Comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa P., Olaciregui M., González N., De Blas I., Gil L. (2019): Comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa. Czech J. Anim. Sci., 64: 59-66.
download PDF

The purpose of this research was to find a suitable protocol to enhance frozen rabbit sperm preservation analysing the role that seminal plasma (SP) plays and the effect of different cryoprotectant agents on sperm quality 0 and 2 h after thawing. Sperm samples were pooled and divided in eight fractions. Four of them were diluted with BotuCrio® (extender A), INRA 96® plus 6% glycerol (extender B), 6% N, N-dimethylformamide (extender C) and 6% N-methyl-2-pyrrolidone (extender D), respectively. The other four fractions were centrifuged and the supernatant was discarded in order to eliminate SP. Each sample was then resuspended with extender A, B, C and D. Samples were cooled progressively, loaded into 0.5 ml freezing straws and frozen with liquid nitrogen vapour. Thawing was performed by placing the straws into a bain-marie at 37°C for 21 s. Straws were dried and sperm samples placed into Eppendorf tubes to be analyzed by ISAS software, vitality test, HOS test and acrosome integrity test. The best motility and velocity parameters were obtained by extender A (P < 0.050) even when the motility parameter was compared with previous studies using other diluents. Additionally, sperm quality decreased over incubation time (P < 0.050) and no differences were found in samples processed with or without SP. This research revealed that BotuCrio® could be used for rabbit sperm cryopreservation and moreover the improvement of the cryopreservation process of rabbit sperm due to the demonstration that SP removing is not required.

Alvarino M.R. (1993): Control of Rabbit Reproduction. Mundi-Prensa, Madrid, Spain. (in Spanish)
Amorim E.A.M., Torres C.A.A., Graham J.K., Amorim L.S., Santos L.V.L. (2009): The hypoosmotic swelling test in fresh rabbit spermatozoa. Animal Reproduction Science, 111, 338-343
Aksoy M., Cankat Lechimcioglu N., Akman O. (2008): Effect of seminal plasma on functional integrity of rabbit sperm membranes during storage at 4°C or freezing. World Rabbit Science, 16, 1–6.
Bezerra Francisco Silvestre B., Castelo Thibério S., Alves Heron M., Oliveira Isabelle R.S., Lima Gabriela L., Peixoto Gislayne C.X., Bezerra Ana Carla S.D., Silva Alexandre R. (2011): Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen. Cryobiology, 63, 263-266
Bjorndahl L. (): Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Human Reproduction, 18, 813-816
Carluccio A., Robbe D., De Amicis I., Contri A., Tosi U., Russo F., Paoletti M. (2004): Artificial insemination in rabbits: Laboratory and field trial with three different semen extenders. World Rabbit Science, 12, 65–79.
Castellini Cesare, Pizzi Flavia, Theau-Clément Michèle, Lattaioli Paolo (2006): Effect of different number of frozen spermatozoa inseminated on the reproductive performance of rabbit does. Theriogenology, 66, 2182-2187
Chalah T., Seigneurin F., Blesbois E., Brillard J.P. (1999): In Vitro Comparison of Fowl Sperm Viability in Ejaculates Frozen by Three Different Techniques and Relationship with Subsequent Fertility in Vivo. Cryobiology, 39, 185-191
Curry M.R., Redding B.J., Watson P.F. (1995): Determination of Water Permeability Coefficient and Its Activation Energy for Rabbit Spermatozoa. Cryobiology, 32, 175-181
Dalimata A.M., Graham J.K. (1997): Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose. Theriogenology, 48, 831-841
Darin-Bennett Annabelle, White I.G. (1977): Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology, 14, 466-470
Fuller Barry, Paynter Sharon (2004): Fundamentals of cryobiology in reproductive medicine. Reproductive BioMedicine Online, 9, 680-691
Gilmore J. A., McGann L. E., Liu J., Gao D. Y., Peter A. T., Kleinhans F. W., Critser J. K. (1995): Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa1. Biology of Reproduction, 53, 985-995
Gogol P. (1999): Cryopreservation of plasma-deprived rabbit semen. Annals of Animal Science, 26, 85–92.
Hall Sally E., Negus Cameron, Johinke Danielle, Bathgate Roslyn, Moreira Nei (2017): Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa. PLOS ONE, 12, e0175965-
Hernandez P.J.E., Fernandez R.F., Rodriguez S.J.L., Negrete R.M., Soto M.Y.G., Garcia R.A.D. (2012): Effect of cryopreservation on viability and the acrosomal state of New Zealand rabbit (Oryctolagus cuniculus) semen. Revista de Salud Animal, 34, 188–191.
Iaffaldano N., Di Iorio M., Rosato M. Pina (2012): The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: Dimethylacetamide versus dimethylsulfoxide. Theriogenology, 78, 1381-1389
Jeyendran R. S., Van der Ven H. H., Perez-Pelaez M., Crabo B. G., Zaneveld L. J. D. (1984): Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. Reproduction, 70, 219-228
Johinke D., de Graaf S.P., Bathgate R. (2014): Investigation of in vitro parameters and in vivo fertility of rabbit spermatozoa after chilled storage. Animal Reproduction Science, 147, 135-143
Kuliková Barbora, Di Iorio Michele, Kubovicova Elena, Kuzelova Lenka, Iaffaldano Nicolaia, Chrenek Peter (2015): The cryoprotective effect of Ficoll on the rabbit spermatozoa quality. Zygote, 23, 785-794
Kulíková Barbora, Oravcová Marta, Baláži Andrej, Supuka Peter, Chrenek Peter (2017): Factors affecting storage of Slovak native rabbit semen in the gene bank. Zygote, 25, 592-600
Lopes K.R.F., Costa L.L.M., Lima G.L., Souza A.L.P., Silva A.R. (2009): Dimethylformamide is no better than glycerol for cryopreservation of canine semen. Theriogenology, 72, 650-654
Lopez F.J., Alvarino J.M.R. (1998): Artificial insemination of rabbits with diluted semen stored up to 96 hours. World Rabbit Science, 6, 251–253.
López F.J, Alvariño J.M.R (2000): Effects of added caffeine on results following artificial insemination with fresh and refrigerated rabbit semen. Animal Reproduction Science, 58, 147-154
Malo C., Gil L., Cano R., Martínez F., García A., Jerez R. A. (2012): Dimethylformamide is not better than glycerol for cryopreservation of boar semen. Andrologia, 44, 605-610
Mocé Eva, Vicente José S. (2009): Rabbit sperm cryopreservation: A review. Animal Reproduction Science, 110, 1-24
Nishijima Kazutoshi, Kitajima Shuji, Koshimoto Chihiro, Morimoto Masatoshi, Watanabe Teruo, Fan Jianglin, Matsuda Yukihisa (2015): Motility and fertility of rabbit sperm cryopreserved using soybean lecithin as an alternative to egg yolk. Theriogenology, 84, 1172-1175
Olaciregui M., Gil L., Montón A., Luño V., Jerez R.A., Martí J.I. (2014): Cryopreservation of epididymal stallion sperm. Cryobiology, 68, 91-95
Pursel V.G., Johnson L.A. (1974): Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation. Theriogenology, 1, 63-68
Rosato Maria Pina, Iaffaldano Nicolaia (2013): Cryopreservation of rabbit semen: Comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology, 79, 508-516
Salmani Hossein, Towhidi Armin, Zhandi Mahdi, Bahreini Majid, Sharafi Mohsen (2014): In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen. Cryobiology, 68, 276-280
Swain J.E., Smith G.D. (2010): Cryoprotectants. In: Chian R.-C. and Quinn P. (eds): Fertility Cryopreservation. Cambridge University Press, 24–38.
download PDF

© 2021 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti