4.2. Effects of Concentrate Supplementation on Pancreatic Development of Yak Calves
Supplemental feeding of concentrate can have a significant effect on the pancreatic weight and pancreatic organ index of yak calves. Appropriate increases in concentrate types of feeds along with the increase of pancreatic secretion and release of insulin from the pancreatic islets of livestock can facilitate the growth and development of the pancreatic gland itself [
19]. Supplementing concentrate also reduced the distribution of intercalated ducts and ducts in the pancreas of yak calves, increased the distribution density of pancreatic acinar and islets and increased the area and proportion of endocrine and exocrine parts of the pancreas. Svensson et al. compared the capillary area in the endocrine and exocrine parenchyma of the pancreas and found that the capillary volume in the islets was about 3.5% [
20], while the volume of capillaries in the exocrine pancreas was significantly lower at 2%. The reason for the discrepancy between these parameters may be the lack of lymphatic capillaries and the relatively small capillary lumen in the pancreatic islets. In particular, there was a significant increase in both the total percentage of area of the exocrine part of the pancreas and the total percentage of area of the endocrine part. The density of islet cells is higher in parts of the pancreas than in the alveolar region [
21], and the number of cells is higher, especially in islets rich in glucagon. The protein content of dairy cows that were given supplemental concentrates affected the exocrine function of the pancreas [
22]. The area ratio of the exocrine part of the pancreas was significantly lower because the supplemented concentrate increased the area of the exocrine part of the pancreas, thus making the area ratio of the endocrine part of the pancreas relatively low, which also reflected that the supplemented concentrate had the greatest effect on the exocrine part of the pancreas. This indicates that supplemental concentrate feeding has a positive effect on the internal alveoli and islets, endocrine and exocrine parts of the pancreas of suckling yak calves and also has a positive effect on the growth and development of the pancreas of yak calves.
This study found that supplementary concentrate can increase the activity of the main digestive enzymes and hormone content in the pancreas of yak calves, especially the activity of pancreatic amylase, pancreatic lipase, pancreatic protease and chymotrypsin and the content of glucagon, insulin and pancreatic polypeptide at significant levels. Supplemental feeding can significantly increase the activity of duodenal pancreatic amylase [
23], pancreatic lipase, pancreatic protease and chymotrypsin in grazing lambs to digest and absorb nutrients in the diet through a series of biochemical reactions and promote the growth, development and production quality of lambs. Since the alveoli of the pancreas secrete the major digestive enzymes such as pancreatic amylase, pancreatic lipase, pancreatic protease and chymotrypsin [
24], Islet A, B, D and PP cells secrete mainly the hormones glucagon, insulin, growth inhibitory hormone and pancreatic polypeptide [
25]. Side by side, it was shown that supplementation with concentrate increased the alveoli and islet cells of the pancreas of suckling yak calves and also relatively increased the density of distribution of their endocrine and exocrine divisions. This is in agreement with the above results and discussion and serves as a good illustration. Thus, it was concluded that supplementation with concentrate not only increased the activity of major digestive enzymes and hormone content of the pancreas of suckling yak calves, but also promoted the digestion and absorption of nutrients in the diet and improved the growth and development of the pancreas of yak calves.
4.3. Effects of Concentrate Supplementation on Pancreatic Metabolomics in Yak Calves
Supplemental feeding of concentrates increased the levels of differential metabolites associated with resistance to oxidative stress in the pancreas of suckling yak calves. D-proline could induce sedation and hypnosis through N-methyl-D-aspartate glutamate receptors and glycine receptors [
26] and could effectively inhibit stress behavior in response to isolation-induced stress. Mammals improve stress tolerance through non-cyclic utilization of purine metabolites, and increased hypoxanthine levels can improve reuse during cyclic metabolism [
27], thus also achieving a reduction in oxidative stress. Supplementation with concentrate increased the levels of the differential metabolites D-proline and hypoxanthine in the pancreas of yak calves in the trial, which effectively reduced the stress behavior of suckling yak calves and facilitated their normal and effective daily feeding and exercise. Acetylcysteine is an acetylated precursor of cysteine, which is involved in the synthesis of glutathione in the body and has antioxidant, anti-inflammatory, antibacterial and fat metabolism regulating effects. Acetylcysteine has positive effects on maintaining the intestinal health of livestock [
28], alleviating oxidative stress and improving their reproductive performance. Supplemental feeding of concentrate increased the acetylcysteine content in suckling yak calves, promoting their good development and healthy growth. Supplemental feeding of concentrates increased gamma-glutamylcysteine levels in the pancreas of suckling yak calves, as found in the trial. In contrast, gamma-glutamylcysteine is abundant in mammals and is involved in the synthesis of glutathione in the presence of its ligase [
29], preventing potential adverse reactions associated with reactive oxygen species and associated redox reactions that may induce oxidative stress and may be linked to innate detoxification processes, ensuring, to some extent, healthy organism growth. Thiazolidine-4-carboxylic acid was modified by bioactivity and inhibited apoptosis caused by oxidative stress by enhancing the activity of catalase and improving the ability to bind to proteins, thereby regulating bioactivity [
30]. The increased thiazolidine-4-carboxylic acid content in the pancreas effectively alleviated the stressful behavior of yak calves, creating favorable conditions for their healthy growth and development. Increased serum levels of piperidine acid enhanced the ability of mammalian cells to resist oxidative stress and also hindered DNA damage and cell growth arrest induced by oxidative stress [
31]. Piperidinic acid, a non-protein amino acid derived from the catabolic metabolism of lysine [
32], is an important modulator of body immunity and piperidinic acid levels increase upon infection with pathogens and by increasing free radical levels with the acquisition of systemic resistance. Supplemental feeding of concentrate increased piperidinic acid levels in the pancreas of yak calves, alleviated cell growth stagnation due to oxidative stress, improved early body immunity and promoted healthy growth and development later in life.
Supplemental feeding of concentrates increased the levels of differential metabolites in the pancreas of suckling yak calves associated with the promotion of their growth and development. Supplemental ellagic acid feeding to suckling foals increased plasma protein concentration and body weight size [
33], improved nutrient metabolism, further regulated their organism health and promoted good growth and development of suckling foals. In this experiment, supplemental feeding of concentrate increased the content of the differential metabolite ellagic acid in the pancreas of yak calves, which was beneficial in promoting the productive performance and growth of suckling yak calves. Nicotinamide promotes pancreatic progenitor cell differentiation through its off-target effect as a kinase inhibitor [
34], while the development of pancreatic progenitor cells and endocrine cells can be induced. By supplementing concentrate to yak calves, the content of nicotinamide in the pancreas increased, which actively regulated the differentiation of pancreatic cells and promoted the good development of the pancreas. Tropolone visualizes the location of genes and cells in vivo based on a dual-isotope SPECT study [
35], and glucagon and insulin positive cells can be observed by immunohistochemistry. Supplemental feeding of concentrate in this experiment increased the level of tropolone in the pancreas of suckling yak calves, corresponding to the previous increase in glucagon and insulin, which is consistent with and validates the previous study. The administration of D-serine to rats improved the positive and negative symptoms and their resulting cognitive dysfunction after schizophrenia [
36]. The increased level of D-serine in the pancreas of yak calves after supplementation with concentrate in the experiment improved the learning and cognitive function of suckling yak calves. Ribulose-5-phosphate is a very important anabolic intermediate in the pentose phosphate pathway [
37], inducing redox metabolism, synthetic glucose metabolism and fatty acid synthesis, and playing a regulatory role in cellular energy metabolism. Supplemental feeding of concentrate in the experiment increased the content of ribulose-5-phosphate in the pancreas of suckling yak calves, indicating that supplemental feeding of concentrate can improve the regulation of glucose metabolism and lipid metabolism in early yak calves and create favorable conditions for their healthy growth. (+/-)5(6)-EET has been shown to have biological effects such as vasodilatory, anti-inflammatory and sodium-urea effects, as well as to stimulate insulin and glucagon secretion from an isolated pancreas [
38], increase insulin sensitivity and lower blood pressure in rats, and increasing (+/-)5(6)-EET levels improved insulin sensitivity and reduced diabetes-related cardiovascular and renal damage. In this experiment, supplementation with concentrate increased (+/-)5(6)-EET levels in the pancreas of suckling yak calves, verified increased insulin and glucagon levels in the pancreas and positively regulated insulin sensitivity and glucose levels during the growth and development of early yak calves. It was found that 2-hydroxycinnamic acid inhibited oleic acid-induced lipid accumulation in HepG2 cells [
39], decreased TC and TG content in cells, enhanced glucose utilization in cells and decreased sterol regulatory elements. The increased levels of 2-hydroxycinnamic acid in the pancreas suggest that supplementation with concentrate can modulate the effects of cellular glucolipid metabolism in suckling yak calves, improve lipid accumulation in cells and increase glucose utilization. Increasing the content of L-phenylalanine promoted the secretion of pancreatic amylase and improved the exocrine capacity of pancreatic alveolar cells by increasing the expression of pancreatic amylase mRNA and forming a protein translation initiation complex [
12]. Supplemental feeding of concentrate in the experiment increased the L-phenylalanine content in the pancreas of suckling yak calves, indicating that supplemental feeding of concentrate can promote the secretion of digestive enzymes from early yak calves’ glandular alveolar cells and the growth and development of the pancreas, as well as improving the digestion and absorption of feed in the small intestine, and increasing its utilization efficiency.
Supplemental feeding of concentrates increased the content of differential metabolites associated with anti-inflammatory preventive diseases in the pancreas of suckling yak calves. The prevalence of diabetes, acromegaly, and pancreatic inflammation were predicted using insulin activity and the ratio of pancreatic lipase concentration to creatinine [
40]. The activity of pancreatic lipase and the content of the differential metabolite creatinine in the pancreas are crucial for the health of the yak calf organism, and, to some extent, they reflect the good growth and development status. Glucocorticoids containing tetrahydrocorticosterone can effectively regulate body homeostasis and glucose homeostasis [
41], where excess glucocorticoids can trigger insulin resistance and type 2 diabetes mellitus. In the present experiment, supplementation with concentrate significantly increased the level of tetrahydrocorticosterone in the pancreas of yak calves, suggesting that it could be used as a potential marker for pancreatic-related metabolic diseases. Pyridoxamine could reduce the accumulation of glycosylation end products in the brain microvascular wall and improve the ischemic and hypoxic state of the type 2 diabetic brain [
42], resulting in a significant reduction in the latency of diabetes. The significant increase in pyridoxamine content in the pancreas of yak calves in this experiment suggests that supplementation with concentrates can prevent potential type 2 diabetes and improve different degrees of behavioral and cognitive dysfunction to some extent. Pancreatic β-cells play an important role in glucose-dependent insulin secretion, and hyper-glycemic glucotoxicity causes pancreatic β-cell dysfunction and decreases insulin secretion [
43]. KUP-1, a xanthine derivative, inhibited the induction of hyperglycemic glucotoxicity, thereby stabilizing β-cell function and increasing insulin secretion. Supplementation with concentrate increased the xanthine content in the pancreas of yak calves, indicating that supplementation with concentrate can effectively control the metabolic syndrome-related diseases induced by hyperglycemia. It was determined that 5-oxoproline could be identified as a candidate diagnostic marker to distinguish mucinous from non-mucinous pancreatic cysts [
44], and its establishment could allow early detection and accurate diagnosis of pancreatic cysts and provide guidance for clinical treatment policy. Compounds synthesized from asparagine conversion exhibited good biological activity in terms of anti-inflammatory, hypotensive and hemostatic properties [
45]. Supplemental feeding of concentrate increased the asparagine content in the pancreas of suckling yak calves, which improved the anti-inflammatory and hemostatic ability of early yak calves. Pro-inflammatory factors in wound inflammation induce the activity of the indoleamine 2,3-dioxygenase IDO1 [
46], and DL-tryptophan is an effective inhibitor of IDO1, thereby accelerating the rate of wound healing. The increase in pancreatic DL-tryptophan content in the experiment suggests that supplemental concentrate feeding improves the ability of suckling yak calves to suppress inflammatory responses and improve self-healing. Indole-3-acrylic acid, a tryptophan metabolite produced by the bacterial species Pepto-coccosis [
47], is a powerful activator of antioxidant mechanisms in cells, promotes intestinal barrier function and decreases inflammation. The increase in indole-3-acrylic acid content in the pancreas suggests that supplementation with concentrate improves the cellular antioxidant capacity of suckling yak calves, reduces the intestinal inflammatory response and promotes healthy growth. Thymine derivatives had strong antioxidant and antibacterial properties [
48], exhibited excellent hematological and biocompatibility and treated wounds with fewer inflammatory cells and faster epithelial tissue regeneration and neointima formation. The increase in pancreatic thymidine content in the trial suggests that supplemental concentrate feeding improved anti-inflammatory and tissue re-generation in suckling yak calves and facilitated early thriving of yak calves. Trehalose ameliorated the abnormal autophagy protein expression caused by the pancreas in diabetic mice by activating the AKT/GSK3β signaling pathway [
49] and increasing the level of autophagy, thus, having a protective effect on the diabetic pancreas. Trehalose reduced the abnormal expression of apoptotic and pyrogenic proteins in the pancreas of diabetic mice and improved the pancreatic damage caused by diabetes mellitus [
50]. The increase in pancreatic trehalose content in the experiment suggests that supplemental concentrate feeding can protect the pancreas of suckling yak calves from damage caused by diabetes and is beneficial to their good pancreatic development and the healthy growth of the organism. Docosahexaenoic acid is a key regulator of cardiomyocyte membranes [
51], responsible for maintaining cholesterol homeostasis and has a direct impact on eicosanoid metabolism. Docosapentaenoic acid and docosahexaenoic acid, as bioactive lipid mediators, have potent anti-inflammatory and immunomodulatory effects in vitro and in vivo. Supplemental feeding of concentrates increased docosapentaenoic acid and docosahexaenoic acid levels in the pancreas of yak calves, indicating that supplemental feeding of concentrates can improve the immunity of suckling yak calves and effectively promote their healthy growth. Branched-chain fatty acid esters of hydroxy fatty acids (FAHFAs) [
52], a product of endogenous synthesis in mammalian tissues, reduced the production of pro-inflammatory cytokines through adipose tissue macrophages and insulin resistance in obese mice. Supplementation of concentrate in the experiment increased the content of FAHFA (18:1/20:3) and FAHFA (18:2/20:4) in the pancreas of yak calves, indicating that supplementation of concentrate enhanced the anti-inflammatory and anti-diabetic properties of early yak calves. In vitro, adrenic acid increases macrophage phagocytosis and cytotoxicity [
53], clears neutrophils from peritonitis in mice and has the potential to prevent and resist inflammation. Supplemental feeding of concentrate in the experiment increased the level of adrenic acid in the pancreas of suckling yak calves, suggesting that supplemental feeding of concentrate can improve the anti-inflammatory and antibacterial capacity of early yak calves. Xanthosine could inhibit inflammatory signaling pathways [
54] antimicrobial gene upregulation and cell adhesion molecules and induce downregulation of inflammatory signals and up-regulation of antimicrobial genes. Supplemental feeding of concentrate increased the content of xanthosine in the pancreas in the experiment, suggesting that supplemental feeding of concentrate can improve the anti-inflammatory and antibacterial ability of suckling yak calves and have a positive effect on their growth and development and production performance.