Effect of Yeast Selenium on Growth Performance, Muscle Selenium Deposition, and Antioxidant Capacity of Juvenile Cherax quadricarinatus

Abstract

This study aimed to investigate the effects of organic selenium-enriched yeast on the growth performance, muscle selenium deposition, and antioxidant capacity of juvenile Cherax quadricarinatus. Five experimental diets supplemented with 0.00 (control), 1.00, 2.00, 3.00, and 4.00 mg/kg of selenium-enriched yeast were fed to crayfish with an average initial body weight of (6.35 ± 1.66) g for 56 days. On days 0, 14, 28, 42, and 56 of the trial, 10 crayfish from each group were randomly sampled for body weight measurement. On day 28, the body weight of the crayfish fed diets containing 1.00, 2.00, 3.00, and 4.00 mg/kg selenium-enriched yeast was significantly higher (p < 0.05) than that of the control group. On day 56, the weight gain rate (WGR) and specific growth rate (SGR) of crayfish fed 1.00, 2.00, and 3.00 mg/kg selenium-enriched yeast were significantly elevated (p < 0.05) compared to those of the control group. Dietary selenium supplementation significantly enhanced muscle selenium deposition (p < 0.05), with deposition levels increasing proportionally to the supplementation levels within the same period. Selenium deposition initially increased, peaked at day 28 (significantly higher than the control), and then declined and stabilized. On day 42, the activities of pancreatic lipase (LPS), total superoxide dismutase (T-SOD), and acid phosphatase (ACP) in the hepatopancreas were significantly higher in the 3.00 and 4.00 mg/kg groups (p < 0.05). In comparison, trypsin activity was significantly increased in the 2.00 mg/kg and 3.00 mg/kg yeast selenium groups (p < 0.05). Alkaline phosphatase (AKP) activity was highly significantly elevated in the 2.00 mg/kg group (p < 0.01). On day 56, glutathione peroxidase (GSH-PX) activity in the hepatopancreas was considerably enhanced in all the selenium-supplemented groups (1.00–4.00) mg/kg (p < 0.05). In conclusion, the appropriate supplementation of selenium-enriched yeast promotes growth performance, enhances muscle selenium deposition, improves hepatopancreatic digestive enzyme activity, and strengthens antioxidant and immune capacities in Cherax quadricarinatus.

1. Introduction

Selenium (Se), an essential trace element for both humans and animals, plays a vital role in growth and metabolic processes [1,2]. Supplementing animal diets with the appropriate selenium levels not only promotes healthy growth in animals but also enhances human dietary selenium intake through selenium-enriched animal protein. Selenium primarily exists in the following two forms: inorganic selenium (e.g., sodium selenite and sodium selenate) and organic selenium (e.g., selenium-enriched yeast and selenomethionine) [3]. Compared to the inorganic forms, organic selenium—as a feed additive—demonstrates significant advantages, including higher bioavailability, reduced environmental contamination, and improved feed utilization efficiency [4]. Studies have shown that dietary selenium-enriched yeast enhances growth performance in aquatic species, thereby optimizing feed conversion ratios and aquaculture profitability [5,6]. Furthermore, selenium-enriched yeast upregulates antioxidant and immune-related factors such as superoxide dismutase (SOD) and interferon-gamma (IFN-γ), thereby strengthening antioxidant defense mechanisms and immune responses [7,8,9,10]. Additionally, it exhibits detoxification properties by antagonizing heavy metal toxicity, such as cadmium exposure [11].

Cherax quadricarinatus, also known as crayfish, belongs to the order Decapoda, the family Parastacidae, and the genus Cherax. This species is characterized by rapid growth, tender meat, high nutritional value, and substantial meat yield, presenting significant potential for large-scale aquaculture development [12,13]. Therefore, this study investigated the effects of dietary selenium-enriched yeast supplementation on the growth performance, muscle selenium deposition, and antioxidant capacity of juvenile Cherax quadricarinatus. The findings aimed to provide a scientific basis for formulating specialized feeds optimized with selenium-enriched yeast for this commercially valuable species.

2. Materials and Methods

2.1. Experimental Design

A total of 450 juvenile Cherax quadricarinatus with intact appendages and no signs of disease were selected, with an average initial body weight of (6.35 ± 1.66) g. The crayfish were randomly allocated into five groups (three replicates per group, 30 individuals per replicate) and fasted for 48 h before the trial. Each group was fed one of five experimental diets—a basal diet supplemented with 0.00 (control), 1.00, 2.00, 3.00, or 4.00 mg/kg selenium-enriched yeast. The experiment adhered to the Guidelines for the Welfare of Experimental Animals in China and was approved by the Animal Ethics Committee of Hunan University of Science and Technology. The experimental crayfish were sourced from Hunan Sumin Agricultural Technology Co., Ltd. (Xiangtan, China) and reared in a recirculating aquaculture system (RAS) under controlled conditions for 56 days.

2.2. Nutrient Composition Content of the Basic Feed

The crude protein, crude fat, and crude ash contents in the experimental diets were determined using the Kjeldahl method (GB/T 5009.5-2016 [14]), Soxhlet extractor method (GB 5009.6-2016 [15]), and gravimetric method (GB 5009.4-2016 [16]), respectively. Calcium and the total phosphorus levels were analyzed according to the Determination of Calcium in Feeds (GB/T 6436-2018 [17]) and Determination of Phosphorus in Feeds—Spectrophotometry (GB/T 6437-2018 [18]) guidelines. The available phosphorus and digestible amino acid contents were calculated based on the nutritional composition of raw materials reported in the Chinese Feed Composition and Nutritional Value Table (31st edition, 2020) and the nutrient profiles of the basal diet used in this trial [19]. The gross energy (GE) of the feed was measured via the bomb calorimetric method, while energy values in excreta were determined using indirect calorimetry.

2.3. Experimental Diet Preparation

The basal diet, formulated with commercial feed (Guangdong Yueda Biological Nutrition Technology Co., Ltd., Kaiping, China) was supplemented with selenium-enriched yeast (Angel Yeast Co., Ltd., Yichang, China; organic selenium content ≥ 98%, total selenium concentration: 2000 mg/kg) at graded levels of 0.00 (control), 1.00, 2.00, 3.00, and 4.00 mg/kg. The actual selenium concentrations measured in the experimental diets were 0.00, 1.85, 2.36, 2.81, and 3.35 mg/kg, respectively. The feed was uniformly spread onto a shallow tray (with a thickness not exceeding 2 cm) and sprayed in three sequential applications, with each application comprising one-third of the total solution volume. Following each spray, the feed was manually agitated for 5 min to ensure complete particle–solution contact. A low-speed fan was employed to facilitate drying for 10 min, minimizing particle agglomeration. The sprayed feed was then transferred to a 40 °C oven for 2 h of drying.

After drying, the feed was sealed in airtight plastic bags with desiccant packets (e.g., silica gel) and stored in a dark refrigerator set at 4 °C to prevent moisture adsorption and selenium degradation. For quality control, twenty feed pellets were randomly selected, subjected to single-step dissolution, and analyzed for selenium content. Standard deviation was calculated from these measurements to characterize within-sample variability.

Yeast selenium was precisely weighed according to the target supplementation levels, dissolved in deionized water at a predetermined ratio, and uniformly sprayed onto the surface of the basal diet using a laboratory-scale coating apparatus. The coated feed was subsequently oven-dried at 45 °C to a constant weight, sealed after drying, and stored under cool, dry conditions until use.

2.4. Feeding and Management

Feeding occurred daily at 18:00, with a ratio equivalent to 3% of the total biomass. Water quality parameters were maintained as follows: temperature 27~31 °C, pH 7.8~8.6, dissolved oxygen > 5.0 mg·L⁻¹, ammonia nitrogen < 0.05 mg·L⁻¹, and nitrite < 0.01 mg·L⁻¹.

2.5. Sample Collection and Data Acquisition

On days 0, 14, 28, 42, and 56 of the trial, 10 juvenile Cherax quadricarinatus were randomly sampled from each group for body weight measurement. On days 14, 28, 42, and 56, three crayfish per group were humanely euthanized. The anesthetic agent MS-222 (ethyl m-aminobenzoate methanesulfonate) was deployed, with the concentration standardized according to crayfish body weight at 250 mg/L. The crayfish were fully submerged in the anesthetic solution, exhibiting a progressive loss of motor function characterized by the cessation of gill ventilation and the absence of escape responses. Upon achieving deep anesthesia, brain destruction was performed by using anatomical needles to access the neurocranial junction at the carapace–cephalothorax suture. Following the procedure, the crayfish were monitored for 15 min to confirm irreversible death. Their abdominal muscle and hepatopancreatic tissues were rapidly dissected, weighed, flash-frozen in liquid nitrogen, and stored at −80 °C in cryovials for the subsequent analysis of muscle selenium content and hepatopancreatic enzyme activity; the remaining individuals were returned to the culture tanks.

2.6. Growth Performance Evaluation

Growth indices, including weight gain rate (WGR), specific growth rate (SGR), dressing percentage (DP), condition factor (CF), and hepatosomatic index (HSI), were calculated using the following formulas:

WGR (%) = (Wd – Wa)/Wa × 100;

SGR (%) = (lnWd – lnWa)/D × 100;

DP (%) = Wm/Wd × 100;

CF (g/cm³) = Wd/Ld³ × 100

HIS (%) = Wh/Wd × 100.

where Wa and Wd are the initial (day 0) and final (day 56) body weights (g); D is the experimental duration (days); Wm is the shell-free abdominal muscle weight (g); Ld is the body length at day 56 (cm); and Wh is the hepatopancreas weight (g).

2.7. Determination of Feed and Muscle Selenium Content

Experimental feeds with different levels of yeast selenium were used as analytical samples, along with muscle tissue from each group at 14, 28, 42 and 56 days of culture. The selenium content in both the feed and muscle tissue was determined by inductively coupled plasma mass spectrometry (ICP-MS) (see GB 5009.268-2016 [20] for measurement criteria).

2.8. Biochemical Assays

The hepatopancreatic digestive enzymes [α-amylase (AMS), lipase (LPS), trypsin (TRY)], antioxidant parameters [catalase (CAT), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC)], immune-related enzymes [acid phosphatase (ACP), alkaline phosphatase (AKP)], and total protein (TP) content were analyzed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.9. Statistical Analysis of the Data

The data from the experiment are expressed as mean ± standard deviation (mean ± SD). For the statistical analysis, the results between each group were analyzed by one-way analysis of variance (One-Way ANOVA) and significant analysis by Duncan’s multiple comparison method. p < 0.05 indicates a significant difference.

3. Results and Analysis

3.1. Effect of Yeast Selenium on Growth, Muscle Deposition, and Digestive Capacity

The effects of yeast selenium on the body weight of crayfish at different time points are presented in Figure 1. With the prolonged breeding time, the weight of the crayfish gradually increased. On day 28, the mean body weight of the groups fed diets with 1.00, 2.00, and 3.00 mg/kg of yeast Se was significantly greater than that of the control group (p < 0.05). Statistical analysis of the effects of dietary yeast selenium supplementation on various growth performance parameters—including weight gain rate (WGR), specific growth rate (SGR), meat yield, and hepatosomatic index (HSI), Statistical analysi—revealed that diets supplemented with 1.00, 2.00, and 3.00 mg/kg yeast selenium significantly enhanced these growth indicators compared to the control group (p < 0.05).

Compared to the control group, on day 14, adding 4 mg/kg of yeast selenium significantly increased muscle selenium content (p < 0.01); on day 28, adding 1 mg/kg, 2 mg/kg, 3 mg/kg, and 4 mg/kg of yeast selenium all significantly increased muscle selenium content (p < 0.01); on day 42, adding 1 mg/kg and 2 mg/kg of yeast selenium significantly increased muscle selenium content (p < 0.05), while adding 3 mg/kg and 4 mg/kg of yeast selenium highly significantly increased muscle selenium content (p < 0.01); on day 56, adding 2 mg/kg of yeast selenium significantly increased muscle selenium content (p < 0.05), and adding 3 mg/kg and 4 mg/kg of yeast selenium highly significantly increased muscle selenium content (p < 0.01). From the perspective of cyclic changes, the addition of 1 mg/kg, 2 mg/kg, 3 mg/kg, and 4 mg/kg of yeast selenium all showed a trend of initially increasing deposition, followed by a decrease, and finally stabilizing, with their maximum value reached on day 28, which was significantly higher than that of the control group (p < 0.01) (Figure 2).

3.2. Effect of Yeast Selenium on the Vitality of Digestive Enzymes and Antioxidant Enzymes in Cherax quadricarinatus

Compared to the control group, on day 14, adding 4 mg/kg of yeast selenium significantly increased liver and pancreatic AMS activity (p < 0.05); on day 42, adding 3 mg/kg and 4 mg/kg of yeast selenium extremely significantly increased liver and pancreatic AMS activity (p < 0.01) (Figure 3A). Compared to the control group, on day 42, adding 3 mg/kg and 4 mg/kg of yeast selenium extremely significantly increased liver and pancreatic TRY activity (p < 0.01); on day 56, adding 3 mg/kg of yeast selenium significantly increased liver and pancreatic TRY activity (p < 0.05) (Figure 3B).

The results of the antioxidant indexes in the liver and pancreatic tissues at different breeding times showed that, on day 42, adding 3 mg/kg and 4 mg/kg of yeast selenium significantly increased the CAT activity in the liver and pancreas (p < 0.05) (Figure 4A). Adding 2 mg/kg and 3 mg/kg of yeast selenium also significantly increased the T-SOD activity in the liver and pancreas (p < 0.05) (Figure 4B). On day 56, the experimental group with 4 mg/kg of yeast selenium in their diet showed significantly higher GSH-PX activity in the liver and pancreas compared to the control group (p < 0.05) (Figure 4C).

3.3. Effect of Yeast Selenium on Immunoenzyme Activity of Cherax quadricarinatus

The determination of immunoenzyme activity at different culture times showed that, on day 42, the activity of ACP in the liver and pancreas was significantly increased by adding 2 mg/kg, 3 mg/kg and 4 mg/kg yeast selenium (p < 0.01) (Figure 5A), and the activity of AKP in liver and pancreas was significantly increased by adding 2 mg/kg yeast selenium (p < 0.01) (Figure 5B).

4. Discussion

4.1. Effect of Yeast Selenium on the Growth Performance of Cherax quadricarinatus

Selenium is an essential trace element for maintaining the growth and metabolism of animals [12,13,19,21]. The addition of an appropriate amount of selenium can improve the growth performance of animals, but the selenium requirements vary across different species. Studies have shown that the addition of 0.6 mg/kg yeast selenium into the feed can effectively promote the growth of the carp (Cyprinus carpio) fish species and improve its feed utilization rate of [13]. The addition of 0.36 to 0.38 mg/kg yeast selenium can significantly promote the growth of yellow cheek (Elopichthys bambusa) juveniles [22]. The addition of 2~4 mg/kg yeast selenium can significantly improve the weight gain rate, final body weight, and feed conversion efficiency of grass carp (Ctenopharyngodon idella) [23]. The addition of 6.73 mg/kg yeast selenium significantly promoted the growth and improved muscle quality of the larval sturgeon (Acipenser schrenckii) [24]. This study found that the rate of weight gain and specific growth were significantly increased at 1.00–3.00 mg/kg (p < 0.05). However, the results in rainbow trout (Oncorhynchus mykiss) and squid (Sepia officinalis) showed that the supplementation of yeast selenium or selenomethionine had no significant effect on their growth [25,26,27,28]. The differences in these results may be caused by species differences, growth stage, farming environment, the form of selenium addition, and the level of addition.

Digestive enzymes mainly include AMS, LPS, and TRY, which can directly reflect the digestive and metabolic capacity of the body [29]. Among them, AMS is the general term for a class of enzymes that hydrolyze starch and sugars, and its secretion determines the ability of animals to decompose sugars in feed [30,31]. LPS can hydrolyze triglycerides into glycerol monoesters and fatty acids, which is more conducive to the absorption and utilization of lipids [32,33]. TRY is a serine protease that hydrolyses peptide bonds at the carboxyl side of the lysine and arginine residues, with a high specificity [34,35,36]. In this test, the test groups with 3.00 and 4.00 mg/kg of yeast selenium and 2.00 and 3.00 mg/kg increased LPS and TRY activities on day 42, indicating that the appropriate amount of yeast selenium in the feed could promote the digestion, absorption, and utilization of carbohydrates, while promoting protein decomposition, which facilitates the growth of crayfish.

4.2. Effect of Yeast Selenium on the Muscle Selenium Content of Cherax quadricarinatus

Selenium supplementation in feed can increase selenium deposition in animal tissues, but different tissues have varying selenium enrichment capacities, and the metabolic pathways and bioavailability of different selenium sources also differ [22,37]. In this study, yeast selenium was the source of selenium. The results show that the deposition of selenium in the muscle of the crayfish increased during the same period, which is consistent with the results of the study by Wang et al. [38]. Studies on Rachycentron canadum, rainbow trout, Atlantic salmon, and other animals have also shown that selenium deposition in muscle tissue gradually increases with the increase in feed selenium content [39,40,41]. The selenium content in body tissues reflects, to some extent, the utilization rate and metabolic status of selenium from the feed. As dietary intake of selenium increases, so does the selenium content in body tissues, indicating that selenium from the feed is assimilated and effectively deposited in the body tissues after ingestion and absorption. From the perspective of a cycle change, muscle selenium deposition in the experimental group fed yeast selenium initially increased, then decreased and stabilized over time. We speculate that there are two primary reasons for this phenomenon. Firstly, it could be due to the imbalance in nutrient synergy; the synergistic effect of vitamin E and selenium in the feed may not be optimized, thereby affecting the absorption and utilization efficiency of selenium [42]. Secondly, it could be because the deposition of selenium in animals does not continually increase. After meeting the demand for selenium in the body, excess selenium will be discharged from the body [37,41] through feces. Therefore, the pattern of selenium deposition in animal tissues may be closely related to the demand for selenium and the metabolic pathway of selenium in animals.

With continuous improvements in living standards, people’s pursuit of food quality is also increasing. In recent years, various selenium-enriched products—including eggs, rice, tea, fruits, and vegetables—have been successively developed, leading to an increase in selenium-rich food options. According to T/HNFX 001-2017, the selenium content in meat and processed products range from 0.15 to 1.20 mg/kg. In this study, maximum muscle selenium deposition in the groups supplemented with 1.00, 2.00, 3.00, and 4.00 mg/kg yeast selenium was observed on day 28. The mean selenium content in the muscle was 0.43, 0.47, 0.55, and 0.65 mg/kg, meeting the selenium content requirements for selenium-rich agricultural products. On days 42 and 56, although selenium deposition in the crayfish muscle decreased, the average selenium content in the muscles of each test group remained between 0.2 and 0.3 mg/kg, still meeting the selenium content requirements for selenium-rich agricultural products. Therefore, supplementation with 1.00, 2.00, 3.00 and 4.00 mg/kg of yeast selenium over a period of 28–56 days can produce selenium-rich crayfish meat.

4.3. Effect of Yeast Selenium on Antioxidant and Immune Enzymes of Cherax quadricarinatus

The antioxidant or free radical defense system in animals can effectively remove lipid peroxides, terminate free radical chain reactions, and relieve lipid peroxidation damage [43,44,45]. Antioxidant enzymes such as T-AOC, CAT, GSH-Px, and T-SOD are important indicators of the antioxidant capacity of the reaction body [46,47]. Among them, CAT has a defensive function and is important for protecting against cell oxidative damage [22]. Selenium is the active center of GSH-Px. It can catalyze the conversion of reduced glutathione (GSH) to transform hydrogen peroxide, lipid peroxides, and other harmful substances into harmless water and alcohol compounds, thereby protecting the cell membrane and the biological macromolecules (such as DNA, protein) from oxidative damage [48]. A study by Wu Lei et al. on juvenile sturgeon showed that the dietary supplementation of 6.73 mg/kg yeast selenium significantly increased hepatic GSH-Px activity [22]. SOD can catalyze the disproportionate reaction of oxygen ions and play a protective role in peroxidation and defense [22]. In this study, the supplementation of yeast selenium significantly increased the viability of CAT, T-SOD, and GSH-P-X in the liver and pancreas (p < 0.05) but had no significant effect on the total antioxidant capacity T-AOC, indicating that supplementation of the appropriate concentration of yeast selenium could improve the antioxidant levels of P. chinensis to some extent.

Nonspecific immunity is the main way through which the immune system of crustaceans can resist bacteria [48]. ACP and AKP are directly involved in phosphate transfer and metabolism and are closely related to the non-specific immune function of shrimp [48]. Among them, ACP is present in a variety of tissues and body fluids, and it is the most representative hydrolase in macrophages, representing the non-specific immune capacity of cells. In an alkaline environment, AKP can hydrolyze phosphomonolipids to ethanol and phosphate, participating in non-specific immune regulation [49,50]. Selenium is present in all immune cells, including T cells, B cells, and NK cells, where it enhances their function and promotes the synthesis of antibodies such as IgG and IgA, thereby improving the body’s specific immune capabilities [51]. Additionally, selenium participates in glycolytic processes within mitochondria, specifically in the tricarboxylic acid cycle, in the form of selenoproteins and selenium-containing amino acids. This involvement helps maintain normal physiological functions and enhances the body’s non-specific immune capabilities [5]. In this study, the supplementation of yeast selenium significantly improved ACP vitality in the liver and pancreas (p < 0.05), indicating that the supplementation of the appropriate amount of yeast selenium in the feed could improve the immunity of Cherax quadricarinatus and help the body resist the invasion of bacteria.

4.4. Effects of Excessive Selenium Intake Have Potential Negative on Organisms

Although selenium from yeast can significantly enhance the growth performance and antioxidant capacity of red mud shrimp, previous studies have shown that excessive selenium can damage multiple physiological functions of aquatic organisms [5,52]. In goldfish, 1.38 mg/kg of selenium nanoparticles increased the MDA levels in seminal plasma and GPx, as well as DNA damage in sperm, reducing sperm quality and disrupting testicular development [53]. Following selenium exposure at 8.9 μg/L, Chironomidae and Gammaridae exhibited marked reductions in population density and biomass compared to controls (0.12 μg/L Se). The treatment groups (1.0 and 8.9 μg/L Se) showed a concomitant decline in invertebrate diversity metrics, with both the Shannon and Simpson indices demonstrating significant decreases relative to the baseline conditions [54].Therefore, the appropriate level of yeast selenium supplementation for different species still requires further specialized research to determine.

In conclusion, with increasing breeding time, the growth performance, muscle selenium deposition, and enzyme activity in the liver and pancreas are also constantly changing. This study shows that yeast selenium supplementation significantly increased muscle selenium deposition, ultimately enhancing the digestive enzymes, antioxidant enzymes, and non-specific immunity. Therefore, further research is needed to explore strategies to enhance growth, as well as antioxidant and immune capacity, through selenium supplementation.

5. Conclusions

Adding an appropriate amount of yeast selenium to the feed can significantly promote the growth of crayfish, increase selenium deposition in the muscle, improve digestive enzyme activity in the liver and pancreas to a certain extent, and enhance antioxidant and immune capacity.