Effects of CU and YSE on growth performance of weaned piglets
CU is a kind of yeast with the characteristics of general yeast. Our results showed that CU had a positive effect on the growth performance of weaned piglets. At present, there are few studies on the application of CU in pigs. Oral active yeast preparation for early weaned piglets can help to relieve diarrhea, promote piglet growth, improve survival rate and growth performance . Active yeast preparation can promote the growth performance and enhance the immune function of piglets, and the mechanism of action may be related to the main components of cell wall, β-glucan, phosphorylated oligosaccharides and intracellular active peptides [11–12]. In this study, we fed CU together with its fermentation medium to piglets. Nucleotides, amino acids, peptides and other flavor substances in yeast culture were important factors to improve the feed intake of piglets. Shen et al. showed that yeast culture could significantly increase the feed intake of piglets. In addition, the researches on piglets and growing-finishing pigs showed that yeast culture could improve the daily gain and feed utilization rate of piglets [14–16]. It may be that yeast culture stimulated intestinal fermentation, increased the yield of volatile fatty acids and the end products of bacterial fermentation, and provided some energy for pigs to improve nutrient utilization[17–19].
The improvement of YSE on the growth performance of pigs is attributed to the fact that YSE saponins reduce the concentration of ammonia and provide a good environment for digestion and absorption of nutrients. It is also the result of antioxidant and anti-inflammatory effects of YSE polyphenols. The health of animals was maintained at a good level, and the energy loss caused by disease was reduced [20, 21]. In comparison, the results of Van, Colina and others were different. The results of these scholars showed that YSE reduced the concentration of ammonia in livestock barn or the content of ammonia nitrogen in feces, but had no significant effect on growth performance[22, 23]. This may be caused by different feed formula, environment, adding amount, physiological stage of pig and so on. The feed composition, especially the content of crude protein, may affect the effect of YSE. Ma et al added YSE to the diets of finishing pigs with crude protein content of 16% and 14% respectively, and the ammonia concentration in the barn was significantly different . Colina et al. showed that the optimum addition amount of YSE in weaned piglets was about 120 ppm, and the ammonia emission and growth performance were ideal. In this study, the addition amount of YSE was 200 ppm, and the actual active ingredient is 120 ppm. The addition amount of YSE in pig diets was different in different physiological stages. Generally speaking, the addition amount of YSE was 50–200 ppm. The diarrhea rate of piglets in the mixed group of CU and YSE was 12.9%, which was 27.9% lower than that of the control group, indicating that the mixed use of CU and YSE had a certain effect on reducing diarrhea in piglets.
Effects of CU and YSE on Organ Index of weaned piglets
With the growth of animals, the visceral organs of animals gradually increase in weight and volume, and their functions tend to be improved, so the organ index can reflect the functional status of animals to a certain extent . Our results showed that oral CU did not affect the organ development of weaned piglets, but had a tendency to promote the development of spleen and mesenteric lymph nodes, and the addition of YSE in diet did not affect the development of heart, liver, thymus and kidney. It can promote the spleen, pancreas and mesenteric lymph nodes. The specific mechanism of CU in promoting immune function is not clear, which may be related to the composition of cell wall. CU, as an antigenic substance, can promote the development of immune organs. Shen et al.  found that a certain proportion of yeast culture increased the concentration of intestinal IFN-γ in weaned piglets, and there was no significant difference between the two groups. YSE may have a dose effect on the development of immune organs. Su et al.  showed that YSE increased the immune organ index at low dose (100 mg / kg and 200 mg / kg), but decreased the immune organ index at high dose.
Effects of CU and YSE on pH in gastrointestinal tract of weaned piglets
Animal gastrointestinal tract is not only an important component of animal digestive system, but also a place for digestion and absorption of nutrients. Therefore, animal intestinal health is very important. The activity of digestive enzymes, the absorption of nutrients and the normal maintenance of intestinal immune function are affected by intestinal acidity. Stress in weaned piglets caused an increase in adrenal corticosterone secretion and a decrease in the content of mucin secreted by digestive tract, resulting in a decrease in the level of intestinal organic acids and a decrease in the number of beneficial lactobacilli, while the decrease in the number of Lactobacillus further increased pH, the increase of pH not only decreased the activity of intestinal digestive enzymes, but also conducive to the reproduction of Escherichia coli and other pathogens, resulting in diarrhea and other intestinal diseases[28–32]. It seriously harms the production performance of piglets [33, 34].
Our results showed that CU had little effect on gastrointestinal pH of piglets. YSE decreased the pH, of stomach, colon, rectum and urine and only decreased the pH value of urine. CU and YSE cooperated with CU extract to reduce the pH of stomach, ileum, rectum and urine. In addition, oral yeast preparation decreased the pH value of intestinal segment, especially the effect of hindgut segment was more obvious [35–37]. Probiotic yeast can inhibit the growth of Escherichia coli, promote the proliferation of Bifidobacterium and Lactobacillus, and cause the increase of intestinal VFA content, while microbial fermentation is mainly in the hindgut, which may be the main reason for the change of pH in hindgut caused by yeast. Tian et al.  pointed out that the YSE could reduce the pH value of rumen fluid of dairy cows. Although the mechanism of reducing ammonia concentration in YSE is controversial, a large number of studies have shown that YSE can inhibit the production of intestinal ammonia. Our results also show the effect of YSE in controlling ammonia gas from another aspect.
Effects of CU and YSE on Plasma biochemical Indexes of weaned piglets
Our results showed that the extract of CU could significantly improve the antioxidant capacity and the contents of plasma glucose, urea nitrogen and total cholesterol in weaned piglets. To a certain extent, plasma glucose and cholesterol reflect the glucose and fat metabolism of the body respectively [39, 40]. Oztasan et al pointed out that the YSE significantly decreased the level of serum glucose in diabetic rats, but CU did not affect the level of plasma glucose . Some studies have shown that the hypoglycemic function of YSE is related to its saponins [42–44]. However, in our study, YSE had no significant effect on plasma cholesterol content, which may be related to the intestinal regulation of cholesterol reabsorption and maintenance of cholesterol level. Blood urea concentration reflects the balance between protein metabolism and amino acids in animals. Our study showed that the addition of YSE to the diet decreased the content of plasma urea nitrogen, and there was a systematic tendency to reduce the content of plasma urea nitrogen between YSE and CU, which was consistent with the results of previous studies . AST and ALT are indexes to evaluate liver function. Reis found that 750 mg / kg significantly increased the content of ALT in animals, and pointed out that high doses of YSE may have side effects on animals . In this study, the addition amount of 120 mg / kg, of CU and the content of ALT of CU were not significantly different from those of the control group, which indicated that this addition was safe for 28-day-old weaned piglets. Even under normal physiological conditions, animal bodies produce free radicals. Excessive free radicals damage the structure and function of sugars, protein nucleic acids and other bioactive molecules and biofilms, resulting in functional and metabolic disorders. MDA is a metabolite of lipid peroxidation, indicating the state of biological stress . SOD exists in a variety of tissues and organs to protect cells from superoxide free radicals, while CAT can hydrolyze hydrogen peroxide into non-toxic water and oxygen . T-AOC is composed of key antioxidant enzymes and is a sign of the total antioxidant capacity of the body. In this study, dietary supplementation of YSE decreased plasma MDA concentration and increased plasma SOD concentration, which may be related to the scavenging effect of YSE on superoxide free radicals or preventing the formation of superoxides or peroxides . Molavian pointed out that YSE scavenges hydrogen peroxide by increasing the level of CAT, which is consistent with the results of this study . However, our study showed that CU increased T-SOD activity, CAT activity, TmurAOC and decreased MDA content, indicating that CU may have more potential in improving animal antioxidant capacity, which may be related to the ability of CU to synthesize glutathione [51, 52]. Among all the experimental groups, CU and YSE synergistically improved the antioxidant capacity of weaned piglets, and the mixed group improved the antioxidant capacity of weaned piglets most obviously.
Effects of CU and YSE on the morphology of ileal mucosa in weaned piglets
Villus height, crypt depth and villus height / crypt depth are important indexes to evaluate the absorption function of small intestine. Our results showed that oral CU and diet supplemented with YSE promoted the development of ileal mucosa, and they synergistically increased villus height / crypt depth. The height of villi was significantly correlated with the number of cells. when the villi were long, the mature cells increased, and the contact area and absorption area with nutrients were enlarged, so the ability to absorb nutrients was enhanced . The tubular structure formed by the sinking of the epithelium at the base of the villi to the lamina propria is the recess. Under the physiological state, the villous epithelial cells fall off normally. The exfoliated cells migrate, differentiate and produce mature villus cells from the base of the crypt to the villus end . The depth of crypt becomes shallower, the maturation rate of epithelial cells increases, and the absorptive capacity of villi increases. Kong et al. added YSE to the diet of piglets to reduce the concentration of ammonia nitrogen in the contents of duodenum, jejunum, ileum, cecum and colon, improve the intestinal villus structure of piglets, increase the villus width of jejunum, increase the villus length and width of jejunum and the ratio of villus length to crypt depth of jejunum, but had no significant effect on the villus morphology of duodenum and jejunum. Yeast culture can be used as a foreign antigen to stimulate the development of intestinal tract. Some studies have shown that the addition of active yeast or yeast and its culture to the diet is beneficial to the development of intestinal mucosa and promote intestinal health [56, 57]. In this study, it was found that YSE and CU increased the villus height of ileal mucosa, villus height / crypt depth, and decreased the crypt depth. In this study, the intestinal villi of piglets in the control group were shedding, while the situation in the experimental group was improved, indicating that YSE and CU can alleviate intestinal health problems caused by weaning stress. This is also one of the reasonable explanations for the reduction of diarrhea rate in the above study.
Effects of CU and YSE on the levels of β-definsin-2 and Occludin in intestinal mucosa of weaned piglets
Intestinal mucosa is the main barrier between the internal and external environment of the body, which can resist the invasion of harmful antigens through the role of mechanical, chemical, immune and microorganisms. Our study showed that oral CU and YSE increased the level of Occludin in jejunum and ileum and the level of β-definsin-2 in jejunum, and increased the level of Occludin in jejunum and ileum together. The main mode of connection between intestinal mucosal epithelial cells is tight junction to maintain the integrity of the mechanical structure and function of the intestinal mucosal barrier. Tight junctions are composed of peripheral cytoplasmic proteins such as occludin, claudins, junction adhesion molecules and closed small ring proteins, in which occludin is a transmembrane protein related to the integrity of tight junctions. The antimicrobial peptides secreted by intestinal mucosa play an important role in resisting the invasion of pathogenic microorganisms. Defensin is one of the most deeply studied and widely existed antimicrobial peptides. On the one hand, it can be lower than the pathogenic microorganisms on the surface of intestinal epithelium. On the other hand, it is involved in immune regulation . Studies have found that probiotics inhibit the translocation of pathogenic bacteria on the intestinal surface by maintaining the integrity of intestinal mucosa and reducing intestinal mucosal permeability, preventing toxins and harmful substances from entering the blood circulation, and pathogens can’t further invade the body. in order to prevent the occurrence of disease . Qamar et al. study found that oral administration of Saccharomyces cerevisiae induced specific immune response in mice infected with Clostridium, significantly increased the level of intestinal mucosal IgA, alleviated diarrhea and intestinal mucosal damage induced by endotoxin produced by Clostridium, indicating that probiotics such as yeast can enhance intestinal immune response and maintain intestinal health. In fact, there are few studies on the effects of CU and YSE on intestinal mucosal tight junction protein or β-defensin, but many studies have proved that they can enhance the immune function of animals. Our research confirms the above results to some extent. The possible mechanisms of probiotics protecting intestinal mucosal barrier are: maintaining the balance of intestinal flora, protecting microbial barrier, promoting mucus secretion, promoting the expression of tight junction protein, strengthening intestinal mucosal mechanical barrier, and stimulating intestinal mucosal immune response. Inhibition of intestinal epithelial cell apoptosis . In this study, YSE increased the levels of jejunal β-defensin and Occludin, which was beneficial to the health of weaned piglets. The effects of YSE and CU on the levels of intestinal β-defensin and Occludin can’t be ignored in addition to the direct immunostimulatory effect on intestinal mucosal structural integrity. In addition, YSE and CU can promote the villus height of ileal mucosa and maintain the normal morphology of intestinal structure, which is essential for the normal secretion of antimicrobial peptides and related proteins. However, CU and YSE showed some differences in the effect on intestine. They both significantly increased the expression of Occludin and β-definsin-2 in jejunal mucosa, but there was no significant difference in protein expression in ileum.
Effects of CU and YSE on cecal microflora of weaned piglets
The species richness of the sample can be reflected indirectly by drawing the dilution curve of the sample. when the curve tends to be smooth, it can be considered that the sequencing depth has basically covered all the species in the sample. Our research shows that the dilution curve height of the control group is lower than that of the other three experimental groups, suggesting that the species richness of the control group may be lower than that of the experimental group. Chao1 index and Shannon index curves tend to be saturated, indicating that the sequencing data are sufficient, most species have been detected, and more OTU can’t be found by further increasing the sequencing data.
The Venn diagram is used to reflect the common and unique species among the samples. Our research showed that the four groups accounted for 84.53% of the total OTU, while the unique OTU control group, CU group, YSE group and CU + YSE group were 0.87%, 0.44%, 0.32% and 0.61%, respectively, indicating that the experimental treatment had little effect on the core species in the intestinal tract of weaned piglets. The intestinal microflora of piglets has been changing since birth until a stable top community is formed . The proportion of OTU shared by the four groups reached 84.63%, which may be related to the stable top community gradually formed after weaning.
Our research showed that at the gate level, the cecal microorganisms of weaned piglets were mainly composed of Bacteroides and Firmicutes, which was consistent with the results of previous studies [62, 63]. YSE and CU had no significant effect on the relative abundance of Bacteroides and Firmicutes bacteria, indicating that the addition of them had no significant effect on the dominant flora of weaned piglets. Proteobacteria are gram-negative bacteria, and many of them are pathogenic bacteria . Our results showed that the YSE had no significant effect on the relative abundance of Proteus, but CU significantly reduced the relative abundance of Proteus, and the effect was better than that of CU alone. Acidobacteria are related to denitrification and nitrogen metabolism, and are greatly affected by nitrogen sources and pH . Our results showed that the YSE decreased the relative abundance of the bacteria, which may be related to the decrease of ammonia in the intestine. Bacteroides can ferment glucose, fructose, galactose, lactose, sucrose and dextrin to produce acid and gas, resulting in a waste of energy. Our study showed that the increase of the relative abundance of Bacteroides was mainly caused by the interaction between YSE and CU, indicating that the interaction between YSE and CU was not ideal in improving the relative abundance of Bacteroides. Lachnospiraceae and Ruminococcaceae can ferment cellulose to produce butyric acid to inhibit colitis, so we usually think that they are beneficial bacteria . Our study showed that oral CU and its culture increased the relative abundance of Lachnospiraceae and Ruminococcaceae in weaned piglets, which was beneficial to the maintenance of intestinal health. We believe that the effect of yeast and its culture on increasing beneficial bacteria and reducing harmful bacteria may be related to oligosaccharides and other active components in yeast culture. these active substances maintain gastrointestinal microecological balance, selectively promote the proliferation of beneficial flora and inhibit the reproduction of harmful bacteria.
In addition, our study showed that the abundance and diversity of cecal microflora in weaned piglets increased with the addition of YSE or CU, alone or in combination. The increase of microbial diversity is usually positively correlated with the stability of microflora and the ability to resist the invasion of pathogens. Our results showed that dietary supplementation of YSE and CU could improve the diversity of cecal microflora and improve the health level of weaned piglets. Beta diversity analysis can support this result. YSE and CU had a certain effect on the structure of intestinal microflora of weaned piglets, which changed the sample distance between the control group and the experimental group in Beta diversity analysis. With the addition of YSE or CU, the change of microflora developed in the same direction, which showed that the samples of the three experimental groups were gathered together. It is suggested that the effects of YSE and CU on intestinal microorganisms may be consistent.