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Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits

  • M. P. Viudes-de-Castro (a1), R. Lavara (a2), H. M. Safaa (a3), F. Marco-Jiménez (a2), G. M. K. Mehaisen (a3) and J. S. Vicente (a2)...


This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.


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Abdelhakeam, AA, Graham, EF, Vazquez, IA and Chaloner, K 1991. Studies on the absence of glycerol in unfrozen and frozen ram semen: development of an extender for freezing: effects of osmotic pressure, egg yolk levels, type of sugars, and the method of dilution. Cryobiology 28, 4349.
Auerbach, AB, Norinsky, R, Ho, W, Losos, K, Guo, Q, Chatterjee, S and Joyner, AL 2003. Strain-dependent differences in the efficiency of transgenic mouse production. Transgenic Research 12, 5969.
Bolet, G, Brun, JM, Monnerot, M, Abeni, F, Arnal, C, Arnold, J, Bell, D, Bergoglio, G, Besenfelder, U, Bosze, S, Boucher, S, Chanteloup, N, Ducourouble, MC, Durand-Tardif, M, Esteves, PJ, Ferrand, N, Gautier, A, Haas, C, Hewitt, G, Jehl, N, Joly, T, Koehl, PF, Laube, T, Lechevestrier, S, López, M, Masoero, G, Menigoz, JJ, Piccinin, R, Queney, G, Saleil, G, Surridge, A, Van der Loo, W, Vicente, JS, Viudes de Castro, MP, Virag, JS and Zimmermann, JM 2000. Evaluation and conservation of European rabbit (Oryctolagus cuniculus) genetic resources. First results and inferences. World Rabbit Science 8, 281315.
Castellini, C, Battaglini, M and Lattaioli, P 1992. Effects of cryoprotectants and freezing on rabbit semen quality. Journal of Applied Rabbit Research 15, 431438.
Chen, Y, Yang, X and Foote, RH 1989. Timed breeding of rabbits with fresh and frozen-thawed semen and evidence of acrosome alteration of following freezing and thawing. Animal Reproduction Science 18, 3541.
Cifre, J, Baselga, M, Garcia-Ximenez, F and Vicente, JS 1998. Performance of a hyperprolific rabbit line I. Litter size traits. Journal of Animal Breeding and Genetics 115, 135138.
Cortell, C and Viudes de Castro, MP 2008. Effect of gelatin addition to freezing extender on rabbit semen parameters and reproductive performance. Paper presented at the 9th World Rabbit Congress, 10–13 June, Verona, Italy, pp. 327–332.
Dalimata, AM and Graham, JK 1997. Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose. Theriogenology 48, 831841.
Fox, RR 1961. Preservation of rabbit spermatozoa: fertility results from frozen semen. Proceedings of the Society for Experimental Biology and Medicine 108, 663665.
Hanada, A and Nagase, H 1980. Cryoprotective effects of some amides on rabbit spermatozoa. Journal of Reproduction and Fertility 60, 247252.
Iaffaldano, N, Di Iorio, M and Pina Rosato, M 2012. The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: dimethylacetamide versus dimethylsulfoxide. Theriogenology 78, 13811389.
Kashiwazaki, N, Okuda, Y, Seita, Y, Hisamatsu, S, Sonoki, S, Shino, M, Masaoka, T and Inomata, T 2006. Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa. Journal of Reproduction and Development 52, 511516.
Lavara, R, Mocé, E, Lavara, F, Viudes de Castro, MP and Vicente, JS 2005. Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits? Theriogenology 64, 11301141.
Lavara, R, David, I, Mocé, E, Baselga, M and Vicente, JS 2013. Environmental and male variation factors of freezability in rabbit semen. Theriogenology 79, 582589.
Long, JA 2006. Avian semen cryopreservation: what are the biological challenges? Poultry Science 85, 232236.
Malo, C, Gil, L, González, N, Cano, R, de Blas, I and Espinosa, E 2010. Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender. Cryobiology 61, 1721.
Mocé, E and Vicente, JS 2009. Rabbit sperm cryopreservation: a review. Animal Reproduction Science 110, 124.
Mocé, E, Vicente, JS and Lavara, R 2003. Effect of freezing-thawing protocols on the performance of semen from three rabbit lines after artificial insemination. Theriogenology 60, 115123.
Mocé, E, Lavara, R and Vicente, JS 2005. Influence of donor male on the fertility of frozen-thawed rabbit semen after artificial insemination of females from different genotypes. Reproduction in Domestic Animals 40, 516521.
Molinia, FC, Evans, G, Casares, PI and Maxwell, WM 1994. Effect of monosaccharides and disaccharides in tris based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. Animal Reproduction Science 36, 113122.
Rosato, MP and Iaffaldano, N 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, 508516.
Sanchez, JP, Theilgaard, P, Minguez, C and Baselga, M 2008. Constitution and evaluation of a long-lived productive rabbit line. Journal of Animal Science 86, 515525.
Songsasen, N and Leibo, SP 1997. Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 35, 255269.
Stranzinger, GF, Maurer, RR and Paufler, SK 1971. Fertility of frozen rabbit semen. Journal of Reproduction and Fertility 24, 111113.
Sztein, JM, Farley, JS and Mobraaten, LE 2000. In vitro fertilization with cryopreserved inbreed mouse sperm. Biology of Reproduction 63, 17741780.
Vicente, JS and Viudes de Castro, MP 1996. A sucrose-DMSO extender for freezing rabbit semen. Reproduction Nutrition Development 36, 485 492.
Vicente, JS, Viudes de Castro, MP, Lavara, R and Lavara, F 2000. Effect of male line on prolificacy from does inseminated with low sperm doses. Paper presented at the 7th World Rabbit Congress, 4–7 July, Valencia, Spain, pp. 1273–1277.
Viudes de Castro, MP and Vicente, JS 1996. A simple method for freezing rabbit semen with successful results on fertility and prolificity. Animal Reproduction Science 44, 195201.
Viudes de Castro, MP and Vicente, JS 1997. Effect of sperm count on the fertility and prolificity rates of meat rabbits. Animal Reproduction Science 46 (3–4), 313319.
Waterhouse, KE, Hofmo, PO, Tverdal, A and Miller, RR 2006. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction 131, 887894.
Woelders, H, Matthijs, A and Engel, B 1997. Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology 35, 93105.


Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits

  • M. P. Viudes-de-Castro (a1), R. Lavara (a2), H. M. Safaa (a3), F. Marco-Jiménez (a2), G. M. K. Mehaisen (a3) and J. S. Vicente (a2)...


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