This study aimed to examine the ultrastructure of spermatogenic stages and mature spermatozoa in the European grayling, Thymallus thymallus. The testes were examined microscopically with a transmission electron microscope to find out details of the structure and morphology of the grayling germ cells, spermatozoa and some somatic cells. The grayling testis has a tubular shape, with cysts or clusters of germ cells within seminiferous lobules. The spermatogenic cells, including spermatogonia, spermatocytes, and spermatids, can be found along seminiferous tubules. There are electron-dense bodies in germ cells from the primary spermatogonia to secondary spermatocyte stages. These undergo mitosis to reach the secondary spermatogonia stage, when they form primary and secondary spermatocytes. Spermatids undergo three different stages of differentiation during spermiogenesis, characterized by the level of chromatin condensation, elimination of cytoplasm, and the occurrence of the flagellum. The midpiece of spermatozoa is short and contains spherical or ovoid mitochondria. The sperm flagellum has an axoneme with nine doublets of peripheral microtubules and two central microtubules. The result of this study is valuable to be used as a standard reference for germ cell development, which is of great importance to get a clear insight into the process of grayling breeding practice.

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

See more in PubMed

Jensen NR, Anders PJ, Hoffman CA, Porter LS, Ireland SC, Cain KD. Performance and macronutrient composition of Age-0 Burbot fed four diet treatments. N Am Aqua. (2011) 73:360–8. doi: 10.1080/15222055.2011.606712 DOI

Kim SH, Lee CH, Song YB, Hur SW, Kim HB, Lee YD. Ultrastructure of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea. Tissue Cell. (2013) 45:261–8. doi: 10.1016/j.tice.2013.03.003, PMID: PubMed DOI

Trabelsi A, Gardeur JN, Teletchea F, Fontaine P. Effects of 12 factors on burbot Lota lota (L., 1758) weaning performances using fractional factorial design experiment. Aquaculture. (2011) 316:104–10. doi: 10.1016/j.aquaculture.2011.03.027 DOI

Grier JJ, Parenti LR. Reproductive biology and systematics of phallostethid fishes as revealed by gonad structure. Env Biol Fishers. (1994) 41:287–99. doi: 10.1007/BF02197850 DOI

Mattei X, Siau Y, Thiaw OT, Thiam T. Peculiarities in the organization of testis of Ophidion sp. (Pisces, Teleostei): evidence for two types of spermatogenesis in teleost fish. J Fish Biol. (1993) 43:931–7. doi: 10.1111/j.1095-8649.1993.tb01167.x DOI

Quagio-Grassiotto I, Carvalho ED. Ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermiogenesis. J Sub Cytol Pathol. (2000) 32:654–9. PubMed

Cinquetti R, Dramis L. Histological, histochemical, enzyme histochemical and ultrastructural investigations of the testis of Padogobius martensi between annual breeding seasons. J Fish Biol. (2003) 63:1402–28. doi: 10.1111/j.1095-8649.2003.00255.x DOI

Uribe MC, Grier HJ, Mejía-Roa V. Comparative testicular structure and spermatogenesis in bony fishes. Spermatogenesis. (2014) 4:e983400. doi: 10.4161/21565562.2014.983400, PMID: PubMed DOI PMC

Rupik W, Huszno J, Klag J. Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)–structural and ultrastructural studies. Micron. (2011) 42:833–9. doi: 10.1016/j.micron.2011.05.006, PMID: PubMed DOI

Fu SY, Jiang JH, Yang WX, Zhu JQ. A histological study of testis development and ultrastructural features of spermatogenesis in cultured Acrossocheilus fasciatus. Tissue Cell. (2015) 48:49–62. doi: 10.1016/j.tice.2015.10.005 PubMed DOI

Almeida FF, Kristoffersen C, Taranger GL, Schulz RW. Spermatogenesis in Atlantic cod (Gadus morhua): a novel model of cystic germ cell development. Biol Reprod. (2008) 78:27–34. doi: 10.1095/biolreprod.107.063669, PMID: PubMed DOI

Medina A, Megina C, Abascal FJ, Calzada A. The sperm ultrastructure of Merluccius merluccius (Teleostei, Gadiformes): phylogenetic considerations. Acta Zool. (2003) 84:131–7. doi: 10.1046/j.1463-6395.2003.00138.x DOI

Quagio-Grassiotto I, Oliveira C, Gosztonyi AE. The ultrastructure of spermiogenesis and spermatozoa in Diplomystes mesembrinus. Fish Biol. (2001) 58:1623–32. doi: 10.1111/j.1095-8649.2001.tb02317.x DOI

Yang Y, Li Y, Wang Y, Hu J, Zhang M, Sun Y, et al. . The ultrastructure of spermatogenic cells and morphological evaluation of testicular development in the silver pomfret (Pampus argenteus). Anat Histol Embryol. (2021) 50:1034–42. doi: 10.1111/ahe.12747, PMID: PubMed DOI

Dadras H, Golpour A, Dzyuba B, Kristan J, Policar T. Ultrastructural feature of spermatogenic cells and spermatozoon in cultured burbot Lota lota. Tissue Cell. (2019) 61:1–7. doi: 10.1016/j.tice.2019.08.005, PMID: PubMed DOI

Jun JC, Chung EY, Yang YC. Ultrastructure of germ cells, cyst epithelial cells and interstitial cells during spermatogenesis of the stone flounder, Kareius bicoloratus. Kor J Ichthyol. (2006) 18:311–8.

Da Cruz C, Orsi AM, Simous K, Vicentini CA. Structural and ultrastructural characteristics of the spermatogenesis of the grey armored catfish Liposarcus anisitsi (Holmberg, 1893) (Teleostei, Siluriformes). Braz J Vet Res Anim Sci. (2005) 42:37–44. doi: 10.11606/issn.1678-4456.bjvras.2005.26452 DOI

Susnik S, Berrebi P, Dovc P, Hansen MM, Snoj A. Genetic introgression between wild and stocked salmonids and the prospects for using molecular markers in population rehabilitation: the case of the Adriatic grayling (Thymallus thymallus L. 1785). Heredity (Edinb.). (2004) 93:273–82. PubMed

Uiblein F, Jagsch A, Honsig-Erlenburg W, Weiss S. Status, habitat use, and vulnerability of the European grayling in Austrian waters. J Fish Biol. (2001) 59:223–47. doi: 10.1111/j.1095-8649.2001.tb01388.x DOI

Szmyt M, Piech P, Panasiewicz G, Niewiadomski P, Pietrzak-Fiecko R. Gonadal steroid fluctuations and reproduction results of European grayling (Thymallus thymallus L.) from broodstock farmed in a recirculation aquaculture system. Anim Reprod Sci. (2021) 224:106648. doi: 10.1016/j.anireprosci.2020.106648, PMID: PubMed DOI

Turek J, Randak T, Velisek J, Podhorec P, Kouril J. The effect of selected ovulation-inducing preparations on post-stripping mortality and reproductive indicators of farmed European grayling (Thymallus thymallus L.). Acta Vet Brno. (2013) 82:381–6. doi: 10.2754/avb201382040381 DOI

Lahnsteiner F, Kletzl M. Suitability of different food types for on-feeding and juvenile production of European grayling, Thymallus thymallus, under intensive farming conditions. J Agric Sci. (2015) 7:161–8. doi: 10.5539/jas.v7n1p161 DOI

Rahimnejad S, Dabrowski K, Izquierdo M, Malinovskyi O, Kolářová J, Policar T. Effects of dietary protein and lipid levels on growth, body composition, blood biochemistry, antioxidant capacity and ammonia excretion of European grayling (Thymallus thymallus). Front Mar Sci. (2021) 8:715636. doi: 10.3389/fmars.2021.715636 DOI

Goryczko K, KuŸmiñski H, Dobosz S. Attempts towards a full cycle of grayling (Thymallus thymallus) production In: Proc. Inter. Conf.: New Fish Species in Aquaculture. Szczecin, Poland: Agriculture University of Szczecin; (1995). 15–8.

Magalhães ALB, Andrade RF, Gomes BVC, Perini VR, Rizzo E, Bazzoli N. Ultrastructure of the semicystic spermatogenesis in the south American freshwater characid Hemigrammus marginatus (Teleostei, Characiformes). J Appl Ichthyol. (2011) 27:1041–6. doi: 10.1111/j.1439-0426.2011.01747.x DOI

Golpour A, Pšenička M, Niksirat H. Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio. J Morphol. (2017) 278:1149–59. doi: 10.1002/jmor.20701, PMID: PubMed DOI

Billard R. Spermatogenesis and spermatology of some teleost fish species. Reprod Nutr Dev. (1986) 26:877–920. doi: 10.1051/rnd:19860601 DOI

Parenti LR, Grier HJ. Evolution and phylogeny of gonad morphology in bony fishes. Integr Comp Biol. (2004) 44:333–48. doi: 10.1093/icb/44.5.333, PMID: PubMed DOI

Grier HJ. Comparative organization of Sertoli cell including the Sertoli cell barrier In: Russell LD, Griswold MD, editors. The Sertoli Cell. Clearwater, FL: Cache River Press; (1993). 704–30.

Grier HJ, Uribe-Aranzábal MC. The testis and spermatogenesis in teleost In: Jamieson BGM, editor. Reproductive biology and phylogeny of fishes (Agnathans and Neotelestomi). Enfield: Phylogeny-Reproductive System-Viviparity-Spermatozoa, Science Publishers Inc. (2009). 119–42.

Grier HJ. Cellular organization of the testis and spermatogenesis in fishes. Am Zool. (1981) 21:345–7. doi: 10.1093/icb/21.2.345 DOI

Lopes DCRJ, Bazzoli N, Brito MFG, Maria TA. Male reproductive system in the south American catfish Conorhynchus conirostris. J Fish Biol. (2004) 64:1419–24. doi: 10.1111/j.0022-1112.2004.00377.x DOI

Muñoz M, Sàbat M, Mallol S, Casadevall M. Gonadal structure and gametogenesis of Trigla lyra (Pisces: Triglidae). Zool Stud. (2002) 41:412–20.

Chung EY, Yang YC, Kang HW, Choi KH, Jun JC, Lee KY. Ultrastructure of germ cells and the functions of Leydig cells and Sertoli cells associated with spermatogenesis in Pampus argenteus (Teleostei, Perciformes, Stromateidae). Zool Stud. (2010) 49:39–50.

Papah MB, Kisiaa SM, Ojooa RO, Makanyaa AN, Wood CM, Kavembe GD, et al. . Morphological evaluation of spermatogenesis in Lake Magadi tilapia (Alcolapia grahami): a fish living on the edge. Tissue Cell. (2013) 45:371–82. doi: 10.1016/j.tice.2013.06.004, PMID: PubMed DOI

Chung EY. Ultrastructure of germ cells, the Leydig cells, and Sertoli cells during spermatogenesis in Boleophthalmus pectinirostris (Teleostei, Perciformes, Gobiidae). Tissue Cell. (2008) 40:195–205. doi: 10.1016/j.tice.2007.11.003, PMID: PubMed DOI

Qiao H, Chen JK, Reynolds A, Höög C, Paddy M, Hunter N. Interplay between synaptonemal complex, homologous recombination, and centromeres during mammalian meiosis. PLoS Gen. (2012) 8:e1002790. doi: 10.1371/journal.pgen.1002790, PMID: PubMed DOI PMC

Zickler D. From early homologue recognition to synaptonemal complex formation. Chromosoma. (2006) 115:158–74. doi: 10.1007/s00412-006-0048-6, PMID: PubMed DOI

Negrao JNC, De Carvalho ED, Foresti F, Quagio-Grassiotto I. Spermatogonia and spermatocyte ultrastructure in Hoplias malabaricus (Teleostei, Characiformes: Erythrinidae). J Zool (Lond). (2002) 257:287–93. doi: 10.1017/S0952836902000882 DOI

Kim JH, Chung JS, Lee KY. Ultrastructural characteristics of the testis, spermatogenesis and taxonomic values of sperm morphology in male Ruditapes philippinarum in Western Korea. Dev. Reprod. (2013) 17:121–32. doi: 10.12717/DR.2013.17.2.121, PMID: PubMed DOI PMC

Gwo JC, Gwo HH, Chang SL. The ultrastructure of the spermatozoon of the teleost fish Acanthopagrus schlegeli (Perciformes, Sparidae). J Morphol. (1993) 216:29–33. doi: 10.1002/jmor.1052160105, PMID: PubMed DOI

Quagio-Grassiotto I, Oliveira C. Sperm ultrastructure and a new type of spermiogenesis in two species of Pimelodidae, with a comparative review of sperm ultrastructure in siluriformes (Teleostei: Ostariophysi). J Comp Zool. (2008) 247:55–66. doi: 10.1016/j.jcz.2007.07.002 DOI

Burns JR, Quagio-Grassiotto I, Jamieson BGM. Ultrastructure of spermatozoa: Ostariophysi In: Jamieson BGM, editor. Reproductive biology and phylogeny of fishes (Agnathans and Neotelestomi). Phylogeny-Reproductive System-Viviparity-Spermatozoa: Science Publishers Inc, Enfield; (2009). 287–388.

Lee TH, Chiang TH, Huang BM, Wang TC, Yang HY. Ultrastructure of spermatogenesis of the paradise fish, Macropodus opercularis. Taiwania. (2006) 51:170–80.

Abou Shabana NM. Ultrastructural study of spermatogenic stages in the protandrous sparid fish Diplodus cervinus cervinus (Lowe, 1838) from the south eastern Mediterranean coast. Afr J Biotechnol. (2012) 11:7270.

Jamieson BGM. Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge: Cambridge University Press; (1991).

Gusmão-Pompiani P, Oliveira C, Quagio-Grassiotto I. Spermatozoa ultrastructure in Sciaenidae and Polynemidae (Teleostei: Perciformes) with some consideration on Percoidei spermatozoa ultrastructure. Tissue Cell. (2005) 37:177–91. doi: 10.1016/j.tice.2004.12.003, PMID: PubMed DOI

Fishelson L, Gon O, Holdengreber V, Delarea Y. Comparative spermatogenesis, spermatocyte ogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei). Anat Rec (Hoboken). (2007) 290:311–23. doi: 10.1002/ar.20412, PMID: PubMed DOI

Vázquez GR, Da Cuña RH, Meijide FJ, Guerrero GA. Spermatogenesis and changes in testicular structure during the reproductive cycle in Cichlasoma dimerus (Teleostei, Perciformes). Acta Zool. (2012) 93:338–50. doi: 10.1111/j.1463-6395.2011.00508.x DOI

Todd PR. Ultrastructure of the spermatozoa and spermatogenesis in New Zealand freshwater eels (Anguillidae). Cell Tiss Res. (1976) 171:221–32. doi: 10.1007/BF00219407 PubMed DOI

Gwo JC, Chiua JY, Lin CY, Sub Y, Yub SL. Spermatozoal ultrastructure of four Sparidae fishes: Acanthopagrus berda, Acanthopagrus australis, Lagodon rhomboids and Archosargus probatocephus. Tissue Cell. (2005) 37:109–15. doi: 10.1016/j.tice.2004.11.003, PMID: PubMed DOI

Maricchiolo G, Laurà R, Genovese L, Guerrera MC, Micale V, Muglia U. Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex. Tissue Cell. (2010) 42:88–96. doi: 10.1016/j.tice.2009.12.002, PMID: PubMed DOI

Gwo JC, Yang WT, Kuo MC, Takemura A, Chengc HY. Spermatozoal ultrastructures of two marine perciform teleost fishes, the goatfish, Paraupeneus spilurus (Mullidae) and the rabbitfish, Siganus fuscescens (Siganidae) from Taiwan. Tissue Cell. (2004) 36:63–9. doi: 10.1016/j.tice.2003.10.001, PMID: PubMed DOI

Gwo JC, Lin CY, Yang WL, Choud YC. Ultrastructure of the sperm of blue sprat, Spratelloides gracilis; Teleostei, Clupeiformes, Clupeidae. Tissue Cell. (2006) 38:285–91. doi: 10.1016/j.tice.2006.06.006, PMID: PubMed DOI

Grier HJ, Linton JR, Leatherland JF, de Vlaming VL. Structural evidence for two different testicular types in teleost fishes. Am J Anat. (1980) 159:331–45. doi: 10.1002/aja.1001590307, PMID: PubMed DOI

Timmermans LPM, Schipper H, Dulos GJ. Peritubular cells in the testis of the common carp (Cyprinus carpio L.); ultrastructure and characterization with acfin and desmin (immuno) cytochemistry. Neth J Zool. (1993) 159:331–45. doi: 10.1002/aja.1001590307 DOI