Skip to main content
NCBI home page
Wiley Open Access Collection logoLink to Wiley Open Access Collection
. 2026 Sep 30;61(Suppl 3):e70260. doi: 10.1111/rda.70260

Comparative Susceptibility of Donkey and Horse Sperm to Liquid‐Storage Temperature

Pilar Vallejo‐Soto 1,✉, Antonio González Ariza 2, Blenda R N Vilela 1, Carlos Montero‐Palma 1, Rocío Rodríguez González‐Miranda 1, Verónica Pérez‐Aguilera 3, Francisco Crespo‐Castejón 3, Manuel Hidalgo 1, Jesús Dorado 1, Isabel Ortiz 1
PMCID: PMC13627757  PMID: 42816916

ABSTRACT

Preservation protocols for donkey sperm are based on those developed for horses, despite potential interspecific differences in cooling tolerance. This study compared horse and donkey sperm during long‐term storage at 5°C and 17°C. Ejaculates from four stallions and four jacks (n = 16) were centrifuged, resuspended in skimmed milk‐based extender, and stored at both temperatures. Sperm motility, plasma and acrosome membrane integrity, and morphology were evaluated after 1, 2 and 7 days. Fresh semen parameters showed no interspecific differences (p > 0.05). At 5°C, donkey sperm showed reduced motility and membrane integrity from Day 1, whereas horse sperm parameters remained stable at both temperatures up to Day 2. After 7 days, horse doses stored at 5°C maintained significantly higher quality. In conclusion, donkey sperm was more sensitive to storage at 5°C than horse sperm, highlighting the need for species‐specific preservation strategies.

Keywords: equine, interspecific differences, jack, semen preservation, stallion

1. Introduction

Artificial insemination (AI) is a widely used reproductive technique that relies on effective semen preservation. Horse semen is commonly stored at 5°C because this temperature slows cellular metabolism, can maintain sperm viability for about 3 days, and reduces bacterial growth (Aurich 2008). In stallions, milder storage temperatures (15°C–22°C) have been shown to maintain sperm fertilizing capacity for up to 7 days. However, their application remains limited by challenges associated with increased metabolic activity, which require very specific extenders and processing techniques (Clulow and Gibb 2022).

In donkeys, few studies have assessed the effects of these higher temperatures (> 15°C) for long‐term storage (Caldevilla et al. 2025; Serres et al. 2002; Vallejo‐Soto et al. 2025). Therefore, semen preservation protocols in this species are still mostly adapted from those developed for horses at 5°C, assuming similar responses to storage conditions (Gobato et al. 2022). However, this approach often results in suboptimal reproductive outcomes compared to horses (Canisso et al. 2019), highlighting the need to evaluate alternative storage strategies for donkeys.

Despite the available literature, most studies on semen preservation in equids are species‐specific, and direct comparisons between horse and donkey sperm under standardized storage conditions remain limited (Catalán et al. 2023; Chaves et al. 2023). Such studies are essential to identify differences in sperm physiology and to optimize preservation protocols.

Therefore, the aim of this study was to compare horse and donkey sperm quality during long‐term liquid storage at 5°C and 17°C over 7 days.

2. Material and Methods

2.1. Semen Collection and Storage

All procedures complied with the guidelines of the Animal Ethics Committee of the University of Cordoba and Spanish legislation on animal welfare.

A total of 16 ejaculates were collected from 4 clinically healthy donkeys and 4 stallions (2 ejaculates per animal, n = 16). Samples were immediately diluted 1:1 (v/v) with INRA96 (IMV, L'Aigle, France) and centrifuged at 400×g for 7 min for donkey samples and 600×g for 10 min for horse samples at 22°C (Consuegra et al. 2019; Ortiz et al. 2015) to remove seminal plasma. The sperm pellets were resuspended in INRA96 to reach a final sperm concentration of 50 × 106 sperm/mL. Each ejaculate was split into two aliquots: one was cooled in a Styrofoam box and stored at 5°C, and the other was directly stored at 17°C.

2.2. Sperm Evaluation

Analyses were performed before processing (Day 0) and after 1, 2, and 7 days of storage. Sperm concentration was assessed with a sperm photometer (Sperma‐Cue, Minitube GmbH, Tiefenbach, Germany). Computer‐assisted sperm analysis (SCA, Microptic, Barcelona, Spain) was used to evaluate total and progressive sperm motility (TM and PM, %) in a 20‐μm four‐chamber slide (Minitube GmbH, Tiefenbach, Germany). Normal sperm morphology was evaluated in formalin‐fixed samples (NF, %) (Brito 2007). Plasma and acrosomal membrane integrity were assessed by flow‐cytometry (Accuri C6 Plus; BD, Franklin Lakes, NJ, USA) using PI/PNA stain (Ortiz et al. 2021), expressed as viable acrosome‐intact sperm (VAI, %).

2.3. Statistical Analysis

Statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC, USA). Data distribution was assessed using the Shapiro–Wilk test. Sperm parameters were analysed using a linear mixed model (PROC MIXED), including temperature, time and species as fixed effects, followed by Tukey's post hoc test. Results were presented as least squares means (LSMeans) ± SEM. Statistical significance was set at p < 0.05.

3. Results

Initial sperm concentration was higher in donkey than in horse ejaculates (343.75 ± 24.24 vs. 181.41 ± 32.73 × 106 sperm/mL). There were no significant differences between horse and donkey sperm parameters of fresh semen (Day 0). During the subsequent days of storage, donkey sperm stored at 5°C showed significantly lower values for TM, PM, and VAI. At 17°C, no significant differences between species were observed for motility parameters; however, membrane integrity was higher in horse sperm (p < 0.05). Sperm morphology did not differ significantly (p > 0.05) on any of the days or temperatures analysed (Figure 1).

FIGURE 1.

FIGURE 1

Open in a new tab

Effect of temperature (5°C vs. 17°C) on donkey and horse sperm parameters in samples stored for 7 days. (LSMean ± SEM). Different superscripts (a–c) indicate differences within each time point (p < 0.05).

4. Discussion

This study provides a direct comparison of horse and donkey sperm stored at 5°C and 17°C. Fresh ejaculates showed no significant differences between species prior to storage, allowing the effects of temperature to be evaluated without initial quality bias. Overall, our results indicate a clear species‐specific response to cooling, with donkey sperm showing greater sensitivity to low temperatures than horse sperm.

Previous studies on long‐term storage of donkey semen at different temperatures have yielded inconsistent results, with no clear superiority of 5°C or 17°C for maintaining sperm quality during extended storage (Caldevilla et al. 2025). However, other studies report that temperatures above 15°C may reduce motility while better preserving membrane integrity (Serres et al. 2002). In line with this, previous work from our group suggests that storage at 17°C may mitigate cold shock and improve the preservation of sperm motility and viability during prolonged liquid storage (Vallejo‐Soto et al. 2025). These species‐specific responses to the temperatures established in this study may be related to physiological differences in sperm membrane composition.

Donkey sperm membranes have greater diversity and a higher amount of lipids, particularly polyunsaturated fatty acids, compared with horse sperm (Chaves et al. 2023). Although these lipids enhance membrane fluidity, their high degree of unsaturation may increase susceptibility to oxidative damage during cooling (Chaves et al. 2025). Interestingly, fresh donkey spermatozoa show relatively high resilience to oxidative stress, likely associated with the antioxidant activity of donkey seminal plasma (Yánez‐Ortiz et al. 2021), even after only brief contact between sperm and seminal plasma before its removal (Catalán et al. 2022), highlighting a complex interaction between membrane lipid composition and redox homeostasis during sperm preservation. Lipid distribution of the plasma membrane has been shown to influence sperm resistance to cryopreservation not only in equine (Cabrera et al. 2018) but also in other species, such as the boar (Pezo et al. 2019). These interspecific differences may explain why no major differences between donkey and horse sperm were observed at 17°C during the first days of storage, as moderate temperatures may not induce sufficient membrane stress to reveal species‐specific differences (Clulow and Gibb 2022). This could also be associated with the flow cytometry findings, as lower VAI values were observed in donkey sperm compared to horse sperm under the same staining protocol. Further studies are needed to determine whether this is related to membrane composition and its influence on tolerance to staining.

The results of this study suggest that storage at 5°C, widely used in horses, may not be suitable for donkey sperm. Further studies, including a larger sample size and additional conditions (temperatures, extenders, cooling methodologies…) are needed to optimize preservation protocols tailored to this species. In this comparative study, a similar processing methodology was followed to allow direct comparisons. Nevertheless, differences in the initial sperm concentration and centrifugation protocols could have influenced the results of this study. Previous studies suggest that higher dilution rates and milder centrifugal forces may be more appropriate for donkey semen (Miró et al. 2009; Serres et al. 2002). Accordingly, species‐specific handling protocols could be further optimized to achieve outcomes comparable to those currently obtained in horses (Clulow and Gibb 2022).

In conclusion, under the conditions evaluated in this study, donkey sperm exhibited greater sensitivity to cooling than horse sperm, with storage at 5°C resulting in reduced sperm quality parameters. On Days 1 and 2, donkey sperm stored at 17°C demonstrated quality comparable to that of horse sperm stored at both 5°C and 17°C. After 7 days of storage, horse sperm maintained at 5°C exhibited the highest overall sperm quality. These findings underscore the importance of developing species‐specific storage protocols and indicate that preservation strategies optimized for stallion sperm may not be directly applicable to donkey sperm.

Author Contributions

Pilar Vallejo‐Soto: conceptualization, methodology, formal analysis, investigation, data curation, writing. Antonio González Ariza: resources, writing – review and editing. Blenda R. N. Vilela: investigation, writing – review and editing. Carlos Montero‐Palma: investigation, writing – review and editing. Rocío Rodríguez González‐Miranda: investigation. Verónica Pérez‐Aguilera: resources, investigation. Francisco Crespo‐Castejón: resources. Manuel Hidalgo: resources, funding acquisition. Jesús Dorado: resources, writing – review and editing, funding acquisition. Isabel Ortiz: conceptualization, methodology, investigation, writing – review and editing.

Funding

This study was supported by the project grant PID2020‐116090RB‐I00 from MICIN (Ministerio de Ciencia e Innovación), which also supported CM‐P. PV‐S was supported by the Spanish FPU fellowship (FPU23/03472) from Ministerio de Universidades. BRNV was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. Funding for open access charge: Universidad de Cordoba / CBUA.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors would like to thank the Military Horse Breeding Centre of Ávila of the Spanish Army and the Agropecuary Provincial Centre of Córdoba for allowing access to their herds.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Aurich, C. 2008. “Recent Advances in Cooled‐Semen Technology.” Animal Reproduction Science 107, no. 3–4: 268–275. 10.1016/j.anireprosci.2008.04.015. [DOI] [PubMed] [Google Scholar]
  2. Brito, L. F. C. 2007. “Evaluation of Stallion Sperm Morphology.” Clinical Techniques in Equine Practice 6, no. 4: 249–264. 10.1053/j.ctep.2007.09.004. [DOI] [Google Scholar]
  3. Cabrera, T. , Ramires‐Neto C., Belaz K. R. A., et al. 2018. “Influence of Spermatozoal Lipidomic Profile on the Cryoresistance of Frozen Spermatozoa From Stallions.” Theriogenology 108: 161–166. 10.1016/j.theriogenology.2017.11.025. [DOI] [PubMed] [Google Scholar]
  4. Caldevilla, M. , Neild D. M., Ferrante A., et al. 2025. “Beyond Extender in Donkey Semen: Evaluation at 5°C and 17°C Over 7 Days.” Journal of Equine Veterinary Science 145: 105270. 10.1016/j.jevs.2024.105270. [DOI] [Google Scholar]
  5. Canisso, I. F. , Panzani D., Miró J., and Ellerbrock R. E.. 2019. “Key Aspects of Donkey and Mule Reproduction.” Veterinary Clinics of North America 35, no. 3: 607–642. 10.1016/j.cveq.2019.08.014. [DOI] [PubMed] [Google Scholar]
  6. Catalán, J. , Yánez‐Ortiz I., Martínez‐Rodero I., et al. 2023. “Comparison of the Metabolite Profile of Donkey and Horse Seminal Plasma and Its Relationship With Sperm Viability and Motility.” Research in Veterinary Science 165: 105046. 10.1016/j.rvsc.2023.105046. [DOI] [PubMed] [Google Scholar]
  7. Catalán, J. , Yánez‐Ortiz I., Tvarijonaviciute A., et al. 2022. “Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.” Antioxidants 11, no. 2: 417. 10.3390/antiox11020417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chaves, M. M. C. , Canisso I. F., Ramires Neto C., et al. 2023. “The Lipidomic Profiles of Donkey Semen Present Greater Diversity and Abundance Than Horse Semen.” Journal of Equine Veterinary Science 125: 104583. 10.1016/j.jevs.2023.104583. [DOI] [Google Scholar]
  9. Chaves, M. M. C. , Ramires Neto C., Freitas‐Dell'Aqua C. P., et al. 2025. “Variation in Semen Lipids During Cryopreservation by Cooling With Milk‐Based Extender in Donkeys.” Journal of Equine Veterinary Science 145: 105275. 10.1016/j.jevs.2024.105275. [DOI] [Google Scholar]
  10. Clulow, J. , and Gibb Z.. 2022. “Liquid Storage of Stallion Spermatozoa—Past, Present and Future.” Animal Reproduction Science 247: 107088. 10.1016/j.anireprosci.2022.107088. [DOI] [PubMed] [Google Scholar]
  11. Consuegra, C. , Crespo F., Dorado J., et al. 2019. “Vitrification of Stallion Sperm Using 0.25 ml Straws: Effect of Volume, Concentration and Carbohydrates (Sucrose/Trehalose/Raffinose).” Animal Reproduction Science 206: 69–77. 10.1016/j.anireprosci.2019.05.009. [DOI] [PubMed] [Google Scholar]
  12. Gobato, M. L. M. , Segabinazzi L. G. T. M., Scheeren V. F. C., et al. 2022. “Ability of Donkey Sperm to Tolerate Cooling: Effect of Extender Base and Removal of Seminal Plasma on Sperm Parameters and Fertility Rates in Mares.” Frontiers in Veterinary Science 9: 1011899. 10.3389/fvets.2022.1011899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miró, J. , Taberner E., Rivera M., et al. 2009. “Effects of Dilution and Centrifugation on the Survival of Spermatozoa and the Structure of Motile Sperm Cell Subpopulations in Refrigerated Catalonian Donkey Semen.” Theriogenology 72, no. 8: 1017–1022. 10.1016/j.theriogenology.2009.06.012. [DOI] [PubMed] [Google Scholar]
  14. Ortiz, I. , Dorado J., Acha D., Gálvez M. J., Urbano M., and Hidalgo M.. 2015. “Colloid Single‐Layer Centrifugation Improves Post‐Thaw Donkey ( Equus asinus ) Sperm Quality and Is Related to Ejaculate Freezability.” Reproduction, Fertility and Development 27, no. 2: 332–340. 10.1071/RD13246. [DOI] [PubMed] [Google Scholar]
  15. Ortiz, I. , Felix M., Resende H., Ramírez‐Agámez L., Love C. C., and Hinrichs K.. 2021. “Flow‐Cytometric Analysis of Membrane Integrity of Stallion Sperm in the Face of Agglutination: The “Zombie Sperm” Dilemma.” Journal of Assisted Reproduction and Genetics 9, no. 38: 2465–2480. 10.1007/s10815-021-02134-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pezo, F. , Romero F., Zambrano F., and Sánchez R. S.. 2019. “Preservation of Boar Semen: An Update.” Reproduction in Domestic Animals 54, no. 3: 423–434. 10.1111/rda.13389. [DOI] [PubMed] [Google Scholar]
  17. Serres, C. , Rodriguez A., Alvarez A., et al. 2002. “Effect of Centrifugation and Temperature on the Motility and Plasma Membrane Integrity of Zamorano‐Leonés Donkey Semen.” Theriogenology 58: 329–332. 10.1016/S0093-691X(02)00876-2. [DOI] [Google Scholar]
  18. Vallejo‐Soto, P. , González Ariza A., León Jurado J. M., Hidalgo M., Dorado J., and Ortiz I.. 2025. “Liquid‐Storage at 17°C as an Alternative to Preserve Donkey Sperm Quality During Long‐Term Storage: Preliminary Results.” Journal of Equine Veterinary Science 145: 105330. 10.1016/j.jevs.2024.105330. [DOI] [Google Scholar]
  19. Yánez‐Ortiz, I. , Catalán J., Mateo‐Otero Y., et al. 2021. “Extracellular Reactive Oxygen Species (ROS) Production in Fresh Donkey Sperm Exposed to Reductive Stress, Oxidative Stress and NETosis.” Antioxidants 10, no. 9: 1367. 10.3390/antiox10091367. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Reproduction in Domestic Animals = Zuchthygiene are provided here courtesy of Wiley

RESOURCES