Semen characteristics and optimisation of sperm cryopreservation protocol of a commercially important minor carp Labeo gonius (Hamilton, 1822)

Hayin  Tamut; Raghvendra Singh; Santosh Kumar; Aditya Kumar; Monika Gupta; Achal Singh; Chandrabhushan  S. Nishad; Rama Shankar Sah; Ajay Kumar  Singh; Arvind Kumar; Kuldeep K. Lal

doi:10.21077/ijf.2025.72.1.152786-15

Authors

Hayin Tamut
Raghvendra Singh ICAR-National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh.
Santosh Kumar
Aditya Kumar
Monika Gupta
Achal Singh
Chandrabhushan S. Nishad
Rama Shankar Sah
Ajay Kumar Singh
Arvind Kumar
Kuldeep K. Lal

https://doi.org/10.21077/ijf.2025.72.1.152786-15

Keywords:

Labeo gonius, sperm cryopreservation, DMSO, ex-situ conservation

Abstract

This study aimed to develop a suitable protocol for the semen cryopreservation of a commercially important minor carp Labeo gonius, to aid in seed production and ex-situ conservation efforts. Semen characteristics were analysed, revealing a pH ranging between 7.4-7.7, an average osmolality of 276±15 mOsmol kg-1 and sperm counts ranging from 25.4×109 to 34.2×109 cells ml-1. Nine extenders were screened and three (8, 9B and 9C) were selected based on their high sperm activation (over 70% with water as activator) and sperm motility time (>40 s) after Principle Component Analysis (PCA). Cryopreservation involved a milt to diluent ratio of 1:6, with 10% DMSO as a cryoprotectant. Diluted milt was loaded into 0.25 ml straws, equilibrated and stored in liquid nitrogen. The selected extenders (8, 9B and 9C) demonstrated hatching rates of 61.1±0.23, 81.3±0.19 and 83.7±2.7%, respectively and survival rates of 44.6±1.2, 45.0±2.64 and 46.7±1.52%, respectively. The control group using fresh sperm had hatching and survival rates of 84.99±0.88 and 48.6±1.52%, respectively. Extenders 9C and 9B, containing 10% DMSO and sugar, showed significantly higher hatching and survival rates than extender 8. However, there were no significant differences in hatching or survival rates between cryopreserved sperm and the control group, except in extender 8. The results clearly indicated that, extenders 8, 9B and 9C supplemented with 10% DMSO can be recommended for semen cryopreservation of L. gonius, contributing to ex-situ conservation and quality seed production.

Keywords: DMSO, Ex-situ conservation, Labeo gonius, Sperm cryopreservation

Downloads

Download data is not yet available.

Author Biographies

Raghvendra Singh, ICAR-National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh.

Scientist, Aquaculture
Monika Gupta

Scientist

ICAR-National Bureau of Fish Genetic Resources, Lucknow - 226 002, Uttar Pradesh, India
Kuldeep K. Lal

Director

ICAR-Central Institute of Brackishwater Aquaculture, Chennai - 600 028, Tamil Nadu, India

References

Afroz, H. and Begum, M. 2014. Analysis of nutritional value and mineral contents of three different parts of body of Cirrhinus reba. International Journal of Scientific and Engineering Research, 5: 2301-2306.

Agarwal, N. K., Saini, V. and Raghuvanshi, S. K. 2013. Characterization and short-term storage of semen of a coldwater Himalayan fish species. Biojournal, 8(1):1-8.

Ahammad, M. M., Bhattacharyya, D. and Jana, B. B. 2003. Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures. Cryobiology, 46(1):2-16. https://doi.org/10.1016/S0011-2240(02)00138-4.

Alavi, S. M. H. and Cosson, J. 2005. Sperm motility in fishes. I. Effects of temperature and pH: a review. Cellular Biology International, 29:101–110. https://doi.org/10.1016/j.cellbi.2004.11.021.

Bozkurt, Y., Yavaş, I. and Yıldız, C. 2016. Effect of extender supplemented with different sugar types on post-thaw motility, viability and fertilizing ability of cryopreserved common carp (Cyprinus carpio) spermatozoa. The Israeli Journal of Aquaculture-Bamidgeh, 68:1-8 https://doi.org/10.46989/001c.20789.

Cabrita, E., Sarasquete, C., Martinez-Paramo, S., Robles, V., Beirao, J., Perez-Cerezales, S. and Herraez, M.P. 2010. Cryopreservation of fish sperm: applications and perspectives. Journal of Applied Ichthyology, 26(5):623-635.http://dx.doi.org/10.1111%2Fj.1439-0426.2010.01556.x.

CAMP., 1998. Report of the workshop on “Conservation assessment and management plan (CAMP) for freshwater fishes of India”. Zoo outreach organization and NBFGR, Lucknow, 156:22-26.

Chondar, S.L. 1999. Biology of Finfish and Shellfish. SCSC Publishers, India, 36: 47-53.

Coser, A. M., Godinho, H. and Ribeiro, D. 1984. Cryogenic preservation of spermatozoa from Prochilodus scrofa and Salminus maxillosus. Aquaculture, 37(4):387-390. https://doi.org/10.1016/0044-8486(84)90303-X.

Das, P., Saharan, N., Pal, A.K., Sahu, N.P., Prakash, C. and Tiwari, V.K. 2010. Labeo gonius (Hamilton, 1822): A potential candidate species for diversification of Indian aquaculture. Aquaculture Europe, 35(3): 3-16.

Das, P.C. and Mishra, B.I. 2016. Multi-species farming of major and minor carps for enhancing fish production in freshwater aquaculture. Indian Journal of Fisheries. 63(2): 55-61.

Diwan, A.D., Ayyappan, S., Lal, K.K. and Lakra, W.S. 2010. Cryopreservation of fish gametes and embryos. The Indian Journal of Animal Sciences, 80(4):109-124. http://krishi.icar.gov.in/jspui/handle/123456789/4990

Durbin, H., Durbin, F.J. and Stott, B. 1982. A note on the cryopreservation of grass carp milt. Aquaculture Research, 13(3):115-117. https://doi.org/10.1111/j.1365 2109.1982.tb00039.x.

FAO. 2014. The State of World Fisheries & Aquaculture. Food and Agriculture Organization of the United Nations. Rome, Italy, 243.

Gupta, S.D., Rath, S.C. and Dasgupta, S. 1995. Fertilization efficiency of cryopreserved carp spermatozoa over four years at different time intervals after thawing. Geobios, 22:208–211.

Huang, Y., Cartlidge, R. E., Walpitagama, M., Kaslin, J., Campana, O. and Wlodkowic, D. 2018. Unsuitable use of DMSO for assessing behavioural end points in aquatic model species. Science of the Total Environment, 615:107–114. https://doi.org/10.1016/j.scitotenv.2017.09.260.

ICAR-NBFGR (2020). Annual Report 2020, ICAR-National Bureau of Fish Genetic Resources, Lucknow - 226002 (U.P.), India.

Jena, J. and Das, P.C. 2011. Grow‐out performance of Kuria labeo, Labeo gonius (Hamilton), with major carps in carp polyculture system. Aquaculture Research, 42(9):1332-1338. https://doi.org/10.1111/j.1365-2109.2010.02721.x.

Jena, J., Das, P.C., Mitra, G., Patro, B., Mohanta, D. and Mishra, B. 2011. Evaluation of growth performance of Labeo fimbriatus (Bloch), Labeo gonius (Hamilton) and Puntius gonionotus (Bleeker) in polyculture with Labeo rohita (Hamilton) during fingerlings rearing at varied densities. Aquaculture, 319(3-4):493-496. https://doi.org/10.1016/j.aquaculture.2011.07.028.

Kalita, D., Dutta, M. and Islam, N. F. 2005. Few plants and animals based folk medicines from Dibrugarh District, Assam. Council of Scientific & Industrial Research, 4(1):81-85. http://nopr.niscpr.res.in/handle/123456789/8488.

Kumar S., Kumar, A., Sah, R. S., Singh, A. K., Yadav, A., Verma, A., Negi, D., Tamut, H. and Lal, K. K., 2023. Optimisation of cryovials and milt dilution ratios to upscale carp sperm cryopreservation for use in hatchery seed production, Indian J. Fish., 70 (3): 90-97. doi:10.21077/ijf.2023.70.3.134401-12.

Kumar, K. 1988. A comparative study of various extenders for cryopreservation of carp spermatozoa. Indian Journal of Animal Sciences. 58(11):1355-1360.

Kurokura, H., Hirano, R., Tomita, M. and Iwahashi, M. 1984. Cryopreservation of carp sperm. Aquaculture, 37(3):267-273. https://doi.org/10.1016/0044-8486(84)90159-5.

Lahnsteiner, F., Berger, B., Horvath, A., Urbanyi, B. and Weismann, T. 2000. Cryopreservation of spermatozoa in cyprinid fishes. Theriogenology, 54(9):1477-1498. https://doi.org/10.1016/S0093-691X(00)00469-6.

Lahnsteiner, F., Weismann, T. and Patzner, R. A. 1997. Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes. Aquaculture Research, 28(6):471-479. https://doi.org/10.2331/suisan.73.1049.

Lakra, W. S. and Krishna, G. 1997. Preliminary trials for cryopreservation of spermatozoa of selected carps and catfishes. Indian Journal of Animal Sciences, 67(1): 90-92.

Mohanta, K. N., Subramanian, S., Komarpant, N. and Saurabh, S. 2008. Alternate carp species for diversification in freshwater aquaculture in India. Aquaculture Asia, 13 (1):11-15. http://hdl.handle.net/1834/19018.

Morisawa, M. 1985. Initiation mechanism of sperm motility at spawning in teleosts. Zoological Science, 2: 605–615.

Nahiduzzaman, M., Hassan, M. M., Khanam, U. H., Mamun, S. N. A., Hossain, M. A. R. and Tiersch, T. R. 2011. Sperm cryopreservation of the critically endangered olive barb (Sarputi) Puntius sarana (Hamilton, 1822). Cryobiology, 62:62-67. https://doi.org/10.1016/j.cryobiol.2010.12.004.

Noor, M. N., Sarder, M. R. I., Al Islam, A. N. A. S., Jahan, D. A. and Islam, M. M. 2018. Development of sperm cryopreservation technique of minor carp, bata (Labeo bata Ham. 1822) for ex-situ conservation. Bangladesh Journal of Fisheries, 30(2):121-133. https://www.researchgate.net/publication/330618740_Development_of_sperm_cryopreservation_technique_of_minor_carp_bata_Labeo_bata_Ham_1822_for_ex-situ_conservation.

Ott, A. G. and Horton, H. F. 1971. Fertilization of steelhead trout (Salmo gairdneri) eggs with cryo-preserved sperm. Journal of the Fisheries Board of Canada, 28(12):1915-1918. https://doi.org/10.1139/f71-289.

Pegg, D.E. 2007. Principles of cryopreservation. In Cryopreservation and freeze-drying protocols. Humana Press. Totowa, New Jersey, 368: 39-57. https://doi.org/10.1007/978-1-59745-362-2_3.

Piironen, J. 1993. Cryopreservation of sperm from the brown trout (Salmo trutta m. lacustris L.) and Arctic charr (Salvelinus alpinus L.) Aquaculture, 116:275-285. https://doi.org/10.1016/0044-8486(93)90014-P.

Pioli, P. D. 2019. Protocol: Hemocytometer cell counting. https://www.med.wmich.edu/sites/default/files/Hemacytometer_Cell_Counting.pdf.

Ponniah, A. G., Lal, K. K., Gopalakrishnan, A. and Srivastava, S. K. 1998. Use of fertilisation protocols to enhance hatching percentage with cryopreserved milt of Cyprinus carpio. National Academy Science Letters, 21(8): 256-260.

Rao, B., Soufir, J. C., Martin, M. and David, G. 1989. Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility. Gamete research, 24(2):127-134. https://doi.org/10.1002/mrd.1120240202.

Routray, P., Choudhary, A. K., Dash, S. N., Verma, D. K., Dash, C., Swain, P. and Sarangi, N. 2006. Cryopreservation of dead fish spermatozoa several hours after death of Indian major carp, Labeo rohita and its successful utilization in fish production. Aquaculture, 261(4): 1204-1211. https://doi.org/10.1016/j.aquaculture.2006.09.029.

Sahu, A. D., Bhol, C., Routray, P. and Sundaray, J. K. 2011. Cryopreservation of male gonad of carps, Labeo calbasu and Labeo rohita for long term storage and its utilization in aquaculture. Indian Journal of Science and Technology, 4(1): 308–309. http://dx.doi.org/10.17485/ijst/2011/v4iS8/31120.

Sharma, A., Ram, R. N. and Prasad, S., 2017. Cryopreservation of sperm in common carp (Cyprinus carpio) and its strain Amur carp (Cyprinus carpio haematopterus): Sperm motility and hatching success of embryos. Indian Journal of Hill Farming, Special, 32(1): 49-53. http://dx.doi.org/10.1006/cryo.2000.2284

Stein, H. and Bayrle, H. 1978. Cryopreservation of the sperm of some freshwater teleosts. In Annales de Biologie Animale Biochimie Biophysique, 18(4): 1073-1076. EDP Sciences. https://eurekamag.com/research/020/795/020795253.php.

Tekin, N., Secer, S., Akcay, E. and Bozkurt, Y. 2003. Cryopreservation of rainbow trout (Oncorhynchus mykiss) semen. The Israeli Journal of Aquaculture Bamidgeh, 55(3):208-212. https://doi.org /10.46989/001c.20345.

Tiersch, T. R. and Green, C. C. 2011. Cryopreservation in aquatic species. World Aquaculture Society, Baton Rouge, LA, 7(1):852-861.

Tsai, S. and Lin, C. 2012. Advantages and applications of cryopreservation in fisheries science. Brazilian archives of biology and technology, 55(3):425-434. https://doi.org/10.1590/S1516-89132012000300014.

Várkonyi, L., Bokor, Z., Molnar, J., Fodor, F., Szari, Z., Ferincz, A. and Bernath, G. 2019. The comparison of two different extenders for the improvement of large‐scale sperm cryopreservation in common carp (Cyprinus carpio). Reproduction in Domestic Animals, 54(3): 639-645. https://doi.org/10.1111/rda.13383.

Verma, D. K., Routray, P., Dash, C., Dasgupta, S., & Jena, J. K. (2009). Physical and biochemical characteristics of semen and ultrastructure of spermatozoa in six carp species. Turkish Journal of Fisheries and Aquatic Sciences, 9(1):67-76 https://dergipark.org.tr/en/pub/trjfas-ayrildi/issue/13280/160481>.

Vuthiphandchai, V., Wilairattanadilok, K., Chomphuthawach, S., Sooksawat, T. and Nimrat, S. 2015. Sperm cryopreservation of silver barb (Barbodes gonionotus): cryoprotectants, cooling rate and storage time on sperm quality. Aquaculture research, 46(10):2443-2451. http://dx.doi.org/10.1111%2Fare.12396.

Warnecke, D. and Pluta, H. J. 2003. Motility and fertilizing capacity of frozen/thawed common carp (Cyprinus carpio L.) sperm using dimethyl-acetamide as the main cryoprotectant. Aquaculture, 215(1-4):167-185. https://doi.org/10.1016/S0044-8486(02)00371-X.

Withler, F. C. 1980. Chilled and cryogenic storage of gametes of Thai carps and catfishes. Canadian Technical Reports Fisheries Aquatic Science. 948:1-15.

Withler, F. C. and Lim, L. C. 1982. Preliminary observations of chilled and deep-frozen storage of grouper (Epinephelus tauvina) sperm. Aquaculture, 27(4): 389-392. https://waves-vagues.dfo-mpo.gc.ca/library-bibliotheque/12906.pdf.

Yang, H., Carmichael, C., Varga, Z. M., Tiersch, T. R. 2007. Development of a simplified and standardized protocol with potential for high-though put for sperm cryopreservation in Zebrafish (Danio rerio). Theriogenology, 68:128-136.