Metabolites, gene expression, and gut microbiota profiles suggest the putative mechanisms via which dietary creatine increases the serum taurine and g-ABA contents in Megalobrama amblycephala

A 90-day experiment was conducted to explore the effects of creatine on growth performance, liver health status, metabolites, and gut microbiota in Megalobrama amblycephala. There were 6 treatments as follows: control (CD, 29.41% carbohydrates), high carbohydrate (HCD, 38.14% carbohydrates), betaine (BET, 1.2% betaine + 39.76% carbohydrates), creatine 1 (CRE1, 0.5% creatine + 1.2% betaine + 39.29% carbohydrates), creatine 2 (CRE2, 1% creatine + 1.2% betaine + 39.50% carbohydrates), and creatine 3 (CRE3, 2% creatine + 1.2% betaine + 39.44% carbohydrates). The results showed that supplementing creatine and betaine together reduced the feed conversion ratio significantly (P < 0.05, compared to CD and HCD) and improved liver health (compared to HCD). Compared with the BET group, dietary creatine significantly increased the abundances of Firmicutes, Bacteroidota, ZOR0006, and Bacteroides and decreased the abundances of Proteobacteria, Fusobacteriota, Vibrio, Crenobacter, and Shewanella in the CRE1 group. Dietary creatine increased the content of taurine, arginine, ornithine, γ-aminobutyric acid (g-ABA), and creatine (CRE1 vs. BET group) and the expression of creatine kinase (ck), sulfinoalanine decarboxylase (csad), guanidinoacetate N-methyltransferase (gamt), glycine amidinotransferase (gatm), agmatinase (agmat), diamine oxidase1 (aoc1), and glutamate decarboxylase (gad) in the CRE1 group. Overall, these results suggested that dietary supplementation of creatine (0.5–2%) did not affect the growth performance, but it altered the gut microbial composition at the phylum and genus levels, which might be beneficial to the gut health of M. amblycephala; dietary creatine also increased the serum content of taurine by enhancing the expressions of ck and csad and increased the serum content of g-ABA by enhancing the arginine content and the expressions of gatm, agmat, gad, and aoc1.


Introduction
Megalobrama amblycephala is one of the most important cultured freshwater fish species in China due to its high nutritional value, rapid growth, and easy breeding (Xu et al. 2016;Zhou et al. 2017). As the cheapest energy source for fish, carbohydrates are incorporated in feed formulation to substitute expensive marine ingredients . However, most fishes have poor carbohydrate-utilization abilities. High-carbohydrate diets cause an increased lipid deposition in carcass and liver ) and increase the prevalence of fatty liver disease in fish . Our research showed that high dietary carbohydrates could significantly decrease the concentration of liver betaine and lead to disturbances in the creatine pathway in M. amblycephala Xu et al. 2018). We also found that betaine supplementation to high-carbohydrate diets could effectively alleviate some negative effects of carbohydrates (Xu et al. 2018). However, previous studies had not reported the effects of dietary creatine supplementation to highcarbohydrate diets on the metabolism of fish.
Creatine, also known as N-methyl guanidine acetic acid or muscarinic acid, is a nitrogenous organic acid that belongs to amino acid derivatives. It is a natural nutrient synthesized by the animal organism, and it is widely used to improve human health and enhance the growth performance of animals due to its important roles in the organism: (i) increasing creatine content in skeletal muscle and improving energy reserves (Fukuda et al. 2010), (ii) increasing protein synthesis (Kreider et al. 1998;Shankaran et al. 2016), (iii) regulating metabolism and reducing lactate production (Ceddia and Sweeney 2004;Roschel et al. 2010), and (iv) improving growth traits (Burns and Gatlin 2019). In humans and mice, creatine is closely linked to lipid metabolism (Kazak et al. 2015;da Silva et al. 2017;Rahbani et al. 2021). A decrease of creatine in cells promotes diet-induced fat accumulation (Rahbani et al. 2021), whereas the enhancement of creatine metabolism facilitates the energy expenditure (Kazak et al. 2015). As a feed additive in aquaculture, the effects of creatine on growth performance differ among different fish species and/or studies. The addition of 0.5-4% creatine to the diet of Sciaenops ocellatus resulted in significant differences in weight gain, feed efficiency, and survival rates compared to the diet without creatine (Burns and Gatlin 2019). However, there were no significant effects on the growth traits of Sciaenops ocellatus (Stites et al. 2020) and the weight gain rate of Sparus aurata (Ramos-Pinto et al. 2019) when they were fed diets with 2% creatine. Up to now, the effects of dietary creatine on growth performance and lipid metabolism of M. amblycephala had not been reported. The objective of this study was to explore the effects of different supplementation levels of dietary creatine on the metabolism of M. amblycephala.
Taurine is a nutritionally essential amino acid for humans (Schaffer et al. 2009). It is widely distributed in the tissues, organs, and cells of animals (HuxTable 1992). Taurine has important roles in humans and other animals: (i) improving the growth performance, (ii) reducing lipid deposition, (iii) regulating osmolality, and (iv) improving immunity and antioxidant capacity (Salze and Davis 2015;Wu 2020). Taurine content is very low in plant-derived feed, and it is commonly used as a feed additive in aquaculture (Li and Wu 2020). The addition of taurine improved the growth performance and immunity of Takifugu rubripes and Clarias gariepinus (Adeshina and Abdel-Tawwab 2020;Shi et al. 2020). Taurine also lowered blood sugar levels in patients with diabetes and regulated glycolipid metabolism to improve the utilization of dietary carbohydrates in hybrid grouper and Monopterus albus (Zheng et al. 2016;Qian et al. 2021;Shi et al. 2022). As nutrients that can regulate metabolism, creatine and taurine together have a potent stimulatory effect on metabolic activity and antidepressant effects in vertebrates (Kim et al. 2020). However, the relationship between taurine and creatine in M. amblycephala has not been studied yet, so another objective of this study was to fill this knowledge gap.γ-Aminobutyric acid (g-ABA) is a non-protein amino acid and major inhibitory neurotransmitter in the central nervous system . g-ABA has important roles in improving human health, such as regulating the intestinal tract, stimulating nerves, and protecting the heart (Diez-Gutierrez et al. 2020). As a feed additive, g-ABA improved the immunity of Gasterosteus aculeatus (Kutyrev et al. 2019) and the growth performance of Ctenopharyngodon idellus (Wu et al. 2016), Litopenaeus vannamei (Xie et al. 2017), and Cyprinus carpio var. Jian . The production of g-ABA by bacteria is influenced by environmental factors and additives (Diez-Gutierrez et al. 2020). Studies had shown that g-ABA could play a role in regulating the blood glucose metabolism in both hyperglycemic and hypoglycemic conditions (Sohrabipour et al. 2018;Zhang et al. 2021). Up to now, the relationship between g-ABA metabolism and creatine metabolism had not been studied.
In this context, the objective of this research was first to explore the appropriate creatine requirement of M. amblycephala fed a high-carbohydrate diet with supplemented betaine according to the growth performance and liver health status. Following this, we set out to explore in detail the metabolic impacts of creatine supplementation by studying the levels of serum metabolites and expression of genes involved in metabolic creatine pathways, as well as the composition of gut microbiota. The ultimate goal of this study was to help elucidate the mechanisms by which dietary creatine increased the serum taurine and g-ABA contents in animals.
After 90 days of the feeding trial, 24 h after the last feeding, fish in all tanks were anaesthetized with MS-222 (Sigma-Aldrich, USA) at the concentration of 100mgL −1 . Twelve fish from each tank were selected randomly to obtain the blood, liver, and gut samples. All animals were handled and experimental procedures were conducted in accordance with the guidelines for the care and use of animals for scientific purposes set by the Ministry of Science and Technology, Beijing, China (No. 398, 2006).
Growth performance, diet composition, and liver H&E analyses The following growth parameters were calculated: weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), feed intake (FI), condition factor (CF), and hepatosomatic index (HSI). Crude protein, crude fat, moisture, and crude ash composition of diets (feeds) were determined by standard methods exactly as previously described . Carbohydrate (nitrogen-free extract) content of diets was analyzed by the 3,5-dinitro salicylic acid method (Yu et al. 1997). Hematoxylin and eosin (H&E) staining analyses were conducted according to the established protocol (Wang et al. 2019).

Quantification of amino acids
To understand the impacts of different dietary creatine levels on the profile of metabolites associated with creatine metabolism in the liver, we measured the content of related amino acids in serum using an Automatic Amino acid analyzer (MembraPure GmbH, Germany). In various amino acid components with different structures, acidity, polarity, and molecular size, the ion-exchange chromatographic column (Li-separation column, MembraPure, Bodenheim, Germany) was adopted, where the amino acids were eluted by a lithium buffer system. The amino acid fractions were eluted down by buffers with different pH ion concentrations and then mixed with ninhydrin reagents one by one. The resultant derivatives were measured by UV detection at two wavelengths of 440 and 570 nm simultaneously and identified and quantified according to their peak time and area when compared with the spectrogram of the standard amino acid samples (Sigma-Aldrich, St. Louis, MO, USA). Relative metabolites (creatine) were quantified using high-performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) on an Ultimate3000 (Dionex, Sunnyvale, USA)-API 4000Q TRAP spectrometer (AB Sciex, Framingham, MA, USA) by the Beijing Mass Spectrometry Medical Research Co. (Beijing, China) as previously described (Xu et al. 2018), with some modifications. One hundred fifty microliters of protein precipitant (containing melatonin) was added to 50 μL of serum solution of each sample, mixed thoroughly, and centrifuged at 13,200 r/min for 4 min, and the supernatant was used for testing. Five microliters of the supernatant was analyzed by the HPLC-MS/MS system. Chromatographic separations were performed on a Sapphire-C18 column (100 × 4.6 mm, 5 μm). The mobile phase A was 1% formic acid in water (v/v), and the organic mobile phase B was 1% acetonitrile (v/v). The full details are described in detail in the Supplementary methods file.

Gut microbiota analysis
Intestinal contents were collected from 12 fish specimens in each tank/repetition and mixed for 16S rRNA sequencing, and there were 3 repetitions in each group. Microbial community genomic DNA was extracted using the cetyltrimethylammonium ammonium bromide (CTAB) method. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHVGGG TWT CTAAT-3′) by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of the 16S rRNA gene was performed as follows: initial denaturation at 95℃ for 3 min, followed by 27 cycles of denaturing at 95℃ for 30 s, annealing at 55℃ for 30 s, and extension at 72℃ for 45 s; finally, an extension at 72℃ for 10 min was followed by holding at 4℃. Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0, and merged by FLASH version 1.2.7 with the following criteria: (i) the 300-bp reads were truncated at any site receiving an average quality score of < 20 over a 50-bp sliding window, the truncated reads shorter than 50 bp were discarded, and reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of the overlap region is 0.2. Reads that could not be assembled were discarded; (iii) samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotide mismatch in primer matching. Operational taxonomic units (OTUs) with a 97% similarity cutoff value were clustered using UPARSE version 7.1, and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database (e.g. Silva v138) using a confidence threshold of 0.7.

Statistical analysis
Data were subjected to a one-way analysis of variance (ANOVA). Significant differences among the group means were further compared using Duncan multiple range tests at the P < 0.05 threshold of statistical significance. All analyses were conducted using SPSS 26 (SPSS, Chicago, USA).

Growth performance and liver histology
After the feeding trial, there were no significant differences in WG, SGR, and CF among all of the experimental groups (Table 2; all P > 0.05). HSI was 258 Fish Physiol Biochem (2023) 49:253-274 significantly higher in the HCD group than in other groups (P < 0.05). FCR values were significantly lower in the CRE group in comparison to the CD and HCD groups (P < 0.05): CRE1 < CRE3 < CRE2 < B ET < HCD < CD (Table 2). However, the FCR value of the CRE group was not significantly different from that of the BET group (P > 0.05).
Livers of the HCD group exhibited a higher number of enlarged hepatocytes and disappearing nuclei than livers of other groups. Livers of the CD, BET, and CRE1 groups had large and round nuclei and exhibited a small number of lipid droplets; while some slightly damaged and disappearing nuclei could be observed in the hepatocytes of these three groups, these symptoms were not as severe as in the HCD group (Fig. 1).
Expression of genes associated with creatine metabolic pathways There were no significant differences in growth performance and liver histology between the CRE1, CRE2, and CRE3 groups, so only the CRE1 group was selected for further analyses.
Compared to the BET group, the expression levels of ck, csad, gamt, gatm, agmat, aoc1, and gad genes were elevated significantly in the CRE1 group, whereas the expressions of arg and odc1 were decreased significantly (all P < 0.05). Compared with the HCD group, the expression of ck, sardh, adoa, adob, csad, gamt, gatm, arg, agmat, aoc1, aldh, and gad genes was elevated significantly, whereas the expression of odc1 was decreased significantly (all P < 0.05), in the CRE1 group. In the BET group, compared with the HCD group, the expression of sardh, adoa, and arg was elevated significantly ( Fig. 2A).

Amino acids and metabolites related to creatine
In the CRE1 group, compared with the BET group, the contents of taurine, arginine, ornithine, γ-aminobutyric acid (g-ABA), and creatine were elevated significantly (P < 0.05), whereas the contents of sarcosine, glycine, cystathionine, and glutamate were decreased significantly (P < 0.05). In the CRE1 group, compared with the HCD group, the contents of glycine, cystathionine, taurine, ornithine, g-ABA, and creatine were all elevated significantly (P < 0.05), whereas the content of glutamate was decreased significantly (P < 0.05). In the BET group, the contents of glycine, serine, cystathionine, ornithine, and g-ABA were elevated significantly (P < 0.05), whereas the contents of sarcosine, taurine, arginine, glutamate, and creatine were not significantly different (P > 0.05) compared with the HCD group (Fig. 2B).

Gut microbiota diversity and composition
A total of 665,409 high-quality DNA sequences were obtained from the 12 samples, and 214 operational taxonomic units (OTU) were identified from the gut DNA samples. We calculated the indices of bacterial diversity (Shannon and Simpson) and richness (Chao and Ace) from the proportion of the OTUs for the calculation of each experimental Values are presented as means of the entire group with standard deviation: CD, control; HCD, high carbohydrate; BET, 1.2% betaine supplementation; CRE1, 1.2% betaine + 0.5% creatine supplementation; CRE2, 1.2% betaine + 1% creatine supplementation; CRE3, 1.2% betaine + 2% creatine supplementation; WG, weight gain; SGR, specific growth rate; FCR, feed conversion ratio; CF, condition factor; HSI, hepatosomatic index. Significant (P < 0.5) differences between groups are indicated by different letters 1.11 ± 0.11 a 1.24 ± 0.08 b 1.10 ± 0.11 a 1.11 ± 0.11 a 1.14 ± 0.10 a 1.10 ± 0.09 a CF 1.97 ± 0.13 1.98 ± 0.12 1.98 ± 0.14 1.98 ± 0.15 2.01 ± 0.09 2.00 ± 0.13 group's bacterial diversity. There was no significant difference in Ace, Chao, Shannon, and Simpson indices among the CD, BET, and CRE1 groups (P > 0.05) (Fig. 3). However, the Shannon index of the CRE1 group was significantly lower than that of the HCD group (P < 0.05) (Fig. 3C). Principal coordinates analysis (PCoA) was conducted according to the proportion of OTUs in different groups, and the results showed that the composition of intestinal microbiota differed between these four groups. In terms of distances between groups, the BET group was closest to the HCD group (the most similar microbial composition), followed by the CD group, and the CRE1 group was farthest away. This suggests that either a high-carbohydrate diet or the addition of creatine influenced the gut microbiota ( Fig. 4A-D) and that exogenous creatine caused the greatest changes in the gut microbial community composition.
Hierarchical clustering tree and community heatmap analysis showed results consistent with the previous PCoA analysis: the HCD and BET groups were compositionally relatively similar among the four groups, whereas the CRE1 group had the most dissimilar gut microbiota pathway composition among the four groups ( Fig. 4E-H).
The bacterial composition at the phylum and genus levels in the four groups is shown in Fig. 4A and B. The microbial community composition analysis showed that the most dominant phyla in all groups were Fusobacteriota, Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteriota. In the CRE1 group, the abundance of Firmicutes and Bacteroidota was significantly increased (P < 0.05), whereas the abundance of Fusobacteriota and Proteobacteria was significantly decreased (P < 0.05), compared to other groups. In the BET group, the abundance of Fusobacteriota was increased significantly (P < 0.05), whereas the abundance of Firmicutes was decreased significantly (P < 0.05) (Fig. 5A), compared with the HCD group.

Growth performance and liver histology
The results of the study indicated that the 0.5-2% creatine supplementation did not significantly improve the growth performance of M. amblycephala (Table 2). This was consistent with studies conducted on Sciaenops ocellatus, Sparus aurata, and pigs (James et al. 2002;Schaffer et al. 2009;Stites et al. 2020), but some other studies found that creatine supplementation improved the growth performance of red tilapia (Wardani et al. 2021), rainbow trout (McFarlane et al. 2001), red drum fish (Burns and Gatlin 2019), and broiler chicken (Michiels et al. 2012). Hence, the effects of creatine on animal growth performance may be dose-specific and species-specific. In our study, the FCR was only nonsignificantly lower in the CRE group (supplemented with 1.2% betaine and 0.5-2% creatine) than in the BET group (supplemented with 1.2% of betaine) and significantly lower in the CRE group in comparison to the CD and HCD groups. Hence, from the viewpoint of growth performance and economic efficiency, we chose the 0.5% creatine supplementation as the optimal level for further studies.
High-carbohydrate diet (HCD) causes liver damage in M. amblycephala, as reflected in elevated HSI values, increased fat accumulation in the hepatocytes, degeneration of many nuclei, and cell membrane rupture (Zhou et al. 2013Prisingkorn et al. 2017). Betaine supplementation can alleviate the negative impacts of HCD, including improving the growth parameters and liver condition in M. amblycephala (Xu et al. 2018). Creatine supplementation can also improve the liver damage caused by a high-fat diet in mice (da Silva et al. 2017). In agreement with these findings, our study provides additional support for the argument that betaine and creatine supplementation can improve the negative impacts of HCD on liver health. Both the BET and CRE groups exhibited a reduced fat accumulation and an increased number of regularly shaped nuclei (Fig. 1). Therefore, it may be important to supplement betaine and creatine together to the feed of M. amblycephala. Following this finding, we selected the CD, HCD, BET, and CRE1 groups for further analyses.

Metabolism of creatine
Exogenous creatine is digested, absorbed, and metabolized in the gut and liver, where it is synthesized into endogenous creatine and then transported to other tissues by the creatine transport proteins (Persky et al. 2003). Creatine can be used to produce creatinine directly through a non-enzymatic reaction, or with the help of creatine kinase, which catalyzes creatine into creatine phosphate (ck), which in turn produces creatinine (Nabuurs et al. 2013). Sarcosine is an intermediate in the creatine metabolic pathway, and creatine can be further metabolized to sarcosine after the production of creatinine (Fig. 6). Sarcosine is in turn metabolized to glycine by sarcosine dehydrogenase (sardh), and subsequently, glycine is converted to serine (Magnusson et al. 2015;Adeva-Andany et al. 2018).
Supplementation of creatine and creatine precursor (guanidineacetic acid) leads to a significant increase in ck levels (Solis et al. 2017). In agreement with these results, dietary creatine supplementation in this study also produced a strong increase in the levels of ck, but the content of sarcosine did not significantly increase compared to the BET group (Fig. 6). The reason might be that sarcosine was promptly metabolized into glycine and further into taurine or g-ABA through intermediate metabolites (serine, cystathionine, cysteine, hypo-taurine, or L-ornithine and guanidinoacetate (GAA), arginine, putrescine/glutamate, etc.). This hypothesis was supported by the increased taurine and g-ABA levels in the CRE1 group compared to the BET group.
Compared to the HCD group, the levels of glycine, cystathionine, and sardh were increased in the CRE1 and BET groups (Fig. 2), but their levels were invariant between the CRE1 and BET groups. These results are consistent with previous studies conducted in our laboratory (Xu et al. 2018), and they indicate that betaine affects the levels of glycine, cystathionine, and sardh, whereas creatine does not. Simultaneously, the cystathionine content in the HCD group was significantly decreased compared with the CD group (Fig. 2), which suggests that high dietary carbohydrates affected cystathionine metabolism. This is also in agreement with our previous results Prisingkorn et al. 2017).
In addition, compared with the BET group, the content of serine in the CRE group showed a significant decrease, which suggested that exogenous creatine could affect the metabolism of creatine via the serine content ( Fig. 2 and Fig. 6).

Endogenous synthesis of taurine
Taurine is closely linked to the metabolism of sulfur-containing amino acids: it can be synthesized from sulfur-containing amino acids such as cysteine, cystine, and methionine through a series of enzymatic reactions (HuxTable 1992). The liver is the most important organ for taurine synthesis (Stipanuk 2004). There are three main pathways for taurine synthesis in animals: the cysteamine (CS) synthesis pathway, the cysteine sulfinic (CSA) synthesis pathway, and the cysteic acid (CA) synthesis pathway (Ueki and Stipanuk 2009;Stipanuk et al. 2011). Taurine can be synthesized from cysteine via the CS pathway and CSA pathway. The CS pathway commonly comprises the oxidation of cysteamine to hypo-taurine by cysteamine dioxygenase (adoa; adob) and then further oxidization of hypo-taurine into taurine (Stipanuk et al. 2011). The CSA pathway is the main pathway for taurine synthesis (Ueki and Fig. 4 The effects of creatine on the intestinal microbiota composition. The microbiota composition of the CD, HCD, BET, and CRE1 groups at the phylum (A) and genus (B) levels. Principal coordinate analyses (PCoA) on the phylum (C) and genus (D) levels. Hierarchical clustering tree analyses on the phylum (E) and genus (F) levels. Community heatmap analyses of gut microbiota on the phylum (G) and genus (H) levels. Red color indicates an increased abundance of a taxon, and blue indicates a decreased abundance of a taxon. The four groups are abbreviated as CD (control), HCD (high carbohydrate), BET (1.2% betaine supplementation), and CRE1 (1.2% betaine + 0.5% creatine supplementation) ◂ Stipanuk 2009). Herein, cysteine is oxidized by the cysteine dioxygenase1 (cdo1) to form CSA, then sulfinoalanine decarboxylase (csad) catalyzes the decarboxylation of CSA to form hypo-taurine, and finally hypo-taurine is oxidized to taurine. In this metabolic process, cdo1 and csad are the two key enzymes that regulate the level of biosynthesis of taurine in animals (Wang et al. 2021a, b). The CA synthetic pathway refers to the partial conversion of cysteine sulfinic acid produced by the CSA pathway to sulfoalanine, which is subsequently converted directly to taurine in a reaction catalyzed by csad (Martin et al. 1974). CA pathway is not the main pathway for taurine synthesis in animals, but it may play an important role in taurine synthesis when CS and CSA pathways are blocked (Martin et al. 1974;Kim et al. 2008).
In previous studies in our laboratory, it was found that betaine could regulate the taurine synthesis of M. amblycephala through the CS and CSA pathways, in which ado and csad are the two key enzymes that determine the level of biosynthesis of taurine. In the present study, although the content of cystathionine was invariant, that of taurine had increased significantly in the CRE group compared to the BET group (Fig. 6). Furthermore, the expression of csad was also elevated significantly in the CRE group compared to the BET group. Moreover, there were no differences in the expression of ado (including a and b genotypes) between the CRE and BET groups. Therefore, the CSA pathway should be the main pathway by which creatine regulates taurine synthesis. Creatine does not increase key metabolites in taurine synthesis, such as serine and cystathionine, but rather increases the expression of csad at the mRNA level.
Creatine supplementation increases the content of g-ABA The experimental results showed that the supplementation of creatine significantly increased the content of g-ABA in the high-carbohydrate diets (Fig. 6). Creatine regulated the content of g-ABA via two mechanisms in this study: (1) it increased the gene expression of glycine amidinotransferase (gatm) and the contents of arginine and ornithine, a by-product in the process of endogenous creatine synthesis, and then it increased the gene expression of glutamate decarboxylase (gad), until the g-ABA was increased; (2) it increased the gene expression levels of agmatinase (agmat) and diamine oxidase1 (aoc1) through the arginine-agmatine-putrescine pathway, resulting in increased g-ABA levels.

Arginine metabolism
Creatine can be obtained from food or synthesized in body tissues from arginine and glycine as precursors  (Borchel et al. 2014). Guanidinoacetate N-methyltransferase (gamt) and gatm are two key enzymes in the process of creatine synthesis (Nabuurs et al. 2013). Endogenous creatine is synthesized from three amino acids: glycine, arginine, and methionine. The complete creatine molecule consists of a complete glycine, an amidine group on arginine, and a methyl group on S-adenosylmethionine (Shao and Hathcock 2006;Brosnan et al. 2011;Moret et al. 2011;Andres et al. 2008). Gatm operates a transfer of an amidino residue from arginine to glycine, resulting in the formation of L-ornithine and guanidinoacetate (GAA) (Nabuurs et al. 2013). GAA produces creatine by the action of gamt. A similarly named gene gatm encodes the rate-limiting enzyme in the process of creatine synthesis. This gene is subject to the negative feedback exerted by high levels of both an intermediate (ornithine) and a product (creatine) of creatine synthesis (Wyss and Kaddurah-Daouk 2000). In our study, the gene expression levels of gatm and gamt were significantly lower in the HCD group compared to the CD group (Fig. 2). This suggested that high carbohydrates affected the synthesis of arginine and endogenous creatine. In the CRE group, exogenous creatine significantly increased the mRNA expression levels of gatm and gamt and the content of arginine compared with the BET group. The results show that exogenous creatine is beneficial to the synthesis of endogenous creatine and arginine in M. amblycephala.
Arginine is one of the most important amino acids in animals, serving as a precursor for the synthesis not only of proteins but also of urea, polyamines, glutamate, creatine, and agmatine (Efron and Barbul   Fig. 6 Overview of the metabolic pathway by which creatine appears to increase the content of taurine and g-ABA in plasma. Red arrows (CRE1 compared to BET), green arrows (CRE1 compared to HCD), and blue arrows (CRE1 compared to HCD) indicate the direction of the change in the expression of genes and metabolites. Up arrows indicate a significant increase, down arrows indicate a significant decrease, and horizontal arrow indicates a non-significant change or unchanged expression. Groups: HCD, BET, and CRE1. ck, creatine kinase; gamt, guanidinoacetate N-methyltransferase; gatm, glycine amidinotransferas; sardh, sarcosine dehydrogenase; adoa adob, cysteamine dioxygenase; csad, sulfinoalanine decarboxylase; arg, arginase; odc1, ornithine decarboxylase1; agmat, agmatinase; aoc1, diamine oxidase1; aldh, aldehyde dehydrogenase; gad, glutamate decarboxylase; GAA, guanidinoacetate; g-ABA, γ-aminobutyric acid 2000). Arginine can be converted to ornithine by arginase (arg) (Bronte and Zanovello 2005), which is then metabolized to produce glutamate (Wang et al. 1995). In our study (Fig. 6), the arginine and ornithine contents of the CRE group were significantly higher than those of the BET group. At the same time, the mRNA expression level of gatm was significantly increased, while the mRNA expression of arg was significantly decreased. This suggested that the increase of serum ornithine might be due to an increase or accumulation of arginine as a by-product of the creatine synthesis pathway. In addition, arginine metabolism is thought to be influenced by diet-induced changes in gut microbes Zhang et al. 2022); thus, arginine metabolism was also possibly influenced by changes in gut microbes caused by creatine.

Putrescine synthesis
Putrescine is a polyamine that is a major inhibitory neurotransmitter in the vertebrate central nervous system, and it has also been implicated in environmental stress tolerance and osmoregulation (Enna 2007;Shelp et al. 2012). There are two synthetic pathways of putrescine: (i) Ornithine generates putrescine via the enzyme ornithine decarboxylase 1 (odc1). (ii) Agmatine produces putrescine via the enzyme agmatinase (agmat) (Nikolaus 1999;Benitez et al. 2018). Agmatine is generated from arginine via the action of arginine decarboxylase, which is a precursor of putrescine (Morris 2006). Compared to the CD group, the expressions of odc1 and agmat were significantly decreased in the HCD group (Fig. 2), which suggested that a high-carbohydrate diet affected the putrescine synthesis. The expression of agmat was significantly higher in the CRE group than in the HCD and BET groups, which suggests that exogenous creatine increased putrescine content by increasing the expression of agmat (Fig. 6).

Synthesis of 4-aminobutyric acid
γ-Aminobutyric acid (g-ABA) can be synthesized from either of the following two precursors: (1) glutamate, via the enzyme gad, and (2) putrescine, via the enzymes diamine oxidase1 (aoc1) and aldehyde dehydrogenase (aldh). g-ABA is typically synthesized from glutamate, but putrescine also serves as an important source of g-ABA under some conditions (e.g. anoxic and hypoxic conditions) in fishes and turtles (Nilsson and Renshaw 2004;Nilsson and Lutz 2004).
In our study (Fig. 6), the level of g-ABA was significantly higher in the CRE1 group compared with the BET and HCD groups. Compared with the BET group, the content of glutamate was not significantly different in the CRE1 group, whereas the gene expression of gad was significantly higher. The results indicated that exogenous creatine increased the g-ABA content by raising the expression of gad. In the putrescine pathway, the aoc1 expression in the CRE1 group was significantly higher than in the BET group. This suggested that exogenous creatine might also increase the g-ABA content by increasing the expression of aoc1 in the putrescine pathway and that aoc1 might be a key enzyme in this pathway.

Exogenous creatine alters the composition of gut microbiota
It is agreed upon by now that gut microbiota composition is important to the health of the host and associated with various physiological and metabolic diseases, including diabetes, obesity, and nonalcoholic fatty liver (Boursier et al. 2016;Boulange et al. 2016). The gut microbiota is regulated by environmental factors, genetic factors, and nutrients in the diet (Maslowski and Mackay 2011). In general, high gut microbial diversity is beneficial to the host's health (Kriss et al. 2018;Mora-Sanchez et al. 2020;Zhang et al. 2020a, b). In our study, microbial diversity did not significantly differ among the CD, BET, and CRE1 groups (Fig. 3). Compared with the HCD group, the Shannon index was significantly lower in the CRE1 group, while the Simpson index did not change. This indicates that creatine did not affect the abundance and diversity of the gut microbial community, while the effect of creatine and betaine together on the diversity of the intestinal microbial community of M. amblycephala under high sugar conditions could not be determined and needs to be further investigated (Fig. 3). This is consistent with other findings that the addition of nutrients to the diet did not significantly change the microbial diversity, although it improved the health of the organism (Wang et al. 2021a, b;Ding et al. 2021). Therefore, the gut microbial composition was analyzed to explore the effect of creatine on the health and metabolism of M. amblycephala.
Bacteroidetes play an important role in a variety of major metabolic activities involving the fermentation of carbohydrates and the utilization of nitrogenous substances . Firmicutes are generally considered to be beneficial microorganisms for fish and other vertebrates (Terova et al. 2019). Compared with the no-carbohydrate and high-carbohydrate diets (20%), the abundance of Firmicutes was significantly increased in Chinese perch fed with a suitable carbohydrate content (10%) . Proteobacteria have been reported to be enriched in environmental pollution areas (Kasemodel et al. 2019), and they can cause an inflammatory response that disrupts the intestinal mucosal barrier (Qiao et al. 2019;Liu et al. 2020). Compared to healthy subjects, patients with inflammatory bowel disease were characterized by an increased proportion of the Proteobacteria accompanied by concomitant depletion of Firmicutes (Mukhopadhya et al. 2012;Shin et al. 2015). Our results showed that exogenous creatine enriched Firmicutes and Bacteroidota and diminished Fusobacteriota and Proteobacteria (Fig. 5A) in the gut of M. amblycephala. Hence, it is reasonable to conclude that creatine might be beneficial to the microbial fermentation of high-carbohydrate diets and fish health. Bacteroidota abundance changes are worthy of further exploration in M. amblycephala.
At the genus level, Cetobacterium was the dominant microbial community in the gut of M. amblycephala (Fig. 5B). This genus was also the dominant member of the gut microbiota in Channa argus, Danio rerio, Ictalurus punctatus, Micropterus salmoides, and Lepomis macrochirus (Roeselers et al. 2011;Larsen et al. 2014;Miao et al. 2018). Cetobacterium is positively associated with protein digestion (Hao et al. 2017), and intestinal Cetobacterium modified glucose homeostasis via parasympathetic activation in zebrafish (Wang et al. 2021a, b). Our results showed that the abundance of Cetobacterium in the BET group was significantly higher than in the other three groups, but it was invariant among the CD, HCD, and CRE1 groups. This suggests that the alteration of Cetobacterium by creatine is within the acceptable range.
Erysipelotrichaceae-unclassified and ZOR0006 both belong to the family Erysipelotrichaceae, which has been associated with metabolic disorders and inflammatory diseases in hosts (Fleissner et al. 2010;Kaakoush 2015;Nagao-Kitamoto et al. 2016). Hu et al. (2019) found that Erysipelotrichaceae-unclassified was negatively correlated with body fat weight. However, some other studies have shown that Erysipelotrichaceae were positively correlated with carbohydrate consumption and that they may also have the capacity to metabolize plant polysaccharides (Cox et al. 2013;Wu et al. 2021). Our results showed that exogenous creatine significantly increased the abundance of ZOR0006, which suggested that ZOR0006 of Erysipelotrichaceae might play an important role in the gut metabolism of M. amblycephala. The extent and the mechanism by which it affects gut metabolism remain unknown.
Bacteroides are one of the main g-ABA-producing bacteria (Strandwitz et al. 2019). As a key enzyme of g-ABA production, the glutamate decarboxylase (Gad)encoding gene has been identified in the bacterial genus abundant in the human intestinal microbiome: Bacteroides (Otaru et al. 2021). Studies had shown that increased abundance of Bacteroides could also increase taurine levels, and this mechanism might be useful in preventing diet-induced metabolic disorders (Nakajima et al. 2020;Chen et al. 2022).This suggests an important contribution of Bacteroides in the regulation of g-ABA and taurine synthesis in the gut. In our study, the content of g-ABA and taurine and the abundance of Bacteroides were also significantly higher in the CRE1 group than in the BET group. Hence, it is possible that exogenous creatine could influence the g-ABA and taurine contents by modulating the abundance of Bacteroides in the gut of M. amblycephala, and the mechanism might be associated with healthier livers in the BET and CRE groups .
Proteocatella is a proteolytic bacterium (Pikuta et al. 2009;Barragan-Trinidad et al. 2017). In our study, the high-carbohydrate diet significantly reduced its abundance. In the CRE1 group, the abundance of Proteocatella was lower than in the BET group and higher than in the HCD group, but it was not significantly different between the CRE1 and CD groups. This indicated that high carbohydrates reduced the abundance of Proteocatella in the intestine of M. amblycephala, while betaine significantly increased its abundance. Although creatine decreased the abundance of Proteocatella, the positive effect of betaine on its abundance annulled the effect, so the abundance was the same as in the control group and even higher than in the HCD group. Therefore, the 268 Fish Physiol Biochem (2023) 49:253-274 alteration of Proteocatella abundance by creatine is within the acceptable range.
Aeromonas, Vibrio, Crenobacter, and Shewanella belong to the Proteobacteria. Certain species of Aeromonas, Vibrio, and Shewanella are opportunistic pathogens that could impair the intestinal immune mechanisms in fish and affect the fish health (Xiong et al. 2015;Li et al. 2016;Firmino et al. 2019;Zhang et al. 2020a, b). In our study, the abundance of Vibrio and Shewanella was decreased significantly in the CRE1 group compared with the BET group. Therefore, it is possible that exogenous creatine could improve the health of the gut microflora.

Limitations of the study and future directions
The findings of this study are based on the supplementation of creatine with 1.2% betaine.
Although three control groups were set up, normal carbohydrate, high carbohydrate, and high carbohydrate + 1.2% betaine, further experiments are needed to determine whether the addition of creatine alone has the same effect on M. amblycephala. As we did not corroborate enzyme activity and gene expression results by measuring the levels of their products (proteins), our results should be interpreted with this limitation in mind. The results showed that 0.5-2% creatine had no significant effect on the growth traits of M. amblycephala (compared with the HCD group). Therefore, future studies should explore the effect of creatine on the growth parameters of M. amblycephala by increasing and decreasing the concentration of creatine, thereby attempting to identify the optimal level for the use of this additive in aquaculture in terms of economic efficiency. Fig. 7 Summary of the effects of creatine supplementation on the metabolites in plasma, expression of genes, growth performance, liver condition, and gut microbiota composition in M. amblycephala. The metabolic pathway by which creatine appeared to increase the content of taurine and g-ABA in the plasma is shown. Red arrows (CRE1 compared to BET), green arrows (CRE1 compared to HCD), and blue arrows (CRE1 compared to HCD) indicate the direction of the change in the expression of genes and metabolites. An up arrow indicates a significant increase, a down arrow indicates a significant decrease, and a horizontal arrow indicates a non-significant change or unchanged expression (P < 0.05). Groups: HCD, BET, and CRE1. ck, creatine kinase; gamt, guanidinoacetate N-methyltransferase; gatm, glycine amidinotransferas; sardh, sarcosine dehydrogenase; adoa adob, cysteamine dioxygenase; csad, sulfinoalanine decarboxylase; arg arginase; odc1, ornithine decarboxylase1; agmat, agmatinase; aoc1, diamine oxidase1; aldh, aldehyde dehydrogenase; gad, glutamate decarboxylase; GAA, guanidinoacetate; g-ABA, γ-aminobutyric acid; FCR, feed conversion ratio

Conclusions
In conclusion, the 0.5-2% creatine supplementation had no significant effect on the growth performance of M. amblycephala. Therefore, from the viewpoint of economic efficiency, the treatment with 0.5% creatine is optimal for the M. amblycephala industry. The results of metabolite and gene expression suggested two putative mechanisms by which creatine regulates taurine and g-ABA contents: (i) The increased content of taurine was metabolically reflected in enhancing the activities of metabolic pathways from creatine to taurine, including the metabolism of creatine and CSA pathways. (ii) Dietary creatine also increased the content of g-ABA via enhancing the activities of metabolic pathways from arginine to g-ABA, including the arginine metabolism, putrescine synthesis, and synthesis of g-ABA (Fig. 7). In addition, dietary creatine altered the gut microbial composition in M. amblycephala. There are weak indications that this may result in a healthier microbial composition at the genus level, but this remains conjectural. Currently, there is limited research about the effects of dietary creatine on the growth, health, and metabolism of fish. In order to improve sustainable aquaculture development, further comprehensive studies of creatine are warranted.