Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi-dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi-dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated 32 P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.

Weaning affects the glycosidase activity towards phenolic glycosides in the gut of piglets.

Phenolic compounds in pig diets, originating either from feed ingredients or additives, may occur as glycosides, that is conjugated to sugar moieties. Upon ingestion, their bioavailability and functionality depend on hydrolysis of the glycosidic bond by endogenous or microbial glycosidases. Hence, it is essential to map the glycosidase activities towards phenolic glycosides present along gut. Therefore, the activity of three key glycosidases, that is α-glucosidase (αGLU), ß-glucosidase (ßGLU) and ß-galactosidase (ßGAL), was quantified in small intestinal mucosa and digesta of piglets at different gastrointestinal sites (stomach, three parts of small intestine, caecum and colon) and at different ages around weaning (10 days before and 0, 2, 5, 14 and 28 days after weaning). Activity assays were performed with p-nitrophenyl glycosides at neutral pH. The αGLU activities in mucosa and digesta were low (overall means 1.4 and 60 U respectively) as compared to ßGLU (15.2 and 199 U) and ßGAL (23.4 and 298 U; p < .001). Moreover, αGLU activity in mucosa was unaffected by age. Conversely, ßGLU and ßGAL activities dropped significantly after weaning. Minimal levels, ranging between 18% and 54% of the pre-weaning values, were reached at 5 days post-weaning. Similarly, in small intestinal digesta, reductions from 60% up to 90% were observed for the three enzyme activities on day five post-weaning as compared to pre-weaning levels. In caecal contents, activities were lowest at 14 days post-weaning, while in stomach and colon no clear weaning-induced effects were observed. Our data suggest that weaning affects the glycosidase activity in mucosa (mainly endogenous origin) and digesta (primarily bacterial origin) with the most pronounced effects occurring 5 days post-weaning. Moreover, differences in activities exist between different glycosidases and between gut locations. These insights can facilitate the prediction of the fate of existing and newly synthetized glycosides after oral ingestion in piglets.

Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors.

This review focuses on phytase functionality in the digestive tract of farmed non-ruminant animals and the factors influencing in vivo phytase enzyme activity. In pigs, feed phytase is mainly active in the stomach and upper part of the small intestine, and added phytase activity is not recovered in the ileum. In poultry, feed phytase activities are mainly found in the upper part of the digestive tract, including the crop, proventriculus and gizzard. For fish with a stomach, phytase activities are mainly in the stomach. Many factors can influence the efficiency of feed phytase in the gastrointestinal tract, and they can be divided into three main groups: (i) phytase related; (ii) dietary related and (iii) animal related. Phytase-related factors include type of phytase (e.g. 3- or 6-phytase; bacterial or fungal phytase origin), the pH optimum and the resistance of phytase to endogenous protease. Dietary-related factors are mainly associated with dietary phytate content, feed ingredient composition and feed processing, and total P, Ca and Na content. Animal-related factors include species, gender and age of animals. To eliminate the antinutritional effects of phytate (IP6), it needs to be hydrolyzed as quickly as possible by phytase in the upper part of the digestive tract. A phytase that works over a wide range of pH values and is active in the stomach and upper intestine (along with several other characteristics and in addition to being refractory to endogenous enzymes) would be ideal.

Role of peptidases of the intestinal microflora and prey in temperature adaptations of the digestive system in planktivorous and benthivorous fish.

Many fish enzymatic systems possess limited adaptations to low temperature; however, little data are available to judge whether enzymes of fish prey and intestinal microbiota can mitigate this deficiency. In this study, the activity of serine peptidases (casein-lytic, mainly trypsin and hemoglobin-lytic, mainly chymotrypsin) of intestinal mucosa, chyme and intestinal microflora in four species of planktivorous (blue bream) and benthivorous (roach, crucian carp, perch) was investigated across a wide temperature range (0-70 °C) to identify adaptations to low temperature. At 0 °C, the relative activity of peptidases of intestinal mucosa (<13%) and usually intestinal microflora (5-12.6%) is considerably less than that of chyme peptidases (up to 40% of maximal activity). The level of peptidase relative activity in crucian carp intestinal microflora was 45% of maximal activity. The shape of t°-function curves of chyme peptidase also differs in fish from different biotopes. Fish from the littoral group are characterized by a higher degree of adaptation of chyme casein-lytic peptidases to functioning at low temperatures as compared to fish from the pelagic group. The role of intestinal microbiota and prey peptidases in digestive system adaptations of planktivorous and benthivorous fish to low temperatures is discussed.

Inclusion of glycerol in forage diets increases methane production in a rumen simulation technique system.

We hypothesised that the inclusion of glycerol in the forage diets of ruminants would increase the proportion of propionate produced and thereby decrease in vitro CH4 production. This hypothesis was examined in the present study using a semi-continuous fermentation system (rumen simulation technique) fed a brome hay (8·5 g) and maize silage (1·5 g) diet with increasing concentrations (0, 50, 100 and 150 g/kg DM) of glycerol substituted for maize silage. Glycerol linearly increased total volatile fatty acids production (P<0·001). Acetate production was quadratically affected (P=0·023) and propionate and butyrate production was linearly increased (P<0·001). Glycerol linearly increased (P=0·011) DM disappearance from hay and silage. Crude protein disappearance from hay was not affected (P=0·789), but that from silage was linearly increased (P<0·001) with increasing glycerol concentrations. Neutral-detergent fibre (P=0·040) and acid-detergent fibre (P=0·031) disappearance from hay and silage was linearly increased by glycerol. Total gas production tended to increase linearly (P=0·061) and CH4 concentration in gas was linearly increased (P<0·001) by glycerol, resulting in a linear increase (P<0·001) in mg CH4/g DM digested. Our hypothesis was rejected as increasing concentrations of glycerol in a forage diet linearly increased CH4 production in semi-continuous fermenters, despite the increases in the concentrations of propionate. In conclusion, this apparent discrepancy is due to the more reduced state of glycerol when compared with carbohydrates, which implies that there is no net incorporation of electrons when glycerol is metabolised to propionate.

Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range.

Freezing or adding trypsin inhibitor to equine intestinal contents extends the lifespan of Clostridium perfringens beta toxin for diagnostic purposes.

Clostridium perfringens type C causes necrotizing enteritis mostly in neonatal animals of several species, including horses. The virulence of C. perfringens type C is mostly mediated by beta toxin (CPB). This toxin is highly sensitive to the action of trypsin and other proteases, which explains the increased susceptibility of neonatal animals to type C infections. Final confirmation of type C disease diagnosis should be based on detection of CPB in the intestinal content of affected animals. However, because CPB is so sensitive to the action of proteases, it is believed that this toxin persists for only a limited period of time in specimens of intestinal content of animals collected for diagnostic purposes. This study was therefore performed to determine the stability of CPB in intestinal content of horses stored at different temperatures and to evaluate the use of trypsin inhibitor to extend the lifespan of CPB in intestinal content of horses. When the intestinal content of horses that had been spiked with different amounts of CPB was tested by a capture ELISA technique to detect CPB, 319 LD(50) of CPB per milliliter was the lowest amount that could be detected. When equine intestinal content spiked with 319 LD(50)/ml was stored at 4 °C, CPB was detected by ELISA until day 8 after spiking. Samples spiked with the same amount of CPB and stored at -20 °C were positive for at least 5 weeks after spiking. When intestinal samples spiked with 319 LD(50)/ml of CPB were mixed with 0.1 mg/ml or 1.0 mg/ml of trypsin inhibitor and stored at 4 °C, all the samples were positive for at least 5 weeks after spiking. This study demonstrates that C. perfringens CPB present in equine intestinal samples stored at 4 °C cannot be detected by ELISA for more than 8 days. Freezing the samples at -20 °C or adding trypsin inhibitor before storage at 4 °C preserves the lifespan of CPB for at least 5 weeks.

A molecular approach to identifying the natural prey of the African creeping water bug Naucoris, a potential reservoir of Mycobacterium ulcerans.

The extra-oral digestion of creeping water bugs (Naucoridae: Hemiptera) hinders the study of their diet using the standard method of identifying prey body parts in the gut. Genetic methods are available, but rely on PCR tests or similar diagnostics to confirm suspected prey. Where the potential prey is unknown and a broad search for all possible prey is desirable, methods that can potentially capture any prey item are required. Naucoris sp. is known to harbor Mycobacterium ulcerans (Actinomycetales: Mycobacteriaceae), the causative bacterium of Buruli ulcer. Outbreaks of Buruli ulcer have been associated with disturbed freshwater habitats, but the mode of transmission to humans remains unclear. Here we examine the diet of Naucoris sp., a dominant aquatic predator in water bodies in Ghana where the prevalence of Buruli ulcer is high. We cloned and sequenced 576 PCR products (mtDNA rrnL, cox1) isolated from the gut of 60 Naucoris sp. individuals to determining diet composition as broadly as possible. Using phylogenetic analysis of newly sequenced clones and 6 potential prey taxa collected from the site, sequences isolated from Naucoris sp. guts matched locally collected Coleoptera (Hydrophilidae). Blastn queries to GenBank of other clone sequences produced matches to (Anura) (n = 1), Rotifera (n = 5), and fungi (n = 4) as additional components of the diet. Our results suggest that sp. in this Buruli ulcer-endemic area feeds on a wide range of prey and body sizes, and that the approach could be successfully applied to studies of aquatic food webs where morphological identification of prey is impossible and where little or no a priori knowledge is available.

Influence of pH upon the activity of glycosidases and proteinases of intestinal mucosa, chyme and microbiota in fish.

It is shown that amylolytic and proteolytic activity of the intestinal mucosa, the chyme and the intestinal flora in the fishes, zander Zander lucioperca (L.), perch Perca fluviatilis L., bream Abramis brama (L.) and roach Rutilus rutilus (L.), belonging according to their feeding habits to different ecological groups at the same pH values as well as in the pH range from 5.0 to 10.0 considerably varies. The glycosidase pH optimum of the mucosa and intestinal microbiota is 7.0, whereas that of the chyme varies from 6.0 (in roach) to 8.0 (in bream). pH optimum of the mucosa proteinases in all fish species is 10.0, whereas that of the chyme and the bacterial flora can be observed in all the range of pH values.

A cysteine protease is critical for Babesia spp. transmission in Haemaphysalis ticks.

Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites.

An evaluation of exogenous enzymes with amylolytic activity for dairy cows.

An experimental (7B) and a commercial (AMA) formulation of enzymes, both primarily with alpha-amylase activity, were evaluated for activity at various pH values, stability in ruminal fluid, the potential to improve in vitro ruminal fermentations, and the potential to improve production performance of lactating cows. When incubated (40 degrees C) in buffer with a pH between 5.4 and 6.0, 7B had about 10 to 25 times greater amylase activity than AMA, and enzyme activity in this range increased by 100% for 7B, whereas activity decreased by about 26% for AMA. Both formulations maintained enzyme activity when they were incubated in in vitro ruminal fermentations for 24 h. After 6 h of ruminal in vitro fermentation, additions of 7B resulted in linear increases in apparent total volatile fatty acid production for flint and dent corn but had no effect on floury corn. In a lactation trial, 28 Holstein cows (68 +/- 31 d in milk, 46.9 +/- 9.1 kg of milk/d) were fed a total mixed ration (TMR) supplemented with nothing (CON), a low dose of 7B [7BL, 0.88 mL/kg of TMR dry matter (DM)], a high dose of 7B (7BH, 4.4 mL/kg of TMR DM), or AMA (0.4 g/kg of TMR DM). The experiment was conducted as a 4. 4 Latin square design with 21-d periods. Cows fed 7BL, 7BH, and AMA ate similar amounts of DM, and cows fed the latter 2 diets consumed more DM than did cows fed CON. Cows fed 7BL produced more milk than cows fed CON and 7BH, but produced similar amounts to cows fed AMA. The production of 3.5% fat-corrected milk was greater from cows fed 7BL and AMA compared with cows fed CON. The percentages of milk fat and milk protein were unaffected by treatment. Total-tract digestion of DM and organic matter were greater for cows fed 7BL compared with those fed CON. The addition of exogenous amylase enzymes to the diets of lactating dairy cows has the potential to improve animal productivity.

Grazing cows are more efficient than zero-grazed and grass silage-fed cows in milk rumenic acid production.

Six rumen-cannulated Holstein cows in early lactation were assigned to 3 treatments: grazing (G), zero-grazing (ZG), and grass silage (GS) harvested from the same perennial rye grass sward in a 3 x 3 Latin square design with three 21-d periods. The objectives of this study were to investigate the underlying mechanisms for the reported elevation in milk rumenic acid (RA) concentration associated with G compared with ZG and GS, and to identify the important variables contributing to the milk RA response. Grazing animals were offered 20 kg of dry matter/cow per day; indoor animals were offered ad libitum grass or silage. A concentrate at a rate of 3 kg/d was also offered to all cows. Rumen, plasma, and milk samples were collected in the third week of each period. Data were analyzed by the MIXED procedure of SAS. Dry matter intakes were less for GS with no difference between G and ZG. Milk yield was greater for G than for ZG or GS. Milk fat and protein contents were less for GS with no difference between G and ZG. The combined intake (g/d) of linoleic and linolenic (18:3n-3) acids was different across the treatments (G: 433; ZG: 327; and GS: 164). Rumen pH was less for G with no difference between ZG and GS. Concentrations of volatile fatty acids and ammonia nitrogen in rumens were not different across the treatments. Wet rumen fill was less for G with no difference between ZG and GS. Vaccenic acid concentrations were different across the treatments in rumen (G: 12.30%, ZG: 9.31%, and GS: 4.21%); plasma (G: 2.18%, ZG: 1.47%, and GS: 0.66%) and milk (G: 4.73%, ZG: 3.49%, and GS: 0.99%). Milk RA concentrations were greater for G (2.07%) than for ZG (1.38%) and GS (0.54%). Milk desaturase index based on the ratio cis-9-14:1/14:0 was not different across the treatments. Milk RA yield per 100 g of linoleic acid and linolenic acid intake (efficiency) was 2.23, 1.50, and 0.62 g in G, ZG, and GS, respectively, suggesting that G cows were more efficient than ZG and GS cows in milk RA production. Stepwise regression analysis of a group of variables revealed that plasma vaccenic acid accounted for 95% of the variation in milk RA production. Milk desaturase index did not enter into the model. Overall findings suggest that substrate intake influenced milk RA production but it was not the only factor involved. There were differences in efficiency of milk RA production, which appears to depend on the factors regulating ruminal vaccenic acid production and its supply to the mammary tissue.

Corn expressing an Escherichia coli-derived phytase gene: residual phytase activity and microstructure of digesta in broiler chicks.

The residual phytase activity, phytic acid P content, and microstructure of the digesta along the gastrointestinal tract (GIT) of broiler chickens fed a corn expressing microbial phytase was studied in a 14-d experiment. The phytase activity of the corn expressing phytase (CBP) was determined to be 660 phytase units/g and was incorporated into broiler diets at varying rates. One hundred forty-four 7-d-old male broiler chickens were grouped by weight into 8 blocks of 3 cages with 6 birds per cage. Three dietary treatments were randomly allotted to the cages within blocks. The corn-soybean meal-based diets consisting of low P and Ca (no added inorganic P) supplemented with 0 (control), 55, or 550 g/kg of CBP (substituting corn) were formulated to contain 0 (control), 36,300, or 363,000 phytase units/kg of phytase activity, respectively. Birds were fed the dietary treatments for 14 d when they were killed and digesta samples from the proventriculus and gizzard, jejunum, and ileum were collected. The residual phytase activity along the GIT increased linearly and quadratically (P < 0.01) with the addition of CBP to the control diets. There was a decrease (P < 0.01) in residual phytase activity as digesta moved distally along the GIT with CBP supplementation. Phytic acid P content significantly decreased (linear, P < 0.01; quadratic, P < 0.05) with CBP supplementation of the control diets. There was substantial degradation (linear and quadratic, P < 0.01) of phytic acid content caudally along the GIT of birds. Extensive cell wall degradation of digesta samples from the proventriculus and gizzard in broilers fed the highest level of CBP compared with birds fed the control diets was observed. The addition of CBP to control diets led to a rapid degradation of the cell walls of digesta and a marked reduction in phytic acid P concentration of digesta in broiler chicks.

Validation of in vitro digestion using simulated small intestinal fluid with specific digestive activity to predict the metabolizable energy of feed ingredients for duck.

Two experiments were conducted to validate a method to prepare simulated small intestinal fluid (SSIF) for in vitro digestion in ducks. Experiment 1 compared the in vitro digestible energy (IVDE) of SSIF to endogenous small intestinal fluid (ESIF) on four feeds. The ESIF 1 or 2 obtained from two groups of jejunal cannulated ducks offered diet 1 (3,050 kcal/kg of ME and 19.95% of CP) or 2 (2,801 kcal/kg of ME and 14.90% of CP) was purified into raw enzyme power (REP) 1 or 2. SSIF 1 to 3 or 4 to 6 were prepared to mimic ESIF 1 or 2, respectively. The enzyme sources were REP 1 for SSIF 1 and 4, REP 2 for SSIF 2 and 5 or reagent enzymes for SSIF 3 and 6, respectively. The IVDE of each feed was determined with SSIF or ESIF. Experiment 2 was to validate whether REP 1 was more effective than only reagent enzymes to prepare SSIF. Ten feeds were determined with pepsin following SSIF 1 or 3 for IVDE 1 or 2, respectively. The accuracy of prediction model of true metabolizable energy (TME) from IVDE 1 or 2 was evaluated to validate the efficacy of SSIF. In experiment 1, higher activities of amylase, trypsin and chymotrypsin were observed in ESIF 1 than ESIF 2 (P < 0.05). The IVDE determined with SSIF 1 and 2 or 3 and 4 were more comparable to that of ESIF 1 or 2 than determinations with SSIF 3 or 6. In experiment 2, the mean IVDE 1 or 2 was 97.22% or 96.23% relative to TME, respectively, and both were highly correlated with TME (P < 0.01; R2 ≥ 0.98). However, the residual SD of TME prediction model with IVDE 1 was less than that generated with IVDE 2 (55 vs. 71 kcal/kg). In conclusion, the IVDE determined with in vitro digestion of pepsin following SSIF prepared with REP can predict accurately TME of feed for ducks.

Digestive and metabolic flexibility allows female degus to cope with lactation costs.

Lactation is the most energetically demanding period in the life cycle of female mammals, and its effects on digestive flexibility and the size of internal organs have been extensively studied in laboratory mice and rats since the early 1900s. However, there have been only two studies on this topic for wild rodent species. Here, we analyzed digestive flexibility--that is, changes in gut content, activity of digestive enzymes, and gut morphology--during lactation in the caviomorph rodent Octodon degus. In addition, we evaluated changes in the size of other internal organs and analyzed their relationship with the resting metabolic rate. We found that gut content, the dry masses of digestive chambers, the dry mass of liver, and resting metabolic rate were greater in lactating than in nonbreeding control females. In contrast, fat stores were higher in control subjects. Maltase and aminopeptidase-N specific activity did not change with lactation, and both enzymes had greater activity values in the middle portion of the small intestine. Thus, our data indicate that the previously reported increase in food assimilation that occurs during lactation in O. degus is related to a mass increase in several central organs, leading, in turn, to higher energetic costs. Fat stores may help to mitigate these costs, but, as expected for small animals, to a limited extent. Our study reveals a complex interplay among energy acquisition, storage, and expenditure processes that ultimately determine an organism's fitness.

[Effect of temperature on proteinase activities in the intestinal chyme and the mucosa of various ecological fish groups].

Effect of temperature on activities of proteinases in intestinal chyme and mucosa was studied in three fish species (pike-perch, zope, roach) belonging to different ecological groups by their type of feeding. There was revealed a significant difference of dependence of enzyme activities in chyme on temperature in the benthophage roach (a higher level of relative activity in the zone of lower temperatures and a larger zone of temperature optimum) as well as of values of apparent energy of activation of the protein hydrolysis process as compared with that in plankto- and ichthyophages--zope and pike-perch, which indicates a significant effect of the enteral microbiota proteinases and of nutrition objects on characteristics of hydrolases functioning in fish intestine.

Effect of roughage source and roughage to concentrate ratio on animal performance and rumen development in veal calves.

Sixty-four male Holstein-Friesian x Dutch Friesian veal calves (46 +/- 3.0 kg) were used to evaluate the effect of the inclusion of different levels and sources of dietary roughage on animal performance and rumen development. Treatments consisted of 1) C100 = concentrate only; 2) C70-S30 = concentrate (70%) with straw (30%), 3) C70-G30 = concentrate (70%) with dried grass (30%), 4) C70-G15-S15 = concentrate (70%) with dried grass (15%) and straw (15%), 5) C70-CS30 = concentrate (70%) with corn silage (30%), 6) C40-CS60 = concentrate (40%) with corn silage (60%), 7) C70-CS30-AL = concentrate (70%) with corn silage (30%) ad libitum, 8) C70-G15-S15-AL = concentrate (70%) with dried grass (15%) and straw (15%) ad libitum. All dietary treatments were provided in addition to a commercial milk replacer. Concentrate was provided as pellets and roughage was chopped. The dietary treatments 1 to 6 were supplied restrictedly to a maximum of 750 g of dry matter (DM) per day, whereas treatments 7 and 8 were offered ad libitum in combination with a reduced amount of milk replacer. Calves were euthanized after 10 wk. Straw supplementation (C70-S30 vs. C70-G30 and C70-CS30) reduced DM intake, and ad libitum supply of concentrate and roughage increased DM intake. Roughage addition did not affect growth performance. Rumen fermentation was characterized by low pH and high total volatile fatty acids and reducing sugar concentrations. Calves fed ad libitum showed lower ruminal lactate concentrations than calves fed restrictedly. Ammonia concentrations were highest in calves fed C-100 and lowest in calves fed ad libitum. The recovery of CoEDTA (added to milk replacer) varied between 20.5 and 34.9%, indicating that significant amounts of milk entered the rumen. Roughage addition decreased the incidence of plaque formation (rumen mucosa containing focal or multifocal patches with coalescing and adhering papillae covered by a sticky mass of feed, hair and cell debris) and the incidence of calves with poorly developed rumen mucosa. However, morphometric parameters of the rumen wall were hardly influenced by the type and level of roughage. Ruminal polysaccharide-degrading enzyme activities reflected the adaptation of the microorganisms to the dietary concentrate and roughage source. Results indicated that in veal calves, the addition of roughage to concentrate diets did not affect growth performance and positively influenced the macroscopic appearance of the rumen wall.

Effects of dietary metabolizable energy and crude protein content on the activities of digestive enzymes in jejunal fluid of Peking ducks.

The response of digestive enzymes activities in the jejunal fluid of Peking ducks to dietary ME and CP content was investigated. In experiment 1, jejunal digesta from 24 cannulated male white Peking ducks of 18 wk of age were collected for 1 h out of every 4 h beginning at 0930 h on d 16, 18, and 20 of the experiment. The activities of amylase, trypsin, and chymotrypsin in jejunal fluid were determined. In experiment 2, 72 male cannulated ducks were randomly sorted into 4 groups. Four treatments consisted of combinations of 3,050 and 2,800 kcal/kg of ME, and 17.50 and 14.40% CP content were available ad libitum. Jejunal digesta samples were collected for 1 h every 4 h from 0930 to 1830 h on d 31, 33, and 35 of the experiment according to the results of experiment 1. The activities of amylase, trypsin, chymotrypsin, lipase, sucrase, and maltase in jejunal fluid were determined. In experiment 1, significant differences were found in the average activities of amylase and chymotrypsin among days. The collection time significantly affected the 3 enzyme activities, and average enzyme activities during the day were higher and more stable than during the night. In experiment 2, the effect of dietary ME content on the 6 digestive enzymes activities was not significant. But the dietary protein content significantly changed amylase, trypsin, and chymotrypsin activities.

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid.

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (beta subunits) from several vertebrates (47-52% and 50-60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa.

Efficacy of supplementation of alpha-amylase-producing bacterial culture on the performance, nutrient use, and gut morphology of broiler chickens fed a corn-based diet.

A trial was conducted to evaluate the efficacy of an Escherichia coli strain producing alpha-amylase of Bacillus stearothermophilus on growth performance, nutrient use, and the morphology of the small intestine of broilers fed a corn-based diet. One hundred thirty-five 1-d-old chicks (Cobb 500) were randomly divided into 3 groups and treated as follows: (i) basal diet (control); (ii) basal diet and water supplemented with an E. coli strain that produced amylase, and (iii) basal diet and water supplemented with an E. coli strain that produced amylase plus bacterial hemoglobin. At 21 d of age, supplementation of E. coli improved daily gain (P < 0.05) and feed conversion (P < 0.01). At the end of the trial, birds supplemented with water containing bacteria consumed more and grew faster (P < 0.05) and had better feed conversion (P < 0.10) than broilers given no bacteria. Also, the presence of bacteria improved apparent digestibility of organic matter (P < 0.01). However, no effects were detected for CP or fat digestibility. Supplementation with E. coli reduced relative pancreas weight (P = 0.06) but did not affect the weight of the liver (P > 0.05) and length of duedonum, jejunum, ileum, and cecum (P > 0.05). Length of the villi and crypts were significantly increased with bacterial supplementation. Presence of the bacterial hemoglobin gene did not cause a significant difference in changes observed. The data indicated that supplementation of an E. coli strain capable of producing alpha-amylase improved digestibility of nutrients and performance of broilers fed a corn-based diet.