Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Paula Domingo; Maite Olaciregui; Noelia González; Ignacio De Blas; Lydia Gil

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Abstract: Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.


cryoprotectant, dimethylformamide, n-methyl-2-pyrrolidone, glycerol, rabbit sperm preservation


Alvarenga MA, Papa FO, Landim-Alvarenga FC, Medeiros ASL. Amides as cryoprotectants for freezing stallion semen: A review. Anim Reprod Sci. 2005;89(1-4):105-13. PMid:16099609.

Alvariño MR. Control de la reproducción del conejo. Madrid: Ed. Mundi-Prensa; 1993.

Amorim EAM, Torres CCA, Graham JK, Amorim LS, Santos LVL. The hypoosmotic swelling test in fresh rabbit spermatozoa. Anim Reprod Sci. 2009;111(2-4):338-43. PMid:18583071.

Bezerra FSB, Castelo TS, Alves HM, Oliveira IRS, Lima GL, Peixoto GCX, Bezerra ACSD, Silva AR. Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen. Cryobiology. 2011;63(3):263-6. PMid:21985769.

Björndahl L, Soderlund I, Kvist U. Evaluation of the one step eosin-nigrosin staining technique for human sperm vitality assessment. Hum Reprod. 2003;18(4):813-6. PMid:12660276.

Carluccio A, Robbe D, Amicis I, Contri A, Tosi U, Russo F, Paoletti M. Artificial insemination in rabbits: laboratory and field trial with three different semen extenders. World Rabbit Sci. 2004;12:65-79.

Castellini C, Cardinali R, Dal Bosco A, Minelli A, Camici O. Lipid composition of the main fractions of rabbit semen. Theriogenology. 2006;65(4):703-12. PMid:16029885.

Chalah T, Seigneurin F, Blesbois E, Brillard JP. In Vitro comparison of fowl sperm viability in ejaculates frozen by three different techniques and relationship with subsequent fertility in vivo. Cryobiology. 1999;39(2):185-91. PMid:10529312.

Curry MR, Redding BJ, Watson PF. Determination of water permeability coefficient and its activation energy for Rabbit spermatozoa. Cryobiology. 1995;32(2):175-81. PMid:7743819.

Futino DO, Mendes MCB, Matos WNL, Mondadori RG, Lucci CM. Glycerol, Methyl-Formamide and Dimethyl-Formamide in canine semen cryopreservation. Reprod Domest Anim. 2010;45(2):214-20. PMid:19019075.

Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod. 1995;53(5):985-95. PMid:8527530.

Hagen DR, Gilkey AL, Foote RH. Spermatozoa velocity and motility and its relationship to fertility in the rabbit inseminated with low sperm numbers. World Rabbit Sci. 2010;10:135-40.

Iaffaldano N, Di Iorio M, Rosato MP. The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of Rabbit sperm: dimethylacetamide versus dimethylsulfoxide. Theriogenology. 2012;78(6):1381-9. PMid:22898020.

Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld LJ. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 1984;70(1):219-28. PMid:6694140.

Johinke D, Graaf SP, Bathgate R. Investigation of in vitro parameters and in vivo fertility of rabbit spermatozoa after chilled storage. Anim Reprod Sci. 2014;147(3-4):135-43. PMid:24852269.

Liu DY, Clarke GN, Baker HWG. Relationship between spermmotility assessed with the Hamilton-Thorne motility analyzer andfertilization rates in vitro. J Androl. 1991;12(4):231-9. PMid:1917688.

Lopes KRF, Costa LLM, Lima GL, Souza ALP, Silva AR. Dimethylformamide is no better than glycerol for cryopreservation of canine semen. Theriogenology. 2009;72(5):650-4. PMid:19541362.

López FJ, Alvariño JMR. Artificial insemination of rabbits with diluted semen stored up to 96 hours. World Rabbit Sci. 1998;6:251-3.

López FJ, Alvariño JMR. Effects of added caffeine on results following artificial insemination with fresh and refrigerated rabbit semen. Anim Reprod Sci. 2000;58(1-2):147-54. PMid:10700652.

Malo C, Gil L, Cano R, Martínez F, García A, Jerez RA. Dimethylformamide is not better that glycerol for cryopreservation of boar semen. Andrologia. 2012;44(Suppl 1):605-10. PMid:21988610.

Mocé E, Vicente JS. Rabbit sperm cryopreservation: a review. Anim Reprod Sci. 2009;110(1-2):1-24. PMid:18805659.

Mota AC Fo., Teles CH, Jucá RP, Cardoso JF, Uchoa DC, Campello CC, Silva AR, Silva LD. Dimethylformamide as a cryoprotectant for canine semen diluted and frozen in ACP-106C. Theriogenology. 2011;76(7):1367-72. PMid:21719085.

Okuda Y, Seita Y, Hisamatsu S, Sonoki S, Shino M, Masaoka T, Inomata T, Kamijo S, Kashiwazaki N. Fertility of spermatozoa cryopreserved with 2% acetamide or glycerol through artificial insemination in the Japanese white rabbit. Exp Anim. 2007;56(1):29-34. PMid:17283888.

Olaciregui M, Gil L, Montón A, Luño V, Jerez RA, Martí JI. Cryopreservation of epididymal stallion sperm. Cryobiology. 2014;68(1):91-5. PMid:24412395.

Pukazhenthi BS, Johnson A, Guthrie HD, Songsasen N, Padilla LR, Wolfe BA, Silva MC, Alvarenga MA, Wildt DE. Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii). Cryobiology. 2014;68(2):205-14. PMid:24508651.

Pursel VG, Johnson L. Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation. Theriogenology. 1974;1(2):63-8. PMid:4219847.

Roca J, Martínez S, Vázquez JM, Lucas X, Parrilla I, Martínez EA. V Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 °C. Anim Reprod Sci. 2000;64(1-2):103-12. PMid:11078971.

Rosato MP, Rebollar PG, Iaffaldano N. Comparación de diferentes diluyentes en las características cualitativas del semen de conejo durante su conservación. In: Abstracts of the XXXI Simposium de Cunicultura; 2006; Lorca, España. Lorca: ASESCU; 2006. p. 9-13.

Sarıözkan S, Özdamar S, Türk G, Cantürk F, Yay A. In vitro effects of L-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage. Cryobiology. 2014;68(3):349-53. PMid:24759298.

Sparr E, Hallin L, Markova N, Wennerström H. Phospholipid-cholesterol bilayers under osmotic stress. Biophys J. 2002;83(4):2015-25. PMid:12324420.

Trejo CA, Meza VVM, Antonio EC, Cotera RJ, Antonio-Cisneros CM. Coconut water (Cocus Nucifera) as a diluent for rabbit fresh semen in artificial insemination. Arch Zootec. 2013;62:299-302.

WHO. Laboratory manual for the examination and processing of human semen. 5th Ed. Geneva: World Health Organization; 2010. p. 26-32.

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