Generation of surrogate goldfish Carassius auratus progeny from common carp Cyprinus carpio parents

Abstract

Surrogate broodstock technology can increase the production efficiency of commercially important fishes that are difficult to breed in confinement and aid the propagation and recovery of endangered populations. In this study, we report the application of germ cell (GC) transplantation (GCT) for increasing the numbers of progeny produced by small-bodied ornamental fishes by using sexually mature adult fish as recipients. The GCs isolated from prepubertal male goldfish (Carassius auratus) donors (n = 5) were transplanted through the genital papilla into the gonads of adult common carp (Cyprinus carpio) recipients. The endogenous GCs of the recipient were depleted using busulfan (40 mg/kg body weight [BW]; in five doses at 2-week intervals) and high-temperature (38 °C) treatments. Within 4 months after GCT, the donor GCs recolonised the recipients’ gonads and resumed gametogenesis. The presence of donor-derived gametes was confirmed through polymerase chain reaction–restriction fragment length polymorphism analysis in all the surrogate common carp males and females. Artificial fertilisation and induced spawning between surrogate males and females yielded pure goldfish progeny; the fertilisation and hatching rates were similar to those of the controls. These results suggest that GCT could also be potentially applied in commercial aquaculture, mainly to increase the numbers of progeny obtained from small-bodied fishes those having low gamete counts.

Introduction

Germ cell (GC) transplantation (GCT) in fish, a powerful artificial reproductive technique originally tested in mammals (Brinster and Zimmermann 1994), involves the transplantation of donor germ cells or primordial germ cells in the blastodisc of blastula stage embryos (Takeuchi et al. 2003; Saito et al. 2011), into the coelomic cavity of hatchlings (Wong et al. 2011; Okutsu et al. 2007), and directly into gonads of adults by surgical or non-surgical (intra-papillar) intervention (Majhi et al. 2009; Lacerda et al. 2010) for rapid and theoretically unlimited production of surrogate gametes. GCT has been widely used in the restoration of fertility in mammals after cytotoxic drugs or radiation treatments (Honaramooz and Yang 2010). This technique is also used in preservation of valuable and endangered genetic resources (Arregui et al. 2008). Furthermore, GCT is also used in basic biological studies, animal transgenesis, and potential gene modification therapy (Zeng et al. 2012; Hamra et al. 2002; Brinster 2002).

However, in teleost fishes, GCT has been widely applied to generate donor-derived gametes and progeny in large-sized fish species (Takeuchi et al. 2003; Morita et al. 2012; Majhi et al. 2014). GCT in fish was first attempted in the rainbow trout (Oncorhynchus mykiss); it involved the grafting of a piece of the testis into isogeneic animals, which resulted in donor-derived spermatogenesis (Nagler et al. 2001). These studies were followed by the development of approaches based on the transplantation of primordial GCs (PGCs) into the coelomic cavity of fish hatchlings and into the blastodiscs of embryos, which lead to generation of viable donor-origin functional gametes (Takeuchi et al. 2003, 2004; Okutsu et al. 2007). Furthermore, studies have shown that in fish models, GCT, which was originally devised for mammals, need not be performed using PGCs because the transplantation of syngeneic or xenogeneic spermatogonia also resulted in the colonisation of the recipient gonads by the transplanted cells and production of donor-derived gametes (Okutsu et al. 2006; Takeuchi et al. 2009; Majhi et al. 2009, 2014; Lacerda et al. 2010). These results confirmed the technical feasibility and the high potential of GCT in aquaculture, mainly to generate progeny of commercially important fishes that are difficult to propagate in captivity.

In the present study, intra-gonadal GCT was explored between donor goldfish (Carassius auratus) and recipient common carp (Cyprinus carpio). Here we report that transplantation of germ cells from C. auratus into C. carpio, previously depleted of endogenous germ cells by Busulfan and high water temperature treatments, resulted in recolonisation of recipient gonads. The transplanted cells successfully developed into functional surrogate sperm and eggs and were used in artificial and natural spawning, resulting into successful fertilisation and production of viable C. auratus progeny.

Materials and methods

Ethics statement

This study was approved by the Animal Ethics Committee of National Bureau of Fish Genetic Resources (#G/CPCSEA/IAEC/2015/2), Lucknow, India. All the fishes used in the experiments were handled according to the prescribed guidelines. During the study period, the fishes were killed using anaesthetic overdose, and the gonads were excised.

Fabrication of cell transplantation platform

The apparatus was fabricated locally (Fig. 1A, B). It consisted of two 50-L glass tanks, placed below and above a metallic frame. The centre of the frame consisted of a glass working platform. A chamber made of wood and foam was placed on the platform. The chamber was fitted with adjustable metallic clamps on both sides to hold the fish in a ventral-side-up position. The chamber had an inlet and outlet for water flow (Fig. 1B). Water containing the anaesthetic agent from the lower tank was raised to the upper tank by using a water pump (0.5 hp); the water descended under gravity, passed through the operculum of the fish that was placed inside the chamber, and flowed back into the lower tank. The apparatus also had racks to hold the electrical switches (for regulating the function of water pump, lights, and computer), camera, and focusing light. The camera was fitted to relay the cell transplantation procedure through a display unit fitted at the top of the metallic frame. The equipment had four metallic wheels at the base to enable movement.

Fig. 1.

View of cell transplantation platform. The platform shown in B was fabricated using sketch shown in A (measurement are in inch ['] and foot ['']). It consisted of two 50-L glass tanks, placed below and above a metallic frame. The centre of the frame consisted of a glass working platform. A chamber made of wood and foam was placed on the platform. The chamber was fitted with adjustable metallic clamps on both sides to hold the fish in a ventral-side-up position. The chamber had an inlet and outlet for water flow. Water containing the anaesthetic agent from the lower tank was raised to the upper tank by using a water pump (0.5 hp); the water descended under gravity, passed through the operculum of the fish that was placed inside the chamber, and flowed back into the lower tank. The apparatus also had racks to hold the electrical switches, camera, and focusing light. The camera was fitted to relay the cell transplantation procedure through a display unit fitted at the top of the metallic frame. The equipment had four metallic wheels at the base to enable movement

Experimental animals and rearing protocol

The 1-year-old adult common carp (Cyprinus carpio) recipients (mean body weight [BW] ± standard deviation [SD]; males: 85 ± 6.5 g and females: 87 ± 4.3 g) (Fig. 2A) were procured from a local pet shop. The carps were stocked in 5000-L tanks at a density of 5.0 kg of fish per m³. They were reared in fresh water (temperature: 25 ± 2 °C; dissolved oxygen: 5.3–6.1 ppm; pH: 7.5–8.0; hardness: 40–45 ppm) under a constant light cycle (12 h light and 12 h darkness [12L12D]) at the National Bureau of Fish Genetic Resources, Lucknow main campus. The fish were acclimated for 2 weeks at 25 ± 2 °C prior to the thermo-chemical treatments. The sexually immature male donor goldfish, Carassius auratus (Fig. 2B), were procured locally and stocked in 100-L tanks at a density of 0.5 kg of fish per m³. The goldfish were reared in fresh water under a constant light cycle (12L12D) at 25 ± 2 °C until use. Both fishes were fed a pelleted commercially available diet two times per day to satiation.

Fig. 2.

Model fish species used in the study. A Common carp Cyprinus carpio (recipient) and, B goldfish Carassius auratus (donor). Scale bar indicates 2 cm

Recipient preparation

The endogenous GCs of the recipient carps were ablated using a protocol described by Majhi et al. 2017. Briefly, the recipients of both sexes were reared at 38 °C and received either treatments of 40 mg/kg BW busulfan (n = 75) or only the vehicle dimethyl sulphoxide (DMSO; n = 30). Busulfan (Sigma Aldrich, St. Louis, MO, USA) was first dissolved in DMSO (HiMedia, Mumbai, India) and diluted with freshwater fish Ringer solution (111 mM NaCl, 5.37 mM KCl, 1 mM CaCl₂, 0.6 mM MgSO₄, 5 mM C₈H₁₈N₂O₄S) to obtain working concentrations. The fish were anesthetised using 2-phenoxyethanol (HiMedia, Mumbai, India), and busulfan was injected intraperitoneally at 1% kg BW using a 1-mL syringe (Hindustan Syringes & Medical Devices Ltd., India) on day 0 and at 2, 4, 6, and 8 weeks; the carps were reared constantly at 38 °C until the termination of experiment at 10 weeks.

Histological analysis of the gonads

For histological analysis of the GC loss and degree of sterilisation, five male and five female carps were randomly sampled at 10 weeks. The carps were killed using an anaesthetic overdose (200 ppm 2-phenoxyethanol), and the gonads were excised. The middle portion of the right and left gonads was immersed in Bouin’s fixative for 48 h and preserved in 70% ethanol. The gonads were processed for light microscopic examination following routine histological procedures up to preparation of 5-µm-thick sections and staining with hematoxylin–eosin. Approximately 100 serial histological sections from each fish were examined under a microscope at magnifications between 10 × and 60 ×.

Isolation and labelling of donor GCs for transplantation

Donor cells from the pre-pubertal male goldfish (n = 5; Fig. 3) were isolated using a protocol described by Majhi et al. (2009). Briefly, the male goldfish were sacrificed using anaesthetic overdose and the gonads were excised and rinsed in phosphate buffered saline (PBS). The gonadal tissue was finely minced and incubated in a dissociating solution containing 0.5% trypsin (Worthington Biochemical Corp., Lakewood, NJ), 5% foetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA), and 1 mM Ca²⁺ in PBS for 2 h at 22 °C. The dispersed gonadal cells were sieved through a nylon screen (mesh size 50 µm) to eliminate non-dissociated cell clumps, suspended in a discontinuous Percoll (Sigma Aldrich, St. Louis, MO, USA) gradient of 50%, 25% and 12%, which was prepared by dilution of the stock solution with PBS (pH 8.2), and centrifuged at 200×g for 20 min at 20 °C. The bottom phase containing predominantly spermatogonia cells was harvested. The cells were rinsed with PBS (pH 8.2) and tested for viability through the trypan blue (0.4% w/v) exclusion assay. The PKH 26 Cell Linker kit (Sigma Aldrich, St. Louis, MO, USA) was used to label the cells for tracking their behaviour inside the recipient gonads. For labelling approximately 1.4 × 10⁸ donor cells, 24 µL of PKH 26 dye was used for 10 min at 24 °C, and the staining was stopped by adding an equal volume of heat-inactivated FBS to the cells. The labelled cells were rinsed with PBS (pH 8.2) three times to remove the unincorporated dye, suspended in Dulbecco Modified Eagle Medium (Life Technologies, Rockville, MD) containing 10% FBS, and stored on ice until transplantation.

Fig. 3.

Histological section of donor goldfish Carassius auratus used in the experiment. B is a high magnification of the box in A. A The male donor fish shows a sexually immature testis containing prominent cyst of spermatogonia cell at the basement membrane (B; indicated with arrows) and absence of spermatid and spermatozoa cells. Scale bar indicates 200 µm (A) and 100 µm (B)

Germ cell transplantation procedure

On termination of the heat and busulfan treatments at 10 weeks, we gradually reduced the water temperature of the experimental tank (1–2 °C/day) to the pre-treatment condition (25 °C), and GCT was performed in 32 male and 33 female carps. Briefly, each carp was anaesthetised in 100 ppm 2-phenoxyethanol and positioned upside down on the cell transplantation platform, where it received a constant flow of oxygenated water containing 80 ppm of the anaesthetic agent (exposure through the gills). To prevent desiccation, the body surface of the carp was moisturised by covering it with wet tissue paper during the transplantation procedure. A 1-mL syringe equipped with a fine glass needle (26 gauge) was used to inject the cell suspension into the gonads through the genital papilla. Each individual was injected with 100 µL of the cell suspension containing approximately 4.5 × 10⁴ cells/µL, at a flow rate of approximately 50 µL/min. Trypan blue (0.4% w/v) was added to the injection medium to allow visualisation of the cell suspension inside the needle and leakage during or after transplantation (Fig. 4A, B). The genital opening of each carp was topically treated with 10% isodine after the procedure, and the carps were resuscitated in clean water (25 °C ± 2 °C) (Fig. 4C).

Fig. 4.

Intra-gonadal transplantation of donor cells into recipient gonads. A The recipients were placed on the platform and received a constant flux of aerated anaesthetic water through the gills during the procedure. B The donor cells were injected through the genital papilla and; C the fish were resuscitated in clean water. Scale bar indicates 3 cm (A, B) and 3.5 cm (C)

Fate of donor cells after transplantation

The fate of the donor cells inside the recipient gonads was assessed through macroscopic and microscopic observation of the PKH 26-labelled cells at week 2, 8, and at 4 months after transplantation. Therefore, five transplanted carps were killed by anaesthetic overdose (200 ppm 2-phenoxyethanol); the gonads were excised and macroscopically observed for the degree of dispersion of the cell suspension (Fig. 5). The gonads were washed in PBS, fixed in 4% formaldehyde (HiMedia, Mumbai, India) overnight at 4 °C, immersed in 15% sucrose (Sigma-Aldrich, St. Louis, MO, USA) for 2–3 h, embedded in optimal cutting temperature compound, frozen by using dry ice, and stored at − 80 °C until actual sectioning. The 8-µm-thick cryostat (Leica CM 1500, Germany) sections were made from representative portions of the gonads and air dried for 30–45 min at room temperature. A cover slip was placed on the section, and the cover slip was sealed using 1–2 drops of scotch instant glue (HiMedia, Mumbai, India). The sections were analysed through fluorescence microscopy. The control sections were prepared using the gonads of the carp that were not subjected to the transplantation procedure.

Fig. 5.

Visualisation of the dispersal of the cell suspension through the gonad after transplantation. A Macroscopic appearance of the testis 2 weeks after germ cell transplantation (note the diffusion of the marker trypan blue through all areas of the testis). B Appearance of a control testis. Scale bars indicate 1 cm

The presence of donor-derived gametes in the GCT recipients was confirmed through molecular (polymerase chain reaction–restriction fragment length polymorphism [PCR–RFLP]) analysis 4 months after transplantation. For the PCR–RFLP analysis of the male carps, 20–30 μL of sperm was manually stripped by applying gentle abdominal pressure on the areas around the genital papilla. For the analysis of the female carps, about 250–300 eggs were collected through cannulation. DNA was extracted using an Invitrogen genomic DNA isolation kit (ThermoFisher Scientific, USA), according to the manufacturer's instructions. The DNA concentrations were measured using a Nanodrop system (Lite Plus, ThermoFisher Scientific, USA) and assessed on an agarose gel (1%). The primer pairs for the RAG2 gene (Forward: 5′-TCCTGCGATAGCTCATGTTG-3′; Reverse: 3′-GGTGGAGTCATCTCCTGCAT-5′; Amplicon size 1286 bp) and 18S rDNA gene (Forward: 5′-AAACGGCTACCACATCCAAG-3′; Reverse: 3′-CCGAGGACCTCACTAAACCA-5′; Amplicon size 1331 bp) were designed based on the available sequences of common carp and goldfish (GenBank accession numbers# HQ615531, FJ710826, HQ366941 and DQ366994). PCR amplifications were performed using 200 μM dNTP, 1.5 mM MgCl₂, 1 × Taq DNA buffer, 0.75 units of Taq Polymerase (ThermoFisher Scientific, USA), 0.1 μM (each) primer, 50 ng of genomic DNA, and autoclaved double-distilled water to make the volume up to 25 μL. The reactions were performed in a thermocycler (ABI, ThermoFisher Scientific, USA) over 30 cycles with the following conditions: 1:25 min at 94 °C, 1:25 min at 52 °C for RAG2 and 54 °C for 18S rDNA, and 1:30 min at 72 °C. The initial denaturation was at 94 °C for 4 min and the final extension was at 72 °C for 10 min. The PCR products were sequenced on ABI Prism™ (Applied Biosystems, ThermoFisher Scientific, USA) using the BigDye^® Terminator v3.1 Cycle Sequencing Kit. The gene sequences were optimised using Bioedit (Hall 1999), version 7.05.3. Restriction maps were analysed to find enzymes producing species-specific RFLP patterns by using the NEBcutter software (Vincze et al. 2003), version 2.0. For PCR–RFLP analysis of RAG2 gene, the enzyme MscI was selected; it made a single cut in the gene of common carp but did not have restriction sites for the gene in goldfish. For the 18S rRNA gene, the enzyme BseYI was used, which made a single cut for the gene in common carp but did not have restriction sites for the gene in goldfish.

Artificial fertilisation and induced spawning

The gametes from the surrogate parents were used in artificial fertilisation together with eggs and sperm from the pure male (32 ± 2.3 g) and female (41 ± 1.6 g) goldfish. For induced spawning trial, surrogate males and females were injected synthetic hormone Ovarim (Congruent pharma, Mumbai, India) at a dose of 0.2 mL/kg BW for male and 0.5 mL/kg BW for female, paired in a 200-L capacity glass aquarium with placing a spawning mop and reared in fresh water (temperature: 25 °C ± 2 °C; dissolved oxygen: 5.3–6.1 ppm; pH: 7.5–8.0; hardness: 40–45 ppm) under a constant light cycle (10L14D). Every morning, between 9:00 and 10:00 AM, the tanks were checked for spawning, and the embryos were collected manually from the spawning mop. The fertilised eggs obtained from each cross were incubated at 25 °C and observed under a light microscope for fertilisation, embryonic development, and hatching. In both the trials, template DNA was extracted from fin clips of the hatchlings after 3 months of nursery rearing by using a PureLink genomic DNA kit (Invitrogen Life Technology, Carlsbad, CA, USA) according to the manufacturer’s protocol and was subjected to PCR analysis using both common carp-specific and goldfish-specific sequences with the conditions listed previously.

Statistical analyses

The body weight and gonado-somatic index measured were compared among the treatments through one-way analysis of variance followed by the Tukey’s multiple comparison test by using the GraphPad Prism software ver. 6.00 (GraphPad Software, San Diego, California, USA). Data are presented as mean ± standard error, and the differences between groups were considered as statistically significant at P < 0.05.

Results

Growth and survival of animals after transplantation

After the GC-transplantation procedure, 62 common carp (n = 32 males and 30 females) survived out of 65. Mortality was only recorded in the female carps 2 weeks after the procedure, and fungal infection was the confirmed cause of mortality. Although the BW decreased significantly during the heat–chemical treatment period (recipient preparation), the carps regained their BW to match that of the control individuals at 4 months after the GCT (Fig. 6).

Fig. 6.

Changes in mean body weight of males (A) and females (B) subjected to heat (38 °C) and busulfan treatments (40 mg/kg, total 5 dosage) between 0 and 10 weeks (before GCT) and 4 months after GCT. Note, at the end of 10-week treatment period there was significant reduction in body weight in males and females. However, 4 months after GCT, the animals regained body weight to match the control group. Means of columns in A and B with different letters differ significantly (ANOVA-Tukey’s multiple comparison test, P < 0.05). The data presented are mean ± standard error

Effects of treatments on GC loss in the recipients

The gonado-somatic index (GSI) of common carp males and females decreased steadily between day 0 and 10 weeks in the heat–chemical-treated group than in the controls (high temperature only) (Fig. 7). The microscopic examination of the gonads recovered at 10 weeks (termination of experiment) from the control males and females revealed minor degeneration of GCs (Fig. 8E, F). By contrast, the degree of germinal degeneration was far more severe in the heat (38 °C)- and chemical (40 mg/kg; five doses)-treated group and 100% of the fish (n = 5 of each sex) were devoid of GCs in all the histological sections examined (Fig. 8B, D); also see Majhi et al. (2017).

Fig.7.

Changes in the gonado-somatic index (GSI) of adult common carp Cyprinus carpio males (A) and females (B) reared at 38 °C water temperature for 10 weeks with 2-week interval injection of vehicle (DMSO only; control) or busulfan (40 mg/kg; treatment). Note, before GCT (during the 10-week treatment period), there was significant reduction in GSI in both males and females. However, 4 months after GCT the GSI significantly increased to match the control group, suggesting the proliferation and differentiation of donor GCs inside the recipient gonads. Means of columns in A and B with different letters differ significantly (ANOVA-Tukey’s multiple comparison test, P < 0.05). The data presented are mean ± standard error

Fig. 8.

Histological changes in the gonads of common carp Cyprinus carpio reared at 38 °C water temperature for 10 weeks with 2-week interval injection of vehicle (DMSO; control) and busulfan (40 mg/kg; treatment). A Testis at the beginning of treatment showing all the stages of germ cells (arrow head indicates large cyst of spermatogonia cell; arrow indicates spermatocytes; asterisk indicates spermatids and circle indicates spermatozoa cells). B Testis recovered at the end of 10 week treatment period showing absence of spermatogonia and all other stages of spermatogenesis with empty niches (arrowhead). C Ovary recovered at the beginning of treatment showing an active oogenesis (arrow indicates stage 2 oocyte. D) Ovary recovered at the end of 10 week treatment period showing absence of oogonia and other types of GCs and considerably shrink ovary. E Control (elevated water temperature only) ovary recovered at 10 week showing minor degeneration of germ cells. F Control testis recovered at 10 week showing minor degeneration of germ cells

Colonisation of donor GCs in recipient's gonads

We observed the processes of colonisation by donor-origin GCs (from goldfish) following their transplantation into the gonads of the recipients (common carp). At 8 weeks after transplantation, the transplanted GCs proliferated and formed cysts, which were visible along the cortical region of the testes; this stage was observed in all five male carps (Fig. 9A, B). After 4 months, the donor GC cysts had undergone differentiation and sperm was collected by gentle abdominal stripping in all the 17 recipients examined. The results of PCR analysis of these GC-transplanted recipients revealed the presence of donor-derived cells in all the recipients (100%, 17 of 17) (Fig. 10).

Fig. 9.

Fate of PKH-26-labelled C. auratus germ cells in recipient C. carpio gonads between 8 weeks and 4 months after germ cell transplantation. A, B Whole-mount preparation of a transplanted testis at 8 weeks showing the presence of transplanted GCs at the blind end of the spermatogenic lobules (arrow; B is a high magnification of the box in A. C, D Whole-mount preparation of a non-transplanted control testis at 8 weeks. E, F Cryostat sections of a transplanted ovary at 8 weeks showing the donor germ cells forming aggregations (arrow; F is a high magnification of the box in E). G, H Cryostat sections of a non-transplanted control female at 8 weeks. I, J Whole-mount preparation of spermatozoa collected from surrogate males 4 months after transplantation. Bright field view of surrogate spermatozoa (I) and corresponding view under fluorescent light (J) showing PKH-26-labelled donor-derived cells (arrows). K, L Whole-mount preparation of oocytes from a transplanted female at 4 months after procedure. Bright field view of surrogate oocytes (K) and corresponding view under fluorescent light (L) showing the presence of fully differentiated donor-derived oocytes (characterised by retention of fluorescent label). Scale bars indicate 200 µm (A, C, E, G, I, J, K and L) and 50 µm (B, D, F and H)

Fig. 10.

PCR–RFLP analysis of sperm and eggs derived from 17 male and 15 female recipients 4 months after germ cell transplantation. Note, sperm derived from all 17 surrogates C. carpio male recipients were pooled and DNA was isolated. Similarly, in case of surrogates C. carpio recipient females, egg samples were pooled and DNA was isolated for further analysis. The primers used in the analysis were from RAG2 gene digested with MscI enzymes (A) and 18S rRNA gene digested with BseYI enzymes (B). Lanes include 100 bp ladder from New England Biolab (M), pure C. carpio (C1), and C. auratus (C2). Donor-derived C. auratus spermatozoa were detected in the sperm of all 17 surrogate C. carpio recipients shown in lanes 1–5 and donor-derived eggs were detected in all 15 surrogate C. carpio recipient shown in lanes 6–10

Similarly, in the female carps, at 8 weeks, the donor germ cells formed clusters in the ovaries of all five recipients (Fig. 9E, F). At 4 months, eggs were collected from all 15 recipients through intra-ovarian cannulation and the PCR analysis results showed the presence of donor-derived cells in all (100%, 15 of 15) the recipients (Fig. 10).

Generation of surrogate progeny

The GC-transplanted common carp recipients, identified through PCR analysis, that produced goldfish spermatozoa (Figs. 9I, J, 10A, B) and eggs (Figs. 9K, L, 10A, B) were then subjected to progeny testing by artificial fertilisation and induced spawning (Fig. 11A–E). These crosses produced viable pure goldfish offspring with normal embryonic development and hatching rates that were comparable with those of the control animals (Tables 1, 2, 3). PCR amplification of DNA from the progeny and RAG2 gene digestion with MscI revealed distinct electrophoretic patterns for common carp (recipient) and goldfish (donor) (Fig. 11F). Similarly, for the 18 s rDNA gene, restriction digestion of the PCR products by using BseYI demonstrated differences in electrophoretic phenotypes between common carp and goldfish (Fig. 11G). The PCR–RFLP analysis results showed that the restriction patterns of the tested progeny samples were 100% similar to those of the donor (goldfish).

Fig. 11.

Artificial fertilisation, induced spawning, and PCR screening of surrogate progeny. A–C Artificial fertilisation of pure C. auratus eggs with sperm derived from surrogate C. carpio males (transplanted with C. auratus germ cells) generates viable C. auratus progeny. D, E Induced spawning between surrogate C. carpio males and females (both transplanted with C. auratus germ cells) generated C. auratus progeny. PCR screening of progeny derived from natural spawning using RAG2 gene digested with MscI enzymes (F) and 18S rRNA gene digested with BseYI enzymes (G) showed all the progeny obtained are of C. auratus origin. Lanes include 100 bp ladder from New England Biolab (M), pure C. carpio (C1), and C. auratus (C2). Donor-derived C. auratus progeny is shown in lanes 1–10

Table 1.

Results of artificial fertilisation between eggs derived from pure C. auratus female and sperm from surrogate C. carpio males (transplanted with C. auratus germ cells)

Surrogate C. carpio males	Eggs derived from wild C. auratus females (n)	Fertilisation (%; n)	Hatching (%; n)	Donor-derived germline transmission (%; n)
#1	65	81.5 (53)	90.5 (48)	100 (48)
#2	73	83.5 (61)	85.2 (52)	100 (52)
#3	54	77.7 (42)	83.3 (35)	100 (35)
#4	80	81.2 (65)	66.1 (43)	100 (43)
#5	65	92.3 (60)	68.3 (41)	100 (41)
#6	70	78.5 (55)	83.6 (46)	100 (46)
#7	58	86.2 (50)	84.0 (42)	100 (42)
#8	43	83.7 (36)	77.7 (28)	100 (28)
#9	76	88.1 (67)	76.1 (51)	100 (51)
#10	60	91.6 (55)	78.1 (43)	100 (43)
Control#1	70	91.4 (64)	82.8 (53)	NA
Control#2	60	80.0 (48)	70.8 (34)	NA

Table 2.

Results of artificial fertilisation of eggs derived from surrogate C. carpio females (transplanted with C. auratus germ cells) with sperm from pure C. auratus males

Pure C. auratus males	Eggs derived from surrogate C. carpio females (n)	Fertilisation (%, n)	Hatching (%, n)	Donor-derived germline transmission (%, n)
#1	65	80.0 (52)	76.9 (40)	100 (40)
#2	48	72.9 (35)	80.0 (28)	100 (28)
#3	52	82.6 (43)	81.3 (35)	100 (35)
#4	63	79.3 (50)	80.0 (40)	100 (40)
#5	75	85.3 (64)	85.9 (55)	100 (55)
#6	70	78.5 (55)	76.3 (42)	100 (42)
#7	63	68.2 (43)	72.0 (31)	100 (31)
#8	86	69.7 (60)	75.0 (45)	100 (45)
#9	62	64.5 (40)	65.0 (26)	100 (26)
#10	55	74.5 (41)	73.1 (30)	100 (30)
Control#1	73	79.4 (58)	68.9 (40)	NA
Control#2	60	71.6 (43)	76.7 (33)	NA

Table 3.

Results of induced spawning trial between surrogate C. carpio males and females (both transplanted with C. auratus germ cells)

Surrogate C. carpio males	Surrogate C. carpio females (n)	Numbers of embryo collected (n)	Fertilisation (%; n)	Hatching (%; n)	Donor-derived germline transmission (%; n)
#1	#1	168	83.3 (140)	91.4 (128)	100 (128)
#2	#2	180	91.6 (165)	78.7 (130)	100 (130)
#3	#3	210	90.4 (190)	81.5 (155)	100 (155)
#4	#4	185	72.9 (135)	77.7 (105)	100 (105)
#5	#5	145	79.3 (115)	85.2 (98)	100 (98)
#6	#6	215	81.3 (175)	76.0 (133)	100 (133)
#7	#7	190	84.2 (160)	75.6 (121)	100 (121)
#8	#8	175	78.8 (138)	79.7 (110)	100 (110)
#9	#9	188	88.2 (166)	75.3 (125)	100 (125)
#10	#10	205	90.2 (185)	77.8 (144)	100 (144)
Control#1		218	77.9 (170)	72.9 (124)	NA
Control#2		165	84.8 (140)	89.2 (125)	NA

Discussion

In the present study, the donor GCs, harvested from the goldfish testes and transplanted into GC-depleted adult common carp through the genital papilla, produced pure functional donor-origin gametes and progeny similar to the previously reported studies using triploid recipients (Okutsu et al. 2007).

GCT has been successfully applied in fishes to generate donor-origin progeny by using various developmental stages of recipients such as embryos, hatchlings, and adults (Takeuchi et al. 2003; Okutsu et al. 2007; Majhi et al. 2014). However, none of these studies have reported production of pure donor-derived gametes and progeny (Takeuchi et al. 2004; Majhi et al. 2009, 2014). In this study, we report for the first time, the production of pure goldfish gametes and progeny from surrogate common carp through intra-gonadal GCT. Notably, the degree of endogenous GC depletion in the recipients affects the production of pure donor-derived gametes (Brinster et al. 2003; Ogawa et al. 1999; Hermann et al. 2007; Majhi et al. 2009). In this study, we completely ablated the endogenous GCs of recipient common carp by heat (38 °C) and chemical (40 mg busulfan/kg BW; five doses at 2-week-intervals) treatments (Majhi et al. 2017). Probably, the complete removal of the endogenous GCs in the common carp increased the niche availability and accessibility to the donor cells inside the recipient gonads and contributed to production of pure donor-derived gametes within 4 months after the cell transplantation (Majhi et al. 2014; Ogawa et al. 1999; Nobrega et al. 2009). One of the possible explanations for rapid proliferation and differentiation of donor cells inside the recipient gonads to produce functional gametes within short span of 4 months might be the rearing of GC-transplanted fish in water temperature of 25 ± 2 °C. The rearing temperature probably represented “continuous summer” that resulted in rapid proliferation and differentiation of gonadal cell (Quintana et al. 2004).

GCT in adult fish, as opposed to that in embryos and hatchlings, has the advantage of a short production time of surrogate gametes and offspring (Lacerda et al. 2010; Majhi et al. 2014). However, performing GCT in adult fish without a proper platform might be a difficult task because the absence of a platform might cause the rupture of vital organs during cell transplantation. Probably, this risk explains why, despite viability of the technology, the GCT approach has not percolated down to entrepreneurs for field application. In this study, we designed and fabricated a completely novel platform for cell transplantation and demonstrated its effectiveness in the generation of surrogate progeny. GCT in embryos and hatchlings as recipients does not require a platform for cell transplantation; instead, the embryos and hatchlings are usually placed on a soft bed due to their small size (Takeuchi et al. 2004; Morita et al. 2012). By contrast, in relatively larger recipients (adult carps in the present study) the body position of the fish must be maintained while transplanting the cells into the gonads mainly to prevent the rupture of soft tissues. This preventive measure is crucial while performing GCT through surgical interventions because cell transplantation by using surgical interventions requires a longer duration than that through the genital papilla. Our previous results revealed that by using our cell transplantation platform the adult common carp could be maintained under a subconscious condition for 45 min. This facilitates the transplantation of the donor cells, irrespective of transplantation approach, with utmost precision.

The process of re-colonisation of the recipient gonads by the transplanted GCs appears to have many similarities across fish species and body sizes. For instance, we observed that when PKH-26-labelled goldfish cells were transplanted into host common carp gonads, the cells (presumably the spermatogonial stem cells) eventually settled and began proliferation and differentiation into functional donor-derived gametes. Notably, a very similar pattern of donor cell migration, colonisation, and differentiation was observed in Atherinopsidae fish (Majhi et al. 2009, 2014) and mice (Nagano et al. 1999) models. These results suggest that regardless of the differences in animal taxa and gonadal structure between the fish model and mammals, the gonadal stem cells have similar patterns of recolonisation.

Notably, the donor-derived gametes obtained from surrogate male and female carps had fertilisation characteristics similar to those of the control gametes. When we performed induced breeding between the surrogate male common carp and wild female goldfish, the donor-derived germ line transmission rate was 100% (Table 3). This rate was significantly higher than the rates reported in previous studies conducted using adult fish as recipient for GCT (Lacerda et al. 2010; Majhi et al. 2014). However, our focus in this study was to apply surrogate broodstock technology for increasing the numbers of goldfish progeny by using surrogate common carp host, which has a considerably higher fecundity rate than goldfish (fecundity: common carp 123 eggs/g BW; goldfish 63 eggs/g BW) (Ortega-Salas and Reyes-Bustamante 2006; Bishai et al. 1974). Thus, when we crossed the surrogate males (common carp with goldfish cells) with surrogate females in a induced spawning trial, we obtained 12,000 ± 450 numbers of goldfish progeny (about 100 progeny/g BW) than with the control (2500 ± 375; about 50 progeny/g BW; cross between pure goldfish parents). These observations suggest that in addition to conservation of endangered fish species, surrogate technology can be effectively applied in commercial aquaculture, mainly to produce a large numbers of progeny of commercially important fish species with low fecundity, such as goldfish.

In conclusion, the most prominent result obtained in this study is production of pure goldfish progeny from surrogate common carp parents. This finding has potential implications in many fields of fishery resource management, particularly in the revival of endangered species and in aquaculture for the generation of offspring in fish species that are difficult to breed in captivity. The approach could also apply in commercial aquaculture, mainly to generate large numbers of progeny in fishes with low gamete counts.

Author contribution

SKM conceptualised and designed the experiment, collected data, analysed the data and prepared the manuscript.

Funding

This work was supported by a Grant-in-Aid from the ICAR-National Bureau of Fish Genetic Resources (Institutional research Grant#IXX10896).

Data availability statement

The data that support the findings of this study are available on request from the author.

Declarations

Conflict of interests

The author declared that no competing interests exist.