Long-Term Dietary Fish Meal Substitution with the Black Soldier Fly Larval Meal Modifies the Caecal Microbiota and Microbial Pathway in Laying Hens.

Feeding laying hens with black soldier fly larval (BSFL) meal improves their performance. However, the beneficial mechanism of BSFL meals in improving the performance of laying hens remains unclear. This study investigated the effects of the BSFL diet on liver metabolism, gut physiology, and gut microbiota in laying hens. Eighty-seven Julia hens were randomly assigned to three groups based on their diets and fed maize grain-and soybean meal-based diets mixed with either 3% fish meal (control diet), 1.5% fish and 1.5% BSFL meals, or 3% BSFL meal for 52 weeks. No significant differences were observed in biochemical parameters, hepatic amino acid and saturated fatty acid contents, intestinal mucosal disaccharidase activity, and intestinal morphology between BSFL diet-fed and control diet-fed laying hens. However, the BSFL diet significantly increased the abundance of acetic and propionic acid-producing bacteria, caecal short-chain fatty acids, and modified the caecal microbial pathways that are associated with bile acid metabolism. These findings indicate that consuming a diet containing BSFL meal has minimal effects on plasma and liver nutritional metabolism in laying hens; however, it can alter the gut microbiota associated with short-chain fatty acid production as well as the microbial pathways involved in intestinal fat metabolism. In conclusion, this study provides evidence that BSFL can enhance enterocyte metabolism and gut homeostasis in laying hens.

Egg quality and laying performance of Julia laying hens fed with black soldier fly (Hermetia illucens) larvae meal as a long-term substitute for fish meal.

The use of insects in animal feed appears to be an efficient approach that contributes to solving the environmental issues related to leftover disposal; however, it has not been approved in some countries due to concerns about pathogenic infections. This study aimed to evaluate the feasibility of long-term substitution of fish meal in poultry feed with organic defatted black soldier fly larvae (BSFL) meal prepared from BSFL raised on leftovers. The 87 Julia laying hens (178-day-old) were allotted in a completely randomized design with three treatments (29 layers in each treatment). The laying hens were fed maize grain and soybean meal-based diet containing either 3% fish meal, 1.5% fish meal and 1.5% BSFL meal, or 3% BSFL meal supplements for 52 wk (541-day-old). Results showed that substituting fish meal with BSFL meal had no effect on the laying rate, feed intake, and feed conversion ratio of laying hens, and only the complete replacement (3% BSFL meal) significantly increased the body weight of laying hens. In terms of egg quality, there was no significant effect on eggshell parameters (weight, thickness, and strength), albumen weight, yolk height, yolk color, and Haugh unit. However, both half (1.5% fish meal and 1.5% BSFL meal) and complete substitution of fish meal increased yolk weight (P < 0.01) and egg weight (P < 0.05). In conclusion, even if BSFL were fed leftovers and the meal was defatted with organic solvents, it can be used as a poultry feed ingredient without any adverse effect. Moreover, the complete substitution of fish meal with BSFL meal may be a feasible way to effectively contribute to the laying hens' performances and poultry farming costs. In addition to fish meal, the replacement of soybean meal with BSFL meal may also needs to be further studied for the extensive BSFL meal application in poultry feed.

Evaluation of Black Soldier Fly (Hermetia illucens) Larvae and Pre-Pupae Raised on Household Organic Waste, as Potential Ingredients for Poultry Feed.

Black soldier fly (BSF) larvae and pre-pupae could be satisfactorily raised on household organic waste and used as poultry feed, offering a potential sustainable way to recycle untapped resources of waste. The present study was conducted to determine if whole (non-defatted) BSF larvae and pre-pupae raised on experimental household waste could substitute soybean meal and oil as ingredients for laying hen diets. While no significant differences in feed intake and the egg-laying rate of hens were observed throughout the experiment, egg weight and eggshell thickness were greater in the pre-pupae-fed group than in the other groups. Moreover, although diversity of the cecal microbiota was significantly higher in the pre-pupae-fed than in the control group, no significant differences in bacterial genera known to cause food poisoning were observed when comparing the treatment groups. Nonetheless, Lactobacillus and Bifidobacterium populations were significantly lower in the treatment than in the control group. Fat content in BSF was possibly related with the changes in the cecal microbiota. Hence, since BSF fat was deficient in essential fatty acids, special attention should be paid to the fat content and its fatty acid composition in the case of regular inclusion of BSF larvae and pre-pupae oil as an ingredient in poultry diets.

Excretion rates of indigestible plastic balls of different specific gravities and diameters in dairy cattle.

We used plastic balls to investigate how their specific gravity and diameter affect excretion rate and rumination in dairy cattle, to develop a capsule that can be used for reaching the lower gastrointestinal tract without physical breakdown and/or degradation in the rumen. Twelve types of indigestible plastic balls composed of a combination of four specific gravities (0.95, 1.19, 1.41, or 2.20) and three diameters (3.97, 6.35, or 7.94 mm) were orally administered to lactating dairy cows, and the balls were collected from feces, after 120 h post-administration, to evaluate the recovery rate. Recovery rate of the balls with specific gravity 1.19 or 1.41 and diameter 6.35 or 7.94 mm was higher than those with specific gravity 0.95 or 2.20 and diameter 3.97 mm. The cumulative recovery rate at 24 and 48 h post-administration was higher for balls with specific gravity 1.19 than that for balls with other specific gravities. These results suggest that specific gravity 1.19 or 1.41 and diameters 6.35-7.94 mm are optimal for use in bypass capsules for administration to cattle. In addition, the passage time of capsules differed between specific gravities 1.19 and 1.41.

Development of three-layered rumen escapable capsules for cattle.

A new rumen escapable tool is presented for cattle in prospect of developing medical treatment or supplementing trace elements for disease prevention. This tool consists of a three-layered capsule that dissolves in the lower digestive tract, but not in the rumen. The capsule was manufactured by capsule-forming techniques through the use of liquid surface tension. This method does not involve high-temperature treatment, so the capsule can contain not only lipophilic substances but also hydrophilic or heat-sensitive substances. Furthermore, the capsule has a specific gravity of 1.3 and diameter of 6.0 mm, which were previously shown to be appropriate to avoid rumination. The objective of this study was to confirm the effectiveness of the capsule pertinent to rumen escaping. In order to validate rumen escape, capsules containing 30 g of water-soluble vitamin (thiamine hydrochloride) per head were administered to four lactating cows assigned in a crossover trial. In the group administered encapsulated thiamine hydrochloride, blood thiamine levels increased from 12.4 ± 1.03 ng/ml before administration to 54.8 ± 2.21 ng/ml at 6 hr following administration, whereas the level remained at 13.3 ± 2.05 ng/ml in the control group administered via aqueous solution. This indicates that the three-layered capsules passed through the rumen and dissolved in the lower digestive tract, thus functioning as a rumen escapable tool.

The effects of administering lactic acid bacteria sealed in a capsule on the intestinal bacterial flora of cattle.

We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 X 10¹¹ colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle.

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells.

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.

Carrageenan inhibits granzyme A-induced detachment of and interleukin-8 release from alveolar epithelial A549 cells.

Granzyme A (GrA) is a lymphocyte serine protease that is believed to enter virus-infected cells and growing tumors and induce apoptosis. We found recently that recombinant rat GrA (rGrA) promotes detachment of and interleukin (IL)-8 release from alveolar epithelial A549 cells and suggested that this protease is involved in the pathogenesis of certain inflammatory lung diseases. In the present study, we found that lambda-carrageenan (a sulfated oligosaccharide constituting the cell walls of seaweeds) potently inhibits rGrA-induced detachment and IL-8 release of A549 cells. This sulfated oligosaccharide might be useful for suppressing the development of inflammatory lung diseases in which GrA is thought to be involved.

Granzyme A causes detachment of alveolar epithelial A549 cells accompanied by promotion of interleukin-8 release.

Granzyme A (GrA) is a serine protease produced in cytotoxic lymphocytes, lung epithelial cells (alveolar type-II cells), and alveolar macrophages. In the present study, recombinant rat GrA (rGrA) was found to cause rounding and detachment of an alveolar type-II epithelial cell line, A549. Also, rGrA stimulated release of a neutrophil chemoattractant, interleukin-8, from the cells, via a mechanism involving microtubule disruption, probably resulting from reduction of cell adhesion to culture dishes. These findings suggest that GrA might be involved in the pathogenesis of certain lung diseases characterized by loss of alveolar wall structures, neutrophil accumulation, and chronic inflammation.

A lymphocyte serine protease granzyme A causes detachment of a small-intestinal epithelial cell line (IEC-6).

Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.

A role of a lymphocyte tryptase, granzyme A, in experimental ulcerative colitis.

In the large-intestinal mucosae of rats orally administered dextran sulfate sodium, which induces an enteritis resembling ulcerative colitis (UC), the activity for granzyme A, a lymphocyte tryptase, increased at an earlier stage than that at which UC markers (growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1 and caspase-3) increased. This suggests involvement of the enzyme in the exacerbation and perpetuation of enteritis.

Evidence for the occurrence of membrane-type serine protease 1/matriptase on the basolateral sides of enterocytes.

MT-SP1 (membrane-type serine protease 1)/matriptase is an epithelial-derived integral membrane enzyme. The purpose of the present study was to examine whether the enzyme exists on the basolateral side of simple columnar epithelial cells, such as enterocytes, of normal adult animals. Using COS-1 monkey kidney cells transiently transfected with rat MT-SP1/matriptase expression plasmids, we found that the enzyme is post-translationally processed by the cleavage between Gly149 and Ser150, that a portion of the C-terminal part (Ser150-Val855) remains in the cells by association with the NTF (N-terminal fragment) (Met1-Gly149), while the other portions are released into the medium and that the release is increased on activation by co-expression with hepatocyte growth factor activator inhibitor type-1. Western-blot analysis of crude membranes prepared from rat jejunum demonstrated the presence of the NTF but negligible or no occurrence of the C-terminal part of the protein. Fractionation of the crude membranes by ultracentrifugation with Percoll followed by Western-blot analysis showed that the fractionation profile of the NTF correlated significantly with that of E-cadherin, an adhesion molecule on the lateral membrane. Immunostaining of the jejunum demonstrated the occurrence of the NTF on the lateral membranes but not on the apical membranes. These results suggest that considerable MT-SP1/matriptase molecules occur on the basolateral sides of normal epithelial cells and support our hypothesis that a possible physiological function of this enzyme is the control of epithelial-cell turnover by regulating cell-cell and/or cell-substratum adhesions.

Sulfated polysaccharides derived from dietary seaweeds increase the esterase activity of a lymphocyte tryptase, granzyme A.

Intake of sulfated polysaccharides, such as fucoidan or lambda-carrageenan extracted from seaweeds, has been shown to enhance immune responses, resulting in inhibition of tumor growth. However, little is known about the mechanisms by which these sulfated compounds mediate the enhancement. In the present study, we examined the effect of sulfated polysaccharides from seaweeds on esterase activity of a lymphocyte tryptase, granzyme A (GzmA), which is believed to induce the production of cytokines in a variety of cells. Inclusion of fucoidan (from Fucus vesiculosus) or lambda-carrageenan (from Gigartina aciculaire and Gigartina) in the reaction mixture increased the hydrolysis of Nalpha-benzyloxy-L-lysine thiobenzyl ester (BLT) by a recombinant rat GzmA in a concentration-dependent manner. Heparin, a sulfated polysaccharide from animal tissues, also increase the BLT hydrolysis, but the effect was less remarkable than those of the polysaccharides from the seaweeds. Hanes-Woolf analysis revealed that the enhancements in the presence of these sulfated compounds from the seaweeds were attributed to the increases in the affinity of the enzyme toward the substrate but not to those in the turnover rate. Chondroitin sulfate A, a sulfated polysaccharide found in animal and plant tissues, showed no positive effect on the hydrolysis. In the present paper, we propose that the enhancement of immune responses by intake of the sulfated polysaccharides from seaweeds can be partially accounted for by their direct effects on GzmA.

Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between (125)I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N -alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.