Effects of Replacement of Fish Meal with Poultry By-Product Meal (PBM) on Growth Performance, Digestive Enzyme, and Immunity of Giant River Prawn ( Macrobrachium rosenbergii )

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Introduction
Protein is an essential component of aqua feed [1] for all commercially important aquaculture species, including prawns, which require 25-42% of the total protein by weight.Fish meal (FM) has traditionally been used as a balanced nutritional source to formulate aquaculture feed [2,3].Nevertheless, due to a variety of factors, such as climate change, overfshing, and the decline of global fsh stocks, developing alternative protein sources is an important research area to enhance aquaculture sustainability [4].
Macrobrachium rosenbergii is known as giant freshwater prawn cultivated in several countries for food [21] because of its ability to grow in captive conditions.A number of studies have been conducted testing alternative protein ingredients, but the complete replacement of FM with PBM has not been studied thoroughly.Hence, the present study was conducted to investigate the inclusion of graded levels of PBM on the growth performance, digestive enzymes, and immunity of M. rosenbergii.

Ethics Statement.
Te study was conducted strictly according to the guidance of the care and use of laboratory animals in China.Te experimental protocol was approved by the Committee on the Ethics of Animal Experiments at Huzhou University.

Formulation and Feed Preparation.
Te fshmeal and PBM were provided by New Hope Liuhe Lited Company, Chengdu, Sichuan, China (Table 1).Five experimental diets were formulated including the control (without PBM).Te main protein source of the control group was 30% of fsh meal, and the fsh meal was replaced by 25%, 50%, 75%, and 100% PBM as groups PBM 25, PBM 50, PBM 75, and PBM 100, respectively.Each experimental diet had four replicates and 50 prawns were distributed into each replicate.Te crude protein and the crude lipids content of all levels of the experimental diet are shown in Table 2.
All the feed ingredients were homogenized, granulated with a granulator, and strained with 60 holes by a squaremeter sieve.All feed ingredients were added accordingly, hand-mixed thoroughly, and mixed with a machine for 20 minutes.Water was added about 30% while continuously mixing to get a good texture for the pellets to be made.Te pellets were made with a 1.5 mm diameter mill.Te pellets were dried in a hot dried air oven at 50 °C for 24 hours.After this, feeds were stored at −4 °C until use.Twenty (20) 300-liter white polyethylene tanks were washed and flled with 250 liters of water each; prawns were acclimatized for two weeks and were fed the control diet.Ten, ffty (50) randomly selected prawns were distributed, making a total of 1000 prawns for the trial.Diets were fed in quadruplicate (50 prawns per replicate) to satiation (3-5% of their body weight).Prawns were fed twice daily (8:00, 17:00).Te dead prawns observed were removed, measured, and registered at any moment.Te fecal matter was regularly removed from each tank's bottom, and adjustments were made in 30-50% of the water.Individual tanks were aerated for 24 hours with more than 5 mg/L dissolved oxygen levels, and water was adjusted regularly.A Handy Polaris ® meter was used to measure the dissolved oxygen and temperature, and a YSI ® pH meter was used to measure pH at 10 cm below the water surface (model pH 100).Te water was in a temperature range (26-30 °C), with an ammonia nitrogen concentration of less than 0.3 mg/L and a pH of 7.8-8.3.

Prawn
Te prawns were fed for 56 days.Ten, the prawns were individually weighed using a scientifc balance, and the standard length was determined using a metric ruler.Te following growth parameters were calculated after the feeding trial: survival rate (SR), weight gain (WG), and specifc growth rate (SGR).

Digestive Enzyme Assay.
At the end of 8 weeks trial, 12 prawns per treatment were randomly selected.Every prawn's carapace cavity was opened from the dorsal region.Forceps were used to extract the hepatopancreas and the gills.Te hepatopancreas and gills were held at 80 °C for further research.Te telson and uropod were cut and separated from the body (abdomen) by cutting slightly through the sixth segment of the prawn's tail.Te intestine

Hemolymph Analysis.
Hemolymph samples were obtained from the third pair of walking legs (pericardial cavity) using 1 mL syringes (needle size 20-25G) and put in a 1.5 ml centrifuge tube before the dissection procedure.Te hemolymph was centrifuged at 4 °C for 20 minutes at 10000 r/ min, and the supernatant was taken and deposited at −80 °C.Te hemolymph was tested for superoxide dismutase (SOD), malondialdehyde (MDA), alkaline phosphatase, and acid phosphatase using Nanjing Jiancheng Bioengineering Co., Ltd., kits.

Te Nutritional Components of the Whole Prawn.
Prawns were randomly selected from each experimental group and stored in the refrigerator at −20 °C for later use after the feeding trial and a 24-hour fast.Proximate composition, moisture, crude protein, and crude fat were measured.Te proportion of crude protein was measured using the Kjeldahl process, the chloroform-methanol method was used to analyze crude fat, and the combustion method at 550 °C was used to determine crude ash.

Statistical Analysis.
Te data were statistically analyzed using the analysis of variance approach (ANOVA).Prior to performing ANOVA, all data were checked for homogeneity of variance.Te impact of various diets on growth parameters (WGR, SGR, and SR), proximate composition of prawns, intestinal digestive enzymes, and immunity were investigated using one-way ANOVA with a signifcant level (P < 0.05).

Efects of Replacement of Fish Meal by the Poultry By-Product Meal on Growth Performance.
Compared with the control group, when the fsh meal was 100% replaced by PBM, the fnal weight, weight gain, and specifc growth rate of prawn were signifcantly reduced (P < 0.05), and the diference between 25%, 50%, and 75% groups was not signifcant (P > 0.05).Te survival rate of prawns in all feed groups was not diferent, as shown in Table 3.

Efects of Replacement of Fish Meal by the Poultry By-Product Meal on Proximate Composition.
Tere was no signifcant diference in the moisture, crude protein, crude fat, and crude ash contents of prawn body composition in diferent proportions of the PBM instead of fsh meal (P > 0.05), as shown in Table 4.

Efects of Replacement of Fish Meal by the Poultry By-Product Meal on
Intestinal Digestive Enzymes.Te 25% PBM replacement of the fsh meal had the highest enzyme activity.Amylase activity was signifcantly increased (P < 0.05) compared to the other groups.Te trypsin and lipase activities of the PBM groups were higher than those of the control group (C), but there was no signifcant diference (P > 0.05), as shown in Table 5.

Efects of Replacement of Fish Meal by the Poultry By-Product Meal on
Immunity.In all experimental diets, there was no signifcant change (P > 0.05) in SOD activity, MDA content, and AKP activity in prawn's hemolymph.However, the ACP activity of all the replacement groups decreased signifcantly (P < 0.05), as shown in Table 6.

Discussion
Te evaluation of ingredients is critical for determining their suitability for aquaculture systems.It is also critical to determine an ingredient's energy and nutrient accessibility and its digestibility to an animal.Tis research revealed that a substantial amount of PBM might be substituted for fsh meal in the feed of M. rosenbergii without afecting growth performance, feed digestibility, and health.M. rosenbergii is a fast-growing species requiring a shorter time to achieve commercial size compared to other aquatic species.Tis shorter culture window means a reduced feed input demand but a greater maintenance expense.PBM has come out as a viable alternative to fsh meal in aquaculture feed formulations for a myriad of purposes, together with its high protein content, low ash content, high palatability, and digestibility, as a great source of cholesterol and phospholipids [22].In addition, PBM is a less expensive protein source than fsh meal and is readily accessible, particularly in poultry-producing areas [23,24].As a result, increasing the amount of PBM in the diets of M. rosenbergii can signifcantly cut feed expenditures.However, to achieve the optimum results, measures for balancing the content of alternative protein sources in the basal feed and promoting M. rosenbergii growth must be implemented.

Efect of Replacement of Fish Meal by the Poultry By-Product Meal on Growth
Performance.When it comes to growth performance, many researchers focus on the nutritional value of proximate composition, nutritional content, and its efect on prawns' growth [25,26].Studies suggested that the total replacement of fsh meal could be realized when using a high-quality PBM [6,27,28].
Te present study showed that the replacement of FM by 25%, 50%, and 75% PBM did not adversely afect prawns' growth parameters.Many research studies support this present study.In diets for Pacifc white shrimp L. vannamei, a high-quality PBM can substitute up to 80% of the fsh meal without the need for amino acid supplements [22] and up to 70% of the fsh meal [29] without noticeably impairing growth.Nik Sin et al. [30] also replaced up to 75% of Macrobrachium rosenbergii's diet without negatively affecting the growth performance.Other studies on diferent PBM inclusion had been studied in crustacean diets without afecting the growth, including 27.7% in P. monodon [31], 29.82% in Macrobrachium nipponense [32], and 21.2% in C. quadricarinatus [33].Another similarity was also observed in Davis and Arnold [34] and Samocha et al. [35], who also reported that the Pacifc white shrimp, L. vannamei did not demonstrate any diference in survival or growth when a PBM product was used to replace 80% FM in the diet.All values are given as the mean ± standard deviation (SD).Means followed by diferent letters in the same column are signifcantly diferent (P < 0.05).All values are given as the mean ± standard deviation (SD).Means followed by diferent letters in the same column are signifcantly diferent t(P < 0.05).

Aquaculture Research
Similarly, the inclusion of 20.1% PBM in the diet of white shrimp produced the best performance [29].Te results of this research are also in line with those found by Cheng et al. [36], Tan et al. [37], and Zhu and Yu [38], with 66%, 80%, and 80% replacement levels, respectively.Te recent study by Nik Sin et al. [30] reported reduced growth performance at 100% PBM replacement in Macrobrachium rosenbergii, which agrees with this present study.Also, Karapanagiotidis et al. [28] reported that 100% replacement of FM by PBM resulted in reduced growth performance in gilthead seabream juveniles due to a lower feed intake and low levels of dietary methionine and lysine.In other studies, tilapia (100% PBM [39]), golden pompano Trachinotus ovatus (up to 100% PBM [40]), and humpback grouper (100% PBM [15]) also had reduced growth performances.Te 100% replacement of fsh meal by PBM agrees with this study because growth performance was signifcantly reduced in the prawns of group PBM100 compared to the control group.PBM protein is characterized by having less methionine and lysine than fsh meal, which is thought to be a growth factor in many species, such as hybrid striped bass [41] and Florida pompano Trachinotus carolinus [42].Amino acid defciency of PBM could be one cause for decreased fsh growth at a greater fsh meal replacement level (100%).Again, poor digestibility can be another cause for poor growth performance in higher replacement (PBM100).Although the apparent digestibility coefcient of the meals in the current research was not tested, it has been reported that many prawns/fsh perform worse in terms of growth because feeds with a lot of poultry by-product meal have a poor digestibility [30,32,43,44].In experiments on humpback grouper [15] and gilthead seabream [6], it was discovered that diets containing 75% and 100% FM protein substituted with PBM protein resulted in reduced growth performance, which was associated with the reduced digestibility of dietary protein.

Efects of Replacement of Fish Meal by the Poultry By-Product Meal on Proximate Composition.
In this present research, replacing fsh meal with PBM had no signifcant efect on the whole-body moisture, crude protein, crude lipid, and crude ash content of prawns.However, the wholebody protein was higher in prawn-fed PBM25 than in the other prawn groups.Tis observation was in agreement with Yang et al. [44] who reported no signifcant diferences in the whole-body moisture and fat, but a trend of increased protein and ash contents of gibel carp fed increasing levels of PBM.Again, the whole-body composition of red sea bream Pagrus major was not afected by diferent groups of dietary PBM [18].Other research studies were on M. rosenbergii [30], L. vannamei [22], L. vannamei [35], L. vannamei [29], M. nipponense [32], C. quadricarinatus, and Pacifc white shrimp [34].Prawns beneft from the fat in PBM because it contains essential nutrients, including phospholipids and cholesterol.Protein and lipid content in the prawn body could be related to changes in their synthesis, deposition rate in muscle, and growth rates [45][46][47].
In contrast, Chinook salmon Oncorhynchus tshawytscha [48] and rainbow trout Oncorhynchus mykiss [27,49] increased whole-body fat of fsh-fed PBM diets.Nengas et al. [6] found that increasing dietary PBM in gilthead seabream resulted in signifcantly decreased carcass lipid, which disagrees with this present study.

Efect of Replacement of Fish Meal by the Poultry By-Product Meal on Intestinal Digestive Enzymes.
Trypsin is a protein-digesting enzyme, amylase is a carbohydratedigesting enzyme, and lipase digests fats and lipids.Te hepatopancreas of the giant freshwater prawn is primarily responsible for amylase, trypsin, and lipase production.Te enzyme amylase is then secreted into the foregut, breaking down glycosidic bonds [50].In this study, the increase in all these enzyme activities in the experimental diets indicates an improved capacity for digesting.Te preponderance of research validated PBM's excellent digestibility as in giant tiger prawn, Penaeus monodon [51], Pacifc white prawn [22,34], Korean rockfsh, Sebastes schlegelii [19], and rainbow trout [52].Factors such as nutritional content, palatability, and digestibility of PBM may function as a moderator of the growth efect of PBM supplementation.In this study, PBM-improved digestive enzymes activity is the important factor for good growth performance and immunity in prawns.

Efect of Replacement of Fish Meal by the Poultry By-
Product Meal on Immunity.Te levels of SOD, MDA, AKP, and ACP can be utilized to assess the prawns' physiological state and immunity response, as is well acknowledged.SOD is the enzyme that converts superoxide anion radicals to hydrogen peroxide and molecular oxygen, and it plays a critical role in regulating cellular reactive oxygen species All values are given as the mean ± standard deviation (SD).Means followed by diferent letters in the same column are signifcantly diferent (P < 0.05).a SOD: superoxide dismutase.b MDA: malondialdehyde.c AKP: alkaline phosphatase.d ACP: acid phosphatase.
Aquaculture Research (ROS) levels [53].Te type of feed intake is also critical for ROS production.Te balance between ROS creation and removal maintains a steady-state ROS level.Malondialdehyde (MDA) is the end product of lipid peroxidation and is widely used as a proxy for oxidative stress [54][55][56].Te alkaline phosphatase (AKP) level in the hemolymph can be used to determine hepatopancreas-related issues.In summary, elevated SOD and MDA mean the health of the prawns has been compromised.Tis study showed no signifcant change in SOD activity, MDA concentration, or AKP activity in prawn hemolymph.Te blood parameter values of prawns fed diferent diets in this study suggested that their condition and health state were normal for prawnfed PBM meals, which agrees with Rio-Zaragoza et al. [57] and Hernández et al. [58].Te substitution of fsh meals with PBM meals had no efect on the body composition of the prawns in this investigation which also showed that prawns were in good health.

Conclusion
Tis study showed that PBM up to 75% replacement group has no efect on growth performance, and all replacement groups have no adverse efect on intestinal digestive enzyme activity and immunity of juvenile stage of M. rosenbergii.Terefore, poultry by-product meal could replace fsh meal by up to 75%.

Table 1 :
Chemical composition and amino acid concentration of the PBM and fsh meal (%).
was removed by carefully pulling the telson and uropod away from the rest of the body.For further research, the intestine was kept at 80 °C.Hepatopancreas and intestines were homogenized in separate tubes of 4 °C precooled deionized water, left in a 4 °C refrigerator overnight, and centrifuged at 4000 r/min for 10 minutes.Te crude enzyme solution was removed from the homogenate and deposited at −80 °C for testing.Trypsin, lipase, and amylase were examined using Nanjing Jiancheng Bioengineering Co., Ltd., kits.

Table 3 :
Te efect of replacement fsh meal by PBM on the growth performance.All values are given as the mean ± standard deviation (SD).Means followed by diferent letters in the same column are signifcantly diferent t(P < 0.05).
a WGR: weight gain rate; b SGR: specifc growth rate; c SR: survival rate.

Table 5 :
Efects of replacement fsh meal by PBM on intestinal digestive enzymes.