Aquaculture 552 2028) 738827 Contents lists available nt ScienceDirect Aquaculture ELSEVIER journal homepage: www.elsevier. comilocate/aquaculture ® Comparison of growth, and non-specific immune of crisp and ordinary sae tilapia (Oreochromis niloticus) Qingqing Li®, Yao Huang", Xi Xie”, Xiaogian Zhai", Meiyan Chen", Dejin Liang‘, Ming Cao", Shaowen Liang", Li Lin" * cumgtrg Petal War Berner od Agua Pros Ser Breer Telegy Research Cer, Cuan Key Laorataryaf Aquat Anal Dias Wanton! Bed, Cole of Anal Scene Tcl, Zhang Unheraty of Ape and Bagi, Cua, Ounpng Poe © Cole of Lie uy and Food, Zhang! of Alar and Engrg, Cuno, Cuando Prove S125, Cina < naa Riga Bren Fane Guns Proc, Guneson, Gump Pov 145% Chie Guana Fup! Sip Chain Mangan Co Le, hogan Ouanpng Pvc 20400, Chon ARTICLE INFO ABSTRACT owns “nia (Ovni ion) en kno wie famed ih a commeny cued amore a & Sr tuned ations The ain deny faba Dean (Wea ab) fr 90-120 proves nse gay sich a8 fa ten ghost ness nein, Howere, fabs bok conan wie sey of nto icra sich — ‘pheno as yp ber acy, ad peal Ainuona atx Quagh ieneles ‘heir metaboleprodues aft the health of fish. This study was compaced growth, atloidant activity, carbon rmetabli enaymtesetivity and nine elated gene expression profes beeen the ordinary a eis tapi. ‘The rests nceated that esp tap (688.00 + 200.83 g) ha significa lower body weight compared with cedar tapi (1078.67 £162.56 g) P< 0.05). Thete wee no sigiicant eiferences in speconide dsmase (SOD) and atioxdanteapacty (T-ADC) berween esp and ordinary tapi in bh serum and liver. The esp ‘lap tecoded signicanly lower sea phospiatse (ACP, and alkaline phosphatase (AKP) in coatast othe ‘diary apa i sera (0.05), bt ot sigan in ve. The ei lpia (serum: 805+ 1.23 Uri, liver: 7.99 1.09 U/ml) expose could sigiieanly Inhibit glucose 6-phorpliatedeiydiogenase (GOPD) a. Uiiles compared with o1dinay Hlapia (Senn: 5.07 £091 Ura, liver: 410 £1.18 U/ak) (P< 005) The Iysoayme (LZM) and eighewide (TG) of ordinary and exsptlapia were sina. The heat shook proeia70 (sp70) and wimor nerensfctor® (TNFa) expression a esp tapia demonstated significa upregulation when compated with eudinay apie Inthe haemoxytes and ler, wheres the Ineveukia18 (metal Tothionela (MD, and chemokine CCMotf Ligand 3 (CCL) expression of csp tapie were sigaldeanty evegulated inthe liver. tn conclusion, the cisptlapia can decrease gowrh, ACP, AKP, and GOPDH activi, laheteas Hsp70 aid TNF expression of exisps demoastaced significant upregulation. The results would conubute f© comprehend the physiolgical parameters ding tapia cisping process and exploring the ‘mechanisn of wala britenes 1. Introduetion production of tilapia rose from 2.6 million tons in 2010 to 4.5 million tons in 2018 (FA0, 2022) The quality of farmed tilapia flesh as dete The second-most farmed fish in the world after carp is dlapia (Oreochromis loricus) since it has a higher stess tolerance in compar Ison to the majority of the species of fishes, with hypoxia and high: density eulture occurring in the practical tilapia culture (Absel Taw wab etal, 20145 Shi et al, 2020). In more than a hundred nations tilapia has been commercially farmed, and the global aquaculture * Conesponding author mal adie ney ia.cdu.n (Le) ps /door/10.1016/Jaquacutuse.2022.738807 Hlorated over time asa result of intense aquaculture, whieh has been & source of public consideration (Ly et al, 2021; Qin et al, 20125 Yilmaz, 2019). Getting better quality fish products while keeping sustainable aquaculture system has become a major industrial goal (ian etl, 2019) ‘Muscle is @ vital component fr fsh quality study since i i the Received 12 Apill 2022; Received in revised for 8 September 2022; Accepted 10 September 2022 ‘Availabe online 15 September 2022 ‘0044 8486/1 2022 Pulsed by lever BYate biggest edible fish element (We! etal, 2020). On the basis ofthe goal of ‘enhancing mouthfeel and profitability, Ulapia dietary faba bean (Vita faba 1.) was differed from ordinary tilapia, exhibiting higher muscle hardness and erispiness, and higher total protein content 928 & 200% Lie a, 20225 Lan et a, 20075 Xie etal, 2021). Faba bean ‘consumption resulted in changes in muscle hardness not justin tilapia but likevsise in other species of fish (Feng el, 2016 Vii tal 2020) ‘Therefore, fish muscle quality improvement by feeding fab bean is gaining inereasing research interest. The special embrittlement feed primarily consisting of faba bean, flour, vitamins, minerals, ete. for Ulapia was developed, whieh can achleve crisp dlapla (Qin al, 20125 Ale eta 2021). However the faba bean seed contain various proportions of antinuritional factors ‘sul as phenolies, annins, trypsin inhibitor activity, and phytic acid (raneis el, 2001). Antinutritionsl factors through themselves or the metabolic products interfere with food wrization and affect the health fof fish (raneis eta, 20015 Meng etal, 2021). Chronic toxicity of ant huivtional factors may lead to lower aceryleholinesterase activity, ‘oxidative stress, and glutamic excitatory intoxication in aquatie animal (Tan and 1, 2006). One of the most serious negative effects of faba bean feeding in fish eultur isthe blocking of the blood ereulation system (Tan and Li, 2000), The previous studies showed that excessive or long term faba bean consimiption has numerous negative impacts on grass ‘arp (Crenopharyngodon idelis), ineliding metmbolie disorders, hemo lysis, and liver damage (a tal, 20202). The faba bean can change the nscle quality of tilapia, although the physiological state of crisp is not ‘elucidated yet in tilapia In this study were investigated grovth, antioxidant capacity, carbon, metabolic enzyme activity, and relative gene expression levels. of immune-selated genes between crisp tlapiefed approximately 60% faba bean and ordinary tilapia. 2. Materials and methods 2.1. Sample collection The crisp and ordinary tilapias were obtained from the farm in ‘hougshan, China. The tapias (mean weight was 500 + 16.65 ) fom. ‘each group were randomly stocked ino three replicate outdoor earthen, pnd for 100 days. The erisp and ordinary tilapins were fed commercial ‘embrittlement feed (Zhongshan Taishan feed Co., Ltd) with approx: rately 6086 faba beans, and formula feed (Zhongshan Taishan Feed Co, {) containing no faba beans respectively. The iapias were fed twice & ‘day at 9:00 nnd 16:00 with fatios of sound 9 9 of total fish biomass, ‘adjusted according to weather, residual feed, and water temperature. After rearing 100 days, the ity fish from each group (risp and or inary) were randomly collected. A digital balance was used 10 record ‘each tlapia's body weight to the closest 0.01, and the body length of blapias was recorded by a digital verniercalliper ta the nearest 0.01 1mm. Fish thar were randomly picked were anesthetised as soon as ‘possible in a 10 L plastic bucket with 20 mg/L clove oil (Iversen etal, 2003). Blood samples were obtained carefully and slowly afer the fish Were starved for one day. Through the use of 2.5 ml plastic injectors, blood samples from each sampled fish were taken through the caudal ‘vein, Blood samples for serum were left a room temperature for three hours before being centrifuged (5000 rpm, 4 °C, 10 min) and stored at ‘80 °G ill the serum biochemical indexes were determined. For blood cells, blood samples were drawn from the caudal vein using an ant: coagulating substance by injector puncture, followed by centrifuging .a€ 5000 rpm and 4 °C for en mins. The obtained cell was stored at 80 °C until further processing. The sampled fish liver vas quickly taken once the blood was draw. The vscern were weighted after being collected and the ratio was eaenlated asa percentage of body weight ‘The condition factor (CF) and viscerosomatic index (VSI) were: Agua 562 (2029) 728627 CConition factor (CP, 2/em") 100 (boty wish, e)/(boty length, cm) Viscerosomatc inex (VSI,%) =100 x (viscera weight g)/ (hole body weit). 22, Antioxidant activity analysis Total antioxidant eapaeity CT-AOC), superoxide dismutase (SOD), malondialdehyde activities (MDA), catalase (CAT), and glutathione peroxidase (GSH.Px) activities were chosen and rested inthe serum and liver uiicng test its as directed by the instructions of tae manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, Chine, 23. Carbon metabolic enzyme activity analysis CConumercil its were used o measure te levels of aid phosphatase (ACP), alkaline phosphatase (AKP), triglyceride (1), and lysozyme (L200 in the liver and serum. Jian Cheng Institute of Biotechnology Nanling, China, provided these kits, Reitman-Franke's approach was wed (0 determine creatinine (Cre), alealne atnotranserese (ALT), aspartate transaminase (AST) levels Inthe liver and serum. ELISA kits wore used to detect the activites of the metabolic enzyme glucose 6 phosphate dehydrogenase (GOPD) in the liver and serum, according to the manufacture’ instructions. MIbio Shanghai, China, provided these kits 2A. Gene expresion analyses ‘Quantitative real-time PCR (qPCR) was used to determine the rel tive mRNA genes love related to mietabolism,astoxidants, aflame tion, and immunity (Table 1). RNAIso Plus reagent (TaKaRa Bio, ne Dalian China) was used (0 extract total RNA fron liver and blood ells Using agarose gel electrophoresis and measuring absorbance values at 260 nny anid 280 nm, the concentration, and quality of total RNA were determined (A260/A280 rato, 1.8- 2.1) 500 ng of total RNA was ut lized to perform reverse transcription (RT) through the use of Prime- Script™ RT Rengent Kit Ver2.0 (TaKaRa, Japan) for synhesising eDNA, fs per the protoros of the manufacturer. The progeam was se to run for one cyele at 95°%C/80s, then 40 cycles at 95 °C/5 5, and 60-69 °C mi. ‘The following is the amplification reaction (25 pL): 12.5 wl of TB Green Premix Ex Taq If Takata), 2 ul of CDNA, 2 iL of eDNA, 8.5 pL of stele Purified 1,0, and 2 pl of eDNA. puactin was employed as a reference ene for calculating the relative mRNA level by the 2° method. “Table 1 presents the tlpin primers used in this study. ‘Table Sequences of primers use in this study. Pres Gee name Seances Paste pact ' CAGUGAGARGATGACOGAGA Lap retin F-cacacccxanacacaace CLS Chemokine COM Linad SF ACAAGCAGEACAACAACAACCAate 25, Statistical analysis SPSS software (version 20.0) was used to calculate the mean = standard deviation (SD) of the data. Student's rest was utilized for ‘examining the disparity among two treatments, in order to establish statistical significance between attributes of crisp and ordinary tilpin. < 0005 was deemed statistically substantial diferences 3 Results 3.1, Tissue inces ‘The ersp tilapia (888.00 + 200.83 g) recorded significantly lower body weight in comparison tothe ordinary tilapia (1078.67 4. 162.56 g) at harvest (P < 0.05). The crisp and ordinary tilapia had similar body length. The CF and VSI of erisp dlapia were lower dhan ordinary tapia, however, no at significant levels (rable 2), 3.2. Antioxidant parameters For the serum, the SOD, T-AOC, and CAT activites of erisp tilapia ‘were higher than ordinary tilapia, but not at significant levels, The crisp tilapia (2.13 + 0.52 nmol/L) recorded significantly lower MDA value in contrast to the ordinary tilapia (8.00 40,69 amol/mL) (P< 0.09). ‘The GSH-Px aetivity of crisp tlapia were higher than ordinary apa, bur nor significant able 5) For the liver, the erisp tilapia (101.28 4. 7.36 U/mg protein) recor: ded significantly higher CAT activities than ordinary tilapia (50.03 11.12 U/mg protein) (® < 0.05). Similarly, the ersp tilapia (18.40 5.96 U/mg protein) recorded significantly higher GSH Px activities than ‘ordinary tilapia (11.12 + 1.81 U/mg protein) (P< 0.05). The evisp and ‘ordinary tilapia had similar T-AOC aad SOD activites, with no statistic dlffeence in MDA value (Table ®) 3, Carbon metabolic ensyme activity For the serum, the crisp tilapia (3.81 + 1.31 king wnit/100 aL) recorded significantly lower ACP compared with ordinary tapi (4.68 241.38 king unlr/100 mL) (P = 0.05). Likewise, the AKP of eispelapia (1.21 + 0.65 king unit/100 mL) was significenty lower in comparison to ordinary tilapia (3.60 + 1.81 king unit/100 ml) P < 0.05). The LZMt and TG of esp tlapia were slightly lower than ordinary apa, but not significant levels. The crisp tilapia (5.53 + 2.40 U/ml recorded significantly lower AST compared with ordinary tilapia (9.81 2.66 U/ mi) (P < 0.05), Similarly, the ALT of ersp tlnpin (9.45 © 3.51 U/mL) ‘was substantially lower then ordinary tilapia (15.48 = 8.36 Umi) (P< 0.05), Whea compared to ordinary tilapia (8.05 4 3.70 U/mL), crisp tilapia (5.07 + 2.58 U/ml.) exposure could dramatically suppress G6PD (@ < 0.05). Compared with the ordinary group (22.98 10.50 nol” IL), Cre was decreased in the crisp group (14.76 + 5:92 umol/m) P< (0.05) Cable 4, For the liver the ACP and AKP of crisp tilapia were slighty lower ‘hn ordinary tilapia, bur not a significant levels. The LZM and 76 of “Table 2 ‘Compasison of growth pesfonnance (mean + SD) between otdinay and esp tilapia Orono Gp 15) ody ig e) 787 = 1 ‘800 120080" Bey ent em) Manas ‘ruts 2.16 con 267 = 048 aati oat ys) 7262239 eaeaaa ‘Nove: data with aifren ees ae sgalieantydifrene (P = 005) berweea treatment, and theno lees ae noe sigalscandy leven. CF, condition actor, I, iserosomatie ie. Agua 562 (2029) 728627 ‘Table ‘Comparison of antioxidant indexes (mean + SD) in serum and liver between inary and rsp tilapia Omochris ics me rds cepa) S00 unt) saa0759 36902738 TA0C U/mt) 5055159 sores car cum arm 1a70 ans DA (ose?) oo eos bias oss 500 Wing pce) e204 543 ones +298 "Toe (Um pete) 2aso. a= 008 (OAT (Ung pei) Soares fotos 7268 MDA CU pein asze06 0762028 (ik (ig prt) asia tado-e sa ‘Nove data with fear ieers ae sgafcantly fren nthe same sues ©0005) between teatments, ad the no lees ae no sigan ferent. SOD, Sperone dimitas; AOC, total antioxidant capacity: CAT, catalase MDA, malondialdehyde activities; GSH. Px, glutathione peroxidase ‘Table a Comparison of carbon metabolic enzyme activgy (mean = SD) in serum and ver betwen ordinary and esp tapi Orvchrams oes Teme ‘rai (5) Spe) aera) ost 268" ssasa40? aArwad 1546-2836" bua $350 ‘c6e0 (uma) Sos = 370" Sar 256 re pmol mt) 7296 + 1050" irereey ACE hing wi100 mt) sss 138" sation KP ung vi100 mL) 60-181" Tat Foss? Tat oem) 88 1840 36 21046 Team) oneom azo Loa AST (u/ prec) 208-1982 aust e116 AUT (Ug pecan) wasnt bere ae {G6PD (mg rte) 7594219" those (i pmol ng pre) poo +60 iso 26 AC (mg pee) host 445.8 fost ear7i ‘A (Ung pete) nse go20 4428 La agra tele) hizsoa tis 095 Te Gamal me ren) 025 £005, 21 s005 ‘Nove: data with tfeear ies ae sigafcanly ifvent nthe same sues “0.05 between teatments and the no letters are not significantly dive AST, aparate transaminase; ALT, alaine aminotransferase; GOPD, glicore 6 Phosphate dehyerogenase; Cre, eteatinne: AC, acid phosphates; AKP, alka Tne phosphatase; L2M, sozyie, TG, uglyesie. ordinary and eispsilapia were similar. The AST and ALT of ersp tilapia were lower than ordinary tilapia, but not at significant levels. The crisp Ulapia (4.10 4 2.35 U/mg protein) exposure could significantly inhibit GGoPD in comparison to te ordinary tilapia (7:59 + 2.19 U/mg protein) (@ < 0.05). The Cre of crisp tilapia were lower than ordinary api, but not a significant level, (Table. 3.4, Immune related genes expression nthe eisp and ordinary ilapia haemoeytes, expression pattems of Immune related genes in haemocyte were studied (Fig. 1A). The Hsp70 and ILIP expression of crisp displayed substantial upregulation compared with ordinary tilapia (P < 0.05) tn contrast tothe ordinary ‘api, the Mand CCL expression were clearly increase inersp tilapia (@ < 0005). Sinilany, the 1FN-y and TNF-x expression of erisp tlapia were significantly higher then ordinary tlapia (P < 0.05). Inthe liver dsstes of crisp and conventional api, mane related genes expression profiles were studied (Pig, 1B). The crisp IL-1 expression revealed substantial downregulation compared with ord raaty dlapia (P < 0.05), whereas Hsp70 expression was signifiemndyate A) Blood TLAB WspT0 CCL3 MT IFNy TNF-a Genes Agua 562 (2029) 728627 B) Liver : 1 Ordinary mm Crisp mRNA relative expression 1p Hsp70 CCL3 MT IFNy TNF-a Genes Fig. 1. qRT-PCR analysis of inmune related gene expression proles in bod (A) and liver (B) benween ordinary and crisp Hapa Orencvamis ios. * indicate sgniieantditernees ((P < 005) upregulated (P< 0.05), Compared with the ordinary tap, the NT and ‘CCLexpression ofthe liver were evidently decreased in crisp tilapia (P< 1005), Cis ish ad considerably more TNF-a expression than ordinary tilapia (P < 0.05) 4. Discussion Compared with soybeans, faba beans are less employed in aquatic ‘animal feed and are solely utilized to improve the muscle quality of fish suchas grass carp, and tlapia. Fish have been raised by faba bean, ‘whose characteristics involve tightened and crisp fresh quality, as well, as delicious taste (Feng etal, 2016), The faba Dean impact on growth Performance, nutrient content, physiological, biochemical index, and digestive enzyme during fish cisping process (A228 et al, 200% Ma ft al, 2020a5 Tan et al, 2019). En tis study, the body weight of eisp tapi was significsntly lower than ordinary tlapin followed by com: mercial embrittlement feed for 100 days. This could be de to the fret that faba bean contains ant-mrtional factors, such as tannins, trypsin Inhibitors, ete. whieh reduce the absorpion and wlization of nutrients in the intestinal, resulting in a decline in fish development performance (\leng etal, 2021). The methionine was the one of limiting essential ‘amino aeid in faba bean for African catfish (Cloriasgorepins} (Piesho ft al, 2021). The previous studies also showed thatthe faba bean re strains the growth performance of tilapia and grass erap (Qin el. 20125 Tian etal, 20195 Tian etal, 2020). IC likely that dhese ant nrients might have impaired the absorption of some essential amino ‘acid components ofthe diets containing faba bean, thus causing growth depression in lap Heemolyss is known to take place as inthe ease of “visi” after dietary raw faba bean (Geviciew et a, 2018). A potentially Iie threatening acute hemolysis manifesting reduced RAC coxnt catsed by low GoPD activity by ingestion of faba Dean. The GOPD activity of crisp ‘lapias inthis study was significantly lower than ordinary tlapis after ‘commercial embrittlement fee for 100 days, suggesting thatthe long tert fed faba bean may distupt carbohydrate metabolism in the Ulapia liver, The previous study also reported that faba bean reduced {GSPD activity inthe grass crap (Fu ta, 2020). GEPD catalyzes the frst and rate limiting step in the pentose phosphate pathviay, which pro ‘duces nicotinamide adenine dinucleotide phosphate (NADPH) (Zhcug ce , 2021). The NADPH enables glutathione reductase (GSSC-GR) t0 ‘convert oxidized glutathione (GSSG) to reduced GSH-Px, which reduces the level of 02 (Hoonyuen et al, 2017). Inthe present study, The GSH- Px activity of tsp tlapla were lower chan ordinary tapi, A decreased Inthe GoPD activity affects the decreased of the GSH-Px and leading to ‘an increased in HO, which indicating oxidative damage and hemolysis may in the blond cells of the crisp tilapia. The excess levels ROS are known to affect the textural quality of fish (ia et al, 20201). Our previous study also showed dhat dietary faba bean cat improve the textural quality of tilapia (xe tl, 2021). There were not signifeant at the SOD, and GAT activities Derween ersp tilapia of ordinary tilapia, further supporting the oxidative damage and hemolysis may in the crisped tilapia. aba bean has been shown to influence metabolic enzymes involved i earbohydrace and lipid atetabolism in fish CY. e< al, 2020), and changes in metabolic enzymes have been ulized to analyse faba bean Coxicity meehenisnis. ACP is a lysosomal enzyme that is involved in the intracellular breakdown of phagocytized antigen and has been euployed 2s # macrophage activation marker in oti Invertebrates and ‘amnvals (Song el, 2000). AKP isan extracellular enzyme that hy- Arolyses phosphoester bonds in earbobydentes, proteins, and lipids. The activity of ARP and ACP, as per our research slightly decreased in the crisp tilapia than ordinary dlapia, but uot significant in the liver, whieh suggested ha the muscle quality may be diferent between esp tapi and ordinary tilapia. This may be due to the faet hat the commercial bmubritlonient feed contains faba benn and other feed ingredients. Tunior necrosis (TNFS) and interleukins (Ls) are examples of eyto kines, which are proteins that regulate signalling amiong several cells result in an immune response, and have @ erucial part in imme mechanism regulation (Lee etl, 2019). The expression Tevels of immunerelated genes in the liver and blood of tilapia have been measured in tls study after fed commercial embritlement feed with about 60% faba beans. Crisp Hsp70 and TNF-a expression were Aramavcally upregulated in the liver a compared to ornary tala, Whereas the 118, MT, and CCL expression of crisp tlaia were signif. leantly deregulated in the liver. The grass carp fed single faba bean, on the other hand, indicated that the expression of te IL-1, Hsp70, and ‘TNF-a demonstrated a similar rise in the liver (et sl, 2015). The ‘TNFa levels were raised in the grass earp testinal fed faba bean (1) cs, 2018). This study pointed ont that the embrittlement feed did cause damage and caused immune upregulation in the liver of tilapia 5. Conclusion In summary, crisp tilapia recorded significantly higher body weight, compared with ordinary dlapia at harvest, There was no significant ditference in the SOD, and T-AQC between crisp and ordinary in both serum as well as he liver. The crisp tilapia recorded lower AST, ALT, and CG6PD netvities compared with ordinary tilapia, The ndings contebuteate to a better understanding of exploring the physiological mechanism of ‘muse ersp in the clap, ‘RediT authorship contribution statement Qingging Li: Conceptuaization, Writing ~ origina draft, Funding acquisition. Yao Huang: Investigation, Data curation. Xi Xie: Software ‘Xiaogian Zhai: Investigation, Meiyan Chen: Formal analysis. Dejin Liang: Supervision. Ming. Gao: Investigation. Shaowen Liang: Re sources. Li Lin: Supervision, Writing ~ review & editing, Funding acquisition. Declaration of Competing Interest All authors declare no conflict of terest. ‘The authors do not have permission fo share data Acknowledgments ‘This work was supported by Guangdong Province Commissioner for ‘Rural Science and Technology in Towns and Village {KTP20210236);, ‘Key Research and Development Program of Guangzhou City 202103000067; Public scence brand and standards establishment for Huadu Fishery Industral Park (21902156) References, Abdel: Tawwab, ML, Hagias A, ape, HAM, Monier MA, 204 Dino ‘ryen cel aod cing ea cen geod anton pyaay, nd et SW Abie A ii Re Ke M2 Eras of fit ans (Vi fob or mina) a epicentre teal in race of vee ie lpi Oreos ae Ager 287, was, Boonen hams, Swangs Tne, Ty Day PJ, Whe Na mon, 2017 A wae off eam ett etyond protein say seri cia mann Be pe thor esha FE, Rtbkel5Sute, DH, Nota. 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