# Long-term effects of high carbohydrate feeding in Nile tilapia broodstock on intermediary and carbohydrate metabolism in offspring through juvenile stage

Linli Luo, Sirijanya Thongchaitriwat, Shenglin Yang, Lucie Marandel, Stéphane Panserat, Surintorn Boonanuntanasarn

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Linli Luo, Sirijanya Thongchaitriwat, Shenglin Yang, Lucie Marandel, Stéphane Panserat, et al. "Long-term effects of high carbohydrate feeding in Nile tilapia broodstock on intermediary and carbohydrate metabolism in offspring through juvenile stage." Aquaculture Reports, 2025, 41, pp.102941. 10.1016/j.aqrep.2025.102941. hal-05141113

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# Long-term effects of high carbohydrate feeding in Nile tilapia broodstock on intermediary and carbohydrate metabolism in offspring through juvenile stage

Linli Luo<sup>a</sup>, Sirijanya Thongchaitriwat<sup>a</sup>, Shenglin Yang<sup>b</sup>, Lucie Marandel<sup>c</sup>, Stephane Panserat<sup>c</sup>, Surintorn Boonanuntanasarn<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Glycolysis

Gluconeogenesis

Amino acid catabolism

Lipogenesis

Nutritional programming

Nile tilapia

## ABSTRACT

Hyperglucidic stimulation involving high-carbohydrate (CHO) feeding in broodstock could be a practical tool for achieving nutritional programming (NP) in fish. Therefore, this study aimed to investigate the effects of high-CHO (HC) feeding stimuli on reproductive performance, and intermediary CHO metabolic responses in broodstock and its long-term impact on CHO and related metabolism in offspring. Two different CHO diets, HC/low-protein (LP) and low-CHO/high-protein (LC/HP), were fed to mature female and male Nile tilapia. The HC/LP diet in female broodstock led to decreased egg weight, but increased fecundity and gonadosomatic index compared with that with the LC/HP diet. In females and males, blood metabolites, hepatic and muscular compositions, and CHO and its related metabolic responses at the molecular level reflected HC feeding. Similar CHO intermediary metabolic responses were found in offspring at 7-days post-hatching and 7 days after the first feeding, suggesting that the parental effects of CHO intermediary metabolism could be transmitted to offspring at early developmental stages. Modulation of intermediary CHO metabolic responses persisted in the juvenile offspring, with increased hepatosomatic index and hepatic triglyceride levels, muscular glycolysis induction, and suppression of hepatic gluconeogenesis and amino acid catabolism. Juvenile offspring fed the HC diet also had more pronounced intermediary CHO metabolism, including glycogenesis, lipogenesis, glycolysis induction, muscular glucose transport, and suppression of hepatic gluconeogenesis and amino acid catabolism. Collectively, the HC diet-mediated hyperglucidic stimuli in broodstock induced NP effects in offspring, which persisted through the juvenile stage.

## 1. Introduction

An increase in the global food supply is required due to the increasing demand for animal-based food, including products from aquatic and terrestrial animals. Livestock and broodstock feed compete for feedstuff resources to develop and improve low-cost, high-quality feed, leading to the exploration of alternative feed ingredients, additives and nutrients (Sandström et al., 2022; Abd El-Naby et al., 2024), and testing their efficient utilisation with minimal negative effects. Carbohydrates (CHOs) are considered the cheapest source of nutrients for energy and commonly incorporated into commercially practical diets to produce low-cost feed. Several studies have focused on establishing the

maximum incorporation of CHOs, and their use for protein retention and growth improvement (Wilson, 1994; Kamalam et al., 2017; Jia et al., 2022; Yang et al., 2023). The effects of dietary CHO on zootechnical performance and intermediary metabolism have been demonstrated. Dietary CHO directly influences the induction of glycolysis and lipo-genesis under high-CHO conditions, and suppresses gluconeogenesis and amino acid catabolism (Azaza et al., 2015; Boonanuntanasarn et al., 2018b, 2018a). Recently, nutritional programming (NP) of CHO has been considered a tool for modulating CHO metabolism and conse-quently improving the efficient use of CHO (Polakof et al., 2012).

NP refers to the impact of nutritional environment stimuli during critical developmental windows, such as gametogenesis, foetal growth,

<footnote><sup>a</sup> School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand</footnote>

<footnote><sup>b</sup> Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China</footnote>

<footnote><sup>c</sup> INRAE, Université de Pau et des Pays de l'Adour, NuMéA, Saint-Pée-sur-Nivelle 64310, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: surinton@sut.ac.th (S. Boonanuntanasarn).</footnote>
and early life, on long-term metabolic responses and health status (Langley-Evans, 2009; Koletzko et al., 2017). NP effects can be mediated through parental provisioning, primarily in mammals, or directly in most fish species, as early embryonic development in fish is largely independent of parental involvement. In particular, nutritional intervention with CHO at an early developmental stage can modulate CHO metabolism later in the life of mammals (Srinivasan et al., 2008). The application of CHO NPs was demonstrated in fish using nutrient intervention or stimuli at early life stages to improve efficient uptake and use when fish were fed a similar dietary nutrient again later in life (Lucas, 1998; Panserat et al., 2019). The effects of NP on CHO varied among fish species and feeding habits, as did the methods of nutritional stimulation. In zebrafish, glucose injection in the late embryonic stage or early high-CHO (HC) feeding in larvae can induce hepatic gluconeogenesis and lipogenesis, muscular gluconeogenesis (juveniles), and hepatic glycolysis (adults) when challenged with the HC diet again (Fang et al., 2014; Rocha et al., 2015). Early HC feeding in rainbow trout, a carnivorous fish, suppresses muscular glucose transport and glycolysis in juveniles fed an HC diet (Geurden et al., 2014). However, early HC feeding in European sea bass larvae did not affect CHO metabolism in juveniles exposed to an HC diet (Zambonino-Infante et al., 2019). Overall, NP of CHO in fish is primarily mediated by direct CHO intervention during early developmental stages, which can modulate CHO metabolism. This modulation varies among fish species.

In aquaculture, tilapia is the second most economically farmed fish after carp. In tilapia farming, Nile tilapia (Oreochromis niloticus) and their feed generally accounts for 50-70% of the total cost. Increasing CHO and decreasing protein content in Nile tilapia feed could be a method to reduce costs. Extensive research on optimal CHO levels (32% CHO) and metabolism in Nile tilapia has been conducted (Azaza et al., 2015; Boonanuntanasarn et al., 2018a, b). As omnivorous fish, Nile tilapia exhibit efficient tolerance and metabolism with a high CHO intake (up to 44% in a commercial diet); however, CHO NP in Nile tilapia could be an effective way to improve the efficient use of an HC diet (up to ~ 66%). Glucose injection into yolk reserve larvae improved growth performance in juvenile, but not adult, stages when they were challenged with an HC diet, while the intermediary CHO metabolism pathway was modulated, including the induction of hepatic and muscular glycolysis in both juveniles and adults (Kumkhong et al., 2020a, 2021). Early HC feeding in Nile tilapia fry for 1-4 weeks could enhance the growth performance and suppression of hepatic amino acid catabolism and induction of muscular glucose transport and glycolysis at juvenile and adult stages after HC feeding (Kumkhong et al., 2020b; Srisakultiew et al., 2022). These findings demonstrated the presence of NP effects of CHO, mediated during early developmental stages in Nile tilapia. In fish, nutrient stimuli can be applied to broodstock to induce parental NP effects. In rainbow trout, female broodstock fed an HC diet for 1 year decreased their gonadosomatic index (GSI) but increased their relative fecundity. Although rainbow trout, which are carnivorous, showed poor utilisation of dietary CHO, HC feeding in female broodstock induced several intermediary CHOs and its related metabolic pathways in offspring in the long term throughout the juvenile stage (Callet et al., 2020, 2021, 2022a, 2022b). The effects of NP on HC feeding in the broodstock of Nile tilapia, an efficient CHO energy source user, might persist and influence CHO metabolism in offspring for the long term.

Therefore, in this study, we aimed to investigate the direct metabolic effects of an HC diet on intermediary CHO metabolic responses in broodstocks. We determined how dietary HC influences reproductive performance regarding fecundity and egg size. Subsequently, to investigate the NP effects at later offspring stages, we measured CHO and its related metabolic responses at the molecular level during early development. In addition, the long-term NP effects of broodstock HC feeding on CHO metabolism in the juvenile offspring were determined to explore whether the NP effects on offspring could persist to the juvenile stage. Furthermore, to examine the capacity of CHO utilisation, the juvenile

offspring fish were subjected to an HC diet.

## 2. Materials and methods

### 2.1. Experimental diet, design, and fish culture

Table 1 shows the experimental diets, including the low-CHO (LC)/ high-protein (HP) and HC/low-protein (LP) diets. The composition of the commercial diets used for acclimatising fish and nursing fry is shown in Table 1. The proximate compositions, including moisture, crude protein (CP), crude fat (CF), crude fibre, and ash, were analysed according to the standard method of the Association of Official Analytical Chemists (AOAC, 1990) shown in Table 1.

To avoid stressful conditions caused by artificial breeding, natural breeding, which is a general practice in Nile tilapia farming, was used for producing fertilised eggs. Suitable stocking rates of 0.2-0.3 kg/m² are recommended for Nile tilapia broodstock in ponds (FAO, 2009). Male Nile tilapia generally exhibits aggressive behaviour, particularly during breeding, as they are highly territorial (Barreto et al., 2011; Rossi et al., 2019). Therefore, the female-to-male ratio also plays an important role in influencing natural reproductive rates. For instance, a sex ratio of 2:1 (female: male) in Nile tilapia broodstock showed the highest number of newly hatched larvae compared with that of sex ratios (female: male) of 3:1, 4:1, and 5:1 (Salama, 1996). In the present study, six females and three males were cultured in a communal pond (breeding pond) to determine their reproductive performance. A completely randomised design with HC/LP and LC/HP diets was used. The scheme of the experimental plan for feeding broodstock and offspring and their sampling are shown in Fig. 1.

Experimental male and female Nile tilapia (O. niloticus) broodstock were obtained from the Suranaree University of Technology Farm (SUT

<caption>Table 1</caption>

Ingredients and chemical composition (g kg⁻¹) of the commercial (diets during nursing) and stimuli diets.

<table><thead><tr><th rowspan="2">Ingredients</th><th colspan="3">Commercial diet</th><th colspan="2">Experimental diets</th></tr><tr><th>Broodstock<br>acclimation<br>(36 %<br>protein)</th><th>Offspring<br>nursing<br>(40 %<br>protein)</th><th>Offspring<br>growth<br>trial<br>(32 %<br>protein)</th><th>LC/<br>HP</th><th>HC/<br>LP</th></tr></thead><tbody><tr><td>Fish meal</td><td>-</td><td>-</td><td>-</td><td>88</td><td>18</td></tr><tr><td>Rice flour</td><td>-</td><td>-</td><td>-</td><td>-</td><td>70</td></tr><tr><td>Fish oil</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7</td></tr><tr><td>Soybean oil</td><td>-</td><td>-</td><td>-</td><td>2</td><td>-</td></tr><tr><td>Gelatin</td><td>-</td><td>-</td><td>-</td><td>8</td><td>-</td></tr><tr><td>Di-calcium<br>phosphate</td><td>-</td><td>-</td><td>-</td><td>0</td><td>3</td></tr><tr><td>Fish premix<sup>a</sup></td><td>-</td><td>-</td><td>-</td><td>2</td><td>2</td></tr><tr><td colspan="6"><strong>Proximate composition (% from wet weight)</strong></td></tr><tr><td>Dry<br>matter</td><td>91</td><td>89</td><td>89</td><td>94.2</td><td>90.2</td></tr><tr><td>Protein</td><td>36</td><td>40</td><td>32</td><td>57.3</td><td>15.3</td></tr><tr><td>Fat</td><td>3.4</td><td>8.0</td><td>3.9</td><td>9.2</td><td>9.0</td></tr><tr><td>Fiber</td><td>7.1</td><td>4.2</td><td>7.3</td><td>0.5</td><td>0.4</td></tr><tr><td>Ash</td><td>12.6</td><td>12.2</td><td>12.2</td><td>22.8</td><td>8.6</td></tr><tr><td>NFE<sup>b</sup></td><td>32.5</td><td>24.3</td><td>34</td><td>4.5</td><td>56.8</td></tr><tr><td>Gross<br>energy (kJ<br>g⁻¹)</td><td>13.13</td><td>14.42</td><td>12.82</td><td>14.40</td><td>15.6</td></tr></tbody></table>

HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein

<footnote>a Vitamin and trace mineral mix provided the following (IU kg⁻¹ or g kg⁻¹ diet): biotin, 0.25 g; folic acid, 0.003 g; inositol, 0.25 mg; niacin, 0.0215 g; pantothenic acid, 0.03 g; vitamin A, 5000 IU; vitamin B1, 0.0025 g; vitamin B2, 0.0012 g; vitamin B6, 0.0075 g; vitamin B12, 0.00005 mg; vitamin C, 1 g; vitamin D3, 1000 IU; vitamin E, 100 IU; vitamin K, 0.008 g; copper, 0.02 g; iron, 0.2 g; selenium, 0.3 mg; zinc, 0.32 g.</footnote>

<footnote>b Nitrogen-free extract = dry matter – (CP + crude lipid + crude fibre + ash).</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Experimental plan of nutritional programming involving dietary high-carbohydrate (HC) stimuli in broodstock and their long-term impacts on carbohydrate (CHO) metabolism in offspring. Mature male and female broodstock were acclimated to a communal breeding pond and fed with commercial diet (36 % crude protein [CP] + 3.4 % crude fat [CF]) for 4 days. Then, broodstock were fed with low-carbohydrate/high-protein (LC/HP) or HC/low-protein (LP) diets for 38 days (total experimental period of broodstock were 42 days [6 weeks]). To determine the number of fertilised eggs, eggs were obtained from the mouth of females after 14 and 28 days of the feeding periods. After 38 days of the feeding period, broodstock were sampled (sampling before another round of natural breeding) to determine fecundity, gonadosomatic index (GSI), and CHO intermediary metabolism. The fertilised eggs obtained at the first ovulation (14 days) were cultured and fed with commercial diets (week 0–7, 40 % CP + 8 % CF; week 8–17, 32 % CP + 4 % CF) through the juvenile stage (week 17). The 7-days post-hatching (7 dph) larvae and 7-days post feeding (7 daf) fry were sampled for determination of CHO metabolism and its related intermediary metabolism. The juvenile fish were challenged with HC/LP diets during weeks 18–21.</figcaption></figure>
Farm, Nakhon Ratchasima, Thailand). To avoid natural diet feeding, the breeding pond was a cement pond (5 × 10 m; water depth 0.8 m) divided into two partitions (5 × 5 m; water depth 0.8 m) for the HC/LP and LC/HP groups. To achieve good natural breeding conditions, six males (BW; 920 ± 16.7 g) and 12 females (BW; 609.6 ± 28.4 g) were transferred to each partition (three males and six females) of the pond according to the optimal stocking density and female to male ratio.

The experimental period for the broodstock was 42 days (6 weeks). To adapt the broodstock to the experimental breeding pond, fish were acclimated to the experimental conditions for 4 days with a commercial diet (36 % CP, 3.4 % CF, 32.5 % CHO). Subsequently, the broodstock were fed either the HC/LP or LC/HP diet for 38 days. The fish were fed twice daily (9 am and 3 pm) at 1.5 % of their body weight. We did not continue culturing for a long time because we observed an impact of dietary HC/LP on the lower size of fertilised eggs. A flow-through water change system was implemented by replacing one-third of the water in each tank with dechlorinated water twice a week.

Healthy Nile tilapia generally produced ovulated eggs every two weeks, therefore, the number of fertilised eggs was examined at 14 and 28 days after feeding. After fertilisation, female tilapia take care the fertilised eggs in their mouth. Therefore, fertilised eggs were individually collected from females, counted, and weighed. Healthy fertilised eggs at the same developmental stage from six females (six replicates) were obtained and transferred into hatching trays with circulating water. The egg size of broodstock fed HC/LP decreased with increasing feeding time. To ensure stimulus exposure without negatively impacting reproductive performance, we used fertilised eggs obtained from broodstock 14 days after feeding to rear to the juvenile stage. After hatching, the larvae at 7 days post-hatching (dph), were transferred to a cage (0.4 × 0.4 × 0.6 m, six cages/broodstock diet) in a cement pond (2 × 2 × 0.8 m; water depth 0.45 m) and fed with commercial diets (Table 1). To avoid confounding effects according to sex dimorphism in growth, fish fry was fed a commercial diet (40 % CP, 8 % CF, 24.3 % CHO) supplemented with 17 α-methyltestosterone (17-MT) at 60 mg/kg five times daily (9 am, 11 am, 1 pm, 3 pm, and 5 pm) for 28 days (Boonanuntanasarn et al., 2018b). Subsequently, during weeks 5–17, fish were transferred to cement ponds (2 × 2 × 0.8 m) and fed commercial feed (weeks 5–7; 40 % CP, 8 % CF, 24.3 % CHO; weeks 8–17; 32 % CP, 4 % CF, 34 % CHO) ad libitum twice daily (9 am and 4 pm) (Fig. 1). Mortality was monitored daily. To assess growth performance, fish were weighed, and feed intake was recorded every 4 weeks throughout the experimental period.

To explore how the offspring of HC-stimulated Nile tilapia broodstock responded to an HC diet at the juvenile stage, two groups of experimental fish were challenged with the HC/LP diet during the end of week 17–21 (Fig. 1). For challenging, one cage (0.6 × 0.6 × 0.9 m) was placed in each cement pond, and six fish (BW: 52.08 ± 2.32 g) were randomly selected and cultured in a cage (six cages replication/treatment, total 12 cages), then fed the HC/LP diet (3 % body weight daily) for 4 weeks at 9 am and 4:30 pm daily.

Throughout the experimental period, the fish were reared in a hatchery using dechlorinated tap water supplied from a reservoir and distributed to each pond under continuous aeration. A flow-through water exchange system was used to replace one-third of the water twice per week. Air and water temperatures were recorded daily during the experimental period, measuring 25–28 °C and 28–32 °C, respectively. Dissolved oxygen content and pH were measured weekly on-site using a Multi-Probe System (YSI 556MPS). The values were found to be within acceptable ranges of 7.10–8.68 and 4.93–6.85 mg L⁻¹, respectively.

## 2.2. Fish sampling

### 2.2.1. Fish sampling in broodstock

At the end of the experimental period (6 weeks), broodstock sampling was performed before another round of natural breeding. Six

females and three males in the communal pond were sampled 5 h after the last meal (based on the postprandial glycaemia peak of Nile tilapia) (Boonanuntanasarn et al., 2018a). For euthanasia, fish were exposed to clove oil at a concentration of 100 mg/L, which corresponds to a double dose of the anaesthetic uses (da Silva et al., 2021). Blood samples were collected from the caudal vein using a hypodermic syringe, mixed with K₂EDTA (1.5 mg mL⁻¹ blood) as an anticoagulant, and centrifuged at 10,000 × g for 5 min at 4 °C to obtain plasma. The plasma was stored at -80 °C until the blood chemistry analysis. After bleeding, livers were collected and weighed to calculate the hepatosomatic index (HSI). Then, the liver and epaxial muscle samples were collected and rapidly frozen in liquid nitrogen and kept at -80 °C for further use in nutritive composition determination and total RNA extraction for analysis of CHO and its related metabolism. In addition, the ovaries and testes of broodstock were weighed to determine the GSI of females and males. The oocytes in the ovary were counted to calculate the fecundity.

### 2.2.2. Fish sampling in offspring

Offspring from the first ovulation (14 days feeding trial in the broodstock) were sampled. Larvae at 7 dph and swim-up fry at 7 days after the first feeding (daf) were sampled for analysis of intermediary glucose metabolism. For sampling, 200 larvae/replication (7 dph) and 100 fry/replication (7 daf) were collected and snap-frozen for proximate composition and glycogen and triglyceride analyses. In addition, 50 larvae/replication (7 dph) and 50 fry/replication (7 daf) were sampled for total RNA extraction and quantitative reverse transcription PCR (qRT-PCR).

For juvenile offspring, at week 17 (before the challenge with the HC/LP diet) and 21 (after the challenge with the HC/LP diet), juvenile fish (six fish per replicate) were sampled 5 h after the last meal. Blood samples were collected using the method described for broodstock sampling. The liver was then collected and weighed to calculate the HSI. In addition, liver and muscle (two fish/replication) as well as the whole body (two fish/replication) were collected and rapidly frozen in liquid nitrogen and kept at -80 °C for determination of nutritive composition. Liver and muscle (two fish/replication) samples were also collected for total RNA extraction to analyse intermediary CHO metabolism.

## 2.3. Blood chemistry analysis

Blood metabolites, including glucose, triglyceride, cholesterol, total protein, and blood urea nitrogen (BUN), were evaluated in broodstock (female, n = 6; male, n = 3 fish/condition) and juvenile offspring (2 fish/replication, 12 fish/treatment). Plasma glucose levels were quantitatively analysed based on glucose formation via the Trinder reaction (Barham and Trinder, 1972). Plasma triglyceride levels were determined colorimetrically using the glycerol-3-phosphate oxidase–sodium N-ethyl-N-(3-sulfopropyl) m-anisidine method, as described by Bucolo and David (1973). Cholesterol levels were evaluated using an enzymatic colorimetric method based on the cholesterol oxidase–phenol + aminophenazone (CHOD-PAP) technique, as described by Flegg (1973). Proteins were analysed using the Biuret method (Gornall et al., 1949). BUN content was determined using a modified indophenol colorimetric method (Weatherburn, 1967). All analytical values were within the linear range of concentrations.

## 2.4. Chemical composition, glycogen, and triglyceride analysis

Broodstock liver and muscle (female: n = 6 fish, male: n = 3 fish/condition); offspring whole-body (7-dph larvae and 7-daf fry; n = 6 per HC/LP and LC/HP groups); and juvenile offspring liver and muscle (2 fish/rep., n = 6, total 24 fish) as well as whole-body (2 fish/rep., n = 6, total 24 fish) samples were used to determine the chemical composition, including CP and CF, according to AOAC (1990), glycogen, and triglyceride.

Glycogen content was analysed according to the method described
by Kirchner et al. (2003), with modifications. The samples (200 mg) were homogenised in 1 mL 1 M HCl, and an aliquot of the homogenate was transferred to a new tube. The homogenate was added to 5 M KOH for neutralisation and subsequently centrifuged at 10,000 × g at 4 °C for 10 min. Free glucose in the supernatant was measured using a plasma glucose kit (Catalogue number: BLT00026, Erba Lachema s.r.o., Karásek Brno, Czechia), according to the manufacturer's instructions. Another aliquot of homogenate solution was then boiled at 100 °C for 2.5 h to hydrolyse glycogen. After boiling, the homogenates were cooled to room temperature and neutralised with 5 M KOH. After centrifugation (10, 000 × g at 4 °C for 10 min), total glucose content (free glucose and glucose obtained from glycogen hydrolysis) in the supernatant was analysed. Using appropriate dilutions, all analytical glucose values were within the linear range of concentrations. Glycogen content was calculated as the amount of glucose after subtracting the total glucose level from the free glucose level.

For the triglyceride content, samples (100 mg) were homogenised with 1 mL of 5 % IGEPAL in deionised water containing 2.8-mm glass beads using Bioprep-24 homogeniser. Samples were heated in a water bath at 90 °C for 10 min and then cooled down to room temperature. Subsequently, samples were centrifuged at 10,000 × g at 4 °C for 10 min, and the supernatant was collected in a new tube and diluted with deionised water. Triglyceride levels with appropriate dilution were measured using a triglyceride kit (Catalogue number: BLT00059, Erba Lachema s.r.o.), according to the manufacturer's instructions.

## 2.5. Total RNA extraction, cDNA synthesis, and qRT-PCR analysis of genes involved in glucose metabolism

Previous studies revealed that the activity of enzymes related to CHO metabolism was correlated with their corresponding mRNA expression levels, both of which reflect CHO metabolic responses (Boonanuntanasarn et al., 2018b). Therefore, this study examined glucose metabolism and related pathways at the molecular level to interpret CHO metabolic responses in both broodstock and offspring. We analysed key enzymes involved in the modulation of hepatic glycolysis (glucokinase [gck], phosphofructokinase [pfklr], and pyruvate kinase [pklr]), hepatic gluconeogenesis (glucose-6-phosphatase [g6pca1 and g6pca2], phosphoenolpyruvate carboxykinase cytosolic [pck1], and phosphoenolpyruvate carboxykinase [pck2], lipogenesis (fatty acid synthase [fasn] and glucose-6-phosphate dehydrogenase [g6pd]), amino acid catabolism in the liver (glutamate dehydrogenase [gdh], alanine aminotransferase [alat], and aspartate amino transferase [asat], glucose utilisation (glucose transporter; glut4), and muscular glycolysis (hexokinase I/II, [hk1 and hk2], phosphofructokinase [pfkma and pfkmb], and pyruvate kinase [pkma]).

To determine the expression of genes related to CHO and its metabolism, qRT-PCR was performed. Total RNA was extracted from the liver (50 mg) and muscle (100 mg) of broodstock; whole-body (100 mg) of fry; and liver (50 mg) and muscle (100 mg) of juvenile offspring using TRIZol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). The total RNA was also verified using 1 % agarose gel electrophoresis. To ensure the reproducibility of mRNA level analysis, duplicate cDNA synthesis reactions along with duplicate qRT-PCR reactions were used. Using the SuperScript III RNAseH-Reverse Transcriptase kit (Invitrogen) with random primers (Promega, Charbonnières, France), cDNA synthesis (duplicate for each sample) was performed from 1 µg of total RNA following the manufacturer's protocol.

The primer sequences used for qRT-PCR are listed in Table S1. To analyse mRNA levels, qRT-PCR of target genes was performed using the Roche Applied Science E-Method, according to Pfaffl (2001). In previous studies, the expression of *ef1α* was used as an internal standard for metabolic analysis at the molecular level (Boonanuntanasarn et al., 2018a,b). In this study, the mRNA of *ef1α* did not differ between

different conditions and tissues; therefore, it was used to normalise gene expression (data not shown). In all cases, the PCR efficiency was measured from the slope of a standard curve generated using serial dilutions of cDNA, and the PCR efficiency values ranged from 1.8 to 2.0.

## 2.6. Statistical analysis

The statistical model utilised was $y_{ij} = \mu + \alpha_{i} + \varepsilon_{ij}$, where $y_{ij}$ was the response, $\mu$ was the general means, $\alpha_{i}$ was dietary (LC/HP and HC/LP) effects, and $\varepsilon_{ij}$ was the random error. All data were analysed using SPSS for Windows version 22 (SPSS Inc., Chicago, IL, USA). An independent sample t-test was performed to analyse the differences between the LC/HP and HC/LP groups. The effects and differences were considered statistically significant at $p < 0.05$.

# 3. Results

## 3.1. Effects of the HC diet on broodstock body weight, reproductive performance, and intermediary metabolism

In this study, we recorded the body weight of the broodstock. There were no significant differences in body weights during the experimental period (Fig. 2A, B). During this period, we collected fertilised eggs twice at day 14 and 28 of the feeding period, and different dietary CHO levels resulted in different reproductive performances, including number of fertilized eggs and egg weight (Fig. 2C, D). At both times of fertilised egg collection, HC/LP-diet females had a lower egg weight and higher number of fertilized eggs, compared with those of LC/HP-diet females ($p < 0.05$). On day 38 of feeding, the ovary and testes were collected from the female and male broodstocks. A higher GSI was observed in females fed the LC/HP diet than that in those fed the HC/LP diet ($p < 0.05$) (Fig. 2E). However, higher fedundity was found in females fed the HC/LP diet ($p < 0.05$) (Fig. 2F). No significant differences were observed in the GSI of the males ($p > 0.05$; Fig. 2E).

After day 38 of the feeding period, plasma metabolites, including glucose, cholesterol, triglycerides, total protein, and BUN were measured to reflect the effects of dietary CHO (Table 2). In both males and females, glucose and triglyceride levels increased in response to the HC/LP stimuli ($p < 0.05$), and the total protein content decreased in the female HC/LP group ($p < 0.05$). However, no significant differences in cholesterol or BUN levels were observed between the experimental groups ($p > 0.05$). Table 2 shows the effects of dietary CHO on liver and muscle composition. Both males and females showed similar trends in the effect of dietary CHO on chemical composition: 1) HC/LP fish exhibited higher HSI, lipid, glycogen, and triglyceride content; and 2) HC/LP fish had lower protein content compared with that in LC/HP fish ($p < 0.05$) (Table 2). Muscular contents were similar to those observed in the liver; significant differences were found only in glycogen and triglyceride contents in females, as well as in protein, lipid, and glycogen in males ($p < 0.05$; Table 2).

The responses at the molecular level of CHO metabolism in the liver and muscle of Nile tilapia broodstock fed different CHO diets are shown in Table 3. The HC diet induced the upregulation of genes related to glycolysis (both male and female; *pfklr* and *pklr*) and lipogenesis (both male and female, *fasn* and *g6pd*) and downregulation of genes associated with gluconeogenesis (female, *pck1*; male, *g6pca2* and *pck1*) and amino acid catabolism (female, *alat*, *asat*, and *gdh*; male, *alat* and *gdh*) in the liver ($p < 0.05$). In the muscle, *glut4* (an enzyme related to glucose transport) was induced in females fed HC/LP ($p < 0.05$). In addition, dietary HC/LP induced muscle glycolysis with the upregulation of *hk1*, *pfkma*, *pfkmb*, and *pkma* in females and *pfkma* in males ($p < 0.05$) (Table 3).
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Final body weight and reproductive performances of experimental Nile tilapia broodstock. Final body weight of female (A) and male (B) broodstock fed with low-carbohydrate/high-protein (LC/HP) and high-carbohydrate/low-protein (HC/LP) diets. For reproductive performances, number of fertilised eggs (C) and egg weight (D) were recorded at initial (at earthen pond of SUT farm) and at 14- and 28- days of LC/HP or HC/LP feeding. Gonadosomatic index (GSI) (E) and fecundity (F) were determined at 38-days of experimental diet feeding. The asterisk (*) denotes a statistically significant difference between LC/HP and HC/LP dietary stimuli at *p* < 0.05.</figcaption></figure>

<caption>**Table 2**</caption>

Plasma metabolites and chemical compositions of broodstock fed experimental diets for 38 days.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Female</th><th colspan="3">Male</th></tr><tr><th>LC/HP</th><th>HC/LP</th><th>p-value<sup>a</sup></th><th>LC/HP</th><th>HC/LP</th><th>p-value</th></tr></thead><tbody><tr><td><strong>Plasma metabolites</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Glucose (mmol L<sup>-1</sup>)</td><td>3.68 ± 0.24</td><td>4.53 ± 0.35</td><td>&lt; 0.001</td><td>3.65 ± 0.29</td><td>4.38 ± 0.21</td><td>0.024</td></tr><tr><td>Triglyceride (mmol L<sup>-1</sup>)</td><td>0.87 ± 0.07</td><td>1.14 ± 0.12</td><td>&lt; 0.001</td><td>0.92 ± 0.08</td><td>1.13 ± 0.07</td><td>0.032</td></tr><tr><td>Cholesterol (mmol L<sup>-1</sup>)</td><td>1.93 ± 0.19</td><td>2.06 ± 0.27</td><td>0.359</td><td>1.80 ± 0.15</td><td>1.90 ± 0.12</td><td>0.383</td></tr><tr><td>Total Protein (g L<sup>-1</sup>)</td><td>36.36 ± 4.26</td><td>30.73 ± 3.31</td><td>0.029</td><td>35.15 ± 4.67</td><td>30.61 ± 4.67</td><td>0.299</td></tr><tr><td>BUN (mmol L<sup>-1</sup>)</td><td>1.67 ± 0.37</td><td>1.55 ± 0.54</td><td>0.661</td><td>1.43 ± 0.71</td><td>1.19 ± 0.41</td><td>0.646</td></tr><tr><td><strong>Liver</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HSI (%)</td><td>1.14 ± 0.22</td><td>3.18 ± 0.31</td><td>&lt; 0.001</td><td>2.02 ± 0.18</td><td>2.86 ± 0.24</td><td>0.009</td></tr><tr><td>Protein (g kg<sup>-1</sup>)</td><td>144.99 ± 2.80</td><td>84.65 ± 2.32</td><td>&lt; 0.001</td><td>109.98 ± 0.81</td><td>76.17 ± 1.21</td><td>&lt; 0.001</td></tr><tr><td>Lipid (g kg<sup>-1</sup>)</td><td>21.40 ± 1.69</td><td>40.50 ± 1.62</td><td>&lt; 0.001</td><td>31.25 ± 3.05</td><td>59.33 ± 0.01</td><td>0.004</td></tr><tr><td>Glycogen (mg g<sup>-1</sup>)</td><td>335.38 ± 55.89</td><td>826.79 ± 72.63</td><td>&lt; 0.001</td><td>392.18 ± 20.81</td><td>898.39 ± 39.41</td><td>&lt; 0.001</td></tr><tr><td>Triglyceride (mg g<sup>-1</sup>)</td><td>20.67 ± 1.77</td><td>24.80 ± 1.93</td><td>0.003</td><td>13.15 ± 1.76</td><td>67.95 ± 11.49</td><td>0.013</td></tr><tr><td><strong>Muscle</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Protein (%)</td><td>209.04 ± 3.37</td><td>205.97 ± 3.63</td><td>0.161</td><td>213.95 ± 4.88</td><td>193.69 ± 2.95</td><td>0.004</td></tr><tr><td>Lipid (%)</td><td>6.69 ± 2.93</td><td>8.48 ± 0.77</td><td>0.201</td><td>5.05 ± 1.91</td><td>9.74 ± 1.69</td><td>0.034</td></tr><tr><td>Glycogen (mg g<sup>-1</sup>)</td><td>23.67 ± 6.26</td><td>55.38 ± 23.76</td><td>0.010</td><td>22.19 ± 7.82</td><td>108.45 ± 14.52</td><td>0.001</td></tr><tr><td>Triglyceride (mg g<sup>-1</sup>)</td><td>1.35 ± 0.08</td><td>1.95 ± 0.48</td><td>0.028</td><td>1.46 ± 0.02</td><td>2.03 ± 0.32</td><td>0.090</td></tr></tbody></table>

mean ± SD; female, n = 6; male, n = 3

## 3.2. Effects of dietary HC in broodstock on growth performance and intermediary CHO metabolism in offspring at early developmental stages

The growth performance of the offspring from the broodstock stimulated with the LC/HP and HC/LP diets is shown in Table 4. The 7-dph

larvae obtained from the HC/LP group were smaller than those obtained from the LC/HP group (*p* < 0.05; Table 4). The 7-dph HC/LP fry demonstrated restored growth rate and appeared to have higher body weight, and the body weight, weight gain, average daily gain, and feed conversion ratio appeared to be similar between the fry of the HC/LP

<footnote><sup>a</sup> An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets). HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein. BUN: blood urea nitrogen. Hepatosomatic index (HSI) = 100 × (liver weight / fish body weight)</footnote>
<table><caption>**Table 3**
mRNA levels of genes related to intermediary CHO metabolism of broodstock fed experimental diets for 38 days.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Female</th><th rowspan="2">-</th><th colspan="3">Male</th></tr><tr><th>LC/HP</th><th>HC/LP</th><th>p-value</th><th>LC/HP</th><th>HC/LP</th><th>p-value</th></tr></thead><tbody><tr><td><strong>Hepatic glycolysis</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>gck</td><td>1.56 ± 0.75</td><td>1.61 ± 0.44</td><td>0.898</td><td></td><td>0.55 ± 0.36</td><td>1.73 ± 1.07</td><td>0.144</td></tr><tr><td>pfklr</td><td>0.43 ± 0.15</td><td>0.86 ± 0.37</td><td>0.036</td><td></td><td>0.22 ± 0.10</td><td>2.06 ± 0.65</td><td>0.037</td></tr><tr><td>pklr</td><td>0.48 ± 0.24</td><td>2.06 ± 0.95</td><td>0.009</td><td></td><td>0.17 ± 0.05</td><td>0.85 ± 0.21</td><td>0.005</td></tr><tr><td><strong>Hepatic gluconeogenesis</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>g6pca1</td><td>1.32 ± 0.25</td><td>1.34 ± 0.25</td><td>0.875</td><td></td><td>0.58 ± 0.04</td><td>0.85 ± 0.25</td><td>0.196</td></tr><tr><td>g6pca2</td><td>1.43 ± 0.23</td><td>1.40 ± 0.20</td><td>0.805</td><td></td><td>0.85 ± 0.06</td><td>0.39 ± 0.08</td><td>0.001</td></tr><tr><td>pck1</td><td>1.55 ± 0.46</td><td>0.31 ± 0.12</td><td>&lt; 0.001</td><td></td><td>0.49 ± 0.17</td><td>0.02 ± 0.01</td><td>0.043</td></tr><tr><td>pck2</td><td>0.84 ± 0.38</td><td>0.67 ± 0.41</td><td>0.468</td><td></td><td>0.75 ± 0.47</td><td>0.26 ± 0.20</td><td>0.171</td></tr><tr><td><strong>Hepatic lipogenesis</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>fasn</td><td>0.16 ± 0.03</td><td>2.55 ± 0.23</td><td>&lt; 0.001</td><td></td><td>0.16 ± 0.01</td><td>1.45 ± 0.16</td><td>&lt; 0.001</td></tr><tr><td>g6pd</td><td>0.20 ± 0.13</td><td>1.68 ± 0.32</td><td>&lt; 0.001</td><td></td><td>0.15 ± 0.06</td><td>1.14 ± 0.09</td><td>&lt; 0.001</td></tr><tr><td><strong>Hepatic amino acid catabolism</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>alat</td><td>1.52 ± 0.32</td><td>1.06 ± 0.17</td><td>0.010</td><td></td><td>1.26 ± 0.13</td><td>0.44 ± 0.06</td><td>0.001</td></tr><tr><td>asat</td><td>2.01 ± 0.42</td><td>0.94 ± 0.09</td><td>&lt; 0.001</td><td></td><td>0.64 ± 0.10</td><td>0.50 ± 0.06</td><td>0.112</td></tr><tr><td>gdh</td><td>1.64 ± 0.30</td><td>0.94 ± 0.10</td><td>&lt; 0.001</td><td></td><td>1.66 ± 0.26</td><td>0.28 ± 0.03</td><td>0.001</td></tr><tr><td><strong>Glucose transport and muscle metabolism</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>glut4</td><td>0.81 ± 0.15</td><td>1.15 ± 0.06</td><td>0.002</td><td></td><td>0.37 ± 0.08</td><td>0.48 ± 0.08</td><td>0.164</td></tr><tr><td>hk1</td><td>0.74 ± 0.11</td><td>1.54 ± 0.15</td><td>&lt; 0.001</td><td></td><td>0.37 ± 0.12</td><td>0.42 ± 0.09</td><td>0.621</td></tr><tr><td>hk2</td><td>3.68 ± 0.49</td><td>4.29 ± 0.50</td><td>0.057</td><td></td><td>1.95 ± 0.22</td><td>2.50 ± 0.51</td><td>0.165</td></tr><tr><td>pfkma</td><td>0.85 ± 0.12</td><td>1.43 ± 0.20</td><td>&lt; 0.001</td><td></td><td>0.29 ± 0.05</td><td>0.63 ± 0.04</td><td>0.001</td></tr><tr><td>pfkmb</td><td>0.58 ± 0.05</td><td>1.00 ± 0.11</td><td>&lt; 0.001</td><td></td><td>1.66 ± 0.01</td><td>1.68 ± 0.37</td><td>0.918</td></tr><tr><td>pkma</td><td>0.96 ± 0.13</td><td>1.49 ± 0.27</td><td>0.002</td><td></td><td>0.97 ± 0.09</td><td>1.05 ± 0.31</td><td>0.677</td></tr></tbody></table>

mean ± SD; female, n = 6; male, n = 3

An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets). HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein

<table><caption>**Table 4**
Growth performances of offspring from Nile tilapia broodstock fed with low-carbohydrate/high-protein (LC/HP) and high-carbohydrate/low-protein (HC/LP) diets.</caption><thead><tr><th></th><th>Broodstock stimulus diets</th><th>Final weight (g)</th><th>Weight gain (g)</th><th>ADG<sup>b</sup><br>(g day<sup>-1</sup>)</th><th>SGR<sup>c</sup> (% day<sup>-1</sup>)</th><th>FCR<sup>d</sup></th></tr></thead><tbody><tr><td rowspan="3">7 days<br>post-hatching (7dph)<sup>a</sup></td><td>LC/HP</td><td>10.39 ± 0.06</td><td></td><td></td><td></td><td></td></tr><tr><td>HC/LP</td><td>10.10 ± 0.03</td><td></td><td></td><td></td><td></td></tr><tr><td>p-value</td><td>&lt; 0.001</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">7 days<br>after first-feeding (7daf)<sup>a</sup></td><td>LC/HP</td><td>33.29 ± 0.49</td><td>23.19 ± 0.56</td><td>3.31 ± 0.08</td><td>17.03 ± 0.34</td><td>0.93 ± 0.02</td></tr><tr><td>HC/LP</td><td>34.17 ± 0.06</td><td>23.99 ± 0.22</td><td>3.43 ± 0.03</td><td>17.30 ± 0.30</td><td>0.90 ± 0.01</td></tr><tr><td>p-value</td><td>0.007</td><td>0.016</td><td>0.016</td><td>0.191</td><td>0.017</td></tr><tr><td rowspan="3">Week 2<br>(14 daf)<sup>a</sup></td><td>LC/HP</td><td>131.05 ± 1.10</td><td>120.94 ± 1.09</td><td>8.64 ± 0.08</td><td>18.30 ± 0.11</td><td>0.58 ± 0.01</td></tr><tr><td>HC/LP</td><td>130.23 ± 0.71</td><td>120.05 ± 0.58</td><td>8.57 ± 0.04</td><td>18.20 ± 0.13</td><td>0.59 ± 0.01</td></tr><tr><td>p-value</td><td>0.160</td><td>0.105</td><td>0.108</td><td>0.167</td><td>0.418</td></tr><tr><td rowspan="3">Week 17 (before challenge)</td><td>LC/HP</td><td>51.25 ± 2.80</td><td>42.81 ± 5.33</td><td>0.36 ± 0.04</td><td>7.17 ± 0.05</td><td>0.98 ± 0.11</td></tr><tr><td>HC/LP</td><td>52.92 ± 1.54</td><td>42.73 ± 1.43</td><td>0.36 ± 0.01</td><td>7.19 ± 0.02</td><td>0.97 ± 0.03</td></tr><tr><td>p-value</td><td>0.230</td><td>0.972</td><td>0.934</td><td>0.397</td><td>0.838</td></tr><tr><td rowspan="3">Week 21<br>(after challenge HC/LP diet)</td><td>LC/HP</td><td>83.54 ± 10.92</td><td>75.10 ± 13.96</td><td>0.51 ± 0.09</td><td>6.13 ± 0.09</td><td>1.38 ± 0.25</td></tr><tr><td>HC/LP</td><td>85.72 ± 8.97</td><td>75.54 ± 8.98</td><td>0.51 ± 0.06</td><td>6.15 ± 0.07</td><td>1.35 ± 0.15</td></tr><tr><td>p-value</td><td>0.713</td><td>0.950</td><td>0.944</td><td>0.783</td><td>0.819</td></tr></tbody></table>

The number of fish samples per replication (n): 7-dph, 200 larvae; 7-daf and 14-daf, 100 fry; Week 17 and Week 21, 12 fish, n = 6 per HC/LP and LC/HP groups.

HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein

<sup>a</sup> 7dph, 7daf and week 2: FW (final weight), mg; WG (weight gain), mg; ADG, mg/day

<sup>b</sup> Average daily gain (ADG) = (final body weight – initial body weight)/experimental days

<sup>c</sup> Specific growth rates (SGR) = 100 × [(ln final body weight – ln initial body weight] / experimental days).

<sup>d</sup> Feed conversion ratio (FCR) = dry feed fed / wet weight gain

and LC/HP broodstock ($p > 0.05$; Table 4).

The chemical compositions of the entire bodies of the offspring are shown in Table 5. Seven days post-hatching, the CF, glycogen, and triglyceride contents were significantly increased in the offspring from broodstock fed the HC/LP diet ($p < 0.05$). After the offspring were fed exogenous feed for the first time for 7 days, increases in glycogen and triglyceride content were still observed. However, in the HC/LP broodstock offspring, the CP content decreased ($p < 0.05$).

The effects of parental HC on glucose and its related metabolic

pathways at the molecular level in 7-dph and 7-daf offspring are shown in Table 6. In 7-dph larvae, upregulation of glycolytic genes (*pfkma* and *pfkmb*) and downregulation of genes related to hepatic gluconeogenesis (*g6pca1*) and amino acid catabolism (*alat* and *gdh*) were observed in offspring from the HC/LP fed broodstock ($p < 0.05$). No significant differences were observed in the expression of *gck*, *pfklr*, *pklr*, *fasn*, *g6pd*, *g6pca2*, *pck1*, *pck2*, *asat*, *glut4*, *hk1*, *hk2*, and *pkma* in the whole body of the offspring according to parental diet ($p > 0.05$). In 7-daf fry, upregulation of genes associated with glycolysis (*pklr*, *hk2*, and *pfkmb*) and
<caption>**Table 5**</caption>

Chemical compositions in whole body of offspring from Nile tilapia broodstock that were fed with experimental diets.

<table><thead><tr><th rowspan="2">Parameters</th><th rowspan="2">Broodstock<br>stimulus diets</th><th colspan="2">Offspring stage</th></tr><tr><th>7 days post-hatching</th><th>7 days after first-feeding</th></tr></thead><tbody><tr><td rowspan="3">Crude protein<br>(g kg<sup>-1</sup>)</td><td>LC/HP</td><td>158.15 ± 6.42</td><td>190.84 ± 6.99</td></tr><tr><td>HC/LP</td><td>167.28 ± 14.29</td><td>168.52 ± 5.13</td></tr><tr><td><i>p</i>-value<sup>a</sup></td><td>0.197</td><td>&lt; 0.001</td></tr><tr><td rowspan="3">Crude fat<br>(g kg<sup>-1</sup>)</td><td>LC/HP</td><td>30.60 ± 6.35</td><td>34.78 ± 7.72</td></tr><tr><td>HC/LP</td><td>64.45 ± 11.85</td><td>37.69 ± 5.50</td></tr><tr><td><i>p</i>-value</td><td>&lt; 0.001</td><td>0.469</td></tr><tr><td rowspan="3">Crude ash<br>(g kg<sup>-1</sup>)</td><td>LC/HP</td><td>15.60 ± 0.92</td><td>15.21 ± 1.24</td></tr><tr><td>HC/LP</td><td>15.18 ± 0.67</td><td>15.78 ± 1.69</td></tr><tr><td><i>p</i>-value</td><td>0.395</td><td>0.520</td></tr><tr><td rowspan="3">Glycogen<br>(mg g<sup>-1</sup> tissue)</td><td>LC/HP</td><td>14.65 ± 3.02</td><td>15.42 ± 5.17</td></tr><tr><td>HC/LP</td><td>33.63 ± 9.20</td><td>34.26 ± 6.19</td></tr><tr><td><i>p</i>-value</td><td>0.003</td><td>&lt; 0.001</td></tr><tr><td rowspan="3">Triglyceride<br>(mg g<sup>-1</sup> tissue)</td><td>LC/HP</td><td>27.71 ± 1.84</td><td>21.21 ± 2.24</td></tr><tr><td>HC/LP</td><td>34.51 ± 2.32</td><td>29.18 ± 4.13</td></tr><tr><td><i>p</i>-value</td><td>&lt; 0.001</td><td>0.003</td></tr></tbody></table>

<sup>a</sup> An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets). HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein

glucose transport (*glut4*) and downregulation of gluconeogenesis-related genes (*g6pca1* and *g6pca2*) were observed in the offspring of broodstock fed the HC diet (*p* < 0.05). However, no significant differences were observed in the expression of *gck*, *pfklr*, *pck1*, *pck2*, *fasn*, *g6pd*, *alat*, *asat*, *gdh*, *hk1*, *pfkma*, and *pkma* (*p* > 0.05).

### 3.3. Effects of the HC diet on broodstock growth performance and intermediary metabolism in juvenile offspring before and after the HC/LP diet challenge

During the juvenile stage (week 17), no significant differences were observed in the growth performances of offspring obtained from the HC/LP and LC/HP broodstocks (*p* > 0.05). However, dietary HC in broodstock significantly increased the HSI in juvenile offspring (*p* < 0.05)

(Table 7). In addition, when juvenile offspring were challenged with the HC diet, the growth performances of offspring from the HC/LP and LC/HP broodstocks was similar (*p* > 0.05) (Table 4).

The plasma metabolites and nutrient compositions of the liver, muscle, and whole-body of 17-week-old offspring are shown in Table 7. No significant difference was noted in plasma metabolites between offspring from the LC/HP and HC/LP broodstocks (*p* > 0.05). Moreover, dietary HC in broodstock significantly increased hepatic triglyceride levels in juvenile offspring (*p* < 0.05) (Table 7). After juvenile offspring were challenged with the HC/LP diet for 4 weeks (week 18–21), the plasma metabolites and proximate compositions of the liver, muscle, and whole body in offspring were assessed. Table 7 shows that the plasma glucose and triglyceride levels of juvenile offspring from the HC/LP-broodstock were significantly higher than those of the offspring from the LC/HP broodstock (*p* < 0.05), whereas plasma total protein was decreased in the offspring from the HC/LP-broodstock (*p* < 0.05). No significant differences in CHO or BUN levels were found between the two experimental groups (*p* > 0.05). In addition, the HSI and the proximate composition of hepatic fat, glycogen, and triglyceride levels were significantly increased in the juvenile offspring of the HC/LP-broodstock (*p* < 0.05) (Table 7). Muscular fat, glycogen, and triglyceride levels were higher in the juvenile offspring of the HC/LP-broodstock than those in the LC/HP-broodstock (*p* < 0.05) (Table 7).

The gene expression responses related to intermediary CHO metabolism in the liver and muscle of juvenile offspring from the LC/HP and HC/LP broodstocks are shown in Table 8. Although the juvenile offspring of the two groups were fed the same diet for 17 weeks, the broodstock stimulated by the HC/LP diet showed downregulation of hepatic gluconeogenesis (*g6pca1*) and amino acid catabolism (*alat*) in their offspring (*p* < 0.05), whereas the mRNA expression of muscular glycolysis genes (*pfkma* and *pfkmb*) was upregulated (*p* < 0.05). The parental HC diet also affected the intermediary metabolism in juvenile offspring when challenged with the HC diet. Table 8 shows that upregulation of genes related to hepatic glycolysis (*gck* and *pklr*) and lipo-genesis (*fasn* and *g6pd*), and downregulation of genes related to hepatic gluconeogenesis (*g6pca2* and *pck2*) and amino acid catabolism (*asat* and *alat*) were detected in offspring derived from the HC/LP-broodstock

<caption>**Table 6**</caption>

mRNA levels of genes related to intermediary CHO metabolism in whole body of offspring from Nile tilapia broodstock that were fed experimental diets.

<table><thead><tr><th rowspan="2">Broodstock history</th><th colspan="3">7 days post-hatching</th><th colspan="3">7 days after first feeding</th></tr><tr><th>LC/HP</th><th>HC/LP</th><th><i>p</i>-value<sup>a</sup></th><th></th><th>LC/HP</th><th>HC/LP</th><th><i>p</i>-value</th></tr></thead><tbody><tr><td>Hepatic glycolysis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>gck</td><td>0.93 ± 0.29</td><td>1.18 ± 0.36</td><td>0.221</td><td></td><td>0.94 ± 0.20</td><td>1.09 ± 0.23</td><td>0.270</td></tr><tr><td>pfklr</td><td>0.93 ± 0.13</td><td>1.00 ± 0.10</td><td>0.321</td><td></td><td>0.83 ± 0.22</td><td>1.21 ± 0.43</td><td>0.082</td></tr><tr><td>pklr</td><td>0.90 ± 0.22</td><td>1.25 ± 0.34</td><td>0.057</td><td></td><td>0.66 ± 0.44</td><td>1.37 ± 0.37</td><td>0.012</td></tr><tr><td>Hepatic gluconeogenesis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>g6pca1</td><td>1.05 ± 0.17</td><td>0.61 ± 0.27</td><td>0.007</td><td></td><td>1.37 ± 0.27</td><td>0.67 ± 0.25</td><td>0.001</td></tr><tr><td>g6pca2</td><td>0.93 ± 0.28</td><td>0.73 ± 0.24</td><td>0.211</td><td></td><td>1.29 ± 0.45</td><td>0.62 ± 0.19</td><td>0.007</td></tr><tr><td>pck1</td><td>1.06 ± 0.45</td><td>1.13 ± 0.66</td><td>0.854</td><td></td><td>1.05 ± 0.31</td><td>0.72 ± 0.24</td><td>0.064</td></tr><tr><td>pck2</td><td>1.16 ± 0.39</td><td>0.93 ± 0.15</td><td>0.209</td><td></td><td>1.12 ± 0.39</td><td>0.79 ± 0.20</td><td>0.098</td></tr><tr><td>Hepatic lipogenesis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>fasn</td><td>0.88 ± 0.10</td><td>0.92 ± 0.08</td><td>0.453</td><td></td><td>0.85 ± 0.28</td><td>1.22 ± 0.41</td><td>0.094</td></tr><tr><td>g6pd</td><td>1.04 ± 0.16</td><td>1.08 ± 0.10</td><td>0.606</td><td></td><td>0.88 ± 0.25</td><td>1.08 ± 0.29</td><td>0.233</td></tr><tr><td>Hepatic amino acid catabolism</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>alat</td><td>1.16 ± 0.28</td><td>0.47 ± 0.12</td><td>&lt; 0.001</td><td></td><td>1.23 ± 0.36</td><td>0.92 ± 0.30</td><td>0.150</td></tr><tr><td>asat</td><td>0.89 ± 0.08</td><td>0.87 ± 0.15</td><td>0.778</td><td></td><td>1.23 ± 0.36</td><td>1.01 ± 0.33</td><td>0.299</td></tr><tr><td>gdh</td><td>0.98 ± 0.09</td><td>0.77 ± 0.18</td><td>0.031</td><td></td><td>1.09 ± 0.19</td><td>0.93 ± 0.15</td><td>0.138</td></tr><tr><td>Glucose transport and muscle metabolism</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>glut4</td><td>0.76 ± 0.24</td><td>0.93 ± 0.14</td><td>0.169</td><td></td><td>0.64 ± 0.14</td><td>1.53 ± 0.28</td><td>&lt; 0.001</td></tr><tr><td>hk1</td><td>1.07 ± 0.19</td><td>1.14 ± 0.10</td><td>0.451</td><td></td><td>0.93 ± 0.19</td><td>1.12 ± 0.40</td><td>0.327</td></tr><tr><td>hk2</td><td>0.96 ± 0.30</td><td>1.27 ± 0.21</td><td>0.063</td><td></td><td>0.74 ± 0.08</td><td>1.34 ± 0.41</td><td>0.016</td></tr><tr><td>pfkma</td><td>0.74 ± 0.13</td><td>0.99 ± 0.19</td><td>0.026</td><td></td><td>0.87 ± 0.28</td><td>1.22 ± 0.33</td><td>0.075</td></tr><tr><td>pfkmb</td><td>0.83 ± 0.14</td><td>1.03 ± 0.13</td><td>0.028</td><td></td><td>0.77 ± 0.11</td><td>1.34 ± 0.32</td><td>0.006</td></tr><tr><td>pkma</td><td>0.89 ± 0.14</td><td>1.03 ± 0.10</td><td>0.082</td><td></td><td>1.05 ± 0.21</td><td>1.16 ± 0.38</td><td>0.528</td></tr></tbody></table>

HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein;

<sup>a</sup> An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets).
<caption>**Table 7**</caption>

Plasma metabolites and proximate composition of liver, muscle, and whole body in juvenile offspring before (week 17) and after (week 21) the HC/LP diet challenge for 4 weeks.

<table><thead><tr><th></th><th colspan="3">Before challenge</th><th colspan="3">After challenge</th></tr></thead><tbody><tr><td>Broodstock history</td><td>LC/HP</td><td>HC/LP</td><td>p-value<sup>a</sup></td><td>LC/HP</td><td>HC/LP</td><td>p-value</td></tr><tr><td>Plasma metabolites</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Glucose (mM)</td><td>4.04 ± 0.45</td><td>4.12 ± 0.31</td><td>0.659</td><td>4.82 ± 0.32</td><td>5.35 ± 0.37</td><td>&lt; 0.001</td></tr><tr><td>Triglyceride (mM)</td><td>1.04 ± 0.33</td><td>1.09 ± 0.23</td><td>0.620</td><td>1.30 ± 0.26</td><td>2.20 ± 0.36</td><td>&lt; 0.001</td></tr><tr><td>BUN (mM)</td><td>0.53 ± 0.26</td><td>0.41 ± 0.28</td><td>0.265</td><td>1.18 ± 0.69</td><td>0.93 ± 0.54</td><td>0.435</td></tr><tr><td>Cholesterol (mM)</td><td>0.94 ± 0.17</td><td>0.99 ± 0.08</td><td>0.513</td><td>1.53 ± 0.25</td><td>1.65 ± 0.29</td><td>0.281</td></tr><tr><td>Total protein (g/L)</td><td>29.93 ± 2.02</td><td>28.42 ± 2.5</td><td>0.231</td><td>35.42 ± 2.07</td><td>32.67 ± 1.58</td><td>0.008</td></tr><tr><td>Liver (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Protein</td><td>8.98 ± 0.73</td><td>8.64 ± 0.53</td><td>0.379</td><td>7.39 ± 0.42</td><td>6.86 ± 0.46</td><td>0.065</td></tr><tr><td>Fat</td><td>2.82 ± 0.33</td><td>3.01 ± 0.26</td><td>0.283</td><td>4.08 ± 0.39</td><td>5.18 ± 0.37</td><td>&lt; 0.001</td></tr><tr><td>Ash</td><td>0.82 ± 0.06</td><td>0.88 ± 0.05</td><td>0.124</td><td>0.86 ± 0.11</td><td>0.92 ± 0.07</td><td>0.331</td></tr><tr><td>Glycogen (mg/g)</td><td>31.45 ± 3.83</td><td>33.92 ± 2.54</td><td>0.257</td><td>43.03 ± 6.01</td><td>54.18 ± 7.58</td><td>0.028</td></tr><tr><td>Triglyceride (mg/g)</td><td>11.32 ± 2.32</td><td>17.68 ± 2.88</td><td>0.003</td><td>23.87 ± 1.85</td><td>27.47 ± 1.28</td><td>0.005</td></tr><tr><td>HSI<sup>b</sup></td><td>1.33 ± 0.12</td><td>1.75 ± 0.17</td><td>0.001</td><td>3.18 ± 0.31</td><td>3.91 ± 0.33</td><td>0.003</td></tr><tr><td>Muscle (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Protein</td><td>16.98 ± 0.45</td><td>17.38 ± 0.63</td><td>0.241</td><td>18.1 ± 0.67</td><td>17.61 ± 0.41</td><td>0.153</td></tr><tr><td>Fat</td><td>1.14 ± 0.25</td><td>1.19 ± 0.21</td><td>0.724</td><td>1.62 ± 0.14</td><td>1.92 ± 0.21</td><td>0.015</td></tr><tr><td>Ash</td><td>1.17 ± 0.10</td><td>1.11 ± 0.10</td><td>0.267</td><td>1.28 ± 0.07</td><td>1.28 ± 0.06</td><td>0.964</td></tr><tr><td>Glycogen (mg/g)</td><td>3.98 ± 0.27</td><td>4.06 ± 0.16</td><td>0.551</td><td>4.97 ± 0.49</td><td>6.55 ± 0.45</td><td>&lt; 0.001</td></tr><tr><td>Triglyceride (mg/g)</td><td>3.02 ± 0.28</td><td>3.12 ± 0.46</td><td>0.695</td><td>4.68 ± 0.23</td><td>5.31 ± 0.37</td><td>0.009</td></tr><tr><td>Whole body (%)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Protein</td><td>11.17 ± 0.36</td><td>11.16 ± 0.43</td><td>0.951</td><td>12.2 ± 1.41</td><td>12.88 ± 0.47</td><td>0.300</td></tr><tr><td>Fat</td><td>3.13 ± 0.25</td><td>3.28 ± 0.19</td><td>0.288</td><td>6.68 ± 0.65</td><td>6.95 ± 0.58</td><td>0.467</td></tr><tr><td>Ash</td><td>3.16 ± 0.34</td><td>3.08 ± 0.35</td><td>0.678</td><td>3.68 ± 0.83</td><td>3.56 ± 0.54</td><td>0.776</td></tr></tbody></table>

HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein

when compared with those in offspring of the LC/HP-broodstock ($p < 0.05$). In addition, a significant upregulation of genes related to muscular glucose transport (*glut4*) and glycolysis (*hk1*, *hk2*, *pfkma*, and *pfkmb*) was observed in the offspring of the HC/LP-broodstock compared with those in the offspring of the LC/HP-broodstock ($p < 0.05$; Table 8).

## 4. Discussion

Nile tilapia are good users of dietary CHO, which could provide protein-sparing effects (Boonanuntanasarn et al., 2018a, b). In addition, the NP concept has been used with Nile tilapia to improve the efficient

<caption>**Table 8**</caption>

mRNA levels of genes related to intermediary CHO metabolism in the liver and muscle of juvenile offspring before (week 17) and after (week 21) the HC/LP diet challenge for 4 weeks.

<table><thead><tr><th></th><th colspan="3">Before challenge</th><th colspan="3">After challenge</th></tr></thead><tbody><tr><td>Broodstock history</td><td>LC/HP</td><td>HC/LP</td><td>p-value<sup>a</sup></td><td>LC/HP</td><td>HC/LP</td><td>p-value</td></tr><tr><td>Hepatic glycolysis</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>gck</td><td>1.47 ± 0.33</td><td>1.61 ± 0.41</td><td>0.512</td><td>1.53 ± 0.13</td><td>1.95 ± 0.29</td><td>0.009</td></tr><tr><td>pfklr</td><td>0.86 ± 0.20</td><td>1.05 ± 0.22</td><td>0.156</td><td>0.89 ± 0.12</td><td>1.08 ± 0.18</td><td>0.051</td></tr><tr><td>pklr</td><td>0.74 ± 0.34</td><td>0.89 ± 0.05</td><td>0.312</td><td>0.48 ± 0.19</td><td>1.27 ± 0.27</td><td>&lt; 0.001</td></tr><tr><td>Hepatic gluconeogenesis</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>g6pca1</td><td>0.61 ± 0.05</td><td>0.22 ± 0.08</td><td>&lt; 0.001</td><td>0.58 ± 0.06</td><td>0.63 ± 0.07</td><td>0.175</td></tr><tr><td>g6pca2</td><td>1.05 ± 0.17</td><td>0.89 ± 0.19</td><td>0.143</td><td>0.60 ± 0.07</td><td>0.43 ± 0.04</td><td>0.001</td></tr><tr><td>pck1</td><td>1.14 ± 0.50</td><td>0.87 ± 0.20</td><td>0.266</td><td>0.55 ± 0.12</td><td>0.46 ± 0.03</td><td>0.135</td></tr><tr><td>pck2</td><td>0.61 ± 0.17</td><td>0.48 ± 0.14</td><td>0.188</td><td>0.66 ± 0.17</td><td>0.45 ± 0.11</td><td>0.032</td></tr><tr><td>Hepatic lipogenesis</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>fasn</td><td>0.72 ± 0.39</td><td>1.05 ± 0.28</td><td>0.129</td><td>0.55 ± 0.21</td><td>0.93 ± 0.13</td><td>0.004</td></tr><tr><td>g6pd</td><td>0.52 ± 0.07</td><td>0.71 ± 0.25</td><td>0.126</td><td>0.52 ± 0.15</td><td>1.15 ± 0.27</td><td>0.001</td></tr><tr><td>Hepatic amino acid catabolism</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>asat</td><td>1.09 ± 0.41</td><td>0.78 ± 0.26</td><td>0.151</td><td>0.83 ± 0.07</td><td>0.65 ± 0.11</td><td>0.005</td></tr><tr><td>alat</td><td>1.14 ± 0.27</td><td>0.74 ± 0.31</td><td>0.041</td><td>1.30 ± 0.29</td><td>0.53 ± 0.14</td><td>&lt; 0.001</td></tr><tr><td>gdh</td><td>0.96 ± 0.23</td><td>0.88 ± 0.12</td><td>0.154</td><td>0.75 ± 0.28</td><td>0.53 ± 0.11</td><td>0.126</td></tr><tr><td>Glucose transport and muscle metabolism</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>glut4</td><td>1.01 ± 0.11</td><td>1.11 ± 0.18</td><td>0.254</td><td>0.85 ± 0.12</td><td>1.00 ± 0.07</td><td>0.029</td></tr><tr><td>hk1</td><td>1.07 ± 0.10</td><td>1.01 ± 0.37</td><td>0.732</td><td>0.87 ± 0.15</td><td>1.05 ± 0.10</td><td>0.031</td></tr><tr><td>hk2</td><td>0.91 ± 0.19</td><td>0.94 ± 0.25</td><td>0.839</td><td>0.65 ± 0.06</td><td>0.99 ± 0.15</td><td>0.001</td></tr><tr><td>pfkma</td><td>0.88 ± 0.15</td><td>1.27 ± 0.26</td><td>0.009</td><td>0.80 ± 0.18</td><td>1.57 ± 0.23</td><td>&lt; 0.001</td></tr><tr><td>pfkmb</td><td>0.70 ± 0.13</td><td>1.25 ± 0.15</td><td>&lt; 0.001</td><td>0.87 ± 0.10</td><td>1.04 ± 0.12</td><td>0.022</td></tr><tr><td>pkma</td><td>1.50 ± 0.16</td><td>1.97 ± 0.69</td><td>0.159</td><td>1.16 ± 0.28</td><td>1.36 ± 0.27</td><td>0.231</td></tr></tbody></table>

<footnote>a An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets).</footnote>

<footnote>b Hepatosomatic index (HSI) = $100 \times$ (liver weight / fish body weight)</footnote>

<footnote>a An independent *t*-test was used to analyse the effects of different stimuli (HP/LC and LP/HC diets). HC, high-carbohydrate; HP, high-protein; LC, low-carbohydrate; LP, low-protein</footnote>
use of dietary CHO through various stimuli methods. For example, glucose injection into the yolk sac of larvae and early feeding with an HC diet in the fry stage improved growth performance and modulated the intermediary CHO metabolism later in juvenile and adult fish challenged with HC feeding (Kumkhong et al., 2020b; Kumkhong et al., 2021, 2020a; Srisakultiew et al., 2022). Broodstock diets have a long-term impact on metabolic modulation in offspring (Izquierdo et al., 2001). Due to their adaptability to dietary CHO (Boonanuntanasarn et al., 2018a, b), this study formulated experimental diets with extremely high and low CHO levels, which consequently resulted in low and high protein contents, respectively. Thus, this study evaluated the molecular-level impacts of CHO as well as other metabolic responses, including amino acid catabolism and lipogenesis. In this study, we provide scientific evidence that dietary CHO stimulation in broodstock may serve as an effective approach for NP in Nile tilapia. The application of NP to improve the utilisation of dietary CHO could offer potential economic benefits in tilapia farming. Additionally, providing a targeted nutritional stimulus to broodstock may support more efficient and practical broodstock management. The persistent effects of the parental HC stimuli diet on intermediary CHO metabolism in juvenile fish have been demonstrated. In particular, the modulation of intermediary CHO metabolism was more pronounced when fish were subjected to a challenging HC diet.

## 4.1. Dietary HC in broodstock does not affect the growth performance but influences the reproductive performance

Nile tilapia are good users of CHO, and the optimum level of dietary CHO for growth depends on the growth stage. In this study, our results showed that Nile tilapia broodstock, both male and female, fed dietary HC/LP for up to 38 days, did not exhibit a decrease in growth performance. Similarly, although rainbow trout is a poor user of CHO (carnivorous), a 2-year-old broodstock fed with up to 35 % dietary CHO for 1 year showed no negative effects on growth performance (Callet et al., 2020). Broodstock fish (both omnivorous and carnivorous) appear to be able to utilise high levels of dietary CHO, at least in the short term, without any negative effects on growth performance. These findings provide a practical stimuli method for broodstock to further investigate the NP of CHO in broodstocks.

Female broodstock had high fecundity, but low egg weight, when fed the HC/LP diet for up to 28 days, demonstrating a trade-off between egg number and size. It was revealed that dietary lipid at a moderate level increased fecundity in Oreochromis karongae broodstock (Nzohabonayo et al., 2017). Additionally, low dietary protein levels in broodstock have been associated with reduced egg size in fish (Masrizal et al., 2015). The HC/LP diet induced an elevation of plasma triglycerides, as well as hepatic and muscular lipids, along with a reduction in plasma protein levels in broodstock, suggesting its potential role in increasing fecundity while concurrently reducing egg weight. A negative relationship between offspring number and size has been demonstrated in cichlids (Kolm et al., 2006). Notably, low GSI was found in female broodstock, but not in males fed the HC/LP diet for 38 days. Coincidentally, rainbow trout females fed a 40 % CP/35 % CHO diet showed higher relative fecundity with no differences in egg size, but lower GSI compared to those fed a 60 % CP/0 % CHO diet (Callet et al., 2020, 2022a). Our data confirm that nutritional status plays an important role in regulating the reproductive performance (Cerdá et al., 1994; Izquierdo et al., 2001; Chong et al., 2004; Iqbal et al., 2021). Thus, dietary CHO influences reproductive performance in fish broodstock, and its effects may vary according to fish feeding habits.

## 4.2. Dietary HC influences intermediary CHO metabolism and its related metabolic pathways in broodstock

Previous reports on the long- (26 weeks) and short-term (4 days) effects of dietary HC on intermediary metabolism and responsive

glucose and related metabolic pathways were demonstrated in Nile tilapia (Boonanuntanasarn et al., 2018a,2018b; Thongchaitriwat et al., 2024). In this study, after we obtained fertilised eggs on days 14 and 28, we decided to perform sampling to determine the effects of dietary HC/LP in the broodstock (at day 38). Our results showed that, compared to LC/HP broodstock, male and female fish showed obvious intermediary metabolic responses to dietary HC in the plasma, liver (increased glycolysis/gluconeogenesis, lipogenesis and decreased amino acid catabolism at a molecular level), and muscle. The metabolic responses at the molecular level were found to be similar to those reported in previous studies (Boonanuntanasarn et al., 2018a,2018b; Thongchaitriwat et al., 2024). According to the optimum sex ratio for natural breeding in Nile tilapia, the number of male individuals was lower than that of females; nonetheless, the CHO metabolic responses were similar to those observed in females. Overall, these findings show that Nile tilapia broodstock responded as expected to dietary HC. Adult Nile tilapia responded to the HC diet after a short feeding period (4 days). To ensure HC stimulus without negative effects on reproductive performance and/or avoid selection bias, this study used offspring from broodstock fed for 14 days to investigate the parental NP effect. Similarly, a 2-week exposure to an HC diet without adverse effects on broodstock reproductive performance or embryogenesis produced an effective parental NP impact in yellow catfish (Xu et al., 2024). Whether HC feeding in the broodstock could affect CHO and its related metabolism in offspring was discussed in the following sections.

## 4.3. Dietary HC in broodstock influences zootechnical parameters and intermediary and CHO metabolism in offspring at early developmental stages

The HC-mediated nutritional stimulation in the early life from first feeding for a short (7 days) and long term (28 days) of Nile tilapia fry lowered the growth of fry; however, compensatory growth occurred and resulted in a similar growth pattern during later development (Kumkhong et al., 2020; Srisakultiew et al., 2022). In this study, compared to the broodstock fed the LC/HP diet, a lower body weight of 7-dph fry was observed in the offspring of broodstock fed the HC/LP diet because of the smaller size of eggs. However, the 14-day offspring of HC/LP exhibited compensatory growth (with superior growth [7-daf fry] of fry of the LC/HP diet broodstock). Taken together, although high dietary CHO stimuli in Nile tilapia broodstock led to reduced embryo size, early larvae and fry compensated for their growth.

Direct HC intervention at early life stages significantly affected CHO metabolic responses, which could suggest the induction of glycogenesis and lipogenesis in Nile tilapia. For example, dietary HC stimulation in Nile tilapia fry at the first feeding for 1 or 4 weeks as well as early CHO intervention by glucose injection into the yolk reserve promoted the accumulation of glycogen and triglycerides in the body (Kumkhong et al., 2020a,2020b, 2021; Srisakultiew et al., 2022). In fish, the maternal diet affects embryonic development (Riddle and Hu, 2021). Thus, maternal metabolic responses reflect the chemical composition of the offspring, particularly in larvae before exogenous feeding. Our results showed that the whole-body composition, including increments of glycogen, triglycerides and CF, and low protein of the two tested stages (7-dph larvae and/or 7-daf fry) might be partly due to maternal accumulation effects and/or ontogenic metabolic responses, influenced by both CHO and protein levels in the broodstock diet. Coincidentally, dietary HC in parents (both female and male) could modulate whole-body composition in offspring, including the following: 1) paternal HC history decreased protein and increased glycogen in offspring; and 2) maternal HC history lowered hepatic glucose content in offspring (Callet et al., 2021). Thus, in fish, CHO and its associated metabolic pathways in offspring (larvae and fry) may be related to the parental HC diet. These findings strongly suggest that the modification of metabolic pathways in the offspring could be partly achieved by the nutrition of parents.

Although the body composition of offspring appeared to be closely
related to that of their parents at the molecular level, a few changes were observed in glucose and its related metabolic pathways. Several modulations of genes indicated CHO metabolism responses according to parental HC such as induction of glycolysis and glucose transport as well as suppression of gluconeogenic pathway and amino acid catabolism were observed in 7-dph larvae and 7-daf fry. These CHO responses were similar to the findings obtained after applying the HC feeding stimuli, from the first feeding for short (7 days) and long (28 days) periods, in whole-body fry (Kumkhong et al., 2020a; Srisakultiew et al., 2022). In zebrafish, 4 days post-fertilisation embryos with a history of glucose injection appeared to induce whole-body glycolysis at the molecular level (Rocha et al., 2015). In yellow catfish, parental NP has been shown to induce the expression of glycolytic (gk, pk, and pfk) and glucose transport (sglt1) genes in the offspring; however, there were no effects on gluconeogenesis (pepck and g6pc) (Xu et al., 2024). Taken together, parental dietary HC feeding could be an effective method for modulating CHO metabolism in offspring, but the degree of metabolic modification might depend on the fish species and experimental condition.

### 4.4. The modulation effects of dietary HC in broodstock on intermediary and CHO metabolism in offspring persisted through the juvenile stage and were more pronounced with HC dietary challenges

Previous studies showed that nutritional stimulation of CHO history in the early life of Nile tilapia through glucose injection led to improved growth performance in juvenile fish, but not in adult fish, suggesting that the NP effects were weaker in the long term (Kumkhong et al., 2020a; Kumkhong et al., 2021). CHO stimulation during first feeding with an HC diet improved the efficient use of the challenged HC diet, resulting in enhanced growth performance during both juvenile and adult stages (Kumkhong et al., 2020b; Srisakultiew et al., 2022). However, in this study, in the presence of compensatory growth in offspring through week 17 with commercial diet, dietary HC stimulated in broodstock did not promote the growth performance in their juvenile offspring challenged with HC diet. The NP benefits of HC utilisation for growth varied. In rainbow trout, HC feeding in male broodstock for 6 months enhanced body weight in their juvenile offspring (24 weeks) fed with commercial diet. Whereas juvenile offspring from broodstock fed HC for 1 year did not show a growth advantage after 3 months of HC-feeding (Callet et al., 2021, 2022). Taken together, the NP benefits of HC efficient utilisation in fish depends on the intervention method (glucose injection in alevin, HC feeding in fry or broodstock). Further investigation is required to explore whether the NP benefits of CHO intervention can be found in later adult stages.

For the NP concept, nutritional intervention with HC in early life was proposed to persistently influence intermediary glucose and its related metabolism. In particular, its effects were expected to be greater when fish were fed an HC diet later in their development. In our study, with persisting effects (increased HSI and hepatic triglyceride, induction of muscular glycolytic and suppression of hepatic gluconeogenic (g6pca1) and amino acid catabolic (alat) pathways) of the HC stimuli in broodstock. Notably, the lower amino acid catabolism observed in the offspring may also be partly impacted by dietary HC/LP stimulation in the broodstock. Greater modulatory effects on CHO intermediary metabolism were observed when the offspring were challenged with the HC/LP diet. These CHO responses were similar to previous reports of hyperglucidic stimuli via glucose injection in alevin and dietary HC in the fry stages of Nile tilapia (Kumkhong et al., 2021, 2020a, 2020b; Srisakultiew et al., 2022). Therefore, the nutritional intervention involving HC feeding in broodstock revealed similar NP effects, although not totally the same as those during the larval and fry stages.

However, the effects of interventions on CHO NPs varied among fish species or feeding habits. In a carnivorous rainbow trout, the effects of HC feeding in broodstock for 1 year led to decreased plasma and hepatic glucose and glycogen in liver, but increased plasma cholesterol as well as downregulation of genes related to glucose transport (glut6) of

juvenile offspring. Nonetheless, when juvenile fish were challenged with the HC/LP diet for 3 months, the whole-body lipid content increased and reinforced hepatic cholesterol biosynthesis (mvdaa and dhcr7a) but decelerated lipid metabolism (acsf2a) (Callet et al., 2021, 2022). HC stimulation in an omnivorous zebrafish broodstock decreased blood glucose levels and expression of hepatic gluconeogenesis (fbp1a, fbp1b, and pck1) in adult offspring challenged with HC diet for 1 week (Lu et al., 2022). In addition, an omnivorous yellow catfish broodstock fed an HC diet showed the upregulation of glycolytic (gk, pk, and pfk) and glucose transport (sglt1) genes in the whole body of the offspring after the HC diet challenge (Xu et al., 2024). Therefore, our findings suggest that, after the application of dietary CHO stimuli in the broodstock, the NP involving CHO metabolic responses in offspring persists up to the juvenile stage and more strengthened when fish were challenged with the HC diet. Likewise, in mammals, parental hyperglycaemic status during pregnancy led to elevated blood glucose levels in the offspring (Srinivasan et al., 2008; Chernausek et al., 2016). In addition, a parental high-fructose diet upregulated the mRNA expression of transcription factors related to de novo lipogenesis and fatty acid synthase in the liver of adult male offspring (Carapeto et al., 2018). Overall, nutritional intervention of HC in broodstock was an effective approach and showed the long-term effects of NP involving CHO in offspring. Further investigations are needed to determine whether these effects are sustained in adult Nile tilapia offspring. In particular, more research into the NP history of broodstock could provide insight into offspring resilience throughout the production cycle, as well as the role of epigenetic regulation in mediating long-term NP effects.

### 5. Conclusions

In summary, without negative effects on growth performance for genitors, the HC diet in the broodstock led to increased fecundity and low egg size. Hatched larvae and fry later gained weight and exhibited compensatory growth. The HC diet modulates several intermediary metabolic pathways of CHO, which are interpreted as increased plasma glucose, lipogenesis, amino acid catabolism, and CHO metabolic responses in broodstock. Metabolic modulation of CHO has also been observed in offspring at the early developmental stages. In addition, HC stimulation in broodstock had long-term effects on glucose and its related metabolism in juvenile offspring, with greater effects when the offspring were challenged with the HC/LP diet. Our data clearly show that the nutrition of the broodstock can have strong impact of the offspring nutrition.

### Author statement

All authors confirm that the manuscript has been read and approved by all named authors. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

### CRediT authorship contribution statement

**Stephane Panserat:** Writing – review & editing, Validation, Super-
vision, Conceptualization.
**Surintorn Boonanuntanasarn:** Writing –
review & editing, Validation, Supervision, Project administration, Data
curation, Conceptualization.
**Srijanya Thongchaitriwat:** Methodol-
ogy, Formal analysis, Data curation.
**Linli Luo:** Writing – original draft,
Methodology, Investigation.
**Lucie Marandel:** Data curation, Concep-
tualization.
**Shenglin Yang:** Data curation, Conceptualization.

### Ethics statement

All experimental procedures involving fish culture and sampling were approved by the Ethics Committee of the Suranaree University of Technology Animal Care and Use Committee (Approval No. SUT-IACUC-001/2023).
Declaration of Generative AI and AI-assisted technologies in the
writing process

The authors did not use any artificial intelligence-assisted technol-
ogies in the writing process.

Funding

We acknowledge the Office of the Permanent Secretary of the Min-
istry of Higher Education, Science, Research and Innovation for the
funding of the research collaboration.

Declaration of Competing Interest

The authors have no conflicts of interest to declare.

Acknowledgements

We would like to thank Mr. Sunai Plymee (Suranaree University of Technology Farm) for maintaining the broodstock fish. This project was funded by National Research Council of Thailand (NRCT) and Suranaree University of Technology (N42A650324). This work was also supported by the Office of the Permanent Secretary of the Ministry of Higher Education, Science, Research and Innovation.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.aqrep.2025.102941.

Data availability

Data will be made available on request.

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