Quality assessment of cryopreserved New Zealand white rabbit spermatozoa in INRA-82 extender containing different cryoprotectants

Aya Mohamed Fadl, Abdel-Raouf Morsy Ghallab, Mostafa Mohamed Abou-Ahmed


The present study aimed to evaluate the effect of supplementation of INRA-82 semen extender with different cryoprotectants (dimethyl sulphoxide; DMSO vs. dimethyl formamide; DMF) on the quality of white New Zealand rabbit buck spermatozoa. We also investigated the possible association between the synergistic action of DMSO and DMF and their relation with INRA-82 extender composition. Semen was collected and pooled from 8 adult rabbit bucks. Pooled semen samples were diluted 1:1 with INRA-82 extender supplemented with DMSO 8%, DMF 8% or a combination of DMSO 4% and DMF 4%. The diluted semen samples were cryopreserved in 0.25 plastic straws. After thawing, progressive motility, sperm viability, sperm abnormalities, membrane integrity, acrosome status, viability index and DNA integrity were evaluated. The results showed that dilution of rabbit buck semen in INRA-82 supplemented with DMSO and DMF (4% each) before freezing significantly (P<0.05) improved sperm motility (42.00%), percentage of live spermatozoa (45.30%), proportions of spermatozoa with intact acrosome (59.75%) and percentage of spermatozoa with non-fragmented DNA (86.04%), compared to those diluted in INRA-82 supplemented either with DMSO 8% (+9, +10, +5 and +7 percentage points, respectively) or with DMF 8% alone (+18 +18, +12 and +9 percentage points, respectively). In conclusion, dilution of rabbit buck semen before freezing with INRA-82 extender supplemented with a combination of DMSO 4% and DMF 4% improved quality of frozen-thawed New Zealand White rabbit spermatozoa. Furthermore, our results also suggest that supplementation of INRA-82 with DMSO or with DMF alone at higher concentrations deteriorates the sperm quality.


rabbit buck; cryopreservation; INRA-82; DMSO;DMF

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Alvarenga M.A., Papa F.O., Landim-Alvarenga F.C., Medeiros A.S. 2005. Amides as cryoprotectants for freezing stallion semen: A review. Anim. Reprod. Sci., 89: 105-113. https://doi.org/10.1016/j.anireprosci.2005.07.001

Ashwood-Smith M.J. 1987. Mechanisms of cryoprotectant action. In: Bowler, K., Fuller, B.J., Temperature and Animal Cells. Biologists, Cambridge, UK., 41: 395-406.

Bezerra F.S.B., Castelo T.S., Alves H.M., Oliveira I.R.S., Lima G.L., Peixoto G.C.X., Bezerra A.S.D., Silva A.R. 2011. Objective assessment of the cryoprotective effects of dimethyl formamide for freezing goat semen. Cryobiology, 63: 263-266. https://doi.org/10.1016/j.cryobiol.2011.09.136

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. https://doi.org/10.1006/cryo.1999.2201

Chan P.J., Corselli J.U., Jacobson J.D., Patton W.C., King A. 1999. Spermac stain analysis of human sperm acrosomes. Fertil. Steril., 72: 124-128. https://doi.org/10.1016/S0015-0282(99)00201-0

Courtens J.L. 1995. La congélation de la semence de lapin. Cuniculture, 123: 101-102.

Cristanelli M.J., Amann R.P., Squires E.L., Pickett B.W. 1985. Effects of egg yolk and glycerol level in lactose–EDTA–egg yolk extender and of freezing rate on the motility of frozenthawed stallion spermatozoa. Theriogenology, 23: 25-38.


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. https://doi.org/10.1006/cryo.1995.1016

Darin-Bennett, A., White, I.G. 1977. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology. 14: 466-470. https://doi.org/10.1016/0011-2240(77)90008-6

Di Iorio M., Rosato M.P., Iaffaldano N. 2012. Effects of semen frozen with DMA and DMSO as cryoprotectants on rabbit reproductive performances. International PhD Workshop on “Welfare, Biotechnology and Quality of Animal Production”, Zielonka, (Poland), 5th-8th September, p.3.

Domingo P., Olaciregui M., González N., De Blas I., Gil L. 2018. Effects of seminal plasma and different cryoprotectants on rabbit sperm preservation at 16°C. Exp. Anim., 67: 413-420. https://doi.org/10.1538/expanim.17-0152

Evans G., Maxwell W.C. 1987. Salamons’ artificial insemination of sheep and goats (No. Ed. 2). Butterworths. Fadl A.M. 2016. Studies on some factors affecting freezability of stallion spermatozoa. M.V.Sc. Thesis, Cairo Univ.

Gao D.Y., Mazur P., Critser J.K. 1997. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK (eds) Reproductive tissue banking. Academic press, San Diego. 263-327. https://doi.org/10.1016/B978-012399770-8/50007-1

Gogol P. 1999. Cryopreservation of plasma-deprived rabbit semen. Ann. Anim. Sci., 26: 85-92.

Graham J.K., Mocé E. 2005. Fertility evaluation of frozen/thawed semen. Theriogenology, 64: 492-504. https://doi.org/10.1016/j.theriogenology.2005.05.006

Hanada A., Nagase. 1980. Cryoprotective effects of some amides on rabbit spermatozoa. J.Reprod. Fertil., 60: 247-252. https://doi.org/10.1530/jrf.0.0600247

Hall S.E, Negus C, Johinke D, Bathgate R. 2017. Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa. PLoS ONE, 12: e0175965. https://doi.org/10.1371/journal.pone.0175965

Holt W.V. 2000. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology, 53: 47-58. https://doi.org/10.1016/S0093-691X(99)00239-3

Kashiwazaki N., Okuda Y., Seita Y., Hisamatsu S., Sonoki S., Shino M., Masaoka T., Inomata T. 2006. Comparison of glycerol, lactamide, acetamide and dimethyl sulfoxide as cryoprotectants of Japanese white rabbit spermatozoa. J. Reprod. Dev., 52: 511-516. https://doi.org/10.1262/jrd.18008

Long J.A. 2006. Avian semen cryopreservation: what are the biological challenges? Poult. Sci., 85: 232-236. https://doi.org/10.1093/ps/85.2.232

Medeiros A.S.L., Gomes G.M., Carmo M.T., Papa F.O., Alvarenga M.A. 2002. Cryopreservation of stallion sperm using different amides. Theriogenology, 58: 273-276. https://doi.org/10.1016/S0093-691X(02)00898-1

Mesa A., Henao G. 2012. Effect of cholesterol and dimethylformamide on the cryosurvival of Colombian creole stallion sperm. Revista Medicina Veterinaria Zootecnia Cordoba, 17: 2908-2915. https://doi.org/10.21897/rmvz.260

Milovanov V.K. 1962. Biology of reproduction and artificial insemination of farm animals. Selihozizdat, Moscow.

Mocé E., Vicente J.S. 2009. Rabbit sperm cryopreservation: a review. Anim. Reprod. Sci., 110: 1-24. https://doi.org/10.1016/j.anireprosci.2008.08.015

Mota Filho A.C., Teles C.H., Juca R.P., Cardoso J.F., Uchoa D.C., Campello C.C., Silva L.D. 2011. Dimethyl formamide as acryoprotectant for canine semen diluted and frozen in ACP- 106C. Theriogenology, 76: 67-72. https://doi.org/10.1016/j.theriogenology.2011.05.010

Olaciregui, M., Gil, L., Montón, A., Luño, V., Jerez, R.A., Martí, J.I. 2014. Cryopreservation of epididymal stallion sperm. Cryobiology 68: 91-95. https://doi.org/10.1016/j.cryobiol.2013.12.009

Partyka A., Niżański W., Ochota M. 2012. Methods of assessment of cryopreserved semen. In: Katkov II (ed) Current Frontiers in Cryobiology. In Tech Open Access Publisher, Rijeka, Croatia. pp 547-574. https://doi.org/10.5772/33565

Pukazhenthi, B.S., Johnson, A., Guthrie, H.D., Songsasen, N., Padilla, L.R., Wolfe, B.A., Coutinho da Silva, M., Alvarenga, M.A., Wildt, D.E. 2014. Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii). Cryobiology 68: 205-214. https://doi.org/10.1016/j.cryobiol.2014.01.013

Sarıözkan S., Tuncer P.B., Bucak M.N., Ulutaş P.A. 2009. Influence of various antioxidants on microscopicoxidative stress indicators and fertilizing ability of frozenthawed bull semen. Acta Vet. Brno.,78: 463-469. https://doi.org/10.2754/avb200978030463

Squires E.L., Keith S.L., Graham J.K. 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology, 62: 1056-1065. https://doi.org/10.1016/j.theriogenology.2003.12.024

Vidament M., Ecot P., Noue P., Bourgeois C., Magistrini M., Palmer E. 2000. Centrifugation and addition of glycerol at 22°C instead of 4°C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology, 54: 907-920. https://doi.org/10.1016/S0093-691X(00)00401-5

Viudes de Castro M.P., Vicente J.S. 1996. A simple method for freezing rabbit semen with successful results on fertility and prolificity. Anim. Reprod. Sci., 44: 195-201. https://doi.org/10.1016/0378-4320(96)01550-3

Watson P.F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci., 60: 481-492. https://doi.org/10.1016/S0378-4320(00)00099-3

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World Rabbit Science  vol: 28  issue: 1  first page: 13  year: 2020  
doi: 10.4995/wrs.2020.12287


 Universitat Politècnica de València


Official journal of the World Rabbit Science Association (WRSA)


e-ISSN: 1989-8886     ISSN: 1257-5011   https://doi.org/10.4995/wrs