Hostname: page-component-8448b6f56d-42gr6 Total loading time: 0 Render date: 2024-04-20T15:54:31.429Z Has data issue: false hasContentIssue false

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

Published online by Cambridge University Press:  13 November 2014

G. S. Yasui*
Laboratory of Theriogenology Dr. O. J. Ginther, Department of Veterinary Medicine – FZEA, University of Sao Paulo, Avenida Duque de Caxias Norte 225, Pirassununga, SP 13630-080, Brazil
J. A. Senhorini
National Center for Research and Conservation of Continental Fish, Chico Mendes Institute of Biodiversity Conservation, Rodovia Pref. Euberto Nemésio Pereira de Godoy, Pirassununga, SP 13630-970, Brazil
E. Shimoda
Department of Pharmacy, Cândido Mendes University, Rua Anita Peçanha 100, Campos dos Goytacazes, RJ 28030-335, Brazil
M. Pereira-Santos
Aquaculture Center, Sao Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil
L. S. O. Nakaghi
Aquaculture Center, Sao Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil
T. Fujimoto
Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, 041-8611, Hakodate, Japan
L. Arias-Rodriguez
Biological Sciences Academic Division, Juarez Autonomous University of Tabasco, C.P. 86150 Villahermosa, Tabasco, México
L. A. Silva
Laboratory of Theriogenology Dr. O. J. Ginther, Department of Veterinary Medicine – FZEA, University of Sao Paulo, Avenida Duque de Caxias Norte 225, Pirassununga, SP 13630-080, Brazil
Get access


In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel® [(D-Ala6, Pro9-NEt) – mGnRH+metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1±4.0%) or Ovopel® (79.5±5.5%) when compared with the control group treated with 0.9% NaCl (55.6±27.2%; P=0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4±7.4%) when compared with untreated male fish (42.1±26.1%; P=0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5°C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20°C when compared with fresh oocytes (P=0.0471), but considering only the stored samples, the optimum temperature was 15°C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.

Research Article
© The Animal Consortium 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)


Babiak, I and Dabrowski, K 2003. Refrigeration of rainbow trout gametes and embryos. Journal of Experimental Zoology 300, 140151.Google Scholar
Barrett, I 1951. Fertility of salmonoid eggs and sperm after storage. Journal of the Fisheries Research Board of Canada 8, 125133.Google Scholar
Bencic, DC, Krisfalusi, M, Cloud, JG and Ingermann, RL 2000. Short-term storage of salmonid sperm in air versus oxygen. North American Journal of Aquaculture 62, 1925.Google Scholar
Billard, R and Cosson, MP 1992. Some problems related to the assessment of sperm motility in freshwater fish. Journal of Experimental Zoology 261, 122131.Google Scholar
Billard, R, Cosson, J, Noveiri, SB and Pourkazemi, M 2004. Cryopreservation and short-term strorage of sturgeon sperm, a review. Aquaculture 236, 19.Google Scholar
Brown, CG and Mims, SD 1995. Storage, transportation and fertility of undiluted and diluted paddlefish milt. Progressive Fish Culturist 57, 6469.Google Scholar
Cosson, J 2004. The ionic and osmotic factors controlling motility of fish spermatozoa. Aquaculture International 12, 6985.CrossRefGoogle Scholar
Coward, K, Bromage, NR, Hibbitt, O and Parrington, J 2002. Gamete physiology, fertilization and egg activation in teleost fish. Reviews in Fish Biology and Fisheries 12, 3358.Google Scholar
Dietrich, MA, Dabrowski, K, Arslan, M, Ware, K and Van Tassell, J 2012. Quantifying quality attributes of walleye eggs prior to fertilization -- Impact of time of ovulation and gametes storage. Journal of Great Lakes Research 38, 445450.Google Scholar
Dreanno, C, Suquet, M, Desbruyères, E, Cosson, J, Le Delliou, H and Billard, R 1998. Effect of urine on semen quality in turbot (Psetta maxima). Aquaculture 169, 247262.Google Scholar
Fauvel, C, Boryshpolets, S, Cosson, J, Wilson Leedy, JG, Labbe, C, Haffray, P and Suquet, M 2012. Improvement of chilled seabass sperm conservation using a cell culture medium. Journal of Applied Ichthyology 28, 961966.Google Scholar
Fujimoto, T, Yasui, GS, Yoshikawa, H, Yamaha, E and Arai, K 2008. Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male. Journal of Applied Ichthyology 24, 430437.CrossRefGoogle Scholar
Fujimoto, T, Kataoka, T, Sakao, S, Saito, T, Yamaha, E and Arai, K 2006. Developmental stages and germ cell lineage of the loach (Misgurnus anguillicaudatus). Zoological Science 23, 977989.CrossRefGoogle ScholarPubMed
Hagedorn, M and Carter, VL 2011. Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success. PLos One 6, e21059.Google Scholar
Harvey, B and Kelley, N 1984. Short-term storage of Sarotherodon mossambicus ova. Aquaculture 37, 391395.CrossRefGoogle Scholar
He, Q, Zhao, E, Lu, Y, Yan, M, Huang, C and Dong, Q 2012. Evaluation of activation and storage conditions for sperm of yellow drum Nibea albiflora . Aquaculture 324–325, 319322.Google Scholar
Jensen, JOT and Alderdice, DF 1984. Effect of temperature on short-term storage of eggs and sperm of chum salmon (Oncorhynchus keta). Aquaculture 37, 251265.Google Scholar
Jing, R, Huang, C, Bai, C, Tanguay, R and Dong, Q 2009. Optimization of activation, collection, dilution, and storage methods for zebrafish sperm. Aquaculture 290, 165171.Google Scholar
Kurokura, H, Hirano, R, Tomita, M and Iwahashi, M 1984. Cryopreservation of carp sperm. Aquaculture 37, 267273.CrossRefGoogle Scholar
Lahnsteiner, F, Berger, B and Weismann, T 2003. Effects of media, fertilization technique, extender, straw volume, and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen. Theriogenology 60, 829841.Google Scholar
Lee, KY, Huang, H, Ju, B, Yang, Z and Lin, S 2002. Cloned zebrafish by nuclear transfer from long-term-cultured cells. Nature Biotechnology 20, 795799.Google Scholar
Lin, S, Long, W, Chen, J and Hopkins, N 1992. Production of germ-line chimeras in zebrafish by cell transplants from genetically pigmented to albino embryos. Proceedings of the National Academy of Sciences 89, 45194523.Google Scholar
Linhart, O, Gela, D, Rodina, M and Rodriguez-Guitierrez, M 2001. Short-term storage of ova of common carp and tench in extenders. Journal of Fish Biology 59, 616623.Google Scholar
Liu, TM, Yu, XM, Ye, YZ, Zhou, JF, Wang, ZW, Tong, JG and Wu, CJ 2002. Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo. Journal of Experimental Zoology 293, 719725.Google Scholar
Nam, YK, Choi, GC and Kim, DS 2004. An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis). Theriogenology 61, 933945.Google Scholar
Nguenga, D, Teugels, GG, Legendre, M and Ollevier, F 2004. Effects of storage and incubation temperature on the viability of eggs, embryos and larvae in two strains of an African catfish, Heterobranchus longifilis (Siluriformes, Clariidae). Aquaculture Research 35, 13581369.Google Scholar
Niksirat, H, Sarvi, K, Mojazi Amiri, B and Hatef, A 2007. Effects of storage duration and storage media on initial and post-eyeing mortality of stored ova of rainbow trout Oncorhynchus mykiss . Aquaculture 262, 528531.Google Scholar
Perchec-Poupard, G, Paxion, C, Cosson, J, Jeulin, C, Fierville, F and Billard, R 1998. Initiation of carp spermatozoa motility and early ATP reduction after milt contamination by urine. Aquaculture 160, 317328.Google Scholar
Piferrer, F, Beaumont, A, Falguiere, J-C, Flajshans, M, Haffray, P and Colombo, L 2009. Polyploid fish and shellfish: production, biology and applications to aquaculture for performance improvement and genetic containment. Aquaculture 293, 125156.CrossRefGoogle Scholar
Poleo, GA, Denniston, RS, Reggio, BC, Godke, RA and Tiersch, TR 2001. Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection. Biology of Reproduction 65, 961966.Google Scholar
Poon, DC and Johnson, AK 1970. The effect of delayed fertilization on transported salmon eggs. Progressive Fish Culturist 32, 8184.Google Scholar
Rizzo, E, Godinho, HP and Sato, Y 2003. Short-term storage of oocytes from the neotropical teleost fish Prochilodus marggravii . Theriogenology 60, 10591070.Google Scholar
Rothbard, S, Rubinshtein, I and Gelman, E 1996. Storage of common carp, Cyprinus carpio L., eggs for short durations. Aquaculture Research 27, 175181.Google Scholar
Sohrabnezhad, M, Kalbassi, MR, Nazari, RM and Bahmani, M 2006. Short-term storage of Persian sturgeon (Acipenser persicus) ova in artificial media and coelomic fluid. Journal of Applied Ichthyology 22, 395399.Google Scholar
Sun, YH, Chen, S-P, Wang, Y-P, Hu, W and Zhu, Z-Y 2005. Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs. Biology of Reproduction 72, 510515.Google Scholar
Suquet, M, Chereguini, O, Omnes, M-H, Rasines, I, Normant, Y, Souto, IP and Quemener, L 1999. Effect of temperature, volume of ova batches, and addition of a diluent, an antibiotic, oxygen and a protein inhibitor on short-term storage capacities of turbot, Psetta maxima, ova. Aquatic Living Resources 12, 239246.Google Scholar
Tanaka, D, Takahashi, A and Ueno, K 2009. Morphometric characteristics and reproductive capacity of nuclear transplants derived from embryonic cells of loach, Misgurnusanguillicaudatus . Journal of Experimental Zoology 311A, 1119.CrossRefGoogle Scholar
Withler, FC and Morley, RB 1968. Effects of chilled storage on viability of stored ova and sperm of sockeye and pink salmon. Journal of the Fisheries Research Board of Canada 25, 26952699.Google Scholar
Yasui, GS, Fujimoto, T and Arai, K 2010. Restoration of the loach Misgurnus anguillicaudatus from cryopreserved diploid sperm and induced androgenesis. Aquaculture 308, S140S144.Google Scholar
Yasui, GS, Arias-Rodriguez, L, Fujimoto, T and Arai, K 2008. Simple and inexpensive method for cryopreservation of fish sperm combining straw and powdered dry ice. CryoLetters 29, 383390.Google Scholar
Yasui, GS, Arias-Rodriguez, L, Fujimoto, T and Arai, K 2009. A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species. Animal Reproduction Science 116, 335345.Google Scholar
Yasui, GS, Fujimoto, T, Sakao, S, Abe, S, Yamaha, E and Arai, K 2011. Production of loach (Misgurnus anguillicaudatus) germline chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres. Journal of Animal Science 89, 23802388.Google Scholar