1 Introduction

The transmission of exogenous genes from one generation to another in transgenic fish shows a higher effectiveness of phenotypic improvement compared with that in nontransgenic fish [1, 2]. The gradual increase in the percentage of CgGH transmission in transgenic mutiara catfish G1 (42.85%), G2 (50%), G3 (70%), and G4 (74%) showed that this catfish could be produced stably in each offspring [3, 4]. The presence of this transgene in each generation indicates that an exogenous gene is permanently inherited in each generation, as shown in zebrafish G3 (containing pCMV-luc) and transgenic salmon G5 (containing opAFP-GHc2) [5, 6]. The genetic superiority of transgenic mutiara catfish G4 must be biologically inherited over G5, indicating that gonadal fertility is an important reproductive characteristic for its mass production [3, 7,8,9]. The reproductive function of transgenic mutiara catfish facilitates the transmission of exogenous growth hormone genes in succeeding generations. The inheritance of CgGH in G5 transgenic mutiara catfish is expected to increase its gonadal fertility compared that of nontransgenic fish under photoperiod manipulation.

The neuroendocrinal reproductive system of fish is related to the responsiveness of photoreceptor cells in the retina and pineal organ to light stimuli (photoperiod); this involves the nervous system as a trigger to release hormones produced in the hypothalamus–pituitary–gonad pathway, which is associated with egg or sperm cell growth [10, 11]. The photoperiod duration influences the maturity of fish ovaries or testes depending on their phototactic nature. Catfish do not like light (negative phototaxis), and gonadal development is more accelerated when there are more dark cycles [12, 13]. Dark conditions induces the nervous system to secrete neurohormones produced by the pineal gland and the eye to respond to photoperiod cues by converting tryptophan to melatonin in both organs. Fluctuations in melatonin levels are closely related to fish spawning season and gonad development stages. Melatonin is a hormone that induces the secretion of gonadotrophin-releasing hormone (GnRH) produced by hypothalamus neurosecretory cells in response to environmental light changes; this is involved in the regulatory pathway for gonadal development stages in nocturnal fish, including catfish [13,14,15,16]. Hence, melatonin is not an antigonadotropic but a neuroendocrinal mechanism of interaction between the nervous system (photoreceptor cells) and endocrine system to treat dark conditions as signals for hormone production. A short photoperiod increases melatonin secretion, which stimulates GnRH secretion. This influences the pituitary gland to secrete gonadotropin hormone (luteinizing hormone, LH) [10, 17,18,19].

Hence, the gonadal development in fish is physiologically regulated by a neuroendocrinal mechanism that regulates gonadotropin signals released by the pituitary gland in response to cyclical dark and light stimuli [18, 20, 21]. The gradual growth of the ovaries of prepubertal catfish is also influenced by the dark cycle, which is related to an increased melatonin production, inducing the secretion of hormones involved in reproduction. An increase in dark cycle induces the secretion of hypothalamic GnRH hormone and pituitary LH hormone as a compensation for an increased expression of GnRH gene receptors (GnRHr) and LH gene receptors (LHr) [10, 17, 22]. An increased expression of GnRHr and LHr causes an increase in liver vitellogenin (Vg) gene expression, which has implications for increasing Vg and estradiol (E2) levels and accelerating ovarian vitellogenesis, leading to the maturation of the gonads of fish [18, 23]. This indicates that a short light duration plays a role in regulating Vg hormone levels in catfish, including G5 transgenic mutiara catfish, during photoperiod induction; this leads to increased Vg hormone levels and ovarian maturity (Fig. 1).

Fig. 1
figure 1

CgGH transmission on G1–G4 [4] and photoperiod induction for reproductive evaluation of G5 transgenic mutiara female catfish

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This suggests that fish reproduction can be regulated by increasing the number of mature gonad broodstocks for aquaculture. The effect of photoperiod induction on female gonadal fertility in G5 transgenic mutiara catfish has not been widely studied. Therefore, it is necessary to evaluate the appropriate induction of light duration on expression levels of GnRHr, LHr, and Vg genes and the response of gonadotropic hormone levels to the growth and development of oocytes of G5 transgenic mutiara catfish.

2 Materials and methods

2.1 Maintenance of G4 transgenic mutiara catfish broodstock for G5 fish production

G5 transgenic mutiara catfish were produced from the spawn of female (1045 g weight, 50 cm total length) and male (weight, 1105 g; total length, 52 cm) transgenic G4 mutiara catfish (Fig. 2). The rearing of G4 transgenic mutiara catfish to become parents has been studied [4]. The breeding of G5 transgenic mutiara catfish broodstock was carried out in a fiberglass tub (diameter, 1.35 m; water depth, 1.05 m). During rearing, the brood fish were given commercial feed Prima Feed 128 (protein content, 38%) at a level of 2% by weight of biomass, and they were fed twice a day until they became prospective broodstock (weight range, 250–325 g). During broodstock maintenance, total water exchange was carried out twice a week at a constant temperature (27 °C ± 1 °C).

Fig. 2
figure 2

Female transgenic G4 fish (A) and male transgenic G4 fish (B)

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2.2 Rearing of G5 fingerlings until broodstock candidate

Fingerlings rearing culture of G5 transgenic and non-transgenic mutiara catfish was performed in separate fiber tanks after RT-PCR analysis of transgenic and non-transgenic fish. Based on the results of the RT-PCR examination, a total of G5 transgenic mutiara catfish was obtained. During maintenance, the water temperature was regulated at a range of 26 °C ± 1 °C using a water heater, photoperiods were set according to treatment and an aeration system was applied to maintain dissolved oxygen levels.

2.3 Broodstock candidates of G5 transgenic mutiara female catfish

Broodstock candidates of G5 transgenic mutiara female catfish were screened through PCR test to be verified as transgenic-positive (containing CgGH 600 bp). RNA samples were taken from the caudal fin and extracted with a Quick-RNA™ Miniprep Plus kit (Zymo Research Corp., Murphy Avenue Irvine, USA). CgGH amplification used primers GH-F and GH-R (Table 1) and My Taq OneStep RT-PCR kit (Bioline, UK, London) to identify G5 transgenic mutiara female catfish. Primers CgßAct-Fw and CgßAct-Rv were used as internal controls.

Table 1 Primers used for screening of G5 transgenic mutiara female catfish
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2.4 Maturation of female broodstock candidates of G5 transgenic mutiara catfish using photoperiods

Six photoperiod treatments (A, B, C, A*, B* and C*) were applied to test the gonadal maturity level of transgenic and nontransgenic catfish using a lamp timer with a Light Emitting Diode light with an intensity of 80 lx. G5 transgenic mutiara and nontransgenic female catfish (5 months old) were exposed to photoperiod treatments, namely A (8 h light; 16 h dark); B (12 h light; 12 h dark); and C (16 h light and 8 h dark) for the transgenic and A* (8 h light; 16 h dark); B* (12 h light; 12 h dark); and C* (16 h light and 8 h dark) for the nontransgenic. Female broodstock candidates treated with photoperiod manipulation for 60 days were reared in circular fiberglass cylindrical tubs with an approximately 1.35-m diameter and 1.05-m tub depth. The number of the female broodstock reared in each treatment tank A, B, C and A*, B*, C* were six female broodsfish used as replicates (n = 6) (Fig. 3). The total number of G5 female broodstock candidates used for gonad maturation was 18 transgenic mutiara catfish and 18 nontransgenic catfish with 5 months of age.

Fig. 3
figure 3

Photoperiod treatment of G5 transgenic mutiara female catfish broodstock (A: 8 h light-16 h dark; B: 12 h light-12 h dark; C: 16 h light-8 h dark); and nontransgenic (*A: 8 h light-16 h dark; B*: 12 h light-12 h dark; C*: 16 h light-8 h dark). 6 fish: replicates (n = 6)

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The water depth required for photoperiod treatment was 70 cm. Water temperature was maintained constantly at 28 °C ± 1 °C, and total water exchange was carried out every 2 days. During maintenance, the broodstock was given commercial feed Prima Feed-128 (38% protein) at a rate of 2% weight of biomass and a frequency three times a day.

2.5 GnRHr, LHr, and Vg expression analysis of G5 fish

To test the expression of genes involved in the reproductive system of G5 transgenic mutiara catfish broodstock candidates, brain, pituitary, and liver tissue samples were taken after each photoperiod treatment. Expression levels of genes involved in the induction of hormone secretion in the hypothalamus (GnRHr gene), pituitary (LHr gene), and liver (Vg gene) axis were analyzed by real-time PCR (rt-qPCR). Brain, pituitary, and liver tissue samples of three G5 female fish were taken from each treatment in each replication. Total RNA was extracted using a Quick-RNA miniprep plus kit (ZymoResearch, UK), and RNA concentrations were measured using a NanoDrop 2000 spectrophotometer (Thermoscientific). cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with gDNARemover (TOYOBO, Osaka, Japan), and it was used as a template in rt-qPCR. rt-qPCR was performed in an Agilent AriaMX Real-time PCR machine (Santa Clara, USA) using 2× SensiFAST SYBR® NO-ROX (Bioline, London, UK), with a final concentration of 100 ng μL−1cDNA; primers qVg-CgF and qVg-CgR, qGnRH-CgF and qGnRH-CgR, and qLHr-CgF, and qLHr-CgR were used. Primers CgβAct-Fw and CgβAct-Rv were used to amplify C. gariepinus β-actin gene as an internal control and to normalize expression levels [26]. qPCR primers for Vg, GnRHr, and LHr genes were designed based on C. gariepinus mRNA sequences in GenBank using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Table 2).

Table 2 Primers used for rt-qPCR (designed from GenBank) using Primer3
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The reaction mixture composition and thermal profile were prepared by quantification using 3 μL of cDNA sample in a final reaction volume of 20 μL containing 10 μL 2X SensiFast SYBR Lo-ROX Mix, 400 nmol of each primer, and 5.4 μL ddH2O. The rt-qPCR program was set as follows: 120 s at 95 °C, 40 amplification phase cycles (5 s at 95 °C, 30 s at 56 °C, and 20 s at 72 °C), and a melting program (30 s at 95 °C, 30 s at 60 °C, and 30 s at 95 °C). Melting curve analysis was performed at the end of the amplification to evaluate the specificity of the reaction. GnRHr and Vg mRNA expression levels were analyzed using the 2−ΔΔCT method after normalization with a β-actin gene [4, 27, 28].

2.6 Measurement of G5 fish vitellogenin and estradiol levels

The serum Vg and estradiol levels of G5 fish were measured using enzyme-linked immunosorbent assay in each post-photoperiod treatment. First, the fish were anesthetized with 2-phenoxyethanol, and about 1.5 mL of blood was taken from their caudal artery using a 2-mL heparinized syringe. The blood samples were centrifuged at 3000 rpm at room temperature for 20 min. Serum was stored at − 20 °C. Vg and E2 levels were analyzed using a Fish Vitellogenin and Estradiol ELISA kit (Bioassay Technology Laboratory, Shanghai, China), following the manufacturer’s protocol.

2.7 Growth performance and gonadosomatic index (GSI) of G5 fish

Observations of the weight of G5 female fish (5 months old) at the beginning and end of the photoperiod treatment (7 months old) for fish maturation induction (Fig. 3) for 60 days were carried out to determine the effect of treatment on the growth level of transgenic fish. The average weight gain of G5 female fish was obtained from the difference in final weight − initial weight of the fish during 60 days of treatment and the growth level of the female G5 transgenic mutiara catfish, which was analyzed for the expression level of the GH insert (CgGH) as confirmation of the growth level of the G5 transgenic fish. rt-qPCR analysis of the CgGH gene of transgenic G5 fish using primers GH-F and GH-R and CgβAct-Fw and CgβAct-Rv with the processing protocol as described above. Average weight gain was calculated using the formula below.

$${\text{Average}}\;{\text{weight}}\;{\text{gain}}\;\left( {\text{g}} \right) = {\text{average}}\;{\text{final}}\;{\text{weight}}\;\left( {\text{g}} \right) - {\text{average}}\;{\text{initial}}\;{\text{weight}}\;\left( {\text{g}} \right).$$

The GSI value of all female G5 fish for each treatment was measured after 60 days of photoperiod treatments. This GSI value is an interpretation of gonad growth in female fish compared to their gonad weight and body weight [29,30,31]. GSI was calculated using the following formula: ovary weight (g)/body weight (g) × 100.

2.8 Histology of G5 female gonads

For histology, 5 μm-thick transverse sections of G5 fish ovaries were taken and immersed in Bouin’s solution for 12 h. Dehydration, clearance, infiltration, planting, cutting, attachment, and hematoxylin–eosin staining was done in G5 female gonad samples [32, 33].

2.9 Statistical analysis

One-way ANOVA with p < 0.05 (SigmaPlot 12.3) was used to detect significant differences in degrees of gonadal maturity between G5 transgenic catfish and nontransgenic fish (analysis of GnRHr, LHr, and Vg gene expression Vg and estradiol levels, and GSI), followed by Duncan’s multiple test.

3 Results

3.1 Detection of CgGH on G4 transgenic broodstock

RT-PCR test showed that female and male G4 transgenic mutiara catfish broodstock, which were screened positive for CgGH (600 bp), could be used for G5 fish production (Fig. 4).

Fig. 4
figure 4

Electropherogram identification of female and male broodstock of G4 transgenic mutiara catfish. CgGH = exogenous growth hormone gene (600 bp, above) and β actin (200 bp, below) as an internal control of gene expression. Broodstock of female = 1, male = 2. pCMV-CgGH plasmid = P, M1 = 1 kb DNA ladder, M2 = 100 bp DNA ladder

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3.2 CgGH transmission in G5 fish

RT-PCR detection of G5 transgenic mutiara catfsh fingerlings (Fig. 5) using the qCgGH-F and qCgGH-R primers (Table 1) revealed that CgGH gene inheritance was up to 89% increased (230/260) compared to G4 (74%), according to results provided previously [4].

Fig. 5
figure 5

Electropherogram of CgGH gene transmission of G5 fingerlings and β-actin of G5 transgenic mutiara catfsh; M, 1 kb DNA ladder; M1, 100 bp DNA ladder; P, pCMV-CgGH; No. 1–26, fish sample number (one well pool of ten different fish fins); N, a sample PCR product without template

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Based on the results of the RT-PCR examination (Fig. 5), a total of G5 transgenic mutiara catfish was obtained. 230 transgenic fish and 30 non-transgenic fish. Furthermore, catfish fingerlings were transferred to fiberglass tank (water volume 2 m3) until 5 months old (broodfish candidate) to check the sex of the broodstock. For maturation purposes using photoperiod treatment, 18 transgenic female and 18 nontransgenic female fish were used to gonadal growth induction (Fig. 3).

3.3 GnRHr expression of G5 fish

The GnRHr expression levels of G5 transgenic catfish different significantly and were higher than those of nontransgenic fish photoperiod treatments 8L-16D; 12L-12D; 16L-8D (Fig. 6A, B).

Fig. 6
figure 6

PCR product of six samples (n = 6, one well pool of two different fish brain tissues) in transgenic of treatments (A1–A3; B1–B3; C1–C3), and nontransgenic of treatments (A*1–A*3; B*1–B*3; C*1–C*3) using primers qGnRHr-CgF and qGnRHr-CgR with semiquantitative reverse transcription PCR (sqRT-PCR) on GnRHr gene (GnRH, 27 cycles) and β-actin as an internal control of expression (β-actin, 25 cycles) (A) and relative expression of G5 transgenic and nontransgenic fish GnRHr mRNA after normalization with β-actin mRNA (B). The data shown are means ± SD of each individual transgenic and nontransgenic fish performed in triplicate PCR. Means followed by different letters indicate significant differences (p < 0.05). Photoperiod treatments 8L-16D; 12L-12D; 16L-8D were exposed to transgenic fish (marked black square) and non-transgenic fish (marked white square). N, sample PCR product without a template. Significance marked with asterisk (*)

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3.4 LHr expression in G5 fish

The expression level of the LHr gene in G5 transgenic catfish was higher than in nontransgenic fish as a consequence of GnRHr gene expression activities that induced pituitary. The LHr expression of transgenic catfish in the short photoperiod treatment (dark cycle > light cycle) was higher than in other treatments (Fig. 7A, B).

Fig. 7
figure 7

PCR product of six samples (n = 6, one well pool of two different fish pitutary tissues) in transgenic of treatments (A1–A3; B1–B3; C1–C3), and nontransgenic of treatments (A*1–A*3; B*1–B*3; C*1–C*3) qLHr-CgF and qLHr-CgR with sqRT-PCR on LHr gene (LHr, 26 cycles) and ꞵ-actin as an internal control of expression (β-actin, 24 cycles) (A) and relative expression of G5 transgenic and nontransgenic fish LHr mRNA after normalization with β-actin mRNA (B). The data shown are means ± SD of each individual transgenic and nontransgenic fish performed in triplicate PCR. Means followed by different letters indicate significant differences (p < 0.05). Photoperiod treatments 8L-16D; 12L-12D; 16L-8D were exposed to transgenic fish (marked black square) and non-transgenic fish (marked white square). N, sample PCR product without a template. Significance marked with asterisk (*)

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3.5 Vg expression of G5 fish

Photoperiod treatment had a significant effect on the expression level of the Vg gene in G5 transgenic catfish and nontransgenic fish; it was higher in G5 transgenic catfish (treatment A, B) than in nontransgenic fish (treatment A*, B*, C*). The expression level in treatment A was higher than in other treatments (Fig. 8A, B). However, the long photoperiod (16L-8D) decreased Vg expression in transgenic and nontransgenic fish (treatments C and C*). Among nontransgenic fish, the expression level of Vg in treatment A* was higher than in treatments B* and C*, whereas in treatments B* and C* the expression levels did not significantly differ.

Fig. 8
figure 8

PCR product of six samples (n = 6, one well pool of two different fish liver tissues) in transgenic of treatments (A1–A3; B1–B3; C1–C3), and nontransgenic of treatments (A*1–A*3; B*1–B*3; C*1–C*3) using primers qVg-CgF and qVg-CgR with sqRT-PCR on Vg gene (Vg, 28 cycles) and β-actin as an internal control of expression (β-actin, 25 cycles) (A) and relative expression of G5 transgenic and nontransgenic fish Vg mRNA after normalization with β-actin mRNA (B). The data shown are means ± SD of each individual transgenic and nontransgenic fish performed in triplicate PCR. Means followed by different letters indicate significant differences (p < 0.05). Photoperiod treatments 8L-16D; 12L-12D; 16L-8D were exposed to transgenic fish (marked black square) and non-transgenic fish (marked white square). N, sample PCR product without a template. Significance marked with asterisk (*)

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3.6 Vitellogenin (Vg) and estradiol (E2) levels of G5 fish

Serum Vg and E2 levels of G5 transgenic mutiara catfish were significantly higher than those of nontransgenic fish (Fig. 9A, B). The short photoperiod (treatment A) induced higher Vg and E2 levels than the other treatments.

Fig. 9
figure 9

Vg (A) and E2 (B) levels of G5 transgenic and nontransgenic fish after photoperiod induction. Means followed by different letters indicate significant differences (p < 0.05). Photoperiod treatments 8L-16D; 12L-12D; 16L-8D were exposed to transgenic fish and non-transgenic fish. Significance marked with asterisk (*)

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3.7 Growth level and gonadosomatic index (GSI) of G5 fish

The average weight gain of G5 transgenic female fish increased higher than that of nontransgenic fish during the photoperiod treatment and the 8L-16D treatment was the highest between transgenic and nontransgenic fish (Fig. 10A). Long photoperiod treatment (16L-8D) gave lower weight growth compared to other treatments, both in transgenic and non-transgenic fish. The increase in weight gain of transgenic fish during the 60 days photoperiod treatment to induce gonad maturity was influenced by the expression of CgGH inserts in transgenic fish and caused their growth to be higher than non-transgenic fish. Analysis of CgGH expression in G5 transgenic fish showed different expression levels in each photoperiod treatment (Fig. 10B), indicating that the increase in weight gain in G5 transgenic fish was influenced by CgGH expression levels in different photoperiods.

Fig. 10
figure 10

Average weight gain of transgenic and non-transgenic G5 catfish in photoperiod treatment (A). CgGH expression levels of G5 transgenic female catfish in different photoperiod treatments (B). Means followed by different letters indicate significant differences (p < 0.05). Significance marked with asterisk (*)

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The gonad growth of female G5 transgenic mutiara catfish was accelerated during the 60-day photoperiod induction, indicating that longer dark cycles (treatments A and B) increased the GSI value compared to other treatments. The increase in light cycle in treatment A, B and C (nontransgenic fish) lowered the GSI value compared to that in transgenic catfish (treatment A, B), indicating that an increase in light cycle slows the gonadal growth of female fish (Fig. 11A, B).

Fig. 11
figure 11

Representative ovary growth (A) and GSI values of G5 fish at different photoperiods (B). The scale line represents 1 cm. Significance marked with asterisk (*)

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4 Discussion

4.1 Expression levels of GnRHr gene in G5 fish

The growth hormone transgenesis in fish effectively showed a higher growth phenotype improvement compared to nontransgenic fish. GH overexpression in G1 transgenic mutiara catfish can increase IGF-1 expression and lead to an increase in the gonad weight of fish compared to that in nontransgenic fish [8]. Hence, fish reproduction can be improved through transgenesis [34]. Further studies on the female gonads of G5 transgenic mutiara catfish indicated that hormone levels and gene expression involved in fish ovary maturation were affected by photoperiod. The expression level of the GnRHr gene in G5 transgenic mutiara catfish (brain tissue) was higher in dark conditions (treatment A) compared with other treatments and the level of GnRHr expression in transgenic catfish in all treatments was higher than that in nontransgenic fish under the short photoperiod, indicating that the insertion of CgGH in the transgenic fish also induced an increase in GnRHr expression (Fig. 6A, B). Consequently, the mean GnRHr expression level of transgenic catfish was higher than that in nontransgenic fish. These findings are similar to research was reported that GH influences expression levels of genes involved in growth and reproduction (including the GnRHr) [35]. Increased dark conditions are thought to induce melatonin secretion, which leads to increased GnRHr expression. Dark conditions (dark cycle ˃ light cycle) induce melatonin secretion as a signal for changing dark and light environment; it acts as a neurohormonal dark signal, triggers GnRH secretion, and activates GnRH gene receptors [13,14,15, 26]. The longer dark cycle in treatments A and B was thought to modulate melatonin secretion and cause an increased GnRHr gene expression of 4.42 and 3.43 in G5 transgenic mutiara catfish, respectively. Hence, melatoninergic and GnRH systems interact in response to environmental photoperiod, involving the interaction of nervous and endocrine systems in the regulation of the fish reproductive system [22, 36, 37]. The decrease in dark cycle caused decrease in GnRHr expression, as shown in treatments C (Fig. 6A, B) when exposed to a long photoperiod (16L-8D) indicating that growth hormone transgenesis in G5 transgenic mutiara catfish treated with 16-h light cycles showed a decrease in the lowest expression level, as shown in nontransgenic fish.

These results indicate that GnRHr expression in transgenic fish (containing CgGH) is affected by bright light duration. GH transgenesis in G3 transgenic mutiara catfish can increase the activity of enzymes involved in breaking down proteins into essential amino acids, including tryptophan, which is an important precursor for melatonin formation [3, 38, 39]. As a compensation, when a shorter light cycle was exposed to G5 transgenic mutiara catfish (treatment A and B), it was suspected that melatonin was formed and GnRHr expression levels remained higher than in nontransgenic fish.

4.2 Expression levels of LHr gene in G5 fish

The level of secretion of reproductive hormones, including LH, in the pituitary pathway is influenced by GnRH induction in the hypothalamus [35]. The increase in LH levels was also accompanied by an increase in LHr gene expression, as shown by the higher photoperiod treatment in G5 transgenic mutiara catfish compared to that in nontransgenic fish (Fig. 7A, B). The highest LHr gene expression among transgenic catfish was found in 16-h dark cycle treatment (treatment A; mean, 5.63), followed by treatment B (12-h dark; mean, 4.86), treatment C (8-h dark; mean, 2.34), and nontransgenic catfish (treatment A, mean, 3.41; treatment B, mean, 2.73; treatment C, mean, 1.38). These LHr gene expression levels were consistent with GnRHr expression levels. These results indicate that the increase in LHr gene expression in G5 transgenic mutiara catfish was induced by the dark cycle; conversely, the induction of a longer light cycle caused the lowest decrease in LHr expression in transgenic and nontransgenic catfish (C treatment).

Similarly, European sea bass (Dicentrarchus labrax) treated with dark cycle (9L-15D) had increased LH levels compared to those treated under 12L-12D photoperiod [17]. This is related to the induction of the dark cycle, which stimulates GnRH neurons via melatonin to stimulate LH secretion and activate LHr, as shown in treatments A and B [40, 41]. In contrast, the lowest LHr expression levels were found in transgenic and nontransgenic fish, which was related to the induction of 16-h light cycle; 8-h dark cycle in this photoperiod treatment caused a decrease in GnRHr expression and had implications for a decreased LHr expression. The decrease in LHr expression levels in G5 transgenic mutiara catfish and nontransgenic (treatment C) was consistent with GnRHr expression in transgenic and nontransgenic catfish. The expression levels of genes involved in regulating reproductive hormones in the hypothalamic-pituitary pathway are interrelated and have consistent expression levels in these pathways [42, 43]. Therefore transgenic catfish under treatment C were exposed to a longer light cycle, causing a decrease in GnRHr expression levels which further inhibits CgGH expression and cause a decline in LHr expression levels, indicating that exogenous GH expression is controlled by GnRHr expression level and duration of bright light; as a consequence, LHr expression decrease in transgenic and nontransgenic fish on long photoperiods. The research on rainbow trout (Oncorhynchus mykiss) showed that LH and FSH secretion required for the continuation of spermatogenesis and oogenesis were induced by growth hormone [44]. This indicates that CgGH in G5 transgenic mutiara catfish played a role in inducing LHr and GnRHr expression when exposed to a short photoperiod.

The short photoperiod treatment (A treatment) in nontransgenic fish increased LHr expression to a level higher than that recorded under treatments B and C, but lower than that in transgenic fish (under A and B treatments). The expression level of LHr in these transgenic fish was similar to that recorded for GnRHr. These results indicate that CgGH is involved in increasing the expression levels of GnRHr and LHr in transgenic fish. This conclusion conforms with that of research was concluded that GnRH secretion is regulated by GH expression and light–dark fluctuations [35].

4.3 Expression levels of Vg gene in G5 fish

The existence of Vg gene in female fish is related to oocyte maturation, especially in vitellus (yolk protein) absorption into the oocyte. This oocyte ripening activity involves the breakdown of vitellus into lipovitellin and phosvitin as yolk materials for oocyte growth [45, 46]. This is followed by an increased Vg gene expression level during vitellogenesis. The mean Vg gene expression in treatment A was 6.67, which was higher than in treatment B (mean, 5.72) and treatment C (mean, 4.49) among fish G5 transgenic mutiara catfish and nontransgenic catfish (treatment A, mean 3.30; B, mean 2.57; C, mean, 2.42). This increase in Vg gene expression (Fig. 8A, B) was similar with LHr gene expression levels in G5 fish (Fig. 7A, B), indicating that a short photoperiod (dark cycle ˃ light cycle) induces an increase in Vg gene expression. The induction of Vg gene expression in the liver of G5 fish was stimulated by pituitary gonadotropin (LH) indicated by the LHr gene. The increased LHr gene expression level during short photoperiods signals LH to increase Vg production and directly increases Vg gene expression. LHr in teleostei fish was found in the brain/pituitary tissue, kidneys, heart, liver, and ovaries of female turbot [49], in the kidneys, ovaries, brain, and pituitary gland of Korean rockfish, and in the kidneys, liver, and ovaries of Clarias macrocephalus fish [47, 48]. This study also showed an induction relation between LHr gene expression in the pituitary and Vg gene expression in the liver of G5 fish, which suggests that an increased LHr gene expression induces an increased Vg gene expression. LHr plays an important role in the reproductive cycle of female turbot, which is involved in early ovarian maturation, particularly in inducing Vg production [49, 50]. This shows that LHr is a reproductive hormone receptor that is important in regulating Vg levels during vitellogenesis for the development of fish ovaries. LHr also influences increase Vg gene expression levels in G5 transgenic mutiara catfish (treatment A, B) higher than nontransgenic fish (Fig. 7B) when reared for 60 days with a short photoperiod (8L-16D and 12L-12D) but in the long photoperiod (16L-8D), LHr expression decreased in both transgenic and nontransgenic fish (treatment C). These results explain that the level of LHr expression in transgenic fish is higher than nontransgenic fish caused by increased LHr expression levels in transgenic fish were associated with increased CgGH expression when exposed to short photoperiods (Fig. 10B). In contrast, during the long photoperiod, the level of LHr expression in transgenic and nontransgenic fish (treatment C) decreased compared to the short photoperiod, indicating that the duration of bright light suppressed both CgGH and LHr expression.

These results indicate that the differences in LHr expression levels during the induction of photoperiod treatments were significantly similar and consistent with differences in Vg expression levels in G5 fish. Vg gene expression in G5 transgenic mutiara catfish under treatment A, B was higher than in nontransgenic fish when induced short photoperiod, because of the effect of CgGH in transgenic catfish on Vg gene expression. Increasing the expression level of GnRHr gene can stimulate GH secretion, and GH affects LH secretion and further modulates Vg gene expression [17, 35, 41, 42, 44, 51]. Hence, GnRH and GH play a role in regulating LH expression, including the LHr gene. Long photoperiod causes decreased of GnRH and GH expression and leads to decreased LHr expression as shown in both transgenic fish (treatment C) and nontransgenic fish and led to decreased Vg gene expression (Fig. 8A, B). So the long photoperiod has an inhibitory effect on the expression levels of GnRHr, LHr, Vg in both transgenic catfish and nontransgenic fish. There was no difference in Vg expression levels in the nontransgenic fish treated with B and nontransgenic fish treated with C and these results were similar with the expression levels of GnRHr in both treatments (Fig. 6A, B). The expression level of the Vg gene in nontransgenic fish treated B and C was also not significantly different and similar indicated by the GnRHr and LHr expression level in nontransgenic fish which was not significantly different showing that the expression level of the Vg gene depended on the GnRHr and LHr expression level. GnRHr and LHr plays a role in regulating the expression level of the Vg gene [49]. So in nontransgenic fish exposed to short or long photoperiods, the Vg expression level is determined by the LHr and GnRHr expression levels, on the other hand in transgenic fish, the increased Vg expression is affected by a short photoperiod which induces an increase in GnRHr and LHr expression, besides that the presence of CgGH is involved in increasing expression GnRHr and LHr when exposed to a short photoperiod.

4.4 Vg and E2 hormone levels

In sea bass, Senegal sole, a short photoperiod (9L-15D) induced broodstock spawning compared to normal photoperiod (12L-12D) and showed higher Vg levels than long photoperiods (15L-9D). This Vg level was similar to the E2 level, which increases during short photoperiod induction (9L-15D). E2 level reaches its peak just before the spawning period. These results indicate that Vg and E2 levels are interconnected and related to oocyte development and growth during photoperiod induction [18, 52, 53]. The Vg and E2 levels of G5 catfish after short photoperiod induction (8L-16D) in treatment A (mean, 852.53 μg/mL and 0.73 ng/mL, respectively) were higher than in other treatments. In contrast, during a long photoperiod (16L-8D), both Vg and E2 levels (mean, 295.16 µg/mL and 0.25 ng/mL, respectively) in transgenic catfish were higher than in non-transgenic fish (mean, 163.54 μg/mL and 0.14 ng/mL, respectively) (Fig. 9A, B). During the pre-vitellogenic period of C. batrachus, Vg levels increased to 400 μg/mL and peaked at 1500 μg/mL during pre-spawning [31, 54].

Similarly, during the pre-vitellogenic period of C. gariepinus, E2 levels increased to 0.2 ng/mL and peaked at 0.5 ng/mL during the pre-spawning period [55]. The high and low levels of Vg and E2 in G5 transgenic mutiara catfish during photoperiod induction were similar to the Vg gene expression levels of these fish, where the longer dark cycle treatment affected the increase in Vg gene expression, followed by an increase in Vg and E2 levels. Similar to the results of this research, in Atlantic croaker fish (Micropogonius undulatus) reared in dark conditions, melatonin production affects LH secretion directly at the pituitary level and indirectly at the brain level. This suggests that the control of reproductive hormone regulation in the hypothalamic-pituitary pathway is influenced by the dark cycle; LH influences the production of Vg and E2, indicating that the dark cycle is required to initiate oocyte maturation [17, 41, 52, 53]. The Vg gene stimulates Vg production by the liver in response to E2 level fluctuations; hence, both Vg and E2 hormones are used for gonadal maturity status of fish and regulated by hormones or reproductive hormone receptors involved in the hypothalamic–pituitary–gonadal pathway. The dark cycle in treatment C (16L-8D) induces GnRH secretion (GnRHr level expression increased), which stimulates the release of pituitary GH, and affects LH, Vg, and E2 secretion [56]. Exogenous GH indirectly induced Vg and E2 levels in transgenic catfish but not in nontransgenic fish. As a consequence, the Vg, and E2 levels in G5 transgenic mutiara catfish under treatment C were higher than in nontransgenic fish (Fig. 9A, B). Pituitary GH acts on liver tissues (to induce the Vg gene) and gonads (to stimulate E2 secretion), indicating that GH plays a role in stimulating vitellogenesis and steroid hormone production needed for gonad maturation in female fish [19, 44]. Although the long photoperiod (16L-8D) has an inhibitory action on GnRHr and LHr gene expression, however the levels of the hormone vitellogenin in transgenic fish are higher than those in non-transgenic fish as a consequence of the higher level of Vg gene expression in transgenic fish compared to nontransgenic fish (Fig. 8A, B) and consistent with the resulted E2 levels (Fig. 9A, B). This indication shows that in the long photoperiod treatment (16L-8D), the effect of inhibiting Vg gene expression on the secretion of vitellogenin and E2 hormone levels in transgenic catfish was lower than in nontransgenic fish due to the presence of CgGH in transgenic fish.

4.5 Growth and gonadosomatic index of G5 fish

The results of measuring the weight of transgenic and nontransgenic G5 female fish in each photoperiod treatment showed that the average increase in weight gain of transgenic fish during treatment was higher than nontransgenic (Fig. 10A). This increase was a consequence of the presence of the CgGH inserts in G5 transgenic catfish and was confirmed based on the relative expression levels of the CgGH in different photoperiod treatments (Fig. 10B). The CgGH expression level in the short photoperiod (8L-16D) was higher than the 12L-12D and 16L-8D photoperiods which had a similar effect on the average weight gain of female fish as compensation for the presence of the CgGH which increased higher when the dark cycle was induced (short photoperiod). The increase in the dark cycle induces melatonin secretion which acts directly on the hypothalamic–pituitary–gonadal axis to modulate GH and prolactin secretion [57, 58]. Short photoperiods increase melatonin secretion which influences hypothalamic processes involved in the control of the GH secretion [59]. This indication was found in the short photoperiod treatment of G5 transgenic female mutiara catfish, that their growth was higher compared to long photoperiod. Furthermore, it was shown that the increase in growth of G5 transgenic catfish was higher than nontransgenic (Fig. 10A) and was also significantly influenced by CgGH expression when treated with a short photoperiod (Fig. 10B). These results indicate that the dark cycle is higher than the light cycle (8L-16D) can stimulate higher growth in transgenic fish compared to nontransgenic fish.

GSI is an early indicator of sexual maturity in female broodstock [60]. The results showed that Vg levels correlated with oocyte growth, as represented by GSI values at different phases of oocyte development [31, 51, 61]. During photoperiod induction with a longer dark cycle (treatments A and B), the GSI value of female G5 transgenic mutiara catfish was higher than that in treatments C and in nontransgenic catfish (treatment A, B, C) (Fig. 10A, B). These results indicate that ovary growth is enhanced by a longer dark cycle. The mean GSI value in transgenic catfish for treatments A (12.24), B (10.23), C (5.14), and in nontransgenic A (3.93), and B (5.81) were significantly different. The mean GSI value of G5 transgenic mutiara catfish under treatment A and B was higher than that of C. gariepinus exposed to 10L-14D (mean, 6.95) during 55 days of rearing [62]. The increase in dark cycle was followed by an increase in GSI value, which was inseparable from the influence of Vg hormone expression at different photoperiods. The increased Vg gene expression in longer dark cycles indirectly induced an increase in GSI values, as shown in treatments A and B; hence, increased Vg levels (including the Vg gene) were correlated with GSI values. Conversely, in longer light cycles, ovary growth in treatment C (nontransgenic catfish) was slower than in transgenic fish (treatment C), and nontransgenic catfish (A and B) which indicates a decrease in GSI value; this is consistent with the Vg gene expression shown in the photoperiod treatments. Similarly, CgGH, through IGF-1 in transgenic catfish, plays a role in the induction of yolk protein into the oocyte, causing an increase in its size and GSI value changes despite being exposed to a long photoperiod.

The increased Vg gene expression leads to increased Vg levels, protein production for oocytes, and ovarian weight; as a consequence, GSI increases [29, 63, 64]. Vg plays an important role in carrying out vitellogenesis processes, which involve yolk protein accumulation, causing oocyte growth, and ovary enlargement, as represented by GSI [65, 66]. In the short photoperiod treatment on G5 transgenic mutiara catfish, the increase in GSI value was associated with the stimulation of Vg gene expression under dark cycle (treatment A and B). Similar results were observed in rainbow trout (Oncorhynchus mykiss) reared in a short photoperiod (9L-15D), increasing the expression of GnRH, GtH, and Vg genes compared to those under normal photoperiods (12L-12D), leading to oocyte maturation [23]. Indirectly, the GSI value in G5 transgenic and non-transgenic mutiara catfish depends on Vg gene expression level, which is influenced by LHr and GnRHr gene expression levels. The yolk protein into the oocytes causes an increase in GSI, indicating a positive correlation between Vg, E2, and GSI [67, 68]. The GSI value of nontransgenic fish in treatment A, B, C was lower than in transgenic fish in treatment A, B and C, indicating that transgenesis-GH could maintain oocyte growth in transgenic fish. GH can induce the Vg gene in liver tissues (via IGF-1) and gonads (stimulating E2 production), indicating that GH is involved in yolk protein synthesis during vitellogenesis and steroid production to initiate oocyte growth during gonadal maturation [8, 19, 44, 69]. Fish reared in a shorter photoperiod matured faster, indicating that photoperiod signals are not needed to initiate the reproductive cycle, but it is needed for oocyte maturation [17, 18, 52].

This indicates that the oocyte development stage of G5 transgenic mutiara catfish can reach early-late vitellogenic stages when treated with short photoperiods (8L-16D and 12L-12D), whereas long photoperiods (16L-8D) slow down oocyte growth (Fig. 12). Similar results were observed in European sea bass reared at a long photoperiod (15L-9D), where gonad maturity was in “immature” stage, and in Senegal sole fish, where a short photoperiod (9L-15D) led to oocyte development reaching the late vitellogenic (pre-spawning) stage [17, 18]. The oocytes in nontransgenic fish (treatment C) were in the primary growth stage, the oogonium stage, characterized by oogonia proliferation, which is a common feature found during early oocyte growth (Fig. 12C*). In contrast, in treatment C, A*, B* an oogonium containing a nucleus was in the middle of an oocyte follicle, and some oogonium continued to develop to form cortical alveoli (Fig. 12C, A*, B*), showing the growth of primary oocytes to secondary oocytes. In treatment B, the oocyte was in pre-vitellogenic stage, marked with the formation of yolk granule (YG) as a stage of yolk accumulation early in the ovary ripening period (Fig. 12B). In treatment A, mature oocytes developed, which were characterized by the formation of theca cells and granulosa cells, which play a role in the aromatization of androgens to E2 to induce Vg production during early to mature oocytes. YG formation increases, causing an enlargement in oocyte size and migration of germinal vesicle toward the periphery known as Germinal Vesicle Break Down (GVBD), indicating that the oocyte is in the mature stage (Fig. 12A). During vitellogenesis before the oocyte matures, induction at the hypothalamic level is required, especially GnRH levels. This includes an increase in GnRHr expression to stimulate an increase in LH levels (including LHr) as a signal to start the formation of maturing induction hormone in the development of secondary oocytes into ootids [53, 70].

Fig. 12
figure 12

Representative histological sections of G5 transgenic and non-transgenic mutiara catfish at different photoperiods. A (8L-16D); B (12L-12D); C (16L-8D); A* (8L-16D); B (12L-12D); B* (12L-12D); C* (16L-8D). CA cortival aveoli, GV germinal vesicle, N nucleus, PG CA primary growth cortical alveoli, P primary growth of oogonium, GC granulosa cell, TC theca cells, YG yolk granules. Scale bar = 100 μm

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5 Conclusion

This study showed that induction with a short photoperiod increased the expression of genes involved in the oocyte maturation of G5 transgenic mutiara catfish in the hypothalamus-pituitary-liver-gonad pathway. A short photoperiod (8L-16D) induced higher GnRHr, LHr, and Vg expression levels, and increased levels of Vg and E2 hormones, which led to increased GSI and oocyte maturation. In contrast, a long photoperiod (16L-8D) caused a decrease in GnRHr, LHr, Vg gene expression levels, Vg and E2 hormone levels, and GSI, and slower oocyte growth in nontransgenic catfish compared to those in transgenic catfish. Exogenous GH (CgGH) in female G5 transgenic mutiara catfish has the potential to maintain oocyte growth during induction with 16L-8D photoperiod.