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Cytometry. Part A : the journal of the International Society for Analytical Cytology2022; 103(6); 479-491; doi: 10.1002/cyto.a.24712

In vitro aging of stallion spermatozoa during prolonged storage at 5°C.

Abstract: Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca ] decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca ] in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.
© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.
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Publication Date: 2022-12-22 PubMed ID: 36519783DOI: 10.1002/cyto.a.24712Google Scholar: Lookup
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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 deterioration in fertility of stallion sperm cells during prolonged cooling, by examining changes in cell function such as calcium balance and plasma membrane fluidity. The results suggest aging processes, i.e., disrupted calcium homeostasis and increased plasma membrane fluidity, as potential reasons for this decline in fertility.

Research Objectives and Methodology

  • The study aimed to understand the changes in stallion sperm cell function during prolonged cooling at 5°C. The specific objective was to determine how factors such as reactive oxygen species (ROS) production, mitochondrial function, calcium balance, and plasma membrane fluidity are affected during storage.
  • The research was conducted using multiparametric flow cytometry, a method that allows for simultaneous measurement of the factors mentioned above in the sperm cells. The viability and acrosome integrity of the sperm cells were also assessed during 96 hours of storage. Seventeen stallions contributed to the data set.

Key Findings

  • After 96 hours of storage, a high proportion (71 ± 8%) of viable and acrosome-intact sperm cells remained. This suggests that the basic integrity of the cells was preserved during the storage period.
  • Above 90% of viable, acrosome-intact sperm cells had active mitochondria and minimal intra-cellular or mitochondrial ROS levels, demonstrating functional mitochondria.
  • Over the storage period, the proportion of viable, acrosome-intact sperm cells with low plasma membrane fluidity and low intracellular calcium levels decreased from 63 ± 16% at 1 hour to 29 ± 18% at 96 hours – a statistically significant reduction (p < 0.05).
  • The intracellular calcium levels in viable sperm cells increased 3.1-fold over the 96-hour storage period (p < 0.05), indicating a disruption in calcium homeostasis.
  • The proportion of sperm cells that exhibited an increase in plasma membrane fluidity in response to bicarbonate stimulation also reduced significantly from 46 ± 17% at 1 hour to 19 ± 12% at 96 hours.

Conclusion and Implications

  • The research concludes that prolonging the storage of stallion semen at low temperatures leads to disturbed calcium homeostasis and increased plasma membrane fluidity. These physiological changes are indicative of in vitro aging processes in the sperm cells.
  • These findings suggest that the decline in fertility of stallion sperm during cooled storage might be related to these in vitro aging changes. This highlights the need for improved preservation strategies that can maintain stallion sperm cell function and fertility during storage.

Cite This Article

APA
Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H. (2022). In vitro aging of stallion spermatozoa during prolonged storage at 5°C. Cytometry A, 103(6), 479-491. https://doi.org/10.1002/cyto.a.24712

Publication

Cytometry. Part A : the journal of the International Society for Analytical Cytology
ISSN: 1552-4930
NlmUniqueID: 101235694
Country: United States
Language: English
Volume: 103
Issue: 6
Pages: 479-491

Researcher Affiliations

Umair, Muhammad
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Claes, Anthony
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Buijtendorp, Marijn
  • Dierenartsenpraktijk Horst e.o., Horst, The Netherlands.
Cuervo-Arango, Juan
  • Equine Fertility Group, Faculty of Veterinary Medicine, Universidad CEU Cardenal Herrera, CEU Universities, Alfara del Patriarca, Valencia, Spain.
Stout, Tom A E
  • Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Henning, Heiko
  • Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.

MeSH Terms

  • Male
  • Animals
  • Horses
  • Semen
  • Calcium / metabolism
  • Reactive Oxygen Species / metabolism
  • Sperm Motility
  • Semen Preservation / methods
  • Spermatozoa / metabolism

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Citations

This article has been cited 4 times.
  1. Brito LFC, Linardi RL, Rosales LAS, Balamurugan NS, Hernández-Avilés C, Ramírez-Agámez L. Evaluation of a Chemically Defined, Long-Term Extender for Liquid Storage of Stallion Semen. Reprod Domest Anim 2025 Sep;60(9):e70126.
    doi: 10.1111/rda.70126pubmed: 41002042google scholar: lookup
  2. Kheawkanha T, Pimprasert M, Boonkum W, Chankitisakul V. Enhancing thai native chicken semen quality and fertility through kaempferia parviflora supplementation during cold storage. Poult Sci 2025 Nov;104(11):105839.
    doi: 10.1016/j.psj.2025.105839pubmed: 40961767google scholar: lookup
  3. Boulbina I, Bekara MEA, AinBaziz H, Mattioli S, Castellini C. Breed-Specific Responses of Rabbit Semen to Chilling Storage: Sperm Quality, Acrosome Status, and Oxidative Stress Biomarkers. Animals (Basel) 2025 Aug 14;15(16).
    doi: 10.3390/ani15162384pubmed: 40867712google scholar: lookup
  4. Garriga F, Maside C, Padilla L, Recuero S, Rodríguez-Gil JE, Yeste M. Heat shock protein 70 kDa (HSP70) is involved in the maintenance of pig sperm function throughout liquid storage at 17 °C. Sci Rep 2024 Jun 11;14(1):13383.
    doi: 10.1038/s41598-024-64488-5pubmed: 38862610google scholar: lookup
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