0～14日龄乳鸽小肠主要消化酶发育规律的研究

以0～14日龄雄乳鸽为研究对象,测定3日龄、8日龄和14日龄乳鸽十二指肠和空肠中淀粉酶、总蛋白水解酶、胰蛋白酶、糜蛋白酶和脂肪酶活性随年龄的变化趋势。结果表明：十二指肠中淀粉酶、总蛋白水解酶、胰蛋白酶和糜蛋白酶活性随日龄增加而呈上升趋势,脂肪酶活性在3～8日龄显著降低,8～14日龄活性升高;空肠中淀粉酶、总蛋白水解酶和脂肪酶活性先降低后升高,而胰蛋白酶和糜蛋白酶活性先升高后降低,并在8日龄时达到峰值。     

大豆分离蛋白对幼建鲤肝胰脏发育及消化道蛋白酶活力的影响

研究了大豆分离蛋白（SPI）对10～35g幼建鲤Cyprinus carpio Var．Jian肝胰脏发育及消化道蛋白酶活力的影响。结果表明：饲料中用SPI替代鱼粉蛋白水平对幼建鲤肝胰指数、肝胰脏中的胰蛋白酶、凝乳蛋白酶活力以及肠道中胰蛋白酶、凝乳蛋白酶活力影响极显著（P＜0．01）或显著（P＜0．05）,随着SPI替代鱼粉蛋白比例的增加,蛋白酶活力均极显著下降（P＜0．01）,肝胰指数显著升高（P＜0．05）。用SPI替代鱼粉蛋白后,可引起幼建鲤消化能力下降,肝胰脏和肠道等消化器官的发育受阻,胰蛋白酶和凝乳蛋白酶的活力降低,饲料利用率下降;SPI中胰蛋白酶抑制因子是引起上述症状的重要因素之一。     

Digestive protease activities and free amino acids in white muscle as indicators for feed conversion efficiency and growth rate in Atlantic salmon (Salmo salar L.)

The aim of the present experiment was to screen several biochemical indices in fish and their interrelations in order to select variables for future studies of growth rate and feed conversion. Several parameters [trypsin activity, chymotrypsin activity, free amino acids (FAA) in plasma and white muscle, and RNA and RNA/protein ratio in the white muscle] were measured together with specific growth rate (SGR), feed intake and feed conversion efficiency (FCE) in four groups of diploid or triploid Atlantic salmon (Salmo salar L.) reared under different light regimes. SGR was measured on individually tagged fish, whereas feed intake and feed conversion was estimated on tank basis. A principal component analysis (PCA) explained 80.6% of the variance in the data, using all measured parameters, regardless of ploidy and light regime. Muscle free hydroxyproline showed the highest correlation, alone explaining 55% of SGR variability. The SGR also significantly correlated with trypsin activity (r=0.34), the activity ratio of trypsin to chymotrypsin (T/C) (r=0.39), plasma essential FAA (EAA) (r=0.39), plasma total FAA (TFAA) (r=0.37), the ratio of essential to non-essential FAA (EAA/NEAA) in the white muscle (r=–0.45), muscle RNA (r=–0.45) and RNA/protein ratio (r=–0.41). Tank FCE correlated positively (r=0.97) with SGR, T/C ratio and muscle free hydroxyproline, and negatively (r=–0.90) with muscle EAA/NEAA. The groups reared under continuous light (LL) regime showed significantly higher SGR than simulated natural photoperiod (SNP) groups, and with an apparently higher FCE. A higher growth rate was associated with either a higher consumption rate and/or a higher feed utilization. A negative correlation between muscle RNA concentration and SGR may indicate that increased growth rate under LL regime was not caused by an increased protein deposition rate.     

Dietary protein:lipid ratio changes growth,digestive enzyme activity,metabolic profile and haematological parameters in hybrid surubim (Pseudoplatystoma fasciatum × Leiarius marmoratus)

Studies investigating improved protein:lipid ratios based on their effects on growth, nutrient utilization, digestive enzyme activities, blood metabolites and erythogram in tropical carnivorous fish are very scarce. This study evaluated the effect of different protein:lipid ratios on these parameters in the hybrid surubim Pseudoplatystoma corruscans × Leiarius marmoratus. Juvenile fish (8.90 ± 0.94 g initial weight) were fed 3% of total biomass for 60 days using four isoenergetic experimental diets with different protein:lipid ratios (9.00, 4.60, 3.54 and 1.78). Higher growth performance parameters were observed at higher protein:lipid ratios. The activities of intestine total alkaline protease, trypsin and lipase (U/mg protein) were not affected by dietary treatments. Chymotrypsin (U/mg protein) was higher in the 4.60 protein:lipid ratio group. Amylase (U/mg protein) was higher in intermediate groups. Blood glucose, total plasma protein, triglycerides, cholesterol and free amino acids decreased as the protein:lipid ratio decreased. Haematocrit was higher at the 9.00 and 4.60 protein:lipid ratios. Hybrid surubim showed metabolic adaptation to the different protein:lipid ratios tested. These results suggest that the 4.60 protein:lipid ratio showed the best protein‐sparing effect of lipids.     

Ontogenic development of digestive enzymes in bigfin reef squid (Sepioteuthis lessoniana)

Bigfin reef squid (Sepioteuthis lessoniana) is an economically important exported aquatic animal in Thailand, but little is known about its husbandry, which needs to be clarified. In this study, the ontogenic development of the main digestive enzymes (pepsin, trypsin, chymotrypsin, lipase and amylase) was investigated within 30 days after hatching (DAH). Fertilized eggs of the wild cuttlefish were hatched and then distributed into four replicate aquaria assigned to varying collection times (0, 5, 10, 15, 20, 25 and 30 DAH). The development of pepsin suggests acidic digestion functionality at 5 DAH, and its specific activity was maintained over the studied period. Specific activities of the serine proteases, trypsin and chymotrypsin, fluctuated significantly with similar pattern, and appear to be the key enzymes contributing ontogenic changes in protein catabolism. The specific activity of lipase was relatively high at 0 DAH, dramatically increased by 5 DAH, and then appeared constant until the end of observations, suggesting important role in utilizing the lipid from yolk at hatching, as well as probably in sparing protein utilization. Dramatic increase in amylase specific activity occurred by 10 DAH after which the activity was constant until 30 DAH, suggesting the utilization of carbohydrate as energy evolves comparatively late. Findings from the current study could be used to manage nutritional protocols according to the digestive enzyme patterns, as well as to develop artificial diets for rearing S. lessoniana.     

Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K⁺ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K⁺ channel blocking activity was also compared.     

Do digestive enzymes set a physiological limit on growth rate and food conversion efficiency in the Atlantic cod (Gadus morhua)?

The objective of this study was to determine the potential sites of maximal growth limitation in Atlantic cod (Gadus morhua). Forty cod were reared in ten sea water tanks. Fish were randomly divided into 5 groups, a control group (injected with saline solution) and 4 experimental groups that received different levels of recombinant bovine somatotropin (rbST: 1.0, 2.0, 4.0 and 10.0 g g fish^–1 2 weeks^–1) by injection in the abdominal cavity. Fish were fed ad libitum 3 times a week during four weeks. We measured individual body mass, growth rate, food intake and food conversion efficiency. At the end of the experiment, we measured the activity of acid proteases in the stomach; trypsin and chymotrypsin in the pyloric caeca; alkaline phosphatase (ALP) and glutamyltransferase (GGT) in the intestine. No significant differences were observed in growth rate, food intake or food conversion efficiency among the five groups. Trypsin, chymotrypsin, ALP and GGT activities when expressed in U g fish^–1 were correlated with growth rate. These enzymes were also correlated with food ingestion except for trypsin when expressed in U mg protein^–1. Trypsin was the only enzyme that showed a significant correlation with food conversion efficiency. Our conclusion is that, at the level of digestion, trypsin is the only enzyme measured that could be suspected to potentially limit growth rate in cod.     

The effect of feeding on the secretion of pepsin,trypsin and chymotrypsin in the Atlantic salmon,Salmo salar L

Pepsin stored in the stomach mucosa of the Atlantic salmon (Salmo salar L.) increases within two days of the onset of starvation. Trypsin and chymotrypsin in the pyloric caeca/pancreas behave similarly, indicating that when no food is present in the gut, digestive enzymes accumulate in the secretory tissues. As a corollary, trypsin and chymotrypsin activities in the gut contents fell during starvation, indicating that secretion is greatly reduced when food is not present. At the onset of feeding, pepsin is rapidly synthesised in the mucosal tissues and then secreted. Twenty four hours after feeding, the pepsin levels of the mucosa are still low, suggesting that synthesis may be a rapid response to the presence of food in the stomach. Secretion of trypsin and chymotrypsin appears to take place as soon as digesta enters the intestine, between 4 and 14h after feeding, and resynthesis of enzyme precursors appears to be complete again within a further 11h. It is suggested that both synthesis and release may be under the control of cholecystokinin.     

Effects of dietary supplementation with feeding stimulants on yellowtail Seriola quinqueradiata (Temminck & Schlegel; Carangidae) protein digestion at low water temperatures

Feeding trials were carried out to examine substances that could be used as supplements in low‐protein/high‐lipid diets to improve protein digestion of yellowtails (Seriola quinqueradiata) during winter. Dietary supplementation with synthetic or natural (krill and squid extracts) feeding stimulants (FS) improved feed intake and growth performance of yellowtails. Moreover, the apparent protein digestibility (APD) and pepsin, trypsin and chymotrypsin secretions in fish fed diets supplemented with the different FS were superior to those in fish fed the control diet. These findings suggest that the dietary inclusion of either synthetic or natural FS is a potential tool for improving protein digestion at low water temperatures, and may provide a promising solution for the winter growth retardation experienced in yellowtail culture.     

Interaction of proteinase inhibitors with Cry1Ac toxicity and the presence of 15 chymotrypsin cDNAs in the midgut of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae)

BACKGROUND: The potential development of resistance to Bacillus thuringiensis (Bt) cotton and surging of non‐targeted insects is a major risk in the durability of Bt plant technology. Midgut proteinases are involved in Bt activation and degradation. Proteinase inhibitors may be used to control a wide range of insects and delay Bt resistance development. Proactive action to examine proteinase inhibitors for synergistic interaction with Bt toxin and cloning of proteinase cDNAs for RNAi is necessary to make transgenic cotton more versatile and durable. RESULTS: A sublethal dose (15 ppb) of Cry1Ac, 0.5% benzamidine and 0.02% phenylmethylsulfonyl fluoride significantly suppressed midgut azocaseinase, tryptic and chymotryptic activities, and resulted in reductions in larval and pupal length and mass of Heliothis virescens. The combination of proteinase inhibitor and Bt suppressed 20–37% more larval body mass and 26–80% more enzymatic activities than the inhibitor only or Bt only. To facilitate knockdown‐resistance‐related proteinase genes, 15 midgut chymotrypsin cDNAs were sequenced. Most predicted chymotrypsins contained the conserved N‐termini IVGG, three catalytic center residues (His, Asp and Ser), substrate specificity determinant (Ser or Gly) and cysteines for disulfide bridges. These putative chymotrypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. CONCLUSION: H. virescens has evolved diverse midgut proteinase genes. Proteinase inhibitors have potential insecticidal activity, and the interaction of Bt with proteinase inhibitors is desirable for enhancing Bt toxicity and delaying resistance development. Intensive sequencing of chymotrypsin cDNAs will facilitate future functional examinations of individual roles in Bt toxicity and resistance development and facilitate targeted control using RNAi and/or proteinase inhibitors. Copyright © 2012 Society of Chemical Industry