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Influence of Thawing Diluents on Vitality, Acrosome Morphology, Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*

Abstract

The present investigation was performed to study the effect of freezing and thawing on boar spermatozoa. Thirty-one ejaculates from four boars were investigated after thawing in three different thawing diluents (seminal plasma, OLEP, isotonic glucose solution).

From each ejaculate one sample of 1 × 109 spermatozoa was thawed in each of the thawing diluents. Each sample was examined in a thermoresistance test in which motility was stimulated with caffeine 30 min. and 3 hrs. after thawing. Furthermore, acrosome morphology and ASAT release from the spermatozoa were investigated for each sample. One ejaculate from the two most frequently used boars was examined by electron microscopy after thawing in each of the thawing diluents.

Differences in the aspects studied appeared between isotonic glucose solution and the other two thawing diluents in the thermoresistance test, in the response to caffeine stimulation 3 hrs. after thawing and in the amount of ASAT released from the spermatozoa. The influence on the acrosome morphology varied between the thawing diluents, but the acrosomal alterations did not seem to be connected with the damage reflected by the thermoresistance test and by the measurement of extracellular ASAT activity.

The ultrastructural investigation showed that all spermatozoa examined had some degree of ultrastructural alteration as compared with freshly ejaculated boar spermatozoa treated in the same way. This alteration could not be related to any of the thawing diluents.

Of the various laboratory tests the thermoresistance test and the measurement of ASAT release are suggested to be sensitive indicators of sperm damage during freezing and thawing. These tests might be useful indicators of variations in sensitivity of spermatozoa to the freezing-thawing procedure.

References

  • Andersen, K.: Morphological abnormalities in the acrosome and nucleus of boar spermatozoa. Nord. Vet.-Med. 1974, 26, 215–218.

    CAS  PubMed  Google Scholar 

  • Bane, A.: Acrosomal abnormality associated with sterility in boar. Proc. IVth Int. Congr. Anim. Reprod., The Hague 1961, 4, 81–817.

    Google Scholar 

  • Bane, A. & L. Nie ander: Electron and light microscopical studies on spermateliosis in a boar with acrosome abnormalities. J. Reprod. Fertil. 1966, 11, 133–138.

    Article  Google Scholar 

  • Boehnke, H. J., R. Hahn, K.-H. Hillman, H. Treu, W. Lorrmann and H. F. Zoder: Zur Tiefgefrierung von Ebersperma. 3 Mitteilung: Weitere Labor- und Besamungsversuche unter Verwendung von TEST und TESNaK-Verdünner sowie verschiedenen Auftau- methoden. (3, Communication: Deepfreezing of boar semen; Laboratory experiments and further field trials using TEST and TESNaK-diluent and different thawing methods). Dtsch. tierärztl. Wschr. 1974, 81, 336–339.

    CAS  Google Scholar 

  • Brown, K. I., B. G. Crabo, E. F. Graham & M. M. Pace: Some factors affecting loss of intracellular enzymes from spermatozoa. Cryobiology 1971, 8, 220–224.

    Article  CAS  Google Scholar 

  • Bryan, J. H. D.: An eosin-fast green-naphtol yellow mixture for differential staining of cytologic components in mammalian spermatozoa. Stain Technol. 1970, 45, 231–236.

    Article  CAS  Google Scholar 

  • The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology: Recommended methods for the determination of four enzymes in blood. Scand. J. clin. Lab. Invest. 1974, 33, 291–306.

    Article  Google Scholar 

  • Crabo, B. & S. Einarsson: Fertility of deep frozen boar spermatozoa. Acta vet. scand. 1971, 12, 125–127.

    CAS  PubMed  Google Scholar 

  • Crabo, B. G. & E. F. Graham: Correlation between some laboratory methods for evaluation of boar semen after freezing. Proc. VHth Int. Congr. Anim. Reprod., Munich 1972, II, 1639–1643.

    Google Scholar 

  • Crabo, B. G., R. E. Bower, K. I. Brown, E. F. Graham & M. M. Pace: Extracellular glutamic-oxaloacetic transaminase as a measure on membrane injury in spermatozoa during treatment. In Current Problems in Fertility, Plenum Press, New York 1971, 33–38.

    Chapter  Google Scholar 

  • Crabo, B. G., K. I. Brown & E. F. Graham: Effect of some buffers on the storage and freezing of boar spermatozoa. J. Animal Sei. 1972, 35, 377–382.

    Article  CAS  Google Scholar 

  • Einarsson, S. & S. Viring: Distribution of frozen-thawed spermatozoa in the reproductive tract of gilts at different time intervals after insemination. J. Reprod. Fertil. 1973, 32, 117–120.

    Article  CAS  Google Scholar 

  • Einarsson, S., O. Soosalu, T. Swensson & S. Viring: On the fertility and survival of deep frozen boar spermatozoa thawed in skim milk:. Acta vet. scand. 1972, 13, 446–448.

    Article  CAS  Google Scholar 

  • Einarsson, S., M. Holtman, O. Soosalu, T. Swensson & S. Viring: Studies on the fertility and survival of deepfrozen boar spermatozoa thawed in four different diluents. Zuchthygiene 1974, 9, 40–45.

    Article  Google Scholar 

  • Garbers, D. L., N. L. First, S. K. Gorman. & H. A. Lordy: The effects of cyclic nucleotide phosphodiesterase inhibitors on ejaculated porcine spermatozoan metabolism. Biol. Reprod. 1973, 8, 599–606.

    Article  CAS  Google Scholar 

  • Graham, E. F. & B. G. Crabo: Some factors influencing the freezing of boar spermatozoa. Proc. Vllth Int. Congr. Anim. Reprod., Munich 1972, II, 1627–1632.

    Google Scholar 

  • Graham, E. F. & M. M. Pace: Some biochemical changes in spermatozoa due to freezing. Cryobiology 1967, 4, 75–84.

    Article  CAS  Google Scholar 

  • Hancock, J. L.: The morphology of boar spermatozoa. J. roy. micr. Soc. 1957, 76, 89–97.

    Google Scholar 

  • Healey, P.: Effect of freezing on the ultrastructure of the spermatozoa of some domestic animals. J. Reprod. Fertil. 1969, 18, 21–27.

    Article  CAS  Google Scholar 

  • Hillman, K.-H. & H. Treu: Zur Tiefgefrierung von Ebersperma 1. Mitteilung: Darstellung und Messung morphologischer und enzymatischer Veränderungen beim Tief gefrieren von Eberspermien. (Deep freezing of boar semen: Demonstration and estimation of morphological and enzyme changes of deep-frozen boar semen). Zuchthygiene 1973, 8, 105–112.

    Google Scholar 

  • Holst, S. J.: Sterility in boars. Nord. Vet.-Med. 1949, 1, 87–120.

    Google Scholar 

  • Jones, R. C.: Changes occurring in the head of boar spermatozoa: Vésiculation or vacuolation of the acrosome? J. Reprod. Fertil. 1973a, 33, 113–118.

    Article  CAS  Google Scholar 

  • Jones, R. C.: The plasma membrane of ram, boar and bull spermatozoa. J. Reprod. Fertil. 1973b, 33, 179–183.

    Article  CAS  Google Scholar 

  • Larsson, K. & S. Einarsson: Fertility and post-thawing characteristics of deep frozen boar spermatozoa. Andrologia 1975, 7, 25–30.

    Article  CAS  Google Scholar 

  • Larsson, K. & S. Einarsson: Fertility of deep frozen boar spermatozoa. Influence of thawing diluents and of boars. Acta vet. scand. 1976, 17, 43–62.

    CAS  PubMed  Google Scholar 

  • Polge, C.: Artificial insemination in pigs. Vet. Rec. 1956, 68, 62–76.

    Google Scholar 

  • Pursel, V. G. & L. A. Johnson: Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing procedure. J. Animal Sci. 1975, 40, 99–102.

    Article  CAS  Google Scholar 

  • Pursel, V. G., L. A. Johnson & G. B. Rampaceck: Acrosome morphology of boar spermatozoa incubated before cold shock. J. Animal Sei. 1972, 34, 278–283.

    Article  CAS  Google Scholar 

  • Pursel, V. G., L. A. Johnson & L. L. Schulman: Effect of dilution seminal plasma and incubation period on cold shock susceptibility of boar spermatozoa. J. Animal Sci. 1973, 37, 528–531.

    Article  CAS  Google Scholar 

  • Romeny, E., K.-H. Hillman & L. Richter: Zur Tiefgefrierung von Eber- samen. 4. Mitteilung: Labor- und Besamungsversuche mit dem Hülsenberg Verdünner IV. (Deep freezing of boar semen. 4. Communication: Laboratory experiments and inseminations with frozen boar semen using the extender Hülsenberg IV). Dtsch. tierärztl. Wschr. 1974, 81, 353–354.

    CAS  Google Scholar 

  • Snedecor, G. W.: Statistical Methods. Ames, Iowa 1966, 5th. Ed.

  • Westendorf, P., L. Richter & H. Treu: Zur Tiefgefrierung von Eber- sperma: Labor- und Besamungsergebnisse mit dem Hülsenberger Pailletten-Verfahren. (Deep freezing of boar semen: Laboratory findings and insemination results with the “Hülsenberger Pailletten” technique). Dtsch. tierärztl. Wschr. 1975, 82, 261–267.

    CAS  Google Scholar 

  • Wilmut, I. & C. Polge: The fertilizing capacity of boar semen stored in the presence of glycerol at 20, 5 and —79°C. J. Reprod. Fertil. 1974, 38, 105–113.

    Article  CAS  Google Scholar 

  • Yasuda, Y. & I. Tanimura: Scanning electron microscopy of boar spermatozoa before and after frozen-thawed treatment. Int. J. Fertil. 1974, 19, 149–158.

    CAS  PubMed  Google Scholar 

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Larsson, K., Einarsson, S. & Nicander, L. Influence of Thawing Diluents on Vitality, Acrosome Morphology, Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*. Acta Vet Scand 17, 83–100 (1976). https://doi.org/10.1186/BF03547945

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