Analyze Diet
Veterinary medicine and science2020; 6(4); 666-672; doi: 10.1002/vms3.315

Effects of cysteine and ascorbic acid in freezing extender on sperm characteristics and level of enzymes in post-thawed stallion semen.

Abstract: Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or none of them were assessed for sperm motility, viability, plasma membrane integrity, morphology and enzymes concentration. The ALP, LDH and GGT were significantly higher in 0-group compared with cysteine and ascorbic acid groups. The sperm motility of frozen-thawed semen with 0-group was significantly better compared with cysteine and ascorbic acid groups. The variation on viability, sperm membrane integrity and morphology were insignificant between all treated groups. Therefore, these enzymes were reduced when using antioxidants in the freezing extender. Results of the present study suggest that concentration of ALP, LDH and GGT enzymes could be used as parameters for prediction of frozen-thawed stallion semen.
Publication Date: 2020-06-30 PubMed ID: 32602662PubMed Central: PMC7738710DOI: 10.1002/vms3.315Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article examines the impact of adding antioxidants, specifically cysteine and ascorbic acid, to the freezing extender used in preserving stallion sperm. The researchers measured the levels of certain enzymes in the post-thawed sperm and assessed its quality by evaluating parameters such as motility, viability, and structure.

Research Methodology

  • The study involved ten ejaculate samples collected from four healthy stallions, which were then cryopreserved using a freezing substance called HF-20. The freezing extender was prepared both without the addition of antioxidants (the control group) or with the addition of either 0.5mg/ml of cysteine or ascorbic acid.
  • After the freezing and thawing process, all samples, which included those with no antioxidants (0-group), those with added cysteine, and those with added ascorbic acid, were examined. The researchers analysed sperm motility (the ability of sperm to move effectively), viability (the proportion of live sperm), plasma membrane integrity (the completeness of the cell’s outer layer), morphology (sperm structure), and the concentration of certain enzymes.

Key Findings

  • The levels of the enzymes alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyl transpeptidase (GGT) were found to be significantly higher in the control group (0-group) compared to those with added cysteine and ascorbic acid.
  • The sperm motility of the group without added antioxidants was significantly better than that of the groups with added cysteine and ascorbic acid.
  • The differences in viability, sperm membrane integrity, and morphology among all groups were found to be insignificant. This showed that the addition of the antioxidants didn’t notably alter these parameters.
  • The findings also indicated a reduction in these enzymes when using antioxidants in the freezing extender, suggesting a possible impact of cysteine and ascorbic acid on the enzymatic activity of post-thawed stallion sperm.

Conclusions

  • The study concludes that the addition of cysteine and ascorbic acid to the freezing extender used in sperm preservation might influence the enzymatic activity of thawed stallion sperm.
  • The research further suggests that the concentration of ALP, LDH, and GGT enzymes could serve as potential indicators for predicting the quality of frozen-thawed stallion semen. However, the specific effects of these antioxidants on critical parameters like sperm motility needs further research.

Cite This Article

APA
Alamaary MS, Haron AW, Hiew MWH, Ali M. (2020). Effects of cysteine and ascorbic acid in freezing extender on sperm characteristics and level of enzymes in post-thawed stallion semen. Vet Med Sci, 6(4), 666-672. https://doi.org/10.1002/vms3.315

Publication

ISSN: 2053-1095
NlmUniqueID: 101678837
Country: England
Language: English
Volume: 6
Issue: 4
Pages: 666-672

Researcher Affiliations

Alamaary, Mohaammed S
  • Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, UPM, Serdang, Malaysia.
  • King Abdulaziz Arabian Horses Center, Ministry of Environment, Water Agriculture, Riyadh, Saudi Arabia.
Haron, Abd W
  • Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, UPM, Serdang, Malaysia.
Hiew, Mark W H
  • Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, UPM, Serdang, Malaysia.
Ali, Mohamed
  • Animal Production and Breeding, Faculty of Agriculture and Veterinary Medicine, Qassim University, Qassim, Saudi Arabia.

MeSH Terms

  • Animals
  • Ascorbic Acid / pharmacology
  • Cryopreservation / veterinary
  • Cryoprotective Agents / pharmacology
  • Cysteine / pharmacology
  • Horses / physiology
  • Male
  • Semen / drug effects
  • Semen / enzymology
  • Semen Preservation / veterinary
  • Spermatozoa / drug effects
  • Spermatozoa / physiology

Conflict of Interest Statement

We declare that there is no conflict of interest in the research.

References

This article includes 38 references
  1. Agarwal A, Nallella KP, Allamaneni SS, Said TM. Role of antioxidants in treatment of male infertility: an overview of the literature.. Reprod Biomed Online 2004 Jun;8(6):616-27.
    doi: 10.1016/S1472-6483(10)61641-0pubmed: 15169573google scholar: lookup
  2. Agrawal YP, Vanha-Perttula T. Glutathione, L-glutamic acid and gamma-glutamyl transpeptidase in the bull reproductive tissues.. Int J Androl 1988 Apr;11(2):123-31.
  3. Aitken RJ. Reactive oxygen species as mediators of sperm capacitation and pathological damage.. Mol Reprod Dev 2017 Oct;84(10):1039-1052.
    doi: 10.1002/mrd.22871pubmed: 28749007google scholar: lookup
  4. Alibawi FNA A, Al‐morshidy SY, Alhuweizi AG. Ascorbic acid oxidation of thiol groups from dithiotreitol is mediated by its conversion. International Conference on Applied Life Sciences 4, 217–222.
  5. Ball BA. Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse.. Anim Reprod Sci 2008 Sep;107(3-4):257-67.
  6. Bucci D, Giaretta E, Spinaci M, Rizzato G, Isani G, Mislei B, Mari G, Tamanini C, Galeati G. Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa.. Theriogenology 2016 Jan 15;85(2):288-295.e2.
  7. Bucci D, Isani G, Giaretta E, Spinaci M, Tamanini C, Ferlizza E, Galeati G. Alkaline phosphatase in boar sperm function.. Andrology 2014 Jan;2(1):100-6.
  8. Bui AD, Sharma R, Henkel R, Agarwal A. Reactive oxygen species impact on sperm DNA and its role in male infertility.. Andrologia 2018 Oct;50(8):e13012.
    doi: 10.1111/and.13012pubmed: 29644708google scholar: lookup
  9. Ciereszko A, Glogowski J, Demianowicz W, Strzezek J. Stimulation of aspartate aminotransferase from farm animal semen by pyridoxal 5′‐phosphate. Animal Reproduction Science 34(3–4), 327–341.
  10. Dogan I, Polat U, Nur Z. Correlations between seminal plasma enzyme activities and semen parameters in seminal fluid of Arabian horses. Iranian Journal of Veterinary Research 10(2), 119–124.
  11. Du J, Cullen JJ, Buettner GR. Ascorbic acid: chemistry, biology and the treatment of cancer.. Biochim Biophys Acta 2012 Dec;1826(2):443-57.
    doi: 10.1016/j.bbcan.2012.06.003pmc: PMC3608474pubmed: 22728050google scholar: lookup
  12. Eugenia J, Maria K, Anita KS, Pietruszka A, Beata M, Dorota N. The relationship between seminal plasma aspartate aminotransferase activity, sperm osmotic resistance test value, and semen quality in boars. Acta Veterinaria 63(4), 397–404.
    doi: 10.2298/AVB1304397Jgoogle scholar: lookup
  13. Frenette G, Dubé JY, Tremblay RR. Origin of alkaline phosphatase of canine seminal plasma.. Arch Androl 1986;16(3):235-41.
    doi: 10.3109/01485018608986946pubmed: 3778020google scholar: lookup
  14. Kaneko T, Whittingham DG, Overstreet JW, Yanagimachi R. Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status.. Biol Reprod 2003 Dec;69(6):1859-62.
    doi: 10.1095/biolreprod.103.019729pubmed: 12904320google scholar: lookup
  15. Kareskoski AM, Reilas T, Sankari S, Andersson M, Güvenc K, Katila T. Alkaline and acid phosphatase, β-glucuronidase and electrolyte levels in fractionated stallion ejaculates.. Reprod Domest Anim 2010 Dec;45(6):e369-74.
  16. Katila T. In vitro evaluation of frozen-thawed stallion semen: a review.. Acta Vet Scand 2001;42(2):199-217.
    pmc: PMC2202319pubmed: 11503365doi: 10.1186/1751-0147-42-199google scholar: lookup
  17. Laudat A, Foucault P, Palluel AM. Relationship between seminal LDH-C4 and spermatozoa with acrosome anomalies.. Clin Chim Acta 1997 Sep 30;265(2):219-24.
    doi: 10.1016/S0009-8981(97)00119-8pubmed: 9385463google scholar: lookup
  18. Mocé E, Graham JK. In vitro evaluation of sperm quality.. Anim Reprod Sci 2008 Apr;105(1-2):104-18.
  19. Morielli T, O'Flaherty C. Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.. Reproduction 2015 Jan;149(1):113-23.
    doi: 10.1530/REP-14-0240pmc: PMC5489333pubmed: 25385721google scholar: lookup
  20. Murcia-Robayo RY, Jouanisson E, Beauchamp G, Diaw M. Effects of staining method and clinician experience on the evaluation of stallion sperm morphology.. Anim Reprod Sci 2018 Jan;188:165-169.
  21. Neild D, Chaves G, Flores M, Mora N, Beconi M, Agüero A. Hypoosmotic test in equine spermatozoa.. Theriogenology 1999 Mar;51(4):721-7.
    doi: 10.1016/S0093-691X(99)00021-7pubmed: 10728997google scholar: lookup
  22. Nishikawa Y. Studies on the preservation of raw and frozen horse semen.. J Reprod Fertil Suppl 1975 Oct;(23):99-104.
    pubmed: 1060880
  23. O'Flaherty CM, Beorlegui NB, Beconi MT. Lactate dehydrogenase-C4 is involved in heparin- and NADH-dependent bovine sperm capacitation.. Andrologia 2002 Apr;34(2):91-7.
  24. O'Flaherty C, Breininger E, Beorlegui N, Beconi MT. Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4.. Biochim Biophys Acta 2005 Oct 30;1726(1):96-101.
    doi: 10.1016/j.bbagen.2005.07.012pubmed: 16112812google scholar: lookup
  25. Paul BD, Sbodio JI, Snyder SH. Cysteine Metabolism in Neuronal Redox Homeostasis.. Trends Pharmacol Sci 2018 May;39(5):513-524.
    doi: 10.1016/j.tips.2018.02.007pmc: PMC5912966pubmed: 29530337google scholar: lookup
  26. Pero ME, Lombardi P, Longobardi V, Boccia L, Vassalotti G, Zicarelli L, Gasparrini B. Influence of γ‐glutamyltransferase and alkaline phosphatase activity on in vitro fertilisation of bovine frozen/thawed semen. Italian Journal of Animal Science 16(3), 390–392.
  27. Pesch S, Bergmann M, Bostedt H. Determination of some enzymes and macro- and microelements in stallion seminal plasma and their correlations to semen quality.. Theriogenology 2006 Jul 15;66(2):307-13.
  28. Purdy PH. A review on goat sperm cryopreservation. Small Ruminant 63(3), 215–225.
  29. Roychoudhury PN, Pareek PK, Gowda HC. Effect of Cold Shock on Glutamic oxaloacetic transaminase (GOT) and Glutamic pyruvic transaminase (GPT) release from bull and ram spermatozoa. Andrologia 6(4), 315–319.
  30. Seligman J, Newton GL, Fahey RC, Shalgi R, Kosower NS. Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of gamma-glutamyl-transpeptidase in sperm maturation.. J Androl 2005 Sep-Oct;26(5):629-37; discussion 638-40.
    doi: 10.2164/jandrol.05040pubmed: 16088041google scholar: lookup
  31. Stefanov R, Abadjieva D, Chervenkov M, Kistanova E, Kacheva D, Taushanova P, Georgiev B. Enzyme activities and motility of boar spermatozoa during 72‐hour lowtemperature storage. Bulgarian Journal of Veterinary Medicine 16(4), 237–242.
  32. Treulen F, Arias ME, Aguila L, Uribe P, Felmer R. Cryopreservation induces mitochondrial permeability transition in a bovine sperm model.. Cryobiology 2018 Aug;83:65-74.
  33. Tuli RK, Schmidt‐Baulain R, Holtz W. Influence of thawing temperature on viability and release of glutamic oxaloacetic transaminase in frozen semen from Boer goats. Animal Reproduction Science 25(2), 125–131.
  34. Tuli RK, Singh M, Matharoo JS. Effect of different extenders on glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) release from frozen buffalo semen.. Theriogenology 1982 Jul;18(1):55-9.
    pubmed: 16725725doi: 10.1016/0093-691x(82)90048-6google scholar: lookup
  35. Turner RM, McDonnell SM. Alkaline phosphatase in stallion semen: characterization and clinical applications.. Theriogenology 2003 Jun;60(1):1-10.
    doi: 10.1016/S0093-691X(02)00956-1pubmed: 12620574google scholar: lookup
  36. Varner DD. Approaches to Breeding Soundness Examination and Interpretation of Results. Journal of Equine Veterinary Science 43, S37–S44.
  37. Žaja IŽ, Samardžija M, Vince S, Majić-Balić I, Vilić M, Đuričić D, Milinković-Tur S. Influence of boar breeds or hybrid genetic composition on semen quality and seminal plasma biochemical variables.. Anim Reprod Sci 2016 Jan;164:169-76.
  38. Zhang W, Yi K, Chen C, Hou X, Zhou X. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa.. Anim Reprod Sci 2012 Jun;132(3-4):123-8.

Citations

This article has been cited 3 times.
  1. Salimi A, Eslami M, Farrokhi-Ardabili F. Influence of trans-ferulic acid on the quality of ram semen upon cold preservation.. Vet Med Sci 2023 May;9(3):1369-1378.
    doi: 10.1002/vms3.1117pubmed: 36913307google scholar: lookup
  2. Shehab-El-Deen M, Ali M, Al-Sharari M. Effects of Extenders Supplementation with Gum Arabic and Antioxidants on Ram Spermatozoa Quality after Cryopreservation.. Animals (Basel) 2022 Dec 28;13(1).
    doi: 10.3390/ani13010111pubmed: 36611720google scholar: lookup
  3. Hossen S, Sukhan ZP, Cho Y, Choi CY, Kho KH. Saccharides Influence Sperm Quality and Expressions of Motility and Fertilization-Associated Genes in Cryopreserved Sperm of Pacific Abalone, Haliotis discus hannai.. Front Cell Dev Biol 2022;10:935667.
    doi: 10.3389/fcell.2022.935667pubmed: 35927989google scholar: lookup