Vet Med - Czech, 2026, 71(1):1-9

Dairy bull sperm subpopulation behaviour in frozen-thawed semen across breed, temperature, and thawing timeOriginal Paper

F Sevilla ORCID...1,2, I Araya-Zuniga ORCID...2, JM Solis ORCID...2, C Corcini ORCID...3, P Cervantes-Acosta ORCID...4, A Hernandez-Beltran ORCID...4, R Molina-Montero ORCID...5, D Pichardo-Matamoros ORCID...6, A Valverde ORCID...2
1 Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE), Instituto Tecnológico de Costa Rica, Universidad Nacional, Universidad Estatal a Distancia, Alajuela, Costa Rica
2 Laboratory of Animal Reproduction, School of Agronomy, Costa Rica Institute of Technology, Alajuela, Costa Rica
3 Veterinary School, Pelotas Federal University, Rio Grande do Sul, Brazil
4 School of Veterinary Medicine and Animal Husbandry, Veracruz University, Veracruz, México
5 Agricultural Production Program, School of Agronomy, Costa Rica Institute of Technology, Alajuela, Costa Rica
6 Animal Husbandry Engineering, National Agrarian University, Chontales, Nicaragua

Optimising dairy cattle reproduction with assisted reproductive technologies, such as artificial insemination, requires standardised semen handling and analysis. This study evaluated the kinematic structure of the spermatozoan subpopulation in frozen-thawed Holstein and Jersey bull semen doses under different thawing protocols. We used frozen semen doses taken from four bulls of each breed. Nine semen doses were collected from each animal, for a total of 72 cryopreserved doses. Straw thawing was performed at three temperatures (35 °C, 37 °C, and 40 °C) and three times (30 s, 40 s, and 45 s). Sperm kinematic patterns were evaluated using a CASA-mot system (ISAS®v1). Sperm kinematic variables for each breed, temperature, and thawing time identified four subpopulations. The analysis revealed an effect of breed and semen thawing protocols (P < 0.05) on sperm subpopulation distribution, sperm movement, and swimming patterns. Subpopulation analyses based on semen assessment are needed to further interpret the relevance and effect on fertility.

Keywords: cryopreservation; dairy cattle; reproduction; spermatozoa; sperm kinematics

Received: August 11, 2025; Revised: November 6, 2025; Accepted: November 7, 2025; Prepublished online: January 26, 2026; Published: January 28, 2026  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
Sevilla F, Araya-Zuniga I, Solis J, Corcini C, Cervantes-Acosta P, Hernandez-Beltran A, et al.. Dairy bull sperm subpopulation behaviour in frozen-thawed semen across breed, temperature, and thawing time. Vet Med - Czech. 2026;71(1):1-9.
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Ahmed H, Andrabi SMH, Jahan S. Semen quality parameters as fertility predictors of water buffalo bull spermatozoa during low-breeding season. Theriogenology. 2016 Oct 1;86(6):1516-22. Go to original source... Go to PubMed...
    2. Alm-Kristiansen AH. Motility subpopulations with distinct motility characteristics using swim-up-selected sperm cells from Norwegian Red bulls: Effects of freezing-thawing and between-bull variation. Biology (Basel). 2023 Aug 3;12(8):1086. Go to original source... Go to PubMed...
    3. Alvarez-Rodriguez M, Tomas-Almenar C, Nieto-Cristobal H, de Mercado E. Evaluation of different thawing protocols on Iberian boar sperm preserved for 10 years at different liquid nitrogen levels. Animals (Basel). 2024 Mar 15;14(6):914. Go to original source... Go to PubMed...
    4. Araya-Zuniga I, Sevilla F, Molina-Montero R, Roldan ERS, Barrientos-Morales M, Silvestre MA, Valverde A. Kinematic and morphometric assessment of fresh semen, before, during and after mating period in Brahman bulls. Animals (Basel). 2024 Jan 3;14(1):132. Go to original source... Go to PubMed...
    5. Azevedo HC, Blackburn HD, Lozada-Soto EA, Spiller SF, Purdy PH. Enhancing evaluation of bull fertility through multivariate analysis of sperm. J Dairy Sci. 2024 Dec;107(12):11774-84. Go to original source... Go to PubMed...
    6. Barquero V, Roldan ERS, Soler C, Yaniz JL, Camacho M, Valverde A. Predictive capacity of boar sperm morphometry and morphometric sub-populations on reproductive success after artificial insemination. Animals (Basel). 2021a Mar 24;11(4):920. Go to original source... Go to PubMed...
    7. Barquero V, Roldan ERS, Soler C, Vargas-Leiton B, Sevilla F, Camacho M, Valverde A. Relationship between fertility traits and kinematics in clusters of boar ejaculates. Biology (Basel). 2021b Jun 28;10(7):595. Go to original source... Go to PubMed...
    8. Bompart D, Vazquez RF, Gomez R, Valverde A, Roldan ERS, Garcia-Molina A, Soler C. Combined effects of type and depth of counting chamber, and rate of image frame capture, on bull sperm motility and kinematics. Anim Reprod Sci. 2019 Oct;209:106169. Go to original source... Go to PubMed...
    9. Cheng M, McCarl B, Fei C. Climate change and livestock production: A literature review. Atmosphere. 2022 Jan 15;13(1):140. Go to original source...
    10. Diskin MG. Review: Semen handling, time of insemination and insemination technique in cattle. Animal. 2018 Jun;12(s1):s75-84. Go to original source... Go to PubMed...
    11. Fernandez-Lopez P, Garriga J, Casas I, Yeste M, Bartumeus F. Predicting fertility from sperm motility landscapes. Commun Biol. 2022 Sep 28;5(1):1027. Go to original source... Go to PubMed...
    12. Ferraz MA, Morato R, Yeste M, Arcarons N, Pena AI, Tamargo C, Hidalgo CO, Muino R, Mogas T. Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls. Theriogenology. 2014 May;81(8):1067-72. Go to original source... Go to PubMed...
    13. Gacem S, Valverde A, Catalan J, Yanez Ortiz I, Soler C, Miro J. A new approach of sperm motility subpopulation structure in donkey and horse. Front Vet Sci. 2021 May 25;8:651477. Go to original source... Go to PubMed...
    14. Garcia-Alvarez O, Maroto-Morales A, Ramon M, del Olmo E, Jimenez-Rabadan P, Fernandez-Santos MR, Anel-Lopez L, Garde JJ, Soler AJ. Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation. Reprod Fertil Dev. 2014 Jun;26(5):725-32. Go to original source... Go to PubMed...
    15. Garcia-Herreros M, Leal CL. Comparative study of sperm washing and selection methods after cryopreservation and its influence on sperm subpopulational structure in a bovine model. Syst Biol Reprod Med. 2014 Dec;60(6):338-47. Go to original source... Go to PubMed...
    16. Goshme S, Asfaw T, Demiss C, Besufekad S. Evaluation of motility and morphology of frozen bull semen under different thawing methods used for artificial insemination in North Shewa zone, Ethiopia. Heliyon. 2021 Oct 13;7(10):e08183. Go to original source... Go to PubMed...
    17. Hidalgo MMT, Almeida ABM, Moraes FLZ, Marubayashi RYP, Souza FF, Barreiros TRR, Martins MIM. Sperm subpopulations influence the pregnancy rates in cattle. Reprod Domest Anim. 2021 Aug;56(8):1117-27. Go to original source... Go to PubMed...
    18. Hidalgo MMT, Marques de Almeida AB, Zito de Moraes FL, Marubayashi RYP, Palhaci RY, Barreiros TRR, Martins MIM. Influence of sperm post-thaw subpopulations of Angus and Nelore bulls on pregnancy rates by fixed-time artificial insemination. Cryo Letters. 2022 May-Jun;43(3):140-9. Go to original source...
    19. Ibanescu I, Leiding C, Bollwein H. Cluster analysis reveals seasonal variation of sperm subpopulations in extended boar semen. J Reprod Dev. 2018 Feb 27;64(1):33-9. Go to original source... Go to PubMed...
    20. Ibanescu I, Siuda M, Bollwein H. Motile sperm subpopulations in bull semen using different clustering approaches - Associations with flow cytometric sperm characteristics and fertility. Anim Reprod Sci. 2020 Apr;215:106329. Go to original source... Go to PubMed...
    21. Kanno C, Sakamoto KQ, Kang SS, Yanagawa Y, Katagiri S, Nagano M. The difference in subpopulation structures in sex-sorted and non-sorted semen by discriminant analysis of bull sperm motility data collected by a Computer-Assisted Sperm Analysis System. Andrologia. 2023;2023:9944344. Go to original source...
    22. Keyer S, Van Der Horst G, Maree L. New approaches to define the functional competency of human sperm subpopulations and its relationship to semen quality. Int J Fertil Steril. 2022 Aug 21;16(3):140-51.
    23. Koch J, Weber LP, Heppelmann M, Freise F, Klingelmann M, Bachmann L. Effect of different thawing methods for frozen bull semen and additional factors on the conception rate of dairy cows in artificial insemination. Animals (Basel). 2022 Sep 7;12(18):2330. Go to original source... Go to PubMed...
    24. Maroto-Morales A, Ramon M, Garcia-Alvarez O, Soler AJ, Fernandez-Santos MR, Roldan ER, Gomendio M, Perez-Guzman MD, Garde JJ. Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in ram ejaculates: Intra- and interindividual variation. Theriogenology. 2012 May;77(8):1529-39. Go to original source... Go to PubMed...
    25. Marti JI, Aparicio IM, Garcia-Herreros M. Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates. Theriogenology. 2011 Jul 1;76(1):97-109. Go to original source... Go to PubMed...
    26. Martinez LB, Crispin RH, Mendoza MM, Gallegos OH, Martinez AA. Use of multivariate statistics to identify unreliable data obtained using CASA. Syst Biol Reprod Med. 2013 Jun;59(3):164-71. Go to original source... Go to PubMed...
    27. Martinez-Pastor F. What is the importance of sperm subpopulations? Anim Reprod Sci. 2022 Nov;246:106844. Go to original source... Go to PubMed...
    28. Martinez-Pastor F, Tizado EJ, Garde JJ, Anel L, de Paz P. Statistical series: Opportunities and challenges of sperm motility subpopulation analysis. Theriogenology. 2011 Mar 15;75(5):783-95. Go to original source... Go to PubMed...
    29. Menon AG, Barkema HW, Wilde R, Kastelic JP, Thundathil JC. Associations between sperm abnormalities, breed, age, and scrotal circumference in beef bulls. Can J Vet Res. 2011 Oct;75(4):241-7.
    30. Mikkola M, Desmet KLJ, Kommisrud E, Riegler MA. Recent advancements to increase success in assisted reproductive technologies in cattle. Anim Reprod. 2024 Aug 12;21(3):e20240031. Go to original source... Go to PubMed...
    31. Muino R, Pena AI, Rodriguez A, Tamargo C, Hidalgo CO. Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls. Theriogenology. 2009 Oct 1;72(6):860-8. Go to original source... Go to PubMed...
    32. Muino R, Tamargo C, Hidalgo CO, Pena AI. Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: Effects of cryopreservation and between-bull variation. Anim Reprod Sci. 2008 Dec;109(1-4):27-39. Go to original source... Go to PubMed...
    33. Murphy EM, Kelly AK, O'Meara C, Eivers B, Lonergan P, Fair S. Influence of bull age, ejaculate number, and season of collection on semen production and sperm motility parameters in Holstein Friesian bulls in a commercial artificial insemination centre. J Anim Sci. 2018 Jun;96(6):2408-18. Go to original source... Go to PubMed...
    34. Ntemka A, Tsousis G, Brozos C, Kiossis E, Boscos CM, Tsakmakidis IA. Breed differences of bull frozen-thawed semen. Reprod Domest Anim. 2016 Dec;51(6):945-52. Go to original source... Go to PubMed...
    35. Pena AI, Adan S, Quintela LA, Becerra JJ, Herradon PG. Relationship between motile sperm subpopulations identified in frozen-thawed dog semen samples and their ability to bind to the zona pellucida of canine oocytes. Reprod Domest Anim. 2018 Nov;53(Suppl_3):14-22. Go to original source... Go to PubMed...
    36. Perez-Marin CC, Arando A, Maroto-Molina F, Marin A, Delgado JV. Sperm subpopulations and quality in fractions obtained after single layer centrifugation in fresh normospermic ram samples. Rev Mex Cienc Pecu. 2021 Aug;12(2):386-401. Go to original source...
    37. Ramon M, Soler AJ, Ortiz JA, Garcia-Alvarez O, Maroto-Morales A, Roldan ER, Garde JJ. Sperm population structure and male fertility: An intraspecific study of sperm design and velocity in red deer. Biol Reprod. 2013 Nov;89(5):110. Go to original source... Go to PubMed...
    38. Santolaria P, Vicente-Fiel S, Palacin I, Fantova E, Blasco ME, Silvestre MA, Yaniz JL. Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep. Anim Reprod Sci. 2015 Dec;163:82-8. Go to original source... Go to PubMed...
    39. Silvestre MA, Soler C, Moce E, Roldan ERS, Yaniz JL. The importance of studying factors that affect the in vitro evaluation of semen quality to predict potential fertility in males. Biology (Basel). 2023 Feb 2;12(2):235. Go to original source... Go to PubMed...
    40. Solis JM, Sevilla F, Silvestre MA, Araya-Zuniga I, Roldan ERS, Saborio-Montero A, Valverde A. Effect of thawing procedure and thermo-resistance test on sperm motility and kinematics patterns in two bovine breeds. Animals (Basel). 2024 Sep 25;14(19):2768. Go to original source... Go to PubMed...
    41. Souza AH, Bessoff HJ, Danzeisen E. Effect of semen type (cooled-fresh vs. frozen-thawed) on fertility of lactating dairy cows. J Anim Sci. 2016 Oct;94(Suppl_5):180. Go to original source...
    42. Valverde A, Arean H, Fernandez A, Bompart D, Garcia-Molina A, Lopez-Viana J, Soler C. Combined effect of type and capture area of counting chamber and diluent on Holstein bull sperm kinematics. Andrologia. 2019 May;51(4):e13223. Go to original source... Go to PubMed...
    43. Valverde A, Arenan H, Sancho M, Contell J, Yaniz J, Fernandez A, Soler C. Morphometry and subpopulation structure of Holstein bull spermatozoa: Variations in ejaculates and cryopreservation straws. Asian J Androl. 2016 Nov-Dec;18(6):851-7. Go to original source... Go to PubMed...
    44. Viquez L, Sevilla F, Araya-Zuniga I, Soler C, Barquero V, Roldan ERS, Valverde A. Morphometric assessment of cryopreserved livestock bull spermatozoa in the tropics. Reprod Domest Anim. 2023 Oct;58(10):1439-47. Go to original source... Go to PubMed...
    45. Yaniz JL, Palacin I, Caycho KS, Soler C, Silvestre MA, Santolaria P. Determining the relationship between bull sperm kinematic subpopulations and fluorescence groups using an integrated sperm quality analysis technique. Reprod Fertil Dev. 2018 Jun;30(6):919-23. Go to original source... Go to PubMed...
    46. Yaniz JL, Vicente-Fiel S, Soler C, Recreo P, Carretero T, Bono A, Berne JM, Santolaria P. Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men. Asian J Androl. 2016 Nov-Dec;18(6):819-23. Go to original source... Go to PubMed...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content