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ORIGINAL PAPER
Effect of initial ejaculate quality on post-thaw parameters in boar semen
1
Instituto Tecnológico de Costa Rica, Universidad Nacional, Universidad Estatal a Distancia, Doctorado en Ciencias Naturales para el Desarrollo (DOCINADE). Alajuela, Costa Rica
2
Instituto Tecnológico de Costa Rica, Escuela de Agronomía, Centro de Investigación y Desarrollo en Agricultura Sostenible del Trópico Húmedo, Laboratorio de Reproducción Animal, Campus Tecnológico Local San Carlos. Alajuela, Costa Rica
3
Universidad Nacional de San Antonio Abad del Cusco, Facultad de Agronomía y Zootecnia, Escuela Profesional de Zootecnia, Laboratorio de Biotecnologías Reproductivas, Centro Experimental La Raya – Cusco, Cusco, Perú
Publication date: 2025-12-01
Corresponding author
A. Valverde
Instituto Tecnológico de Costa Rica, Escuela de Agronomía, Centro de Investigación y Desarrollo en Agricultura Sostenible del Trópico Húmedo, Laboratorio de Reproducción Animal, Campus Tecnológico Local San Carlos. Alajuela, Costa Rica
Instituto Tecnológico de Costa Rica, Escuela de Agronomía, Centro de Investigación y Desarrollo en Agricultura Sostenible del Trópico Húmedo, Laboratorio de Reproducción Animal, Campus Tecnológico Local San Carlos. Alajuela, Costa Rica
KEYWORDS
TOPICS
ABSTRACT
The initial ejaculate sperm quality influences its subsequent
freezability, which varies significantly between individuals. The objective of this
study was to determine whether non-viable, static, or morphologically altered
sperm affect the freezability of motile, viable sperm with normal morphology.
Semen samples were collected from five sexually mature boars (≥3 ejaculates
per animal) and allocated to four treatment groups based on the proportion
of viable, motile, morphologically normal sperm: 100% (control), 75%, 50%,
and 25%. Samples were diluted in TRIS-egg yolk extender, cooled to 5 °C,
then further diluted with a glycerol-containing freezing medium and loaded to
0.5-ml straws. The cryopreservation protocol involved two controlled cooling
stages, followed by storage in liquid nitrogen for seven days. After thawing at
37 °C for 20 s, sperm quality was evaluated using computer-assisted semen
analysis (CASA) for motility and flow cytometry for membrane and acrosome
integrity with triple fluorescent staining (Hoechst 33342, propidium iodide,
and PNA-FITC). Assessments were carried out before freezing and at 30 and
150 min post-thaw. The percentage of non-viable sperm in the ejaculates prior
to freezing significantly affected post-thaw sperm quality (P < 0.05), reducing
motility and structural integrity in proportion to their initial presence.
ACKNOWLEDGEMENTS
The authors thank the Costa Rica Institute
of Technology (ITCR) for funding this research
through its Vice-Chancellor’s office of Research
and Extension (VIE) under project VIE-2151083.
The authors also would like to thank the
university’s Postgraduate Department. The funders
were not involved in study design, data collection
and analysis, decision to publish, or manuscript
preparation.
FUNDING
This research was funded by the Costa Rica
Institute of Technology (Vice-Chancellor’s office
of Research and Extension; VIE (Vicerrectoría de
Investigación y Extensión), as part of the research
project VIE-2151083, ‘Optimización de la conservación
y búsqueda de parámetros de la fertilidad
en espermatozoides de animales de interés productivo’).
The APC were also funded by Costa Rica
Institute of Technology (Vice-Chancellor’s office
of Research and Extension); VIE (Vicerrectoría de
Investigación y Extensión).
CONFLICT OF INTEREST
The Authors declare that there is no conflict of
interest.
REFERENCES (65)
1.
Alahmar A., 2019. Role of oxidative stress in male infertility: An updated review. J. Hum. Reprod. Sci. 12, 4, https://doi.org/10.4103/jhrs.J....
2.
Al-Kass Z., Morrell J.M., 2024. Freezing stallion semen-What do we need to focus on for the future? Vet. Sci. 11, 65, https://doi.org/10.3390/vetsci....
3.
Araya-Zúñiga I., Sevilla F., González J.A., Matamoros K., Valverde A., 2025. The cryopreservation of the germplasm of livestock species: A step towards sustainability. Agron. Mesoam. 36, 61375, https://doi.org/10.15517/am.20....
4.
Bang S., Tanga B.M., Fang X. et al., 2022. Cryopreservation of pig semen using a quercetin-supplemented freezing extender. Life 12, 1155, https://doi.org/10.3390/life12....
5.
Barquero V., Roldan E.R.S., Soler C., Vargas-Leitón, B., Sevilla, F., Camacho, M., Valverde, A., 2021. Relationship between fertility traits and kinematics in clusters of boar ejaculates. Biology 10, 595, https://doi.org/10.3390/biolog....
6.
Bolarin A., Berndtson J., Tejerina F., Cobos S., Pomarino C., D’Alessio F., Blackburn H., Kaeoket K., 2024. Boar semen cryopreservation: State of the art, and international trade vision. Anim. Reprod. Sci. 269, 107496, https://doi.org/10.1016/j.anir....
7.
Bolton R.L., Mooney A., Pettit M.T., Bolton A.E., Morgan L., Drake G.J., Appeltant R., Walker S.L., Gillis J.D., Hvilsom C., 2022. Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking. Reprod. Fertility 3, R121, https://doi.org/10.1530/RAF-22....
8.
Borate G.M., Meshram A., 2022. Cryopreservation of Sperm: A Review. Cureus 14, e31402, https://doi.org/10.7759/cureus....
9.
Caamaño J.N., Tamargo C., Parrilla I. et al., 2021. Post-thaw sperm quality and functionality in the autochthonous pig breed gochu asturcelta. Animals 11, 1885, https://doi.org/10.3390/ani110....
10.
Capra E., Frigerio R., Lazzari B. et al., 2024. Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen. Front. Vet. Sci. 11, 1437410, https://doi.org/10.3389/fvets.....
11.
Cheng Q., Li L., Jiang M., Liu B., Xian Y., Liu S., Liu X., Zhao W., Li F., 2022. Extend the survival of human sperm in vitro in non-freezing conditions: Damage mechanisms, preservation technologies, and clinical applications. Cells 11, 2845, https://doi.org/10.3390/cells1....
12.
Chicaiza-Cabezas N., Garcia-Herreros M., Aponte P.M., 2023. Germplasm cryopreservation in bulls: Effects of gonadal tissue type, cryoprotectant agent, and freezing-thawing rates on sperm quality parameters. Cryobiology 110, 24–35, https://doi.org/10.1016/j.cryo....
13.
Costa J., Braga P.C., Rebelo I., Oliveira P.F., Alves M.G., 2023. Mitochondria quality control and male fertility. Biology 12, 827, https://doi.org/10.3390/biolog....
14.
Crowe A.D., Lonergan P., Butler S.T., 2021. Invited review: Use of assisted reproduction techniques to accelerate genetic gain and increase value of beef production in dairy herds. J. Dairy. Sci. 104, 12189–12206, https://doi.org/10.3168/jds.20....
15.
de Andrade A.F.C., Balogun K., Machaty Z., Knox R.V., 2023. Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa. Theriogenology 200, 33–42, https://doi.org/10.1016/j.ther....
16.
Ďuračka M., Benko F., Tvrdá E., 2023. Molecular Markers: A new paradigm in the prediction of sperm freezability. Int. J. Mol. Sci. 24, 3379, https://doi.org/10.3390/ijms24....
17.
Dutta S., Majzoub A., Agarwal A., 2019. Oxidative stress and sperm function: A systematic review on evaluation and management. Arab. J. Urol. 17, 87, https://doi.org/10.1080/209059....
18.
Engdawork A., Belayhun T., Aseged T., 2024. The role of reproductive technologies and cryopreservation of genetic materials in the conservation of animal genetic resources. Ecol. Genet. Genom. 31, 100250, https://doi.org/10.1016/j.egg.....
19.
Flowers W.L., 1997. Management of boars for efficient semen production. J. Reprod. Fertil. Suppl. 52, 67–78 Fraser L., Zasiadczyk Ł., Mogielnicka-Brzozowska M., 2025. Boarto-boar variations in quality characteristics of sperm from different ejaculates following freezing-thawing. Cells 14, 212, https://doi.org/10.3390/cells1....
20.
Godde C.M., Mason-D’Croz D., Mayberry D.E., Thornton P.K., Herrero M., 2021. Impacts of climate change on the livestock food supply chain; a review of the evidence. Glob. Food. Sec. 28, 100488, https://doi.org/10.1016/j.gfs.....
21.
Goldberg A., Argenti L.E., Faccin J.E., Linck L., Santi M., Lourdes- Bernardi M., Cardoso M.R., Wentz I., Bortolozzo F.P., 2013. Risk factors for bacterial contamination during boar semen collection. Res. Vet. Sci. 95, 362–367, https://doi.org/10.1016/j.rvsc....
22.
Góngora A., Holt W. V., Gosálvez J., 2024. Sperm human biobanking: An overview. Arch. Med. Res. 55, 103130, https://doi.org/10.1016/j.arcm....
23.
Guthrie H.D., Welch G.R., 2006. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescenceactivated flow cytometry. J. Anim. Sci. 84, 2089–2100, https://doi.org/10.2527/jas.20....
24.
Halaweh W., Khnissi S., Ben-Souf I., Salman M., M’Hamdi N., 2025. Impact of mating methods and semen preservation on reproductive and growth performances in Palestinian Assaf sheep. Biology 14, 80, https://doi.org/10.3390/biolog....
25.
Halliwell B., Gutteridge J.M.C., 1990. Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186, 1-85, https://doi.org/10.1016/0076-6....
26.
Henning H., Luther A.M., Höfner-Schmiing L., Waberski D., 2022. Compensability of an enhanced incidence of spermatozoa with cytoplasmic droplets in boar semen for use in artificial insemination: a single cell approach. Sci. Rep. 12, 21833, https://doi.org/10.1038/s41598....
27.
Hirai M., Boersma A., Hoeflich A., Wolf E., Foll J., Aumüller T.R., Braun J., 2001. Objectively measured sperm motility and sperm head morphometry in boars (Sus scrofa): relation to fertility and seminal plasma growth factors. J. Androl. 22, 104–110 https://doi.org/10.1002/j.1939....
28.
Holt W.V., Medrano A., Thurston L.M., Watson P.F., 2005. The significance of cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope. Theriogenology 63, 370-382, https://doi.org/10.1016/j.ther....
29.
Iaffaldano N., Di Iorio M., Cerolini S., Manchisi A., 2016. Overview of turkey semen storage: Focus on cryopreservation - A review. Annals of Anim. Sci. 16, 961–974, https://doi.org/10.1515/aoas-2....
30.
Jakop U., Müller K., Müller P., Neuhauser S., Rodríguez I.C., Grunewald S., Schiller J., Engel K.M., 2022. Seminal lipid profiling and antioxidant capacity: A species comparison. PLoS One 17, e0264675, https://doi.org/10.1371/journa....
31.
Johnson L.A., Weitze K.F., Fiser P., Maxwell W.M.C., 2000. Storage of boar semen. Anim. Reprod. Sci. 62, 143–172, https://doi.org/10.1016/S0378-....
32.
Jovičić M., Chmelíková E., Sedmíková M., 2020. Cryopreservation of boar semen. Czech J. Anim. Sci. 65, 115–123, https://doi.org/10.17221/47/20....
33.
Juyena N.S., Stelletta C., 2012. Seminal plasma: An essential attribute to spermatozoa. J. Androl. 33, 536–551, https://doi.org/10.2164/jandro....
34.
Khan I.M., Cao Z., Liu H., Khan A., Rahman S.U., Khan M.Z., Sathanawongs A., Zhang Y., 2021. Impact of cryopreservation on spermatozoa freeze-thawed traits and relevance OMICS to assess sperm cryo-tolerance in farm animals. Front. Vet. Sci. 8, 609180, https://doi.org/10.3389/fvets.....
35.
Krupa E., Wolfová M., Krupová Z., Žáková E., 2020. Estimation of economic weights for number of teats and sperm quality traits in pigs. J. Anim. Breed. Genet. 137, 189–199, https://doi.org/10.1111/jbg.12....
36.
Krzastek S.C., Farhi J., Gray M., Smith R.P., 2020. Impact of environmental toxin exposure on male fertility potential. Transl. Androl. Urol. 9, 2792813, https://doi.org/10.21037/tau-2....
37.
Li X., Wang L., Li Y., Zhao N., Zhen L., Fu J., Yang Q., 2016. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro. Anim. Reprod. Sci. 172, 39–51, https://doi.org/10.1016/j.anir....
38.
Mańkowska A., Brym P., Paukszto Ł., Jastrzębski J.P., Fraser L., 2020. Gene polymorphisms in boar spermatozoa and their associations with post-thaw semen quality. Int. J. Mol. Sci. 21, 1902, https://doi.org/10.3390/IJMS21....
39.
Mazur P., 1977. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 14, 251–272, https://doi.org/10.1016/0011-2....
40.
Mellagi A.P.G., Will K.J., Quirino M., Bustamante-Filho I.C., Ulguim R. da R., Bortolozzo F.P., 2023. Update on artificial insemination: Semen, techniques, and sow fertility. Mol. Reprod. Dev. 90, 601–611, https://doi.org/10.1002/mrd.23....
41.
Monteiro M.S., Torres M.A., Passarelli M. da S. et al., 2022. Impact of cryopreservation protocols (one- and two-step) on boar semen quality at 5 °C and post-thawing. Anim. Reprod. Sci. 247, 107093, https://doi.org/10.1016/j.anir....
42.
Nowicka-Bauer K., Szymczak-Cendlak M., 2021. Structure and function of ion channels regulating sperm motility-An overview. Int. J. Mol. Sci. 22, 3259, https://doi.org/10.3390/ijms22....
43.
Paschoal A.F., Luther A.M., Jakop U., Schulze M., Bortolozzo F.P., Waberski D., 2021. Factors influencing the response of spermatozoa to agitation stress: Implications for transport of extended boar semen. Theriogenology 175, 54–60, https://doi.org/10.1016/j.ther....
44.
Pezo F., Romero F., Zambrano F., Sánchez R.S., 2019. Preservation of boar semen: An update. Reprod. in Domest. Anim. 54, 423–434, https://doi.org/10.1111/rda.13....
45.
Pomeroy K.O., Comizzoli P., Rushing J.S., Lersten I.L., Nel- Themaat L., 2022. The ART of cryopreservation and its changing landscape. Fertil. Steril. 117, 469–476, https://doi.org/10.1016/j.fert....
46.
Ren Q., Gong Y., Su P. et al., 2025. How Developments in genebanks could shape utilization strategies for domestic animals. Agriculture 15, 133, https://doi.org/10.3390/agricu....
47.
Sabeti P., Pourmasumi S., Rahiminia T., Akyash F., Talebi A.R., 2016. Etiologies of sperm oxidative stress. Int. J. Reprod. Biomed. 14, 231, https://doi.org/10.29252/ijrm.....
48.
Schulze M., Nitsche-Melkus E., Jakop U., Jung M., Waberski D., 2019. New trends in production management in European pig AI centers. Theriogenology 137, 88–92, https://doi.org/10.1016/j.ther....
49.
Sciorio R., Tramontano L., Adel M., Fleming S., 2024. Decrease in sperm parameters in the 21st century: Obesity, lifestyle, or environmental factors? An updated narrative review. J. Personalized Med. 14, 198, https://doi.org/10.3390/jpm140....
50.
Shepherd M.J., Gonzalez-Castro R.A., Herickhoff L.A., 2024. Application of antioxidants in extender on bull sperm cryopreservation to reduce the male effect in dairy fertility. J. Dairy Sci. 107, 10027–10040, https://doi.org/10.3168/jds.20....
51.
Tanga B.M., Qamar A.Y., Raza S., Bang S., Fang X., Yoon K., Cho J., 2021. Semen evaluation: methodological advancements in sperm quality-specific fertility assessment - A review. Anim. Biosci. 34, 1253, https://doi.org/10.5713/ab.21.....
52.
Thurston L., Watson P., Mileham A., Holt W., 2001. Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation. J. Androl. 22, 382–394, https://doi.org/10.1002/j.1939....
53.
Thurston L., Watson P., Holt W., 2002. Semen cryopreservation: a genetic explanation for species and individual variation? Cryo Letters 23, 255–262.
54.
Tsujii H., Ohta E., Miah A.G., Hossain S., Salma U., 2006. Effect of fructose on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa. Reprod. Med. Biol. 5, 255–261, https://doi.org/10.1111/j.1447....
55.
Vahedi-Raad M., Firouzabadi A.M., Tofighi-Niaki M., Henkel R., Fesahat F., 2024. The impact of mitochondrial impairments on sperm function and male fertility: a systematic review. Reprod. Biol. Endocrinol. 22, 83, https://doi.org/10.1186/s12958....
56.
Valverde A., Arenán H., Sancho M., Contell J., Yániz J., Fernández A., Soler C., 2016. Morphometry and subpopulation structure of Holstein bull spermatozoa: Variations in ejaculates and cryopreservation straws. Asian J. Androl. 18, 851–857, https://doi.org/10.4103/1008-6....
57.
Valverde A., Barquero V., Carvajal V., 2021. Applied biotechnology to the study of the boar semen motility. Agron. Mesoam. 32, 662–680, https://doi.org/10.15517/am.v3....
58.
Valverde A., Madrigal-Valverde M., Zambrana-Jiménez A., 2018. Assessment of boar sperm kinetics and motility in tropical conditions. Actas Iberoamericanas de Conservación Animal 12, 125–132.
59.
Walke G., Gaurkar S.S., Prasad R., Lohakare T., Wanjari M., 2023. The impact of oxidative stress on male reproductive function: Exploring the role of antioxidant supplementation. Cureus 15, e42583, https://doi.org/10.7759/cureus....
60.
Watson P.F., 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891, https://doi.org/10.1071/RD9950....
61.
Wiebke M., Hensel B., Nitsche-Melkus E., Jung M., Schulze M., 2022. Cooled storage of semen from livestock animals (part I): boar, bull, and stallion. Anim. Reprod. Sci. 246, 106822, https://doi.org/10.1016/j.anir....
62.
Woelders H., Matthijs A., Besten-Den M., 1995. Boar variation in „Freezability” of the semen. Reprod. Domest. Anim. 31, 153–159, https://doi.org/10.1111/j.1439....
63.
Wolf J., Smital J., 2009. Quantification of factors affecting semen traits in artificial insemination boars from animal model analyses. J. Anim. Sci. 87, 1620–1627, https://doi.org/10.2527/jas.20....
64.
Yánez-Ortiz I., Catalán J., Rodríguez-Gil J.E., Miró J., Yeste M., 2022. Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep. Anim. Reprod. Sci. 246, 106904, https://doi.org/10.1016/j.anir....
65.
Yeste M., 2015. Recent advances in boar sperm cryopreservation: State of the art and current perspectives. Reprod. Domest. Anim. 50, 71–79, https://doi.org/10.1111/rda.12....
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