Seminal plasma interference in the kinetics and viability of cryopreserved canine sperm




Canine semen; Freezing; Sperm analysis.


Most researchers have frozen dog semen using methodologies described for other species. Consequently, these studies have shown that the thawed semen of dogs is of low quality, with conception rates lower than that of other species. Therefore, in this study, we evaluated different freezing protocols for canine semen, using three adult Bulldog Campeiro males, aged 2 to 5 years, with proven fertility. Five semen samples were collected from each animal using the penile bulb digital manipulation method. The collected samples were divided into: group 1, the samples were diluted directly in Botudog® commercial freezing medium (Botupharma Biotecnologia Animal) and group 2, the samples were centrifuged at 600 g for 10 min and the pellet was resuspended in Botudog®, totaling a concentration end of 200 x 106 sperm per ml. The samples were packaged in 0.5 mL straws at a concentration of 100 x 106 viable sperm. The samples remained for 1 h in stabilization at 4 °C and transferred to nitrogen vapor for 20 min, immersed in nitrogen, stored in a cryogenic cylinder, and thawed at 46 °C for 20 s. It was found that the total motility (MT, %), path speed (VAP, μm/s), progressive motility (PM, %), progressive linear speed (VSL; µm/s), curvilinear speed (VCL; µm/s), linearity (LIN, %), percentage of fast sperm (RAP, %), and plasma and acrosomal membrane integrity were higher in group 1, with samples not being centrifuged. These data demonstrate that the canine semen freezing protocol, using the Botudog® diluent, does not recommend the centrifugation of the ejaculate, prior to freezing.


Amann, R. P. (1981). A critical review of methods for evaluation of spermatogenesis from seminal characteristics. Journal of Andrology, 2(37), 5 8.

Aurich, C. (2005). Factors affecting the plasma membrane function of cooled-stored stallion spermatozoa. Animal Reproduction Science, 89: 65-75.

Crespilho, A. M., Nichi, M., Guasti, P. N., Freitas-Dell´Aqua, C. P., Sá Filho, M. F., Maziero, R. R., Dell´Aqua, J. A., Papa, F. O. (2014). Sperm fertility and viability following 48h of refrigeration: Evaluation of different extenders for the preservation of bull semen in liquid state. Animal Reproduction Science, 146, 126-133.

Cunha, I. C. N., Lopes, M. D. (1999). Centrifugation effects on canine semen quality. Veterinary Brazilian Reproduction Animals, 23(3): 306-308.

Davis, M. J., Davis, M., Minter, L. (2007). Detection of reactive oxygen species in canine semen. Theriogenology, 68, 492-518.

England, G. C. W. (1993). Cryopreservation of dog semen: a review. Journal of Reproduction and Fertility, 47, 243-255.

Goericke-Pesch S., Klaus, D., Failing, K., Wehrend, A. (2012). Longevity of chilled canine semen comparing different extenders. Animal Reproduction Science, 135, 97-105.

Gunzel‐Apel, A. R., Ekrod, B. (1991). Influences of seminal plasma and extender on sperm motility, ATP‐concentration, and the activity of acid and alkaline phosphatases of beagle dog semen. Reproduction in Domestic Animals, 26, 31-41.

Hori, T., Masuda, T., Kobayashi, M., Kawakami, E. (2017). Role of prostatic fluid in cooled canine epididymal sperm. Reproduction in Domestic Animals, 52, 655-660.

Jarosz, L., Gradzki, Z., Kalinowski, M. (2016). Quality of fresh and chilled-stored raccoon dog semen and its impact on artificial insemination efficiency. BMC Veterinary Research, 12, 224.

Kutzler, M. A. (2005). Semen collection in the dog. Theriogenology, 64, 747-754.

Lima J. S., Guaitolini, C. R. F., Crespilho, A. M., Freitas-Dell´Aqua, C. P., Martins, M. I. M., Rigoto, R. P., Marques, A. B., Hidalgo, M. M. T., Maziero, R. R. D. (2019). Effect of diluting media on canine semen freezability. Brazilian Journal of Development, 5(9), 15841-15855.

Mascarenhas, R. M., Xavier, P. R., Marques Junior, A. P. (2014). Ejaculados individuais e pools de sêmen: diferenças em condições experimentais. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 66(1), 22-26.

Olar, T. T., Amann, R. P., Pickett, B. W. (1983). Relationship among testicular size, daily production and output of spermatozoa and extragonadal reserves of the dog. Biology of Reproduction, 29, 1114-1120.

Ochsendorf, F. R. (1999). Infections in the male genital tract and reactive oxygen species. Human Reproduction Update, 5: 399-420.

Platz, C. C., Seager, S. W. (1977). Successful pregnancies with concentrated frozen canine semen. Laboratory Animal Science, 27(6), 1013-1016.

Puja, I. K., Sawitri, N. M., Maharani, N., Heryani, L. G. S. S., Dharmayudha, A. A. G. O; Gunawan, I. W. N. F. (2019). Preservation of semen from Kintamani Bali dogs by freezing method. Journal of Advanced Veterinary Animal Research, 6(2), 158-162.

Rijseelaere, T., Soom, A. V., Maes, D., Kruif, A. (2002). Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. Theriogenology, 57, 1669-1681.

Rota, A., Strom, B., Linde-Forsberg, C. L. (1995). Effects of seminal plasma and three extenders on canine semen stored at 4°C. Theriogenology, 44, 885-900.

Rowson, L. E. A. (1954). Infertility of cow, sow and bitch. Irish Veterinary Journal, 8, 216-221.

Santos, J. F. P., Gosmes, E. T., Siqueira, A. K. M., Cardoso, R. C. S. (2016). Andrologia e criopreservação de sêmen. Revista Brasileira de Reprodução Animal, 40(4), 167-179.

Silva, A. R., Cardoso, R. C. S., Silva, L. D. M. (2000). Congelação de sêmen canino com diferentes concentrações de gema de ovo e glicerol em diluidores à base de tris e água de coco. Ciência Rural, 30(6): 1021-1025.

Silva, A. R., Cardoso, R. C. S., Uchoa, D. C., Silva, L. D. M. (2003). Quality of canine semen submitted to single or fractionated glycerol addition during the freezing process. Theriogenology, 59, 821-829.

Silva, L. D. M., Verstegen, J. P. (1995). Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa. Theriogenology, 44, 571-579.

Soares, M. P., Rosse, C. A. R., Mezzalira, A., Cecim, M. (2002). Etileno glicol na criopreservação de sêmen canino. Ciência Rural, 32(4): 119-127.

Souza, F. F., Barreto, C. S., Lopes, M. D. (2007). Characteristics of seminal plasma proteins and their correlation with canine semen analysis. Theriogenology, 68(1), 100-106.

Thirumala, S., Ferrer, M. S., Al-Jarrah, A., Eilts, E. E., Paccamonti, D. L., Devireddy, R. V. (2003). Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents. Cryobiology, 47(2), 109-124.

Threlfall, W. R. (2003). Semen collection and evaluation. In: Kustritz, M.V.R. The practical veterinarian: Small Animals Theriogenology. Elsevier Science, USA, 97-124.

Treulen, F., Sánchez, R., Risopatrón, J. (2012). Effects of seminal fluid fractions on plasma and acrosome membrane integrity and mitochondrial membrane potential determined by flow cytometry in chilled canine spermatozoa. Reproduction in Domestic Animals, 47: 1043–1048.

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 an in vivo studies. Theriogenology, 64, 720-733.



How to Cite

Rigoto, R. P., Guaitolini, C. R. de F., Crespilho, A. M., Cruz, G. P. da, Tramontin, M. L. D., Dell´´´´´ Aqua, C. P. F. ., Martins, M. I. M., & Maziero, R. R. D. . (2020). Seminal plasma interference in the kinetics and viability of cryopreserved canine sperm . Research, Society and Development, 9(11), e309119459.



Agrarian and Biological Sciences