Effect of Shilajit on Freezing Rooster Semen

Abstract

Reactive oxygen species (ROS) are created in excess during the cryopreservation process, which speeds up the rate of lipid peroxidation (LPO). This negatively impacts spermatozoa functions and reduces their capacity to fertilize. The spermatozoon plasma membrane consists of significant amounts of polyunsaturated fatty acids (PUFA), which can be easily oxidized by ROS and produce harmful agents that are toxic to cells. The plasma membrane of rooster spermatozoa contains high concentrations of polyunsaturated fatty acids and very small amounts of mitochondria, cytoplasm, and cytoplasmic antioxidants. Cryopreservation of rooster semen has been associated with adverse effects, including increased lipid peroxidation, structural damage in the mitochondria and acrosomal area, changes in the integrity and permeability of the spermatozoon plasma membrane, and severe damage to DNA. In the study, semen taken from 20 Plymouth Rock roosters were pooled to eliminate individual differences. By adding 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL and 25 μg/mL shilajit to Beltsville Poultry Semen Extender diluent, 5 experimental and 1 control groups were formed and frozen in 0.25 mL straws. After thawing in a water bath at 37oC, spermatologic parameters were analyzed with the CASA system. Viability evaluations were made with eosin – nigrosin stain and morphological evaluations were made with Hancock method. Sperm DNA integrity was examined with the COMET assay. As a result, it was concluded that the addition of 10, 15, 20 μg/mL shilajit to rooster semen extender improves semen quality parameters and DNA integrity of semen after cryopreservation.

Keywords

• Cryopreservation
• Rooster
• Shilajit
• Sperm

Horoz Spermasının Dondurulmasında Shilajit'in Etkisi

Abstract

Reaktif oksijen türleri (ROS), kriyoprezervasyon sürecinde aşırı miktarda oluşmakta ve lipid peroksidasyonu (LPO) hızını artırmaktadır. Bu durum, spermatozoa fonksiyonlarını olumsuz etkilemekte ve fertilizasyon yeteneğini azaltmaktadır. Spermatozoon plazma membranı, önemli miktarda çoklu doymamış yağ asidi (PUFA) içermekte ve ROS tarafından kolayca oksitlenebilmekte ve hücrelere toksik olan zararlı maddeler üretebilmektedirler. Horoz spermatozoon plazma membranı, yüksek konsantrasyonlarda çoklu doymamış yağ asitleri ve çok az miktarda mitokondri, sitoplazma ve sitoplazmik antioksidan içermektedir. Horoz spermasının kriyoprezervasyonu, artmış lipid peroksidasyonu, mitokondri ve akrozomal bölgede yapısal hasar, spermatozoon plazma membranının bütünlüğü ve geçirgenliğinde değişiklikler ve DNA'da ciddi hasar gibi olumsuz etkilerle ilişkilendirilmiştir. Çalışmada, 20 Plymouth Rock ırkı horozdan alınan sperma bireysel farklılıkları ortadan kaldırmak için bir araya getirildi. Beltsville Poultry Semen Extender (BPSE) Sulandırıcısına 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL ve 25 μg/mL miktarlarda shilajit eklenerek 5 deney ve 1 kontrol grubu oluşturuldu ve 0.25 mL payetler içerisinde donduruldu. 37°C su banyosunda çözdürüldükten sonra spermatolojik parametreler CASA sistemi ile belirlendi. Spermatozoa canlılık oranı eosin – nigrosin boyama metodu ile, morfolojik değerlendirmeler ise Hancock yöntemi ile yapıldı. Spermatozoonn DNA bütünlüğü COMET analiz yöntemi ile değerlendirildi. Sonuç olarak, horoz sperma sulandırıcısına 10, 15, 20 μg/mL shilajit eklenmesinin, kriyoprezervasyon sonrası spermatolojik parametreleri ve DNA bütünlüğünü olumlu yönde etkilediği sonucuna varıldı.

Keywords

• kriyoprezervasyon
• horoz
• shilajit
• sperma

References

1. Alvarez, J. G., & Storey, B. T. (1992). Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. Journal of andrology, 13(3), 232-241. https://doi.org/https://doi.org/10.1002/j.1939-4640.1992.tb00306.x Bilodeau, J. F., Chatterjee, S., Sirard, M. A., & Gagnon, C. (2000). Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development: Incorporating Gamete Research, 55(3), 282-288.
2. Blesbois, E., Grasseau, I., & Seigneurin, F. (2005). Membrane fluidity and the ability of domestic bird spermatozoa to survive cryopreservation. Reproduction, 129(3), 371-378.
3. Chatterjee, S., de Lamirande, E., & Gagnon, C. (2001). Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Molecular Reproduction and Development: Incorporating Gamete Research, 60(4), 498-506.
4. Fraser, L., & Strzezek, J. (2004). The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C. Folia Histochemica et Cytobiologica, 42(1), 49-55. Ghosal, S. (1990). Chemistry of shilajit, an immunomodulatory Ayurvedic rasayan. Pure and Applied Chemistry, 62(7), 1285-1288.
5. Gliozzi, T., Zaniboni, L., Iaffaldano, N., & Cerolini, S. (2017). Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders. British poultry science, 58(5), 578-584.
6. Gliozzi, T. M., Zaniboni, L., & Cerolini, S. (2011, 2011/06/01/). DNA fragmentation in chicken spermatozoa during cryopreservation. Theriogenology, 75(9), 1613-1622. https://doi.org/https://doi.org/10.1016/j.theriogenology.2011.01.001
7. Gundogan, M., Yeni, D., Avdatek, F., & Fidan, A. (2010). Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage. Animal reproduction science, 122(3-4), 200-207.
8. Halliwell, B., & Gutteridge, J. M. (2015). Free radicals in biology and medicine. Oxford university press, USA. Hughes, C. M., Lewis, S. E., McKelvey-Martin, V. J., & Thompson, W. (1996). A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay. MHR: Basic science of reproductive medicine, 2(8), 613-619.

9. Hughes, C. M., Lewis, S. E., McKelvey-Martin, V. J., & Thompson, W. (1997). Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 374(2), 261-268.
10. Joyce, D. A. (1987). Oxygen radicals in disease. Adverse Drug Reaction Bulletin, 127(1), 476-479.
11. Kathiravan, P., Kalatharan, J., Karthikeya, G., Rengarajan, K., & Kadirvel, G. (2011). Objective sperm motion analysis to assess dairy bull fertility using computer‐aided system–a review. Reproduction in Domestic Animals, 46(1), 165-172.
12. Klaude, M., Eriksson, S., Nygren, J., & Ahnström, G. (1996). The comet assay: mechanisms and technical considerations. Mutation Research/DNA Repair, 363(2), 89-96.
13. Kong, Y., But, P., Ng, K., Cheng, K., Cambie, R., & Malla, S. (1987). Chemical studies on a nepalese panacea-shilajit (I). International Journal of Crude Drug Research, 25(3), 179-182.
14. Long, J. (2006). Avian semen cryopreservation: what are the biological challenges? Poultry science, 85(2), 232-236.
15. Lotfi, S., Mehri, M., Sharafi, M., & Masoudi, R. (2017). Hyaluronic acid improves frozen-thawed sperm quality and fertility potential in rooster. Animal reproduction science, 184, 204-210.
16. Maneesh, M., Jayalekshmi, H., Dutta, S., Chakrabarti, A., & Vasudevan, D. (2005). Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats.
17. Masoudi, R., Sharafi, M., Pourazadi, L., Davachi, N. D., Asadzadeh, N., Esmaeilkhanian, S., & Dirandeh, E. (2020). Supplementation of chilling storage medium with glutathione protects rooster sperm quality. Cryobiology, 92, 260-262.
18. McKelvey-Martin, V., Melia, N., Walsh, I., Johnston, S., Hughes, C., Lewis, S., & Thompson, W. (1997). Two potential clinical applications of the alkaline single-cell gel electrophoresis assay:(1) human bladder washings and transitional cell carcinoma of the bladder; and (2) human sperm and male infertility. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 375(2), 93-104.
19. Mehdipour, M., Daghigh Kia, H., Najafi, A., Mohammadi, H., & Álvarez-Rodriguez, M. (2020). Effect of crocin and naringenin supplementation in cryopreservation medium on post-thaw rooster sperm quality and expression of apoptosis associated genes. Plos one, 15(10), e0241105.
20. Najafi, A., Kia, H. D., & Hamishehkar, H. (2021). Does alpha-lipoic acid–loaded nanostructured lipid carriers improve post-thawed sperm quality and ameliorate apoptosis-related genes of rooster sperm? Poultry science, 100(1), 357-365.
21. Najafi, A., Kia, H. D., Mehdipour, M., Hamishehkar, H., & Álvarez-Rodríguez, M. (2020). Effect of quercetin loaded liposomes or nanostructured lipid carrier (NLC) on post-thawed sperm quality and fertility of rooster sperm. Theriogenology, 152, 122-128.
22. Najafi, A., Taheri, R. A., Mehdipour, M., Martínez-Pastor, F., Rouhollahi, A. A., & Nourani, M. R. (2019). Improvement of post-thawed sperm quality in broiler breeder roosters by ellagic acid-loaded liposomes. Poultry science, 98(1), 440-446.
23. Najafi, D., Taheri, R. A., Najafi, A., Shamsollahi, M., & Alvarez-Rodriguez, M. (2020). Effect of astaxanthin nanoparticles in protecting the post-thawing quality of rooster sperm challenged by cadmium administration. Poultry science, 99(3), 1678-1686.
24. Nandre, R. (2007). Effect of preservation of spermatozoa at sub-zero temperature on DNA integrity by comet assay
25. Ommati, M., Zamiri, M., Akhlaghi, A., Atashi, H., Jafarzadeh, M., Rezvani, M., & Saemi, F. (2013). Seminal characteristics, sperm fatty acids, and blood biochemical attributes in breeder roosters orally administered with sage (Salvia officinalis) extract. Animal production science, 53(6), 548-554.
26. Partyka, A., Łukaszewicz, E., & Niżański, W. (2012). Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen. Theriogenology, 77(8), 1497-1504.
27. Partyka, A., Niżański, W., & Łukaszewicz, E. (2010). Evaluation of fresh and frozen-thawed fowl semen by flow cytometry. Theriogenology, 74(6), 1019-1027.
28. Polge, C. (1951). Functional survival of fowl spermatozoa after freezing at− 79° C. Nature, 167(4258), 949-950.
29. Rezaie, F. S., Hezavehei, M., Sharafi, M., & Shahverdi, A. (2021). Improving the post-thaw quality of rooster semen using the extender supplemented with resveratrol. Poultry science, 100(9), 101290.
30. Safa, S., Moghaddam, G., Jozani, R. J., Kia, H. D., & Janmohammadi, H. (2016). Effect of vitamin E and selenium nanoparticles on post-thaw variables and oxidative status of rooster semen. Animal reproduction science, 174, 100-106.
31. Schäfer, S., & Holzmann, A. (2000). The use of transmigration and Spermac™ stain to evaluate epididymal cat spermatozoa. Animal reproduction science, 59(3-4), 201-211.
32. Shanmugam, M., Kannaki, T., & Vinoth, A. (2016). Comparison of semen variables, sperm DNA damage and sperm membrane proteins in two male layer breeder lines. Animal reproduction science, 172, 131-136. Sharma, R. K., & Agarwal, A. (1996). Role of reactive oxygen species in male infertility. Urology, 48(6), 835-850.
33. Siari, S., Mehri, M., & Sharafi, M. (2022). Supplementation of Beltsville extender with quercetin improves the quality of frozen-thawed rooster semen. British poultry science, 63(2), 252-260.
34. Sikka, S. C., Rajasekaran, M., & Hellstrom, W. J. (1995). Role of oxidative stress and antioxidants in male infertility. Journal of andrology, 16(6), 464-468.
35. Singh, N. P., McCoy, M. T., Tice, R. R., & Schneider, E. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell research, 175(1), 184-191.
36. Singh, N. P., Muller, C. H., & Berger, R. E. (2003). Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertility and sterility, 80(6), 1420-1430.
37. Srivastava, R. S., Kumar, Y., Singh, S. K., & Ghosal, S. (1988). PC68 SHILAJIT: ITS SOURCE AND ACTIVE PRINCIPLES. International Symposium on the Chemistry of Natural Products, 1988, 524. https://doi.org/10.24496/intnaturalprod.1988.0_524
38. Sultan, J., Awan, M. A., Rakha, B. A., Waqar, S. A., Ansari, M. S., Naz, S., Iqbal, S., & Akhter, S. (2021). Asphaltum Improves the Post-thaw Quality and Antioxidant Status of Nili Ravi Buffalo Bull Sperm. Biopreservation and Biobanking, 19(3), 194-203.
39. Tarif, A., Bhuiyan, M. M. U., Ferdousy, R. N., Juyena, N. S., & Mollah, M. B. R. (2013). Evaluation of semen quality among four chicken lines. IOSR J. Agric. Vet. Sci, 6(5), 7-13.
40. Twigg, J., Fulton, N., Gomez, E., Irvine, D. S., & Aitken, R. J. (1998). Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Human reproduction (Oxford, England), 13(6), 1429-1436.
41. Wang, A. W., Zhang, H., Ikemoto, I., Anderson, D. J., & Loughlin, K. R. (1997). Reactive oxygen species generation by seminal cells during cryopreservation. Urology, 49(6), 921-925.
42. Xiao, Y., Wu, Z., & Wang, M. (2018). Effects of fulvic acids on goat sperm. Zygote, 26(3), 220-223.