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. 1999 Mar 1;40(1):1–9. doi: 10.1186/BF03547036

Sperm Viability in Ram Semen Diluted and Stored in Three Different Extenders

Spermieviabilitet hos baggsperma spädd i tre olika spädningsvätskor

A López 1,2, L Söderquist 1,✉, H Rodriguez-Martinez 1
PMCID: PMC8043155  PMID: 10418191

Abstract

Semen was collected with an artificial vagina from 4 one-year-old rams, in order to study the changes in sperm motility and membrane integrity of spermatozoa split-diluted and stored at 5 °C during 7 days in sodium citrate, Tris, and milk-based extenders, respectively. Sperm motility was assessed subjectively and sperm membrane integrity was determined using the fluorescent probes Calcein-AM and Ethidium homodimer. Representative samples were studied using scanning electron microscopy (SEM). The average incidence of sperm motility decreased over time in all the extenders (p<.001). The incidence of spermatozoa showing progressive motility and intact plasma membrane was significantly higher in semen diluted with sodium citrate than in the other 2 extenders following 4 days of dilution until the end of the study. Evaluation with SEM confirmed the findings obtained with the supra vital fluorescent dyes. The results of the present study indicated that there were no differences between sodium citrate-, Tris- or milk-based extenders when ovine liquid semen was stored at 5 °C during a short period (2 days). However, when semen was stored for longer time, spermatozoa in the sodium citrate-based extender sustained its viability better.

Keywords: liquid, cooled, membrane integrity, motility, storage, sodium citrate, Tris

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References

  1. Althouse GC, Hopkins SM. Assessment of boar sperm viability using a combination of two fluorophores. Theriogenology. 1995;43:595–603. doi: 10.1016/0093-691X(94)00065-3. [DOI] [PubMed] [Google Scholar]
  2. Amann RP, Hay SR, Hammerstedt RH. Yield, characteristics, motility and cAMP content of sperm isolated from seven regions of ram epididymis. Biol. Reprod. 1982;27:723–733. doi: 10.1095/biolreprod27.3.723. [DOI] [PubMed] [Google Scholar]
  3. Blackshaw AW. The effects of milk diluents on the viability of ram spermatozoa and their revival after freezing. Aust. Vet. J. 1960;36:432–435. doi: 10.1111/j.1751-0813.1960.tb03729.x. [DOI] [Google Scholar]
  4. Dacheux JL, Paquignon M: Effects of caffeine on ram and boar spermatozoa: influence of their stage of maturation and the medium; initiation of progressive motility of testicular spermatozoa. In: Testicular development, structure and function, 1980. Raven Press, New York, USA.
  5. Eriksson B, Rodriguez-Martinez H: Assessment of membrane damage in frozen/thawed boar spermatozoa. Reprod. Dom. Anim. 1996, 31, 285– 286.
  6. Garner DL, Pinkel D, Johnson LA, Cooke YD. Assessment of spermatozoa function using dual fluorescent staining and flow cytometric analyses. Biol. Reprod. 1986;34:127–138. doi: 10.1095/biolreprod34.1.127. [DOI] [PubMed] [Google Scholar]
  7. Harrison RAP, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fert. 1990;88:343–352. doi: 10.1530/jrf.0.0880343. [DOI] [PubMed] [Google Scholar]
  8. Januskauskas A: Studies on the assessment of postthaw sperm viability in Swedish dairy A.I. bulls. 1995; Thesis, Uppsala. 28pp.
  9. Januskauskas A, Rodriguez-Martinez H. Assessment of sperm viability by measurement of ATP, membrane integrity and motility in frozen/thawed bull semen. Acta vet. scand. 1995;36:571–574. doi: 10.1186/BF03547671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson LA, Maxwell WMC, Dobrinsky JR, Welch GR. Staining sperm for viability assessment. Reprod. Dom. Anim. 1996;31:37–47. doi: 10.1111/j.1439-0531.1995.tb00004.x. [DOI] [Google Scholar]
  11. Langford GA, Fiser PS: Influence of storage temperature and duration of storage on the fertilizing capacity of extended ram semen. J. Anim. Sci. 1980, 51 (Suppl. 1), 295.
  12. Maxwell WMC, Salamon S. Liquid Storage of ram semen: a review. Reprod. Fertil. Dev. 1993;5:613–638. doi: 10.1071/RD9930613. [DOI] [PubMed] [Google Scholar]
  13. Maxwell WMC, Stojanov T. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod. Fertil. Dev. 1996;8:1013–1020. doi: 10.1071/RD9961013. [DOI] [PubMed] [Google Scholar]
  14. Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 1996;42:55–65. doi: 10.1016/0378-4320(96)01544-8. [DOI] [Google Scholar]
  15. Rodriguez-Martinez H, Larsson B, Pertoft H. Evaluation of sperm damage and techniques for sperm clean-up. Reprod. Fertil. Dev. 1997;9:297–308. doi: 10.1071/R96081. [DOI] [PubMed] [Google Scholar]
  16. Salamon S. Studies on the artificial insemination of Merino sheep. III The effect of frequent ejaculation on semen characteristics and fertilising capacity. Aust. J. Agric. Res. 1962;13:1137–1150. [Google Scholar]
  17. Salamon S, Maxwell WMC, Firth JH. Fertility of ram semen after storage at 5 °C. Anim. Reprod. Sci. 1979;2:373–385. doi: 10.1016/0378-4320(79)90024-1. [DOI] [Google Scholar]
  18. Salamon S, Maxwell WMC: Frozen storage of ram semen. I Processing, freezing, thawing and fertility after cervical insemination. Anim. Reprod. Sci. 1995a, 37, 185–249.
  19. Salamon S, Maxwell WMC: Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim. Reprod. Sci. 1995b, 38, 1–36.
  20. Salamon S, Robinson TJ. Studies on the artificial insemination of Merino sheep. II. The effects of semen diluents and storage on lambing performance. Aust. J. Agric. Res. 1962;13:271–281. [Google Scholar]
  21. Schindler H, Amir D. Longevity of ram sperm in various diluents and at different dilution rates. J. Agric. Sci. 1961;56:183–189. doi: 10.1017/S002185960002462X. [DOI] [Google Scholar]
  22. Statistical Analysis System Institute: SAS/STAT Guide for Personal Computers, Version 6 ed., 1993; SAS Institute, Cary, NC.
  23. Söderquist L, Madrid-Bury N, Rodriguez-Martinez H. Assessment of ram sperm membrane integrity following different thawing procedures frozen in mini straws. Theriogenology. 1997;48:1115–1125. doi: 10.1016/S0093-691X(97)00344-0. [DOI] [PubMed] [Google Scholar]
  24. Vishwanath R, Shannon P. Do sperm cells age? A review of the physiological changes in sperm during storage at ambient temperature. Reprod. Fertil. Dev. 1997;9:321–331. doi: 10.1071/R96088. [DOI] [PubMed] [Google Scholar]
  25. Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 1995;7:871–891. doi: 10.1071/RD9950871. [DOI] [PubMed] [Google Scholar]
  26. White IG. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod. Fertil. Dev. 1993;5:639–658. doi: 10.1071/RD9930639. [DOI] [PubMed] [Google Scholar]
  27. Woelders H: Overview of in vitro methods for evaluation of semen quality. Reprod. Dom. Anim. 1991; Suppl 1 (Johnson, L A & Rath, D, eds), pp. 145–164, Paul Parey, Hamburg.

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