Dimethyl-β-propiothetine (DMPT) supplementation under the all-plant protein diet enhances growth performance, digestive capacity and intestinal structural integrity for on-growing grass carp (Ctenopharyngodon idella)

Highlights

• All-plant protein diets compromised fish feed intake, growth and digestive capacity.

• Appropriate DMPT supplementation under the all-plant protein diet reversed the adverseness.

• DMPT increased FI and digestive enzyme activities was partially related to NPY content and CCK content, respectively.

• DMPT relieved oxidative damage and improved antioxidant ability by modulation of Nrf2 signalling.

• The appropriate DMPT level for growth of on-growing grass carp under this diet were estimated to be 282.78 mg/kg diet.

Abstract

This study aimed to investigate the effects of dimethyl-β-propiothetine (DMPT) supplementation under the all-plant protein diet on the growth, digestive capacity, oxidative damage and intestinal structural integrity of on-growing young grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp (216 ± 0.29 g) were randomly divided into six groups with three replicates and fed one fish meal (FM) diet and five all-plant protein diets with graded levels of DMPT (0 to 520 mg/kg diet) for 60 days. The results indicated that compared to the FM diet group, the all-plant protein diet without DMPT supplementation (all-plant protein diet group) significantly reduced feed intake (FI), specific growth rate (SGR), percentage weight gain (PWG), and digestive enzyme activities (trypsin, chymotrypsin, lipase and amylase activities) as well as damaged the intestinal histological structure of fish. However, these parameters could be restored to levels equal or superior to the FM diet group at DMPT supplementation levels under the all-plant protein diet up to 260.00 mg/kg diet. This is the first study to show that (1) DMPT supplementation under the all-plant protein diet increased FI and digestive enzyme activities in the intestine were partly related to improved neuropeptide Y content and cholecystokinin content, respectively. (2) DMPT supplementation under the all-plant protein diet enhanced the intestinal structural integrity of fish partially by alleviating oxidative damage and partially by up-regulating antioxidant enzyme activities as well as the mRNA levels of antioxidant enzyme (except CuZnSOD), which were partially related to [Keap1a (not Keap1b)/Nrf2] signalling. (3) Finally, based on the quadratic regression analysis for optimal growth performance indices (FI and PWG) and oxidative damage indices (protein carbonyl and malondialdehyde content), the appropriate levels of DMPT supplementation under the all-plant protein diet for on-growing grass carp were estimated as to be 278.30, 282.78, 326.52 and 299.25 mg/kg diet, respectively. In summary, DMPT supplementation under the all-plant protein diet improved the growth performance and digestive capacity and strengthened the intestinal structural integrity of on-growing grass carp. Simultaneously, it also provided a practical and effective approach to the highly efficient utilisation of plant protein and a reference for the non-fish meal diet formulation of fish.

Introduction

Recently, plant protein sources have been extensively used to replace fishmeal in aquafeeds, due to stagnant fish meal supply and exorbitant price (Anderson et al., 2016). However, studies have reported that the all-plant protein diet lead to some unsatisfactory results such as low FI and poor growth in red sea bream (Pagrus major) (Biswas et al., 2019) and low digestive capability in blunt snout bream (Megalobrama amblycephala) (Abasubong et al., 2019). These adverse effects were ascribed to poor palatability, high antinutritional factors, essential amino acid deficiencies and so on in the diet (Francis et al., 2001; Gatlin Iii et al., 2007). Among these factors, poor palatability is an important factor (Freitas et al., 2011). Studies showed that feeding stimulants are widely used to solve the diet palatability issue and enhance the animal appetite and growth in the all-plant protein diet, such as krill meal in Nile tilapia (Oreochromis niloticus) (Gaber, 2010), and taurine in zebrafish (Danio rerio) (Guimarães et al., 2018). Thus, it is of great necessity to use feeding stimulant to increase FI and improve animal growth performance in an all-plant protein diet.

At present, dimethyl-β-propiothetine (DMPT) is highly commended among feeding stimulants. Previous studies have reported that DMPT improved FI and promoted growth of gold fish (Carassius auratus) (Nakajima, 1996), as well as enhanced digestive ability by improving protease, lipase and amylase activities in the intestine of Gibel carp (Carassius auratus gibelio) (Xue et al., 2007). Nevertheless, a previous report demonstrated that DMPT could react with the hydroxyl radical (·OH) and decrease the free radical content in marine algae (Sunda et al., 2002). Another study indicated that the decreased free radical content could reduce oxidative damage in gerbils (Lu et al., 2012). Moreover, digestive function has been found to depend in part on the intestinal structural integrity, which could be influenced by oxidative damage to some extent (Chiva et al., 2003; Shoji et al., 2007; Wei et al., 2018). According to the above studies, DMPT, apart from enhancing animals' FI, we speculate that it might relieve oxidative damage to the intestines of fish. However, there is hardly any report regarding DMPT on this intriguing topic, which is worthy of investigation.

To our knowledge, oxidative damage can be relieved by antioxidant systems which benefit in scavenging excess levels of free radicals in fish (Martínez-Álvarez et al., 2005). Fish antioxidant systems are composed of non-enzymatic compounds and antioxidant enzymes (Jiang et al., 2018). Different antioxidant enzyme genes play different roles or functions in regulating antioxidant ability in animals (Jiang et al., 2017). Glutathione peroxidase type 1(GPx1) is responsible for protecting cells from damage caused by hydrogen peroxide, whereas GPx4 mainly inhibits the membrane phospholipid peroxidation (Chen et al., 2012; Ran et al., 2004). Glutathione S-transferase omega (GSTO) conjugates the several products of oxidative stress, glutathione S-transferase P (GSTP) detoxifies a range of compounds such as DNA and RNA hydroperoxides, and glutathione S-transferase R (GSTR) is responsible for conjugating 4-hydroxynonenal produced by lipid peroxidation (Espinoza et al., 2013). Copper zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) are located in the cytoplasm and the matrix of mitochondria in fish, respectively (Umasuthan et al., 2012). Furthermore, it was reported that antioxidant enzyme genes expression could be regulated by NF-E2-related factor 2 (Nrf2) signalling pathway in fish (Yao et al., 2007). However, until now, studies concerning the effect of DMPT on oxidative damage, antioxidant ability and the related signalling pathways in animal intestine are scarce. A previous study has indicated that DMPT provided a methyl donor and participated in methionine synthesis in rats (Dubnoff and Borsook, 1948). Our laboratory study showed that methionine enhanced non-enzymatic compound glutathione (GSH) content and activities of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) in the intestines of grass carp (Ctenopharyngodon idella) (Wu et al., 2017). Wang et al. (2014) reported that DMPT improved the muscle protein level of taimen (Hucho taimen). In human, the improved protein level was shown to increase glucagon-like peptide 1 (GLP-1) release (Geraedts et al., 2010), which could activate the Nrf2 signalling in endothelial cells (Pujadas et al., 2015). Thus, these results indicated a possible relationship between DMPT and fish intestinal oxidative damage, antioxidant ability and the related Nrf2 signalling pathway, which is worthy of further investigation.

Grass carp is an herbivorous fish with the largest production in global freshwater aquaculture (He et al., 2017), and the fish meal proportion in the current commercial formulation of this species was approximately 3.0–5.0% (Chen and Zhang, 2019; Wu et al., 2018). At present, the appropriate DMPT supplementation level for grass carp has only been investigated in the juvenile stage of grass carp under low fish meal diet (Zhang et al., 2011). However, the appropriate supplementation levels of feeding stimulants may vary with content of fish meal in diets and different growth stages, such as taurine in rainbow trout (Oncorhynchus mykiss) (Gaylord et al., 2010) and turbot (Scophtalmus maximus) (Qi et al., 2012). Until now, there has been no study investigating the appropriate levels of DMPT supplementation for on-growing grass carp under the all-plant protein diet, which requires investigation.

Taken together, this study is the first to evaluate the appropriate level of DMPT supplementation under the all-plant protein diet for the on-growing grass carp, which may provide a reference for the commercial non-fish meal diet formulation and healthy breeding of this fish. Meanwhile, using grass carp as a research model, apart from investigating the effects of dietary DMPT supplementation on growth performance and digestive capacity, we investigated the relationship between DMPT and oxidative damage and antioxidant ability as well as the related Nrf2 signalling, which could provide a partial theoretical basis and molecular mechanisms for DMPT regulating the structural integrity in the intestines of fish and animals, for the first time. This study also provided new ideas for the study of feeding stimulant.