Effects of extenders on the functional activity of chilled canine spermatozoa during prolonged storage
Abstract
Preserving the quality and providing a high fertile capacity of spermatozoa are some of the main objectives of successful fertilization of dogs. Insemination using chilled semen produces higher pregnancy parameters and larger litters, compared with using frozen semen. Therefore, this practice is in high demand. Nevertheless, there is an issue of prolonged storage of chilled semen, especially relevant during transportation to long distances. Considering that high-quality parameters of spermatozoa directly depend on a medium in which they are stored, the objective of this study was determining the effects of different types of extenders on the functional characteristics of chilled canine semen during prolonged storage. We studied the effects of the extenders on the motility, ability to move in a straight line, integrity of the membrane, and DNA fragmentation of canine spermatozoa. The second fraction of ejaculate was collected from five dogs using the masturbation method. The semen was centrifuged to achieve settlement of the spermatozoa and remove the plasma. To the spermatozoa sediment, one of the following extenders was added, which had been heated up to the room temperature: Т – TRIS + citric acid + glucose (base content); Т-EG – base content + egg yolk; Т-BSA – base content + BSA; T-EDTA – base content + EDTA in a calculation of 150×10 6 spermatozoa in 5 cm 3 of extender. After pipetting, the samples were cooled to 4 °C with subsequent daily monitoring until the tenth day of storage. The results of the study revealed that the extender Т-BSA provided the highest parameters of the general motility and straight-line movement of the spermatozoa over the storage period. In the descending order, the effectiveness of extenders in preserving the sperm motility at a 50% level was as follows: Т-BSA (7 days); T and T-EDTA (6 days); Т-EG (5 days). At the same time, according to the percentage of gametes with straight-line movements, the results were as follows: Т-BSA (7 days); T and Т-EG (5 days); T-EDTA (4 days). The percentage of spermatozoa with damaged membrane over the period of storage increased (by 13.4 – 16 .0 %, compared with day zero), although no effect of the extender composition on this parameter was observed. The level of DNA fragmentation in the chilled canine sperm increased insignificantly (up to 0.4% by day 10) over the storage period, without significant effect of the extender on this parameter. In summary, the extender Т-BSA demonstrated the highest effectiveness in the preservation of sperm motility at a 4 °C temperature, maintaining the general motility at the level of 37.4 ± 5.9% and the straight-line move ment at the level of 31.2 ± 6.6 % on day 10 of storage. This allows us to consider Т-BSA an optimal compos i tion for long storage of dog semen in a chilled state.References
Álvarez-Rodríguez, M., Nieto-Cristobal, H., & de Mercado, E. (2024). Bovine serum albumin inclusion in the thawing extender improves boar sperm membrane and acrosomal integrity. Reproduction in Domestic Animals, 59(S3), e14592.
Bailey, J. L., Bilodeau, J. F., & Cormier N. (2000). Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. Journal of Andrology, 21(1), 1–7.
Ballester, J., Fernández-Novell, J. M., Rutllant, J., García-Rocha, M., Jesús Palomo, M., Mogas, T., Peña, A., Rigau, T., Guinovart, J. J., & Rodríguez-Gil, J. E. (2000). Evidence for a functional glycogen metabolism in mature mammalian spermatozoa. Molecular Reproduction and Development, 56(2), 207–219.
Bencharif, D., Amirat-Briand, L., Le Guillou, J., Vitelli, C., Anton, M., Schmitt, E., Desherces, S., Barriere, P., & Tainturier, D. (2013). Refrigeration of canine sperm at + 4 °C: Comparative study of four different extenders for the refrigeration of canine sperm at +4 °C: LDL, Tris egg yolk, Equex®, and INRA96®. Revue de Médecine Vétérinaire, 164(5), 252–262.
Bergeron, A., Crête, M. H., Brindle, Y., & Manjunath, P. (2004). Low-density lipoprotein fraction from hen's egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biology of Reproduction, 70(3), 708–717.
Birkhead, T. R., & Møller, A. P. (1993). Sexual selection and the temporal separation of reproductive events: sperm storage data from reptiles, birds and mammals. Biological Journal of the Linnean Society, 50(4), 295–311.
Blank, M., Soo, L., & Britten, J. S. (1976). Adsorption of albumin on rabbit sperm membranes. The Journal of Membrane Biology, 29(4), 401–409.
Bousseau, S., Brillard, J. P., Marguant-Le Guienne, B., Guérin, B., Camus, A., & Lechat, M. (1998). Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology, 50(5), 699–706.
Bozzini, G., Seveso, M., Bono, P., De Francesco, O., Mandressi, A., Taverna, G. (2016). FRII-03 Lazzaro Spallanzani (1729–1799): The first successful artificial insemination experiments. Journal of Urology, 195(4S), e521.
Bucci, D., Isani, G., Spinaci, M., Tamanini, C., Mari, G., Zambelli, D., & Galeati, G. (2010). Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa. Reproduction in Domestic Animals, 45(2), 315–322.
Bucci, D., Spinaci, M., Bustamante-Filho, I. C., & Nesci, S. (2023). The sperm mitochondria: Clues and challenges. Animal Reproduction, 19(4), e20220131.
Bustani, G. S., & Baiee, F. H. (2021). Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Veterinary World, 14(5), 1220–1233.
Calabria, A., Del Prete, C., Roberto, C., Longobardi, V., Spada, S., Alfano, M. T., De Felice, D., Gasparrini, B., & Cocchia, N. (2023). Effect of crocin supplementation in the extender on the quality of chilled canine semen. Animal Reproduction Science, 259, 107374.
Carr, D. W., Usselman, M. C., & Acott, T. S. (1985). Effects of pH, lactate, and viscoelastic drag on sperm motility: A species comparison. Biology of Reproduction, 33(3), 588–595.
Contri, A., Gloria, A., Robbe, D., Valorz, C., Wegher, L., & Carluccio, A. (2013). Kinematic study on the effect of pH on bull sperm function. Animal Reproduction Science, 136(4), 252–259.
Cormier, N., & Bailey, J. L. (2003). A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa. Biology of Reproduction, 69(1), 177–185.
Deco-Souza, T., Paula, T. A. R., Araujo, G. R., Bergo, L. C. F., Carazo, L. R. B., Vasconcelos, G. S. C., & Silva, M. C. C. (2020). Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 72, 2119–2126.
Del Prete, C., Calabria, A., Longobardi, V., Palumbo, V., Merlo, B., Iacono, E., Tafuri, S., Carotenuto, D., Ciani, F., Damiano, S., Ciarcia, R., Cocchia, N. (2022). Effect of aqueous extract of maca addition to an extender for chilled canine semen. Animals, 12(13), 1638.
Divar, M. R., Azari, M., Mogheiseh, A., & Ghahramani, S. (2022). Supplementation of melatonin to cooling and freezing extenders improves canine spermatozoa quality measures. BMC Veterinary Research, 18(1), 86.
du Plessis, S. S., Agarwal, A., Mohanty, G., & van der Linde, M. (2015). Oxidative phosphorylation versus glycolysis: What fuel do spermatozoa use? Asian Journal of Andrology, 17(2), 230–235.
Eilts, B. E. (2005). Theoretical aspects of canine semen cryopreservation. Theriogenology, 64(3), 692–697.
Fernández, J. L., Muriel, L., Rivero, M. T., Goyanes, V., Vazquez, R., & Alvarez, J. G. (2003). The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. Journal of Andrology, 24(1), 59–66.
Fernández-Novell, J. M., Ballester, J., Medrano, A., Otaegui, P. J., Rigau, T., Guinovart, J. J., & Rodríguez-Gil, J. E. (2004). The presence of a high-Km hexokinase activity in dog, but not in boar, sperm. Federation of European Biochemical Societies Letters, 570(1–3), 211–216.
Flores, A. J., Fernández, A. V., Huamán, U. H., Ruiz, G. L., & Santiani, A. A. (2010). Refrigeration of canine semen using glucose, fructose, trehalose or sucrose to extend sperm survival. Revista de Investigaciones Veterinarias del Perú, 21(1), 26–34.
Foutouhi, A., & Meyers, S. (2022). Comparative oxidative metabolism in mammalian sperm. Animal Reproduction Science, 247, 107095.
Goericke-Pesch, S., Klaus, D., Failing, K., & Wehrend, A. (2012). Longevity of chilled canine semen comparing different extenders. Animal Reproduction Science, 135(1–4), 97–105.
Hermansson, U., Johannisson, A., & Axnér, E. (2021). Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender. Veterinary Medicine and Science, 7(3), 812–819.
Hori, T., Yoshikuni, R., Kobayashi, & M., Kawakami, E. (2014). Effects of storage temperature and semen extender on stored canine semen. Journal of Veterinary Medical Science, 76(2), 259–263.
Iguer-Ouada, M., & Verstegen, J. P. (2001). Long-term preservation of chilled canine semen: Effect of commercial and laboratory prepared extenders. Theriogenology, 55(2), 671–684.
Kasimanickam, V. R., Kasimanickam, R. K., Memon, M. A, & Rogers, H. A. (2012). Effect of extenders on sperm mitochondrial membrane, plasma membrane and sperm kinetics during liquid storage of canine semen at 5 ˚C. Animal Reproduction Science, 36(1), 139–145.
Kustritz, M. R. (2007). The value of canine semen evaluation for practitioners. Theriogenology, 68(3), 329–337.
Kutzler, M. A. (2005). Semen collection in the dog. Theriogenology, 64(3), 747–754.
Layek, S. S., Mohanty, T. K., Kumaresan, A., & Parks, J. E. (2016). Cryopreservation of bull semen: Evolution from egg yolk based to soybean-based extenders. Animal Reproduction Science, 172(9), 1–9.
Lee, K. B., Kim, M. K., & Park, B. K. (2013). The semen property and preservation in Shih Tzu dogs. Journal of Embryo Transfer, 28(2), 121–125.
Linde-Forsberg, C. (1991). Achieving canine pregnancy by using frozen or chilled extended semen. Veterinary Clinics of North America: Small Animal Practice, 21(3), 467–485.
Lopes, G., Simoes, A., Ferreira, P., Martins-Bessa, A., & Rocha, A. (2009). Differences in preservation of canine chilled semen using different transport containers. Animal Reproduction Science, 112(2), 158–163.
Mahi, C. A., & Yanagimachi, R. (1978). Capacitation, acrosome reaction, and egg penetration by canine spermatozoa in a simple defined medium. Gamete Research, 1, 101–109.
Martínez-Barbitta, M., & Rivera Salinas, C. (2022). Evaluation of chilled dog semen extended with sperm activator. Frontiers in Veterinary Science, 8, 764750.
Matsuoka, T., Imai, H., Kohno, H., & Fukui, Y. (2006). Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa. Journal of Reproduction and Development, 52(5), 675–683.
Michael, A. J., Alexopoulos. C., Pontiki, E. A., Hadjipavlou-Litina, D. J., Saratsis, P., Ververidis, H. N., & Boscos, C. M. (2009). Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa. Animal Reproduction Science, 112(1–2), 119–135.
Nguyen, V., Ponchunchoovong, S., Kupittayanant, S., & Kupittayanant, P. (2019). Effects of egg yolk and soybean lecithin on sperm quality determined by computer-assisted sperm analysis and confocal laser scanning microscope in chilled canine sperm. Veterinary Medicine and Science, 5, 345–360.
Nizański, W. (2006). Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: Use of an infusion pipette and the Osiris catheter. Theriogenology, 66(2), 470–483.
Phillips, P. H. (1939). Preservation of bull semen. Journal of Biological Chemistry, 130(1), 415.
Ponglowhapan, S., Essén-Gustavsson, B., & Linde-Forsberg, C. (2004). Influence of glucose and fructose in the extender during long-term storage of chilled canine semen. Theriogenology, 62(8), 1498–1517.
Prete, C. D., Ciani, F., Tafuri, S., Pasolini, M. P., Valle, G. D., Palumbo, V., Abbondante, L., Calamo, A., Barbato, V., Gualtieri, R., Talevi, R., & Cocchia, N. (2018). Effect of superoxide dismutase, catalase, and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs. Journal of Veterinary Science, 19(5), 667–675.
Prinosilova, P., Rybar, R., Zajicova, A., & Hlavicova, J. (2012). DNA integrity in fresh, chilled and frozen-thawed canine spermatozoa. Veterinární Medicína, 57(3), 133–142.
Puja, I. K., Sawitri, N. M., Maharani, N., Gunawan, I. W. N. F., Heryani, L. G. S. S. (2018). A comparative study on the effects of coconut water based extenders on the quality of Kintamani dog semen preserved at 4 ˚C. Advances in Animal and Veterinary Sciences, 6(5), 192–196.
Raheja, N., Choudhary, S., Grewal, S., Sharma, N., & Kumar, N. (2018). A review on semen extenders and additives used in cattle and buffalo bull semen preservation. Journal of Entomology and Zoology Studies, 6(3), 239–245.
Rahman, M. A., Park, S. H., & Yu, I. J. (2017). Effect of monosaccharides in glycerol-free tris extender on reactive oxygen species and apoptosis in dog sperm cryopreservation. Cryo Letters, 38(1), 51–57.
Rigau, T., Farré, M., Ballester, J., Mogas, T., Peña, A., & Rodríguez-Gil, J. E. (2001). Effects of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculates. Theriogenology, 56(5), 801–815.
Risopatrón, J., Catalán, S., Miska, W., Schill, W. B., & Sánchez, R. (2002). Effect of albumin and polyvinyl alcohol on the vitality, motility and acrosomal integrity of canine spermatozoa incubated in vitro. Reproduction in Domestic Animals, 37(6), 347–351.
Rodenas, C, Parrilla, I., Roca, J., Martinez, E. A., & Lucas, X. (2014). Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates. Theriogenology, 82(4), 621–666.
Rota, A., Ström, B., & Linde-Forsberg, C. (1999). Effects of seminal plasma and three extenders on canine semen stored at 4 °C. Theriogenology, 44(6), 885–900.
Sandal, A. İ., Şenlikci, H., Baran, A., & Özdaş, Ö. B. (2020). Effects of semen extender supplemented with bovine serum albumin (BSA) on spermatological traits of Saanen buck semen stored at +4 °C. Kafkas Universitesi Veteriner Fakultesi Dergisi, 26(4), 515–520.
Schäfer-Somi, S., Binder, C., Burak, J., Papadopoulos, N., Ilas, J., Boersma, A., Aurich, C. (2021). Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase. Cryobiology, 100, 63–71.
Serrano, R., Solar Málaga, S., González-Fernández, L., Gervasi, M. G., García-Marín, L. J., Bragado, M. J., & Martin-Hidalgo, D. (2025). Glucose prevents the acquisition of the capacitated state in pig spermatozoa. Andrology, 13(3), 637–649.
Simopoulos, A. P., & Salem Jr., N. (1992). Egg yolk as a source of long-chain polyunsaturated fatty acids in infant feeding. American Journal of Clinical Nutrition, 55(2), 411–414.
Songsasen, N., Yu, I., Murton, S., Paccamonti, D. L., Eilts, B. E., Godke, R. A., & Leibo, S. P. (2002). Osmotic sensitivity of canine spermatozoa. Cryobiology, 44(1), 79–90.
Sumayya, S. R., Suchitra, B. R., Renukaradhya, G. J., Kalmath, G. P., Narasimha, M., & Mohan, H. (2024). Comparative study on effects of soy lecithin and egg yolk-based extenders on canine semen preservation at refrigeration temperature. The Indian Journal of Animal Sciences, 94(12), 1078–1083.
Suzuki, H., Watanabe, H., & Abe, Y. (2022). Assisted reproductive techniques for canines: Preservation of genetic material in domestic dogs. Journal of Reproduction and Development, 68(1), 1–11.
Teixeira, D. O., Silva, H. V. R., Brito, B. F., Barbosa, B. S., Tabosa, B. E. A., & da Silva, L. D. M. (2022). Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS. Animal Reproduction, 19(3), e20210069.
Thérien, I., Moreau, R., & Manjunath, P. (1999). Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biology of Reproduction, 61(3), 590–598.
Thomassen, R., & Farstad, W. (2009). Artificial insemination in canids: A useful tool in breeding and conservation. Theriogenology, 71(1), 190–199.
Uysal, O., Bucak, M. N., Yavas, I., & Varisli, O. (2007). Effect of various antioxidants on the quality of frozen-thawed bull semen. Journal of Animal and Veterinary Advances, 6(12), 1362–1366.
Verstegen, J. P., Onclin, K., & Iguer-Ouada, M. (2005). Long-term motility and fertility conservation of chilled canine semen using egg yolk added Tris-glucose extender: In vitro and in vivo studies. Theriogenology, 64(3), 720–733.
Yildiz, C., Kaya, A., Aksoy, M., & Tekeli, T. (2000). Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology, 54(4), 579–585.
Yu, L. J. (2006). Evaluation of glucose-BSA as extender of chilled canine spermatozoa for long-term storage. Korean Journal of Embryo Transfer, 21(4), 323–330.
Yurchuk, T., Pavlovich, O., & Petrushko, M. (2022). Using dextran instead of egg yolk in extender for cryopreservation of spermatozoa of dogs of different ages. Animals, 12(24), 3480.
Zhang, X. G., Yan, G. J., Hong, J. Y., Su, Z. Z., Yang, G. S., Li, Q. W., Hu, J. H. (2015). Effects of bovine serum albumin on boar sperm quality during liquid storage at 17 °C. Reproduction in Domestic Animals, 50(2), 263–269.
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