Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.
Abstract: The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.
Copyright © 2019 Elsevier Inc. All rights reserved.
Publication Date: 2019-10-17 PubMed ID: 31732114DOI: 10.1016/j.jevs.2019.102801Google Scholar: Lookup
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Summary
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The research investigated the impact of four different freezing methods on the quality of equine epididymal sperm. The result revealed the slow cooling rate with controlled rate freezer enhances the sperm quality after thawing, and equilibration in a cooled environment is advantageous when using a floating rack in nitrogen vapor as an alternate freezing method.
Methodology
- The researchers aimed at assessing the implications of diverse freezing methods on sperm quality parameters including motility, viability, acrosomal status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity.
- Sperm samples were collected from 10 healthy stallions by retrograde flushing after testectomy.
- These samples were then diluted with a semen extender containing specific milk proteins and a freezing extender involving egg yolk and glycerol.
- The samples were cooled down using four different freezing pathways employing a programmable freezer and a floating rack implementing a slow or a swift cooling speed.
Results
- The research found reduced total post-thaw motility and slow sperm values when process 4 was used, compared to processes 1 and 2.
- Progressive motility was lower in process 4 than process 1 after thawing and after an hour of incubation.
- The number of fast sperms was decreased when process 4 was used directly post thawing and after an hour of incubation.
- Healthy sperm values were boosted in processes 1 and 2 compared to process 4 after an hour’s incubation.
- However, more vital sperm with high MMP were detected when using process 3 immediately following thawing.
- Least number of sperms containing calcium were observed in process 3 after thawing and in process 4 after one hour of incubation.
- Process 3 produced a higher subpopulation of viable/hMMP/lCa sperm immediately after defrosting compared to process 2. However, the least amount of this subpopulation was noted in process 4 after one hour of incubation.
- All the four freezing procedures showed no major differences in terms of DNA integrity of the thawed sperm.
Conclusion
- The research concluded that a slow cooling rate with a controlled rate freezer results in optimal sperm quality after thawing.
- The research also suggested that using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment would be beneficial.
Cite This Article
APA
Neuhauser S, Bollwein H, Siuda M, Handler J.
(2019).
Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.
J Equine Vet Sci, 82, 102801.
https://doi.org/10.1016/j.jevs.2019.102801 Publication
Researcher Affiliations
- Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Bad Saarow, Germany.
- Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
- Clinic of Reproductive Medicine, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.
- Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Bad Saarow, Germany. Electronic address: johannes.handler@fu-berlin.de.
MeSH Terms
- Animals
- DNA
- Freezing
- Horses
- Male
- Membrane Potential, Mitochondrial
- Semen Preservation / veterinary
- Spermatozoa
Citations
This article has been cited 2 times.- 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).
- Kumaresan A, Das Gupta M, Datta TK, Morrell JM. Sperm DNA Integrity and Male Fertility in Farm Animals: A Review.. Front Vet Sci 2020;7:321.
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