Fish and Shellfish Immunology 88 (2019) 567–577 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Effects of different dietary protein sources on the immunological and T physiological responses of marron, Cherax cainii (Austin and Ryan, 2002) and its susceptibility to high temperature exposure ∗ Ishaaq Saputraa,e, Ravi Fotedara, Sanjay K. Guptaa,b, , Muhammad A.B. Siddika,c, ∗∗ Md Javed Foysala,d, a School of Molecular and Life Sciences, Curtin University, Bentley, WA, 6102, Australia b ICAR-Indian Institute of Agricultural Biotechnology, Ranchi, Jharkhand, India c Department of Fisheries Biology and Genetics, Patuakhali Science and Technology University, Patuakhali, 8602, Bangladesh d Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh e Fish Quarantine and Inspection Agency, Cilegon, Banten, Indonesia ARTICLE INFO ABSTRACT Keywords: A two phased feeding trial was conducted to evaluate the effects of alternative protein sources on the im- Marron munophysiological responses of marron. During the phase I, marron were fed with five alternative protein Immune supplemented diets for 90 days, while in phase II, the same marron were exposed to elevated temperature (30 °C) Condition indices and their immunophysiological responses were investigated post exposure. Five isoproteic (crude protein 30%) Gut micro villi and isoenergetic diets were prepared by containing fishmeal, poultry by-product meal, feather meal, lupin meal, Temperature and meat and bone meal as the main protein source. A hundred and fifty juvenile marron (Cherax cainii) of the average weight 9.09 ± 0.21 g were randomly distributed into 15 tanks (three replicates per feeding treatments). In the Phase I, general immune response parameters, such as, total haemocyte count (THC), proportion of hyaline cells, neutral red retention time (NRRT), phagocytic rate (PR), heamolymph bacteraemia, and condition indices of marron were investigated. The highest (P < 0.05) THC among dietary protein sources was obtained in marron fed with PbM at the end of experiment. Marron fed with FeM protein sources resulted in the highest survival rate followed by PbM fed group. Longer microvilli length (3.83 ± 0.18 μm) was demonstrated in marron fed with PbM diet. Diets containing FM and PbM protein sources revealed significantly (P < 0.05) lower number of microvilli/group than diets containing FeM and LM. The results demonstrated that different dietary protein sources in the marron diets did not detect significant (P > 0.05) change of the condition indices throughout the experiment period, however highest Hiw and Hid was recorded in marron fed with PBM at day 45. The PR of marron fed dietary protein from PbM did not change significantly after temperature exposure. Increased NRRT, PR and haemolymph bacteraemia was observed with dietary feeding of FM at the end of the trial. However, results revealed that PbM could be an alternative protein source for culture of marron as reflected in terms of increased THC, longer microvillus length and improved susceptibility to high temperature exposure. Overall, result could serve as useful baseline data in developing cost effective potential diets for marron aquaculture. 1. Introduction constitutes one of the major protein ingredients in crustacean aqua- culture. In past, studies have demonstrated the potential of total or As disease outbreaks are still continuing to be the major threat to partial substitution of fishmeal with alternative protein sources in the aquaculture industry, the use of conventional antibiotics to elim- crustaceans [4] but with variable success. Previous research outcomes inate pathogens have been significantly reduced [1]. Simultaneously, revealed that several plant and animal based-protein sources have the the concert for depleting fishmeal supply is on the rise [2,3] which potential to substitute fishmeal protein in the diets of some decapod ∗ Corresponding author. School of Molecular and Life Sciences, Curtin University, Bentley, WA, 6102, Australia. ∗∗ Corresponding author. School of Molecular and Life Sciences, Curtin University, Bentley, WA, 6102, Australia. E-mail addresses: [email protected] (S.K. Gupta), [email protected] (M.J. Foysal). https://doi.org/10.1016/j.fsi.2019.03.012 Received 9 November 2018; Received in revised form 6 March 2019; Accepted 7 March 2019 Available online 11 March 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved. I. Saputra, et al. Fish and Shellfish Immunology 88 (2019) 567–577 crustaceans [5,6]. Furthermore, the increase of non-fishmeal inclusion Table 1 in the diets was observed to have detrimental effects on the physiolo- Feed ingredients and proximate composition (% dry weight). gical responses of signal crayfish, Pascifastacus leniusculus [7,8] and Diets black tiger prawn, Penaeus monodon [9]. However, no studies have in- vestigated the changes in the immunophysiological responses of the FM PbM LM FeM MbM marron fed diets containing alternative protein sources to fishmeal. Ingredients (% dry weight) Marron, Cherax cainii, an esteemed aquaculture commodity; are an Fish Meala 30.15 0.00 0.00 0.00 0.00 iconic freshwater species to Western Australia [10,11] which generated Feather Meala 0.00 0.00 0.00 22.00 0.00 AUD 1.5 million per year through its farming [12]. Over and above, the Lupin Meala 0.00 0.00 36.40 0.00 0.00 a significant increase in commercial demand of marron has led toboost Poultry by-Product Meal 0.00 29.50 0.00 0.00 0.00 Meat and Bone meala 0.00 0.00 0.00 0.00 24.30 the research on improvement of immune responses to tackle down the b Soybean Meal 10.80 10.80 21.00 15.65 16.60 disease outbreaks. Innate immunity in crayfish is similar to other Wheat (10 CP) b 48.13 50.88 28.67 48.31 43.13 crustaceans, which involves circulating blood cells or haemocyte to Corn/wheat starchb 4.80 4.80 4.01 5.50 5.50 b eliminate both pathogen and particles from the body through cellular Betacaine 1.20 1.20 3.50 1.02 3.50 b activity [13–15]. Several attempts to improve innate immunity have Cod liver oil 4.20 2.10 3.45 4.80 4.00 Calcium carbonateb 0.02 0.02 0.02 0.02 0.02 been reported in previous studies in crayfish [16–18] and in tiger prawn Ascorbic Acidb 0.05 0.05 0.05 0.05 0.05 [19]. The immunophysiological responses measured by alterations in Vitamin premixb 0.15 0.15 0.15 0.15 0.15 total haemocytes counts (THC), differential haemocyte counts (DHC), Cholesterolb 0.25 0.25 0.25 0.25 0.25 b neutral red dye retention technique (NRRT), phagocytic rate (PR), Casein 0.25 0.25 2.50 2.25 2.50 bacteraemia and condition indices could be used as indicators of im- Proximate composition (g Kg−1 dry weight basis) c mune competence of several crustaceans [16,20,21], including marron, DM% 89.07 87.86 88.46 87.85 87.26 C. tenuimanus [17] in order to understand the underlying mechanism of Ash% 8.24 4.05 6.21 3.18 10.13 GE MJ/kg 18.63 18.57 18.59 18.39 18.34 improved health status. Apart from this, research investigations on CP% 30.13 30.88 30.84 30.46 30.18 improvement of the clinical signs of the digestive system has also been Lipid%c 7.17 7.53 7.26 7.16 7.63 carried out to evaluate the health status of cray fish [22,23]. Fiber%c 1.22 1.35 1.29 1.07 1.27 Marron have gained significant attention as a potential candidate DM (Dry matter); GE (Gross energy); MJ (Mega joule); CP (Crude protein). species for prospective growth of aquaculture owing to its large harvest a Glen Forest Specialty Feeds, Western Australia. size (up to 2 kg), higher price, non-burrowing behavior, simple life b Talloman, Freemantle, Western Australia. cycle and ease of live transport [10,11,24]. Consistent efforts are in c Data were obtained from database. progress to translocate crayfish species from natural habitat to other locations for globalization, therefore the effects of high temperature on 2.2. Experimental animals and design physiological responses necessitates further investigations. Rouse & Kartamulia (1992) investigated the temperature tolerance of marron, Two hundred marron juveniles 0 + years old, average wt. Cherax tenuimanus before its domestication process in the US [10]. (9.09 ± 0.21 g) were procured from Aquatic Resource Management Numerous investigations on effects of temperature in crayfish physio- Pty. Ltd., Manjimup, Western Australia and transported to Curtin logical responses have been carried out [25–31]. However, the effects Aquatic Research Laboratory (CARL), Technology Park, Curtin of temperature exposure on the immunophysiological responses of University, Western Australia. Marron juveniles were then acclimated marron following supplementation with different dietary protein source for two weeks in three cylindrical holding tanks (200 l capacity), which are yet to be revealed. were equipped with continuous aerations and an adequate number of Therefore, the aim of this study was to evaluate the efficacy of hides in the form of PVC pipes. Each acclimation tank was equipped dietary protein sources on the immunophysiological responses and gut with eight PVC pipes (55 mm in diameter, 150 mm long) and a filtra- micrograph of marron. In addition, the susceptibility to the high tem- tion unit. Marron were fed with a commercial marron diet (Glenn perature exposure of marron was delineated in terms of im- Forrest, Australia) with a composition of 28% crude protein, 9% crude munophysiological responses. fat and 5% crude ash, calcium (1.5%), phosphorous (1%), and digestion energy 11 MJ/kg at 3% of body weight during the acclimation period.