The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Chinese herbal medicine is rich in active ingredients that can promote growth and enhance immune function. In this study, Lycium barbarum, Panax ginseng, Astragalus membranaceus and Phragmitis rhizoma were crushed and mixed to prepare a compound Chinese herbal medicine. The basic feed of Megalobrama amblycephala was supplemented with 0 (control group), 1% (T1), 2% (T2) and 4% (T3) of this compound medicine. After raising for 90 days, in the T1 and T2 experimental groups, the length and width of intestinal villi and the activities of amylase, trypsin and lipase in the intestine were significantly higher than those in the control group. The weight gain rate and specific growth rates were highest and the feed coefficient was lowest in the T2 experimental group. In the control group, a large number of dilated hepatic sinusoids were detected, while this number significantly decreased in the T1 experimental group and they were not detected at all in the T2 and T3 experimental groups. The spleen and liver body indices were highest in the T2 experimental group. In all experimental groups, the Lys content and the activities of T-SOD, CAT, ACP, AKP and GSH-PX in serum were significantly higher than those of the control group. The expression of IgM, C3, TNF-ɑ and IL-1β in the head kidney; C3, TNF-ɑ and IL-1β in the spleen; C3 and IL-1β in the gills; IgM, C3 and IL-1β in liver; and IL-1β in the intestine was highest in the T2 experimental group. After challenge with Aeromonas hydrophila, the cumulative mortality rate of M. amblycephala was lowest in the T2 experimental group. The results of this study indicated that this compound Chinese herbal medicine could significantly enhance immunity, increase the activity of intestinal digestion-related enzymes and promote the growth of M. amblycephala. The appropriate addition amount of this compound Chinese herbal medicine in the basic feed of M. amblycephala was 2%.

Immunoglobulin M (IgM) is widely recognized as the major isotype of teleost immunoglobulins. Its role in protecting fish against a number of pathogens is critical. In the current study, the IgM heavy chain gene of Siniperca kneri Garman was cloned for the first time. Its IgM heavy chain cDNA is 1952 bp in length and encodes a polypeptide of 583 amino acid residues, which includes a signal peptide, a variable region (VH) and four constant regions (CH1, CH2, CH3 and CH4). This confirms the secretory pattern of IgM (sIgM). The sIgM amino acid sequence shares high identity with those of Siniperca chuatsi (88.57%), Lateolabrax japonicus (65.73%) and Latris lineata (64.92%). Analysis of phylogenetic trees shows that the sIgM of S. kneri Garman is clustered with perciformes fish homologues and constructed as one clade. QRT‐PCR analysis found the highest sIgM expression in the head kidney, with higher expressions in the spleen and liver than in the brain, intestine, mesonephros, gill, skin and heart. After Aeromonas hydrophila injection, the sIgM expression pattern was up‐regulated and peaks occurred in the head kidney, spleen, intestine and gill on days 5, 7 and 21 post‐challenge. Changes in the peak expression of sIgM compared to controls were most significant in the head kidney (38.59‐fold) and spleen (23.75‐fold), with smaller differences in the gill (9.54‐fold) and intestine (7.75‐fold). This suggests that it is possible that sIgM is of greater importance to systemic immunity than mucosal immunity. Its importance to the adaptive immune response is thereby confirmed.

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide involved in the regulation of reproductive function in vertebrates. It is able to inhibit the synthesis and secretion of GnRH and Gth in the HPG axis. However, the regulatory role and mechanism by which current gonadal steroid hormones regulate GnIH are still unclear. In this study, transcription factor binding site analysis was performed on the promoter sequence of zebrafish GnIH. Whereafter, transgenic zebrafish (GnIH: mCherry) accurately labeled GnIH and esr2b knockout zebrafish, which were constructed previously, were used to explore the regulation between estrogen and GnIH. In vitro exposure of wild-type zebrafish embryos and transgenic zebrafish embryos to estradiol showed that 10 μM estradiol significantly increased the transcription level of GnIH. However, both 10 μM and 50 μM estradiol significantly increased the transcription level of GnIH in esr2b knockout zebrafish. We compared the expression levels of GnIH in esr2b knockout zebrafish and wild-type zebrafish at different developmental stages (48 hpf–120 hpf). The results showed that from 96 hpf, the expression level of GnIH in esr2b knockout zebrafish was significantly higher than that in wild-type zebrafish, indicating that esr2b is involved in the negative regulation of GnIH, and this regulatory relationship is established on the fourth day of zebrafish development.