Resumption of spermatogenesis in senescent goldfish Carassius auratus (Linnaeus, 1758) through spermatogonial cell therapy

In recent times, stem cell research has gained considerable prominence because of its applications in assisted reproductive technology and the treatment of deadly diseases. In teleost fishes, spermatogonial stem cells have been effectively used to produce progeny of difficult-to-breed fish species and/or commercially valuable species through the surrogacy technique. The present study is the first report of an innovative application of stem cell therapy in teleostean fish species for revitalising the reproductive competence of senescent individuals. Senescent male goldfish, Carassius auratus aged approximately 10 years were procured from an ornamental fish-breeding farm and were reared locally for an additional 2 years. The senescence of the fish was evaluated and confirmed using histological analysis, gonadal index assessment, and germ-cell specific vasa gene expression. Analyses revealed the absence of spermatogonial cells and other germ cells in the testes of the senescent fish (n = 5). Spermatogonial cells from a prepubertal C. auratus male donor were isolated using discontinuous percoll gradients, labelled with the fluorescent dye PKH-26, and transplanted into the gonads of senescent C. auratus males through the urogenital papilla. Six months after the therapy, spermatozoa from males were collected through applying gentle manual pressure on the abdomen and were observed under the microscope. All the senescent therapy-treated C. auratus males produced spermatozoa from the transplanted cells; this was confirmed by retention of PKH-26 in the spermatozoa and diagnostic SSR locus. The senescent males were crossed with gravid C. auratus females through artificial insemination and natural spawning, and viable progeny was produced. These observations suggest that the reproductive competence of senescent individuals of commercially valuable and/or endangered fish species can be revitalised and extended through spermatogonia stem cell therapy to produce functional gametes.


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Stem cells are described as undifferentiated cells that have the potential to renew 54 themselves and differentiate into a single cell type or multiple specialised cell types [1]. This 55 implies that they can undergo numerous cycles of cell division while maintaining their 56 undifferentiated state and they have the capacity to differentiate into specialised cell types. 57 Currently, stem cell research is one of the upcoming areas of research in science. Since their 58 discovery and the subsequent establishment of protocols for successful isolation and culture 59 [2] researchers have used them for various purposes, including treatment of chronic diseases 60 and reproductive ailments [3]. Stem cell therapy has also shown the potential to revitalise or Male goldfish (C. auratus) (n = 60; mean body weight ± standard deviation [SD] of 87 230 ± 20.5 g) were procured from Maharashtra, India and reported to be approximately 10 88 years old by the providers. The age of the fish was evaluated and confirmed by counting the 89 growth rings located at scale (the growth rings from the farm raised fish with known age was 90 used as reference). The fish were stocked in 500L tanks at a density of 5.0 kg of fish per m 3 91 and reared in flow-through fresh water system (Temperature: 25°C ± 2°C; dissolved oxygen: 92 5.3-6.1 ppm; pH: 7.5-8; hardness: 40-45 ppm) under a constant light cycle (12 h light and 12 93 h dark). The fish were reared for an additional 2 years prior to the confirmation of their 94 senility through histological analysis and germ-cell-specific vasa gene expression studies.

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The prepubertal donor C. auratus males (2-3 months old) were produced at the rearing 96 facilities of the National Bureau of Fish Genetic Resources, Lucknow. Both groups of 97 animals were fed a pelleted commercially available diet twice per day to satiation.       Fig. 6E). At 6 months after the therapy, the sperm could be collected by 223 applying gentle pressure on abdomen in all the 25 senescent C. auratus males (Fig. 6F). The 224 sperm density, which was not detectable before the therapy in the senescent C. auratus males, 225 significantly increased after the therapy and was comparable with that of sexually mature 226 control males (Fig. 7).

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Six months after cell therapy, the senescent C. auratus males were found to resume 229 spermatogenesis and produce spermatozoa from the transplanted cells; the origin of the 230 spermatozoa (from the transplanted cells) was confirmed by the existence of two different 231 genotypes and presence of red fluorescent labels in the cells (Fig. 6F). Locus J60* 232 differentiated amplicons from blood (recipient) and sperm (donor cell) DNA (Fig. 8). These  (Table 1). In addition, when the senescent males were coupled with wild C. auratus 239 females for natural spawning, the crosses resulted in 95.5%-99.5% hatching with normal 240 embryonic development (Fig. 9) and viable progeny; oppose to 97.7%-98.4% in control 241 ( Table 2). These observations suggest the viability of the proposed approach in revitalising 242 the reproductive competence of commercially valuable fish species that become senescent 243 with age.

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In the present study, donor spermatogonial cells harvested from prepubertal C. compatible with the gonadal environment of the senescent C. auratus males. Moreover, the 302 progeny produced using the spermatozoa derived from therapy-treated C. auratus males 303 exhibited a similar growth pattern to that of the progeny produced from the control animals 304 (data not shown). These observations suggest that the reproductive competence of senescent 305 C. auratus males could be successfully revitalised using spermatogonial cell therapy.

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Currently, we are examining the feasibility of the cell-therapy approach in revitalising the 307 reproductive competence of female fish.

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In conclusion, the most remarkable achievement of this study is the production of