Skip to main content Accesibility Help

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

  • D. Fernández Abella (a1) (a2), M. Da Costa (a2), Y. Guérin (a3) (a4) (a5) (a6) and J. L. Dacheux (a3) (a4) (a5) (a6)

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.

Corresponding author
Hide All
Alvarez, M, Tamayo-Canul, J, Martinez-Rodriguez, C, Lopez-Uruena, E, Gomes-Alves, S, Anel, L, Martinez-Pastor, F and de Paz, P 2012. Specificity of the extender used for freezing ram sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis). Animal Reproduction Science 132, 145154.
An, TZ, Wada, S, Edashige, K, Sakurai, T and Kasai, M 1999. Viable spermatozoa can be recovered from refrigerated mice up to 7 days after death. Cryobiology 38, 2734.
Colas, G 1975. Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen. Journal of Reproduction and Fertility 42, 277285.
Dacheux, JL 1980. An ‘in vitro’ luminal perfusion technique to study epididymal secretion. IRCS Medical Science 8, 137.
Dacheux, JL and Dacheux, F 2014. New insights into epididymal function in relation to sperm maturation. Reproduction 147, R27R42.
Dacheux, JL, Belghazi, M, Lanson, Y and Dacheux, F 2006. Human epididymal secretome and proteome. Molecular and Cellular Endocrinology 250, 3642.
Dacheux, JL, Belleannee, C, Jones, R, Labas, V, Belghazi, M, Guyonnet, B, Druart, X, Gatti, JL and Dacheux, F 2009. Mammalian epididymal proteome. Molecular and Cellular Endocrinology 306, 4550.
Ehling, C, Rath, D, Struckmann, C, Frenzel, A, Schindler, L and Niemann, H 2006. Utilization of frozen-thawed epididymal ram semen to preserve genetic diversity in Scrapie susceptible sheep breeds. Theriogenology 66, 21602164.
El Amiri, B, Karen, A, Sulon, J, Melo de Sousa, N, Alvarez-Oxiley, AV, Cognie, Y, Szenci, O and Beckers, JF 2007. Measurement of ovine pregnancy-associated glycoprotein (PAG) during early pregnancy in Lacaune sheep. Reproduction in Domestic Animals 42, 257262.
Fernandez-Santos, MR, Dominguez-Rebolledo, AE, Esteso, MC, Garde, JJ and Martinez-Pastor, F 2009a. Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation. Reproduction in Domestic Animals 44, 212220.
Fernandez-Santos, MR, Martinez-Pastor, F, Matias, D, Dominguez-Rebolledo, AE, Esteso, MC, Montoro, V and Garde, JJ 2009b. Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa. Animal Reproduction Science 111, 93104.
Foote, RH 2000. Fertilizing ability of epididymal sperm from dead animals. Journal of Andrology 21, 355.
Fournier-Delpech, S, Colas, G, Courot, M, Ortavant, R and Brice, G 1979. Epididymal sperm maturation in the ram: motility, fertilizing ability and embryonic survival after uterine artificial insemination in the ewe. Annales de Biologie Animale, Biochimie, Biophysique 19, 597605.
Garcia-Macias, V, Martinez-Pastor, F, Alvarez, M, Garde, JJ, Anel, E, Anel, L and de Paz, P 2006a. Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog. Theriogenology 66, 19211930.
Garcia-Macias, V, Martinez-Pastor, F, Alvarez, M, Borragan, S, Chamorro, CA, Soler, AJ, Anel, L and de Paz, P 2006b. Seasonal changes in sperm chromatin condensation in ram (Ovis aries), Iberian red deer (Cervus elaphus hispanicus), and brown bear (Ursus arctos). Journal of Andrology 27, 837846.
Garde, JJ, Ortiz, N, Garcia, AJ, Gallego, L, Landete-Castillejos, T and Lopez, A 1998. Postmortem assessment of sperm characteristics of the red deer during the breeding season. Archives of Andrology 41, 195202.
Hall, SH, Yenugu, S, Radhakrishnan, Y, Avellar, MC, Petrusz, P and French, FS 2007. Characterization and functions of beta defensins in the epididymis. Asian Journal of Andrology 9, 453462.
Hishinuma, M, Suzuki, K and Sekine, J 2003. Recovery and cryopreservation of sika deer (Cervus nippon) spermatozoa from epididymides stored at 4 degrees C. Theriogenology 59, 813820.
Hori, T, Hagiuda, K, Kawakami, E and Tsutsui, T 2005a. Unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs. Theriogenology 63, 15731583.
Hori, T, Hagiuda, K, Endo, S, Hayama, A, Kawakami, E and Tsutsui, T 2005b. Unilateral intrauterine insemination with cryopreserved caudal epididymal sperm recovered from refrigerated canine epididymides. The Journal of Veterinary Medical Science 67, 11411147.
Jin, YZ, Bannai, S, Dacheux, F, Dacheux, JL and Okamura, N 1997. Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis. Molecular Reproduction and Development 47, 490496.
Jishage, K, Uead, O and Suzuki, H 1997. Fertility of mouse spermatozoa from cauda epididymis preserved in paraffin oil at 4°C. Journal of Mammalian Ova Research 14, 4548.
Jones, RC 1998. Evolution of the vertebrate epididymis. Journal of Reproduction and Fertility Supplement 53, 163181.
Kaabi, M, Paz, P, Alvarez, M, Anel, E, Boixo, JC, Rouissi, H, Herraez, P and Anel, L 2003. Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem. Theriogenology 60, 12491259.
Kikuchi, K, Nagai, T, Kashiwazaki, N, Ikeda, H, Noguchi, J, Shimada, A, Soloy, E and Kaneko, H 1998. Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C. Theriogenology 50, 615623.
Kishikawa, H, Tateno, H and Yanagimachi, R 1999. Fertility of mouse spermatozoa retrieved from cadavers and maintained at 4 degrees C. Journal of Reproduction and Fertility 116, 217222.
Krishnakumar, S, Whiteside, DP, Elkin, B and Thundathil, JC 2011. Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison). Theriogenology 76, 252260.
Lubicz-Nawrocki, CM, Lau, NI and Chang, MC 1973. The fertilizing life of spermatozoa in the cauda epididymidis of mice and hamsters. Journal of Reproduction and Fertility 35, 165168.
Martinez-Pastor, F, Garcia-Macias, V, Alvarez, M, Chamorro, C, Herraez, P, Paz, PD and Rodriguez, LA 2006. Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer. Theriogenology 65, 471485.
Martins, CF, Rumpf, R, Pereira, DC and Dode, MN 2007. Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production. Animal Reproduction Science 101, 326331.
Martins, CF, Driessen, K, Costa, PM, Carvalho-Neto, JO, de Sousa, RV, Rumpf, R and Dode, MN 2009. Recovery, cryopreservation and fertilization potential of bovine spermatozoa obtained from epididymides stored at 5 degrees C by different periods of time. Animal Reproduction Science 116, 5057.
Maxwell, WM and Salamon, S 1993. Liquid storage of ram semen: a review. Reproduction, Fertility, and Development 5, 613638.
McLachlan, RI 1998. The use of epididymal spermatozoa in assisted reproduction. Journal of Reproduction and Fertility Supplement 53, 277284.
Salamon, S and Maxwell, WM 2000. Storage of ram semen. Animal Reproduction Science 62, 77111.
Soler, AJ and Garde, JJ 2003. Relationship between the characteristics of epididymal red deer spermatozoa and penetrability into zona-free hamster ova. Journal of Andrology 24, 393400.
Soler, AJ, Perez-Guzman, MD and Garde, JJ 2003. Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility, viability, and morphological integrity. Journal of Experimental Zoology Part A Comparative Experimental Biology 295, 188199.
Songsasen, N, Tong, J and Leibo, SP 1998. Birth of live mice derived by in vitro fertilization with spermatozoa retrieved up to twenty-four hours after death. The Journal of Experimental Zoology 280, 189196.
Tamayo-Canul, J, Alvarez, M, Mata-Campuzano, M, Alvarez-Rodriguez, M, de Paz, P, Anel, L and Martinez-Pastor, F 2011a. Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa. Animal Reproduction Science 129, 188199.
Tamayo-Canul, J, Alvarez, M, Lopez-Uruena, E, Nicolas, M, Martinez-Pastor, F, Anel, E, Anel, L, de Paz, P 2011b. Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes. Animal Reproduction Science 126, 7682.
Tesh, JM and Glover, TD 1969. Ageing of rabbit spermatozoa in the male tract and its effect on fertility. Journal of Reproduction and Fertility 20, 287297.
Varisli, O, Uguz, C, Agca, C and Agca, Y 2009. Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures. Animal Reproduction Science 110, 256268.
Vieira, LA, Gadea, J, Garcia-Vazquez, FA, Aviles-Lopez, K and Matas, C 2013. Equine spermatozoa stored in the epididymis for up to 96h at 4 degrees C can be successfully cryopreserved and maintain their fertilization capacity. Animal Reproduction Science 136, 280288.
White, WE 1933. The duration of fertility and the histological changes in the reproductive organs after ligation of the vas efferentia in the rat. Proceedings of the Royal Society of London 113, 544553.
Young, WC 1929. A study of the function of the epididymis II. The importance of an aging process in sperm for the length of the period during which capacity is retained by sperm isolated in the epididymis of the guinea-pig. Journal of Morphology 48, 475491.
Yu, I and Leibo, SP 2002. Recovery of motile, membrane-intact spermatozoa from canine epididymides stored for 8 days at 4 degrees C. Theriogenology 57, 11791190.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 1751-7311
  • EISSN: 1751-732X
  • URL: /core/journals/animal
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed