Effects of three positively buoyant dietary supplements on the buoyancy of feces,growth and intestinal health of Tilapia,Oreochromis niloticus×O.aureus

Hang Yang,Xiaoqin Li,Dianyuan Huan,Zhen Xu,Yi Zhang,Xiangjun Leng,*

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

bCentre for Research on Environmental Ecology and Fish Nutrition(CREEFN)of the Ministry of Agriculture,Shanghai Ocean University,Shanghai 201306,China

cShanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding,Shanghai Ocean University,Shanghai,201306,China

dDepartment of Animal Science,Xichang University,Xichang 615000,China

1.Introduction

In intensive aquaculture, fishes excrete lots of feces that cause a tremendous pressure on the health of the water treatment system and environment.Quickly and efficiently removing the feces from the water is an effective way to stabilize the farming system and ensure successful production.In the culture of Atlantic salmon(Salmo salar),Hillestad,Åsgård,and Berge(1999)reported that the percentage of excreted fecal nutrients into water system,accounting for the ingested nutrients were 13%(protein),8%(fat),40%(carbohydrate),17%(organic matter),50%(ash)and 23%(dry matter).The property of feces,including the density,stability and size,significantly affects the treatment efficiency of feces(Unger&Brinker,2013a).These physical characteristics of feces are closely related with the feed characteristics and composition.Dias,Huelvan,Dinis,and Metailler(1998)found that dietary cellulose increased the feces firmness of European seabass(Dicentrarchus labrax).In Nile tilapia(Oreochromis niloticus),a high inclusion of starch in diet increased the viscosity of digesta and the removal efficiency of feces(Amirkolaie,Verreth,&Schrama,2006).Guar gum has been found to increase the water content in digesta of rainbow trout(Oncorhynchus mykiss)(Storebakken,1985).

In a small-scale feeding system,the excreted feces by fish can be easily removed by siphon,fecal collectors and nets,while a cone bottom clarifier is usually designed to remove the wastes in a largescale feeding system.These methods are designed to clean sinking feces and are not convenient for the rearing enterprise.Unger and Brinker(2013b)found that supplementing 2%cork powder in rainbow trout diets could generate floating feces,which made the feces removal easier and may be a new alternative method to treat water quality in intensive aquaculture.

Buoyant materials can decrease the density of fish feces,but should not affect fish growth,feeding efficiencies and intestinal health.To evaluate the effects on intestinal health and digestion,intestinal histology and evacuation velocity are usually conducted.Thus,the buoyant materials should be indigestible and non-toxic with a low density and high floating rate.Cork,expanded and vitrified micro ball and expanded vermiculite could be considered as the possible candidates for this supplemental material.Cork is the bark of the cork oak(Quercus suberL.),and it has a peculiar cellular structure with low density and good elasticity,and is impermeable to gases and liquids(Anjos,Pereira,&Rosa,2008;Gil,2009;Silva et al.,2005).Vermiculite is a natural mineral,and its volume can expand 8—20 times after heating.Expanded vermiculite(abbreviated as vermiculite)is a chemically inert and fire resistant material with low density(El-Gamal,Hashem,&Amin,2012;Sutcu,2015).Expanded and vitrified micro ball(abbreviated as micro ball)is made from a special type of perlite mineral with porous structure and low density,which is also chemically stable and resistant to fire(Fang,Mukhopadhyaya,Kumaran,&Shi,2011;Ping,Yi,&Gong,2009).

As the second most farmed fish group worldwide,tilapia has provided the increasing worldwide demand for protein sources(Ng&Romano,2013).So,in this study,three buoyant materials including cork,micro ball and vermiculite with fine particles(a diameter of 250—450μm,40—60 mesh,trial 1)orcoarse particles(a diameter of 450—830μm,20—40 mesh,trial 2)were supplemented with varying levels into a basal diet to investigate the effects on growth performance,intestinal histology,and fecal properties of tilapia,to inform a possible strategy for improving the waste treatment efficiency in intensive aquaculture.

2.Materials and methods

2.1.Experimental materials

Three materials,cork,micro ball and vermiculite of two sizes,fine particles with a diameter of 250—450μm(40—60 mesh)and coarse particles with a diameter of 450—830μm(20—40 mesh),were used in trial 1 and trial 2,respectively.The cork was a brown color with a bulk density of 0.12 g/cm3for coarse particle and 0.20g/cm3for fine particle.The microball had a gray color with a bulk density of 0.10 g/cm3(coarse particle)and 0.20g/cm3( fine particle).The vermiculite had a brown color with a bulk density of 0.15 g/cm3(coarse particle)and 0.30g/cm3( fine particle).Cork,microball and vermiculite were supplied by Xinxin Cork Products Factory(Zhejiang),Wangda Building Materials Factory(Jiangxi)and Yanxi Minerals Processing Plant(Hebei),China,respectively(Fig.1).

2.2.Experimental design and diets

The study consisted of two trials:Trial 1 was designed for the fine particles of buoyant materials and tilapia fry and trial 2 for the coarse particles and tilapia juveniles.

Trial 1:Fine particles(250—450μm,40—60 mesh)of cork,micro ball and vermiculite were supplemented into basal diet(control)with 1%,2%,and 3%levels,respectively,and then 10 diets were obtained.The soybean meal and wheat bran inclusion was appropriately adjusted to balance the formula composition.Feed ingredients were ground and then screened through a 40-mesh sieve.The mixture of all ingredients was granulated into pellets with a diameter of 2.0 mm by a single-screw extruder(SLP-45,Fishery Machinery and Instrument Research Institute of the Chinese Academy of Fishery Sciences,Shanghai,China).The pelleting temperature was 90—95°C.All diets were air-dried and stored at 4°C until use.The diet formulation and proximate composition are shown in Table 1.

Trial 2:Larger particles and higher supplementation levels of buoyant materials than in trial 1 were conducted in trial 2.Coarse particles(450—830μm,20—40 mesh)of cork,microball and vermiculite were supplemented into basal diet(control)with 3%,4%,and 5%levels,respectively.The diets were prepared as detailed in trial 1.The diet formula and proximate composition are shown in Table 2.

2.3.Experimental fish and feeding management

Trial 1:Nine hundred tilapia(Oreochromis niloticus×O.aureus)fry with an initial body weight of 5.0±0.1g were allocated randomly into 30 cages(1.5 m×1.0 m×1.2 m)with 30 fish per cage.Ten cages from the ten diets were randomly placed in one indoor cement pool without direct sunshine,and three pools were used.The fish were fed with one of the ten diets three times a day(8:00,12:30 and 17:00)with a daily feeding rate of 5%—10%of body weight.The feed consumption was recorded daily.The feed intake was adjusted appropriately according to the feeding behavior and water temperature to ensure to satiation and no feed residue was left after feeding(in 5min).All cages were maintained with a similar amount of feed intake.During the feeding period,about one third of the cultured water was renewed with pond water after filtration and dark sedimentation every 7 d.The waste in the pools was cleared by siphon every 7 d and water was continuously aerated.Water temperature,dissolved oxygen and ammonia nitrogen levels were 26—32°C,＞5 mg/L,and ＜0.2mg/L,respectively.The feeding trial was conducted at Binhai Aquaculture Station of Shanghai Ocean University(Pudong New District,Shanghai,China)and lasted for 30 days.

Trial 2:Four hundred and fifty fish with an initial body weight of 55.0±1.0 g were allocated randomly into 30 cages with 15 fish per cage.The feeding trial lasted for 21 days.Water temperature,dissolved oxygen and ammonia nitrogen were 24—26°C,＞5mg/L,and＜0.2mg/L,respectively.The cages distribution and feeding management were the same as in trial 1 except that the daily feeding rate was 3%—5%of body weight.

2.4.Measurement indicators and methods

2.4.1.Growth performance and physical indices

At the end of the feeding trial,the total weight of fish per cage were weighed after withholding feed for 24h,to calculate weight gain(WG)and feed conversion ratio(FCR).Three fish per cage were selected randomly to measure body weight and body length individually to calculate condition factor(CF)and then were dissected to weigh the whole intestine(including stomach,but not including liver and other organs)without chyme in digestive tract,to calculate intestine body index(IBI).

2.4.2.Proximate composition of diets

Crude lipid,crude protein,moisture and ash of diets were analyzed following the method of AOAC(1995).The moisture content was estimated by drying the samples to a constant weight at 105°C in a drying oven.The crude protein content was estimated using the Kjeldahl system method(2300 Auto analyser;FOSS Tecator,AB,Hoganas,Sweden).The crude lipid content was determined after ether extraction using Soxtherm(SOX 416 Macro,Gerhardt,Germany).The ash content was determined by combusting samples in a muf fl e furnace at 550°C for 6h.

2.4.3.The histology of anterior intestine

After measuring the intestine weights of the three fish in trial 2,the anterior intestine with a length of 1cm was sampled and immersed in Bouin's solution for 24h,and then transferred into 70%ethyl alcohol.The tissue was dehydrated in a series of alcohol solutions and embedded in paraffin.Then sections(7μm)were cut and stained with hematoxylin-eosin and sealed with a neutral gum.The morphological structures of the tissues were observed using an imaging microscope(Nikon YS100,Japan).The image was analyzed with the Image J14.0 image analysis software,and villus height,cryph depth and muscular thickness were measured.

2.4.4.Sinking velocity of feed and feces

Fifteen pellets per treatment were individually put into a measuring cylinder(a height of 20 cm and a diameter of 14cm)full of distilled water,and the time of pellet sinking from the water surface to the bottom was recorded to calculate the sinking velocity as follows(Chen et al.,2011).

For the measurement of fecal sinking velocity,ten pieces of intact sinking feces per cage was collected by siphoning from the bottom of the cage at the 2nd h after feeding and processed using the method above.

2.4.5.Chyme evacuation velocity

After the feeding trial,18 fish per treatment were stocked in cage and fed the original diet for 3 days to acclimate the environment,and then the fish were deprived feed for 24h.At the 6th,12th,24th,30th,48th and 72nd h after the fish were fed to satiation,three fish per treatment were dissected and the location of chyme was recorded to calculate intestine evacuation velocity by using Wang's method(Wang,Liu,&Wang,2006)as follows.Intestine evacuation velocity(%)=100×The length of empty part before the chyme in intestine/Total length of intestine

Fig.1.The buoyant materials used in the present study(before sieving).

Table 1 Ingredients and proximate composition of experimental diets in Trial 1(as fed basis%).

Table 2 Ingredients and proximate composition of experimental diets in Trial 2(as fed basis%).

2.5.Statistical analysis

All the data were analyzed by one-way analysis of variance(ANOVA)using SPSS version 22.0 software.When ANOVA detected a difference among groups,Duncan's multiple range test was used to identify the difference in the means.Data are presented as mean±standard deviation of the mean(SD).Statistical significance was determined at P＜.05.

3.Results

3.1.Sinking velocity of diets and feces

As shown in Fig.2(trial 2),dietary cork(3%—5%)significantly decreased the sinking velocity of diet and feces and the sinking velocity decreased with the increasing cork level in diets.Dietary microball(3%—5%)significantly decreased the sinking velocity of diet,but not feces,and dietary vermiculite(4%—5%)significantly decreased the sinking velocity of feces,but not diet,when compared to the control.

Fig.2.Effects of buoyant material supplementation on sinking velocity(cm/s)of feeds and feces.

During the feeding period(trial 2),the fish fed diets containing vermiculite(3%—5%)produced few floating feces and the floating feces in micro ball groups accounted for a very small part of the total feces.In cork groups,the ratio of floating feces to total feces increased with the increasing cork level and it was estimated to be 15%—20%.

3.2.Growth performance and physical in dices

In trial 1(Table 3),no mortality was recorded during the feeding period.The weight gain decreased and FCR increased with the increasing buoyant materials in diets.Weight gain of 2%,3%cork groups,3%micro ball and 3%vermiculite were significantly lower,and FCR of 3%cork and 3%vermiculite groups were significantly higher than those of control(P＜.05).At the same supplementation level,the WG and FCR showed no significant differences among cork,micro ball and vermiculite groups(P＞.05).

In trial 2(Table 4),all fish survived during the feeding period.The supplementation of the three materials(3%—5%)significantly decreased WG and increased FCR(P＜.05)when compared to the control.In the groups of micro ball and vermiculite,there were no significant differences in WG and FGR(P＞.05).In the cork groups,the WG tended to decrease and FCR tended to increase with the cork level increasing from 3%to 5%in diets.At the inclusion level of 4%,or 5%,but not at 3%,the cork group showed lower WG and high

Table 3 Effects of buoyant materials supplementation on growth of tilapia(Trial 1).

Table 4 Effects of buoyant materials supplementation on growth of tilapia(Trial 2).

Fig.3.Effects of buoyant material supplementation on intestine evacuation velocity of tilapia(Trial 2).

Table 5 Effects of buoyant materials supplementation on histology of anterior intestine of tilapia(Trial 2).

FCR than microball and vermiculite groups(P＜.05).

3.3.Intestine evacuation velocity

As shown in Fig.3,the chyme in all groups stayed in the stomach or the joint part of stomach and intestine with an evacuation rate of 0%at 6h after feeding.At 72 h,the chyme of control group reached the end of the intestine or excreted out of the body with an evacuation rate of 100%and all buoyant material-supplemented groups showed significantly lower evacuation velocity than the control(P＜.05).In supplemented groups,the intestine evacuation velocity decreased(P＜.05)with increasing buoyant material levels from 3%to 5%.At 12 h,the intestine evacuation velocity of microball groups(3%—5%)were 0%,significantly lower than other groups(P＜.05).At 24,36,48 and 72 h,the groups of 4%and 5%microball showed the lowest evacuation velocity among groups.

3.4.The histology of anterior intestine

As shown in Table 5,villus height,cryph depth and muscular thickness tended to decrease(P＜.05)with the increased supplementation.Supplemented groups showed significantly lower villus height than the control group.The villus height,cryph depth and muscular thickness of the 5%microball group were the lowest among all groups.Fig.4 shows the histology of anterior intestine of control,5%cork,5%microball,and 5%vermiculite groups.

4.Discussions

When rainbow trout were fed diets supplemented with 1%—2%cork or 2%microball for 10 weeks,the growth performance showed no significant differences(Unger&Brinker,2013b).In the present study,the low supplementation level of buoyant materials(1%—2%)did not affect the growth performance of tilapia(trial 1),but the high supplementation significantly decreased the WG,and increased FCR(Table 3,Table 4)(trial 1 and trial 2).Therefore,the negative effects of buoyant materials on growth are closely related to the supplemental level.The buoyant materials cannot be digested by fish and the high supplementation may decrease the nutrients'digestibility and even impair the structure and function of digestive tract.The present study showed that buoyant materials supplemented groups had lower villus height(Table 5),lower feed utilization and growth than the control(Table 4).In trial 2,the cork groups showed lower growth than microball and vermiculite groups,when the supplemental level was 4%and 5%.There are many micropores inside the cork and some nutrients may be embedded in these micropores,producing negative effects on the digestion and absorption.

Two particle sizes of buoyant materials (250—450μm,450—830μm)were conducted in trial 1 and trial 2,respectively.At the same inclusion of 3%,the WG of cork,vermiculite and micro ball groups were 6.2%,6.0%and 4.2%lower in trial 1( fine particle),and 11.8%,4.7%,and 4.9%lower in trial 2(coarse particle)than that of the control group,respectively.Such a result indicates that the coarse cork particles(450—830μm)produce a more negative impact on growth of tilapia than the fine particles,but for vermiculite and micro ball,the particle size,coarse(450—830μm)or fine(250—450μm),shows similar negative effects on the growth.In addition,high inclusion of buoyant materials(5%)significantly decreased the indicator of CF,which was concerned with the growth of these groups.

Fig.4.Anterior intestine histology of tilapia fed control,5%cork,5%vermiculite,or 5%microball diet after 21 days feeding.

In intestine evacuation trial,all the three materials significantly decreased the evacuation velocity and vermiculite groups showed lower values than the other groups.On the one hand,a lowered evacuation velocity means more residence time for digestion and absorption in digestive tract,but on the other hand,the negative effects(including a decreased villus height)caused by buoyant material are more serious,which may be related to a finally lowered digestive ability caused by the intestinal response.Martínezllorens,Baezaarin~o,Nogalesmerida,Jovercerda,and Tomasvidal(2012)found that lower villus height had a lower intestinal protection though the mucus production which acts as a lubricant in the alimentary tract.In addition,these buoyant materials are indigestible and could reduce the moving speed of chyme in digestive tract.Similarly,Adamidou et al.(2009)once reported longer passage times in digestive tract caused by dietary legumes with high insoluble fiber level.

When rainbow trout were fed a diet supplemented with 2.5%cork granules(0.5—1mm),the average single-pass removal by a specially developed surface separator accounted for 78.3%of floating solids,which accounted for 35.4%of total system solids(Unger,Schumann,&Brinker,2015).The study of Schumann,Unger,and Brinker(2016)showed that the feces removing efficiency was increased by 236%,by the supplementation of 2.5%cork granules(0.5—1 mm)in the diet of rainbow trout,when compared to the control.In the present study,the cork groups also produced some floating feces and the amount of floating feces increased with the increasing cork level.As we could not successfully collect all the floating feces,the exact amount of floating feces could not be obtained and it was just estimated to be 15%—20%of total feces,which was lower than the report by Ungeret al.(2015).The difference may be concerned with the size of cork and the supplementation of binding materials.The diameter of cork granules in the study of Ungerand Brinker(2013b)was0.5—1 mm,whileit was 0.25—0.45mm(trial 1)and 0.45—0.83 mm(trial 2)in this study.The bigger cork granules mean more micro pores inside the granules,which could produce more buoyancy.As no additional binding agents supplementation,some cork granules were observed to float in this study.It is possible that some cork granules in feces were freed from feces due to the weak bond of cork granules and other components in feces,leading to the sinking of the feces.Brinker(2007)reported that the fecal stability was significantly improved by the supplementation of 0.3%guar gum in the diet of rainbow trout.The study by Unger and Brinker(2013b)and Unger et al.(2015)also showed that dietary guar gum(0.3%)promoted the formation of floating feces.In the future studies,coarse granules and binding agents should be jointly included in the diet to produce more floating feces.

Dietary cork can generate floating feces,which is related to the physical property of cork.The low density of cork is mainly due to a high porosity and tiny closed cells of suberin that are filled with an air-like gas(Gibson,Easterling,&Ashby,1981).The porosity of cork is relevant with the producing area and environment(Pereira,1988;Costa,Pereira,&Oliveira,2003;Silva et al.,2005).Suberin is a major component in cork which looks like a wax,and it can prevent the water from crossing.A good cork should have a good water resistance and a good elasticity(Zhang,Lei,&Chang,2009),which could produce more floating feces and means the less inclusion level of cork in diet.

During the feeding period,dietary vermiculite produced few floating feces and dietary microball generated only a few floating feces.The floating feces and sinking feces produced in microball groups showed a property of liquid feces,which mean that some visible liquid existed in the feces.It seemed that dietary microballs were destroyed during the extrusion process and irregular fragments were produced.These irregular fragments may irritate the intestine wall,resulting in liquid feces.Unger and Brinker(2013b)supplemented 2%perlite microperl(0.25—0.5 mm and 0.5—1mm)and 1%—2%glass bubbles(0.2—0.85mm and 0.3—1.15 mm)in diets and found that most of the microperl and bubbles were broken by the extrusion due to the high pressure in diet processing.With flexible and elastic properties,cork is not easily broken,or prone to form sharp fragments during the feed processing.

Different materials in diets have different effects on sinking velocity of feed and feces.In the process of extrusion,dietary cork was seldom destroyed and the properties of low density and strong positive buoyance were still kept,thus the sinking velocity of feed and feces were the slowest among groups.Micro ball and vermiculite might be destroyed during the extrusion and then stimulated the intestine to generate liquid feces.Tissue fluid in liquid feces decreased the feces density and reduced the sinking velocity of feces in microball groups.

5.Conclusion

Dietary cork,microball and vermiculite(3%—5%)negatively affect the growth performance and intestinal histology of tilapia,but the supplementation of cork in diet could decrease the density of feed and feces to produce floating feces.

Acknowledgements

This work was financially supported by the Shanghai University Knowledge Service Platform,Shanghai Ocean University Aquatic Animal Breeding Center(ZF1206).

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