The honey solution effect as a natural cryoprotectant on sperm quality of botia, Chromobotia macracanthus Bleeker 1852

Abinawanto Abinawanto, Siti Z Musthofa, Retno Lestari, Anom Bowolaksono


The purpose of study was to obtain the best combination of natural cryoprotectant (honey solution) and synthetic cryoprotectant (methanol) on the quality of spermatozoa Chromobotia macracanthus Bleeker 1852, after freezing at -80 ℃ for 48 hours. The combination of 10% methanol with various concentrations of honey solution (0%, 0.1%, 0.3%, 0.5%, 0.7% and 0.9%) was tested in this study. Ringer's solution was used as an extender. The diluted sperm was then equilibrated for 25 minutes at 4 ℃, then frozen at -80 ℃ for 48 hours. The sperm was then thawed at 40 ℃ for 13 seconds. Viability, motility and percentage of fertilization were evaluated. The results of the analysis of variance showed that the combination of 10% methanol with several concentrations of honey solution had a significant effect (P <0.05) on the viability of spermatozoa and the percentage of fertilization, but had no effect on sperm motility (P> 0.05). The combination of 0.1% honey solution and 10% methanol showed the highest percentage of both motility (89.4 ± 5.45%), viability (85.75 ± 4.79%), and the percentage of fertilization (98.55 ± 1.69%). %). The conclusion of this study is that 0.1% honey solution combined with 10% methanol in Ringer's solution is the best cryoprotectant for C. macracanthus spermatozoa stored at -80 ℃ for 48 hours


botia fish; honey; methanol; preservation of spermatozoa; Ringer's solution

Full Text:



Abinawanto, Nurman K, Lestari R. 2012a. The effect of sucrose on sperm quality of Goramy fish, Osphronemus goramy Lacepede, 1801 two days post cryopreservation. Journal of Agricultural Science and Technology. B., 2(2B):204-207.

Abinawanto, Anindita I, Lestari R. 2012b. Cryopreservation of spermatozoa of Osphronemus goramy fish using skim milk. International Journal of Engineering and Innovation Technology, 2(5): 62-4.

Abinawanto, Pratiwi IA, Lestari R. 2017a. Sperm motility of giant gourami (Osphronemus goramy, Lacepede, 1801) at several con-centrations of honey combined with DMSO after short-term storage. AACL Bioflux, 10(2): 156-163.

Abinawanto A, Putri PE. 2017b. Goramy spermatozoa quality after subzero freezing: the role of coconut water as the cryopro-tectant. Cell Biology and Development, 1(1): 1-5.

Abinawanto, Rahayu S, Lestari R. 2013. Cryopreservation of Java barb (Barbonymus gonionotus) spermatozoa using egg yolk as a cryoprotectant. Global Veterinaria, 10(3): 318-321.

Abinawanto, Zuraida, Lestari R. 2016. The effect of skim milk combined with 5% of metanol on motiliy, viability, and abnormality of Java barb, Barbonymus gonionotus spermatozoa after 24 hours freezing. AACL Bioflux, 9(2): 326-333.

Afros S, Ahmed N. 2016. Effect of degraded ecosystem on fish biodiversity in the Old Brahmaputra River, Bangladesh and Its conservation measures. IOSR Journal of Environmental Science, Toxicology and Food Technology, 10(9):37-43.

Agarwal NK. 2011. Cryopreservation of fish semen. In: Bhatt JP, Thapliyal M, Thapliyal A (Editors). Himalayan Aquatic Biodiv Conservation & New Tools in Biotechnology. Transmedia Publication, Srinagar (Garhawal) Uttarakhand, India. pp. 104-127.

Anil S. 2013. Development of invitro culture and cryopreservation protocol for Zebrafish (Danio rerio) ovarian tissue fragments. PhD thesis. University of Bedfordshire. 198 p.

Anil S, Ghafari F, Zampolla T, Rawson DM, Zhang T. 2011. Studies on cryoprotectant toxicity to zebrafish (Danio rerio) ovarian tissue fragment. CryoLetters, 32(1): 40-50.

Akcay E, Bozkurt Y, Secer S, Tekin N. 2004. Cryopreservation of mirror carp semen. Turkish Journal of Veterinary and Animal Sciences, 28(5): 837-43.

Bernáth G, Bokor Z, Żarski D, Várkonyi L, Hegyi A, Staszny A, Urbányi B, Ifj JR, Horváth, A. 2016. Commercial-scale out-of-season cryopreservation of Eurasian perch (Perca fluviatilis) sperm and its application for fertilization. Animal Reproduction Science, 170:170-177.

Best BP. 2015. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Re-search, 18(5): 422-36.

Boryshpolets S, Sochorová D, Rodina M, Linhart O, Dzyuba B. 2017. Cryopreser-vation of carp (Cyprinus carpio L.) sperm: impact of seeding and freezing rates on post–thaw outputs. Biopreservation and Biobanking, 15(3): 234-40.

Bozkurt Y, Seçer S, Tekin N, Akçay E. 2005. Cryopreservation of rainbow trout (On-corhynchus mykiss) and mirror carp (Cypri-nus carpio) sperm with glucose based exten-der. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi, 1(1): 21-25.

Cabrita E, Sarasquete C, Martines-Paramo S, Robles V, Beirao J, Peres-Carezales S, Herraes P. 2010. Cryopreservation of fish sperm: Applications and perspectives. Jour-nal of Applied Ichthyology, 26(5): 623-635.

Carmichael C, Westerfield M, Varga ZM. 2009. Cryopreservation and in vitro fertilization at the Zebrafish International Resource Center. Methods in Molecular Biology 546: 45-65.

Chew C, Zulkafli AR. 2012. Sperm cryopreser-vation of some freshwater fish spesies in Malaysia. In: Igor I. Katkov (editor). Current Frontiers in Cryopreservation. Intech., 269–293.

Ciereszko A, Dietrich GJ, Nynca J, Dobosz S, Zalewski T. 2014. Cryopreservation of rain-bow trout semen using a glucosemethanol extender. Aquaculture, 420: 275-281.

Dash SN, Routray P, Dash C, Guru BC, Swain P, Sarangi N. 2008. Use of the nontoxic cryoprotectant trehalose enhances recovery and function of fish embryonic stem cells following cryogenic storage. Current Stem Cell Research & Therapy, 3(4): 277-287.

Dey A, Barat S. 2015. Spawning biology and captive breeding of vulnerable loach Botia histrionica (Blyth) in Cooch Behar, West Bengal, India. Journal of Experimental Bio-logy, 5(10): 46-48.

Dey A, Sarkar D, Barat S. 2015. Spawning biology, embryonic development and captive breeding of vulnerable loach Botia dario (Hamilton). Journal of Entomology and Zoology Studies, 3:183–188.

Figueroa E, Farias JG, Lee-Estevez M, Valdebenito I, Risopatrón J, Magnotti C, Romero J, Watanabe I, Oliveira RPS. 2018. Sperm cryopreservation with supplemen-tation of α-tocopherol and ascorbic acid in freezing media increase sperm function and fertility rate in Atlantic salmon (Salmo salar). Aquaculture, 493: 1-8.

Figueroa E, Valdebenito I, Merino O, Ubilla A, Risopatrón J, Farias JG. 2016. Cryopreser-vation of Atlantic salmon Salmo salar sperm: effects on sperm physiology. Journal of Fish Biology, 89(3):1537-1550.

Gil HW, Lee TH, Park IS. 2017. Effects of Cryoprotectants and Diluents on the Cryo-preservation of Spermatozoa from Far Eastern Catfish, Silurus asotus. Development & Reproduction, 21(1): 71.

Gupta N, Sivakumar K, Mathur VB, Chadwick MA. 2015. Terrestrial protected areas and managed reaches conserve threatened fresh-water fish in Uttarakhand, India. Parks, 21(1): 89-101.

Harvey B. 1983. Cryopreservation of Sarothe-rodon mossambicus spermatozoa. Aquacul-ture, 32(3-4): 313–20.

Harvey B, Ashwood-Smith MJ. 1982. Cryopro-tectant penetration and supercooling in the eggs of salmonid fishes. Cryobiology, 19(1): 29−40.

Hezavehei M, Mohsen S, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, Shahverdi A. 2018. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reproductive BioMedicine Online, 37(3): 327-339.

Horvath L, Miskolczi E, Urbanyi B. 2003. Cryopreservation of common carp sperm. Aquatic Living Resources, 16(5):457-460.

Horvath Á, Miskolczi E, Mihálffy S, Ősz K, Szabó K, Urbányi B. 2007. Cryopreservation of common carp (Cyprinus carpio) sperm in 1.2 and 5 ml straws and occurrence of haploids among larvae produced with cryopreserved sperm. Cryobiology, 54(3): 251-257.

Hossain Md Y, Hossen Md A, Ahmed ZF, Yahya K, Rahman Md M, Ahmed F, Ohtomi J. 2015. Threatened fishes of the world: Botia dario (Hamilton, 1822) (Cypriniformes: Cobitidae). Croatian Journal of Fisheries, 73(2): 86 – 88

Hossain Md Y, Hossen Md A, Pramanik Md NU, Nawer F, Rahman Md M, Sarmin, S, Khatun D, Bahkali AH, Ergoban AM, Yahya K. 2017. Life-History Traits of the Endangered Carp Botia dario (Cyprinidae) from the Ganges River in Northwestern Bangladesh. Pakistan Journal of Zoology, 49(3): 801-809

Hossen Md A, Hossain Md Y, Pramanik Md NU, Nawer F, Khatun D, Parvin MF, Rahman Md M. 2016. Morphological characters of Botia lohachata. Journal of Coastal Life Medicine, 4(9): 689-692

Indonesian Biodiversity Strategy and Action Plan (IBSAP). 2003. National document. National Development Planning Agency (Bappenas).: Indonesia 160 p

Islam Md A, Asif A Al, Samad Md A, Sarker B, Ahmed M, Satter AMA, Hossain AA. 2017. Comparative study on fish biodiversity with conservation measures of the Bhairabriver, Jessore, Bangladesh. Journal of Medical and Biological Research, 3(3): 357-67.

Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, Choi CW, Lee SR, Han J. 2017. Cryopreservation and its clinical applications. Integrative Medicine Research, 6(1): 12-8.

Kottelat M. 2013. The fishes of the inland waters of southeast asia: a catalogue and core bibliography of the fishes known to occur in freshwaters, mangroves and estuaries. The Raffles Bulletin Supplement Zoology, 27: 1–663

Kottelat M, Whitten AJ, Kartikasari SN, Wirjo-atmodjo S. 1996. Freshwater Fishes of Western Indonesia and Sulawesi. Periplus Ltd. Jakarta: 221 p

Kutluyer F, Kayim M, Öğretmen F, Büyükleblebici S, Tuncer PB. 2014. Cryo-preservation of rainbow trout Oncorhynchus mykiss spermatozoa: effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility. Cryobiology, 69(3): 462-466.

Lahnsteiner F, Mansour N, Weismann T. 2002. The cryopreservation of spermatozoa of the burbot, Lota lota (Gadidae, Teleostei). Cryobiology, 45(3): 195–203.

Lakra WS, Sarkar UK, Kumar RS, Pandey A, Dubey VK, Gusain OP. 2010. Fish diversity, habitat ecology and their conservation and management issues of a tropical River in Ganga basin, India. The Environmentalist, 30(4): 306-19.

Legendre M, Satyani D, Subandiyah S, Sudarto, Pouyaud L, Baras E, Slembrouck J. 2012. Biology and culture of the clown loach Chromobotia macracanthus (Cypriniformes, Cobitidae): 1-Hormonal induced breeding, unusual latency response and egg production in two populations from Sumatra and Borneo Islands. Aquatic Living Resources, 25(2): 95–108

Magyary I, Urbanyi B, Horvath L. 1996. Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization. Journal of Applied Ichthyology, 12(2):117-9.

Matthews JL, Murphy JM, Carmichael C, Yang H, Tiersch T, Westerfield M, Varga ZM. 2018. Changes to extender, cryoprotective medium, and in vitro fertilization improve zebrafish sperm cryopreservation. Zebrafish, 15(3): 279-290.

Muchlisin ZA. 2005. Current status of extenders and cryoprotectants on fish spermatozoa cryopreservation. Biodiversitas Journal of Biological Diversity. 2005; 6(1): 66-69.

Muchlisin ZA, Hashim R, Chong, AS. 2004. Preliminary study on the cryopreservation of tropical bagrid catfish (Mystus nemurus) spermatozoa; the effect of extender and cryoprotectant on the motility after short-term storage. Theriogenology, 62(1-2): 25–34.

Muchlisin ZA, Nadiah WN, Nadiya N, Fadli N, Hendri A, Khalil M, Siti-Azizah MN. 2015. Exploration of natural cryoprotectans for cryopreservation of African catfish, Clarias gariepinus, Burchell 182 (Pisces: Clariidae) spermatozoa. Chezch Journal of Animal Sciences, 60(1): 10-5.

Muchlisin ZA, Nadiya N, Nadiah WN, Musman M, Siti-Azizah MN. 2010. Preliminary study on the natural extenders for artificial breeding of African catfish Clarias gariepinus (Burchell, 1822). AACL Bioflux, 3(2):119-124.

Musthofa SZ, Wulandari R, Abinawanto A. 2018. Spawning biology and fertility of Clown Loach (Chromobotia macracanthus Bleeker 1852) in captivity. Proceeding 3rd Int Symp Curr Progress in Math and Sci. In AIP Conference Proceedings 2018. AIP Publishing LLC., 4 p.

Muthmainnah CR, Eriani K, Hasri I, Irham M, Batubara AS, Muchlisin ZA. 2018. Effect of glutathione on sperm quality after short-term cryopreservation in seurukan fish Osteo-chilus vittatus (Cyprinidae). Theriogeno-logy, 122: 30-34.

Negus MT. 2008. Salmonid sperm cryopreser-vation techniques. Minnesota Department of Natural Resources. Division of Fish and Wildlife, Section of Fisheries. 167 p.

Ng PKL, Tan HH. 1997. Freshwater fishes of Southeast Asia: potential for the aquarium fish trade and conservation issues. Aquarium Sciences and Conservation. 1997, 1(2): 79-90.

Olanrewaju AN, Kareem OK, Orisasona O. 2015. Cryopreservation: A Viable Tool for Sustainable Catfish Aquaculture Industry in Nigeria. Journal Fisheries Livestock Pro-duction, 3(149): 4 p.

Omitogun OG, Olaniyan OG, Oyeleye OF, Ojiokpota OO, Aladele C, Odofin SE, Odoflin WT. 2010. Potentials of short term and long term cryopreserved sperm of the African giant catfish (Clarias gariepinus Burchell, 1822) for aquaculture. African Journal of Biotechnology, 9(41): 6973-6982

Onrizal, Kusmana C, Saharjo BH, Handayani IP, Kato T. 2005. Social Environmental Issues of Danau Sentarum National Park, West Kalimantan. Biodiversitas, 6(3): 220-223.

Perchec G, Jeulin C, Cosson J, André F, Billard R. 1995. Relationship between sperm ATP content and motility of carp spermatozoa. Journal of Cell Science, 108(2): 747-53.

Permana A, Alimuddin, Hadi W, Priyadi A. 2015. Growth response of clown loach (Chromobotia macracanthus Bleeker 1852) juveniles immersed in water containing recombinant growth hormone. Indonesian Aquaculture Journal, 10(2): 125-130.

Putra HFE, Sugianto S, Rahardjo P, Permana A. 2019. The artificially spawning of botia Fish (Chromobotia macracanthus Bleeker) with HCG (Human Chorionic Gonadothropin) and LHRH-a (Luteinizing Hormone Releasing Hormone Analog) injection. Journal of Aquaculture and Fish Health, 6(3): 101-6.

Raghavan R, Dahanukar N, Tlusty M, Rhyne A, Kumar K, Molur S, Rosser AM. 2013 Uncovering an obscure trade: Threatened freshwater fishes and the aquarium pet markets. Biological Conservation, 164: 158-69.

Rebocho SRDMV. 2018. Development of a new ultrafast freezing procedure for zebrafish sperm cryopreservation (Doctoral disserta-tion).

Riesco MF, Oliveira C, Soares F, Gavaia PJ, Dinis MT, Cabrita E. 2017. Solea senegalensis sperm cryopreservation: New insights on sperm quality. PLoS One, 12(10): 1-19.

Robles V, Cabrita E, Cuñado S, Herráez MP. 2003. Sperm cryopreservation of sexreversed rainbow trout (Oncorhynchus mykiss): parameters that affect its ability for freezing. Aquaculture, 224(1-4): 203-212.

Sakri FM. 2015. Honey and its efficacy: Healthy supplement without side effect. 1st Print. Diandra Indonesian Library, Yogyakarta. 84 p

Satyani, D., H. Mundriyanto, S. Subandiyah, Chumaidi, Sudarto, P. Taufik, J. Slembrouck, M. Legendre & L. Pouyaud. 2006. Teknologi Pembenihan ikan hias botia (Chromobotia macracanthus Bleeker) skala laboratorium. Loka Riset Ikan Air Tawar. Depok: 19 hlm.

Sieme H, Oldenhof H, Wolkers WF. 2016. Mode of action of cryoprotectants for sperm pre-servation. Animal Reproduction Science, 169: 2-5.

Stoss J, Donaldson EM. 1983. Studies on Cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (On-chorchynchus kisutch). Aquaculture, 31(1): 51–65.

Sunarma A, Hastuti DW, Sistina Y. 2007. Combination effect of honey with different cryoprotectant on spermatozoa of the Indonesian shark minnow, Osteochillus hasseltii Valenciennes, 1842] after cryopre-servation. Proceeding Indonesian Aqua-culture Conference 2007, Surabaya, June 5–7, 2007. Indonesian Aquaculture Society, 1-9.

Szurek EA, Eroglu A. 2011. Comparison and avoidance of toxicity of penetrating cryoprotectants. PloS One, 6(11): 27604.

Tan HH, Kottelat M. 2009. The fishes of the Batang Hari drainage, Sumatra, with description of six new species. Ichthyological Exploration of Freshwaters, 20(1): 13-69.

Tekin N, Secer S, Akcay E, Bozkurt Y. 2003. Cryopreservation of rainbow trout (Oncorhynchus mykiss) semen. Israeli Journal of Aquaculture, 55(3): 208-212.

Tiersch TR. 2008. Strategies for commer-cialization of cryopreserved fish semen. Revista Brasileira de Zootecnia, 37: 1-19.

Tlusty MF, Dowd S, Raghavan PR. 2008. Saving forest through fisheries-ornamental Fisheries as means to avoid deforestation. Ornamental Fish International Journal, 56: 21-5.

Tsai S, Lin C. 2012. Advantages and applications of cryopreservation in fisheries science. Brazilian Archives of Biology and Techno-logy, 55(3): 425–34.

Ugwu SI, Kowalska A, Morita M, Kowalski RK. 2018. Application of glucosemethanol ex-tender to cryopreservation of Mozambique tilapia (Oreochromis mossambicus) sperm. Turkish Journal of Fisheries and Aquatic Sciences, 19(1): 41-50.

Viveiros ATM, So N, Komen J. 2000. Sperm cryopreservation of African catfish, Clark garieninus: Cryoprotectants, freezing, and sperm dilution ratio. Theriogenology, 54(9): 1395-l408.

Withler FC. 1982. Cryopreservation of spermatozoa of some freshwater fishes cultured in South and Southeast Asia. Aquaculture, 26(3-4): 395-398.

Yang H, Tiersch TR. 2009. Current Status of Sperm Cryopreservation in Biomedical Research Fish Models: Zebrafish, Medaka, and Xiphophorus. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 149(2): 224-232.

Article Metrics

   Abstract views: 234   PDF views or download: 13


Copyright (c) 2020 Jurnal Iktiologi Indonesia
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.


  • There are currently no refbacks.