Safety Evaluation Of Benzoic Acid In Piglets

Benzoic acid,as an organic acidifier and preservative,has been widely used in the feed and food industry in recent years.Dietary proper benzoic acid supplementation can protect health,improve growth performance and keep intestinal microorganism balance of livestock.However,few studies have found that excesssive benzoic acid intake may impair the health of animals and humans.Besides,it is unknown whether dietary excessive benzoic acid supplementation would have toxicity on piglets.Therefore,it is necessary to evaluate the safety of benzoic acid that was used in diets of piglets.The research was conducted to investigate effects of dietary benzoic acid supplementation on growth performance,blood components and histopathology in piglets,as well as evaluating the possible reasons of toxic effects in piglets resulted by excess of benzoic acid,and make it full use in feed industry.Experiment one Effects of benzoic acid on growth performance,blood components and histopathology of pigletsThe experiment was conducted to investigate effects of benzoic acid on growth performance,blood constituents and histopathology of piglets in order to assess its safety in piglets.A total of 120 cross barrows[(Yorkshire × Landrace)× Duroc]with initial average BW of 8.16 ± 0.09 kg(28 ± 1 d of age)were randomly allotted to four groups receiving basal diet and diets supplementing 0.5%.2.5%and 5.0%benzoic acid for 56 d.Phase 1:days 1-28,4 treatments.6 replicates(pens)per treatment,5 pigs per pen;phase 2:days 29-56.4 treatments.6 replicates(pens)per treatment.4 pigs per pen.One pig with a weight close to the average BW per pen was selected to obtain blood samples via the jugular veins after 12 h fasting in the morning of day 1,day 29 and day 57.After blood collection,these pigs were slaughtered.The results were as follows:(1)When compared with the control group.dietary 0.5%benzoic acid supplementation did not affect the growth performance of piglets;dietary 2.5%benzoic acid supplementation decreased ADFI and ADG during 1-28 d and 1-56 d(P<0.05).When compared with other three groups,dietary 5.0%benzoic acid supplementation significantly decreased ADFI and ADG(P<0.01).increased F/G(P<0.01),and resultedin ten pecent of mortalities.(2)When compared with other three groups,dietary 5.0%benzoic acid supplementation deceased HCT and MCV on 28th day(P<0.05),increased RDW-CV value(P<0.01)and tended to decrease hemoglobin on 28th day(P = 0.10)and 56th day(P=0.08),and supplementing 2.5%or 5.0%benzoic acid in the diet also decreased WBC level on 56th day(P<0.01).(3)When compared with control group and 0.5%benzoic acid group,dietary 2.5%benzoic acid supplementation decreased serum GLOB,BUN and CR levels(P<0.05).increased serum ALB level on 56th day(P<0.05);dietary 5.0%benzoic acidsupplementation significantly decreased serum GLOB.BUN.CR and TC levels on 28th and 56th day(P<0.05).increased serum ALT activity on 28th day(P<0.01).and increased serum AST activities on 28th day and 56th day(P<0.05)and serum AKP activity on 56th day(P<0.01).(4)When compared with control group,dietary 0.5%benzoic acid supplementation increased serum and liver CAT activities on 28th day(P<0.05).increased liver GSH-Px activity on 56th day(P<0.05);dietary 5.0%benzoic acid supplementation significantly reduced serum T-SOD and CAT activities on 28th day(P<0.01).and reduced serum and liver T-SOD and GST activities on 56th day(P<0.05).(5)When compared with other three groups,dietary 5.0%benzoic acid supplementation decreased villus heights and crypt depths in duodenum,.jejunum and ileum(P<0.05),and tended to decrease the ratio of villus height/crypt depth in ileum on 28th day(P = 0.08);dietary 5.0%benzoic acid supplementation also decreased villus heights and crypt depths in jejunum and ileum(P<0.05).and tended to decrease villus height in duodenum on 56th day(P = 0.06).(6)When compared with control group,dietary 2.5%benzoic acid supplementation increased heart and kidneys indices on 56th day(P<0.05),dietary 5.0%benzoic acid supplementation significantly increased heart,liver,kidneys,lung and pancreas indices on 56th day(P<0.01).(7)Dietary no less than or equal to 2.5%benzoic acid supplementation did not impair liver histological structure;dietary 5.0%benzoic acid supplementation impaired liver tissues,caused severe granular degeneration on 28th day,even vesicular degeneration on 56th day.Dietary no less than 0.5%benzoic acid supplementation did not impair spleen histological structure,when the dose is increased to 2.5%or 5.0%,the spleen appeared to mild congestion or severe congestion on 28th and 56th day,and accompanied by a reduction of lymphocyte in white pulp region.The histopathology structures of heart,kidneys and brain were not affected by dietary benzoic acid supplementation both on 28th and 56th day.(8)When compared with the control group and 0.5%benzoic acid group,dietary 2.5%benzoic acid supplementation resulted in higher benzoic acid residue in kidneys on 28th day(P<0.05),and increased higher benzoic acid residues in liver and kidneys on 56th day(P<0.05).Supplementing 5.0%benzoic acid in the diet also increased higher benzoic acid residue in liver and kidneys both on 28th and 56th day(P<0.01).Experiment two An intensive research about the toxic effects resulted by excess of benzoic acid in pigletsThe experiment was an intensive research about the toxic effects resulted by high dose of benzoic acid in piglets to obtain the possible reasons of its toxic action in the body.A total of 24 cross barrows[(Yorkshire × Landrace)× Duroc]with initial average BW of 7.94 ± 0.10 kg weaned at 21 ± 2 d were randomly allotted to three groups and were fed basal diet and diets supplemented 0.5%and 5.0%benzoic acid for 28 d.Each treatments had 8 replicates(cages),each replicates had 1 pigs.and single cage rearing.All pigs were selected to obtain blood samples via the jugular veins after 12 h fasting in the morning of day 1 and day 29.After blood collection,these pigs were slaughtered.The results were as follows:(1)When compared with control group and 0.5%benzoic acid group,pigs fed by the diet supplementing 5.0%benzoic acid had lower ADFI and ADG(P<0.01),and a higher F/G(P<0.01).Dietary 0.5%benzoic acid supplementation did not affect the growth performance of piglets.The growth performance results were similar with the growth datain experiment one.(2)When compared with control group and 0.5%benzoic acid group,pigs fed by the diet supplementing 5.0%benzoic acid had higher serum ALT activity(P<0.01).When compared with control group,pigs fed by the diet supplementing 0.5%benzoic acid had lower ALT activity(P<0.05).There were no difference on serum AST activity between these three groups.(3)When compared with control group and 0.5%benzoic acid group,pigs fed by the diet supplementing 5.0%benzoic acid had a larger number of CD4+ T lymphocytes(P<0.05),but had a smaller number of CD8+ T lymphocytes(P<0.01),and a higher ratio ofCD4+/CD8+(P<0.01).When compared with control group,pigs fed by the diet supplementing 5.0%benzoic acid also had lower serum IgG and IL-10 levels(P<0.01).(4)Dietary 0.5%benzoic acid supplementation did not impair liver,spleen and lung cell ultrastructure,had no adverse impact on cell membranes,organelles and nucleus.However,Dietary 5.0%benzoic acid supplementation could impair liver structure,impaired cell membrane integrity and cytoplasm,reduced organelles and ribosomes,decreased nuclear chromatin,and resulted in cell necrosis.In addition,dietary 5.0%benzoic acid supplementation could impair spleen structure,leaded to adverse bleeding in intercellular space,reduced organelles in mesh lining cell,dissolved mitochondrion,but did not impair cell membrane.Dietary 5.0%benzoic acid supplementation could impair lung structure,caused alveolar congestion and impaired air-blood barrier,deformed the Ⅱtype epithelial cells,and impaired the nuclear membrane in the cells.(5)When compared with control group,dietary(0.5%or 5.0%benzoic acid supplementation did not affect relative expressions of the oxidative stress genes(Nrf-2.HO-1 and NQO-1)and the apoptosis genes(Bax,Bcl-2 and Caspase-3)in the liver.spleen and lung.In conclusions,our results have demonstrated that:(1)Dietary 0.5%benzoic acid supplementation had no negative effect on growth performance,blood components and organ histopathology of piglets,and benefited to antioxidant capacity in the liver.Dietary 2.5%benzoic acid supplementation caused poor growth performance,changed blood components and impaired spleen structure.Dietary 5.0%benzoic acid supplementation had adverse effects on piglets growth and health,disrupted blood normal physiological status,impaired antioxidant and immune function,and do harm to small intestine morphology and resulted in organs(liver,spleen and lung)damage.(2)excess of dietary benzoic acid supplementation causing growth retardation of piglets can be derived from impairing feed intake,blood components,antioxidant capacity and organ structure,and increasing metabolic load.The process of organ injury was not affeced by the apoptosis-related genes(Bax,Bcl-2 and Caspase-3)and oxidative stress-related genes(Nrf-2,HO-1 and NQO-1).(3)In our study,the safety dose of dietary benzoic acid supplementation in piglets was 0.5%.