Effects of sodium butyrate and poly-β-hydroxybutyrate on the intestinal resistance and recovery from ammonia nitrogen stress in grass carp (Ctenopharyngodon idella)

doi:10.21203/rs.3.rs-4899676/v1

To study the effects of dietary butyrate supplementation on the resistance and recovery from ammonia nitrogen stress in the intestine, grass carp (Ctenopharyngodon idella) were administered four distinct diets for 56 days: control diet (CK group), 600 mg butyrate /kg powdered sodium butyrate (PSB group), microencapsulated sodium butyrate (MSB group) and poly-β-hydroxybutyrate (PHB group). After feeding, the fish were exposed to ammonia nitrogen (10.47 ± 0.67 mg/L) for 96 hours, followed by a 15- day recovery in pristine water. Our results revealed that dietary PSB, MSB and PHB primarily increased intestinal crypt depth and goblet cell count, and PHB also elevated the ratio of villus area to intestinal area. Moreover, three additions significantly increased the levels of acid phosphatase (ACP), lysozyme (LZM), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and β defensin (β-DF) of the intestine. Meanwhile, these supplements significantly decreased the intestinal contents of interleukin-1β (IL-1β) and malondialdehyde (MDA). In acute ammonia nitrogen stress and recovery experiment, the addition of PSB, MSB and PHB decreased lipid peroxides (MDA) and enhanced antioxidant capacity (T-SOD and T-AOC), thereby effectively mitigating ammonia-induced oxidative stress. These additives further enhanced intestinal non-specific immunity, as evidenced by the increased levels of ACP, LZM and β-DF, and simultaneously alleviated inflammatory response (IL-1β) induced by ammonia nitrogen. Furthermore, the integrated biomarker response analysis revealed that MSB group exhibited a superior effect. In conclusion, dietary supplementation of 600 mg/kg butyrate demonstrated an enhanced ability to mitigated ammonia nitrogen stress and recovery in the intestine of grass carp.

Ammonia nitrogen

Powder sodium butyrate

Microencapsulated sodium butyrate

Poly-β-hydroxybutyrate

Stress recovery

Sodium butyrate (PSB, MSB, PHB) improve grass carp growth, muscle protein, intestinal morphology.

These additives increase intestinal antioxidants and immunity, reducing IL-1β and MDA.

These additives alleviate ammonia stress by increasing antioxidants and non-specific immunity.

MSB exhibited a superior effect in resisting and recovering from ammonia nitrogen stress.

Ammonia is a prevalent pollutant in aquatic environments (Randall and Tsui 2002; Parvathy et al. 2023). In recent years, the rapid development of intensive aquaculture has resulted in a continuous rise of ammonia concentration (Romano and Zeng 2013; Yu et al. 2019). In the aquaculture ponds of Andhra Pradesh, ammonia levels in 78% of the samples exceeded 0.5 mg/L as recommended by the World Health Organization (Nagaraju et al. 2023). Notably, aquatic animals exhibit higher sensitivity to ammonia, and the acute aquatic life criteria for ammonia is set at 10.24 mg/L (pH 7.0 and 20 ℃) (Fan et al. 2021). Previous investigations have demonstrated that ammonia inhibits the growth and development of fish, impacting physiological and immune responses, and ultimately increasing their susceptibility to diseases (Esam et al. 2022; Guo et al. 2022; Zhang et al. 2022). Recent research has reported that ammonia can induce adverse effects on the intestines, including the hyperplasia of intestinal goblet cells, disruption of normal immune-enzyme activities, the modulation of inflammatory cytokine expression and alteration of intestinal microbial composition, which collectively impair intestinal health (Qi et al. 2017; Kuang et al. 2023). Therefore, ammonia stress constitutes a significant hazard to the health and safety of fish in aquaculture environments, necessitating specific strategies to mitigate the harmful impact of ammonia on aquatic animals.

Implementing dietary strategies is deemed effective for stimulating immune function and enhancing the overall health of aquatic species to safeguard against diseases (Kiron 2012; Mendivil 2021; Jiao et al. 2022). There is an expanding emphasis on research and application of dietary short chain fatty acids to promote the health of aquatic animals within the aquaculture industry (Jesus et al. 2019, 2021; Yamamoto et al. 2021). Sodium Butyrate (SB), its powdered form (PSB) and microencapsulated form (MSB), along with poly-β-hydroxybutyrate (PHB), are all derivatives of short chain fatty acid-butyrate. Several studies has indicated that the supplementation of SB and PHB into feed has multiple benefits for aquatic animals, such as promoting the growth (Silva et al. 2016; Zhang et al. 2020), enhancing muscle nutrient contents (Guo 2017), and improving intestinal immunity (Tian et al. 2017). In terms of intestinal health, Partanen and Mroz (1999) have demonstrated that SB provides energy for epithelial cells, fosters the maturation of intestinal cells and facilitates the recovery of damaged villi. Zhao et al. (2021) reported that incorporating PSB into the diet can enhance resistance to ammonia stress of juvenile yellow catfish (Pelteobagrus fulvidraco) by modulating antioxidant status and immune response in the gut. PHB has been found to has similar functions to SB and PSB in promoting the growth of intestinal probiotics, since it can undergo enzymatic or chemical hydrolysis to produce β-hydroxybutyric acid in the gut of aquatic animals (Halet et al. 2007; De Schryver et al. 2011; Raza et al. 2018). Deng et al. (2015) have documented that dietary PHB supplementation results in higher lzm gene expression levels in gills and hepatopancreas of Litopenaeus vannamei after ammonia nitrogen stress, indicating that PHB can improve the ability of non-specific immunity system to react to ammonia stress. Nevertheless, the efficacy of the same actual added level of SB or PHB in alleviating ammonia stress by regulating fish intestinal health remains unclear. In addition, although most studies have focused on enhancing the ability of aquatic animals to resist ammonia stress through nutritional regulation, the post-stress health status is often overlooked.

The gut of aquatic animals serves as an site for microbial colonization and proliferation, thereby playing a pivotal role in both intestinal immunity and nutritional management (Han et al. 2010; Tran et al. 2017; Wang et al. 2018). In 2022, the production of grass carp (Ctenopharyngodon idella) constituted 18% of China’s total freshwater fish production as the 2023 reported by National Bureau of Statistics of China, which made it as one of the most popular farmed fish species in our country. However, grass carp is a relatively sensitive species to ammonia nitrogen and often suffers from ammonia stress (IJAZ et al. 2011; Zhao et al. 2019; Cao et al. 2021). Based on this, equivalent levels of PSB, MSB and PHB were incorporated the diet to evaluate their effects on the growth performance and muscle nutrition in grass carp, as well as the immune and antioxidant responses in the intestinal under ammonia nitrogen stress and recovery. The findings of this study provide valid data for management aimed at diminishing the hazard of ammonia nitrogen in fish.

2.1 Chemicals and Experimental Diets

The ingredients and proximate composition of experimental diets were shown in Table 1. The PSB (purity ≥ 98%) and MSB (purity ≥ 98%, with a 30% effective content of sodium butyrate) were purchased from Hangzhou King Techina Feed Co., Ltd (Hangzhou, China). The PHB (purity ≥ 98%) was sourced from Ningbo Tianan Biologic Material Co., Ltd (Ningbo, China). According to studies from Tian et al. (2017), Qiao et al. (2019) and our preliminary experiments, the basal diet was respectively supplemented with PSB, MSB and PHB at a level of 600 mg butyrate/kg, and the basal diet was used as the control (CK) in this study. The diets were produced using an extrusion machine to form pellet sizes, then air-dried and stored at -20 ℃.

Table 1

The ingredients and proximate composition of experimental diets
Item	CK	PSB	MSB	PHB
Ingredients (%)
Fish meal	10.00	10.00	10.00	10.00
Soybean meal	27.00	27.00	27.00	27.00
Rapeseed meal	27.00	27.00	27.00	27.00
Wheat gluten	5.00	5.00	5.00	5.00
Wheat middling	18.00	18.00	18.00	18.00
Soybean oil	3.60	3.60	3.60	3.60
Vitamin premix^a	1.00	1.00	1.00	1.00
Mineral premix^b	2.00	2.00	2.00	2.00
Ca(H₂PO₄)₂	2.00	2.00	2.00	2.00
Cr₂O₃	1.00	1.00	1.00	1.00
Choline chloride	0.30	0.30	0.30	0.30
Ethoxy quinoline	0.10	0.10	0.10	0.10
Microcrystalline cellulose	3.00	2.9	2.8	2.94
PSB	0	0.1	0	0
MSB	0	0	0.2	0
PHB	0	0	0	0.06
Proximate composition (%)
Moisture	15.37	17.16	17.17	15.22
Crude protein	32.64	32.07	32.59	32.85
Crude fat	4.06	4.03	4.14	4.19
Crude ash	8.28	8.18	8.17	8.13
Notes: a. one kilogram of vitamin premix contained the following components: vitamin D3, 0.480 g; vitamin E, 20.00 g; vitamin K3, 0.22 g; vitamin B12,0.1 g; D-biotin, 5 g; folic acid, 0.52 g; vitamin B1, 0.13 g; vitamin C, 7.16 g;nicotinic acid, 2.58 g; inositol, 52.33 g; Ca-pantothenate, 3.07 g; riboflain,0.99 g; pyridoxine, 0.75 g; cornstarch, 906.67 g. b. one kilogram of mineral premix contained the following components: MgSO4.7H20, 350.91 g; FeSO4.7H2O, 38.07 g; CuSO4.5H2O, 0.60 g; ZnSO4.7H2O, 6.67 g; MnSO4.H2O, 2.04 g; KI, 1.45 g; Na2SeO3, 2.50 g; cornstarch, 597.76 g.

2.2 Feeding experimental procedure

The same cohort of breeding and healthy grass carp were provided by Chonghu fish farm in Hubei, China. These grass carp were preliminarily raised for two weeks in an indoor re-circulating system, and were fed with the basal diet twice daily at 9:00 and 17:00 until they were satiated. The culture conditions were maintained at a temperature range of 24–28 ℃, a pH range of 7.4–7.9, an ammonia nitrogen concentration ≤ 0.2 mg/L, and a dissolved oxygen level > 5 mg/L.

For formal feeding test, a total of 240 grass carp, with an average weight of 58.79 ± 2.31 g, were randomly assigned to four groups: a control group, a PSB group, a MSB group and a PHB group. Each treatment group consisted of three replicate cylindrical aquariums with a capacity of 300-L. During the 56-d feeding experiment, fish in each tank were regularly weighed. At the end of breeding experiment, 6 individual fish from each group were randomly sampled, anesthetized with MS-222 solution, and their intestines were dissected and weighed. The fore, mid and distal intestines of three fish were respectively fixed with 4% paraformaldehyde for histological observation. The remaining intestines and muscles were rapidly frozen in liquid nitrogen and kept at -80°C.

2.3 Ammonia Stress and Recovery Tests

Fish were starved for 48 h following the conclusion of the experiment and then exposed to 10 mg/L total ammonia nitrogen (TAN) for 96 h (about 50% 96-h LC₅₀, a preliminary result in our laboratory). The solution of ammonia exposure was prepared by adding a stock solution of 10 g/L NH₄Cl. During the exposure period, TAN concentrations were measured every 24 h using the Nessler's Reagent Spectrophotometry method (HJ535-2009). The actual measured TAN was 10.47 ± 0.67 mg/L, and the estimated free ammonia was 0.28 ± 0.03 mg/L. After a 96-hour exposure to ammonia nitrogen, the fish were transferred to clean water for subsequent 15-d recovery period. The culture conditions were the same as those of the feeding trial, and no mortality of grass carp occurred during ammonia stress and the subsequent recovery tests. At the end of the ammonia exposure and recovery periods, the intestines of grass carp were collected and stored at -80°C.

2.4 Growth performance calculation and adopted growth models

The weight gain rate (WGR), specific growth rate (SGR), condition factor (CF), hepatosomatic index (HIS), viscerasomatic index (VSI), spleensomatic index (SSI) and intestinesomatic index (ISI) of grass carp were calculated. The detailed calculation procedures were provided in the Supporting Information (Supplementary Text S1).

Three nonlinear growth models, the Logistic, Gompertz and Von Bertalanffy were applied to conduct a nonlinear regression analysis on the body weight data of grass carp from four treatment groups (Table S1). The parameter estimates of A, B and K and the fitting degree (R²) were obtained for each group (Table S2). Among which, R² was used to evaluate the optimal growth curve fitting models for different groups.

2.5 Muscle proximate composition

The determination of fish muscle composition referred to the description by Hu et al. (2023). Moisture was determined by drying the muscle with a 105°C constant weight method. Crude ash was measured using the 550°C burning method, in which the dried muscle were first carbonized until smokeless, then heated at 550°C for 9 hours and weighted. Crude protein content is determined by digesting the muscle with sulfuric acid followed by the Kjeldahl method. Crude lipid was determined by Soxhlet extraction, where dried muscle was wrapped in paper and wool followed by distillation with anhydrous ether.

2.6 Histopathological evaluation

The fore, mid and distal intestine samples were fixed overnight in 4% paraformaldehyde. Subsequently, samples underwent dehydration in a series of ethanol solutions (70–100%), followed by hyalinization in a xylene-ethanol mixture(v/v, 1:1) for 15 minutes and in 100% xylene for another 15 minutes. Samples were embedded in paraffin wax (56–58 ℃), sectioned into 5 µm slices, and stained with hematoxylin and eosin (H&E). Histological analysis was conducted using an optical microscope (Nikon, Japan). The crypt depth, villus area and intestinal area were measured using ImageJ 1.53 C (National Institutes of Health, Bethesda, MD, USA), while goblet cells were enumerated.

2.7 Determination of antioxidant and immune parameters

Intestinal samples were homogenized in ice-cold physiological saline (1:9 w/v). After centrifugation for 10 min at 3,000 r/min and 4 ℃, the supernatant was isolated for the detection of immune and antioxidant parameters (T-AOC, T-SOD, MDA, LZM, ACP, IL-1β and β-DF) following the respective kit manufacturer’s instructions. The manufacturer and item number of the kits were provided in the Supporting Information (Supplementary Text S2). All experiments were conducted by six biological replicates fish per treatment group.

2.8 Integrated Biomarker Response Analysis

IBR analysis was utilized to integrate all measured biomarkers into a comprehensive index (Beliaeff and Burgeot 2002). In the study, IBR analysis was used to evaluate the effects of dietary supplementation with PSB, MSB and PHB on the intestinal resistance and recovery from ammonia nitrogen stress in grass carp. The detailed calculation procedure is described in the Supporting Information (Supplementary Text S3).

2.9 Statistical Analysis

Data normality and variance homogeneity were assessed with the Kolmogorov-Smirnov test and Leven’s test using SPSS 27.0 (SPSS Inc., USA). Significant differences among groups (P-value < 0.05) were calculated using one-way ANOVA followed by Duncan’s test. Results are presented as mean ± standard error, and graphics were created with Origin 2021 software.

3.1 Growth parameters

As shown in Table 2, in the PSB, MSB and PHB groups, the WGR and SGR of grass carp increased significantly compared to the CK group (P < 0.05). Following the 56-day feeding test, the changes observed in CF, VSI, HSI and SSI across all treatment groups were not statistically significant (P > 0.05). In addition, the ISI for grass carp in the MSB group was significantly higher than that in CK group (P < 0.05).

Table 2

Effects of dietary sodium butyrate and poly-β-hydroxybutyrate on the growth performance of grass carp
Index	CK group	PSB group	MSB group	PHB group
WGR (%)	111.27 ± 6.47^a	126.57 ± 4.89^b	123.55 ± 4.05^b	128.28 ± 8.94^b
SGR (%)	1.34 ± 0.06^a	1.46 ± 0.04^b	1.44 ± 0.03^b	1.47 ± 0.07^b
CF (%)	1.69 ± 0.07	1.83 ± 0.01	1.72 ± 0.18	1.89 ± 0.09
VSI (%)	8.85 ± 0.47	9.05 ± 1.16	9.64 ± 1.30	9.20 ± 2.26
HSI (%)	1.56 ± 0.30	1.72 ± 0.12	1.70 ± 0.38	1.58 ± 0.03
SSI (%)	0.11 ± 0.01	0.13 ± 0.02	0.12 ± 0.01	0.12 ± 0.02
ISI (%)	3.08 ± 0.24^a	3.73 ± 0.69^ab	4.33 ± 0.68^b	3.13 ± 0.27^a
Notes: Data are express as means ± SE (n = 6). Different superscript letters in a line show significant difference between different treatments (P < 0.05).

The fitting parameters of the Logistic, Gompertz and Von Bertalanfy growth models in grass carp are shown in Supporting Information (Table S2). Based on the R2 values, the growth curve of grass carp was fitted by logistic model. Then, the parameters were substituted into the logistic model to obtain the growth curve equation of grass carp in all groups, as follows:

CK group: Y=154.8/(1+1.685*exp(-0.038t)),

PSB group: Y =165.9/(1+1.840*exp(-0.038t)),

MSB group: Y =157.3/(1+1.729*exp(-0.039t)),

PHB group: Y = 171.5/(1+1.919*exp(-0.036t)),

Among them, t is the day of breeding, Y is the weight of fish on the t day of breeding. The results showed a trend in weight gain for grass carp across all treatment groups, which could be ranked as follow: PHB group > PSB group > MSB group > CK group (Fig. 1).

3.2 Proximate composition

The effects of dietary PSB, MSB and PHB on proximate composition of fish muscle were presented in Table 3. No notable variations in muscle moisture level were detected across all treatment groups (P > 0.05). Compared to the control diet, crude protein levels of grass carp muscle were significantly increased in PSB and MSB groups (P < 0.05), while crude lipid levels in the muscle of grass carp were significantly decreased in all treatment groups (P < 0.05). The crude ash levels in PSB and PHB groups were significantly higher than those of the CK group (P < 0.05).

Table 3

Effects of dietary sodium butyrate and poly-β-hydroxybutyrate on the proximate composition of grass carp
Index	CK group	PSB group	MSB group	PHB group
Moisture	75.90 ± 0.53	75.95 ± 0.66	76.33 ± 0.49	76.01 ± 0.34
Crude protein	18.25 ± 0.80^a	19.44 ± 0.64^b	19.00 ± 0.37^b	18.82 ± 0.34^ab
Crude lipid	3.13 ± 0.23^a	2.41 ± 0.22^b	2.45 ± 0.27^b	2.29 ± 0.21^b
Crude ash	0.94 ± 0.09^a	1.05 ± 0.11^b	1.04 ± 0.08^ab	1.12 ± 0.11^b
Notes: Data are express as means ± SE (n = 6). Different superscript letters in a line show significant difference between different treatments (P < 0.05).

3.3 Intestinal morphological assessment

The intestinal images displayed distinct differences among the three treatment groups compared to the CK group, characterized by increased crypt depth, goblet cell numbers and villus area. In the PSB group, the crypt depths of the fore and mid intestines were significantly increased (P < 0.05)(Fig. 2D). Similarly, both the MSB and PHB groups showed an increased crypt depth in the mid and distal intestines (P < 0.05) (Fig. 2D). The number of goblet cells per unit area in the fore and mid intestines of the PSB group, and the fore and distal intestines of MSB group, as well as the mid and distal intestine of PHB group were significantly elevated (P < 0.05) (Fig. 2E). The villus areas was significantly higher in the fore intestine in the PSB group, the distal intestine in the MSB group, and the mid and distal intestine in PHB group than in the control group (P < 0.05) (Fig. 2F).

3.4 Intestinal immunity and oxidative-antioxidant status

The effects of dietary supplementation with PSB, MSB and PHB on the intestine oxidative-antioxidant status and non-specific immunity were presented in Fig. 3. Compared to the CK group, the PSB group exhibited a significant increase in the activities of ACP (in FI and MI), T-SOD (in FI and MI) and T-AOC (in FI) as well as the content of β-DF (in FI and MI), while the contents of IL-1β (in FI and MI) and MDA (in FI) were significantly decreased (P < 0.05). In the MSB group, there are a significant increase in the activities of LZM (in MI and DI), ACP (in FI and DI) and T-SOD (in MI and DI) along with the content of β-DF (in FI, MI and DI), whereas the contents of IL-1β (in MI and DI) and MDA (in FI, MI and DI) were markedly reduced (P < 0.05). The PHB group also showed an increase in the activities of LZM (in MI and DI), ACP (in MI and DI) and T-SOD (in MI and DI) as well as the content of β-DF (in DI), but a decrease in the contents of IL-1β (in MI and DI) and MDA (in DI) (P < 0.05). Furthermore, the IBR index for intestinal oxidative-antioxidant and non-specific immune parameters in grass carp followed this order: MSB (IBR/n = 0.64) > PHB (IBR/n = 0.49) > PSB (IBR/n = 0.38) > CK (IBR/n = 0.00).

3.5 Intestinal non-specific immunity and oxidative-antioxidant status under ammonia stress

The effects of ammonia stress on the intestinal oxidative-antioxidant status and non-specific immunity of fish, following dietary supplementation with PSB, MSB and PHB, were depicted in Fig. 4. In the PSB group, compared to the control group, there was a significant increase in the activities of LZM (in FI), ACP (in FI and MI), T-SOD (in FI and MI) and T-AOC (in FI) as well as the content of β-DF (in FI and MI), while the contents of IL-1β (in FI and MI) and MDA (in FI) were significantly decreased (P < 0.05). In the MSB group, the activities of LZM (in FI, MI and DI), ACP (in MI and DI), T-SOD (in FI, MI and DI) and T-AOC (in MI and DI) and the content of β-DF (in FI, MI and DI) were significantly increased (P < 0.05). Meanwhile, the contents of IL-1β (in DI) and MDA (in FI, MI and DI) were significantly reduced (P < 0.05). In the PHB group, the activities of LZM (in MI and DI), ACP (in MI and DI) and T-SOD (in FI, MI and DI) and T-AOC (in MI and DI) and the content of β-DF (in MI and DI) were significantly elevated, but the contents of MDA (in MI and DI) showed a significant decrease compared to the CK group (P < 0.05). In addition, the IBR index for intestinal oxidative-antioxidant and non-specific immune parameters in grass carp exhibited the following trend: PHB (IBR/n = 0.44) > MSB (IBR/n = 0.42) > PSB (IBR/n = 0.23) > CK (IBR/n = 0.00).

3.6 Intestinal oxidative-antioxidant status and non-specific immunity following ammonia nitrogen stress recovery

The effects of dietary supplementation with PSB, MSB and PHB on the intestinal oxidative-antioxidant status and non-specific immunity of fish following ammonia nitrogen stress recovery are presented in Fig. 5. Compared to the CK group, significant increases were observed in the PSB group for the activities of LZM (in FI), ACP (in FI), T-SOD (in FI) and T-AOC (in FI) as well as the content of β-DF (in FI and MI), while the contents of IL-1β (in FI) and MDA (in FI and MI) significantly decreased (P < 0.05). In the MSB group, there were significant increases in the activities of LZM (in FI and DI), ACP (in MI and DI) and T-SOD (in FI and DI) and the content of β-DF (in FI, MI and DI), with a significant reduction in the contents of IL-1β (in MI and DI) and MDA (in MI and DI) (P < 0.05). The PHB group also showed significant increases in the activities of LZM (in MI and DI), ACP (in DI) and T-SOD (in DI) and the content of β-DF (in MI and DI), whereas the levels of IL-1β (in MI and DI) and MDA (in DI) were significantly lower compared to the CK group (P < 0.05). Moreover, the IBR index for intestinal oxidative-antioxidant and non-specific immune parameters in grass carp exhibited the following order: MSB (IBR/n = 0.38) > PSB (IBR/n = 0.29) > PHB (IBR/n = 0.17) > CK (IBR/n = 0.00).

The excessive presence of ammonia nitrogen in aquaculture water threatens the health and safety of aquatic animals (Vaage and Myrick 2021; Zhang et al. 2022). This investigation provided evidence that dietary supplementation of butyrate (PSB, PMB and PHB) could promote growth of grass carp, and improve the intestinal resistance and recovery to ammonia stress, and the effect of MSB was better than that of others.

4.1 Effects of PSB, MSB and PHB on the growth performance and proximate composition

Previous studies have demonstrated that adding 500 and 1000 mg/kg SB to the diet for 56 d remarkably gains weight in Pelteobagrus fulvidraco (Zhao et al. 2021). Similarly, the inclusion of PHB in the diet tends to increase the weight gain of Nile tilapia and Gibel carp (Situmorang et al. 2016; Liu et al. 2022). In our present study, the WGR and SGR of grass carp in the PSB, MSB and PHB groups were significantly increased, indicating that these three feed additives can promote the growth performance of grass carp. Our results also showed that PSB, MSB and PHB supplementation significantly increased intestinal crypt depth and villus area. Similarly, the supplementation of 2.0 g/kg coated SB (effective content 50%) significantly elevated the intestinal villus height and improved intestinal morphology of Oncorhynchus mykiss (Lin et al. 2023). The supplementation with SB and PHB enhanced the length and circumference of intestinal villi in L. vannamei, which promoted the absorption of nutrients (Silva et al. 2016). Thus, as short-chain fatty acids additive, PSB, MSB and PHB in our study can respectively be degraded into butyrate and β-hydroxybutyrate in the intestine, which contribute to enterocyte proliferation, enhance nutrient digestion and absorption, and thus promote the growth of grass carp.

Fish body composition, including protein, lipid, moisture and so on, serves as a dependable predictor of muscle quality, nutritional value and physiological condition (Venugopal and Shahidi 1996; Breck 2014). In this study, the addition of PSB, MSB and PHB increased the crude protein level but decreased the crude lipid level in grass carp muscle. Benedito-Palos et al. Benedito-Palos et al. (2016) and Yan et al. (2021) also found that dietary sodium butyrate increased the protein content of fish muscle, which were due to the down-regulation of the expression of genes (such as cdh15, capn3) related to protein degradation. Dietary supplementation with PSB, MSB and PHB has been reported to reduce the content of crude fat in muscle of grass carp and siberian sturgeon (Acipenser baerii) (Najdegerami et al. 2015; Zhou et al. 2019; Yan et al. 2021). And the reason for that is probably because short-chain fatty acid can mediate the activation of GPR43 to suppress insulin signaling in adipocytes, thereby inhibiting fat accumulation (Kimura et al. 2013). Thus, our results suggest that the addition of PSB, MSB and PHB can promote the growth and improve muscle quality of grass carp .

4.2 Protective effects of PSB, MSB and PHB on intestinal immunity and antioxidant capacity

The intestine of fish is a complex, multi-functional organ, that is not only responsible for digestion and absorption of nutrients but also playing a critical role in immunity (Rombout et al. 2011). Fish intestinal immunity is closely related to non-specific immune antimicrobial substances such as LZM, ACP and β-DF. LZM play a direct antimicrobial role and can modulate the host immune response to the infection (Bols et al. 2001; Ragland and Criss 2017). Defensins are a group of small antimicrobial peptides that serve the first line of host defense against invading pathogens and contribute to the adaptive anti-microbial immunity (Bulet et al. 2004; Zou et al. 2007). Cytokines, such as IL-1β, are key regulators of the immune system, and the expression of different cytokine genes is an important indicator of intestinal health (Wang and Secombes 2013). Previous studies have confirmed that short-chain fatty acids like butyric acid can regulate inflammatory responses by inhibiting the production of pro-inflammatory factors (Cavaglieri et al. 2003; Estensoro et al. 2016). Mirghaed et al. (2019b) found that SB improved intestinal immune responses of Oncorhynchus mykiss. Increased expression of antimicrobial peptides in PHB-fed European sea bass post-larvae indicated that PHB could stimulate the immune system during the early life stages of fish (Franke et al. 2017). In our study, higher activities of intestinal LZM and ACP, as well as greater β-DF content were detected in three treatment groups compared to the CK group, while IL-1β content was lower, indicating that these three feed additives can enhance intestinal nonspecific immunity and reduce inflammation of grass carp. This result is also supported by the increased intestinal goblet cells after supplement of PSB, PMB and PHB. Goblet cells and the mucus they secrete serve as crucial barriers, effectively preventing pathogens from infiltrating the mucosa and inducing intestinal inflammation (Knoop and Newberry 2018; Yang and Yu 2021).

MDA content is an indicator of the degree of lipid peroxidation and cellular damage (Valavanidis et al. 2006). The changes in T-AOC level provides a comprehensive reflection of the body’s antioxidant status (Aliko et al. 2018). SOD functions to convert superoxide free radicals into hydrogen peroxide (H₂O₂), acting as a primary defense against antioxidant stress (Yasui and Baba 2006). Previous investigations have shown that dietary supplementation with SB can enhance the expression of sod gene in the gut of rainbow trout, indicating that sodium butyrate contributes to the health of rainbow trout. Sui et al. (2016) found that the activity of SOD in the serum of Eriocheir sinensis increased with the increment of PHB supplementation level. In our study, the intestinal MDA content of grass carp in three treatment groups was reduced compared to the CK group, while SOD activity and T-AOC levels were elevated, indicating that the inclusion of PSB, MSB and PHB could reduce the degree of lipid damage of intestinal cells and improve the antioxidant capacity of grass carp. The results of IBR index further showed that the MSB group had the highest overall improvement in health status, followed by the PSB and SB groups.

4.3 Effects of PSB, MSB and PHB on intestinal immunity and antioxidant capacity under ammonia stress and recovery

Ammonia exposure disrupts the equilibrium between the generation of reactive oxygen species and antioxidant system in fish (Li et al. 2016; Jia et al. 2017; Mirghaed et al. 2019a), emphasizing the need for effective strategies to mitigate ammonia toxicity in aquaculture. In a recent study by Gao et al. (Gao et al. 2022), the supplementation of SB following a high-fat diet was found to significantly increase T-SOD activity and decrease MDA content in the liver of grass carp, indicating a potential of SB in reducing oxidative stress. Similarity, continuous feeding with 4% PHB for 30 d enhanced the resistance of gibel carp to viruses and bacteria (Liu et al. 2022). In our study, the increased T-AOC and decreased MDA observed during periods of ammonia stress and recovery revealed that the addition of PSB, MSB and PHB can enhance the fish’s antioxidant capacity to reduce reactive oxygen species and lipid peroxides, ultimately alleviating ammonia-induced oxidative stress.

Changes in immune responses are recognized as an important indicator for identifying the toxic effects of ammonia exposure in fish (Xu et al. 2021). The gastrointestinal immune system is crucial in maintaining immune homeostasis (Lee et al. 2021). Previous studies have shown that ammonia nitrogen exposure can result in intestinal mucosal goblet cell hyperplasia and increased levels of LZM and β-DF, suggesting a decline in the innate immunity of fish intestines (Kuang et al. 2023). (Qi et al. (2017) further found no significant difference was found in kidney immune-related gene expression (such as tnf-α and il-1β) in carassius auratus after a 15-day treatment without ammonia, indicating that the immune response caused by ammonia nitrogen stress in fish might be reversible. Zhao et al. (2021) found that the supplementation of SB can activated the intestinal antioxidant defense of yellow catfish, thereby weakening the inflammatory response induced by ammonia. Deng et al. (2015) also reported that PHB could improve the ability of the immune system of Litopenaeus vannamei to cope with ammonia nitrogen stress. In the current study, during the periods of acute ammonia stress and subsequent recovery, the activities of LZM and ACP and the contents of β-DF were higher in the intestinal of grass carp treated with PSB, MSB and PHB compared to the control group, while the levels of IL-1β were lower. These findings suggested that these three additives (PSB, MSB, and PHB) contribute to enhance the intestinal non-specific immunity of grass carp and alleviate inflammatory responses caused by ammonia nitrogen. Furthermore, the IBR analysis corroborates these findings, providing evidence of the beneficial effects of the three additives on the intestinal health of grass carp under ammonia stress and recovery. It should be noted that the MSB group showed a more pronounced effect in enhancing the intestinal immune response compared to the other treatments.

This current study demonstrated that the dietary supplement of PSB, MSB and PHB has multiple beneficial effects on the growth, muscle quality, intestinal structure and health of grass carp. During acute ammonia nitrogen stress and subsequent recovery periods, the inclusion of PSB, MSB, and PHB also improved the intestinal anti-ammonia nitrogen stress and recover capacities, which was demonstrated by improvements in the antioxidant and non-specific immune functions, with MSB showing a particularly strong effect. Overall, the use of these three supplements might be an effective approach for the prevention, alleviation, and restoration of ammonia nitrogen stress in aquaculture practices.

Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

All authors contributed to the study conception and design. Yingying Wang: Conceptualization, Investigation, Data curation, Validation, Formal analysis, Writing-original draft, Writing editing. Kang Ou-Yang: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Software. Ya He: Investigation. Hui Yang: Data curation. Liangmou Wang: Investigation. Dapeng Li: Resources, Supervision. Li Li: Funding acquisition, Project administration, Supervision, Methodology, Writing – review & editing.

Ethics statement

The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Huazhong Agricultural University, Wuhan, China, with the approval number (HZAUFI-2023-0051).

Funding

This work was jointly supported by the National Key R&D Program of China (2023YFD2400501), the National Natural Science Foundation of China (32071621), the China Agriculture Research System of MOF and MARA (CARS-45-24).

Author Contribution

Yingying Wang: Conceptualization, Investigation, Data curation, Validation, Formal analysis, Writing-original draft, Writing editing.Kang Ou-Yang: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Software. Ya He: Investigation. Hui Yang: Data curation. Liangmou Wang: Investigation. Dapeng Li: Resources, Supervision. Li Li: Funding acquisition, Project administration, Supervision, Methodology, Writing – review & editing.

Data availability

Data will be made available on request.

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No competing interests reported.

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Graphical Abstract

Effects of sodium butyrate and poly-β-hydroxybutyrate on the intestinal resistance and recovery from ammonia nitrogen stress in grass carp (Ctenopharyngodon idella)

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1 Introduction

2 Materials and methods