Dietary aflatoxin B1 induces abnormal deposition of melanin in the corium layer of the chicken shank possibly via promoting the expression of melanin synthesis-related genes

WANG Yong-li ,HUANG Chao ,YU Yang ,CAl Ri-chun ,SU Yong-chun ,CHEN Zhi-wu ,ZHENG Mai-qing,CUl Huan-xian

1 State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2 Guangxi Jinling Agriculture and Animal Husbandry Group Co., Ltd., Nanning 530049, P.R.China

Abstract San-Huang chicken is a high-quality breed in China with yellow feather,claw and break.However,the abnormal phenomenon of the yellow shank turning into green shank of San-Huang chicken has been a concern,as it seriously reduces the carcass quality and economic benefit of yellow-feathered broilers.In this study,the cause of this abnormal green skin in shank was systematically investigated.Physiological anatomy revealed that the abnormal skin in shank was primarily due to the deposition of melanin under the dermis.After analyzing multiple potential causes such as heredity (pedigree and genetic markers),environment (water quality monitoring) and feed composition (mycotoxin detection),excessive aflatoxin B1 (AFB1) in feed was screened,accompanied with a higher L-dihydroxy-phenylalanine(L-DOPA) (P<0.05) and melanin content (P<0.01).So it was speculated that excessive AFB1 might be the main cause of abnormal green skin in shank.Subsequently,the further results showed that a high concentration of AFB1 (>170 μg kg–1)indeed induced the abnormal green skin in shank compared to the normal AFB1 content (<10 μg kg–1),and the mRNA levels of TYR,TYRP1,MITE,MC1R and EDN3 genes related to melanin deposition would significantly up-regulate(P<0.01) and the content and activity of tyrosinase (TyR) significantly increased (P<0.05).At the same time,the content of L-DOPA and melanin deposition also increased significantly (P<0.01),which also confirmed the effect of excessive AFB1 on melanin deposition in skin of shank.Results of additional experiments revealed that the AFB1’s negative effect on melanin deposition in skin of shank could last for a longer time.Taken together,the results of this study explained the occurrence and possible mechanisms of the abnormal AFB1-related green skin in shank of chickens.Excessive AFB1 in diets increased the L-DOPA content and melanin abnormal deposition in the chicken shank possibly via promoting TyR content and activity,and the expression of melanin synthesis-related genes.Furthermore,our findings once again raised the alarm of the danger of AFB1 in the broiler production.

Keywords: aflatoxin B1,melanin deposition,skin color in shank,chicken,negative effect

1.lntroduction

Mycotoxin residues in meat,eggs,milk and other animal products threaten human health (Eaton and Groopmanet al.1994;Alonsoet al.2013;Adegbeyeet al.2020).Mycotoxins,such as aflatoxins (AFTs),are toxic secondary metabolites produced by fungi in the physiological processes of growth and metabolism (Wang and Groopmanet al.1999;Huanget al.2009).The major mycotoxins affecting the safety of feed include AFTs,zearalenone (ZEN),trichothecenes (TS),deoxynivalenol(DON),fumonisins B (FB) and ochratoxin (OT).Among them,aflatoxin B1 (AFB1) is one of the most toxic mycotoxins found to date,with mutagenic,teratogenic and carcinogenic properties (Coulombeet al.1993;Smelaet al.2001;Mishra and Das 2003),and is primarily active in liver (Dohnalet al.2014;Rajputet al.2017).Poultry are extremely sensitive to AFB1 with reactive doses as low as 15–30 μg kg–1.In fact,the European Union limit for mycotoxins in poultry whole feed ingredients is 20 μg kg–1.Excessive AFB1 can lead to immune suppression,damage to the reproductive system,liver and kidney diseases and other problems in livestock and poultry,affecting their growth and development,and even causing death (Komsky-Elbazet al.2017;Rajputet al.2017;Yuet al.2018;Jahanianet al.2019),resulting in huge economic losses to animal husbandry (Zaghiniet al.2005).

Lutein is a carotenoid that cannot be synthesized by poultry.The total feed lutein is the main source of skin color in carcass (Perez-Vendrellet al.2001).Besides the above-mentioned harmful effects on the broilers,mycotoxins in feed have side effects on the absorption and metabolism of carotenoids (Schaefferet al.1988).Aflatoxin blocks the fatty process of carotenoids and lutein in the intestine,inhibits the absorption and transport of lutein,increases the amount of lutein in the liver,and then transforms into a form that is difficult to use,hindering the absorption of lutein (Reddyet al.2006;Leeet al.2012).

After the H7N9 avian influenza outbroke in 2013,the chilled listing of yellow-feathered broilers has become an inevitable trend in market development.The chicken shank is a skin derivative and is one of the important reference indexes for broiler chicken coming into the market.Its color mainly depends on whether theepidermisanddermisof the tibia contain melanin and yellow pigments and the level of pigments,and the deposition of melanin and yellow pigments is mainly controlled by by genetic factors (Erikssonet al.2008;Xuet al.2017;Liet al.2020).

Melanins have also radioprotective and antioxidant properties that can effectively protect living organisms from ultraviolet radiation (Fraseset al.2006).Some actinomycetes bacteria are capable of producing darkbrown substances in the culture media,generally referred to as melanin or melanoid pigments (Dastageret al.2006).The biosynthesis of melanin pigment in microorganisms is similar to that of higher organisms.Tyrosinase can transform tyrosine into 3,4-dihydroxyphenyl-L-alanine(L-DOPA),which is further converted into dopachrome and autooxidized to indol-5,6-quinone.The latter is spontaneously polymerized into DOPA-melanin.Based on the above results,we speculated that mycotoxins,especially AFB1,might be associated with the production or metabolism of pigments.

San-Huang chicken is a high-quality local chicken breed in China,but abnormality characterized by the change of the normal yellow skin in shank into green was found,accompanied by liver injury and growth inhibition,which seriously reduced the carcass quality of yellowfeathered broilers.Considering all of the above research progresses,we hypothesized that the AFB1 in the diet might be an important reason for the abnormal green shanks of broilers due to the melanin deposition in the shank,and it might enhance the synthesis and deposition of the melanin in the shankviaregulating the expression and activity of related enzymes and genes to the melanin synthesis and finally cause the green shanks,and also this effect might be not easy to be removed.Therefore,the objective of the current study was to firstly carry out a systematic survey investigation on the anatomical characteristics of thedermisof broilers with abnormal green shank,the melanin content in thedermis,the related genetic background,the contents of bacteria and metal elements in drinking water,and the content of mycotoxins in the diet taken during broiler production.Then,according to the survey investigation results,a further experimental investigation was conducted to observe the effect of dietary AFB1 supplementation on the incidence of abnormal green shank,the content of L-DOPA and melanin,the expression and activity of related enzymes and genes in thedermisof the shank of broilers as well as the duration time of the above effect,so as to confirm the above scientific hypothesis.

2.Materials and methods

2.1.Survey investigation

Animals and dietsA total of 200 San-Huang chickens(half male and half female) aged 120 days with abnormal green skin in shank (group 1,n=100) or normal yellow skin in shank (group 2,n=100) were used in this study.All chickens were obtained from Guangxi Jinling Agriculture and Animal Husbandry Group Co.,Ltd.(Nanning,China),and were givenadlibitumaccess to the feed and water during the experiment.Complete diets for yellowfeathered broiler breeders were formulated based on NY/T 33-2004 (2004).

Animals dissection and collection of shank corium layer samplesFor sample collection,each 10 representative yellow-feathered broilers (half male and half female) aged 120 days with normal yellow skin in shank or abnormal green skin in shank from groups 1 and 2 were used to investigate the biochemical origin of the abnormal green skin in shank.All chickens were individually euthanized by carbon dioxide anesthesia and exsanguination by severing the carotid artery,and the feather was removed.Also,theepidermisanddermislayers of the shank were separated,and thedermislayer was dissected in depth for the purpose of observation.The samples of shankcoriumlayer were collected and stored at–80°C for subsequent determination of L-DOPA and melanin content.

Design of the investigation of the cause of the abnormal green skinTo determine the cause of the abnormal green shank,we performed a systematic investigation of every possible key factor associated with the green skin,including genetic,water quality and feed composition,and the details are as follows:

(1) Research of family tree history.Using all the 200 chickens,we retrospectively investigated the family tree history of their parents and grandparents.Then,after recording the data of the abnormal green skin in shank of their parents and grandparents,the data were analyzed to trace the familial inheritance of the trait of skin color in shank.

(2) Detection of the single nucleotide polymorphisms(SNPs) of theBCO2gene associated with chicken yellow skin in shank.Blood samples were collected from the wing vein of the 200 individual birds with the abnormal green skin in shank or normal yellow skin in shank for DNA extraction.Genomic DNA was extracted from uncoagulated blood by the phenol-chloroform method and used for PCR amplification.The specific primers for the beta-carotene oxygenase 2 (BCO2) gene were designed according to the reference sequences (chromosome 24:6 140 068–6 160 788;GRCg6a:CM000116.5 to amplify the sequences of theBCO2gene to screen for SNPs.Every PCR product was directly sequenced to screen different genotypes.The sequencing results in the chromatogram form were analyzed using the DNAMAN Software (version 6.0;Lynnon Corporation,San Ramon,CA,USA).The evaluation criteria were that: the two homozygous types should have one peak for a single nucleotide base,and the heterozygous type should have double peaks for two different nucleotides.Thus,the sequence mutations were identified between individuals,and the three different genotypes of every SNP were recorded.

(3) Water quality analysis.We performed the analysis of the quality of the drinking water for chickens to investigate the possible involvement of the water quality in the cause of the abnormal green skin in shank.Two of each 1-L secondary water supplies from the water supply terminal and stored in a glass or metal container were collected to assay the indicators of bacteria and metal ions.The assays were performed by the Beijing Qingxi Technology Research Institute according to GB/T 5750.12-2006 (2006).Using plate counting method to detect the total number of bacterial counts,total coliforms,thermotolerant coliform bacteria andEscherichiacoliindicators were determined by multiple tube technique(GB/T 5750.12-2006 2006).Aluminum,selenium,arsenic and mercury were determined by atomic fluorescence spectrophotometry.Hexavalent chromium,lead,cadmium,iron,manganese,copper and zinc were determined by atomic absorption spectrophotometry(GB/T 5750.6-2006 2006).Ultimately,the results were contrasted to determine whether these indicators exceeded the standard according to the government standard.

(4) Collection of diet samples and detection of mycotoxins in diets.Diet samples of 500 g each for yellow-feathered broiler breeders at different feeding stages (d 0–49,d 50–140 and d 141–180) were collected according to the method of Feed Sampling(GB/T 14699.1-2005 2005) to determine the levels of mycotoxins.First,the simple quick detection of the major mycotoxins (including AFB1,ZEN,DON,OT and FB) was performed using the fungal toxin rapid qualitative test strips (H016645,H016647,H016648,H016649,and H016650),which were purchased from the Qingdao Pribolab Biological Engineering Co.,Ltd.(Qingdao,China).Then,the content of AFB1 was further quantitatively determined by referring to GB/T 30955-2014 (2014).

2.2.Experimental investigation

Animals,diets and sample collectionsA total of 300 yellow-feathered broilers (half male and half female)at the day of hatching with normal yellow skin in shank were selected and randomly assigned to two groups(Experimental group,n=200;Control group,n=100).All chickens were obtained from Guangxi Jinling Agriculture and Animal Husbandry Group Co.,Ltd.Two groups were set with 10 or 20 replicate cages of 10 chickens for each replicate cage (half male and half female).Basal diets with low AFB1 contents (d 0–49,6.80 μg kg–1;d 50–120,4.70 μg kg–1) was formulated in line with NY/T 33-2004 (2004).Ingredients and composition of basal diets are shown in Table 1.The amount (170 μg kg–1) of the added AFB1 in the experimental group was selected according to the content of AFB1 in the diets from the previous survey investigation.The above amount of AFB1 (Shanghai Acmec Biochemical Co.,Ltd.,China)with a purity of 98% was added to the basal diets.And the concentrations of AFB1 in the experimental group were analyzed to be 176.8 μg kg–1(d 0–49),and 174.7 μg kg–1(d 50–120),respectively.Feed and water were givenadlibitumduring the experiment.All chickens were fed for four weeks,and the number of individuals with abnormal green skin in shank of every group were recorded.And then,10 chickens with abnormal green skin were randomly selected from the experimental group,and another 10 chickens with normal yellow skin were selected from the control group (half male and half female).After death by air injection,the samples of shankcoriumlayer were collected and stored at–80°C for subsequent total RNA extraction and determination of L-DOPA content,melanin content,TyR content and activity.After that,the residual individuals at the age of 28 d in the experimental group with abnormal green skin in shank were fed with the normal basal diet for up to 120 d for further investigation of skin color in shank.

Table 1 The composition and nutrient levels of the basal diets for yellow-feathered broiler breeders at different stages (as-fed basis,%)

Determination of L-DOPA,melanin content,TyR content and activityFrozen shankcoriumlayer of four chickens were randomly selected from abnormal green skin group and normal yellow skin group.The specific ELISA kits (Shanghai Enzyme-linked Biotechnology Co.,Ltd.,China) were used to determine melanin content,tyrosinase(TyR) content and activity and L-DOPA content (Jiangsu MEIMIAN Industrial Co.,Ltd.,China).A sample of 0.1 g of shankcoriumlayer was fully homogenized with 900 μL PBS and centrifuged (4°C,3 000×g,25 min),the supernatantwas used for melaninm and TyR measurement.In addition,take samples of equal quality and 900 μL of 70%methanol solution was added for the same treatment for the determination of L-DOPA.The assay was performed according to the manufacturer’s instructions.

qPCRTotal RNA (2.0 μg) was extracted from the frozen shankcoriumlayer of 10 chickens from the group with abnormal green skin in shank and 10 chickens from the group with normal yellow skin in shank and used to synthesize cDNA in a final volume of 20 μL using the GoScript? Reverse Transcription System (Promega Corporation,Madison,WI,USA) according to the manufacturer’s instructions.All PCR primers (Table 2)of melanocortin 1 receptor (MC1R),microphthalmiaassociated transcription factor (MITF),tyrosinase (TYR),tyrosinase related protein 1 (TYRP1) and endothelin-3(EDN3) were designed at or just outside exon/exon junctions to avoid amplification of residual genomic DNA,and specificity was determined using the Primer Premier 5.0 Software (Premier Biosoft,Palo Alto,CA,USA).The qPCR reaction was performed in a 20-μL final volume containing 10 μL of 2× iQ? SYBR Green Supermix,0.5 μL (10 mmol L–1) of each primer and 100 ng of cDNA.The qPCR reactions were performed on an ABI 7500 Real-Time PCR Detection System (Applied Biosystems Inc.,Foster City,CA,USA).A melting curve was constructed to verify that only a single PCR product was amplified.Samples were assayed in triplicate with standard deviations of the CTvalues not exceeding 0.5 on a within-run basis.

Table 2 The specific primers for PCR in this study

2.3.Statistical analyses

All statistics were analyzed by SPSS Software version 22.0 (IBM Corp.,Armonk,NY,USA).Statistical differences between groups were evaluated using the Student’st-test.P<0.05 (＊) or <0.01 (＊＊) was considered statistically significant.Individual chicken was the experimental unit for tissues and molecular biology assays.

3.Results

3.1.Survey investigation

Anatomical analysis of abnormal green skin inshank of yellow chickenThe results of the anatomical analysis showed that the abnormal green skin in shank disappeared and there was a black area in the whitedermisafter removing the yellowepidermislayer,as shown in Fig.1-A and B.Further examination showed that the melanin pigments were deposited on the inner side of thedermisin the black area,accompanied with the lower melanin deposition in skin of carcass (Fig.1-C).This finding revealed that the abnormal green skin in shank of San-Huang chickens was formed in the overlay of the yellowepidermislayer in the black area in the whitedermis.Further,the L-DOPA and melanin content in shankcoriumlayer of chickens from abnormal green skin group and normal yellow skin group was detected.These measurements reflected that the content of L-DOPA(5.17 pg mL–1) in the green shank was significantly higher(P<0.001) than those of the yellow shank (2.63 pg mL–1),and melanin (370.00 pg mL–1) content in the green shank was significantly higher (P=0.036) than yellow shank(110.00 pg mL–1),as shown in Fig.1-D and E.Thus,it was confirmed that the actual cause of the abnormal green skin in shank was the abnormal deposition of melanin in the epidermal layer of the skin in shank of chickens.

Fig.1 Anatomical analysis and L-DOPA and melanin contents of abnormal green skin and normal yellow in shank of yellow chicken.A,visual observation of external features.B and C,observation of the outside side and the inner side of the dermis in shank (n=10).D,L-dihydroxy-phenylalanine (L-DOPA) content of shank corium layer.E,melanin content of shank corium layer.Data are presented as the mean±SE (n=4 for D and E).＊,P<0.05;＊＊,P<0.01.

Analysis of the cause of the abnormal green colorTo identify the factors leading to the abnormal deposition of melanin,we conducted several research experiments from three perspectives,namely inheritance (genealogy and genetic markers),environment (water quality monitoring)and feed composition (mycotoxin detection).First,we investigated the family tree history and the SNP variations to determine whether there was mixing of foreign lineages in chickens with the trait of abnormal green skin in shank.The results indicated that there was no corresponding genetic relationship between the individuals with the trait of abnormal green skin in shank and their parents and grandparents.Moreover,we identified one SNP in theBCO2gene related to the skin color in shank of the used population,and the evidence revealed by the SNP analysis showed that the mutation sites led to the same phenotype in chickens with abnormal green skin in shank and normal yellow skin in shank (Fig.2).Also,the results of the water quality monitoring indicated that all samples were within the normal standard range of all the indicators of bacteria and heavy metal ions (Table 3).

Fig.2 Identification of the single nucleotide polymorphism(SNP) in the BCO2 gene.Using the PCR amplification and directly sequencing,we identified one SNP in the BCO2 gene(n=200).G,green shank;Y,yellow shank.

Table 3 Bacteria numbers and mineral contents in the drinking water

Considering the easy contamination of feed with mold in the hot and humid weather in the south of China,the content of mycotoxins in feed was also determined.The simple quick measurement of major mycotoxins indicated that both the chick feed and breeder feed had abnormally high content of AFB1,while the contents of ZEN,DON,OT and FB were normal (Table 4).Furthermore,as expected,an additional quantitative analysis performed by referring to GB/T 30955-2014 (2014) confirmed the presence of an excessive amount of AFB1 content in both the chick(d 0–49) feed and bred (d 50–140) feed with the 173.50 and 172.80 μg kg–1,respectively.However,the AFB1 content in the breeder (d 141–180) feed was up to standard (14.90 μg kg–1) (Fig.3).

Fig.3 Content of aflatoxin B1 (AFB1) in feeds at different stages of chickens.The excessive amount of AFB1 content in the chick feed and bred feed was 173.5 and 172.8 μg kg–1,respectively (n=3).

Table 4 Detection of major mycotoxins in feed using the fungal toxin rapid qualitative test strips

3.2.Experimental investigation

Evaluation of the effect of AFB1 on melanin deposition and related indicesWe also conducted an experiment to confirm the relationship between AFB1 and abnormal melanin deposition in the skin of shank of chickens fed with the excessive AFB1 (170 μg kg–1),the chick fed with AFB1 within the normal range was used as control.After breeding for four weeks,the abnormal green skin in shank was found to be associated with the melanin deposition in all individuals of the experimental groups fed with the feed containing excessive AFB1 exceeding the standard of mycotoxin content,but not in the normal group (Fig.4-A).

Fig.4 Changes of shank skin color,L-dihydroxy-phenylalanine (L-DOPA) and melanin content,tyrosinase (TyR) content and activity,and gene expression level of related genes for the additional excess aflatoxin B1 (AFB1).With an excessive AFB1 exceeding the standard content (170 μg kg–1),the yellow-feathered chickens were fed for 4 weeks.A,observation of external feature on the skin color in shank;n=292 (8 dead individuals were removed).B,the changes of mRNA levels of the MC1R,MITF,TYR,TYRP1 and EDN3 genes.C,TyR content and activity of shank corium layer.D,the content of L-DOPA and melanin of shank corium layer.Data are presented as the mean±SE (n=10 for B;n=4 for C and D).＊,P<0.05;＊＊,P<0.01.

The qPCR analysis of the expressions of five representative genes on controlling the melanin deposition was performed.As shown in Fig.4-B,the results revealed that the mRNA levels of theMC1R,MITF,TYR,TYRP1andEDN3genes were significantly up-regulated in thedermisof the skin in shank of experimental group compared with those of the control group.In addition,the content of TyR in thedermisof shank skin in the experimental group was significantly increased (P=0.040),and the activity of TyR was also significantly increased(P=0.045) (Fig.4-C).It was worth mentioning that we further detected the content of L-DOPA and melanin,and found that the content of L-DOPA (P=0.024) in the experimental group increased significantly,and the melanin content was also significantly higher than that in the control group (P=0.001) (Fig.4-D).

Long-term influence of melanin deposition on the skin color in shankAll individuals from the experimental groups,aged four weeks with abnormal green skin in shank,were continuously fed with the normal AFB1 standard feed to investigate the influence cycle of AFB1 on the abnormal green skin in shank.At up to 120 d,the number of chickens with green skin on the shank was still up to 96%.These results suggested that AFB1 would have a long-term negative influence on the abnormal skin color in shank.

4.Discussion

The results of this study have supported our above hypothesis.Overall,our survey investigation results showed that the abnormal green shank of chicken determined by anatomy was caused by abnormal melanin deposition in thedermisand the increased content of melanin and L-DOPA probably due to the excessive AFB1 in the diets.The results from the experimental investigation further confirmed that additional excessive AFB1 could promote the expression ofMC1R,MITF,TYR,TYRP1andEDN3genes,and increase the contents of melanin and L-DOPA,and the content and activity of TyR.And the negative effect on melanin deposition in the skin of shank could last for a longer time.These findings are helpful to better understand the abnormal melanin deposition caused by the excessive AFB1 and possible mechanisms.Solving these problems will help to prevent the occurrence of abnormal green skin and improve the carcass quality of yellow-feathered broilers.

In this study,the anatomical analysis revealed that the abnormal deposition of melanin in the epidermal layer of the skin led to the abnormal green skin in shank of chickens.The cause of the abnormal green skin in shank was investigated by first performing a search of the pedigree to exclude the influence of consanguinity.TheBCO2gene was identified as the key gene related to skin color in shank (Erikssonet al.2008;Wuet al.2016),thus the SNP variations ofBCO2also were examined and the genetic variations in skin color did not found.We also focused on evaluating two aspects (water quality and feed composition) in terms of the plausibility of harm related to mold growth due to the humidity and hot conditions in the sourth of China.It was found that environmental and genetic factors were not associated with the abnormal tibial skin color,but a high level of mycotoxin AFB1 was found in chicks and growing feed,accompanied with the significantly higher L-DOPA and melanin contents,so we speculated that excessive AFB1 might cause the melanin deposition and induce abnormal green skin in shank.

Since it has been reported that bacteria can cause abnormal green skin in shank by regulating melanin deposition in thedermisof chicken (Mcgibbon 1979;Pavanet al.2020),we focused on investigating the effect of mycotoxin AFB1 on melanin deposition.Further experiments confirmed that different degrees of melanin deposition can occur in all chickens after feeding for a period of time with excessive AFB1 exceeding the standard level,and the formation of abnormal green skin in shank was reproduced.Therefore,this result supported the notion that excessive levels of the AFB1 toxin can cause the abnormal green skin in shank of yellow-feathered chickens.Subsequently,the mRNA expression ofMC1R,MITF,TYR,TYRP1andEDN3genes related to melanin deposition and the content and activity of TyR which directly controls melanin synthesis,changed accordingly (Dorshorstet al.2011;Yuet al.2013;Zhouet al.2018;Pavan and Sturm 2019),with concomitant increases in L-DOPA and melanin content.This provided new ideas and strategies for preventing AFB1 induces the occurrence of abnormal melanin deposition in shank of chicken.

These findings reveal that besides causing immune suppression,reproductive system damage,liver and kidney diseases and affecting the growth and development of livestock and poultry (Rajputet al.2017;Komsky-Elbazet al.2018;Jahanianet al.2019;Liuet al.2019;Taranuet al.2019),mycotoxin concentration exceeding the standard level can also affect the deposition of melanin and negatively affect the carcass quality of chickens.In addition,we also conducted an extended observation experiment to study whether melanin deposition will be eliminated with the growth time.The results suggest that the effect of AFB1 on melanin deposition in the lower leg is a long-term process,and there is no effective quick treatment to reverse this process.This finding represents an alert for broiler producers and helps to improve the consciousness of prevention among breeders.Besides,in the whole experiment,we paid attention to the color change of chicken shank skin,without statistical growth performance data.In future research,we will supplement the statistics of growth performance observation indicators.

Although this study explained the cause of the green shank in yellow-feathered chickens as the result of abnormal melanin deposition,more in depth research needs to be performed to avoid more economic losses in the production of poultry,such as the levels of melanin induced by AFB1,or the possible molecular mechanism by which AFB1 regulates melanin deposition.

5.Conclusion

In the present study,we demonstrated that the abnormal green shank in the broiler production was caused by deposition of melanin indermisprobably due to the excessive AFB1 in diets,and dietary excessive AFB1 resulted in increased contents of L-DOPA and melanin,as well as increased content and activity of TyR and expression levels of genes related to melanin synthesis.The findings of this study also indicated that the abnormal green skin in shank caused by exposure to AFB1 had a certain long-term persistence,once again raising the alarm of the danger of AFB1 in the broiler production.

Acknowledgements

This research was funded by the grants from the China Agriculture Research System of MOF and MARA(CARS-41) and the Agricultural Science and Technology Innovation Program,China (ASTIP-IAS04).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was conducted in accordance with the guidelines for experimental animals developed by the Ministry of Science and Technology of China.All animal experimental protocols were approved by the Science Research Department (in charge of animal welfare issues)at the Institute of Animal Sciences,Chinese Academy of Agricultural Sciences (Beijing,China) (No.IAS2021-21).