). The results indicated that good activation rates can be achieved with either fresh or frozen-thawed horse sperm, without the need for additional activation treatments.
Research Methodology and Process
- The research involved collecting oocytes from ovaries obtained from an abattoir.
- These oocytes, specifically those with expanded cumulus cells, were matured in a culture medium for 24-26 hours.
- After maturation, oocytes with a first polar body (indicative of readiness for fertilization) were selected for sperm injection.
- For sperm injection, both fresh and frozen-thawed sperm from the same stallion were used.
- The sperm were then injected directly into the oocytes using a special drilling device known as a Piezo drill.
- After the sperm was injected, the resultant zygotes (fertilized oocytes) were either grown in a culture medium or implanted into the oviducts of recipient mares.
Results and Observations
- The research revealed that there were no significant differences in the fertilization rate between fresh and frozen-thawed sperm in either horse or bovine oocytes.
- However, the number of pronucleus formations (which indicates successful fertilization) in bovine oocytes was significantly higher than in equine oocytes.
- No significant differences were observed in the cleavage rate (cell division) or the average number of nuclei at 96 hours post-injection in horse oocytes injected with either fresh or frozen-thawed sperm.
- Embryos that developed in vivo had a significantly higher number of nuclei compared to those cultured in vitro.
Conclusions and Implications
- The outcomes of this study suggest that activation rates after injecting either fresh or frozen-thawed equine sperm are robust, negating the need for any additional activation treatment.
- The results also suggest that injecting frozen-thawed equine sperm result in similar embryo development as with fresh equine sperm.
- It was also noted that in vivo culture within the oviduct achieved better results than in vitro culture in terms of cell population.
- Finally, the results indicated bovine oocytes could serve as a useful model for assessing sperm function in horses.
Cite This Article
APA
Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.
(2002).
Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa.
Reproduction, 123(3), 455-465.
Publication
Researcher Affiliations
- Departments of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466, USA.
MeSH Terms
- Animals
- Blastocyst / physiology
- Cattle
- Cells, Cultured
- Cryopreservation
- Embryo Transfer
- Embryonic and Fetal Development
- Female
- Horses
- Male
- Oocytes
- Oogenesis
- Semen Preservation
- Sperm Injections, Intracytoplasmic / methods
Citations
This article has been cited 7 times.- Sun YD, An T, Liang R, Luo YW, Xia HZ, Fu L, Han S, Zhu YX, Song ZY, Bai XY, Fu Y, Fu XW, Hou YP, Lu Q. Precise mimicry of physiological Ca(2+) oscillations for mammalian oocyte activation by nanosecond pulsed electric field. Bioeng Transl Med 2026 Jan;11(1):e70094.
- de Oliveira RA, Alonso MA, Fonte JS, Fernandes CB. Equine ICSI: an update on semen perspective. Anim Reprod 2024;21(4):e20240015.
- Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II. Animals (Basel) 2021 Nov 20;11(11).
- Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE. Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. Animals (Basel) 2021 Jan 3;11(1).
- Gambini A, Duque Rodríguez M, Rodríguez MB, Briski O, Flores Bragulat AP, Demergassi N, Losinno L, Salamone DF. Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern. PLoS One 2020;15(9):e0238948.
- Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Carnevale EM. Cytoskeletal alterations associated with donor age and culture interval for equine oocytes and potential zygotes that failed to cleave after intracytoplasmic sperm injection. Reprod Fertil Dev 2015 Jul;27(6):944-56.
- Lange Consiglio A, Dell'Aquila ME, Fiandanese N, Ambruosi B, Cho YS, Bosi G, Arrighi S, Lacalandra GM, Cremonesi F. Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse. Reprod Biol Endocrinol 2009 Oct 16;7:113.
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