Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results.
Abstract: The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.
Publication Date: 2001-03-14 PubMed ID: 11245266DOI: 10.1016/s0093-691x(01)00444-7Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research sought to develop a method for successful freezing of cooled horse sperm, to enhance the easy transportation of stallion semen to specialized facilities without requiring the stallions to leave their homes. The experiments revealed that optimum cryopreservation is achieved when the semen is centrifuged at 25°C to remove seminal plasma, diluted to a concentration of 250 million/mL, kept at 5°C for 12 hours, and then frozen.
Study Methodology
- The research involved three different experiments.
- The first experiment compared various spermatozoal extenders, packaging sizes, time after collection, and how long sperm can be cooled before being frozen.
- The second experiment evaluated the difference in spermatozoa centrifuged before and after cooling, and the effect of cooling duration before freezing.
- The third experiment examined spermatozoa centrifuged for seminal plasma removal, resuspended at different concentrations, cooled to 5 degrees Celsius for 12 hours, and then frozen.
Key Findings
- In the first experiment, researchers found that semen frozen 2.5 hours after cooling had a higher proportion of progressively motile (active and moving in a forward direction) spermatozoa than semen frozen 24 hours after cooling.
- Semen frozen in extenders containing egg yolk showed higher percentages of progressively motile spermatozoa compared to those frozen in extenders containing just skim milk.
- The study found no significant difference in sperm quality when frozen in either 0.5- or 2.5-mL straws.
- The second experiment showed that sperm centrifuged before cooling had higher progressively motile spermatozoa percentages than those centrifuged after cooling.
- Spermatozoa that were centrifuged at room temperature (around 25 degrees Celsius) first and then cooled to 5 degrees Celsius for 12 hours had better post-thaw progressive motility.
- In the final experiment, researchers discovered that samples with sperm concentrations of 50 or 250 million/mL had higher percentages of progressively motile spermatozoa after freezing than samples with a concentration of 500 million spermatozoa/mL.
Recommendations
- Based on the results, the authors suggest that for optimal sperm preservation, semen should be centrifuged at approximately 25 degrees Celsius to remove seminal plasma.
- The sperm should then be diluted to a concentration of 250 million spermatozoa/mL and kept at 5 degrees Celsius for 12 hours before being frozen.
Cite This Article
APA
Crockett EC, Graham JK, Bruemmer JE, Squires EL.
(2001).
Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results.
Theriogenology, 55(3), 793-803.
https://doi.org/10.1016/s0093-691x(01)00444-7 Publication
Researcher Affiliations
- Department of Physiology, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Animals
- Centrifugation / veterinary
- Cold Temperature
- Cryopreservation / veterinary
- Horses / physiology
- Male
- Semen Preservation / methods
- Semen Preservation / veterinary
- Sperm Motility
- Spermatozoa / physiology
- Time Factors
Citations
This article has been cited 6 times.- Colombo M, Morselli MG, Franchi G, Schäfer-Somi S, Luvoni GC. Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport. Animals (Basel) 2022 Mar 23;12(7).
- Neila-Montero M, Riesco MF, Alvarez M, Montes-Garrido R, Boixo JC, de Paz P, Anel-Lopez L, Anel L. Centrifugal force assessment in ram sperm: identifying species-specific impact. Acta Vet Scand 2021 Nov 4;63(1):42.
- Fernandes M, Hernández PR, Simões J, Barbas JP. Effects of Three Semen Extenders, Breeding Season Month and Freezing-Thawing Cycle on Spermatozoa Preservation of Portuguese Merino Sheep. Animals (Basel) 2021 Sep 7;11(9).
- Di Iorio M, Rusco G, Lauriola F, Antenucci E, Roncarati A, Cerolini S, Schiavitto M, Iaffaldano N. Validating Sperm Concentration in Rabbit Cryopreservation Protocol: Implications for Fertility, Litter Size, and Offspring Growth. Vet Sci 2025 Jul 18;12(7).
- Spanner EA, de Graaf SP, Rickard JP. A multivariate model for the prediction of pregnancy following laparoscopic artificial insemination of sheep. Sci Rep 2024 Nov 11;14(1):27556.
- Di Iorio M, Lauriola F, Rusco G, Antenucci E, Schiavitto M, Iaffaldano N. Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol. Vet Sci 2023 Dec 22;11(1).
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