Effects of dietary glutamine supplementation on growth performance, intestinal digestive ability, antioxidant status and hepatic lipid accumulation in Xenocypris davidi Bleeker.

doi:10.21203/rs.3.rs-2975524/v1

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Effects of dietary glutamine supplementation on growth performance, intestinal digestive ability, antioxidant status and hepatic lipid accumulation in Xenocypris davidi Bleeker.

https://doi.org/10.21203/rs.3.rs-2975524/v1

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The effects of different concentrations of glutamine on host performance are important in the context of aquaculture. Here, four diets were fed to Xenocypris davidi Bleeker, with 0, 3, 6, and 9 g/kg of glutamine supplementation, for a period of 8 weeks. The results showed that supplementation with 6 and 9 g/kg of glutamine significantly increased the final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) of the fish (P < 0.05). Conversely, there was a decreasing trend observed in the condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI), and feed conversion ratio (FCR) with increased glutamine supplementation. Dietary glutamine was found to improve the intestinal structure and function of X. davidi. This was achieved through an increase in the digestive and antioxidant enzyme activities in the intestine, as well as an increase in the muscle layer thickness (MT), villus height (VH), and villus width (VW) of the intestine. These effects were particularly evident in the 6 and 9 g/kg glutamine supplementation groups. This finding was further corroborated by oil red O staining and the relative areas of lipid droplets. A gradual increase in plasma total protein (TP) concentrations with increasing glutamine supplementation was observed, and was considerably higher in the 9 g/kg group than in the control group (P < 0.05). In contrast, plasma total cholesterol (TC) and triglyceride (TG) concentrations were decreased significantly after fish were fed with diets with appropriate glutamine supplementation levels. Notably, we further constructed the correlation analysis to find and verify the important factors that affect the growth performance of host after feeding glutamine. In the present study, we observed that PEP and AMS (belong to digestive enzymes), MT, VH and VW (belong to intestinal morphology), crude protein (belong to proximate composition) showed significant positive correlation with FBW, WGR, SGR (belong to growth performance, P < 0.05). Overall, these findings suggest that dietary glutamine can enhance host performance in X. davidi, particularly when supplemented at 9 g/kg. This may have important implications for the aquaculture industry, as it can improve the health and growth of farmed fish.

glutamine

Xenocypris davidi Bleeker

growth performance

intestinal healthy status

hepatic lipid accumulation

Glutamine is regarded as nutritionally nonessential amino acids for fish (Li, et al., 2020a). However, glutamine has been classified as a “functional amino acid” because of its several functions in animals, including promoting growth, enhancing intestinal health, and immunological modulation (Teng, et al., 2021; Qu, et al., 2019). Although the physiological requirements for glutamine are particularly high in all cultured animals, including livestock, poultry, and fish, conventional protein sources in feeds such as soybean meal (18 mg/kg) and fishmeal (12.3 mg/kg) contain only low amounts of glutamine (Li, et al., 2020a; Li, et al., 2020b). It is therefore necessary to supplement the feed of cultured animals with appropriate amounts of glutamine. Over the decades, numerous studies in livestock and poultry have demonstrated that supplementing the diet with appropriate levels of glutamine has a variety of benefits, including promoting growth, maintaining intestinal function, and improving immunity (Ji, et al., 2019; Shan, et al., 2012; Drackley, et al., 2006; Yi, et al., 2005). In recent years there has been an increase in research on the application of glutamine to aquatic species. Previous studies have shown that the appropriate supplementation of glutamine to feed can improve the performance, intestinal health, and disease resistance of aquatic species such as yellow catfish (Pelteobagrus fulvidraco), tilapia (Oreochromis niloticus), half-smooth tongue sole (Cynoglossus semilaevis Günther), and Jian carp (Cyprinus carpio var. Jian) (Hu, et al., 2015; Liu, et al., 2015; Ye, et al.; Silva, et al., 2010). Glutamate has been demonstrated to reduce intestinal inflammation and protect intestinal epithelial cells from oxidative stress in studies on yellow catfish (Jiao, et al., 2021) and Jian carp (Chen, et al., 2009). These findings support the use of glutamine in aquatic animals to improve growth performance and intestinal structure.

X. davidi is a kind of fish with strong disease resistance, fast growth rate and strong adaptability, and is favoured by consumers for its tender, nutritious meat and high meat content (Hu, et al., 2012). It is omnivorous, similar to grass carp(Ctenopharyngodon idella), with low dietary costs and plant-based protein sources being used extensively in the diet (Xu, et al., 2011). High levels of plant-based proteins in aquatic animals' diets may promote fat storage in fish on the one hand, and structural damage owing to anti-nutritional substances causing enteritis on the other(Ye, et al., 2019; Yin, et al., 2018; Yaghoubi, et al., 2016). It is not clear whether the supplementation of glutamine in the diet can improve the intestinal health and fat metabolism of X. davidi. Consequently, this study focused on the effects of dietary glutamine on intestinal morphology, digestive capacity, antioxidant status, hepatic lipid accumulation, and growth performance of X. davidi.

2.1. Ethical statement

The study protocol and procedures were approved by the ethics committee of Dalian Ocean University. The use and care of animals tally with China's animal protection law.

2.2. Experimental fish and rearing conditions

X. davidi were collected from the local hatchery of Dandong in China and temporary reared in key laboratory of applied biology and aquaculture of northern fishes in Liaoning province. Before the feeding trial, the fish were fed with a control diet for two weeks to adapt to the experimental conditions. Then, fish were fasted for 24 h, and similar-sized individuals (2.94 ± 0.14 g) were randomly distributed into 12 tanks containing 100 L of water and with a recirculation filtration system (0.90 L/min), at a density of 20 fish per tank, for 56 d. Fish were manually fed twice daily for the feeding trail, at 8:00 a.m. and 16:00 p.m., at a rate of 4.0% body weight per day. Every two weeks, the fish were weighed in batches, and the daily feed supply was adjusted in accordance with the results. Water temperature, dissoloved oxygen and pH in this trial were 26 ± 1 °C, 6 ± 0.5 mg/L and 7.9 ± 0.3, respectively.

2.3. Experimental diets

The test feed formulations are shown in Table 1. Four groups of isonitrogenous and isolipid feeds were formulated with fish meal and soybean meal as the main protein sources and soybean oil as the main fat source. Glutamine (99% purity, Beijing Dingguo Changsheng Biotechnology Co., Ltd, Beijing, China) was added at 0, 3, 6 and 9 g/kg respectively. All ingredients were fully mixed before being fabricated into a single pellet size (1.0 mm) using a press machine (JLA-125; Pinzheng Equipment Co. Ltd.). Pellets were dried in a ventilated oven (DK-s26; Shanghai Samsung Laboratory Instrument Co. Ltd, Shanghai, China) to a moisture content of approximately 10%. All diets were packaged and stored at -20 ℃ until used.

Table 1 Composition and nutrient levels of experimental diets (dry matter).

Ingredients	Levels of dietary glutamine (g/kg)
	0	3	6	9
Fish meal	60	60	60	60
Corn protein meal	70	70	70	70
Soybean meal	400	400	400	400
Rapeseed meal	60	60	60	60
Wheat flour	330.1	330.1	330.1	330.1
Soybean oil	40	40	40	40
Sodium chloride	3	3	3	3
Calcium bis	20	20	20	20
Choline chloride	1.5	1.5	1.5	1.5
Vitamin C	0.4	0.4	0.4	0.4
Vitamin mixture¹	1	1	1	1
Mineral mixture²	5	5	5	5
Glutamine	0	3	6	9
Glycine	9	6	3	0
Total	1000	1000	1000	1000
Nutrient compositions (g/kg dry basis except moisture)
Moisture	106.3	102.5	105.6	103.3
Crude protein	328.4	328.1	327.7	327.6
Crude lipid	54.1	54.5	55.4	56.2
Ash	66.7	63.8	67.5	69.4

¹Vitamin mixture (per kg of premix): vitamin A, 1,000,000 IU; vitamin D₃, 300,000 IU; vitamin E, 4,000 IU; vitamin K₃, 1,000 mg; vitamin B₁, 2,000 mg; vitamin B₂, 1,500 mg; vitamin B₆, 1,000 mg; vitamin B₁₂, 5 mg; nicotinic acid, 1,000 mg; vitamin C, 5,000 mg; Ca pantothenate, 5,000 mg; folic acid, 100 mg; inositol, 10,000 mg; carrier glucose; H₂O ≤100 g/kg.

²Mineral mixture (0.025mg/g of premix): NaCl, 107.79; MgSO₄⋅7H₂O, 380.02; NaHPO₄⋅2H₂O, 241.91; KH₂PO₄, 665.20; Ca(H₂PO₄)⋅2H₂O, 376.70; Fe citrate, 82.38; Ca lactate, 907.10; Al(OH)₃, 0.52; ZnSO₄⋅7H₂O,9.90; CuSO₄, 0.28; MnSO₄⋅7H₂O, 2.22; Ca (IO₃)₂, 0.42; CoSO₄⋅H₂O, 2.77.

2.4. Sample collection

At the end of the trial, all fish were starved for 24 hours before the sampling. Then the fish were anesthetized with 50 mg/L MS222 (ScanVac, Sigma Aldrich Shanghai Trading Co., Ltd, Shanghai, China) and each fish was weighed and length measured respectively for calculating the growth performance. Four fish from each tank (twelve fish per treatment) were randomly chosed and stored at -20 °C for whole-body composition analyses. Blood was collected intravenously from five fish in each tank using heparinized (1,600 U/ml; Nakalai Tesque) syringes and centrifuged at 5000 r/min for 10 min. The liver and intestine of five fish from each tank were rapidly removed and weighted for calculating the indexes of viscera and then frozen in liquid nitrogen and stored at -80 °C for analysis of enzyme activity. Three fish per tank were chosed to clect their midgut and liver, flushed with saline, and fixed in 4% paraformaldehyde solution for morphological analyses.

2.5. Growth performance

At the end of the feeding trial, all fish were fasted for 24 h and weighed to measure the growth parameters. Growth parameters were calculated as follows: BWG (%) = 100 × (final mean body weight − initial mean body weight) / initial mean body weight; SGR (%/d) = (ln final mean body weight − ln initial mean body weight) / feeding days; FCR = feed mean intake / (final mean body weight − initial mean body weight); CF (g/cm³)= (body weight∕body length³) × 100; HSI (%) = (hepatic weight∕body weight) × 100; ISI (%) = (intestinal weight∕body weight ) × 100; Viscero-somatic index (VSI) = (viscera weight / body weight) ×100; SR (%) = 100 × (final fish number) / (initial fish number).

2.6. Biochemical analysis

The AOAC standard method (AOAC, 1990) was used for proximate analysis of the diet, the fish whole-body, and the lipid contents in the liver. Crude protein was determined by Kjeldahl method (Kjeltec KDN-1000 Auto Analyzer, Shanghai, China). Crude lipid was measured by ether extraction using Soxhlet method. Ash content was examined using a muffle furnace (Shanghai Yuejin Medical Instrument Co., Ltd., Shanghai, China) at 550 °C for 6 h. Moisture content was determined in an oven at 105 °C until a constant weight was reached.

The activities of digestive enzymes and antioxidant enzymes in the intestine, including lipase (LPS), amylase (AMS), pepsin (PEP), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPX), and malondialdehyde (MDA), were measured by kits from the Jiancheng Bioengineering Institute (Nanjing, China).

Histology of midgut was performed according to Zeng et al. (2021). The intestinal sample was fixed using 4% paraformaldehyde solution at 4°C for 24h and dehydrated using a series of graded ethanol concentrations. Then to prepare the slices for hematoxylin-eosin (HE), standard paraffin embedding methods were utilized. Measurement using S-viewer software and observation with a light microscope equipped with an image analyzer. To assess muscle layer thickness (MT), villus height (VH), and villus width (VW), at least 10 intact villi were chosen and averaged for each sample.

Fresh liver tissue was preserved in 4% paraformaldehyde solution at 4°C for 24 h, then stained with Oil Red O as previously reported (Zang et al., 2015; Pan et al., 2018). At least 10 microscope fields were randomly examined for each sample for statistics of relative areas of lipid droplets in oil-red O staining.The Oil Red O-positive relative area was quantiﬁed using Image Pro Plus software.

Plasma biochemical parameters including total protein (TP), total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured by kits from the Jiancheng Bioengineering Institute (Nanjing, China).

2.7. Statistical analysis

The experimental data were analyzed using one-way ANOVA with the software SPSS 25.0 (SPSS, Chicago, Illinois), while data were expressed as mean values ± standard error of mean (SEM). Data from each group were compared by the Duncan test and a significant difference was set at P < 0.05. The figures were created with GraphPad Prism 8. Orthogonal polynomial contrasts were used to test linear and quadratic response of dietary glutamine levels as described by Guo et al. (2022).

2.8. Correlation analysis

The correlation analysis between growth performance and digestive enzymes, intestinal morphology and proximate composition were calculated by the Spearman correlation coefficient. The analysis was performed using the “psych” and “reshape2” package in R (Jiao, et al., 2020). All analyses were conducted in R (version 3.5.1, R Development Core Team) unless otherwise stated.

3.1. Growth performances

The growth performances of X. davidi fed the experimental diets for 56 d are shown in Table 2. There was no significant difference in the survival rates (SR) among groups (P > 0.05). The final body weight (FBW), weight gain rate (WGR) and specific growth rate (SGR) of X. davidi were significantly increased at glutamine supplementation levels of 6 g/kg and 9 g/kg compared to the control group (P < 0.05). As the level of glutamine supplementation increased, there was a decreasing trend in the condition factor (CF), viscerosomatic index (VSI), hepatosomatic index (HSI), and feed conversion ratio (FCR) of X. davidi. The CF and FCR were significantly lower in the 6 g/kg and 9 g/kg groups than in the control group (P < 0.05). The 9 g/kg group showed considerably lower VSI and HSI than the control group (P < 0.05).

Table 2 Effects of dietary glutamine supplementation on growth performance of Xenocypris davidi

Parameters	Levels of dietary glutamine (g/kg)				Liner		Quadratic
	0	3	6	9	R²	P-value	R²	P-value
IBW (g)	2.92±0.04	2.94±0.14	2.97±0.06	2.96±0.03	0.169	0.103	0.141	0.204
FBW (g)	15.15±0.32^a	15.64±0.19^a	16.58±0.20^b	18.31±0.27^c	0.854	<0.001	0.910	<0.001
WGR (%)	418.26±11.19^a	431.86±3.19^ab	458.36±6.80^b	518.47±9.20^c	0.813	<0.001	0.895	<0.001
SGR (%/d)	2.94±0.04^a	2.98±0.01^ab	3.07±0.02^b	3.25±0.03^c	0.811	<0.001	0.884	<0.001
CF (g/cm³)	0.78±0.03^b	0.73±0.01^ab	0.72±0.02^a	0.72±0.01^a	0.225	0.068	0.339	0.063
VSI (%)	7.77±0.43^b	7.11±0.12^ab	7.05±0.20^ab	6.71±0.22^a	0.377	0.020	0.334	0.065
HIS (%)	0.99±0.03^b	0.92±0.06^ab	0.90±0.07^ab	0.70±0.03^a	0.395	0.017	0.386	0.045
ISI (%)	2.68±0.12	3.08±0.21	3.09±0.26	3.27±0.28	0.191	0.087	0.127	0.221
FCR	1.79±0.01^c	1.74±0.02^c	1.65±0.01^b	1.56±0.03^a	0.894	<0.001	0.892	<0.001
SR (%)	95.33±0.02	97.67±0.02	95.33±0.05	97.67±0.02	-0.085	0.721	-0.206	0.941

Values (Means±SE) (n=3) in the same row with different superscripts show significant difference (P < 0.05).

IBW: initial body weight, FBW: final body weight, WGR: weight gain rate, SGR: specific growth rate, CF: condition factor, VSI: viscerosomatic index, HSI: hepatosomatic index, ISI: Intestinesomatic index, FCR: feed conversion ratio. SR: Survival rate.

3.2. Digestive enzymes activities

Effects of dietary glutamine supplementation on digestive enzymes activities in the intestine of X. davidi are shown in Table 3. Pepsin (PEP) activity in the 3 g/kg, 6 g/kg, and 9 g/kg groups was significantly higher than that of the control group (P < 0.05). The 6 g/kg and 9 g/kg groups showed considerably higher amylase (AMS) and lipase (LPS) activities than the control group (P < 0.05).

Table 3 Effects of dietary glutamine supplementation on digestive enzymes activities of Xenocypris davidi

Parameters	Levels of dietary glutamine (g/kg)				Liner		Quadratic
	0	3	6	9	R²	P-value	R²	P-value
PEP (U /mg prot)	7. 25±0.17^a	8.05±0.48^b	9.08±0.93^c	9.56±0.25^c	0.909	<0.001	0.906	<0.001
AMS (U/mg prot)	2.34±0.11^a	2.44±0.15^a	3.24±0.07^b	3.50±0.17^b	0.784	<0.001	0.767	0.001
LPS (U/g prot)	11.30±0.61^a	11.76±0.86^a	13.63±1.37^b	13.77±0.58^b	0.555	0.03	0.510	0.016

Values (Means±SE) (n=3) in the same row with different superscripts show significant difference (P < 0.05).

PEP: pepsin, AMS: amylase, LPS: lipase.

3.3. Intestinal histomorphometry

The effects of dietary glutamine on the intestinal (midgut) morphology of X. davidi are presented in Figure 1 and Figure 2. As the level of glutamine supplementation increased, there was an increasing trend in the muscle layer thickness (MT), villus height (VH), and villus width (VW) of X. davidi. The MT and VW of the intestine of X. davidi rose considerably in the 6 g/kg and 9 g/kg groups compared to the control group (P < 0.05), whereas no significant variations were observed in the 3 g/kg group (P > 0.05). Increased dietary glutamine supplementation substantially enhanced intestinal VH, with VH significantly higher in the 3 g/kg, 6 g/kg, and 9 g/kg groups than in the control group (P < 0.05).

3.4. Whole-body composition

The effects of dietary glutamine supplementation on whole body proximate composition of X. davidi are presented in Figure 3. Whole-body moisture and ash content of X. davidi among groups was not signiﬁcantly affected by dietary glutamine levels (P > 0.05). With increasing dietary glutamine levels, the whole-body crude protein content of X. davidi rose, and was considerably higher in the 9 g/kg group than in the control group (P < 0.05). However, as the level of glutamine in the diet increased, the crude lipid content of whole-body X. davidi decreased, although the difference was not substantial (P > 0.05).

3.5. Intestine antioxidant indices

The effects of dietary glutamine supplementation on intestine antioxidant indices of X. davidi are shown in Table 4. With increasing dietary glutamine levels, the total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT), and glutathione peroxidase (GPX) activities in the intestine of X. davidi gradually increased, and were considerably higher in the 6 g/kg and 9 g/kg groups than in the control group (P < 0.05). The levels of malondialdehyde (MDA) in the intestine of X. davidi in the 6 g/kg and 9 g/kg groups were dramatically lower than in the control group.

Table 4 Effects of dietary glutamine supplementation on intestine antioxidant indices of Xenocypris davidi

Parameters	Levels of dietary glutamine (g/kg)				Liner		Quadratic
	0	3	6	9	R²	P-value	R²	P-value
T-AOC/(U•mg^-1prot)	64.81±1.85^a	69.94±1.32^ab	74.47±2.06^b	76.08±1.96^b	0.682	0.001	0.684	0.002
T-SOD/(U•mg^-1prot)	128.58±2.00^a	132.62±5.37^a	147.18±3.34^b	150.91±2.40^b	0.704	<0.001	0.672	0.03
CAT/(U•mg^-1prot)	45.07±2.17^a	51.28±2.13^a	59.36±1.84^b	63.81±2.48^b	0.822	<0.001	0.806	<0.001
MDA/(nmol•mg^-1prot)	1.29±0.05^b	1.15±0.04^ab	1.05±0.07^a	1.00±0.05^a	0.625	0.001	0.616	0.005
GPX/(U•mg^-1prot)	22.53±1.78^a	25.01±0.98^ab	28.28±1.56^bc	32.26±1.82^c	0.694	<0.001	0.670	0.003

Values (Means±SE) (n=3) in the same row with different superscripts show significant difference (P < 0.05).

T-AOC: total antioxidant capacity, T-SOD: total superoxide dismutase, CAT: catalase, MDA: malondialdehyde, GPX: glutathione peroxidase.

3.6. Hepatic lipid accumulation

As shown in Figure 4, the proportion of lipid droplets with Oil Red O stain decreased in the 3 g/kg, 6 g/kg, and 9 g/kg groups compared with the control group. Fewer lipid droplets were present in the liver tissue of the 9 g/kg group, and the relative areas of lipid droplets were substantially lower than those of the control group (P < 0.05). The relative areas for lipid droplets in Oil Red O staining backed up the findings even more (Figure 5a).

The effects of dietary glutamine supplementation on liver lipid contents of X. davidi are shown in Figure 5b. The liver lipid contents were affected by dietary glutamine levels; compared with the control group, liver lipid contents were significantly lower in the 9 g/kg group.

3.7. Plasma biochemistry

The effects of dietary glutamine supplementation on the plasma biochemical parameters of X. davidi are shown in Table 5. Among dietary treatments, there was no signiﬁcance in AST and ALT activities. However, a gradual increase in plasma total protein (TP) concentrations with increasing glutamine supplementation was observed, and was considerably higher in the 9 g/kg group than in the control group (P < 0.05). In contrast, plasma total cholesterol (TC) and triglyceride (TG) concentrations were decreased significantly after fish were fed with diets with appropriate glutamine supplementation levels.

Table 5 Effects of dietary glutamine supplementation on the plasma biochemical parameters of Xenocypris davidi

Parameters	Levels of dietary glutamine (g/kg)				Liner		Quadratic
	0	3	6	9	R²	P-value	R²	P-value
TP (g /L)	15.41±0.22^a	15.56±0.31^a	16.14±0.33^ab	16.86±0.09^b	0.638	0.001	0.651	0.004
TC (mmol/L)	4.84±0.03^c	4.71±0.11^bc	4.50±0.09^ab	4.30±0.05^a	0.746	<0.001	0.725	0.001
TG (mmol/L)	0.53±0.01^b	0.52±0.03^b	0.51±0.06^b	0.48±0.09^a	0.702	<0.001	0.739	0.001
AST (U/L)	14.77±0.33	14.59±1.32	15.15±1.05	14.94±1.21	0.046	0.245	-0.002	0.409
ALT (U/L)	4.13±0.26	4.09±0.03	4.17±0.06	4.09±0.13	0.157	0.112	0.175	0.170

Values (Means±SE) (n=3) in the same row with different superscripts show significant difference (P < 0.05).

TP: total protein, TC: total cholesterol, TG: triglyceride, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase.

3.8. Correlation analysis

Based on the strong interactions between growth performance and digestive enzymes, intestinal morphology and proximate composition, further analysis of the correlation was constructed to find the important factors that affect the growth performance of host after feeding glutamine (Figure 6). These results showed that FBW, WGR, and SGR show significant positive correlations with digestive enzymes (PEP and AMS), intestinal morphology (MT, VH, and VW) and proximate composition (crude protein), while FCR showed a significant negative correlation with proximate composition (crude lipid, P < 0.05).

VW: villus width, VH: villus height, MT: muscle layer thickness, AMS: amylase, PEP: pepsin, FBW: final body weight, WGR: weight gain rate, SGR: specific growth rate, CF: condition factor, VSI: viscerosomatic index, HSI: hepatosomatic index, FCR: feed conversion ratio.

In the present study, X. davidi fed with 6 g/kg and 9 g/kg of glutamine showed significant improvements in growth performance, which indicate that the appropriate levels of glutamine can promote fish growth. Similar findings have been reported on half-smooth tongue sole (Cynoglossus semilaevis Günther) (Jingwei, et al., 2015), grass carp (Ctenopharyngodon idella) (Qu, et al., 2019), Jian carp (Cyprinus carpio var. Jian) (Hu, et al., 2015). These promotion of growth performance in fish was associated with increased glutamine reserves in muscle and liver, and glutamine promoted muscle protein synthesis while decreasing its degradation (Wang, et al., 2011). Glutamine increased the efficiency of the fish to utilise nutrients on their own, which was a key factor for the improved growth performance. Moreover, glutamine can improve fish intestine function with the improvement of intestinal structure and enhances the release of digestive enzymes (Chen, et al., 2009; Pohlenz, et al., 2012; Qu, et al., 2019), such like the way 6 g/kg and 9 g/kg group did. However, the growth performance of sea cucumber (Apostichopus japonicus Selenka) (Yu, et al., 2016) and Nile tilapia (Oreochromis niloticus) (Pereira, et al., 2017) was found to be reduced after feeding diets supplemented with high level of glutamine. Which indicate that high levels of glutamine may cause neurotoxic effects and abnormal amino acid metabolism (Garlick, 2001; Jingwei, et al., 2015). These findings further suggest that a diet supplemented with 9 g/kg glutamine may be optimal for promoting growth performance in X. davidi.

Due to the crucial roles in digestive process of aquatic animals, including facilitating the digestion and utilisation of feeds in intestine, measuring the activity of digestive enzymes is a commonly used method to assess the digestive ability of fish (Cahu, et al., 1998; Yu, et al., 2016). Herein, 6 g/kg and 9 g/kg group demonstrated significantly higher activity of intestinal pepsin, amylase, and lipase compared to the control group, indicating that the appropriate levels of glutamine could improve the digestive capacity of X. davidi. Similar results have been observed in sea cucumbers (Yu, et al., 2016), grass carp (Qu, et al., 2019) and white shrimp (Penaeus vannamei) (Cui, et al., 2021). The observed improvements in intestinal digestive enzymes activity could potentially be attributed to the beneficial effect of glutamine on the morphology of the intestine. For example, compared to the control group, the with 6 g/kg and 9 g/kg groups exhibited significantly higher MT (mucosal thickness), VH (villus height), and VW (villus width), along with thicker and denser intestinal villi. This implies that the appropriate levels of glutamine could increase the intestinal surface area, thereby improving nutrient absorption and utilization through the intestine. These improvements were also observed in sea cucumber (Yu, et al., 2016) and grass carp (Qu, et al., 2019). Previous studies had shown that glutamine is the primary energy substrate for intestinal epithelial cell division, which plays an essential process that plays a vital role in increasing the height of the intestinal villi and maintaining the integrity of the intestinal tissues (Gu, et al., 2021; Kim and Kim, 2017; Wu, 2013). Consequently, the improvement of digestive enzyme activities and intestinal function of the experimental groups may contribute to the growth promotion of X. davidi.

In this study, as dietary glutamine levels increased, the whole-body crude protein content of X. davidi also increased, notably, the 9 g/kg group exhibited significantly higher than the control group. It is worth noting that although the difference was not significant, the crude lipid content of whole-body X. davidi showed a decreasing trend as the level of glutamine in the diet increased, with the similar results in juvenile gilthead seabream (Sparus aurata) (Caballero-Solares, et al., 2015). In terrestrial animals, glutamine supplementation can increase the protein synthesis and inhibit the protein breakdown in skeletal muscle (MacLennan, et al., 1988; Wu and Thompson, 1990). In fish, glutamine could also increase in crude protein content and decreases in crude fat content in whole fish composition, such as Nile tilapia(Pereira, et al., 2017). Based on the analysis of liver lipid content and liver oil red O staining, we have deduced that dietary glutamine may be effective in lowering the overall lipid content of the body by modulating hepatic lipid metabolism.

Previous studies have demonstrated the efficacy of glutamine in safeguarding intestinal epithelial cells of Jian carp against oxidative stress damage by H₂O₂ (Chen, et al., 2009). Here, the intestinal antioxidant capacity of X. davidi improved and the oxidative stress damage to the intestine was gradually reduced, with the amount of glutamine in the diet increased. Similar positive effects of dietary glutamine on antioxidant capacity were also observed in half-smooth tongue (Liu, et al., 2015), gilthead sea bream (Sparus aurata) (Coutinho, et al., 2016), and sea cucumber (Yu, et al., 2016). Glutamine serves as a precursor for the synthesis of glutathione, which is the primary endogenous antioxidant scavenger in the body. Therefore, the observed improvement in the intestinal antioxidant capacity of X. davidi can also be attributed, at least in part, to the increased levels of glutamine in the diet. In addition, the enhanced antioxidant capacity of the intestine may have a positive impact on growth by reducing oxidative damage and promoting the development of intestinal structure to some extent.

Lipid accumulation in the liver can lead to oxidative damage and cell death, particularly as the liver is a crucial organ for lipid metabolism (Ye, et al., 2019; Zhao, et al., 2019; Christian, et al., 2013). Previous studies have revealed that glutamine could improve liver fat metabolism (Qi, et al., 2020), promote the expression of lipid-lowering genes in the liver (Meng, et al., 2009), and regulate glycolysis and lipogenesis (Caballero-Solares, et al., 2015). Similarly, dietary supplementation of glutamine has been found to significantly reduce the buildup of lipids in the liver of X. davidi, particularly in 9 g/kg group. Oil red O staining and measurement of the relative area of lipid droplets provided further support for this finding. These results provide further evidence that glutamine has an impact on the buildup of lipids in the liver of cultured fish.

Plasma biochemical parameters are useful indicators of tissue cell permeability and the overall metabolic state of the body, and are therefore considered appropriate markers for assessing the health status of fish (Zohreh, et al., 2018). Protein deposition and growth in fish could be promoted by higher plasma total protein levels (Hu, et al., 2018). As expected, an incremental rise in plasma TP concentrations was noted as the level of glutamine supplementation increased, with the highest level in the 9 g/kg group. Which suggested that glutamine could improve the protein metabolism of X. davidi. Similar effects were also observed in juvenile hybrid sturgeon (Acipenser schrenckii ♀ × Huso dauricus ♂) (Zhu, et al., 2010) and mirror carp (Song, et al., 2016). Appropriate levels of glutamine supplementation were associated with a significant decrease in plasma TC and TG concentrations in X. davidi, suggesting an improvement in lipid metabolism. This result was also consistent with the improvement of hepatic lipid accumulation by glutamine in terrestrial vertebrates (Xi, et al., 2007). Moreover, supplementing with suitable levels of glutamine in the diet did not damage the liver of X. davidi, which demonstrated that glutamine could serve as the first major energy source to protect and support liver function (Li, et al., 2020b; Jia, et al., 2017).

Notably, we further constructed correlation analysis to identify important factors affecting host growth performance after glutamine feeding. During the current study, we observed that PEP and AMS (belong to digestive enzymes), MT, VH and VW (belong to intestinal morphology), crude protein (belong to proximate composition) showed significant positive correlation with FBW, WGR, SGR (belong to growth performance, P < 0.05). The finding is consistent with the results of present study, which indicate an improvement in digestive process and intestinal morphology may lead to improved host growth (Yu, et al., 2016; Cahu, et al., 1998). In contrast, crude lipid showed significant negative correlation with FBW, WGR, SGR (belong to proximate composition, P < 0.05), Ash and Moisture showed no correlation with most growth performance index. These results further clarify the complexity and variable of the important factors that impact host growth performance. Consequently, our finding indicated that feeding glutamine to fish can improve their growth by enhancing their digestive process and intestinal morphology .

In summary, our study found that adding glutamine to fish feed at appropriate levels can have a range of beneficial effects. This includes increased digestive enzyme activity, enhanced intestinal development, improved antioxidant capacity, reduced liver lipid accumulation, improved plasma biochemical indices, and better growth performance. The supplementation of the diet with 6 g/kg and 9 g/kg of glutamine resulted in significant improvements in growth performance of X. davidi, These improvements may be attributed to the enhancement of intestinal digestive enzyme activity and the development of the intestinal morphological structure. Based on our experimental conditions, we recommended a dietary glutamine supplementation of 9 g/kg for X. davidi.

ACKNOWLEDGMENTS This work was supported by Dalian Youth Star project of Science and Technology [grant number 2019RQ137]; Liaoning Province Science and Technology Major Special Project [grant number 2022JH1/10200002] and Blue Talents Project of Dalian Ocean University.

CONFLICT OF INTEREST The authors declare no competing interests.

ETHICAL STATEMENT All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors

DATA AVAILABILITY STATEMENT

All data generated or analysed during this study are included in this published article.

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Effects of dietary glutamine supplementation on growth performance, intestinal digestive ability, antioxidant status and hepatic lipid accumulation in Xenocypris davidi Bleeker.

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