Common carp (Cyprinus carpio) is the fourth most-produced fish species in aquaculture and frequently used model species with significant effort invested in development of biotechnological applications. In present study, we attempted to establish an in vitro germ cell culture condition for short term cell culture, which could facilitate further applications such as surrogacy or gene manipulation. Basal media and different types of feeder cells were investigated to optimize carp germ cell culture condition to favor maintenance of mitotic proliferation. Results indicated that germ cells cultured with hESC media and RTG2 cell line as feeder possessed significantly higher proliferation and survival rate compared to that cultured with StemPro media and Sertoli cell line as feeder. In addition, we compared two dissection strategies to compare risk of cell culture contamination and body cavity was open from dorsal part or from ventral part. As a result, carp open from the dorsal side can minimize the risk of contamination. In summary, this is the first study to optimize the cultivation of germ cells in common carp. This opens up new opportunities for the application of specific techniques in the breeding of those species with high commercial value and frequent use as a model fish. Results obtained in this study are important for implementation of new strategies in common carp breeding, conservation of genetic resources, restoration of lines or development of clonal and isogenic carp lines.

Department of Genetics The Silberman Institute The Hebrew University of Jerusalem Jerusalem Israel

Department of Gynecology and Obstetrics Xijing Hospital of Airforce Military Medical University Xi'an China

Department of Neurosurgery Tangdu Hospital of 4th Military Medical University Xi'an China

Research Institute of Fish Culture and Hydrobiology South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in Ceské Budějovice Ceské Budějovice Czechia

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Hulata G. A review of genetic-improvement of the common carp (Cyprinus carpio L.) and other cyprinids by crossbreeding. hybridization and selection. Aquaculture. (1995) 129:143–55. 10.1016/0044-8486(94)00244-I DOI

Kocour M, Mauger S, Rodina M, Gela D, Linhart O, Vandeputte M, et al. . Heritability estimates for processing and quality traits in common carp (Cyprinus carpio L.) using a molecular pedigree. Aquaculture. (2007) 270:43–50. 10.1016/j.aquaculture.2007.03.001 DOI

Prchal M, Bugeon J, Vandeputte M, Kause A, Vergnet A, Zhao J, et al. . Potential for genetic improvement of the main slaughter yields in common carp with in vivo morphological predictors. Front Genet. (2018) 9:283. 10.3389/fgene.2018.00283 PubMed DOI PMC

Flajšhans M, Linhart O, Šlechtová V, Šlechta V. Genetic resources of commercially important fish species in the Czech Republic: present state and future strategy. Aquaculture. (1999) 173:471–83. 10.1016/S0044-8486(98)00477-3 DOI

Flajšhans M, Kocour M, Rodina M, Boryshpolets S, Kašpar V, Hulák M, et al. . On-farm conservation of fish genetic resources and testing of performance traits. In. World Aquaculture Society Conference AQUA 2012. Prague: Prague Congress Centre. (2012). p. 356.

Linhart O, Rodina M, Cosson J. Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos. Cryobiology. (2000) 41:241–50. 10.1006/cryo.2000.2284 PubMed DOI

Horváth A, Miskolczi E, Mihálffy S, Osz K, Szabó K, Urbányi B, et al. . 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. (2007) 54:251–57. 10.1016/j.cryobiol.2007.02.003 PubMed DOI

Franěk R, Tichopád T, Steinbach C, Xie X, Lujić J, Marinović Z, et al. . Preservation of female genetic resources of common carp through oogonial stem cell manipulation. Cryobiology. (2019) 87:78–85. 10.1016/j.cryobiol.2019.01.016 PubMed DOI

Franěk R, Kašpar V, Shah MA, Gela D, Pšenička M. Production of common carp donor-derived offspring from goldfish surrogates. Aquaculture. (2021) 534:736252. 10.1016/j.aquaculture.2020.736252 DOI

Anil S, Rawson D, Zhang T. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in-vitro culture in cryopreservation studies. Cryobiology. (2018) 83:75–83. 10.1016/j.cryobiol.2018.05.004 PubMed DOI

Tsai S, Rawson DM, Zhang T. Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies. Theriogenology. (2010) 74:290–303. 10.1016/j.theriogenology.2010.02.013 PubMed DOI

Okutsu T, Suzuki K, Takeuchi Y, Takeuchi T, Yoshizaki G. Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proc Natl Acad Sci U S A. (2006) 103:2725–9. 10.1073/pnas.0509218103 PubMed DOI PMC

Jin YH, Robledo D, Hickey JM, McGrew MJ. Surrogate broodstock to enhance biotechnology research and applications in aquaculture. Biotechnol Adv. (2021) 49:107756. 10.1016/j.biotechadv.2021.107756 PubMed DOI PMC

Sakai N. In vitro male germ cell cultures of zebrafish. Methods. (2006) 39:239–45. 10.1016/j.ymeth.2005.12.008 PubMed DOI

Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, et al. . Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci USA. (2004) 101:8011–6. 10.1073/pnas.0308668101 PubMed DOI PMC

Shikina S, Yoshizaki G. Improved in vitro culture conditions to enhance the survival, mitotic activity, and transplantability of rainbow trout type A spermatogonia. Biol Reprod. (2010) 83:268–76. 10.1095/biolreprod.109.082123 PubMed DOI

Xie X, Li P, Pšenička M, Ye H, Steinbach C, Li C. Optimization of in vitro culture conditions of sturgeon germ cells for purpose of surrogate production. Animals. (2019) 9:106. 10.3390/ani9030106 PubMed DOI PMC

Iwasaki-Takahashi Y, Shikina S, Watanabe M, Banba A, Yagisawa M, Takahashi K, et al. . Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout. Commun Biol. (2020) 3:308. 10.1038/s42003-020-1025-y PubMed DOI PMC

Su S, Jing X, Zhang C, Hou Y, Li Z, Yang X, et al. . Interaction between the intestinal microbial community and transcriptome profile in common carp (Cyprinus Carpio L). Front Microbiol. (2021) 12:659602. 10.3389/fmicb.2021.659602 PubMed DOI PMC

Schulz RW, de França LR, Lareyre JJ, Le Gac F, Chiarini-Garcia H, Nobrega RH. Spermatogenesis in Fish. Gen Comp Endocrinol. (2010) 165:390–411. 10.1016/j.ygcen.2009.02.013 PubMed DOI

Skaar KS, Nobrega RH, Magaraki A, Olsen LC, Schulz RW, Male R, et al. . Proteolytically activated, recombinant anti-müllerian hormone inhibits androgen secretion, proliferation, and differentiation of spermatogonia in adult zebrafish testis organ cultures. Endocrinology. (2011) 152:3527–40. 10.1210/en.2010-1469 PubMed DOI

Miura T, Chiemi M, Kohei Y. Spermatogenesis in the Japanese Eel. In: Eel Biology. London: Springer. (2003). p. 319–29.

Ohta H, Yomogida K, Dohmae K, Nishimune Y. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-Kit and its ligand SCF. Development. (2000) 27:2125–131. 10.1242/dev.127.10.2125 PubMed DOI

Vincent S, Segretain D, Nishikawa S, Nishikawa SI, Sage J, Cuzin F, et al. . Stage-Specific expression of the kit receptor and its ligand (KL) during Male gametogenesis in the mouse: a kit-KL interaction critical for meiosis. Development. (1998) 125:4585–93. 10.1242/dev.125.22.4585 PubMed DOI

Conrad S, Hossein A, Maryam H, Mikael K, Michael B, Jörg H, Markus R. Differential gene expression profiling of enriched human spermatogonia after short- and long-term culture. editor, Irma Virant-Klun. BioMed Res International. Histon: The Company of Biologists. (2014). p. 138350. PubMed PMC

França Luiz R, Rafael H, Nóbrega Roberto DVS, Morais Luiz H, De Castro A, Rüdiger W, et al. . Sertoli cell structure and function in anamniote vertebrates. In: Sertoli Cell Biology. Amsterdam: Elsevier. (2015). p. 385–407.

Meng X, Lindahl M, Hyvönen ME, Parvinen M, de Rooij DG, Hess MW, et al. . Regulation of Cell Fate Decision of Undifferentiated Spermatogonia by GDNF. Science. (2000) 287:1489–93. 10.1126/science.287.5457.1489 PubMed DOI

Nakajima S, Hayashi M, Kouguchi T, Yamaguchi K, Miwa M, Yoshizaki G. Expression patterns of Gdnf and Gfrα1 in rainbow trout testis. Gene Expr Patterns. (2014) 14:111–20. 10.1016/j.gep.2014.01.006 PubMed DOI