Guito Ding,Songlin Li,b,c,*,An Wng,Nisong Chen,b,c,**

a National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai,201306,China

b Research Centre of the Agriculture Ministry on Environmental Ecology and Fish Nutrition,Shanghai Ocean University,Shanghai,20136,China c Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding,Shanghai Ocean University,Shanghai,201306,China

A R T I C L E I N F O

Keywords:

Chicken haemoglobin meal

Growth

Feed utilization

Non-specific immunity

Largemouth bass

A B S T R A C T

A 12-week feeding trial was conducted to evaluate the effects of replacing fish meal by chicken haemoglobin powder in largemouth bass diets.Four isonitrogenous(48%)and isolipidic(12%)diets were formulated to replace 0%(control),9.80%,19.61% and 29.41% of fish meal with chicken haemoglobin powder.Each diet was fed to triplicate groups of fish(initial weight:49.50±0.07 g)twice daily.The fish specific growth rate was significantly reduced when diet replacement level was up to 19.61%,which may be related to the feed intake and apparent digestibility coefficient of protein and amino acids.Meanwhile,the feed efficiency ratio and protein efficiency ratio were not significantly decreased until the replacement level up to 29.41%.The activity of CCP was significantly reduced when 19.61% of fish meal was replaced.Meanwhile,the activity of lysozyme and serum protein content was significantly altered in fish fed with the diets up to 29.41% replacement.Additionally,the red blood cell count and haemoglobin content was significantly decreased when the replacement level was up to 19.61%.In overall,the diet with 9.80% of chicken haemoglobin powder was more suitable for largemouth bass.

1.Introduction

Fish meal is a major protein source in the aquafeeds for carnivorous fish,which have higher dietary protein requirements than omnivorous and herbivorous fish,due to their balanced amino acid profile and palatability(NRC,2011).However,the shortages of fish resources and the increasing price of fish meal have a serious impact in fish aquaculture,and several efforts have been conducted to identify other sustainable protein sources.Plant based diets are alternative sources but when compared to animal protein sources they have limited used due to their relative low protein content and palatability and the presence of anti-nutritional factors and unbalanced amino acids profiles(Bureau,Harris,& Cho,1999;Gatlin et al.,2007;Glencross,Booth,& Allan,2007;Oliva-Teles,Enes,& Peres,2015;Moutinho et al.,2017).

Haemoglobin powder is a by-product obtained from separating haemoglobin from the un-coagulant blood of farmed animals via the low-temperature processing method.This protein possess a high digestible protein content,high lysine and leucine content and excellent pellet binding capacity(Hertrampf&Piedad-Pascual,2000).Therefore,haemoglobin powder may be an alternative protein source for fish aquafeeds and porcine haemoglobin powder has been used to partly replace fish meal in Japanese eel(Lee&Bai,1997a),Nile Tilapia(Lee&Bai,1997b)and hybrid grouper(Yao et al.,2018)diets.Chicken haemoglobin powder is richer in nucleotides when compared to porcine haemoglobin due to the presence of nucleated chicken red blood cell.In some fish species,it has been shown that nucleotide rich diets improve growth performance and disease resistance and enhance immunity(Li&Gatlin,2006;Peng et al.,2013;Tahmasebi-Kohyani,Keyvanshokooh,Nematollahi,Mahmoudi,& Pasha-Zanoosi,2011)and therefore,chicken haemoglobin may be a good protein source to fish aquafeeds.

The largemouth bass(Micropterus salmoides)is widely cultured in China due to its fast growth rate,efficient feed conversion and high market value.The total culture production of China in 2017 was up to 457 thousand tons(Yearbook,2018,p.22).Some studies have been conducted to explore the nutrient requirement of this carnivorous fish,such as protein and lipid(Huang et al.,2017),amino acids(Dairiki,Ctdoss,&Jep,2007;Zhou,Chen,Qiu,Zhao,&Jin,2012)and vitamins(Chen et al.,2015;Li,Lian,Chen,Wang,&Sang,2018)and information on the use of the alternative protein sources to replace fish meal in diets is still limited.Therefore,the present study aims to evaluate the effect of chicken haemoglobin powder in the diet of the largemouth bass by looking at fish growth performance,feed utilization,immunity and haematological index.

Table 1Formulation and chemical composition of the experimental diets(% dry matter).

2.Material and methods

2.1.Experimental diets

Four isonitrogenous(48% crude protein)and isolipidic(12% crude lipid)diets were formulated to replace 0%(control),9.80%,19.61% and 29.41% of the fish meal with chicken haemoglobin(Table 1).Fish oil was added to make up the loss of lipid caused by fish meal replacement.Meanwhile,crystalline lysine and methionine were added to obtain an equivalent indispensable amino acid profile(Table 2),and chromic oxide was incorporated as inert digestibility marker.

All the low-lipid ingredients were mixed thoroughly after being grounded to fine powder through 75μm mesh.Subsequently,the mixed lipid ingredients(fish meal,soybean oil and soybean phospholipid)were mixed thoroughly with the low-lipid mixture and water was added to produce a stiffdough.The dough was extruded through a 2.5 mm die by a pelleting machine.The extruded pellets were dried in a ventilated oven at 55°C after cooked in an oven at 105°C for 15min for starch gelatinization.The obtained diets were stored at－20°C until used.

2.2.Experimental procedures

The fish used in the present study were obtained from a commercial hatchery in Suzhou(Jiangsu,China),and acclimated for 2 weeks in an indoor temperature-controlled re-circulating freshwater system(Shanghai,China)with commercial diet(crude protein,48%;crude lipid,12%).After fasted for 24 h,fish with similar body weights(49.50±0.07 g)were randomly distributed into 12 tanks(800 L)with30 individuals per tank and triplicate tanks were used for each experimental condition.The fish were fed to satiation for 12 weeks twice daily(08:00 and 16:00)with the experimental diets.During the rearing period,all tanks were maintained under a natural photoperiod and were provided with a continuous flow of sand-filtered freshwater(2.0 L/min)and aeration(dissolved oxygen,≥6 mg/L).Water temperature was maintained at 27±1°C,and pH was 7.2±0.2.

Table 2Amino acid compositions(%)of the experimental diets(% dry matter)a.

2.3.Sample collection

Prior to the feeding trials,twenty juveniles were randomly collected for the analysis of initial body composition.After three weeks,faeces samples were collected once daily according to Lee(2002).Briefly,faeces were collected from the faecal collection device attached to the tank before the morning feed,and immediately filtered and stored at－20°C until freeze-dried.Dried faecal samples were ground to powder and sieved to remove scales.At the end of the feeding trials,fish were fasted for 24 h and weight recorded.Fifteen juveniles per tank were randomly selected for further sampling.Before manipulations fish were anesthetized with eugenol(1:10,000;Shanghai Reagent,China).Five fish were collected for body composition analysis,and serum samples were obtained from the remaining ten fish by centrifuging blood samples(4000 g,10 min,4°C)collected from the caudal vasculature using 1-mL heparinized syringes.Viscera and liver weight were measured to calculate viscerosomatic index(VSI)and hepatosomatic index(HSI),respectively,and the liver and muscle samples were collected for proximate composition analysis.The remaining fish(15 in each tank)were fed with the experimental diets for additional five days and from six juveniles per tank blood samples were collected for haematological index analysis using ethylenediaminetetraacetic acid(EDTA)-containing Vacutainers.

2.4.Composition analysis

Moisture was measured by drying samples to constant weight in a oven at 105°C.Crude protein was measured following the Kjeldahl method(N×6.25)(Kjeltec 2200,FOSS,Denmark),and ash determined by combustion to constant weight in muffle furnace at 550°C(AOAC,2003).Crude lipids were determined using the chloroform-methanol extraction method(Folch,Lees,& Sloane Stanley,1957)according toPeng et al.(2014).The amino acid level was determined with the acid hydrolysis method using the L-8900 amino acid analyser(Hitachi,Japan).Chromic oxide was determined with the wet-acid digestion method to determine the nutrient digestibility coefficient(ADC)(Furukawa & Tsukahara,1966).

Table 3Growth performance and feed utilization of largemouth bass fed with chicken haemoglobin powder diets for 12 weeks*.

2.5.Immunity index

The activity of lysozyme was measured using the turbidimetric assay method(Ellis,1990)following Zuo et al.(2012).Briefly,the suspension ofMicrococcus lysodeikticuswas used as the substrate,and the absorbance was spectrophotometrically measured after 0.5 and 4.5 min,respectively.Each unit was defined as the amount of serum sample that caused a decrease in absorbance of 0.001 per minute.The activity of classical complement pathway(CCP)was measured according to the method by Inglis,Radziwon,and Maniero(2008)described in Zhou et al.(2012).Sheep red blood cells were chosen to measure the haemolysis of CCP,and the serum volume that caused 50% haemolysis(CH50)was determined and the number of CH50 unit per mL serum calculated.The total serum protein content was determined according to the Bradford method(Bradford,1976)using bovine serum albumin as standard.

2.6.Haematological index

Blood samples were diluted with Grower's solution(Allan,2000)and concentration of red blood cells was measured using the Neubauer haemocytometer chamber.Haematocrit was determined using microhematocrit technique according to Sandnes,Lie,and Waagbø(1988),and the content of haemoglobin was measured at 540 nm using a spectrophotometer according to the cyanmethemoglobin method(Bradford,1976).

2.7.Calculations and statistics

The following variables were calculated:

The results were expressed as mean±standard error of the mean(SEM).Statistical significance of the data was inferred using the oneway analysis of variance(ANOVA)with the software of SPSS 19.0.Tukey's multiple range test was chosen as multiple comparison test and a significance level of 5% was used.

3.Results

3.1.Growth performance and feed utilization

The SR was not altered with the inclusion in the diet of chicken haemoglobin(P＞0.05).The SGR was not significantly reduced in fish fed with the 19.61% chicken haemoglobin diet(P＜0.05)(Table 3).The inclusion of chicken haemoglobin meal significantly decreased HSI(P＜0.05).Meanwhile,the VSI significantly decreased in the 0%(control)and 9.80% chicken haemoglobin diets(P＜0.05),and thereafter reached a plateau(P＞0.05)(Table 3).

The FI remained slightly reduced when 19.61% fish meal was replaced(P＞0.05)and then decreased significantly with the further increase of inclusion level(P＜0.05)(Table 3).Meanwhile,the 29.41% chicken haemoglobin diet produced a significant lower FER and PER when compared with other treatments(P＜0.05)(Table 3).Although the ADC of protein was significantly reduced with the inclusion of chicken haemoglobin(Table 4),the fish that were fed the 9.80% chicken haemoglobin diet had the highest protein retention PRR(Table 3).The ADC of Thr,Met,Leu,Phe,Lys and Arg decreased significantly with the introduction of chicken haemoglobin(P＜0.05)but the ADC of Val,Iso and His was only significantly decreased with the 29.41% chicken haemoglobin diet(P＜0.05)(Table 4).

Table 4Apparent digestibility coefficient of protein and essential amino acids in fish largemouth bass fed with chicken haemoglobin powder diets for 12 weeks*.

3.2.Proximate composition analysis

Chicken haemoglobin diets had no significant effect on moisture and ash content of the fish carcass(P＞0.05)(Table 5).The crude protein content in fish fed the control diet(without chicken haemoglobin)was significantly higher than in fish fed with the 18.61% chicken haemoglobin diet(P＜0.05),while fish fed with 9.80% chicken haemoglobin diet showed the lowest crude lipid content(P＜0.05)(Table 5).The crude protein content in muscle followed a similar pattern to that of carcass,while led to a significant increase of crude lipid,moisture and ash content(P＜0.05)(Table 5).The replacement of 9.80% of the fish diet significantly elevated the liver moisture content when compared to the control(P＜0.05),and the further increase from 19.61 to 29.41% significantly decreased the moisture content(P＜0.05)(Table 5).Fish fed with the 9.80% chicken haemoglobin diet possessed the highest crude protein content but the lowest crude lipid content(Table 5).Meanwhile,the liver ash content was remarkably reduced with the increase of chicken haemoglobin(P＜0.05)(Table 5).The content of Thr and Arg was significantly reduced with the degree of fish meal replacement(P＜0.05)(Table 6).Meanwhile,the increase of chicken haemoglobin significantly decreased the Ser,Gly,Ala and Pro content(P＜0.05),but significantly increased the Cys content(P＜0.05)(Table 6).

Table 5Proximate composition(% wet weight)of largemouth bass fed with of chicken haemoglobin powder diets for 12 weeks*.

Table 6Amino acids composition(% dry matter)in carcass of largemouth bass fed with chicken haemoglobin powder diets for 12 weeks*.

3.3.Immunological parameters and haematological parameters

The activity of lysozyme was not significantly altered in fish fed altered until the inclusion of chicken haemoglobin up to 29.41%(P＜0.05)(Table 7).The serum protein content followed a similar trend than the lysozyme activity(Table 7).Meanwhile,the activity of CCP was significantly reduced in fish fed with the 19.61% chicken haemoglobin diet(P＜0.05)(Table 7).The red blood cell count and haemoglobin content significantly decreased when the replacement level was up to 18.61%(P＜0.05)(Table 7).Meanwhile,the haematocrit significantly decreased in all chicken haemoglobin diets(P＜0.05)(Table 7).

4.Discussion

Haemoglobin powder has been considered a good alternative protein source in fish diets due to its high digestible protein content and function in binding pellet.It has been reported that porcinehaemoglobin powder could partly replace the used of fish protein in diets for Japanese eel(Lee & Bai,1997a),Nile tilapia(Lee & Bai,1997b),gilthead sea bream(Martínez-Llorens et al.,2008)and hybrid grouper(Yao et al.,2018).Similarly,in the present study the SGR was not significantly modified until introduction of chicken haemoglobin powder up to 19.61%,and based on the fish growth performance the 9.80% replacement may be a good alternative for largemouth bass.

Table 7Non-specific immunity and haematological parameters of largemouth bass fed with chicken haemoglobin powder diets for 12 weeks*.

In the present study,the FI was progressively decreased in fish fed with the 9.80%-29.41% chicken haemoglobin diets suggesting the unpalatability of high haemoglobin powder content.The reduced FI may be associated with the reduced SGR as a consequence of the introduction of chicken haemoglobin.Previously studies in the hybrid grouper also showed a remarkable reduced of feed palatability and fish appetite with the increase use of porcine haemoglobin(Yao et al.,2018).The utilization of blood in the diets significantly reduced the FI of hybrid tilapia(Fasakin,Serwata,& Davies,2005),nonetheless the FER was not significantly decreased until the inclusion of 29.41% chicken haemoglobin.Meanwhile,the FER and PRR were only significantly reduced with the replacement level of 29.41%.Therefore,haemoglobin powder could partly replace the use of fish protein in largemouth bass feeds and addition of feed attractant would further improve the use of haemoglobin powder in aqua feeds.

In general,the amino acid profile affects the growth performance and feed utilization of farmed fish.Despite supplementation of crystalline lysine and methionine to achieve an equivalent indispensable amino acid profile of the diets,the ADC of amino acids was progressively reduced with the introduction of chicken haemoglobin powder.However,in previous studies the whole-body crude protein was not significantly affected when using porcine haemoglobin powder in fish diets(Lee&Bai,1997a,b;Martínez-Llorens et al.,2008).In the present study,the HSI was not changed with the inclusion level up to 19.61%.However,the introduction of porcine haemoglobin in gilthead sea bream(Martínez-Llorens et al.,2008)and hybrid grouper(Yao et al.,2018)diets increased HSI,probably due to the conversion of amino acids to glycogen or lipid(Marcouli,Alexis,Andriopoulou,&Iliopoulou-Georgudaki,2004).However in Nile tilapia and Japanese eel no significant influence on HSI occurred(Lee & Bai,1997a;b).

Chicken haemoglobin powder is enriched in nucleotides,which has a potential role in improving growth performance and enhancing immunity and disease resistance in some fish species(Li & Gatlin,2006;Peng et al.,2013;Tahmasebi-Kohyani et al.,2011).In the present study,the activity of lysozyme and protein plasma content was significantly reduced only when the replacement level was up to 29.41%.Meanwhile,the activity of CCP was significantly reduced in fish feed with the 19.61% diet.The haematological parameters in response to the inclusion of haemoglobin powder was also determined to reflect the health condition of fish(Kim et al.,2017).The red blood cell count and haemoglobin content was not significantly reduced in the fish fed with the diets until the inclusion level up to 19.61%.Therefore,the replacement of 9.80% fish meal with chicken haemoglobin powder did not produce negative effects on the non-specific(innate)immunity and haematological parameters of largemouth bass.

In conclusion,at least 9.80% of the fish feeds for largemouth bass may be replaced by chicken haemoglobin powder.Higher introduction of chicken haemoglobin powder significantly reduces the growth performance,feed utilization and health of largemouth bass.

Conflicts of interest

All authors declared no conflict of interest.

Acknowledgement

This work was financially supported by China Agricultural Research System(CARS-46).