Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

Instituto de Oceanografía y Cambio Global IOCAG Universidad de Las Palmas de Gran Canaria Las Palmas de Gran Canaria Spain

Nishiura Station South Ehime Fisheries Research Center Ehime University Matsuyama Japan

Research Institute of Fish Culture and Hydrobiology South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses University of South Bohemia Ceske Budejovice Ceské Budějovice Czechia

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Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol Reprod. (1976) 15:467–70. 10.1095/biolreprod15.4.467 PubMed DOI

Goto K, Kinoshita A, Takuma Y, Ogawa K. Fertilisation of bovine oocytes by the injection of immobilised, killed spermatozoa. Vet Rec. (1990) 127:517–20. PubMed

Cochran R, Meintjes M, Reggio B, Hylan D, Carter J, Pinto C, et al. . Live foals produced from sperm-injected oocytes derived from pregnant mares. J Equine Vet Sci. (1997) 18:736–40. 10.1016/S0737-0806(98)80504-2 DOI

Kolbe T, Holtz W. Birth of a piglet derived from an oocyte fertilized by intracytoplasmic sperm injection (ICSI). Anim Reprod Sci. (2000) 64:97–101. 10.1016/S0378-4320(00)00204-9 PubMed DOI

Catt SL, Gomez MC, Maxwell WMC, Evans G, Catt JW. Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm. Vet Rec. (1996) 139:494–95. 10.1136/vr.139.20.494 PubMed DOI

Deng M, Yang X. Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection. Mol Reprod Dev. (2001) 59:38–43. 10.1002/mrd.1005 PubMed DOI

Otani S, Iwai T, Nakahata S, Sakai C, Yamashita M. Artificial Fertilization by Intracytoplasmic Sperm Injection in a Teleost Fish, the Medaka (Oryzias latipes). Biol Reprod. (2009) 80:175–83. 10.1095/biolreprod.108.069880 PubMed DOI

Parmar MS, Pant C, Karuppanasamy K, Mili B, Upadhyay D, Kant V, et al. . Intracytiplasmic Sperm Injection (ICSI) and its applications in veterinary sciences: an overview. Sci Int. (2013) 8:266–70. 10.17311/sciintl.2013.266.270 DOI

Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil Steril. (1995) 63:343–9. 10.1016/S0015-0282(16)57366-X PubMed DOI

Sato M, Ochi T, Nakase T, Hirota S, Kitamura Y, Nomura S, et al. . Mechanical tension-stress induces expression of bone morphogenetic proteins BMP-2 and BMP-4, but not BMP-6, BMP-Z and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res. (1999) 14:1084–10. 10.1359/jbmr.1999.14.7.1084 PubMed DOI

Markoulaki S, Kurokawa M, Yoon SY, Matson S, Ducibella T, Fissore R, et al. . Comparison of Ca2+ and CaMKII responses in IVF and ICSI in the mouse. Mol Hum Reprod. (2007) 13:265–72. 10.1093/molehr/gal121 PubMed DOI

Swann K. The role of Ca2+ in oocyte activation during in vitro fertilization: Insights into potential therapies for rescuing failed fertilization. Biochim Biophys Acta Mol Cell Res. (2018) 1865:1830–7. 10.1016/j.bbamcr.2018.05.003 PubMed DOI

Caperton L, Murphey P, Yamazaki Y, McMahan CA, Walter CA, Yanagimachi R, et al. . Assisted reproductive technologies do not alter mutation frequency or spectrum. PNAS. (2007) 104:5085–90. 10.1073/pnas.0611642104 PubMed DOI PMC

Palermo G, Joris H, Devroey P, Van Steirteghem A. Pregnancies after intracytoplasmatic injection of single spermatozoon into an oocyte. Lancet. (1992) 340:17–8. 10.1016/0140-6736(92)92425-F PubMed DOI

Palermo GD, O'Neill CL, Chow S, Cheung S, Parrella A, Pereira N, et al. . Intracytoplasmic sperm injection: state of the art in humans. Reproduction. (2017) 154:F93–F110. 10.1530/REP-17-0374 PubMed DOI PMC

Salamone DF, Canel NG, Rodríguez MB. Intracytoplasmic sperm injection in domestic and wild mammals. Reproduction. (2017) 154:F111–24. 10.1530/REP-17-0357 PubMed DOI

Sansinena MJ, Taylor SA, Taylor PJ, Schmidt EE, Denniston RS, Godke RA, et al. . In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: Effect of chemical activation treatments and culture conditions. Anim Reprod Sci. (2007) 99:342–53. 10.1016/j.anireprosci.2006.05.020 PubMed DOI

O'Brien JK, Crichton EG, Evans KM, Schenk JL, Stojanov T, Evans G, et al. . Sex ratio modification using sperm sorting and assisted reproductive technology—a population management strategy. In ‘The 2nd International Symposium of Assisted Reproductive Technologies for the Conservation and Genetic Management of Wildlife, Omaha, Nebraska. Omaha, NE: Henry Doorly Zoo; (2002). p. 224–31.

Poleo GA, Godke RA, Tiersch TR. Intracytoplasmic sperm injection using cryopreserved, fixed, and freeze-dried sperm in eggs of Nile tilapia. Mar Biotechnol. (2005) 7:104–11. 10.1007/s10126-004-0162-5 PubMed DOI

Ringleb J, Waurich R, Wibbelt G, Streich W, Jewgenow K. Prolonged storage of epididymal spermatozoa does not affect their capacity to fertilise in vitro-matured domestic cat (Felis catus) oocytes when using ICSI. Reprod Fert Dev. (2011) 23:818–25. 10.1071/RD10192 PubMed DOI

Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction. (2011) 142:529–38. 10.1530/REP-11-0145 PubMed DOI

Anzalone DA, Palazzese L, Iuso D, Martino G, Loi P. Freeze-dried spermatozoa: an alternative biobanking option for endangered species. Anim Reprod Sci. (2018) 190:85–93. 10.1016/j.anireprosci.2018.01.010 PubMed DOI

Fernández-González L, Hribal R, Stagegaard J, Zahmel J, Jewgenow K. Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection. Theriogenology. (2015) 83:995–9. 10.1016/j.theriogenology.2014.11.037 PubMed DOI

Kishikawa H, Tateno H, Yanagimachi R. Fertility of mouse spermatozoa retrieved from cadavers and maintained at 4 degrees C. J Reprod Fertil. (1999) 116:217–22. 10.1530/jrf.0.1160217 PubMed DOI

Wildt DE, Howard JG, Hall LL, Bush M. The reproductive physiology of the clouded leopard. I Electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. Biol Reprod. (1986) 34:937–47. 10.1095/biolreprod34.5.937 PubMed DOI

Roth TL, Howard JG, Donoghue AM, Swanson WF, Wildt DE. Function and culture requirements of snow leopard (Panthera unciaI) spermatozoa in vitro. J Reprod Fertil. (1994) 101:563–9. 10.1530/jrf.0.1010563 PubMed DOI

Lillie FR. Studies of fertilization. VI The mechanism of fertilization in arbacia. J Exp Zool. (1914) 16:523–90. 10.1002/jez.1400160404 DOI

Hiramoto Y. Microinjection of the live spermatozoa into sea urchin eggs. Exp Cell Res. (1962) 27:416–26. 10.1016/0014-4827(62)90006-X PubMed DOI

Tesarik J. Fertilization of oocytes by injecting spermatozoa, spermatids and spermatocytes. Rev Reprod. (1996) 1:149–52. 10.1530/ror.0.0010149 PubMed DOI

Kroll KL, Amaya E. Transgenic Xenopus embryos form sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development. (1996) 122:3173–83. 10.1242/dev.122.10.3173 PubMed DOI

Sparrow DB, Latinkic B, Mohun TJ. A simplified method of generating transgenic Xenopus. Nucleic Acids Res. (2000) 28:E12–E12. 10.1093/nar/28.4.e12 PubMed DOI PMC

Moro LN, Sestelo AJ, Salamone DF. Evaluation of cheetah and leopard spermatozoa developmental capability after interspecific ICSI with domestic cat oocytes. Reprod Domest Anim. (2014) 49:693–700. 10.1111/rda.12355 PubMed DOI

Yamashita M, Onozato H, Nakanishi T, Nagahama Y. Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii. Dev Biol. (1990) 137:155–60. 10.1016/0012-1606(90)90016-C PubMed DOI

Poleo GA, Denniston RS, Reggio BC, Godke RA, Tiersch TR. Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection. Biol Reprod. (2001) 65:961–6. 10.1095/biolreprod65.3.961 PubMed DOI

Yasui GS, Saito T, Zhao Y, Fujimoto T, Yamaha E, Arai K, et al. . Intra-ooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae). Zygote. (2018) 26:408–16. 10.1017/S0967199418000448 PubMed DOI

Liu T, Liu L, Wei Q, Hong T. Sperm nuclear transfer and transgenic production in the fish medaka. Int J Biol Sci. (2011) 7:469–75. 10.7150/ijbs.7.469 PubMed DOI PMC

The I. U. C. N . IUCN Red List of Threatened Species. Version 2022-1, (2022). Available online at: http://www.iucnredlist.org (accessed September 05, 2022).

Fatira E, Havelka M, Labbé C, Depincé A, Iegorova V, Pšenička M, et al. . Application of interspecific Somatic Cell Nuclear Transfer (iSCNT) in sturgeons and an unexpectedly produced gynogenetic sterlet with homozygous quadruple haploid. Sci Rep. (2018) 8:5997. 10.1038/s41598-018-24376-1 PubMed DOI PMC

Fatira E, Havelka M, Labbé C, Depincé A, Pšenička M, Saito T, et al. . A newly developed cloning technique in sturgeons; an important step towards recovering endangered species. Sci Rep. (2019) 9:10453. 10.1038/s41598-019-46892-4 PubMed DOI PMC

Psenicka M, Kaspar V, Alavi SMH, Rodina M, Gela D, Li P, et al. . Potential role of the acrosome of sturgeon spermatozoa in the fertilization process. J Appl Ichthyol. (2011) 27:678–82. 10.1111/j.1439-0426.2010.01642.x DOI

Ginsburg AS, Dettlaff TA. The Russian Sturgeon Acipenser gueldenstaedtii. Part I. gametes and early development up to time of hatching In: Animal Species for Developmental Studies. editors, Dettlaff TA, Vassetzky G. Boston, MA: Springer; (1991) p. 15–66.

Iegorova V, Pšenička M, Lebeda I, Rodina M, Saito T. Polyspermy produces viable haploid/diploid mosaics in sturgeon. Biol Reprod. (2018) 99:695–706. 10.1093/biolre/ioy092 PubMed DOI PMC

Glogowski J, Kolman R, Szczepkowski M, Horvath A, Urbanyi B, Sieczynski P, et al. . Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol. Aquaculture. (2002) 211:367–73. 10.1016/S0044-8486(02)00003-0 DOI

Havelka M, Fujimoto T, Hagihara S, Adachi S, Arai K. Nuclear DNA markers for identification of Beluga and Sterlet sturgeons and their interspecific Bester hybrid. Sci Rep. (2017) 7:1694. 10.1038/s41598-017-01768-3 PubMed DOI PMC

Havelka M, Boscari E, Sergeev A, Mugue N, Congiu L, Arai K, et al. . A new marker, isolated by ddRAD sequencing, detects Siberian and Russian sturgeon in hybrids. Anim Genet. (2018) 50:115–6. 10.1111/age.12733 PubMed DOI

Börk K, Drauch A, Israel JA, Pedroia J, May B. Development of new microsatellite primers for green and white sturgeon. Conserv Genet. (2008) 9:973–9. 10.1007/s10592-007-9417-9 DOI

Welsh AB, Blumberg M, May B. Identification of microsatellite loci in lake sturgeon, Acipenser fulvescens, and their variability in green sturgeon, A. medirostris. Mol Ecol Notes. (2003) 3:47–55. 10.1046/j.1471-8286.2003.00346.x DOI

King TL, Lubinski BA, Spidle AP. Microsatellite, DNA variation in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and cross-species amplification in the Acipenseridae. Conserv Genet. (2001) 2:103–19. 10.1023/A:1011895429669 DOI

McQuown EC, Sloss BL, Sheehan RJ. Microsatellite analysis of genetic variation in Sturgeon: new primer sequences for Scaphirhynchus and Acipenser. Trans Am Fish Soc. (2000) 129:1380 10.1577/1548-8659 DOI

Havelka M, Hulák M, Bailie DA, Prodöhl PA, Flajšhans M. Extensive genome duplications in sturgeons: new evidence from microsatellite data. J Appl Ichthyol. (2013) 29:704–8. 10.1111/jai.12224 DOI

Dettlaff TA, Ginsburg AS, Schmalhausen OI. Sturgeon Fishes: Developmental Biology and Aquaculture. New York, US: Springer-Verlag; (1993). p. 300.

Tsekov A, Ivanova P, Angelov M, Atanasova S, Bloesch J. Natural sturgeon hybrids along bulgarian black sea coast and in danube river. Acta Zool Bulg. (2008) 60:311–6.

Havelka M, Kašpar V, Hulák M, Flajšhans M. Sturgeon genetics and cytogenetics: a review related to ploidy levels and interspecific hybridization. Folia Zool. (2011) 60:93–103. 10.25225/fozo.v60.i2.a3.2011 DOI

Shen L, Shi Y, Zou YC, Zhou XH, Wei QW. Sturgeon aquaculture in China: status, challenge and proposals based on nation-wide surveys of 2010–2012. J Appl Ichthyol. (2014) 30:1547–51. 10.1111/jai.12618 DOI

Beridze T, Boscari E, Scheele F, Edisherashvili T, Anderson C, Congiu L, et al. . Interspecific hybridization in natural sturgeon populations of the Eastern Black Sea: the consequence of drastic population decline? Conserv. Genet. (2022) 23:211–6. 10.1007/s10592-021-01413-7 DOI

Nagy ZP, Liu J, Joris H, Verheyen G, Tournaye H, Camus M, et al. . The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod. (1995) 10:1123–9. 10.1093/oxfordjournals.humrep.a136104 PubMed DOI

Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenetic cells: its biology and applications in human and animals. Reprod Biomed Online. (2005) 10:247–88. 10.1016/S1472-6483(10)60947-9 PubMed DOI

Brun RB. Studies on fertilization in Xenopus laevis. Biol Reprod. (1974) 11:513–8. 10.1095/biolreprod11.5.513 PubMed DOI

Pocherniaieva K, Güralp H, Saito T, Pšenička M, Tichopád T, Janko, et al. . The timing and characterization of maternal to zygote transitionand mid-blastula transition in sterlet acipenser ruthenus and A.ruthenus x Acipenser gueldenstaedtii Hybrid. Turk J Fish Aquat Sci. (2019) 19:167–74. 10.4194/1303-2712-v19_2_09 DOI

The Practice Committee of the American Society for Reproductive Medicine . Endometriosis and infertility. Fertil Steril. (2004) 81:1441–6. 10.1016/j.fertnstert.2004.01.019 PubMed DOI

Palermo G, Nagy Z. Manual of Intracytoplasmic Sperm Injection in Human Assisted Reproduction: With Other Advanced Micromanipulation Techniques to Edit the Genetic and Cytoplasmic Content of the Oocyte. Cambridge: Cambridge University Press; (2021).

Ciereszko A, Glogowski J, Dabrowski K, Tiersch TR, Mazik PM. Cryopreservation of Aquatic Species. Baton Rouge, LA: World Aquaculture Society; (2000). p. 20–48.

Johnston LA, Lacy RC. Genome resource banking for species conservation: selection of sperm donors. Cryobiology. (1995) 32:68–77. 10.1006/cryo.1995.1006 PubMed DOI

Billard R, Cosson J, Noveiri SB, Pourkazemi M. Cryopreservation and short- term storage of sturgeon sperm, a review. Aquaculture. (2004) 236:1–9. 10.1016/j.aquaculture.2003.10.029 DOI

Psenicka M, Hadi Alavi SM, Rodina M, Cicova Z, Gela D, Cosson J, et al. . Morphology, chemical contents and physiology of chondrostean fish sperm: a comparative study between Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus). J Appl Ichthyol. (2008) 24:371–7. 10.1111/j.1439-0426.2008.01139.x DOI

Sakkas D, Urner F, Bianchi PG, Wagner I, Jaquenoud N, Manicardi G, et al. . Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod. (1996) 11:837–43. 10.1093/oxfordjournals.humrep.a019263 PubMed DOI

Dumoulin JCM, Coonen E, Bras M, Bergers-Janssen JM, Ignoul-Vanvuchelen RCM, van Wissen LCP, et al. . Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure. Hum Reprod. (2001) 16:306–12. 10.1093/humrep/16.2.306 PubMed DOI

Hajiyavand AM, Saadat M, Abena A, Sadak F, Sun X. Effect of injection speed on oocyte deformation in ICSI. Micromachines. (2019) 10:226. 10.3390/mi10040226 PubMed DOI PMC

Rouillon C, Depincé A, Chênais N, Le Bail PY, Labbé C. Somatic cell nuclear transfer in non-enucleated goldfish oocytes: understanding DNA fate during meiosis resumption and first cellular division. Sci Rep. (2019) 9:12462. 10.1038/s41598-019-48096-2 PubMed DOI PMC

Maxwell WMC, Evans G, Hollicshead FK, Bathgate R, Graaf SP, et al. . Integration of sperm sexing technology into the ART toolbox. Anim Reprod Sci. (2004) 82:79–95. 10.1016/j.anireprosci.2004.04.013 PubMed DOI