Effects of applying nutritional programming at different early stages of Largemouth bass (Micropterus salmoides, Lacepède) development on growth and dietary plant protein utilization.

Plant protein (PP) utilization in fish is limited due to lower digestibility compared to fishmeal (FM) and the presence of antinutritional factors. Its utilization can be improved by nutritional programming (NP), a method wherein a fish is provided a nutritional stimulus early in life which can alter their physiology. NP has been shown to be effective but methods of applying NP are varied and have been applied at different stages of development with different outcomes. To find the optimal timeframe to perform NP in fish early stages Largemouth bass (Micropterus salmoides, Lacepède) were nutritionally programmed at three different ages in early development. In this study bass were programmed with: (1) live food enriched with soybean meal (SBM) from 6 to 15 days post-hatch (dph) (NPL), (2) SBM-based formulated diet from 16 to 25 dph (NPD1) and (3) formulated SBM-diet from 26 to 35 dph (NPD2). After programming, each group was fed FM-diet before being refed SBM-diet from 100 to 172 dph. A positive control (PC) was fed FM-diet throughout. Final average body weight of PC was significantly higher than NPD1 and NPD2 but did not significantly differ from NPL. Overall NPL showed much improved growth and utilization of PP compared to NPD1 and was similar to growth achieved by PC. This study showed an optimum window of time exists wherein NP of Largemouth bass yields the best impact on growth in the larval stage and later in life when fed SBM-diet. Programming should be performed right after mouth opening using enriched live food and can result in growth similar to non-programmed fish fed FM-based diet. Programming effects similar to that of the live food approach can be achieved with formulated diet, however it is crucial that Largemouth bass are of a proper age and sufficiently developed when programmed with dry food or severe impacts on growth can occur.

The use of dipeptide supplementation as a means of mitigating the negative effects of dietary soybean meal on Zebrafish Danio rerio.

Soybean meal (SBM) inclusion in aquaculture diets has been found to negatively affect growth and induce intestinal inflammation in fish. The objective of this study was to determine the effect of health-promoting dipeptide supplementation into SBM-based feeds on growth performance, intestinal health, and muscle free amino acid composition, an indicator of dietary amino acid availability, in a zebrafish model. There were five treatment groups in this study. The first group ((+) Control) received a fishmeal-based diet. The second group ((-) Control) received SBM-based diet. The last three groups (Ala-Glu, Car, and Ans) were fed SBM-based diets, supplemented with alanyl-glutamine, carnosine, and anserine respectively. The Ala-Glu and Car groups experienced a significantly higher weight gain than the (-) Control group, weighing 35.38% and 33.96% more, respectively at the conclusion of the study. There were no significant differences in gene expression among the groups, but Ala-Glu had the highest expression of both nutrient absorption genes measured, PepT1 and fabp2. Ala-Glu had significantly longer intestinal villi, and a significantly higher villus length-to-width ratio than the (-) Control group. The Car group had a significantly higher post-prandial tissue concentration of lysine, compared to the (-) Control group. The increase in villus surface area and expression of nutrient absorption genes represent an improvement in intestinal absorptive capacity in the Ala-Glu group. The results from this study provide support for the use of alanyl-glutamine and carnosine supplementation as a means of improving growth performance of zebrafish fed with a high level SBM-based diet.

A Novel Approach in the Development of Larval Largemouth Bass Micropterus salmoides Diets Using Largemouth Bass Muscle Hydrolysates as the Protein Source.

This study's objectives were to determine the effect of Largemouth Bass (LMB) muscle hydrolysates obtained using same-species digestive enzymes and the degree of LMB muscle hydrolysis when included in the first feeds of growth performance and survival, skeletal development, intestinal peptide uptake, and muscle-free amino acid composition of larval LMB. LMB muscle was mixed with digestive enzymes from adult LMB, and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, the intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. Those diets were characterized by their level of each hydrolysate (presented as a ratio of 1.5, 3, and 6 Ts hydrolysates): 1:1:1, 1:3:6, 1:3:1, 6:3:1 for diets A, B, C, and D, respectively. To account for gut development, one group of larval LMB was fed a weekly series of diets B, C, and D to provide an increasing molecular weight profile throughout development. This group was compared against others that received either; (1) diets D, C, and B; (2) diet A; or (3) intact diet. The initial inclusion of the hydrolysates significantly improved the total length of the larval LMB; however, neither the hydrolysate inclusion nor the series of dietary molecular weight profiles improved the overall growth of larval LMB. The inclusion of hydrolysates significantly decreased the occurrence of skeletal deformities. The degree of hydrolysis did not have a significant effect on the parameters measured, except for intestinal peptide uptake, which was increased in the group that received the most hydrolyzed diet at the final time of sampling. The lack of overall growth improvement suggests that while the hydrolysates improve the initial growth performance, further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds for larval LMB.

The Effects of Plant Protein-Enriched Live Food on Larval Zebrafish Growth and the Status of Its Digestive Tract Development.

Live food is necessary for the proper development of zebrafish larvae, providing nutrition in a form that is easily digestible and available to the larvae. Live food is commonly enriched to increase the dietary content of certain nutrients. However, little research has been done on protein-based enrichments, especially those of plant origin. This study sought to examine how different quality protein enrichments affected the composition of live food as well as growth and digestive tract development of larval zebrafish, Danio rerio. Larval zebrafish were fed from 3 to 22 days posthatch (dph) with one of six live food (rotifers Brachionus plicatilis and Artemia spp.) treatments: (1) live food with no enrichment (starved; control); (2) live food enriched with commercially used Spirulina spp. algae; (3) live food enriched with soybean meal (SBM); (4) live feed enriched with soy protein concentrate (SPC); (5) live feed enriched with a fishmeal hydrolysate; and (6) live feed enriched with intact fishmeal (FM). Proximate composition of live food was significantly affected by enrichment, in particular, protein content of rotifers was significantly increased by enrichment with SBM. Zebrafish fed SBM-enriched live food showed longer total body length than all other groups, except SPC. Zebrafish in the SBM group also showed increased gene expression of chymotrypsin in the intestine, possibly indicating improved intestinal development and extracellular digestion, which likely contributed to improved growth. Conversely, zebrafish fed hydrolysate-enriched live food showed reduced gene expression of alkaline phosphatase, possibly indicating a less developed intestinal tract, correlating with reduced growth compared to SBM group. Overall, plant protein was shown to be a promising source of live food enrichment for improving larval zebrafish growth.

The use of live food as a vehicle of soybean meal for nutritional programming of largemouth bass Micropterus salmoides.

Nutritional Programming (NP) has been studied as a means of improving dietary plant protein (PP) utilization in different fish species. This study investigated the use of enriched live feed as a vehicle for NP in larval fish. The objective of this study was to determine the effect of NP induced during the larval stage via PP-enriched live feed on: (1) growth performance; (2) expression of genes associated with inflammation and any morphological changes in the intestine; and (3) muscle free amino acid composition in largemouth bass (Micropterus salmoides) during its later life stages. Two diets were used in this study, a fish meal (FM)-based diet, and a soybean mean (SBM)-based diet, serving as the PP diet. There were 4 groups in this study. The two control groups, ( +) Control and (-) Control, were not programmed and received the FM-diet and SBM-diet, respectively throughout the whole trial after the live feed stage (27-122 days post hatch (dph). The next group, programmed, was programmed with SBM-enriched Artemia nauplii during the live feed stage (4-26 dph) and challenged with the SBM-diet during the final stage of the study (79-122 dph). The final group, non-programmed, did not receive any programming and, was challenged with the SBM-diet during the final stage of the study. The programmed group experienced a significantly higher (%) weight gain during the PP-Challenge than the non-programmed group. In addition, the live feed programming resulted in significantly longer distal villi, and a higher villi length to width ratio, compared to the non-programmed group. No significant effects on free amino acid composition and gene expression were observed between the programmed and non-programmed group, except for an increased post-prandial concentration of free proline in the programmed group. The results of this study support use of live feed as a vehicle for nutritional programming and improving the growth performance of largemouth bass fed with a SBM-based diet.

Fish muscle hydrolysate obtained using largemouth bass Micropterus salmoides digestive enzymes improves largemouth bass performance in its larval stages.

The present study utilized digestives tracts from adult largemouth bass (LMB) to hydrolyze Bighead carp muscle and obtain an optimal profile of muscle protein hydrolysates that would be easily assimilated within the primitive digestive tract of larval LMB. Specifically, muscle protein source was digested for the larva using the fully developed digestive system of the same species. The objectives of this study were: 1) to develop an optimal in vitro methodology for carp muscle hydrolysis using LMB endogenous digestive enzymes, and 2) to evaluate the effect of dietary inclusion of the carp muscle protein hydrolysate on LMB growth, survival, occurrence of skeletal deformities, and whole-body free amino acid composition. The study found that the in vitro hydrolysis method using carp intact muscle and LMB digestive tracts incubated at both acid and alkaline pH (to mimic digestive process of LMB) yielded a wide range of low molecular weight fractions (peptides), as opposed to the non-hydrolyzed muscle protein or muscle treated only with acid pH or alkaline pH without enzymes from LMB digestive tracts, which were comprised of large molecular weight fractions (polypeptides above 150 kDa). Overall, the dietary inclusion of the carp muscle hydrolysate improved growth performance of larval LMB in terms of final average weight, weight gain, DGC, SGR, and body length after 21 days of feeding compared to fish that received the diet based on non-hydrolyzed carp muscle. The study also found that hydrolysate-based feed significantly reduced skeletal deformities. The positive growth performance presented by fish in the hydrolysate-fed group possibly resulted from matching the specific requirements of the larvae with respect to their digestive organ development, levels of digestive enzymes present in the gut, and nutritional requirements.

Nutritional Programming with Dietary Soybean Meal and Its Effect on Gut Microbiota in Zebrafish (Danio rerio).

Nutritional programming (NP) is considered a promising approach that can counteract the negative effects of dietary plant protein (PP) by introducing PP to fish in the early developmental stages. Therefore the objective of our study was to assess the effect of NP on PP utilization and the gut microbiome in zebrafish Danio rerio. The study included four treatment groups: (1) a positive control group that received a fishmeal (FM) diet throughout the entire trial (+ control); (2) a negative control group that received PP diet throughout the entire trial (- control); (3) an NP group that received dietary PP during the larval stage followed by FM-based diet during the juvenile stage and PP diet again during a PP challenge in the grow-out phase (NP-PP); and (4) an FM-group that received FM-based diet during the larval and juvenile stages and was challenged with a PP diet during the grow-out phase (NP-FM). During the PP challenge, the NP-PP group achieved the highest weight gain compared to the (-) control and NP-FM groups. The relative abundance of certain phyla such as Chloroflexi, Planctomycetes, and Bacteroidetes presented higher values in some groups at early juvenile stage. The fish gut microbiome also presented differences throughout the study.

Nutritional programming improves dietary plant protein utilization in zebrafish Danio rerio.

Nutritional Programming (NP) has been shown to counteract the negative effects of dietary plant protein (PP) by introducing PP at an early age towards enhancement of PP utilization during later life stages. This study explored the effect of NP and its induction time on growth, expression of appetite-stimulating hormones, and any morphological changes in the gut possibly responsible for improved dietary PP utilization. At 3 days post-hatch (dph) zebrafish were distributed into 12 (3 L) tanks, 100 larvae per tank. This study included four groups: 1) The control (NP-FM) group received fishmeal (FM)-based diet from 13-36 dph and was challenged with PP-based diet during 36-66 dph; 2) The NP-PP group received NP with dietary PP in larval stage via live food enrichment during 3-13 dph followed by FM diet during 13-36 dph and PP diet during 36-66 dph; 3) The T-NP group received NP between 13-23 dph through PP diet followed by FM diet during 23-36 dph and PP diet during 36-66 dph; and 4) The PP group received PP diet from 13-66 dph. During the PP challenge the T-NP group achieved the highest weight gain compared to control and PP. Ghrelin expression in the brain was higher in T-NP compared to NP-FM and NP-PP, while in the gut it was reduced in both NP-PP and T-NP groups. Cholecystokinin expression showed an opposite trend to ghrelin. The brain neuropeptide Y expression was lower in NP-PP compared to PP but not different with NP-FM and T-NP groups. The highest villus length to width ratio in the middle intestine was found in T-NP compared to all other groups. The study suggests that NP induced during juvenile stages improves zebrafish growth and affects digestive hormone regulation and morphology of the intestinal lining-possible mechanisms behind the improved PP utilization in pre-adult zebrafish stages.

Can intestinal absorption of dietary protein be improved through early exposure to plant-based diet?

Nutritional Programming (NP) has been studied as a means of mitigating the negative effects of dietary plant protein (PP), but the optimal timing and mechanism behind NP are still unknown. The objectives of this study were: 1) To determine whether zebrafish (Danio rerio) can be programmed to soybean meal (SBM) through early feeding and broodstock exposure to improve SBM utilization; 2) To determine if NP in zebrafish affects expression of genes associated with intestinal nutrient uptake; 3) To determine if early stage NP and/or broodstock affects gene expression associated with intestinal inflammation or any morphological changes in the intestinal tract that might improve dietary SBM utilization. Two broodstocks were used to form the six experimental groups. One broodstock group received fishmeal (FM) diet (FMBS), while the other was fed ("programmed with") SBM diet (PPBS). The first ((+) Control) and the second group ((-) Control) received FM and SBM diet for the entire study, respectively, and were progeny of FMBS. The last four groups consisted of a non-programmed (FMBS-X-PP and PPBS-X-PP) and a programmed group (FMBS-NP-PP and PPBS-NP-PP) from each of the broodstocks. The programming occurred through feeding with SBM diet during 13-23 dph. The non-control groups underwent a PP-Challenge, receiving SBM diet during 36-60 dph. During the PP-Challenge, both PPBS groups experienced significantly lower weight gains than the (+) Control group. NP in early life stages significantly increased the expression of PepT1 in PPBS-NP-PP, compared to PPBS-X-PP. NP also tended to increase the expression of fabp2 in the programmed vs. non-programmed groups of both broodstocks. The highest distal villus length-to-width ratio was observed in the dual-programmed group, suggesting an increase in surface area for nutrient absorption within the intestine. The results of this study suggest that NP during early life stages may increase intestinal absorption of nutrients from PP-based feeds.

Prevalence of Theileria equi and Babesia caballi infection in horses from northern Italy.

Babesia caballi and Theileria equi are the causative agents of equine piroplasmosis. In this epidemiological study, 294 horses reared in a rural area of northern Italy were studied. During January 2008-January 2009, blood samples were taken for serology (indirect fluorescent antibody test) and for polymerase chain reaction (PCR). Data on the geographical area, sex, and age were collected for statistical analysis of risk factors associated with infection. A seroprevalence of 8.5% was found: 8.2% of the animals were positive for anti-T. equi antibodies and 0.3% for anti-B. caballi antibodies. No dual infections were observed. Of those horses with positive serology to T. equi, 33% were also positive in PCR, whereas none of the seropositive horses for B. caballi was positive in PCR. No significant correlation between sex or age was found for infection status.