Date of Award
5-1-2024
Degree Name
Doctor of Philosophy
Department
Zoology
First Advisor
Kwasek, Karolina
Abstract
The heavy reliance on live feeds is currently restricting the growth and sustainability of the aquaculture industry, therefore, the overall goal of this research was to improve the utilization of formulated dry diets at first feeding of larval fish. This was done with a specific focus on the production and provision of the optimal dietary protein form and composition. Chapter 2 aimed to provide an efficient protein source for larval fish by using same-species muscle and endogenous enzymes to produce hydrolysates and by providing a series of diets with increasing molecular weight protein fragments through larval development. Largemouth Bass (Micropterus salmoides) (LMB) muscle was mixed with the digestive enzymes from adult LMB and hydrolyzed for 1.5, 3, and 6 h, respectively. Five diets were produced, an intact diet containing non-hydrolyzed muscle and four diets with 37% muscle hydrolysate inclusion. The molecular weight profile of those diets were formulated to vary based on the inclusion level of each hydrolysate. To account for gut development, one group of larval LMB was fed a weekly series of diets with an increasing molecular weight profile. 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 results from this study suggest that the inclusion of same-species hydrolysates can improve the initial growth of first-feeding LMB, but further research is necessary to determine the optimal molecular weight profile, hydrolysate inclusion level, and physical properties of feeds to improve the overall growth performance during the larval stage. Chapter 3 compared the effect of dietary inclusion of a fish muscle hydrolysate produced from species-specific muscle and enzymes to hydrolysates produced from those of a different species, in diets for larval Walleye (Sander vitreus). Four intact and hydrolyzed protein products were produced from each combination of Walleye muscle and endogenous enzymes, and muscle and endogenous enzymes from Nile Tilapia (Oreochromis niloticus). The hydrolyzed products were continuously mixed for 3 h during the hydrolysis, (at 22oC and 28oC for Walleye and Tilapia enzymes, respectively), and the pH was adjusted throughout the process to mimic gastric and intestinal digestion conditions. Four diets were produced with the dietary protein supplied as a 50/50 ratio of the intact and hydrolyzed muscle from the respective muscle/enzyme combination. There was a significant interaction effect between muscle and enzyme source on the growth of larval Walleye. At the conclusion of the study, the larval Walleye that received the diet with muscle hydrolysate produced with Walleye muscle and Walleye endogenous enzymes had a significantly higher average weight than all other groups, and significantly higher postprandial levels of total free amino acids and indispensable amino acids in the muscle. Each hydrolysate-based diet led to a significant reduction in skeletal deformities and survival, compared to a group fed with a commercial diet. The results from this study suggest that species-specific muscle and enzymes produce a more optimal dietary protein source for larval fish than non-species-specific products, but further research should focus on improving the physical properties of the formulated diets to improve survival of fish larvae. Chapter 4 proposed a practical controlled hydrolysis method to utilize the endogenous enzymes within the fish body for the breakdown of tissues proteins, and to produce a species-specific meal that is tailored to the nutritional requirements and absorptive capacity of fish larvae. Four Zebrafish (Danio rerio) meals were produced from whole-body adult Zebrafish, three hydrolysates that were hydrolyzed for 1, 2, and 3 h, respectively, and an unhydrolyzed meal. From these meals, three diets were produced, each defined by their supply of dietary protein. The Unhydro diet was solely based on the unhydrolyzed Zebrafish meal. The 50% Hydro diet was based on 50% Zebrafish hydrolysate mix and 50% unhydrolyzed Zebrafish meal. The 100% Hydro diet was 100% based on the Zebrafish meal hydrolysate. The hydrolysate mix contained equal parts of the 1, 2, and 3 h hydrolysates. Proteomic analysis showed that the proposed hydrolysis method was able to efficiently hydrolyze the protein within Zebrafish body. The feeding trial found no significant differences in the final weight, total length, or survival between the Unhydro, 50% Hydro, and 100% Hydro groups, but the 50% Hydro group did express a significant upregulation of PepT1 at 24 h after feeding, compared to the Unhydro group. The growth results paired with PepT1 gene expression potentially indicate Zebrafish larvae to be adapted to dry feeds at first feeding and able to utilize dietary protein in different molecular forms efficiently for growth. Overall, the proposed hydrolysis method provides a practical and cost-effective approach to producing species-specific fishmeal hydrolysates. Further research is necessary to determine whether the produced hydrolysates can improve the growth of larval fish in other fish models. Further insight into behavioral and physiological responses in fish to imbalanced dietary amino acid profiles was provided in Chapter 5. The objective of this study was to determine how stomachless fish respond to diets deficient in the main limiting IDAA (lysine, methionine, and threonine), using Zebrafish as a model species. Six semi-purified diets were formulated for this study. The CG diet contained casein and gelatin as its only protein sources, while FAA50 diet had 50% of is dietary protein supplied with crystalline amino acids. Both were formulated to contain identical, balanced amino acid profiles. The remaining diets were supplied with the same amino acid mix as the FAA50 diet, but with minor adjustments to create deficiencies of the selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the free amino acid (FAA) mix, respectively, and the Def diet was deficient in all three. The fish were fed to apparent satiation three times a day, and each feeding was carefully observed to ensure all feed added to the tanks was consumed. The results showed that although the singular deficiency of the three main limiting amino acids did not induce significant changes in feed intake, the combined deficiency of the three IDAA significantly increased the feed intake of juvenile Zebrafish. This increased feed intake prevented the IDAA deficiencies from significantly reducing growth, however, the feeding efficiency was also reduced. There was also an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group, compared to the FAA50 group. The outcomes of this study provide insight into the behavioral and physiological response to dietary amino acid imbalances of stomachless fish and suggests stomachless fish increase their feed intake when challenged with IDAA-deficient diets, and that the regulation of NPY might play a role in this response. Chapter 6 assessed the postprandial FAA dynamics in the plasma, liver, and muscle of three species; 1) Largemouth Bass – warm-water, stomach-possessing carnivorous species; 2) Walleye – cool-water, stomach-possessing carnivorous species; and 3) Zebrafish– tropical, stomachless omnivorous species. Two diets were formulated for this study, a diet based on intact casein and gelatin (CG), and a diet with 50% of its protein supplied in FAA form (FAA50). Forty-two fish from each species were utilized, with one group of 21 receiving the CG diet, and the other 21 receiving the FAA50 diet. All fish were starved for 24 hours prior to the final feeding before sampling. Three fish were sampled at each time point, with three samples (plasma, liver, and muscle) taken from each fish. Samples were taken prior to feeding (0 h) and then at 0.5, 1, 2, 3, 6, and 12 h after feeding, for all species. A significant three-way interaction was observed between the diet, species, and postprandial time on the total FAA, IDAA, and DAA levels in the plasma, liver, and muscle, indicating that the postprandial FAA patterns were significantly different between species and in response to the different diets. In stomach-possessing species, dietary amino acids from the FAA50 diet were absorbed more rapidly than those from the CG diet, resulting in fewer correlations with the dietary IDAA profiles. The absorption of FAA in cool-water Walleye was more gradual and prolonged than the warm-water LMB, leading to more significant correlations with the dietary IDAA and more sustained peaks. The postprandial peaks of FAA typically occurred at the same time in the stomachless Zebrafish fed with the CG or FAA50 diet. The levels of FAA were noticeably lower after feeding with the FAA50 diet in Zebrafish, compared to the CG diet. These results provide a reference for differences in the FAA dynamic patterns of three species with differing physiological characteristics, when fed diets with intact protein or supplemented with FAA. The findings presented in this dissertation provide support and novel methods for the production and inclusion of species-specific protein hydrolysates as an ideal protein source in formulated diets for first-feeding larval fish. This research contributes to the development of larval diets that can release the limitations of growth placed on the aquaculture industry by the reliance on live feeds, particularly within the hatchery sector. This research also provides further understanding of dietary protein utilization and delivers new fish nutrition knowledge that will benefit the aquaculture industry as a whole.
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