Effects Of β-glucanase And Xylanase Compounds On Growth Axis Hormones And Approach To The Mechanism In Weaned Piglets

The study was conducted to investigate the effects of supplementing a barley-based diet with exogenousβ-glucanase and xylanase compounds (GXC) on growth axis hormones of weaned piglets by determining gastrin (GAS) gene expression, growth hormone (GH) and insulin-like grwoth factorâ… (IGF-â… ) gene expression, GH pulsatile secretion, serum IGF-â… levels and other growth-releated hormones as well as serum biochemical indexes, endogenous enzymes activity and microbiota in large intestine. The objective of this study was to approach to the mechanism that exogenous GXC affecting the growth of piglets and supply scientific foundations for utilization of exogenous GXC in swine production.The special primers for GAS, GH, IGF-â… and P-actin were designed respectively according to the gene sequences reported previously in Genebank. Then total RNA was extracted from antral tissue, pituitary gland and liver of pigs and the target gene mRNA amplified through RT-PCR with special primers. The cDNA fragments of GAS, GH, IGF-â… and P-actin were obtained. The PCR product was cloned into pGEM-T easy Vector and sequenced. Sequence analysis suggested that the gene fragment gained in this study shared 100%,99.60%,100% and 100% homology with the reported cDNA sequence (NM₀01004036, M22761, M31175 and U07786) of pig in Genebank, respectively. Based on the gene cloning, the suitable annealing temperature, Mg2+ concentration, and cycles in PCR system were discussed and the optimal semi-quantitative RT-PCR methods were constructed to determine the effects of GXC supplementation on mRNA expression of GAS, GH and IGF-â… in weaned piglets fed a barley-based diet. The results showed that the suitable annealing tempreture for GAS,GH,IGF-â… andβ-actin was 62℃,62℃,61℃and 60℃, respectively. Mg2+ concentration was 1.5mmol/L for all genes and the cycles 29.A total of 36 crossbred (Duroc×Landrace×Yorkshire, weaned at age of 28 d) weaned piglets with body weight 13.10±1.38 kg were randomly assigned to two groups with three pens based on sex and mass. Each group was fed on the diet based on barley (the content of barley was 65% in basal diet) with or without added GXC (1.5 g/kg). The enzyme activities ofβ-glucanase and xylanase were 10 000 U/g and 80 000 U/g, respectively. The feeding experiment lasted 28 days after a 7-day adaptation period. Feed and water were offered ad libitum throughout the experiment. At the end of the feeding trial,12 pigs (six pigs from each dietary treatment with sex balance) were randomly selected to collect blood samples for the determination of GH pulsatile secretion. After 1 h of feed removal, blood samples were obtained through jugular catheters every 20 min for 3 h (from 10:00 to 13:00 h). Pigs were then slaughtered, samples of serum, liver, pitutary gland, antrum, pancreas, duodenum, jejunum, ileum and cecum were collected for subsequent analysis. Meanwhile, in vitro trial primary cultured porcine pars distalis cells from pituitary gland and hepatocytes were used to investigate the effects of gastrin with different concentration grades on GH and IGF-I release. The main results were as follows:1. Results of feeding trials indicated that supplementation with GXC (1.5 g/kg) enhanced average daily gain (ADG) by 13.99%(P<0.05), decreased feed gain ratio (FGR) by 6.32%(P<0.05), but had no efftect on average daily feed intake (ADFI) (P>0.05), although an increasing tendency was observed. The results also showed that enzymes addition markedly decreased the diarrhoea frenquency of piglets by 41.80% (P<0.05).2. Analysis of serum biochemical indexes and some selected gowth-related hormone levels showed that additin of GXC decreased the activity of glutamic-pyruvic transaminase (GPT) and serum urea nitrogen (SUN) by 26.46% (P<0.05) and 21.27%(P<0.01) respectively, whereas increased the content of total protein (TP) and glucose (Glu) by 22.97%(P<0.01) and 8.22%(P<0.05) respectively, but unaffected the activity of glutamic-oxalacetic transaminase (GOT) and the contents of cholesterol (CHL) and triglycerides (TG) (P>0.05). Enzymes supplementation markedly enhanced the serum levels of free triiodothyronine (FT3) and insulin by 43.42%(P<0.05) and 32.07%(P<0.05). respectively. The contents of thyroid-stimulating hormone (TSH) and free thyroxine (FT4) exhibited an increasing trend, but no significant difference was observed(P>0.05).3. Results from analysis of antral GAS mRNA abundance and serum GAS levels revealed that exogeous enzymes supplementation greatly upregulated the gene expression of antral GAS and serum GAS levels by 64.04%(P<0.01) and 48.41% (P<0.01).4. Analysis of GH pulsatile secretion, serum IGF-I levels and gene expression of pituitary gland GH and hepatic IGF-I indicated that supplementation with enzymes significantly enhanced basal GH level, mean GH level and pulse amplitude by 27.27%(P<0.05),34.62%(P<0.05) and 34.69%(P<0.05), but unaffected pulse frequency and pulse duration (P>0.05). GXC addition markedly upregulated GH mRNA abundance in pituitary gland and IGF-I mRNA abundance in liver as well as serum IGF-I levels by 36.90%(P<0.05),33.43%(P<0.01) and 43.91%(P<0.01).5. Results from in vitro cell culture indicated that supplementation of pentagastrin (pGAS, an in vitro synthetized analogue of GAS) with the concentration of 10-10 mol/L,10-8 mol/L and 10-6 mol/L stimulated the proliferation of primary cultured porcine pars distalis cells(P<0.05) and hepatocytes (P<0.05) and induced significant enhancement of GH release (P<0.05) from pars distalis cells and and IGF-I compositing (P<0.05) in hepatocytes after 12 h,24 h,36 h and 48 h treatment of pGAS, respectively, and enhanced the number of these two kind of cells in S phase(P<0.05) and cell proliferation index (P<0.05) after 24 h treatment of different dosage of pGAS. In addition, the correlation between the proliferation rates of pars distalis cells and GH release, and that between the proliferation rates of hepatocytes and IGF-I compositing differ significantly (P<0.05).6. Results from analysis of endogenous digestive enzyme activity and digestive organs morphology indicated that enzymes supplementation had no effect on the activities of pancreatic digestive enzyme and pepsin (P>0.05), but an increasing trend of enzyme activity was observed. The results also showed that supplementation of enzyme compounds enhanced the activities of maltase, sucrase and y-glutamyl transpeptidase (y-GT) by 103.33%(P<0.05),145.35%(P<0.05) and 94.74%(P<0.05) in jejunal mucosa and 149.50%(P<0.05),136.00%(P<0.05) and 93.33%(P<0.05) in ileal mucosa, respectively. Supplementation with exogenous GXC improved the morphology and structure of liver, pancreas, antral pyloric glands and the mucosa villi in small intestine, inceased the intestinal villi height(P<0.01) and made them more orderly and decreased recess depth (P<0.05).7. Analysis of microbiota, the activities of bacterial enzymes and the total content of short-chain fatty acids (SCFA) in cecal contents of piglets indicated that supplementation with enzymes enhanced the number of Bifidobacteria (P<0.05) and Lactobacillus (P<0.05) in cecal contents, whereas decreased the number of Salmonella (P<0.05) and E. Coli (P<0.05). The activity of P-glucuronidase was decreased by 23.38%(P<0.05), while the activity ofβ-galactosidase was elevated by 19.31%(P<0.05). The results also showed that GXC supplementation enhanced the contents of lactic acid, butyric acid and total SCFA by 7.49%(P<0.05),27.72% (P<0.05) and 15.15%(P<0.05), respectively, decreased the pH values of the cecal contents (P<0.05).The results of the current study implicated:â‘ Supplementation of GXC could enhance growth and feed conversion efficienct in piglets.â‘¡Exogenous enzymes addition could upregulate gene expression of antral GAS, markedly increase serum IGF-I levels, improve the morphology and structure of pyloric glands and mucosa villi in the small intestine and significantly elevate the activities of disaccharidase.â‘¢GXC addition could upregulate gene expression of GH in pituitary gland and GAS in antrum and enhance GH pulsatile secretion and serum IGF-I levels significantly.â‘£Results of primary cultured porcine cells indicated that GAS could upregulate the proliferation of pars distalis cells and hepatocytes and enhance the release of GH from pars distalis cells and of IGF-I in hepatocytes.⑤Supplementation of GXC could change microflora in cecal contents, markedly increase the number of Bifidobacteria and Lactobacillus, significantly decrease the number of Salmonella and E. Coli, and significantly decrease the diarrhoea frequency of weaned piglets.