1. Introduction
γ-aminobutyric acid(GABA), a non-protein amino acid with four carbon atoms [
1], plays an essential role as a neurotransmitter in the mammalian nervous system [
2]. While GABA is widely found in nature [
2], its concentration in natural feed ingredients is typically insufficient to support optimal biological functions [
3,
4]. Therefore, dietary supplementation with exogenous GABA is necessary for animals to confer a range of health benefits. Zhang et al. [
1] have demonstrated that GABA supplementation can enhance the performance and physiological condition of laying hens, and relieve heat stress through regulating hormone secretion, antioxidant and immune activities, and maintaining electrolyte balance. Wang et al. [
5] discovered that dietary GABA supplementation improved feed intake, performance, and antioxidant status in perinatal dairy cows. Guang et al. [
6] reported that the addition of GABA to the diet enhanced the growth performance of yellow-feathered broilers aged between 36 and 49 days under high-temperature conditions. This supplementation reduced serum intracellular enzyme activity, which contributed to the improved health of the broilers’ organs and the maintenance of the integrity of their intestinal morphology [
6]. Mokhtar et al. [
7] reported that GABA could regulate damage related to stress in broilers. Ngo et al. [
2] proved that GABA decreased the production of pro-inflammatory mediators and ameliorated inflammatory symptoms, thereby inhibiting inflammation.
High ambient temperatures can reduce feed intake, feed conversion efficiency, and growth rates in livestock and poultry [
8]. Mink, which naturally thrive in cold regions like North America and Northern Europe, prefer cooler climates over heat [
9]. High ambient temperatures not only induce heat stress reactions in mink but also lead to a range of health problems, such as decreased appetite, weight loss, and reduced immune function [
10,
11]. Therefore, it is crucial to implement effective measures to ensure the health and productivity of mink under high-temperature environments.
The increasing recognition of GABA’s potential as a feed additive has promoted extensive research into its application in the diets of poultry and ruminant animals [
5,
6,
7]. However, the effects of GABA on growing minks are not well documented. This experiment aimed to assess the impact of GABA on growth performance, immunity, and intestinal microbiota in growing minks, and to determine the optimal level of GABA supplementation for their development (
Figure 1).
2. Materials and Methods
2.1. Experimental Animals, Design and Management
The experiment was conducted at a commercial mink farm located in Haiyang, a region in the eastern part of China, from July to September in 2022. A total of 100 Silverblu minks, at approximately 12 weeks of age and with an average body weight of 1134.47 ± 22.76 g, were randomly selected from the farm’s animal stock for the study. All the minks were divided into five groups, each consisting of 10 male minks and 10 female minks. The minks in these groups were fed a basal diet supplemented with GABA at 0 (control), 10, 20, 30, and 40 mg of diet, respectively (
Figure 1).
The GABA (99% purity) used in the present experiment was provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The experiment lasted for 8 wk after one week of adaptation. The diets used in the experiment were formulated according to the commercial standards. The composition of the diets (as-fed basis) and nutrient levels on an air-dry basis are detailed in
Table 1.
All the minks were housed in an open-type barn with two rows of wire mesh cages. Each experimental mink was housed individually in a wire mesh cage. The cage included an attached straw-bedded nest box, which had an entrance for the mink to enter and exit.
Fresh mink diet was manufactured and provided by the farm twice a day (6:30 AM and 5:30 PM, respectively). Fresh water was supplied by a drinker in the cage. Both diet and water were available ad libitum during the experiment. The feed consumption and feces condition of the experimental minks were observed to evaluate the health of the minks daily throughout the study, and the death of the minks was recorded.
The ambient temperature was 25.53 ± 0.07 °C, relative humidity was 83.16 ± 0.24%, and the light schedule was a natural light regime throughout the study.
2.2. Sampling and Measurements
All the minks in the five groups were individually weighed at the beginning (week 0) and end (week 8) of the study to determine the initial and final body weights. The average daily gain (ADG) was then calculated. The apparent feed intake of the minks was monitored for three consecutive days (days 3 to 5) each week during the experiment. The given and remaining rations were weighed daily during the 3d monitoring period, respectively. Then, the average daily feed (ADFI) and feed/gain ratio (F/G) were calculated.
At the end of the study, six male and six female minks from each group were selected for blood collection via cardiac puncture. Following the blood collection, these minks were euthanized to obtain samples of the jejunal mucosal tissue and rectal content. The blood was centrifuged at 3000× g at 4 °C for 10 min, and subsequently, the serum was obtained. Jejunal mucosal tissue homogenate was prepared with 9 mL of 0.9% NaCl and 1 g of jejunal mucosal tissue. The tissue was centrifuged at 3500× g at 4 °C for 10 min, and the supernatant was obtained. The blood and the jejunal mucosal tissue supernatant were stored in a −20 °C refrigerator for further assessment, and the rectal content samples were stored in a −80 °C refrigerator for flora analysis.
2.3. Biochemical Parameters in Serum
The contents of albumin (ALB), blood urea nitrogen (BUN), and total protein quantitative (TP) in serum were determined by colorimetry. The absorbance of ALB, BUN, and TP was determined by a UV-VIS spectrophotometer (UV-6200, Shanghai, China). The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were determined by the micro-plate method with a full-wavelength micro-plate reader. All these indicators were measured using kits manufactured by Jiancheng Biological Engineering Research Institute (Nanjing, China).
2.4. Immunity Analysis
The collected serum and jejunal mucosa samples were assayed for immune indices according to the methods of Li et al. [
12]. The concentration of immunoglobulins (IgA, IgM, and IgG) in serum, and SIgA and cytokines (IL-4, IL-10, IL-2, IL-12, TNF-α, and IFN-γ) in jejunal mucosa were assayed using a commercial kit (Jiancheng Biological Engineering Research Institute, Nanjing, China) and read using a full-wavelength micro-plate reader.
2.5. Antioxidant Capacity Analysis
The serum total antioxidant capacity (T-AOC), activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and malondialdehyde (MDA) were quantitatively assessed using a colorimetric assay.
2.6. Intestinal Flora Analysis
The collected samples from the rectal contents were extracted DNA to determine intestinal flora according to the previous method [
13]. The extracted genomic DNA from rectal contents was detected, amplified (ABI GeneAmp®, 9700, Applied Biosystems, Foster City, CA, USA), purified, recovered, quantified, and sequenced [
14].
2.7. Statistical Analysis
The Proc Mixed model of the SAS 9.4 software (SAS Inst. Cary, NY, USA) was used to analyze the data of growth performance and, immunohistochemical and antioxidant capacity. The model included GABA, gender, and their interactions as fixed effects, and the cage as the experimental unit. The least square means were separated by Tukey adjustment. Differences were considered significant when p < 0.05.
The data of intestinal flora were analyzed on the I-Sanger cloud platform. The FLASH 1.2.11 software performs pair-end double-ended sequence splicing. The Spearman correlation coefficient was adopted to analyze the correlation of the intestinal flora with the immunity of minks.
4. Discussion
The results of the experiment indicated that GABA had no effects on BW and ADG of growing minks. This is the same opinion as the consequences of the previous studies on pigs that GABA had no effects on the BW and ADG of growing-finishing pigs [
15]. However, the supplementation of GABA at 10, 20, 30, or 40 mg/kg of diet increased the feed intake of minks. Especially in the case of high temperatures, the increase in feed intake can mitigate the suppressive action of heat stress in terms of feed intake. As suggested by Jonaidi et al. [
16], GABA stimulated feed intake by interaction with neuropeptide Y (NPY). In addition, GABA is an important inhibitory neurotransmitter and sedative, which agents in the central nervous system and can improve the feed intake and absorption of animals by regulating feed intake center, promoting gastric acid secretion, inhibiting cholecystokinin secretion, and suppressing stomach satiety center [
1].
The present study proved that GABA at 20, 30, and 40 mg/kg of diet increased TP, and GABA at 10, 20, 30, and 40 mg/kg of diet decreased the BUN concentrations. Additionally, GABA tended to decrease the AST activity in serum. These findings are consistent with the results reported by Ncho et al. on chickens [
17]. GABA inhibits glutamic acid decarboxylation by reducing the activities of alkaline phosphatase and aminotransferase, and reacts with α-ketoglutaric acid to produce glutamic acid, thereby reducing the content of nitrogen in serum, subsequently reducing the content of nitrogen excreted in urine [
18], and promoting the deposit of protein. According to Zhang et al. [
19], TP is the combination of a range of proteins, which is synthesized by the liver. In addition, AST as an intracellular enzyme is determined to assess liver necrosis. The elevation of AST in serum indicates that AST is released into the blood by the damaged hepatocyte [
20]. Both the reduction in the AST activity and the elevation in the TP concentration in serum are associated with the better functioning of the liver [
17]. Thus, the present study suggested that GABA supplementation is beneficial to liver function.
The experimental results confirmed that supplemental GABA at 20, 30, and 40 mg/kg of diet increased the serum IgA and IgM concentrations, and GABA at 30 mg/kg of diet enhanced the IgG concentrations in serum. Several studies proved that GABA could improve the concentrations of IgA, IgM, and IgG [
21,
22]. IgM is mediated by the activation of the complement system as a defense against foreign substances. IgA drives mucosal responses against pathogens. IgG is involved in secondary reactions against toxins and viruses [
23]. GABA promotes T lymphocyte proliferation and reduces macrophage apoptosis by inhibiting somatostatin and adrenal corticosteroid hormone secretion [
24,
25,
26], thereby enhancing immunoglobulin production and improving the humoral immunity of the body.
The first line of defense against any pathogens in the intestine is the intestinal immune system [
27]. In this study, the minks in the GABA groups had greater levels of IL-4 and IL-10 in the mucosa. The minks in the 30 mg/kg GABA group had lower levels of mucosal IL-12 and IFN-γ. Additionally, the minks in the 20 and 30 mg/kg GABA groups had higher levels of SIgA. The findings suggest that 30 mg/kg GABA is more effective in modulating intestinal immunity. As suggested by Chen et al. [
28], dietary GABA promoted the gene expression of SIgA, IL-4, and IL-10 in weaning piglets. Intestinal mucosa can express inflammatory cytokines produced by the intestinal immune system, SIgA produced by plasma cells, and other immune factors [
29,
30]. SIgA is the effector component of the mucosal immune system [
31], and promotes SIgA secretion by promoting polymeric immunoglobulin receptor (pIgR) expression [
27,
32]. Tanga et al. [
33] observed that a decrease in TLR4 leads to a decrease in pro-inflammatory cytokine expressions such as IL-12 and IFN-γ. GABA may suppress the expression of the TLR4/NF-κB pathway and inhibit the activation of TLR4 by activating GABARs, thus affecting downstream inflammatory processes [
15]. Multiple studies proved that IL-10 signaling plays a positive role in the maintenance of appropriate epithelial permeability in the gut epithelium [
34,
35,
36]. In addition, GABA regulates T cell proliferation and promotes IL-10 production [
36,
37,
38]. It is probable that GABA modulates the expression of immune factors by triggering M1 and M2 immune responses and signaling pathways (IRFs, STATs, NF-κB, and SOCS) [
39].
In agreement with previous studies [
15,
40], dietary GABA at 20, 30, and 40 mg/kg of diet increased the activities of T-AOD and GSH-Px. T-AOC is a critical indicator of the functional status of the body’s antioxidant system [
41]. GSH-Px catalyzes the reduction in H2O2 and lipid peroxides [
42]. The exposure of the gut to foreign substances and microbial pathogens produces ROS, and the excessive production of ROS can affect intestinal health, resulting in various intestinal diseases [
43]. Dietary GABA supplementation can enhance antioxidant enzymes and the activities of GSH, thereby reducing ROS production and lipid peroxidation [
1]. GABA is involved in reducing the production of ROS by inhibiting the expression of the enzymes involved in NF-κB signaling, thereby blocking the occurrence of inflammatory reactions, and improving the antioxidant capacity of animals under stress [
15,
43].
The study demonstrated that GABA markedly modulated the compositions and diversity of intestinal flora. It suggests that GABA performs an essential function in preserving intestinal microbial homeostasis. The comparative analysis revealed a distinct relative abundance of the microbiota between the male and female subjects at both the phylum and genus taxonomic levels. However, Firmicutes was the most dominant in both the males and females. This finding is consistent with the study by Bahl et al. on mink colon microbiota [
44]. Most of the bacteria in the phylum Firmicutes are beneficial [
45]. At the genus level, the relative abundance of
Weissella and
Streptococcus increased, while that of
Lactobacillus and
Escherichia-Shigella decreased, which differs from the results reported by Chen et al. [
27]. This discrepancy may be due to minks being different from other domestic animals. The genus
Weissella has probiotic properties and biotechnological potential [
46].
Clostridium sensustricto (
cluster I) is a type of bacteria capable of using glucose to produce short-chain fatty acids [
47].
Advenella is a potential degrader of organic pollutants [
48]. GABA effectively lowers gut pH by increasing the concentrations of short-chain fatty acids, particularly propionic and butyric acids, and consequently suppresses the proliferation of pathogenic bacteria [
49,
50].
Previous studies have noted that GABA could potentially influence host metabolism through the modulation of the gut microbiota [
51,
52]. The supplementation of GABA has been shown to modulate the gut microbiota in ETEC-infected piglets by promoting the richness and diversity of the microbial community [
27]. Our study investigated the interaction between the intestinal microbiota genera and gut immune indicators. We discovered a positive association between the genus
Psychrobacter and SIgA and IL-12.
Psychrobacter has potential antibiofilm properties [
53]. Previous research has indicated that
Psychrobacter possesses the ability to modulate immune system function, bolster antioxidant capacity, and augment disease resistance, thereby exerting a prebiotic effect on the health of exopalaemon carinicauda [
54]. We also discovered that
Lactobacillus was positively associated with IgM and T-AOC, but negatively associated with IFN-γ, SIgA, and GSH-Px. These findings suggest that
Lactobacillus may not contribute positively to mink health. These results indicate that GABA can be used as a regulator of intestinal microbiota to mediate metabolism and the immune system.