This experiment was undertaken in accordance with the guidance, care, and use of laboratory animals in China with approval from the Committee on the Ethics of Animal Experiments of Guangdong Ocean University.

Two-year-old female spotted scats were bought from the Zhuhai HengDa Cultivation Base (Guangdong, China). The sex ratio of spotted scats is approximately 1:1 (Mustapha et al., 2021). The female spotted scats were empirically selected from the mixed-sex fish by the sexual morphological difference in the head, anal fin, body color, body weight, and genital pore (Barry and Fast, 1992). The fish were transferred to the Donghai Island experimental base, Guangdong province, China. The fish were reared in a concrete pond (12 m × 5 m × 2 m) and adapted to the rearing condition for 2 weeks. Aeration was supplied to the tank through air stones, and the average water temperature was 31.1 ± 0.8°C. The salinity was 8‰, while pH and dissolved oxygen ranged from 7.5–8.5 and 6.0–7.0 mg/L, respectively. Fish were fed 2.5% of their body weight with commercial fish pellets consisting of 43% crude protein and 12% crude lipid. Fish were fed two times a day at 06:00 and 18:00 h throughout the adaptation period. The pond water was not recirculated. Approximately, 20% of the water in the pond was drained off and replaced with fresh water once in 3 days.

After the adaptation period, 100 females (average initial weights of 242.83 ± 50.90 g, and length of 19.48 ± 1.13 cm) were randomly distributed into two treatment groups (50 fish in each group) containing different levels of fish oil (0% and 8%; ingredients are listed in Table 1). The diet containing 8% fish oil was marked as the FO group, while the diet of the control group containing 8% soybean oil marked the SO group. Each treatment group was duplicated and contained 25 fish in each net cage (5 m × 3.5 m × 1.8 m). Two independent concrete ponds (12 m × 5 m × 2 m) were used; one concrete pond had two net cages for both FO and SO treatments. The fish were fed with the two diets for 40 days before sampling. The culturing condition was consistent with the adaptation stage.

At the termination of the experiment, the spotted scats were sampled after 24 h starvation. Five fish per cage were sacrificed for sampling. The final number of fish, body weight, body length, gut weight, liver weight, and gonad weight were taken individually. The survival rate (SR), condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI), and gonadosomatic index (GSI) were calculated using the following formulas:

SR (%) = final number of fish/initial number of fish × 100;

CF = final body weight (g)/final body length (cm) ³ × 100;

VSI (%) = final viscera weight (g)/final body weight (g) × 100;

HIS (%) = final liver weight (g)/final body weight (g) × 100;

GSI (%) = final gonad weight (g)/final body weight (g) × 100.

The liver and ovary samples of the fish were excised and quickly frozen in liquid nitrogen. Blood samples were collected from the caudal vein of the fish. The blood samples were stored at 4°C overnight and centrifuged (at 3,000 rpm for 10 min at 4°C), and the upper layer serums were collected. All samples were subsequently stored at −80°C for further analysis.

The total lipid from the ovary and liver (n = 6 per group) and feed were extracted using diethyl ether after acid hydrolysis, whereas lipid from the serum (n = 6 per group) was extracted with chloroform according to the procedure of the National Standards of the People’s Republic of China (5009.168—2016). Extracted lipid was saponified with NaOH-methanol. Their constitutive fatty acids were methylated through the action of boron trifluoride-methanol, separated using Agilent 7890B gas chromatograph (Agilent Technologies, Santa Clara, CA, United States) on a capillary column (SPTM-2560; 100 m × 0.25 mm × 0.25 mm), and quantified using glyceryl triundecanoate as an internal standard.

To evaluate the effects of fish oil on the genes expressed in the liver, the transcriptome analysis was carried out. First, total RNA (n = 6 per group) from the two groups was extracted from fish liver using RNeasy^® Mini Kit reagents according to the instructions of the manufacturer (Qiagen GMBH, Hilden, Germany). The RNA quality was determined and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States) and checked using the RNase-free agarose gel electrophoresis. The mRNA was enriched by oligo (dT) beads; the mRNA was interrupted. The first strand of cDNA was synthesized in M-MuLV reverse-transcriptase system, and the second strand was synthesized from dNTPs in DNA polymerase I system. Then, the cDNA fragments were ligated to Illumina sequencing adapters. The ligation products were size selected using agarose gel electrophoresis, PCR amplified, and sequenced using novaseq 6000 by Guangzhou Genedenovo Biotechnology Co. (Guangzhou, China). All clean libraries of sequencing data were submitted to the NCBI Sequence Read Archive (SRA) database (Accession No: PRJNA765892).

To ensure that high-quality data are achieved, the original data were filtered before analyzing. The raw reads were filtered using fastp (version 0.18.0). The adapters read with more than 10% unknown nucleotides and over 50% low-quality nucleotides (Q-value ≤ 20) were removed. The ribosome RNA (rRNA) reads were removed with the alignment tool Bowtie2 (version 2.2.8). Then, these clean reads were mapped to the spotted scat reference genome (Huang et al., 2021) with HISAT2.2.4. Genes with unknown transcripts or in intergenic regions were defined as novel genes. The fragments per kilobase of transcript per million mapped reads (FPKM) method was used to estimate the gene expression levels. Differentially expressed genes (DEGs) in two groups were identified using the EdgeR software package. In comparison, to avoid some important genes being filtered out, genes with | log2 (fold change)| > 1 and P < 0.05 were identified as significant DEGs. DEGs were annotated with the enrichment analysis of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The KEGG pathway with P < 0.05 was regarded as a significant enrichment pathway.

To validate the gene expression profiles from the RNA-seq analysis, 24 differentially expressed genes related to fat metabolism and ovarian development were selected for the real-time PCR analysis. Real-time PCR was performed using the PerfectStart^TM Green qPCR SuperMix (TRAN, China). The PCR reaction was carried out in a total volume of 20 μl with 10 μl of 2 × PerfectStart^TM Green qPCR SuperMix, 0.4 μl of each primer, and 2 μl cDNA. The thermal cycling program included an initial denaturation at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 60°C for 15 s, and 72°C for 10 s. The reference gene, β-actin, was used to normalize the expression values. The 2^–ΔΔCt method was used to calculate the relative expression of the target genes. Each group contains 10 independent samples, and each sample was detected in triplicate. Primer sequences used for real-time PCR are listed in Supplementary Table 1.

The content of serum Vtg was detected using Grouper Vitellogenin ELISA Kit according to the instructions of the manufacturer (Cusabio, Wuhan, China). The serum samples from 10 fish in each group were analyzed.

All data are expressed as means ± standard error (SE), and an independent sample t-test was used with the significance level at P < 0.05. The statistical analysis of the data was performed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, United States).

The DHA, EPA, and ARA contents in FO diet are higher than that of the SO diet. On the contrary, the oleic acid (C18:1n-9) and linoleic acid (C18:2n-6) contents in FO diet are lower than that of the SO diet (Table 2). The differences are mainly attributed to their different oil sources.

Table 3 shows fatty acid composition in different tissues of female spotted scat. In the ovary, fish oil treatment significantly increased (P < 0.05) the EPA concentration. Also, in the liver, fish oil treatment significantly decreased (P < 0.05) the C18:3n-6 concentration and increased the EPA concentration. In the serum, fish oil treatment significantly increased (P < 0.05) C14:0, C15:0, C16:1n-7, C17:0, C22:1n-9, EPA, C24:0, and DHA concentration, while fish oil treatment significantly decreased (P < 0.05) linoleic acid, C18:3n-6, C20:2-6, and C20:3n-6 concentration.

At the end of the experiment, no fish died in both FO and SO groups. The final body weight, body length, CF, GSI, and VSI of the SO and FO fish are similar (P > 0.05, Table 4). The HSI of fish in FO group was significantly lower than that of the SO group (P < 0.05).

Twelve cDNA libraries were constructed from livers from the SO and FO groups. In this experiment, a total of 534,586,540 (i.e., 289,243,632 in the SO group and 245,342,908 in the FO group) clean reads were obtained from the transcriptome sequencing. The percentages of Q30 bases were more than 93% for all the samples, indicating a high-quality sequence (Table 5). The results showed a total mapped reads of 497,792,649, and the percentage of mapped reads for each library ranged from 91.21 to 94.36% (Table 5). A total of 20,574 genes were detected, including 20,238 known genes and 336 novel genes, accounting for 83.44% of the reference genomes. Supplementary Table 2 shows the number of genes detected in each sample.

In this study, a total of 764 genes were annotated to be DEGs (P < 0.05, | log2(fold change)| > 1). Compared with the SO group, 497 genes were upregulated in the FO group, while 267 genes were downregulated in the FO group (Figure 1).

In this study, GO and KEGG enrichment analyses were carried out to explore the biological functions of the DEGs. The DEGs were annotated into GO categories of the cellular component, biological process, and molecular function (Figure 2). In the cellular component categories, organelle (61), cell (74), cell part (74), membrane part (45), and membrane (50) were the most enriched GO terms. In the biological process categories, cellular process (144), single-organism process (128), metabolic process (124), biological regulation (91), and response to stimulus (60) were the most enriched GO terms. In the molecular function categories, binding (140), catalytic activity (89), molecular transducer activity (22), and nucleic acid binding transcription factor activity (17) were the most enriched GO terms.

The top 20 biological pathways revealed in KEGG analysis are shown in Figure 3. The DEGs are mainly classified into cellular processes, environmental information processing, metabolism, organismal systems, and human diseases. Key genes for steroid biosynthesis (i.e., Lss, Hsd17b7, Tm7sf2, and Nsdhl, etc.), oocyte meiosis (i.e., Ccnb2, Ppp2ca, etc.), cell cycle (i.e., Cdc14ab, Myc, Cdkn1a, and Gadd45b, etc.), FoxO signaling pathway (i.e., Pck1, Foxo1a, Sgk3, Bnip3, and Bcl6, etc.), MAPK signaling pathway (i.e., Dusp8, Jund, Hsp70, Epha2, Jun, Fos, Relb, Mapk8ip1, and Fgfr1a, etc.), PPAR signaling pathway [i.e., Acsbg2, Stearoyl-CoA desaturase 1b (Scd1b), and Fabp2, etc.], phagosome (i.e., Calr, Mr1, etc.), as well as additional crucial genes that play roles in amino sugar and nucleotide sugar metabolism (i.e., Cmah, Chia, etc.) were identified. In addition, DEGs fell into the categories of the interaction of cytokine-cytokine receptor (i.e., tumor necrosis factor receptor superfamily member 6B-like isoform X2, Ackr3, etc.), Herpes simplex infection [i.e., Socs3, aryl hydrocarbon receptor nuclear translocator-like protein 2 (Arntl2), etc.], and progesterone-mediated oocyte maturation (i.e., Hsp90a.1, etc.).

Twenty-four genes related to ovarian development and fat metabolism from RNA-seq were verified using real-time PCR (Figure 4). The results showed that the expression levels of the selected genes were consistent with RNA-seq, suggesting that the RNA-seq results are reliable.

The mRNA expression levels of Vtga and Vtgb in livers of the FO group are higher than that of the SO group. The Vtg protein level in the serum was detected. The results showed that the serum Vtg level in the fish from the FO group was significantly higher than that of the SO group (P < 0.05; Figure 5). The results at the mRNA and protein levels are consistent.

The growth of the fish was not affected by the oil source during the 40-day experimental stage. In sharpsnout seabream (Diplodus puntazzo), replacing fish oil with vegetable oil did not affect growth after 3 months of feeding (Piedecausa et al., 2007). However, in cobia (Rachycentron canadum), the proportional replacement of fish oil with soybean oil gradually increased (i.e., 0% SO, 33% SO, 67% SO, and 100% SO) and resulted in a gradual decrease in weight gain (Trushenski et al., 2011). The discrepancy could be related to the differences in fatty acid requirement among species (Xu et al., 2015). The current study suggests that spotted scat can be fed with a diet containing lower fish oil for normal growth. That is probably because spotted scat is omnivorous fish species. Of course, we could not conclude that the spotted scat does not need LC-PUPA for normal growth, as residual fish oil in fish meal could supply the LC-PUPA in the SO group. Decreased HSI in the FO group reveals that a high level of dietary n-3 PUFAs significantly reduced fat accumulation in the liver. It is well known that adult zebrafish suffer from hepatic adipose infiltration, which might influence reproduction and offspring (Virote et al., 2020). The liver with less fat tissue should be healthier, and the healthy liver might have a stronger ability to synthesize the proteins and lipids, such as the Vtg and Zp proteins required for gonadal development. In female spotted scat, the higher GSI values were observed in the reproduction season, indicating the maturing of the ovary (Gandhi et al., 2014). Consistently, the GSI in the FO group is relatively higher than that of SO group, although there was no statistical difference (P = 0.306). The possible reason for the large standard error between groups is the genetic background of the fish affected the regulation of lipid metabolism (Morais et al., 2011). These results suggest that a high level of dietary n-3 LC-PUFA might be beneficial to a healthy liver and promote ovarian development in female spotted scat.

The liver transcriptome analysis was carried out to confirm whether the high level of LC-PUPA would benefit the reproduction via influencing the liver genes. Vitellogenesis- and zonagenesis-related genes are induced by the estrogen in the liver to synthesize Vtg and zona radiata proteins (Zrp), respectively. Vtg and Zrp are secreted and transported from blood to the ovary and are taken up by the maturing oocytes (Arukwe and Goksøyr, 2003; Levi et al., 2009). Vtga, Vtgb, and Vtgc are all expressed increasingly during the ovary maturation from phase II to phase IV in the female liver of spotted scat (Cui et al., 2017). In this study, both transcriptome and real-time PCR data showed that Vtga and Vtgb are highly expressed in the FO group. Consistently, the serum Vtg protein is also higher in the FO group than that of the SO group. Zp4 is one of the genes controlling the making of Zp (in mammals) or vitelline envelope (in fish, amphibians, and birds), a glycoprotein layer surrounding the oocytes (Laine et al., 2019). In addition, Zp4 expression is significantly upregulated in the livers of FO fish. Taken together, the fish oil might promote ovary development by upregulating genes critical for vitellogenesis (i.e., Vtga and Vtgb) and zonagenesis (i.e., Zp4) in spotted scat.

The estrogen signaling pathway is mediated by the nuclear receptors. Three estrogen receptors, erα, erβ1, and erβ2, were characterized in many fish species, including spotted scat (Cui et al., 2017). Although, the three estrogen receptors are expressed in the female livers, their expressions were not affected by the high level of LC-PUPA in the presents study. In contrast, the liver estrogen level might be increased by the upregulation of the Hsd17b7. Hsd17b7 could transform estrone to estradiol in vitro (Ohnesorg et al., 2006). Similarly, fish oils upregulate 17β-hsd gene expression in the testicular tissues of rats (Mohammad et al., 2015). The regulatory mechanism of fish oil on Hsd17b7 expression remains to be elucidated.

Hsp90 is a ubiquitously expressed molecular chaperone of estrogen receptors and is involved in the signal transduction process of estrogen receptors (Chang et al., 2014). Herein, the Hsp90 expression was downregulated in the FO group. Whether less Hsp90 would either promote or inhibit the estrogen signaling pathway is still unclear. Considering the upregulation of Vtgs and Zp4 expressions in the FO group, the results suggested that less Hsp90 might enhance the estrogen signaling pathway. The possible mechanism is that decreasing Hsp90 would increase the number of free estrogen receptors. Those free receptors would combine with estrogen to be translocated into the nuclear to take action. The aryl hydrocarbon nuclear translocator (Arnt) has been found to be a coactivator of erα- and erβ-dependent transcription (Brunnberg et al., 2003). The Arntl2 gene has been considered a candidate gene in the molecular regulation of reproduction seasonality (Zhao W. et al., 2020). However, its expression was decreased in the FO group. So, the n-3 LC-PUPA might have a diverse influence on the expression of the genes involved in the network of the estrogen signaling pathway. The total output of the estrogen signaling pathway in the female liver should be enhanced in the group that contains higher level n-3 LC-PUPA.

The forkhead transcription factor Foxo1 is a critical regulator of hepatic lipid and glucose metabolism, and it functions in hepatic lipid homeostasis (Shin et al., 2012). The DHA supplemented diets reduced the expression of Foxo1 in the liver of pigs after 30-day feeding experiment (Chen et al., 2012). In contrast, EPA was more effective in reducing Foxo1 expression than DHA in rats after an 8-week feeding experiment (Hong et al., 2019). DHA or EPA alone or together could reduce the expression of Foxo1 in 3T3-L1 adipocytes in vitro (Martins et al., 2020). In mice, gain and loss of function proved that Foxo1 could promote the insulin sensitivity and lipid synthesis (Matsumoto et al., 2006). Foxo1a was highly expressed in insulin-responsive tissues (e.g., adipose tissue, liver, and muscle) in grass carp (Ctenopharyngodon idella) (Sun et al., 2017). In contrast, the addition of bovine insulin in vitro stimulates Vtg expression in hepatopancreatic explants from the mud crab (Scylla paramamosain) (Huang et al., 2017). The decreasing of the HIS in the FO group could be explained by the DHA/EPA in the fish oil decreasing the Foxo1a expression and reducing the lipid synthesis. Whether the increase of Vtg in FO group is affected by Foxo1-mediated insulin needs further verification.

Leptin (Lep) is a protein hormone that regulates body weight and energy homeostasis (Paolucci et al., 2020). In fish, the liver seems to be the primary organ for Lep synthesis (Frøiland et al., 2010). In large yellow crookeder (Larimichthys crocea), Lep levels in blood were significantly lower in the FO group than in palm oil (Wang et al., 2019). Similarly, the liver Lep mRNA in the fish of FO group was significantly lower than that of SO group in the present study. The high level of LC-PUPA might decrease the Lep expression level in the liver in fish. However, there was a contrary relationship between liver lipid content and liver Lep mRNA expression in Arctic charr (Salvelinus alpinus) (Frøiland et al., 2010). The upregulation of the Lep mRNA expression in the SO group containing high HIS might be a negative feedback mechanism to regulate the liver lipid. The E2-induced Vtg mRNA transcript was influenced negatively by recombinant Lep in the rainbow trout (Oncorhynchus mykiss) in vitro (Paolucci et al., 2020). Thereafter, the induction of the Vtgs expression by higher n-3 LC-PUPA level might also depend on the decreasing of Lep.

The Ppar-related gene expression could be regulated by dietary fat that affects hepatic lipid storage and maintenance of metabolic health (Soni et al., 2015). The fatty acid-binding protein (Fabp) is the DEG enriched by the KEGG analysis in the PPAR signaling pathway (ko03320). The long-chain fatty acids are assisted by some fatty acid transporters to move through the cell membrane and are then transported by Fabps to the corresponding metabolic target sites (Lei et al., 2019). A Fabp2^–/– zebrafish decreased intestinal LC-PUPA and increased hepatic lipid accumulation compared with wild-type zebrafish. At the same time, transgenic overexpression Fabp2 significantly increased intestinal LC-PUPA (Zhao Y. et al., 2020). These results suggested that Fabp2 also promoted LC-PUPA absorption in fish. Consistently, Fabp2 expression was upregulated in the FO group, which contains high LC-PUFA than the SO group in the present study. In golden pompano (Trachinotus ovatus), the liver expressions of the Fabp -1, -4, and -6 were higher in the 2.1% LC-PUFA than that of 1.0% LC-PUFA feed group, suggesting that Fabps may be involved in LC-PUFA metabolism in this species (Lei et al., 2019). Taken together, Fabp gene family members may be essential for the LC-PUFA uptake and deposition in fish, while different subtypes of Fabp may be used for gene duplication and functional diversification in different species. In addition, the major facilitator superfamily domain-containing 2a (Mfsd2a) is confirmed as a transporter of DHA as lysophosphatidylcholine-DHA and is highly expressed in the liver (Pauter et al., 2017). Consistently, Mfsd2ab is also upregulated in the FO group in spotted scat. Therefore, the n-3 LC-PUFA supplementation affected the genes involved in LC-PUFA metabolism in spotted scat; hence, the regulator mechanism of these genes should be studied in the future.