Trends in rabbit insemination extenders for fresh and frozen semen. A review




artificial insemination, rabbit, extender composition


Artificial insemination (AI) has become a popular technique in rabbit farms worldwide. This report discusses the progress made on semen extenders used in rabbit AI, setting out the latest innovations. Fresh and frozen semen have different requirements, so the extender composition will vary depending on the type of semen used. We discuss the endocrine supplementation of extenders for ovulation induction, the use of active molecules as an alternative to conventional antibiotics and the extenders developed for rabbit sperm cryopreservation.


Download data is not yet available.

Author Biographies

María P. Viudes-de-Castro, Instituto Valenciano de Investigaciones Agrarias (IVIA)

Centro de Investigación y Tecnología Animal (CITA)

José S. Vicente , Universitat Politècnica de València

Institute of Science and Animal Technology (ICTA)


Abdelnour S.A., Hassan M.A.E., Mohammed A.K., Alhimaidi A.R., Al-Gabri N., Al-Khaldi K.O., Swelum A.A. 2020. The effect of adding different levels of curcumin and its nanoparticles to extender on post-thaw quality of cryopreserved rabbit sperm. Animals, 10: 1508.

Abdelnour S.A., Hassan M.A.E., El-Ratel I.T., Essawi W.M., El-Raghi A.A., Lu Y., Sheiha A.M. 2022. Effect of addition of l-carnitine to cryopreservation extender on rabbit post-thaw semen parameters, antioxidant capacity, mitochondrial function, apoptosis and ultrastructure changes. Reprod. Domest. Anim., 57: 902-911.

Adams G.P., Ratto M.H., Silva M.E., Carrasco R.A. 2016 Ovulation-inducing factor (OIF/NGF) in seminal plasma: a review and update. Reprod. Dom. Anim., 51: 4-17.

Amann R.P., Waberski D. 2014. Computer-assited sperm analysis (CASA): capabilities and potential development. Theriogenology, 81: 5-17.

Bahar A., Ren D. 2013. Antimicrobial Peptides. Pharmaceuticals, 6: 1543-1575.

Bradshaw R.A., Mobley W., Rush R.A. 2017. Nerve growth factor and related substances: A brief history and an introduction to the International NGF Meeting Series. Int. J. Mol. Sci., 18: 1143.

Bromfield J.J. 2016. A role for seminal plasma in modulating pregnancy outcomes in domestic species. Reproduction, 152: R223-32.

Casares-Crespo L., Vicente J.S., Talaván A.M., Viudesde-Castro M.P. 2016a. Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?. Theriogenology, 85: 928-932.

Casares-Crespo L., Talaván A.M., Viudes-de-Castro M.P. 2016b. Can the genetic origin affect rabbit seminal plasma protein profile along the year? Reprod. Dom. Anim., 51: 294-300.

Casares-Crespo L., Fernández-Serrano P., Vicente J.S., Mocé E., Castellini C., Stabile A.M., Viudes-de-Castro M.P. 2018a. Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance. Theriogenology, 105: 61-65.

Casares-Crespo L., Fernández-Serrano P., Viudes-de-Castro M.P. 2018b. Protection of GnRH analogue by chitosandextran sulphate nanoparticles for intravaginal application in rabbit artificial insemination. Theriogenology, 116: 49-52.

Casares-Crespo L., Fernández-Serrano P., Vicente J.S., Marco-Jiménez F., Viudes-de-Castro M.P. 2018c. Rabbit seminal plasma proteome: The importance of the genetic origin. Anim. Reprod. Sci., 189: 30-42.

Casares-Crespo L., Fernández-Serrano P., Viudes-de-Castro M.P. 2019. Proteomic characterization of rabbit (Oryctolagus cuniculus) sperm from two different genotypes. Theriogenology, 128: 140-148.

Castellini C. 2008. Semen production and management of rabbit bucks. In Proc.: 9th World Rabbit Congress, 10-13 June, 2008. Verona, Italy.

Castellini C., Lattaioli P., Moroni M.M., Minelli A. 2000. Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa. Anim. Reprod. Sci., 63: 275-282.

Castellini C., Mattioli S., Cotozzolo E., Pistilli A., Rende M., Bartolini D., Di Sante G., Menchetti L., Dal Bosco A., Stabile A.M. 2022. The Effect of interaction NGF/p75NTR in sperm cells: a rabbit model. Cells, 11: 1035.

Correa A.F., Bastos I.M.D., Neves D., Kipnis A., Junqueira-Kipnis A.P., de Santana J.M. 2017. The activity of a hexameric M17 metallo-aminopeptidase is associated with survival of Mycobacterium tuberculosis. Front. Microbiol., 8: 504.

Da Costa P.M., Loureiro L., Matos A.J.F. 2013. Transfer of multidrug-resistant bacteria between intermingled ecological niches: the interface between humans, animals and the environment. Int. J. Environ. Res. Public Health, 10: 278-294.

Dal Bosco A., Rebollar P.G., Boiti C., Zerani M., Castellini C. 2011. Ovulation induction in rabbit does: Current knowledge and perspectives. Anim. Reprod. Sci., 129: 106-117.

Dal Bosco A., Cardinali R., Brecchia G., Rebollar P.G., Fatnassi M., Millán P., Mattioli S., Castellini C. 2014. Induction of ovulation in rabbits by adding lecirelin to the seminal dose: In vitro and in vivo effects of different excipients. Anim. Reprod. Sci., 150: 44-49.

Dickneite G., Schorlemmer H.U., Hofstaetter T., Sedlacek H.H. 1985. Immunostimulation as a therapeutic principle in bacterial infections: the effect of the immunomodulator bestatin on the experimental chronic E. coli urinary tract infection. In: Harzmann R., Jacobi G.H., Weissbach L. (eds) Experimentelle Urologie. Springer, Berlin, Heidelberg. p. 229-235.

Di Lorio M., Colonna M.A., Miranda M., Principe P., Schiavitto M., Cerolini S., Manchisi A., Iaffaldano N. 2018. Initial cooling time before freezing affects post-thaw quality and reproductive performance of rabbit semen. Anim. Sci. J., 89: 1240-1244.

Druart X., Rickard J.P., Mactier S., Kohnke P.L., Kershaw-Young C.M., Bathgate R., Gibb Z., Crossett B., Tsikis G., Labas V., Harichaux G., Grupen C.G., de Graaf S.P. 2013. Proteomic characterization and cross species comparison of mammalian seminal plasma. J. Proteomics, 91: 13-22.

European Food Safety Authority, European Centre for Disease Prevention and Control. 2020. The European Union Summary Report on Antimicrobial Resistance in zoonotic and indicator bacteria from humans, animals and food in 2017/2018. EFSA Journal, 18: 6007.

Fadl A.M., Ghallab A.R.M., Abou-Ahmed M.M., Moawad A.R. 2021. Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa. Reprod. Domest. Anim., 56: 103-111.

Fernández-Serrano P., Casares-Crespo L., Viudes-de-Castro M.P. 2017. Chitosan-dextran sulphate nanoparticles for GnRH release in rabbit insemination extenders. Reprod. Domest. Anim. 52: 72-74.

Finnegan S., Percival S.L. 2015. EDTA: An antimicrobial and antibiofilm agent for use in wound care. Adv. Wound Care, 4: 415-421.

Gil J., Lundeheim N., Söderquist L., Rodríguez-Martínez H. 2003. Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen. Theriogenology, 59: 1241-1255.

Gogol P. 2016. Reproductive Performance of rabbit does artificially inseminated with semen supplemented with GnRH Analogue [des-Gly10, D-Ala6]-LH-RH Ethylamide. Pol. J. Vet. Sci., 19: 659-661.

Gupta S., Gabrani R., Ali J., Dang S. 2011. Exploring Novel Approaches to Vaginal Drug Delivery. Recent Pat. Drug Deliv. Formul., 5: 82-84.

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.

Harper G.P., Glanville R.W., Thoenen H. 1982. The purification of nerve growth factor from bovine seminal plasma. Biochemical characterization and partial amino acid sequence. J. Biol. Chem., 257: 8541-8548.

Holt W.V. 2000. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology, 53: 47-58.

International Rabbit Reproduction Group. 2005. Guidelines for the handling of rabbit bucks and semen. World Rabbit Sci., 13: 71-91.

Jitendra S., Sharma P.K., Bansal S., Banik A. 2011. Noninvasive routes of proteins and peptides drug delivery. Indian J. Pharm. Sci., 73: 367-75.

Kershaw-Young C.M., Druart X., Vaughan J., Maxwell W.M. 2012. Beta-nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas. Reprod. Fertil. Dev., 24: 1093-1097.

Kuliková B., Di Iorio M., Kubovicova E., Kuzelova L., Iaffaldano N., Chrenek P. 2014. The cryoprotective effect of Ficoll on the rabbit spermatozoa quality. Zygote, 23: 785-794.

Kubovicova E., Makarevich A.V., Balazi A., Vasicek J., Chrenek P. 2022. Factors affecting rabbit sperm cryopreservation: a mini-review. Zygote; 30: 1-8.

López-Gatius F., Sances G., Sancho M., Yániz J., Santolaria P., Gutiérrez R., Núñez M., Núñez J., Soler C. 2005. Effect of solid storage at 15°C on the subsequent motility and fertility of rabbit semen. Theriogenology, 64: 252-260.

Maranesi M., Petrucci L., Leonardi L., Piro F., Rebollar P., Millán P., Cocci P., Vullo C., Parillo F., Moura A., Mariscal G., Boiti C., Zerani M. 2018. New insights on a NGF-mediated pathway to induce ovulation in rabbits (Oryctolagus cuniculus). Biol. Reprod., 98: 634-643.

Mahlapuu M., Håkansson J., Ringstad L., Björn C. 2016. Antimicrobial peptides: An emerging category of therapeutic agents. Front. Cell. Infect. Microbiol., 6: 194.

Marco-Jiménez F., Puchades S., Mocé E., Viudes-de-Castro M.P., Vicente J.S., Rodríguez M. 2004. Use of powdered egg yolk vs. fresh egg yolk for the cryopreservation of ovine semen. Reprod. Dom. Anim., 39: 438-441.

Marco-Jiménez F., Borrás S., Garcia-Dominguez X., D’Auria G., Vicente J.S., Marin C. 2020. Roles of host genetics and sperm microbiota in reproductive success in healthy rabbit. Theriogenology, 158: 416-423.

Mattioli S., Maranesi M., Castellini C., Dal Bosco A., Arias-Álvarez M., Lorenzo P.L., Rebollar, P.G., García-García R.M. 2021. Physiology and modulation factors of ovulation in rabbit reproduction management. World Rabbit Sci., 29: 221-229.

Mocé E., Vicente J.S., 2009. Rabbit sperm cryopreservation: A review. Anim. Reprod. Sci. 110: 1–24.

Mocé E., Blanch E., Talaván A.M., Viudes-de-Castro M.P. 2014. Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation. Theriogenology, 82: 1049-1053.

Mocé E., Blanch E., Talaván A.M., Viudes-de-Castro M.P. 2015. Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa. Reprod. Fertil. Develop., 27: 846-851.

Moretti E., Capitani S., Figura N., Pammolli A., Federico M.G., Giannerini V., Collodel G. 2009. The presence of bacteria species in semen and sperm quality. J. Assist. Reprod. Genet., 26: 47-56.

Muiño-Blanco T., Pérez-Pé R., Cebrián-Pérez J.A. 2008. Seminal plasma proteins and sperm resistance to stress. Reprod. Dom. Anim., 43: 18-31.

Munari C., Ponzio P., Alkhawagah A.R., Schiavone A., Mugnai C. 2019. Effects of an intravaginal GnRH analogue administration on rabbit reproductive parameters and welfare. Theriogenology, 125: 122-128.

Nagy Sz., Sinkovics Gy., Kovács A. 2002. Viability and acrosome integrity of rabbit spermatozoa processed in a gelatinsupplemented extender. Anim. Reprod. Sci., 70: 283-286.

Nishijima K., Kitajima S., Matsuhisa F., Niimi M., Wang C.C., Fan J. 2021. Strategies for highly efficient rabbit sperm cryopreservation. Animals, 11: 1220.

Ondruška L., Parkányi V., Rafay J., Chlebec I. 2008. Effect of LHRH analogue included in seminal dose on kindling rate a prolificacy of rabbits artificially inseminated. 9th World Rabbit Congress, 10-13 June, 2008. Verona, Italy.

Paiva L., Silva M., Carrasco R., Ratto M.H. 2022. The ovulatory and luteotropic actions of the male-derived beta-nerve growth factor in South American camelids. Animal Front., 12: 87-94.

Quintela L.A., Pena A.I., Vega M.D., Gullon J., Prieto M.C., Barrio M., Becerra J.J., Maseda F., Herradón P.G. 2004. Ovulation induction in rabbit does submitted to artificial insemination by adding buserelin to the seminal dose. Reprod. Nutr. Dev., 44: 79-88.

Quintela L.A., Pena A.I., Vega M.D., Gullon J., Prieto C., Barrio M., Becerra J.J., Herradón P.G. 2009. Reproductive performance of rabbit does artificially inseminated via intravaginal administration of [des-gly 10, D-Ala6]-LHRH ethylamide as ovulation inductor. Reprod. Domest. Anim., 44: 829-833.

Rabea E.I., Badawy M.E.T., Stevens C.V., Smagghe G., Steurbaut W. 2003. Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules, 4: 1457-1465.

Ratto M.H., Leduc Y.A., Valderrama X.P., van Straaten K.E., Delbaere L.T., Pierson R.A., Adams G.P. 2012. The nerve of ovulationinducing factor in semen. Proc. Natl. Acad. Sci., 109: 15042-15047.

Rebollar P.G., Dal Bosco A., Millán P., Cardinali R., Brecchia G., Sylla L., Lorenzo P.L., Castellini C. 2012. Ovulating induction methods in rabbit does: The pituitary and ovarian responses. Theriogenology, 77: 292-298.

Rodríguez-Martínez H. 2003. Laboratory semen assessment and prediction of fertility: still utopia?. Reprod. Dom. Anim., 38: 312-318.

Rodríguez-Martínez H., Kvist U., Ernerudh J., Sanz L., Calvete J.J. 2011. Seminal plasma proteins: what role do they play? Am. J. Reprod. Immunol., 66: 11-22.

Rosato M.P., Iaffaldano N. 2011. Effect of chilling temperature on the long-term survival of rabbit spermatozoa held either in a Trisbased or a jellified extender. Reprod. Dom. Anim., 46: 301-308.

Rosato M.P., 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: 508-516.

Rudramurthy G.R., Swamy M.K., Sinniah U.R., Ghasemzadeh A. 2016. Nanoparticles: Alternatives against drugresistant pathogenic microbes. Molecules, 21: 836.

Sahariah P., Másson M. 2017. Antimicrobial chitosan and chitosan derivatives: A review of the structure-activity relationship. Biomacromolecules, 18: 3846-3868.

Sánchez-Rodríguez A., Abad P., Arias-Álvarez M., Millán P., Rebollar P.G., Bautista J.M., Lorenzo P.L., García-García R.M. 2019. Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits. PLoS ONE, 14: e0223091.

Trapani A., Lopedota A., Franco M., Cioffi N., Ieva E., García-Fuentes M., Alonso M.J. 2010. A comparative study of chitosan and chitosan/cyclodextrin nanoparticles as potential carriers for the oral delivery of small peptides. Eur. J. Pharm. Biopharm., 75: 26-32.

Ubeda J.L., Ausejo R., Dahmani Y., Falceto M.V., Usan A., Malo C., Pérez-Martínez F.C. 2013. Adverse effects of members of the Enterobacteriaceae family on boar sperm quality. Theriogenology, 80: 565-570.

Vicente J.S., Lavara R., Marco-Jiménez F., Viudes-de-Castro M.P. 2008. Rabbit reproductive performance after insemination with buserelin acetate extender. Livest. Sci., 115: 153-157.

Vicente J.S., Lavara R., Marco-Jiménez F., Viudes-de-Castro M.P. 2011. Detrimental effect on availability of buserelin acetate administered in seminal doses in rabbits. Theriogenology, 76: 1120-1125.

Viudes-de-Castro M.P., Lavara R., Marco-Jiménez F., Cortell C., Vicente J.S. 2007. Ovulation induced by mucosa vaginal absorption of buserelin and triptorelin in rabbit. Theriogenology, 68: 1031-1036.

Viudes-de-Castro M.P., Mocé E., Lavara R., Marco-Jiménez F., Vicente J.S. 2014a. Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate. Theriogenology, 81: 1223-1228.

Viudes-de-Castro M.P., Lavara R., Safaa H.M., Marco-Jiménez F., Mehaisen G.M.K., Vicente J.S. 2014b. Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits. Animal 8: 765–770. s1751731114000135.

Viudes-de-Castro M.P., Casares-Crespo L., Marco-Jiménez F., Vicente J.S. 2017. Physical effect of insemination cannula on ovulation induction in rabbit doe. AIDA, XVII Jornadas sobre Producción Animal, Zaragoza, España, 380-382.

Viudes-de-Castro M.P., Marco-Jiménez F., Vicente J.S., Marin C. 2021a. Antibacterial activity of some molecules added to rabbit semen extender as alternative to antibiotics. Animals, 11: 1178.

Viudes-de-Castro M.P., Talaván A.G., Vicente J.S. 2021b. Evaluation of dextran for rabbit sperm cryopreservation: Effect on frozen-thawed rabbit sperm quality variables and reproductive performance. Anim. Reprod. Sci., 226: 106714.

Watson P.F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci., 60-61: 481-492.

Zapletal D., Pavlik A. 2008. The effect of lecirelin (GnRH) dosage on the reproductive performance of nulliparous and lactating rabbit does. Anim. Reprod. Sci., 104: 306-315.

Zhang Y.Q., Qin Y. 2012. Reproductive performance of rabbit does by adding leuprorelin in semen to induce ovulation. In Proc.: 10th World Rabbit Congress, 3-6 September, 2012. Sharm El-Sheikh, Egypt.

Zhu Z., Fan X., Lv Y., Zhang N., Fan C., Zhang P., Zeng W. 2015. Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action. PLoS ONE, 10: e0145383.

Zhu Z., Ren Z., Fan X., Pan Y., Lv S., Pan C., Lei A., Zeng W. 2017a. Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages. PLoS ONE, 12: e0181110.

Zhu Z., Fan X., Lv Y., Lin Y., Wu D., Zeng W. 2017b. Glutamine protects rabbit spermatozoa against oxidative stress via glutathione synthesis during cryopreservation. Reprod. Fertil. Dev., 29: 2183-2194.

Zhu Z., Li,R., Lv Y., Zeng W. 2019. Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress. Cryobiology, 88: 1-8.




Funding data