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Article

Effects of Exogenous Hormones on Spawning Performances, Serum Gonadotropin and Sex Steroid Hormone in Manchurian Trout (Brachymystax lenok) during Sexual Maturation

by
Yucai Pan
1,2,
Gefeng Xu
1,*,
Tianqing Huang
1,
Wei Gu
1,
Enhui Liu
1 and
Gaochao Wang
1
1
Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture and Rural Affairs, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China
2
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Fishes 2024, 9(7), 269; https://doi.org/10.3390/fishes9070269 (registering DOI)
Submission received: 31 May 2024 / Revised: 2 July 2024 / Accepted: 4 July 2024 / Published: 8 July 2024
(This article belongs to the Special Issue Use of Hormones in Fish Farming)
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Abstract

:
The objective of this study was to investigate the effects of salmon gonadotropin releasing hormone analog (S-GnRH-A) and human chorionic gonadotropin (HCG) on the serum levels of gonadotropins (GtHs) and sex steroid hormones in Manchurian trout during sexual maturity. Females in the treatment group were injected with 20 µg/kg S-GnRH-A and 400 IU/kg HCG, whilst males were injected with half the amount. Blood samples were collected at 0, 5, 10, and 20 days post injection (dpi). The results showed an increase in female follicle-stimulating hormone (FSH) levels in the treatment group at 5 dpi, and from 5 dpi onwards, a difference was observed between the groups, with higher FSH levels in the treatment group compared to the control group. In males, FSH levels showed group differences at only 5 dpi (p < 0.05) and were higher in the treatment group. In females, luteinizing hormone (LH) peaked at 10 dpi in the treatment group, and there were differences between the two groups at 10 dpi and 20 dpi. In males, LH was significantly higher in the treatment group than in the control group at 5 dpi and 10 dpi (p < 0.05). In females, estradiol (E2) was significantly higher in the treatment group than in the control group at 5 dpi and 10 dpi (p < 0.05), whereas in males, the difference between the two groups was only observed at 5 dpi (p < 0.05). The female testosterone (T) level in the treatment group was significantly higher than that of the control group at 5 dpi and 10 dpi (p < 0.05), while T levels were significantly higher in the male treatment group than in the control group at 5 dpi (p < 0.05). The level of 17α–hydroxyprogesterone (17α-OHP) in the females of the treatment group was significantly higher than that of the control group at 5 dpi and 10 dpi (p < 0.05), and 17α-OHP levels were significantly higher in the male treatment group than in the control group at 10 and 20 dpi (p < 0.05). The fish were successfully spawned after S-GnRH-A and HCG injections, and brood amount, fertilization rate, and hatching rate were significantly increased (p < 0.05). This study provides a framework for a better understanding of the mechanisms of exogenous hormone-mediated control of reproduction in Manchurian trout.
Keywords:
Brachymystax lenok; exogenous hormones; gonadotropins; sex steroid hormones; spawning performances
Key Contribution: In this study, S-GnRH-A and HCG were injected into sexually mature Manchurian trout to systematically measure in serum gonadotropins (GtHs); sex steroid hormone levels and spawning performances. The results showed S-GnRH-A and HCG promoted reproduction in Manchurian trout; increasing their egg production; fertilization rate; and hatching rate. In addition, S-GnRH-A and HCG regulated the release of FSH, LH, E2, T, and 17α-OHP in the serum of sexually mature Manchurian trout.

Graphical Abstract

1. Introduction

Fish reproduction is regulated by their internal mechanisms and external environmental factors [1]. Environmental factors (e.g., water temperature, photoperiod, etc.) trigger the action of internal mechanisms. In addition, hormones can also regulate reproductive processes in fish, as represented by the increased synchronization of spawning and accelerated gamete production [2,3]. The internal mechanism controlling the reproductive process in fish is the hypothalamus–pituitary–gonadal axis (HPG) [1,4]. External stimuli activate the HPG axis, which in turn, synthesizes and releases gonadotropin-releasing hormone (GnRH, a decapeptide) via the hypothalamus and converts it into an endocrine signal [5,6,7]. The GnRH stimulation of the pituitary gland leads to the synthesis and release of the protein hormones, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) [6]. During the early stages of spermatogenesis and vitellogenesis, the pituitary gland and blood are predominantly filled with FSH, which stimulates the production of estradiol (E2) and testosterone (T), and regulates gametogenesis [8,9,10]. During gonadal maturation, spermatogenesis, and ovulation, the level of LH in the pituitary gland and blood increases several times compared with FSH, stimulating the production of 17α–hydroxyprogesterone (17α-OHP) [11,12]. 17α-OHP is the precursor of 17α-20β-dihydroxy-4-pregnen-3-one (17-20β-P), which is considered to be the most potent MIH (maturation-inducing hormone) in most teleosts, and MIH promotes the final maturation of sperm and oocytes as well as spermatogenesis and ovulation [13].
Currently, much attention is being paid to hormone induction and artificial insemination in terms of their applicability in farms, as they allow better control of the various steps of the fish reproductive cycle [14,15,16]. For example, human chorionic gonadotropin (HCG) hormone successfully induced and accelerated spawning in African catfish (Clarias gariepinus), and improved reproductive performance with increasing doses of HCG [16]. Previous studies have shown that gonadotropin-releasing hormone analogue (GnRH-A) and HCG stimulate germinal vesicle breakdown (GVBD) in several fish oocytes, and stimulate vitellogenesis and steroid production in mature follicles [16,17]. Therefore, GnRH-A and HCG are widely used for artificial fish reproduction [18,19,20]. However, a great deal of work is needed to perfect the technique of exogenous hormone-induced reproduction in salmonids.
Manchurian trout (Brachymystax lenok), Salmonidae, live in cold waters and are mainly distributed in Northeast China in the Heilongjiang River basin [21]. In natural environments, Manchurian trout tend to live in mountainous rivers, which usually originate high in the mountains and have fast, pure water [22]. Therefore, Manchurian trout require high-altitude conditions for reproduction, and cannot reproduce properly under cultured conditions. Juvenile Manchurian trout grow and mature sexually slowly, reaching a length of about 13–16 cm at 1 year of age and sexual maturity at 3–5 years of age [23]. In the wild, females generally spawn in May–June, the amount of eggs carried is generally 4000–8000 [24]. However, the captive rearing of Manchurian trout usually fails to synchronize spawning, resulting in uneven levels of cultivation, which makes it difficult to ensure the survival rate of fry, which may be due to the inability of the pituitary gland to properly release GtHs to stimulate sex steroid hormone production [6,25,26]. Previously, we induced spawning in wild and artificial cultured mature Manchurian trout by hormone injections and counted the differences between the two in terms of growth rate, number of eggs, and eyestalking rate [27]. Nevertheless, steroid hormone regulation by exogenous hormones in artificial cultured mature Manchurian trout remains unknown. GnRH-A and HCG are effective in stimulating gonadal development and ovulation/spermatogenesis in salmon [8,28]. Therefore, in the current study, salmon GnRH-A (S-GnRH-A) and HCG were injected into sexually mature Manchurian trout to systematically measure in serum gonadotropins (GtHs), sex steroid hormone levels, and spawning performances. This study provides a scientific basis for improving the efficiency of artificial reproduction in trout, including Manchurian trout, and for implementing exogenous hormone stimulation to achieve high spawning and fertilization rates.

2. Materials and Methods

2.1. Experimental Fish

Experimental fish were bred and grown at the Bohai Experimental Station of the Heilongjiang River Fisheries Research Institute (Harbin, China). All the fish were disease-free and sexually mature (Figure 1). The experimental conditions were kept constant at pH 7.3 ± 0.2, 8.5 ± 0.1 mg/L dissolved oxygen, and at a temperature of 10 ± 0.8 °C. The photoperiod was 15 h:9 h light/dark. Fish were fed a commercial diet (TR-2242; VitaCare, Chile). Male (mean weight of 1.42 ± 0.3 kg, n = 18) and female (mean weight of 2.55 ± 0.5 kg, n = 18) Manchurian trout were randomly selected and temporarily reared in a tank with running water for two weeks.

2.2. Hormone Treatment, Fertilization, and Sampling

After acclimation, a total of 36 Manchurian trout were randomly collected from the tank (3500 L) and equally allocated between the two groups. There were 18 fish (9 males and 9 females) in each group. All fish were anesthetized (100 mg/L MS-222; Sigma, St. Louis, MO, USA) during the injection treatment, gamete collection and blood sampling. In the control group, males and females were injected with 1.0 mL/kg 0.75% NaCl. In treatment group, females were injected with 20 µg/kg S-GnRH-A and 400 IU/kg HCG (both hormones were purchased from Ningbo Hormone Products Factory, Zhejiang Province, China); males were injected with half the amount (Figure 2). S-GnRH-A and HCG were injected together, one injection only, at a dose referenced from Mou et al. (2013) [27]. The control and treatment groups were injected with the same doses. The hormones were injected, intraperitoneally through the ventral (bottom) part of the fish behind the pelvic fin. After injection, the fish were stocked into 3500 L tanks and observed for spawning performances.
The bottom of the fish tanks was checked regularly for the presence of released eggs after hormone injections, which indicated the approximate spawning time [29]. For fertilization trails, the males were stripped first. Collect sperm by applying gentle pressure on the abdomen of the fish with a syringe without a needle, the fast movement time (FT) of sperm after water activation was 35.10 ± 6.60, and the life time (LT) was 68.52 ± 5.04. The ovulated fish were anesthetized and stripped by slight pressure on the abdominal region. Three egg samples (300 eggs per sample) from each female were dry fertilized with 0.1 mL of pooled sperm taken from at least four males. After fertilization, the eggs were transferred to an incubator. The incubation conditions were kept constant at pH 7.3 ± 0.2, 8.5 ± 0.1 mg/L dissolved oxygen, and at a temperature of 8 ± 0.5 °C. The photoperiod was 0 h: 24 h light/dark. According to [30], blood samples were collected from males (n = 9) and females (n = 9) at 0, 5, 10, and 20 days post injection (dpi). The blood was collected from the tail vein, and the volume of blood collected was 1 mL per fish. The blood samples were clotted at 4 °C for 4 h, and the serum was precipitated naturally and stored at −25 °C for the determination of FSH, LH, E2, T, and 17α-OHP levels.

2.3. Spawning Performance

The latency period was evaluated as the time between hormone injection and spawning; brood amount was used as an important parameter to evaluate the spawning capacity of female fish [29]. The fertilization rate was calculated as the percentage of the number of fertilized eggs divided by the number of spawned eggs by examining eggs at the cell division stage [31]. Eggs were cleaned in a solution of glacial acetic acid and saline (1:20 v/v), then examined under a stereomicroscope [32]. Cleaved eggs were classified as fertilized. The hatching rate is the percentage of the number of newly hatch larvae divided by the number of spawned eggs; the survival rate was the number of fry that survived after the eggs broke the membrane and surfaced divided by the total number of eggs [33].

2.4. Detecting Steroid Hormone Levels

The E2 (ml003452), T (ml025781), 17α-OHP (ml003449), FSH (ml003455), and LH (ml003456) levels in the Manchurian trout serum were assessed using the enzyme-linked immunosorbent assay (Fish-specific ELISA; MLBIO, Shanghai, China), as used by [34]. The substance to be measured and the biotin-labeled antibody are first incubated simultaneously. After washing, affinity-labeled horseradish peroxidase (HRP) was added. Further incubation and washing removes unbound enzyme conjugates, then substrates A and B are added and act simultaneously with the enzyme conjugates. Color is produced. The depth of color is proportional to the concentration of the substance to be measured in the sample. All samples and standards were run in triplicates (both the CV of inter-assay and intra-assay were less than 10%).

2.5. Statistical Analysis

All data are expressed as the mean ± standard error of the mean (SEM). All data were analyzed using the SPSS statistical package version 22.0 (IBM, Armonk, NY, USA). The Mann–Whitney U test was used to identify the differences between hormones in the control and treated groups. After log-transformation of the hormone data met the assumptions of the test (normality of residuals), hormone data were analyzed using linear mixed models (LMMs). LMMs were used to determine relationships between sex, groups, time, and hormone concentrations. The differences detected were considered significant at p < 0.05. Normality and homogeneity of variances were tested by the Kolmogorov–Smirnov and Bartlett methods, respectively.

3. Results

3.1. Spawning Performances

Fish spawned successfully 4.7 ± 0.6 days post-injection with a fertilization rate of 81.4 ± 1.6% and a hatching rate of 76.7 ± 1.2%, and the Latency period was significantly reduced compared to the control group. The average brood amount of female fish reaches 1780.0 ± 45.6 eggs. After breaking the film, the survival rate of fish fry reaches 96% (Table 1). The brood amount, fertilization rate, and hatching rate of the control group were significantly lower than those of the hormone-treated group (p < 0.05), while there were no differences in survival rates between the two groups (p > 0.05; Table 1).

3.2. Results of Linear Mixed Effects Model

We used LMM to determine the effects of groups, time, and sex on hormone levels. Table 2 shows that the groups, time, and sex had a significant effect on FSH, LH, and E2 (p < 0.05). Time and sex had a significant effect on T (p < 0.05). The groups and time had a significant effect on 17α-OHP (p < 0.05). Overall, the groups and time factors had a greater effect on these five hormones in Manchurian trout.

3.3. Serum Concentrations of FSH

At 5, 10, and 20 dpi, FSH levels of serum were significantly higher in female fish in the treatment group than in the control group (p < 0.05), and FSH levels peaked at 5 dpi; at 0 dpi, the difference in FSH levels between the groups was not significant (Figure 3A; p > 0.05). In males, serum FSH levels were significantly higher in the t treatment group than in the control group at 5 dpi. The differences in serum FSH levels between the two groups of males were not significant at 0 dpi, 10 dpi, and 20 dpi (Figure 3B; p > 0.05).

3.4. Serum Concentrations of LH

In female Manchurian trout, serum LH levels peaked at 10 dpi, and were significantly elevated in the treatment group (p < 0.05); at 20 dpi, serum LH levels decreased in the treatment group but remained significantly higher than those in the control group (Figure 4A; p < 0.05). In males, serum LH levels were significantly higher in the treatment group than in the control group at 5 dpi (p < 0.05), whereas at 10 dpi, serum LH levels peaked in the treatment group and were significantly higher than those in the control group (p < 0.05; Figure 4B).

3.5. Serum Concentrations of E2

In females, serum E2 levels increased and then decreased, with a peak at 5 dpi in the treatment group (Figure 5A). The E2 levels in the treatment group were significantly higher than those in the control group at 5 and 10 dpi (Figure 5A; p < 0.05). In males, serum E2 levels changed moderately during exogenous hormone injections, whereas at 5 dpi E2 levels were significantly higher in the treated group than in the control group (Figure 5B; p < 0.05).

3.6. Serum Concentrations of T

In females, serum T levels peaked at 5 dpi in the treatment group and were significantly higher than those in the control group (Figure 6A; p < 0.05). Similarly, T levels in the treatment group were significantly higher than those in the control group at 10 dpi (p < 0.05), and decreased to a minimum at 20 dpi (Figure 6A). In males, serum T levels peaked at 5 dpi in the treatment group and were significantly higher than those in the control group (Figure 6B; p < 0.05).

3.7. Serum Concentrations of 17α-OHP

In females, serum 17α-OHP levels were significantly higher in the treatment group than in the control group at 5 and 10 dpi (p < 0.05), and equalized between the groups at 20 dpi (Figure 7A). In males, the serum 17α-OHP levels of the treatment group tended to increase gradually, and were significantly higher than those in the control group at 10 and 20 dpi (Figure 7B; p < 0.05).

4. Discussion

In salmonids, GnRH promotes the secretion of FSH and LH, which acts on the gonads to promote the release of sex steroid hormones, and sex steroid hormones can also affect the synthesis and secretion of FSH and LH through negative feedback on the brain and pituitary gland [35,36,37]. The synthesis of sex steroid hormones is promoted by both FSH and LH, with the difference being that FSH can affect vitellogenesis and spermatogenesis, while LH promotes follicular maturation and ovulation [5,36]. In general, gonad development, maturation, gamete release, and fertilization naturally occur in the natural environment of wild Manchurian trout. However, when Manchurian trout are transferred to captive environments, their reproduction can be limited [22,27]. Therefore, elucidating the exogenous GnRH-induced endocrine regulatory mechanisms of gonadal development and maturation in Manchurian trout is important for their reproduction and breeding. This study is the first to have quantified the effect of exogenous hormones on the changes in the serum concentrations of GtHs and sex steroids over time in sexually mature Manchurian trout.
The results of the present study showed that, compared to the control group, serum levels of FSH and LH in the treatment group significantly increased in females and peaked at 5 and 10 dpi, respectively, suggesting that S-GnRH-A synergistic HCG exert a GnRH-like function by inducing the release of FSH and LH from the pituitary through their receptors. Furthermore, female LH levels peaked at 10 dpi, which was consistent with the positive correlation between LH levels and spawning in response to the exogenous hormone stimulation found in previous reports [38,39]. The serum FSH levels in the male treatment group were significantly higher than those in the control group at 5 dpi, which is similar to that reported by Woo et al. (2021) [30], suggesting that S-GnRH-A synergistic HCG also has a contributory effect on FSH secretion in males. Compared to the control group, LH levels in males increased significantly in the treatment group and peaked at 10 dpi, indicating that LH was induced by S-GnRH-A synergistic HCG stimulation, thus promoting sperm maturation. Mylonas et al. (1997) [40] found that injecting striped bass (Morone saxatilis) with the exogenous hormone GnRH resulted in a sustained increase in serum LH levels, which is similar to the results of the present study.
In teleosts, sex steroid hormones play important reproductive regulatory functions, such as E2, 17α-OHP, and T, which regulate several reproductive processes [41]. The results of the present study showed that serum E2 levels in the treatment group peaked at 5 dpi in females, and increased significantly compared to the control group. E2 is a steroid hormone secreted by the ovaries and is known to be the main estrogen responsible for regulating the sexual characteristics and ovulatory cycle of female fish [42]. A similar study performed in grass carp (Ctenopharyngodon idellus) reported that treatment with S-GnRH-A and HCG resulted in a substantial increase in serum E2 levels, along with accelerated oocyte maturation and successful spawning induction [43]. Ovarian E2 levels are also significantly increased in eel (Anguilla japonica) after exogenous hormone treatment [44], and these findings are similar to those of the present study, indicating that the S-GnRH-A synergistic HCG may have activated the sex hormone regulatory pathway in Manchurian trout at sexual maturation, which in turn induced the proliferation of Manchurian trout oocytes. The changes in serum E2 levels in females, in the present study, were similar to the changes in the serum levels of FSH and LH, suggesting that E2 may function synergistically with hormones secreted by the pituitary gland [45]. Previous studies have shown that E2 is not only an important female sex steroid hormone but is also indispensable in male reproduction, as it regulates the physiological functions of male spermatogonia and sertoli cells [41,46]. In this study, E2 levels were significantly higher in the male treatment group than in the control group at 5 dpi. A similar situation was observed in exogenous hormone-induced reproduction in Pseudorasbora parva, suggesting that S-GnRH-A synergistic HCG treatment affects male E2 production [47]. In males, E2 is typically present in the serum at low levels and appears to increase during the first few months of the reproductive cycle, followed by a gradual decrease [48]. Currently, little is known about the role of E2 in regulating spermatogenesis in fish [49], although it is thought to regulate the renewal of spermatogonial stem cells [50].
In the present study, serum T levels in female Manchurian trout were significantly elevated and peaked at 5 dpi after exogenous hormone injection, which is generally consistent with previous studies on grass carp [43], goldfish (Carassius auratus) [14], and gilt-head seabream (Sparus aurata) [41]. It was previously hypothesized that the sex hormone T plays a key role in stimulating the growth of small follicles in fish oocytes as it increases the oocyte diameter and promotes development in eels and Atlantic cod (Gadus morhua) [51,52]. Thus, the rapid increase in T at 5 dpi suggests a positive effect on the growth and development of Manchurian trout oocytes, but this requires further study and validation. In the present study, T and E2 changes in female Manchurian trout showed a similar trend, which may be due to S-GnRH-A synergistic HCG-inducing T production in the follicular membrane layer through a cascade reaction (HPG axis), thus stimulating E2 production in intact follicles at the central germinal vesicle (GV) stage [6]. However, the level of T started to decrease in the late injection period (20 dpi), and the difference between the treatment and control groups was not significant, which may be related to ovarian degeneration after S-GnRH-A synergistic HCG stimulation of ovulation in Manchurian trout. In parallel with female T serum levels, male T levels were also significantly elevated at 5 dpi, similarly to the results reported for most salmonids [6,35,39], suggesting that S-GnRH-A synergistic HCG can increase the level of T in male Manchurian trout, which in turn promotes them for gametogenesis. During this period, the release of male Manchurian trout T into the bloodstream may be involved in feedback effects in the brain and pituitary gland and as a progenitor for the synthesis of other steroid hormones directly involved in the control of spermatogenesis [53]. However, we observed that serum T levels in the treatment group were significantly reduced at 20 dpi, as described in gilt-head seabream (Sparus aurata), which may be related to the degradation of the spermathecae in male fish after sperm discharge [41,54].
17α-OHP is the precursor of 17-20β-P, known as the hormonal steroid that induces the final maturation of oocytes [13]. In the present study, the changes in serum 17α-OHP levels of females in the treatment group showed a tendency to increase and then decrease, and were significantly higher than the control group at 5 dpi. Previous studies have reported elevated levels of 17α-OHP following the in vitro treatment of fully vitellogenic oocytes of the longchin goby (Chasmichthys dolichognathus) with diethylstilbestrol (DES) [55]. In addition, synthetic salmon GnRHa, gonadorelin, and carp pituitary extract (CPE) all significantly increased the 17α-OHP levels in mature female tambaqui (Colossoma macropomum) and induced ovulation [56], which is similar to the results of the present study. Similar results were also reported in rainbow trout (Oncorhynchus mykiss) [57]. This suggests that 17α-OHP may enhance the maturation of oocytes and ovulation in female Manchurian trout under the stimulation of S-GnRH-A synergistic HCG. Previous studies have reported that blood levels of 17α-OHP were significantly higher in mature male eels from HCG- and CPE-treated groups than in the control group, and that 17α-OHP levels in the treated groups peaked at the time of sperm release and decreased 10 days after sperm release [58]. In this study, S-GnRH-A and HCG promoted the production of 17α-OHP in male Manchurian trout, which would contribute to spermatogenesis.
In the present study, the overall spawning performance of the control group was lower than that of the hormone-treated group. When S-GnRH-A and HCG were given in combination, the latency period was 4.7 ± 0.6 days, the average brood amount was 17,800.0 ± 450.6, and the fertilization rate was 81.4 ± 1.6%. Thus, combined S-GnRH-A and HCG injections appear to be available for future reproduction and aquaculture applications, as saline treatment does not appear to induce the release of sufficient amounts of gonadotropins for optimal spawning, and therefore resulted in a lower spawning success. It has been shown that the latency period varies greatly among species and depends on both intrinsic and environmental parameters, as well as the type of hormone or dose [33,59]. This is supported by the secretion of sex steroid hormones, where FSH, E2, and 17α-OHP were significantly elevated in females after S-GnRH-A and HCG treatments. In addition, the longer latency period in the female fish treated with saline group may be related to the fact that sex steroid hormone secretion was low, and therefore, why spawning was delayed. Consistent with our findings, reduction in the latency period resulting from exogenous hormone treatment has been reported in other species, such as meager (Argyrosomus regius) and Levantine scraper (Capoeta damascina) [33,60]. In addition, brood amount, fertilization rate, and hatching rate were significantly increased by the induction of S-GnRH-A and HCG. The effects of mGnRH and HCG on spawning induction is in accordance with our study, showing that these gonadotrophs increased spawning, fertilization and hatching rates in golden rabbitfish (Siganus guttatus) [32]. In Levantine scraper, the injection of S-GnRH-A +domperidone also increased the female brood amount. These same findings have also been reported for other species [14,30,61].

5. Conclusions

We found that S-GnRH-A and HCG can promote the reproduction of Manchurian trout and improve the brood amount, fertilization rate, and hatching rate. Also, S-GnRH-A and HCG had a stimulatory effect on the release of FSH and LH in the serum of sexually mature female Manchurian trout, and similarly on the secretion of LH in males. In addition, S-GnRH-A and HCG effectively stimulated the generation of sex steroid hormones (E2, T and 17α-OHP) in females, and also promoted the generation of T and 17α-OHP in males.

Author Contributions

Y.P.: Writing—original draft, writing—review and editing, formal analysis, data curation. G.X.: funding acquisition, investigation, resources. T.H.: project administration, resources. W.G.: conceptualization, data curation. E.L.: investigation, methodology. G.W.: resources, software. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by China Agriculture Research System of MOF and MARA (Grant/Award Number: CARS-46) and Central Public-interest Scientific Institution Basal Research Fund (Grant/Award Number: NO. 2020TD32).

Institutional Review Board Statement

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the Animal Care and Use Committee of the Chinese Academy of Fishery Sciences (ACUC-CAFS) (approval code: 2019-03-15) approval has been received. Fish handling and blood sampling procedures were conducted in accordance with the standard principles for laboratory animals, and the study was performed in accordance with the European Communities Council Directive (86/609/EEC). All fishes involved in this research were bred according to the guidelines of the Animal Husbandry Department of Heilongjiang, PR China.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fishes 09 00269 g001
Figure 1. The sexually mature Manchurian trout: (A) Male Manchurian trout for experiments; (B) Female Manchurian trout for experiments; (C) The abdomen of the male Manchurian trout; and (D) The abdomen of the female Manchurian trout.
Figure 1. The sexually mature Manchurian trout: (A) Male Manchurian trout for experiments; (B) Female Manchurian trout for experiments; (C) The abdomen of the male Manchurian trout; and (D) The abdomen of the female Manchurian trout.
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Figure 2. The Manchurian trout were intraperitoneally injected with S-GnRH-A and HCG.
Figure 2. The Manchurian trout were intraperitoneally injected with S-GnRH-A and HCG.
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Figure 3. Serum levels of FSH in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are the mean ± SEM. Different uppercase letters represent significant difference along the time series and different lowercase letters represent significant differences across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
Figure 3. Serum levels of FSH in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are the mean ± SEM. Different uppercase letters represent significant difference along the time series and different lowercase letters represent significant differences across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
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Figure 4. Serum levels of LH in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
Figure 4. Serum levels of LH in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
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Figure 5. Serum levels of E2 in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote a significant difference if p < 0.05. dpi, days post injection.
Figure 5. Serum levels of E2 in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote a significant difference if p < 0.05. dpi, days post injection.
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Figure 6. Serum levels of T in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
Figure 6. Serum levels of T in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent a significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
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Figure 7. Serum levels of 17α-OHP in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are the mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
Figure 7. Serum levels of 17α-OHP in female (A) and male (B) Manchurian trout at 0, 5, 10, and 20 dpi. Values are the mean ± SEM. Different uppercase letters represent a significant difference along the time series and different lowercase letters represent significant difference across treatments within the same time point. Letters denote significant difference if p < 0.05. dpi, days post injection.
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Table 1. Spawning performances of Manchurian trout injected with S-GnRH-A and HCG, given in combination.
Table 1. Spawning performances of Manchurian trout injected with S-GnRH-A and HCG, given in combination.
HormonesLatency Period (Days)Brood Amount (Eggs)Fertilization Rate (%)Hatching Rate (%)Survival Rate (%)
Control (saline)7.5 ± 0.8 a1236.0 ± 35.6 b39.7 ± 1.3 b42.7 ±1.3 b94.8 ± 1.3 a
S-GnRH-A and HCG4.7 ± 0.6 b1780.0 ± 45.6 a81.4 ± 1.6 a76.7 ± 1.2 a96.5 ± 1.5 a
Note: The Mann–Whitney U test was used to determine the difference between the control and treatment groups. Different letters indicate significant differences between treatment and control groups (p < 0.05).
Table 2. Effect of groups, time, and sex on hormone secretion.
Table 2. Effect of groups, time, and sex on hormone secretion.
HormonesPredictorsEstimateSEtp
FSHGroups−1.6700.339−4.920<0.001
Time (dpi)−0.4400.151−2.902<0.001
Sex0.4980.3321.4680.006
LHGroups−16.3220.497−3.3270.002
Time (dpi)4.6440.2192.1170.040
Sex12.8381.932.6170.012
E2Groups−11.0920.529−2.0940.042
Time (dpi)4.8151.3642.0320.048
Sex17.0801.2913.2240.002
TGroups−41.0440.902−2.1580.038
Time (dpi)1.8200.8500.2140.008
Sex−64.781.921−3.4060.001
17α-OHPGroups−1.5130.338−4.473<0.001
Time (dpi)−0.5810.151−3.843<0.001
Sex0.1950.3510.5780.567
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Pan, Y.; Xu, G.; Huang, T.; Gu, W.; Liu, E.; Wang, G. Effects of Exogenous Hormones on Spawning Performances, Serum Gonadotropin and Sex Steroid Hormone in Manchurian Trout (Brachymystax lenok) during Sexual Maturation. Fishes 2024, 9, 269. https://doi.org/10.3390/fishes9070269

AMA Style

Pan Y, Xu G, Huang T, Gu W, Liu E, Wang G. Effects of Exogenous Hormones on Spawning Performances, Serum Gonadotropin and Sex Steroid Hormone in Manchurian Trout (Brachymystax lenok) during Sexual Maturation. Fishes. 2024; 9(7):269. https://doi.org/10.3390/fishes9070269

Chicago/Turabian Style

Pan, Yucai, Gefeng Xu, Tianqing Huang, Wei Gu, Enhui Liu, and Gaochao Wang. 2024. "Effects of Exogenous Hormones on Spawning Performances, Serum Gonadotropin and Sex Steroid Hormone in Manchurian Trout (Brachymystax lenok) during Sexual Maturation" Fishes 9, no. 7: 269. https://doi.org/10.3390/fishes9070269

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