Bacillus subtilis Effects on Growth Performance and Health Status of Totoaba macdonaldi Fed with High Levels of Soy Protein Concentrate.

T. macdonaldi is a carnivorous species endemic to the Gulf of California. Indiscriminate exploitation has put totoaba at risk, inducing the development of aquaculture procedures to grow it without affecting the wild population. However, aquafeeds increasing cost and low yields obtained with commercial feeds have motivated researchers to look for more nutritious and cheaper alternatives. Soybean (SB) is the most popular alternative to fishmeal (FM); however, antinutritional factors limit its use in carnivorous species. In this study, we analyzed B. subtilis 9b probiotic capacity to improve growth performance and health status of T. macdonaldi fed with formulations containing 30% and 60% substitution of fish meal with soy protein concentrate (SPC). In addition, we investigated its effect on internal organs condition, their capacity to modulate the intestinal microbiota, and to boost the immunological response of T. macdonaldi against V. harveyi infections. In this sense, we found that T. macdonaldi fed with SPC30Pro diet supplemented with B. subtilis 9b strain and 30% SPC produced better results than SPC30C control diet without B. subtilis and DCML commercial diet. Additionally, animals fed with SPC60Pro diet supplemented with B. subtilis 9b strain and 60% SPC doubled their weight and produced 20% more survival than SPC60C control diet without B. subtilis. Thus, B. subtilis 9b improved T. macdonaldi growth performance, health status, modulated intestinal microbiota, and increased animal's resistance to V. harveyi infections, placing this bacterium as an excellent candidate to produce functional feeds with high levels of SPC.

Soy protein concentrate effects on gut microbiota structure and digestive physiology of Totoaba macdonaldi.

AIMS: Examine the effect of soy protein concentrate (SPC) on allochthonous microbiota, hindgut integrity, and liver tissue of totoaba (Totoaba macdonaldi). METHODS AND RESULTS: Four diets were prepared: control diet (100% fishmeal) and experimental diets containing partial substitution of fishmeal by SPC (15%, 30% and 45% SPC). After 90 days, samples of the hindgut contents were taken to determine the taxonomic composition of the allochthonous microbiota through sequencing of the V3-V4 region of the 16S rRNA gene. Simultaneously, liver and hindgut samples were collected for examination by histological approaches. The SPC modulated the richness and abundance of the accessory microbiota, of which the main operational taxonomic unit showed an increase corresponding to the Phylum Firmicutes (Bacillales and Lactobacillales). With the increase in SPC, a slight decrease in mucosal fold width, a decrease in goblet cells and a slight distortion of the villi in the hindgut were observed. In the liver, SPC was observed to influence hepatocytes morphology through irregular and enlarged nuclei. CONCLUSION: The study demonstrates that Proteobacteria dominated the allochthonous microbiota of subadult totoaba, regardless of the diet. However, the SPC modulated the accessory bacteria communities and caused slight effects on the liver and gut of fish. SIGNIFICANCES AND IMPACT OF THE STUDY: To our knowledge, this is the first study that analyses the effects of SPC on allochthonous microbiota of subadults T. macdonaldi through new generation techniques such as DNA sequencing for metagenomic analysis.

Soybean protein concentrate as a protein source for totoaba (Totoaba macdonaldi) juveniles: Effect on intermediary metabolism and liver histological organization.

This study aimed to investigate the effects of replacing fish meal (FM) with soybean protein concentrates (SPC) on the intermediary metabolism and health of Totoaba macdonaldi juveniles. Fish (initial weight 50 ± 1 g) were fed for 60 days with eight diets: a reference diet (RD) and seven experimental diets where FM was replaced gradually with 15 to 100% SPC (SPC15, SPC30, SPC45, SPC60, SPC75, SPC90, and SPC100, respectively). Hexokinase (HK), glucokinase (GK), and alanine aminotransferase (ALT) enzyme activities showed highly significant differences (p < 0.01) between fish fed RD (0% SPC) compared to fish fed the diets with 60, 75, 90, and 100% SPC. The ALT enzyme shows a highly significant (p < 0.01) decrease in activity for fish fed 75, 90, and 100% SPC inclusions compared to fish fed the RD. The aspartate aminotransferase AST/ALT ratio showed a significant increase in activity for fish fed 100% soybean compared only with fish fed the control diet. The histological organization of the liver in totoaba juveniles fed RD, SPC15, SPC30 and SPC45 diets were similar. Totoaba fed with SPC90 and SPC100 showed histological alterations in hepatic and pancreatic parenchyma. Overall, according to the findings in this study, 45% of dietary FM could be replaced by SPC without causing adverse changes in metabolism, histological organization of liver, and health of juveniles of totoaba when cultured for 60 days. However, levels greater than 60% of SPC could compromise the health status of fish.

Tryptophan supplementation helps totoaba (Totoaba macdonaldi) juveniles to regain homeostasis in high-density culture conditions.

High-density culture brings with it chronic stress situations that affect fish welfare. In order to evaluate the effect of tryptophan (Trp) levels on the response to stress, Totoaba macdonaldi juveniles were stocked at low (13.5 kg m-3) and high (27.0 kg m-3) densities (32.5 and 56.4 kg m-3, respectively, at the end of the experiment) in 100-L tanks and fed for 63 days with experimental diets containing different Trp levels: control diet CD0.42 (0.42%) and three supplemented diets with 0.99, 1.55 and 2.19% (0.99Trp, 1.55Trp and 2.19Trp, respectively) (three tanks × density × diet). The high-density stocking fed with CD0.42 diets showed significantly increased blood parameters. Trp decreased catalase (CAT) activity in low- and high-density stocking, while the superoxide dismutase (SOD) activity showed no difference. Serotonin (5-hydroxytryptamine, 5-HT) content decreased, and the serotonin turnover ratio (5-HIAA:5-HT) increased in the brains of fish fed with the CD0.42 diet. Indeed, Trp-supplemented diets helped to restore homeostasis in high-density growth conditions as evaluated by the hematological and plasma parameters as well as the serotonergic activity. When the fish were provided a diet containing moderate Trp levels, plasma cortisol increased under high-density conditions. However, no differences were observed among stock densities when totoaba were fed with the 2.19Trp diet. Notably, survival was unaffected by both Trp or densities, but weight gain (WG) decreased with the dietary Trp levels in the high density culture. In sum, Trp supplementation decreased the parameter values linked to stress response on totoaba juveniles cultured at high stock densities.

Digestive system development and study of acid and alkaline protease digestive capacities using biochemical and molecular approaches in totoaba (Totoaba macdonaldi) larvae.

The present study aimed to describe and understand the development of the digestive system in totoaba (Totoaba macdonaldi) larvae from hatching to 40 days post-hatch (dph) from morphological and functional perspectives. At hatch, the digestive system of totoaba was undifferentiated. The anus and the mouth opened at 4 and 5 dph, respectively. During exogenous feeding, development of the esophagus, pancreas, liver and intestine was observed with a complete differentiation of all digestive organs. Expression and activity of trypsin and chymotrypsin were observed as early as at 1 dph, and increments in their expression and activity coincided with changes in food items (live and compound diets) and morpho-physiological development of the accessory digestive glands. In contrast, pepsin was detected later during development, which includes the appearance of the gastric glands between 24 and 28 dph. One peak in gene expression was detected at 16 dph, few days before the initial development of the stomach at 20 dph. A second peak of pepsin expression was detected at day 35, followed by a peak of activity at day 40, coinciding with the change from live to artificial food. Totoaba larvae showed a fully morphologically developed digestive system between 24 and 28 dph, as demonstrated by histological observations. However, gene expression and activity of alkaline and acid proteases were detected earlier, indicating the functionality of the exocrine pancreas and stomach before the complete morphological development of the digestive organs. These results showed that integrative studies are needed to fully understand the development of the digestive system from a morphological and functional point of views, since the histological organization of digestive structures does not reflect their real functionality. These results indicate that the digestive system of totoaba develops rapidly during the first days post-hatch, especially for alkaline proteases, and the stomach becomes functional between 20 and 24 dph allowing the weaning process to begin at this age.

Effect of fishmeal replacement by soy protein concentrate with taurine supplementation on growth performance, hematological and biochemical status, and liver histology of totoaba juveniles (Totoaba macdonaldi).

The effect of dietary inclusion of soy protein concentrate (SPC) and simultaneous supplementation with taurine on the growth, hematology, blood biochemistry, and liver histology of totoaba (Totoaba macdonaldi) juveniles was assessed. Four isoproteic and isolipidic diets were formulated containing either 30 or 60% of SPC (diets S30 and S60), supplemented or not with 1% of taurine (diets S30T and S60T). A fishmeal-based diet formulated for totoaba nutritional requirements, without SPC and taurine supplementation, was used as a reference diet. Triplicate groups of 32 totoaba juveniles (average body weight 7.5 ± 0.6 g) were fed these diets for 45 days. Results showed that growth performance in fish fed S30, S30T, and S60T was similar to fish fed the reference diet. Red blood cells and hematocrit in fish fed with supplemented taurine in both levels of SPC (S30T and S60T) were similar to the fish fed the RD; the addition of taurine improved the state of hydration of totoaba. Plasmatic hemoglobin in fish fed the lower SPC level was similar to fish fed the RD. The mean corpuscular hemoglobin concentration in fish fed S30T was similar to fish fed the RD, taurine supplementation prevented the development of hypochromic anemia in this group of fish. Plasmatic albumin in fish fed S30 was similar to fish fed the RD. Plasmatic total protein and globulin concentration increased and AL:GLB (albumin:globulin ratio) decreased in fish fed the SPC-based diets despite taurine supplementation. The protein profile showed that taurine supplementation did not prevent a possible inflammatory process (increased globulins, decreased AL:GLB) in juvenile totoaba fed both levels of SPC. Glucose concentration was similar in fish fed S30, S30T, and S60T. The histological hepatic index was highest in fish fed S60. These results suggest that with an appropriate nutritional level, taurine may play an important modulatory role in the hematology and blood biochemistry status in totoaba fed SPC-based diets, contributing to the enhancement of an overall healthy growth performance.

Expression and activity of trypsin and pepsin during larval development of the spotted rose snapper Lutjanus guttatus.

The present study aimed to describe and understand the development of the digestive system in spotted rose snapper (Lutjanus guttatus) larvae from hatching to 40 days post-hatch (dph). The mouth opened between 2 and 3 dph, at that moment the digestive tract was barely differentiated into the anterior and posterior intestine, although the liver and pancreas were already present. Gastric glands were observed until 20 dph, followed by the differentiation of the stomach between 20 and 25 dph. Trypsinogen expression and trypsin activity were detected at hatching, increasing concomitantly to larval development and the change in the type of food. Maximum levels of trypsinogen expression were observed at 25 dph, when animals were fed with Artemia nauplii, and maximum trypsin activity was detected at 35 dph, when larvae were fed with an artificial diet. On the other hand, pepsinogen gene expression was detected at 18 dph, two days before pepsin enzymatic activity and appearance of gastric glands. Maximum pepsin activity was also observed at 35 dph. These results suggest that in this species weaning could be initiated at an earlier age than is currently practiced (between 28 and 30 dph), since larvae of spotted rose snapper develop a functional stomach between days 20 and 25 post-hatch.