What is the effect of reduced glutathione on the growth of turbot?

 Scophthalmus max imus, commonly known as turbot, is native to the northeastern coast of the Atlantic Ocean. It has a fast growth rate, adapts to low water temperature, accepts feeds easily and has a high conversion rate [1], which makes it particularly suitable for factory farming along the northern coast of China [2]. With the expansion of industrial scale and the change of culture mode, the factory farming mode has also brought some impacts on turbot culture while increasing the production. The deterioration of the water environment caused by high-density culture[3] , the oxidative rancidity of feeds due to improper preservation during processing, storage and transportation[4] , diseases, and operational stresses caused by transportation and other management aspects[5] can cause oxidative stress to turbot, resulting in damage to its growth and development, and even its death.

 

Studies have shown that nutritional regulation can be used to eliminate or alleviate the stress caused by stress in aquatic animals, and non-nutritive immune enhancers (immunoglycans, antioxidants, etc.) can promote their growth and stress resistance[3] .

 

Glutathione (usually referred to as reduced glutathione) is an active peptide formed by the peptide bonding of glutamic acid, cysteine and glycine, which plays an important role in a wide range of cellular activities[6] . Reduced glutathione is a non-protein, low molecular weight thiol that is a non-enzymatic antioxidant, and it performs a variety of functions such as scavenging of oxygen radicals, detoxification, maintenance of DNA biosynthesis, and cellular immunity through the transfer of electrons and protons[7] . The most important function is to maintain the dynamic balance between oxidation and antioxidant[8] .

 

Currently, studies on the application of reduced glutathione in aquafeeds have shown that the addition of appropriate amounts of reduced glutathione to feeds can significantly improve the performance of Oreo-chromis niloticus GIFT[9] , rainbow trout Oncorhy nchus mykiss[10] , shrimp Litopenaeus v. annamei[11] , O. niloticus × O. aureus[12] , brown toothfish Paralichthys oliveaceus[13] , and other species of flounder, including O. niloticus × O. aureus[14] . annamei)[11] , Oncorhynchus mykiss (Oncorhynchus mykiss)[10] , Litopenaeus v. annamei (Litopenaeus v. annamei)[11] , O. niloticus × O. aureus (O. niloticus × O. aureus)[12] , and Paralichthys oliveaceus (Paralichthys oliveaceus)[13] , whereas the addition of reduced glutathione to the diets of grass carp Ctenopharyngodon idella (Ctenopharyngodon idella) did not have any significant effect on growth and bait coefficient. In grass carp (Ctenophary ngodon idella), the addition of reduced glutathione to the diet had no significant effect on growth and bait coefficient, but significantly increased the total antioxidant capacity of the liver and decreased the levels of reactive oxygen species (ROSs) in the liver and serum[14] , and the addition of reduced glutathione to the diet of the abalone H aliotis discus hannailna (H aliotis discus hannailna) did not have any significant effect on the rate of increase in mass, but it did have an improvement in the antioxidant system[15] .

The author chose turbot as the test object, and added different levels of reduced glutathione to the basic feed to study its effect on the growth and development and antioxidant capacity of turbot, and to determine the optimal amount of reduced glutathione in turbot feed, so as to provide a theoretical basis for the application of reduced glutathione in turbot feed and the healthy aquaculture of turbot.

 

1 Materials and Methods

1 .1 Test feed

Fishmeal and soybean meal were used as the main protein sources, high-gluten wheat flour as the main sugar source, and fish oil and soy lecithin as the main fat source to formulate nitrogen- and energy-equivalent basic feeds. The formula and nutritional composition of the basal feed are shown in Table 1. Reduced glutathione 0, 100, 200, 400 and 600 mg/kg were added to the basal diets to formulate five kinds of experimental diets.

The raw materials are crushed and passed through a 60-mesh sieve, then expanded and mixed step by step according to the test ratios, and then extruded into pellets with a particle size of 2 mm by a pelletizer, then naturally air-dried to a moisture of about 10% and put into self-sealing bags, and then placed in a -20 ℃ refrigerator for freezing and storing.

 

1 .2 Feeding and management

Turbot juveniles were purchased from Dalian Tianzheng Industrial Co., Ltd. and fed with basic feed for two weeks, and then randomly grouped into individuals without disease or trauma and with an initial mass of (23.08± 0.09) g after they had adapted to the feed and aquaculture environment.

Fifteen 60 cm × 45 cm × 40 cm aquariums (actual water consumption: 90 L) were used, each aquarium was used as one culture unit, and five treatments were set up with three replicates of each treatment, each replicate was stocked with 14 fishes, and the culture experiment lasted for 8 weeks. The aquariums were fed twice a day (8:00 and 18:00) under natural light, and the residual bait was collected after 30 min of feeding. The water temperature ranged from 14 to 18.5 ℃, and the dissolved oxygen was >6 mg/L. The water was changed once/d, and the water volume ranged from 33.3% to 50%.

 

1 .3 Sample Collection

Before the end of the experiment, turbot was fasted for 24 h, and each tank was weighed and counted separately. Five fish were randomly taken from each aquarium, weighed and measured, and the livers were collected, weighed and frozen in liquid nitrogen at -80 ℃. The mass gain rate and specific growth rate were calculated according to the following formula:

Mass increase rate/% = (m2 - m1)/m1 × 100%

Specific growth rate/% - d-1 = (lnm2 - lnm1 ) / t × 100%

where m1 is the initial body mass (g), m2 is the final body mass (g), and t is the incubation time (d).

 

1 .4 Sample Determination

The collected liver tissues were mechanically homogenized by adding pre-cooled 0.86% physiological saline as homogenizing medium (m/V=1/9) in an ice-water bath, and then centrifuged at 4 ℃ and 3000 r/min for 10 min, and the supernatant was extracted for the determination of the indexes.

For the determination of total protein, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Kaumas Brilliant Blue Protein Quantification Test Kit was used; for the determination of total antioxidant capacity, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Total Antioxidant Capacity Test Kit was used, which utilized the colorimetric measurement of antioxidant substances by reacting with Fe2+ and pheophytin; for the determination of malondialdehyde, Nanjing Jianjian Institute of Biological Engineering (NJIBE) Malondialdehyde Test Kit was used, which utilized the colorimetric measurement of the red product formed by condensation of malondialdehyde and thiobarbituric acid. The determination of malondialdehyde was done by colorimetric reaction between malondialdehyde and thiobarbituric acid; the determination of superoxide dismutase was done by superoxide dismutase test kit of Nanjing Jianjian Institute of Biological Engineering; the determination of reduced glutathione was done by micro enzyme assay using micro reduced glutathione test kit of Nanjing Jianjian Institute of Biological Engineering; the determination of glutathione peroxidase and glutathione reductase was done by glutathione peroxidase of Nanjing Jianjian Institute of Biological Engineering; the determination of glutathione peroxidase was done by glutathione peroxidase of Nanjing Jianjian Institute of Biological Engineering. Glutathione peroxidase and glutathione reductase were determined using a glutathione peroxidase kit from Nanjing Jianjian Institute of Biological Engineering, using the reaction of dithiodinitrobenzoic acid with sulfhydryl compounds for colorimetric determination; glutathione sulfotransferase was determined using a glutathione sulfotransferase kit from Nanjing Jianjian Institute of Biological Engineering, using glutathione sulfotransferase catalyzed by the binding of reduced glutathione with 1-chloro 2,4-dinitrobenzene substrate for colorimetric determination.

 

1 .5 Statistical analysis

The experimental data were expressed as mean ± standard deviation. The data were analyzed by one-way ANOVA using SPSS 21.0 software. Duncan's multiple comparisons were used to test for differences between groups if the differences were significant (P < 0.05).

 

2 Results

2 .1 Effect of reduced glutathione on the growth of turbot (Scophthalmus maximus)

The addition of reduced glutathione to the feed increased the mass gain rate and specific growth rate of turbot to different degrees, and the mass gain rate and specific growth rate of turbot showed a tendency of increasing and then decreasing with the increase of the added amount of reduced glutathione (Table 2). In the experimental group with 200 mg/kg of reduced glutathione, the mass gain rate and specific growth rate of turbot were significantly higher than those of the other groups (P < 0.05), while the differences among the other groups were not significant (P > 0.05). The predictive model was established by linear regression analysis, and the regression equation showed that the maximum specific growth rate of turbot reached 1.67% when the dietary reduced glutathione was added at 189.70 mg/kg (Figure 1).

 

2.2 Effect of reduced glutathione on malondialdehyde content, total antioxidant capacity and superoxide dismutase activity in turbot liver

The addition of reduced glutathione did not significantly affect the malondialdehyde content, total antioxidant capacity and superoxide dismutase activity in the liver of turbot (P＞0.05) (Figs. 2-4), and the malondialdehyde content in the liver of turbot showed a tendency of decreasing and then increasing with the increase of the added amount of reduced glutathione, among which the control group had the highest malondialdehyde content and the test group had the lowest malondialdehyde content when the added amount of reduced glutathione was 200 mg/kg. The control group had the highest malondialdehyde content and the test group with 200 mg/kg reduced glutathione had the lowest. The total antioxidant capacity and superoxide dismutase activity in the liver of turbot showed a tendency of increasing and then decreasing with the increase of reduced glutathione, with the highest levels in the control group and the lowest level in the test group at 200 mg/kg of reduced glutathione, which were (1.99± 0.12) and (99.32± 3.09) U/mg, respectively.

 

2.3 Effects of reduced glutathione on reduced glutathione content and glutathione peroxidase activity in liver of turbot (Scophthalmus maximus)

With the increase in the amount of reduced glutathione added to the feed, the reduced glutathione content and glutathione peroxidase activity in the liver of turbot showed a tendency to increase and then decrease (Figs. 5 and 6). Reduced glutathione content in the liver of turbot was highest when 200 mg/kg of reduced glutathione was added to the feed, and the liver content of reduced glutathione was significantly higher in the groups with 200 and 400 mg/kg of reduced glutathione than in the control group (P < 0.05). The glutathione peroxidase activity in the liver of turbot was highest at 200 mg/kg of reduced glutathione, but the difference between the experimental group and the control group was not significant (P＞0.05).

 

2.4 Effect of reduced glutathione on glutathione sulfotransferase and glutathione reductase activities in turbot liver

The activities of glutathione sulfotransferase and glutathione reductase in the liver of turbot showed a decreasing and then increasing trend with the increase of reduced glutathione in the feed (Figs. 7 and 8). When 200 mg/kg of reduced glutathione was added to the diet, the activities of glutathione sulfotransferase and glutathione reductase in the liver of turbot were the lowest, which were (38.08± 5.68)U/mg and (6.87± 0.87)U/g, respectively, and were significantly lower than those in the control group (P<0.05).

 

3 Discussion

3.1 Effects of reduced glutathione on the growth of turbot (Scophthalmus maximus)

Studies have shown that the addition of reduced glutathione to feed can have a positive effect on the growth performance of aquatic animals, and can promote the growth of aquatic animals[16] , and the mechanism of action is the result of the coordination of multiple systems. Liu Xiaohua et al[11] showed that the addition of a certain amount of reduced glutathione to feed can improve the quality increase rate and feed efficiency of shrimp Vannamei, and pointed out that reduced glutathione can destroy the disulfide bond of the growth inhibitory molecules through the intermediate metabolite cysteamine, to release the growth inhibitory hormone control on growth hormone, and to promote growth hormone in the existing level, so as to promote the growth of the organism. Zhao Hongxia et al.[17] showed that the addition of reduced glutathione to feed can promote the growth of grass carp by regulating the level of growth hormone and increasing the level of insulin-like growth factor I. Zhou Tingting et al.[18] showed that the addition of reduced glutathione to feed can increase the level of insulin-like growth factor I in the growth hormone of grass carp. Zhou Tingting et al.[9] showed that the addition of reduced glutathione to feed could promote protein synthesis and increase the intake of Jifu tilapia. These findings have been confirmed in experiments with rainbow trout[10] , brown turbot[13] , Pelteoba grus fulv idraco[18] and Onirofus tilapia[19] .

 

In this experiment, different doses of reduced glutathione were added to the basic feed formula, and the results showed that the addition of reduced glutathione to turbot feed could significantly improve the growth performance of turbot, and the rate of increase in mass and specific growth rate of turbot in the experimental group with the addition of reduced glutathione was significantly higher than that of the control group with no reduced glutathione, and reached a significant level at the addition rate of 200 mg/kg. The significant level was reached at 200 mg/kg, which is consistent with the results of the above experiments. In addition, it has been shown that reduced glutathione in the intestinal lumen of animals can protect the intestinal mucosa by scavenging peroxides[20] , and in a study by Venurini[21] , it was shown that reduced glutathione can enhance the feeding response of Hydra attenuate. Thus, the growth-promoting effect of reduced glutathione on turbot is the result of a series of mechanisms, and the specific growth mechanism needs to be further investigated.

 

3.2 Effect of dietary reduced glutathione on the antioxidant capacity of turbot (Scophthalmus maximus)

In practice, there are many factors that cause oxidative stress to organisms, resulting in the production of a large number of reactive oxygen radicals (RORs), which lead to the production of reactive oxygen species (RORs) exceeding their decomposition rate and causing damage to organisms and even to their growth and development. Lipid peroxidation leads to the production of secondary products, such as malondialdehyde, which is an important indicator of oxidative stress damage caused by reactive oxygen species in different marine organisms[22] . In this experiment, malondialdehyde decreased and then increased with the addition of reduced glutathione to the diets, and the levels of malondialdehyde in the reduced glutathione group were lower than those in the control group, and the lowest level was found in the reduced glutathione group at 200 mg/kg, but did not reach a significant level. The exogenous addition of reduced glutathione reduced the oxidative damage of cells to a certain extent.

 

In order to maintain the balance between oxidation and reduction reactions, organisms have a whole set of antioxidant system. The antioxidant system continuously scavenges free radicals, regulates the level of reactive oxygen species in the organism, and at the same time participates in a variety of biochemical reactions to effectively regulate the oxidative stress of the organism, thus ensuring the stability of the internal and external cellular environment of the organism and its normal physiological functions. With the increase of reduced glutathione in the feed, the malondialdehyde content in the liver of turbot was lower than that of the control group, and the increase of total antioxidant capacity and superoxide dismutase activity also proved that the moderate addition of reduced glutathione in the feed could improve the antioxidant capacity of turbot to a certain extent.

 

The changes in glutathione peroxidase activity after the addition of reduced glutathione showed the same trend as that of reduced glutathione, total antioxidant capacity (TAC) and superoxide dismutase (SOD) activity, but did not reach a significant level. The decrease of glutathione reductase activity in this experiment also proved that the dynamic balance of reduced glutathione/oxidized glutathione could be regulated by the addition of exogenous reduced glutathione, but not by the endogenous oxidized glutathione. It is not necessary to convert endogenous oxidized glutathione to reduced glutathione catalyzed by glutathione reductase to increase the reduced glutathione content in the body. This result is similar to that of brown turbot[13] , but different from that of rainbow trout[10] and jiffy tilapia[23] , so the mechanism of glutathione reductase in turbot needs to be further investigated.

 

Glutathione sulfotransferase exists in large quantities in hepatocytes, and when hepatocytes are damaged, glutathione sulfotransferase will be rapidly released into the blood, so glutathione sulfotransferase can also be used as a sensitive indicator of liver injury[24] . In this experiment, malondialdehyde showed a decreasing and then increasing trend with the increase of reduced glutathione, with the highest in the control group and the lowest in the experimental group with 200 mg/kg of reduced glutathione, and the glutathione sulfotransferase showed an opposite trend to malondialdehyde. It was further proved that the moderate addition of reduced glutathione could reduce the oxidative damage in the cells. In addition, when the glutathione peroxidase of turbot showed a decreasing trend, the glutathione sulfotransferase showed a corresponding increasing trend. This result is similar to that of brown turbot[13] and rainbow trout[25] . The results suggest that glutathione sulfotransferase can also be used as an antioxidant protection mechanism when the activity of glutathione peroxidase is low.

 

3.3 Optimal levels of reduced glutathione in turbot feeds

It has been shown that reduced glutathione, although an important scavenger of free radicals in the body, can be a pro-oxidant at concentrations of up to 1 mmol/L in living organisms, causing DNA damage[26] . In addition, excessive accumulation of reduced glutathione as a precursor of oxidants can be toxic[27] , and some compounds can be converted to cytotoxic, genotoxic, or mutagenic metabolites by combining with reduced glutathione[28] .

 

The present study showed that the addition of reduced glutathione to feed increased the accumulation of reduced glutathione in the liver to a certain extent, which was significantly different from that of the control group, which was consistent with the results of Wang Fangqian et al[13] . This result was consistent with that of Wang Fangqian et al[13] . However, He Fen et al[29-30] showed that the addition of reduced glutathione to feed did not have a significant effect on the liver reduced glutathione content, which may be related to the different types of test subjects, and the exact mechanism needs to be further investigated.

 

It was also found that the growth performance and antioxidant capacity of turbot showed a tendency of increasing and then decreasing with the increase of reduced glutathione content, and the malondialdehyde content in the liver showed a tendency of increasing after the addition of 200 mg/kg, which indicated that the antioxidant capacity of the organism declined and a certain degree of oxidative damage appeared when the added amount of reduced glutathione was higher than a certain level. The results were similar to those of the shrimp Penaeus vannamei[11] , brown turbot[13] and Pelteobagrus fulvidraco[18] . The results showed that only when the amount of reduced glutathione was added in the appropriate range, it had the effect of promoting growth and antioxidant capacity; if it was added in excess, it would lead to the excessive accumulation of reduced glutathione in turbot, resulting in oxidative damage, which could have toxic effects on fish. The optimal level of reduced glutathione in aquatic feeds varies according to the target species. The optimal level of reduced glutathione in feeds for rainbow trout is 200 mg/kg[10] , in feeds for shrimp, Penaeus vannamei, the optimal level is 174.13 mg/kg[11] , in feeds for brown flounder, the optimal level is 368.92 mg/kg[13] , in feeds for yellow croaker, the optimal level is 368.92 mg/kg[14] , in feeds for catfish, Pelteobagrus fulvidraco, and in feeds for catfish, Pelteobagrus fulvidraco, the optimal level is 368.92 mg/kg[15] . 13]; the optimum level of reduced glutathione in Pelteobagrus fulvidraco feed is 357.69 mg/kg[18]; the optimum level of reduced glutathione in Jiffy tilapia juvenile feed is 355.13 mg/kg[23]; in the present experiment, the specific growth rate was used as the evaluation index, and a prediction model was established by linear regression analysis, through which the optimum level of reduced glutathione in turbot feed was determined to be 189.13 mg/kg[24]. The optimal level of reduced glutathione in turbot feed was 189.70 mg/kg[23] .

 

4 CONCLUSIONS

Addition of appropriate amount of reduced glutathione to turbot feed can promote the growth of turbot, improve the ability of turbot to resist oxidative stress, remove oxygen radicals in the body, and alleviate the oxidative damage to turbot in the process of aquaculture, and the optimal amount of reduced glutathione added to the feed is 189.70 mg/kg.

 

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