by Dr Karthik Masagounder, Head Aqua and Swine Research, Evonik Nutrition and Care GmbH, Germany and Dr D Allen Davis, Professor, Auburn University, USA


An in-vitro study based on the enzymes extracted from shrimp hepatopancreas showed that shrimp are able to completely cleave the four available isomers of DL-methionyl-DL-methionine (AQUAVI® Met-Met) but at different rates. This allows delayed liberation and absorption of D- and L- Methionine (Met) in the digestive tract. However, no in-vivo data were published showing changes in the postprandial profile of Met in the hemolymph – a fluid analogous to the blood in vertebrates – of shrimp fed different Met sources.

We therefore instigated a study to investigate and compare postprandial Met levels in relation to other amino acids in the hemolymph of whiteleg shrimp fed diets supplemented with AQUAVI® Met-Met or DL-Met. The effects of different sources on the Met levels of shrimp hemolymph were evaluated under two feeding frequencies (once per day and three times per day) to understand if the feeding frequency would influence the dietary effects on the hemolymph Met levels.

Met is typically the first limiting amino acid in the practical shrimp feed and therefore its supplementation is essential. Previous studies showed that AQUAVI® Met-Met is a more efficient Met source (≥ 200%) for shrimp compared with DL-Met (Xie et al. 2017; Niu et al. 2018). Better bioavailability of AQUAVI® Met-Met versus DL-Met is attributed mainly to (i) its less water solubility and leaching and (ii) slow and long release of Met from different sterioisomers (DL-Met-Met, LD-Met-Met, LL-Met-Met, DD-Met-Met).

Trial design to test different methionine sources and feeding frequencies

Basal diet was formulated to contain ~37 percent protein using soybean meal as the primary protein source, and eight percent lipid and ~20 MJ/kg gross energy using wheat and fish oil as the main energy sources. The basal diet was formulated to meet the known nutritional requirements of whiteleg shrimp with the exception of Met. Two test diets were formulated by replacing 0.30 percent glycine from the basal diet with 0.30 percent DL-Met (DL-Met diet) or 0.31 percent AQUAVI® Met-Met (Met-Met diet).

All the diets were pressure pelleted using a meat grinder and a 3mm die at the feed laboratory of Auburn University. After pelleting, diets were dried to a moisture content of 8-10 percent and stored at 4°C. Analysed Met and other amino acid levels in the three diets showed close values as those of planned.

In trial one, 54 tanks, each tank containing four shrimps (~17g average body weight), were used. Each diet was offered to 18 tanks (80l volume) in total. This allowed triplicate samples of hemolymph (one hemolymph sample pooled from four shrimp per tank) at each time point for six time points (0, 15, 30, 45, 60 and 120 min) for each dietary group (three replicates x six time points x three diets = 54 tanks). No tank was sampled more than once. Shrimp were preconditioned to the experimental diets at one feeding per day for four days before hemolymph sampling.

Feeding for different tanks were staggered between 8 and 10am to allow time for samplings in different tanks at the targeted time points. Prior to the sampling day, shrimp were fasted overnight and basal hemolymph samples were taken from three tanks in each dietary group at zero minute just before feeding. Shrimp in the remaining tanks were offered an excess of the respective experiment diets for 15 minutes, tanks were siphoned to remove any excess feed, and shrimp were fasted thereafter during hemolymph collection.

In trial two, shrimp were conditioned to the diets for four days before hemolymph collection. Each diet was offered to 15 tanks (80l volume) containing four shrimp (~21g average body weight) per tank. First feeding for different tanks was staggered between 7am and 9:30am to accommodate hemolymph samplings on the sampling day at the targeted time points. A three-hour interval was given between the two meals during the day. Hemolymph samples from triplicate tanks of shrimp were planned after the third meal up to 60 min for every 15 min interval.

On day five, shrimp fasted overnight were fed two times each lasting for one hour, following the same feeding schedule used in the previous days. After each feeding, tanks were siphoned for any uneaten feed and shrimp were fasted for two hours. Before the third feeding, basal hemolymph samples were taken at zero minute from three tanks. Shrimp in the remaining tanks were offered an excess of the respective diets for 15 minutes and were fasted thereafter during the subsequent samples. Hemolymph samples were taken at 15, 30, 45, and 60 min, each time from triplicate tanks of shrimp in each treatment group.

Collected hemolymph samples were centrifuged and decanted to obtain free amino samples. Samples were then freeze dried and analysed for individual amino acid content.

Absolute quantity of Met and total amino acids (TAA) were evaluated for each dietary treatments and time points. In order to eliminate the influence of variation in the amount of feed consumed by individual shrimp on the Met levels at a given time point, Met level was expressed in percent of TAA measured. This value was then compared among treatments and different time points for the two different feeding frequency. Data were subjected to two-way ANOVA to measure the effects of diets and different time points on the hemolymph TAA (μg/ml) and Met levels (μg/ml, (%TAA) of shrimp. Additionally, combining both the trial data, hemolymph Met (%TAA) level was also subjected to three-way ANOVA to evaluate the effects of diet, time and feeding frequency (time point 120 min was excluded for 1x feeding).

During the study, water temperature was maintained at around 30°C. Dissolved oxygen was maintained near saturation using air stones in each aquarium and the sump tank using a common airline connected to a regenerative blower. Water salinity was maintained at around nine ppt.

Impacts of diet (methionine sources), time post feeding and feeding frequency in the hemolymph

Results of both main and interaction effects of diets and time on the hemolymph amino acid levels of shrimp are summarised in Table 2. In trial one, where shrimp were fed one time per day, diet affected only hemolymph Met levels (μg/ml, %TAA), but not TAA. Both TAA and Met levels were affected by time. However, no interaction effect due to diet and time were detected on TAA or Met. Hemolymph Met levels were higher in shrimp fed Met-Met diet relative to the basal (μg/ml) or both basal and DL-Met diets (%TAA). Hemolymph Met level expressed in %TAA showed higher levels in the hemolymph for the DL-Met diet than for the basal diet. Across diets, Met and TAA levels in the hemolymph of shrimp measured at 15, 30- and 45-min post feeding were generally higher than those measured in other time points (0, 60 and 120 min).

In trial two, where shrimp were fed three times per day, absolute quantity of TAA and Met levels (μg/ml) in shrimp hemolymph showed interaction effects due to diet and time (See Table 2). However, no particular trend could be detected due to diet x time interaction. Furthermore, post-hoc tukey comparison showed no significant differences between any mean combinations. Variations in the peak of amino acid levels within the dietary groups over time were likely because of differences in feed intake among individuals. Degree of satiation among individuals seem to vary more when shrimp are fed more often.

Examining hemolymph Met levels as %TAA partially corrects the confounding effects of variations in feed intake on the diet effects. Met levels in %TAA showed effects due to diet or time with no interaction between them. Met levels (%TAA) were higher in shrimp fed Met-Met diet than those fed the basal diet, while shrimp fed DL-Met diet showed intermediate no-significant Met levels. Met levels (%TAA) reached its peak at 15 min post feeding and then declined over time. Similar time effects were also observed on the TAA and Met levels (μg/ml) when examined at absolute quantity.

Subjecting hemolymph Met data (%TAA) obtained from both the trials through a three-way ANOVA showed significant effects due to diet, time, and feeding frequency without any interaction effects. Shrimp fed the Met-Met diet showed significantly higher hemolymph Met levels than those fed the basal or DL-Met diet, while shrimp fed the DL-Met diet showed higher Met levels than those fed the basal diet. As detected before, Met level (%TAA) in the hemolymph reached its peak at 15 min post feeding and was significantly better than that observed at 0, 45 or 60 min post feeding. Feeding shrimp three times a day resulted in higher Met level in the hemolymph than those fed one time a day. Data analysis with three-way ANOVA clearly show increase in the hemolymph Met levels with the Met-Met diet versus the basal or DL-Met diet in both the feeding frequency.

Overall, study results clearly demonstrate that feeding AQUAVI® Met-Met supplemented diet compared with the DL-Met supplemented diet results in higher Met (% TAA) in the hemolymph of shrimp over a long time. and thereby, can support for higher body protein synthesis and better growth performance. This in-vivo study provides evidence for the higher biological efficiency observed in the previous studies for AQUAVI® Met-Met vs. DL-Met.

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