1. Introduction
Transport stress is an important factor in the production of stress in animals, and it inhibits the immune system, causes changes in body metabolism [
1], and even causes serious damage to animal tissues and organs [
2]. It also leads to weight loss and the suppression of the immune system in animals, as well as disrupting the stability of the gastrointestinal ecosystem of animals, causing changes in the bacterial community, releasing endotoxins, and weakening disease resistance, thereby increasing the risk of gastrointestinal infections [
3]. It also affects animal welfare. Wei [
4] found that transport stress could induce intestinal oxidative stress in pigs, destroy the antioxidant defense system, damage the intestinal barrier function, increase the risk of bacterial translocation, and lead to the incidence rate increasing. Therefore, the question of how to take effective measures to prevent and alleviate transport stress is crucial to improving animal immune function, reducing the incidence rate and improving animal welfare. So, the prevention and treatment of transport stress has become the focus of research. Currently, the drugs used to prevent and treat transport stress mainly include vitamins and antibiotics. Other researchers [
5,
6,
7] found that adding a certain amount of vitamin C and electrolyte to the diet could alleviate anorexia, indigestion and diarrhea, and decrease fat and immunity and intestinal inflammatory reactions caused by stress in animals. It could effectively alleviate stress. In clinical practice, neomycin has good preventive and therapeutic effects on various animal diseases caused by bacteria. Transport stress often leads to a decrease in the immunity of animals, which can lead to a series of diseases after infection by pathogenic microorganisms. Therefore, the prevention and treatment of transport stress is extremely important. At present, the effect and mechanism of electrolytic multivitamin and neomycin in the immunity and intestinal barrier function of transported lambs are unclear.
Therefore, this experiment aimed to investigate the effects of different treatments on the stress hormone levels in sera, immune function and intestinal health of lambs by adding an electrolytic multivitamin before and after transportation, and adding neomycin after transportation. This experiment can provide a reference for the selection and application of anti-stress additives. It is meaningful for the rational utilization of animal resources and the improvement of animal welfare.
4. Discussion
Animals are affected by various stressors during transportation: their catabolism increases, nutrient consumption accelerates, and immunity decreases. Gou et al. [
14] found a positive correlation between transportation time and weight loss. Furthermore, Hongjian et al. [
15] found that vitamin D could significantly improve animal production and alleviate stress symptoms. In this experiment, the weight of lambs in the EG and NG was significantly higher than that in the CG. On day 7, the weight in the EG increased compared to the CG. This indicated that electrolytic multivitamin and neomycin could effectively increase the weight of lambs and alleviate transport stress, and the electrolytic multivitamin had a better effect than neomycin. The possible reason was that the supplementation of vitamins could improve the immunity of lambs [
16]. In addition, with the extension of time, the weight of lambs gradually increased within 0–14 days, and transport stress was gradually alleviated.
The relative weight of immune organs represents the development status of immune organs [
17], which affects the immune function of the body. It was found that the liver weight of calves decreased significantly with 11 h transportation, which was due to an increase in vitamin consumption by stress [
18]. The electrolytic multivitamin had a better effect on increasing immune organ indices than neomycin, and the possible reason was that the energy consumption increased after lambs were transported, which increased demand for vitamins in lambs; the electrolytic multivitamin could supplement vitamins to the body, so the immune organ indices increased [
19,
20].
Under stress, the function of the hypothalamic pituitary adrenal axis (HPA axis) in animals enhances, leading to an increase in the synthesis and secretion of related hormones such as ACTH, COR, and E [
21]. Zeng et al. [
22] found that COR levels in serum reduced significantly and the anti-stress of cows was improved by adding vitamins to the diet. In this experiment, the concentrations of ACTH, COR, and E in the EG were the lowest, and the concentration of E in the NG was significantly lower than that in the CG. This indicated that the electrolytic multivitamin and neomycin could alleviate transport stress effectively, and the effect of the electrolytic multivitamin was better than neomycin. With the extension of time, the concentrations of stress hormones such as ACTH, E, and COR in the sera of lambs decreased gradually, and transport stress alleviated gradually.
Inflammatory factors are important mediators in the neuroendocrine and immune networks. They participate in and regulate the physiological functions of the body, and play an important role in regulating immune and inflammatory responses [
23]. It was found that various stress factors could induce the secretion of inflammatory factors such as IL-1β, IL-6, and TNF-α, which could lead to inflammatory reactions in the body [
24]. Shojadoost et al. [
19] found that adding an appropriate amount of vitamins to the diet could reduce the expressions of intestinal inflammatory factors, and it helped to keep a balance of intestinal inflammatory factors and improve the immune function of body. In this experiment, after adding the electrolytic multivitamin, the concentrations of IL-1β, IL-6, and IFN-γ in EG decreased significantly, which indicated that the electrolytic multivitamin could reduce the inflammatory reaction, which was helpful to alleviate transport stress. In addition, with the extension of time, the concentrations of IL-2, IL-6, IL-12, and IFN-γ in sera decreased gradually, and the concentration of IL-4 increased gradually. This indicated that there was still a serious inflammatory reaction in the lamb within 7 days after transportation, and the inflammatory reaction weakened and the stress alleviated gradually on day 14.
Immunoglobulins such as IgA, IgM, and IgG are the most common indicators for evaluating humoral immune function. Amaral et al. [
25] found that stress could lead to the decrease of immunoglobulin in serum and the increase of the incidence rate in animals. Liu et al. [
26] found that adding vitamin E to the diet could increase the content of immunoglobulin in the sera of chicken. In this experiment, the concentrations of IgA in the EG and NG were significantly higher than that in the CG. IgA plays an important role in intestinal mucosal immune response [
27]. This indicated that the electrolytic multivitamin and neomycin could block the penetration of pathogenic microorganisms into the intestinal mucosa and exert immune regulatory effects. The concentration of IgA was the highest in EG, which indicated that the electrolytic multivitamin had a better effect than neomycin. It was also found that the concentration of IgG in the EG was significantly higher than those in NG and CG. This indicated that the electrolytic multivitamin could enhance the immunity of lambs effectively. The electrolytic multivitamin and neomycin had no effect on the concentration of IgM in sera; the specific reasons need further research. Stanger et al. [
28] found that the immune cells returned to normal levels after the adult cattle landed for 6 days after they had been transported for 72 h. In this experiment, the concentrations of IgA, IgG, and IgM increased within 0–14 days, the transportation stress of lambs was gradually relieved, and the immune function of lambs enhanced gradually.
The VFA in intestinal contents is the degraded product of carbohydrates by intestinal microorganisms [
29], which can regulate intestinal pH, inhibit the proliferation of harmful bacteria [
30], protect the intestinal mucosal barrier [
31], and provide energy for the body and intestinal cells [
32]. In this experiment, the content of colonic propionate in the EG and NG increased significantly, and the possible reasons were related to the types of microorganisms in the intestine [
33], the addition levels of the electrolytic multivitamin and neomycin, and the pH of the intestine. The addition of the electrolytic multivitamin and neomycin had no effect on the contents of acetate, butyrate, isovalerate in the jejunum and colon, as well as the propionate in the jejunum. It is possible that the majority of VFA was absorbed in the rumen, so the change in the intestine was not significant. With the extension of time, the contents of acetate, propionate, butyrate, and isovalerate in the jejunum and colon increased gradually. The digestive function of lambs enhanced gradually, and the stress response alleviated gradually.
The intestine is an important site for digestion, absorption, and nutrient metabolism. It can easily become a target organ for transport stress [
34], which results in a series of digestive system diseases. Yu et al. [
35] found that there was significant damage to the intestine during heat stress in pigs, including damage to the top of the intestinal villi, reductions in villi height, and a deepening of the crypt depth. Xiao et al. [
36] found that vitamins could promote intestinal development, maintain the integrity of intestinal barriers, and improve the digestion and absorption of nutrients. In this experiment, the crypt depth of the jejunum in the EG decreased, and the V/C and villus height increased. Moreover, the crypt depth of the jejunum in the NG decreased, and the V/C increased. This indicated that the electrolytic multivitamin and neomycin could promote jejunal development, and the electrolytic multivitamin had more effects than neomycin. In the colon, the villus height and V/C of EG and NG increased, and the crypt depth in NG decreased. This indicated that the electrolytic multivitamin and neomycin could maintain the colonic morphological integrity of the lambs. The electrolytic multivitamin could improve the morphology in the jejunum and colon of lambs, and it improved the nutrient utilization efficiency and growth and development of lambs. This was consistent with the result showing that the electrolytic multivitamin could promote the weight of lambs.
Tight junction proteins play an important role in maintaining intestinal homeostasis [
37], and the decrease of their expression levels results in an increase in diarrhea rate [
38]. The mucous layer is the first line of defense against foreign pathogens, and the decrease in the content of mucin results in intestinal barrier dysfunction [
39]. Carol et al. [
40] found that vitamin D could increase the expressions of tight junction proteins such as Claudin, Occludin, and ZO-1 in intestinal mucosal epithelial cells, which protected the intestinal mucosal barrier. In this experiment, the mRNA expressions of Occludin and MUC1 of the jejunal mucosa in EG, as well as the protein expression of Occludin in EG, increased. This indicated that the electrolytic multivitamin could increase the mRNA expressions of Occludin and MUC1 and protein expressions of Occludin in the jejunal mucosa of lambs; it potentially enhanced tight junctions. In the colonic mucosa, the mRNA expressions of ZO-1 and Occludin in the EG, as well as the protein expression of Occludin in the EG, increased. This indicated that the electrolytic multivitamin had a potential effect on improving the barrier function of colonic mucosa. With the extension of time, the mRNA expressions of ZO-1, Occludin, MUC1, and MUC2, and the protein expressions of Occludin and MUC2 in jejunal and colonic mucosa, increased gradually, the tight junction recovered gradually, and the barrier function of the jejunal and colonic mucosa was enhanced. The electrolytic multivitamin had a better effect on protecting the intestinal digestive function, which is consistent with the change in the intestinal morphology of lambs. Therefore, it was speculated that the electrolytic multivitamin could maintain and improve physical and chemical barrier functions of the intestine by affecting the morphology of intestinal tissue, intestinal tight junction proteins, and mucins.
The TLRS/MyD88/NF-kB signaling pathway is an important pathway in the occurrence and development of inflammation, and it participates in biological processes such as immune regulation [
41]. Yang et al. [
24] found that the sustained activation of the TLR4/MyD88/NF-kB signaling pathway led to excessive immune inflammatory responses and ultimately damaged tissue. The expression levels of related signaling molecules in the TLR4/MyD88/NF-kB signaling pathway changed if an inflammatory response occurred. The electrolytic multivitamin downregulated the mRNA expressions of TRAF6, TLR4, MyD88, and NF-kB, and the protein expression of NF-kB p65 in the jejunum, and neomycin also downregulated the mRNA expression of TRAF6 and the protein expression of NF-kB p65. Meanwhile, the electrolytic multivitamin downregulated the mRNA expressions of TRAF6, TLR4, and NF-kB in the colon. This indicated that the electrolytic multivitamin could alleviate inflammation in the colon effectively. The mRNA expression levels of TRAF6, TLR4, MyD88, and NF-kB, and the protein expressions of TLR4, MyD88, and NF-kB p65 in the jejunum and colon, decreased along with increasing time, and the stress response of the jejunum and colon alleviated gradually. There was a consistent trend of change in the expression levels of TRAF6, TLR4, NF-kB, and MyD88 in the jejunum and colon, and the concentrations of IL-1β, IL-6, and IFN-γ in the sera of lambs. The electrolytic multivitamin was more beneficial than neomycin in reducing the inflammatory reaction. It is speculated that the electrolytic multivitamin may inhibit the expression levels of important genes in the TLR4/MyD88/NF-kB signaling pathway in the jejunum and colon of lambs, thereby inhibiting the production of inflammatory factors.