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Optimization of cryopreservation protocols for cooled-transported stallion semen.
Abstract: Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.
Copyright © 2020 Elsevier B.V. All rights reserved.
Publication Date: 2020-08-20 PubMed ID: 32891911DOI: 10.1016/j.anireprosci.2020.106581Google 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
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This research article focused on optimizing the process of cryopreserving cooled-transported stallion semen, through experimentation with different cooling and freezing conditions, extenders, and freezing methods that may affect sperm motility and integrity. The study recommended that Botucrio and lactose-EDTA extenders should be adopted in conventional cryopreservation.
Objective of the Study
The study aims to simplify and optimize the protocols associated with freezing cooled-transported semen. This is aimed at providing standardization in the process which lacks uniform freezing and shipping protocols. The objective is to enhance the quality and performance of the thawed sperm post the freezing protocol.
Experiment 1: Centrifugation temperature impact on semen quality
- The first experiment revolved around testing the impact of centrifugation temperature on the quality of semen post freezing. The semen was centrifuged either at room temperature or at 5°C. The variables including motility, membrane integrity, acrosome integrity and membrane fluidity of the sperm were assessed before and after freezing
- The results showed that the temperature of centrifugation did not have any impact on the quality of semen post thawing. Hence it can be performed at room temperature or 5 °C
Experiment 2: Cryopreservation in different semen freezing extenders
- In the second experiment, the cooled-transported semen was cryopreserved in three different semen freezing extenders after centrifuging it at room temperature
- The extenders included the improved modified French formula, Botucrio, and the improved lactose-EDTA formula
- The results depicted that semen cryopreserved in the improved modified French formula had less total and progressive motility post thawing as compared to the other two extenders. It also had lesser percentage sperm with intact membranes as compared to lactose-EDTA and a greater percentage of sperm showing capacitation-like changes as compared tp Botucrio
Experiment 3: Conventional freezing versus ultra-freezing
- The third experiment focused on comparing the conventional freezing method with direct placement in a -80°C ultra-freezer, with the semen diluted in each of the three extenders
- The results indicated that freezing in an ultra-freezer resulted in lesser post-thaw sperm motility. However, it had no impact on membrane and acrosome integrity and capacitation-like changes
Conclusion and Recommendations
- Based on the results, the study concluded that centrifugation and the addition of freezing extenders to cooled transported semen can be performed at room temperature or at 5 °C
- Additionally, for conventional cryopreservation of cooled-transported stallion semen, the use of Botucrio and lactose-EDTA extenders is recommended compared to the modified French formula
Cite This Article
APA
Ferrer MS, Canisso IF, Ellerbrock RE, Podico G, Lister BN, Hurley DJ, Kline K, Palomares RA.
(2020).
Optimization of cryopreservation protocols for cooled-transported stallion semen.
Anim Reprod Sci, 221, 106581.
https://doi.org/10.1016/j.anireprosci.2020.106581 Publication
Researcher Affiliations
- Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA. Electronic address: msferrer@uga.edu.
- Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA.
- Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
- Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, IL, USA.
- Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
- Department of Animal Sciences, College of Agricultural, Environment and Nutritional Sciences, Urbana, IL, USA.
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- Cryoprotective Agents / pharmacology
- Freezing
- Horses / physiology
- Male
- Semen / drug effects
- Semen Preservation / veterinary
- Specimen Handling / methods
- Specimen Handling / veterinary
Citations
This article has been cited 4 times.- Jurado-Campos A, Soria-Meneses PJ, Arenas-Moreira M, Alonso-Moreno C, Rodríguez-Robledo V, Soler AJ, Garde JJ, Del Rocío Fernández-Santos M. Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams. J Anim Sci Biotechnol 2023 Aug 10;14(1):106.
- Akhtar MF, Ma Q, Li Y, Chai W, Zhang Z, Li L, Wang C. Effect of Sperm Cryopreservation in Farm Animals Using Nanotechnology. Animals (Basel) 2022 Sep 2;12(17).
- Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K. Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes. Biol Reprod 2025 May 13;112(5):867-879.
- Halo M Jr, Tirpák F, Massányi M, Dianová L, Lenický M, Slanina T, Zemanová J, Matušková A, Greń A, Halo M, Massányi P. Calcium affects stallion spermatozoa parameters in different incubation temperatures. Heliyon 2024 Aug 30;10(16):e35879.
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