Comparative Studies On The Utilization Of Plant Protein Of Japanese Seabass (Lateolabrax Japorticus) And Siberian Sturgeon(Acipenser Baeri Brandt), The Effects On Growth Performance And GH/IGF-I Axis

Both of the teleostean species Japanese seabass (Lateolabrax japonicus) and the chondrosteanspecies Siberian sturgeon (Acipenser baeri Brandt) are important aquaculture species in the world, andthey were classified as euryhaline carnivorous varieties in Fishbase (www.fishbase.org). The presentstudy aimed to study the effects of partial or total fishmeal substitution by plant protein blend (PPB,Wheat gluten meal and Soybean meal) on growth performance, flesh quality and GH/IGF-I axis inJapanese seabass or Siberian sturgeon. In order to find the best lever of plant protein blend (PPB) fortwo species. There are four parts in this study, the results were respectively summarized as follows:Molecular Cloning and Character Analysis of cDNAs GH and GHR gene in Japanese seabass andSiberian sturgeonThe somatotropic axis (GH/IGF-I) is an important endocrine axis for fish, including growth hormone(GH), GH receptor (GHR) and Insulin-like growth factor-I(IGF-I), with the primary function ofcontrolling the growth of animals. The full sequence of cDNA for the IGF-I genes of Japanese seabassand Siberian sturgeon had been listed in Genbank, and the registry number were JQ327805.1andFJ428828.1, respectively. RT-PCR and rapid amplification of cDNA ends (RACE) method were used inthis experiment to clone the full sequence of cDNA for the GH gene and GHR of Japanese seabass andSiberian sturgeon. The full-length GH cDNA of Japanese seabass (JQ995145) was949bp, consisting ofa5’-terminal untranslated region (UTR) of72bp, a3’-terminal UTR of262bp, and an open readingframe (ORF) of615bp encoding a polypeptide of204amino acids with a theoretical molecular weightof23.0624kDa and an estimated isoelectric point of7.27, and shared high similarity with other GHgenes (33.2～90.2%). The full-length GHRI cDNA of Japanese seabass (JX402001) was3070bp,consisting of a5’-terminal untranslated region (UTR) of227bp, a3’-terminal UTR of932bp, and anopen reading frame (ORF) of1911bp encoding a polypeptide of637amino acids with a theoreticalmolecular weight of70.7948kDa and an estimated isoelectric point of4.37, and shared high similaritywith other GHRI genes (32.2～86.2%).The full-length GHRII cDNA of Japanese seabass (Q995146)was2926bp, consisting of a5’-terminal untranslated region (UTR) of252bp, a3’-terminal UTR of925bp, and an open reading frame (ORF) of1749bp encoding a polypeptide of582amino acids with atheoretical molecular weight of64.4164kDa and an estimated isoelectric point of4.77, and shared highsimilarity with other GHRII genes (30.8～81.4%). The full-length GH cDNA of Siberian sturgeon(JX003684) was999bp, consisting of a5’-terminal untranslated region (UTR) of53bp, a3’-terminalUTR of301bp, and an open reading frame (ORF) of645bp encoding a polypeptide of214amino acidswith a theoretical molecular weight of24.1429kDa and an estimated isoelectric point of5.76, andshared high similarity with other GH genes (38.3～70.1%). The full-length GHR cDNA of Siberiansturgeon (JX003685) was2238bp, consisting of a5’-terminal untranslated region (UTR) of377bp, a3’-terminal UTR of190bp, and an open reading frame (ORF) of1716bp encoding a polypeptide of 571amino acids with a theoretical molecular weight of63.4096kDa and an estimated isoelectric pointof4.92, and shared similarity with other GHR genes (36.6～49.2%). Phylogenetic analysis revealed thatthe GH and GHR of Siberian sturgeon were closer related to mammals, but not teleostean, such asJapanese seabass. The full-length GH and GHR cDNAs of two species will be used to further study onthe regulatory mechanism of the somatotropic axis (GH/IGF-I) and the evolution of fish.The effects of total fishmeal substitution by plant protein blend (PPB) on short-term feeding,growth performance and GH/IGF-I axis in Japanese seabassA3-week feeding trial was conducted to study the effect of totally replaced dietary low temperaturesteam dried fish meal (LT-FM) by PPB (Soybean meal/wheat gluten meal=1:4.843, CP,71.47%) onshort-term feeding rate, growth performance and the expression of GH/IGF-I axis genes in Japaneseseabass (Initial weight,65.01±0.07g). In control diet,51.4%of LT-FM was used as sole protein sourceto formulated the diets at40.35%of protein levels for Japanese seabass, with named as FM. The PPBdiet named as PPB100. Two diets are isonitrogenous and isoenergetic, and the PPB100groupsupplemented with DL-methionine, Lysine-HCl, L-Threonine and Ca(H2PO4)2to keep the limitingamino acid and available phosphorus balance. Four replicates for each treatment with20seabass in eachtank. The experimental fish were stocked in a recirculating system, fed twice per day at8:00and20:00for40min exactly to form a feeding rhythm. Results showed that: fish fed the control diet hadsignificantly higher FR、FBW、SGR、FCR and WGR than PPB100group (P<0.05); There was nosignificantly different of SR between the two groups (P>0.05), At postprandial3h, the content ofplasma leptin (Lep) and ghrelin (Ghr) of the two groups were not different (P>0.05), but theNeuropeptide Y family of peptides (NPY) of PPB100group was significantly higher than LJ-FMgroup(P<0.05). The GH-3h, GH-6h, GH-24h, IGF-I-3h, IGF-I-24h content in plasma were not different(P>0.05), but the IGF-I-6h of LJ-FM group was significantly higher than those of PPB100group(P<0.05). At postprandial6h, the expression of GHRI was significantly decreased in fish of PPB100group (P<0.05); In postprandial24h, the expressions of GHRI, GHRII and IGF-I gene in livers of FMgroup were significantly higher than those of PPB100group (P<0.05); Although the expression of GHgene in pituitary was not different (P>0.05), the expression of GH gene for FM group was lower thanthat of fish fed PPB100diet. The results demonstrated that totally replacement of FM by PPB wouldinhibit the short-term feed intake, the expression of GH/IGF-I axis significantly down regulated withthe absent nutrients intake, which was the main reason for lower growth performance of Japaneseseabass.Comparative studies on the utilization of plant protein of Siberian sturgeon and Japaneseseabass, the effects on growth performance and GH/IGF-I axisTwo16-week growth trials were conducted to comparatively study the abilities of the two species toutilize the plant proteins. Five experimental diets were prepared for each species. In control diet,56.9%and51%of low-temperature steam dried fishmeal (LT-FM) were used as sole protein source to formulated the diets at40%and36%of protein levels for Japanese seabass and Siberian sturgeon, andnamed as LJ-FM and AB-FM, respectively. The other two groups of four diets were prepared to replace25%,50%,75%and100%of fishmeal by the PPB (soybean meal and wheat gluten meal at1:1.67) andnamed as LJ-PPB25, LJ-PPB50, LJ-PPB75, LJ-PPB100and AB-PPB25, AB-PPB50, AB-PPB75,AB-PPB100, respectively. All the diets are isonitrogenous and isoenergetic, and the treated groupssupplemented with DL-methionine, Lysine-HCl, L-Threonine and Ca(H2PO4)2to keep the limitingamino acid and available phosphorus balance. Four replicates for each species with30seabass(7.34±0.01g) or22sturgeon （49.48±1.00g）in each tank. All the experimental fishes were stocked in arecirculating system, fed twice for Japanese seabass or three times for Siberian sturgeon per day. Theresults showed that:1) Both for Japanese seabass and Siberian sturgeon, the fish fed diet LJ/AB-PPB25indicated highest growth performance. However, there were no difference for Siberian sturgeon fedvarious diets, but Japanese seabass showed significantly reduced trend for survival and growth withhigher plant protein levels (≥50%).2) The content of blood respiratory burst acitivity (expressed asNBT reaction), plasma myeloperoxidase (MPO) and alanine aminotransferase(ALT) were significantlyincreased with increasing of PPB levels for Japanese seabass (P<0.05); the diets of higher plant proteinwould inhibite the immunity response of the Japanese seabass might induce liver function damage.However, these were not happened in Siberian sturgeon.3) During the0-12week, the results of SR,FR,and SGR of Japanese seabass was significantly increased with increasing of PPB levels (P<0.05); butduring the12-16week, the fish showed feeding adaptation to the higher plant protein diets, and therewere no differences in five groups (p>0.05); during the stage, the SGR of LJ-FM group was nosignificantly differences with LJ-PPB25, LJ-PPB50and LJ-PPB75group (p>0.05)，but significantlyhigher than LJ-PPB100group (P<0.05) After12week, Japanese seabass could feed high levels of plantprotein diet normally, and the expression of GH/IGF-I axis increasing as the nutrients intake increasing，and growth performance recover. But for Siberian sturgeon, high levels of plant protein diet didn’taffect the feeding and growth performance at all, the content of GH or IGF-I in plasma and expressionof GH/IGF-I axis during all the period of this trail, indicate that Siberian sturgeon could use plantprotein diet effectively.Effects of Replacement of Fish Meal by Plant Protein Blend on Flesh Quality in Japanese Seabassand Siberian SturgeonTwo16-week growth trials were conducted to the evaluate the effects of replacement of fishmeal by aplant protein blend (PPB,soybean meal and wheat gluten meal at1:1.67) on flesh quality of Japaneseseabass and Siberian sturgeon. The experimental diets, experimental systems and feeding andmanagement are same as pre-metioned experiment. The results showed that the moisture and ashcontent of whole body were increased but the crude lipid contents were decreased on Japanese seabasswith the increased replacing levels of PPB. The whole body nutrient compositions of Siberian sturgeonwere not different. Generally, the muscle free amino acids (FAA) content of Siberian sturgeon were much higher than that of Japanese seabass. When PPB replaces75%or lower level of FM, the flavorfree amino acids content in muscle were increased for both of species. However, PPB utilizationincreased the free histidine (the precursor of histamine) of Japanese seabass, but decreased that inmuscle of Siberian sturgeon.Texture profile analysis (TPA) of fillets in Japanese seabass and Siberiansturgeon did not affect by fed with various level of PPB diet, and neither on drop loss of fillets ofJapanese seabass, but drop loss value significantly decreased for Siberian Sturgeon (P<0.05).Furthermore, the content of collagen in muscle for both of species increased in groups of PPB25, butsignificantly decreased for high lever plant protein (P<0.05). High lever (≥50%) of plant protein blendinduce to the flesh quality of Japanese seabass significantly decreased (P<0.05). Totally replacement ofFM did not affect the growth of Siberian sturgeon, but consider the flesh quality, the replacing levelwould not higher than75%.