Adjusted risk of one-year graft loss in the low-risk recipient group was similar to that of patients with younger donors; results were consistent using validation dataset. Our results show that a system of careful recipient selection can reduce the risks of graft loss associated with older donor age.Advanced age of liver donor is a risk factor for graft loss after transplant. We sought to identify recipient characteristics associated with negative post-liver transplant (LT) outcomes in the context of elderly donors. Using 2014–2019 OPTN/UNOS data, LT recipients were classified by donor age: ≥70, 40–69, and <40 years. Recipient risk factors for one-year graft loss were identified and created a risk stratification system and validated it using 2020 OPTN/UNOS data set. At transplant, significant recipient risk factors for one-year graft loss were: previous liver transplant ; mechanical ventilation ; portal thrombus ; serum sodium <125 mEq/L ; and Karnofsky score 10–30% , 40–60% . Using those risk factors and multiplying HRs, recipients were divided into low-risk (  The need for donor livers currently exceeds the number of organs available for transplantation in the US . For exaPrevious studies have investigated recipient risk factors for poor liver transplantation outcomes when using livers from older donors -10; thesIn this study, we hypothesized that using liver from older donors could be optimized by carefully considering the medical and surgical conditions of recipients. We sought to identify recipient characteristics associated with negative outcomes after receipt of organs from elderly donors and to create a risk stratification system based on these characteristics that would reduce the risk of graft loss. The primary endpoint was set for one-year graft loss which includes patient death.This study used data from the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) Standard Transplant and Research (STAR) files for LT. The study period was set from 1 January 2014 to 31 December 2019, with 1 year of post-transplant observation for each patient. Study procedures were approved by the Henry Ford Health System Institutional Review Board; the requirement for written informed consent was waived due to the deidentified nature of the data. Patients who were 18 years or older at the time of transplant were eligible for this study. Patients who received a partial/split graft or combined organ transplant with thoracic organs, kidney, intestine, and/or pancreas or patients for whom donor age was unknown were excluded. Also, if patients who had one or more missing data which was evaluated in this study, those were excluded . To asse2)  . Continu2) ; serum b2) , 1.8 and2) ; and int2) , 1.8 andRisk of one-year graft loss after receipt of an organ from the ≥70 donor group was compared to the 40–69 and <40 donor groups. Graft loss was defined as death or re-transplantation. Analyses were adjusted for recipient demographic  and clinical characteristics  at the time of transplantation. Analyses were also adjusted for donor  and organ characteristics .Recipient risk factors for one-year graft loss were determined with multivariable Cox regression. The total risk score for each patient was calculated by multiplying the adjusted hazard ratios (aHRs) of recipient risk factors according to a previously used methodology . If one p value less than 0.157 in univariable analyses   years. TAdvanced recipient age is also a known risk factor for liver graft loss , 4. HoweIn the past, many transplant centers would not accept DCD donors older than 60 years old, as there were reports of higher risk of graft loss with older DCD donors -27. MoreThere are a number of limitations to our analysis. This is a retrospective study using the OPTN/UNOS registry, which lacks detailed post-transplant clinical data, such as surgical complications after transplantation. We were also limited by the small proportion of donors ≥70 years in the dataset. Although we were able to detect a number of significant risk factors despite the relative small sample size, it is possible that a larger sample size would have provided more precision in our estimates. The primary outcome examined in this study (one-year graft loss) was a short-term outcome and may not be applicable to mid-to long-term outcomes. Three-year and 5-year graft survival were evaluated, which demonstrated that the negative impact of recipient risk factors was more prominent in the first year post-transplant, then the survival curves became parallel after 1 year between the low and high risk groups. Also, we could not evaluate the impact of grafts after DCD in elderly donors due to the absence of such donors in the dataset. Despite these limitations, the scoring system could be useful to determine suitable recipient selection when using the liver graft from older donors. Our scoring system would not be used to regulate organ acceptance practice. Transplant physicians and centers could use it to estimate its risk and should decide indications at their discretion if those risks are acceptable for each case.In conclusion, our risk stratification system using the following recipient factors, history of the previous liver transplant, low Karnofsky Performance Status score, need for mechanical ventilation, presence of portal vein thrombosis, and hyponatremia, might be useful for recipient selection who are eligible for liver grafts from older donors. This could lead to further expansion of the donor pool without compromising outcomes.


Lactobacillus, reduced that of Bacillus and Weissella, and significant correlations were observed between fermentative qualities and bacterial abundances. However, decreased diversity of bacterial community and the contents of LA implied that shifts in bacterial community might exert negative effects on silage fermentation. Our results suggest that bloom wax could alter the microbial community composition of ensiled sweet sorghums, which thus influence the fermentation qualities.Epicuticular wax, as secondary metabolites (hydrophobic compounds) covering plant surface, plays important roles in protecting plants from abiotic and biotic stresses. However, whether these compounds will influence fermentation process of silage is still not clear. In this study, two sweet sorghum cultivars with varying epicuticular wax on sheath (bloom), Yajin 2 , and Jintian , were harvested at flowering and maturing stages, and ensiled with or without bloom, aiming to evaluate the effects of bloom on fermentation quality, feed nutrition and microbial community. The bloom was collected manually with de-waxed cotton and extracted with chloroform. The results showed that the bloom reduced the concentrations of water-soluble carbohydrate and crude protein of the two cultivars at both stages, reduced lactic acid (LA) for YJ at both stages and for JT at flowering stage, and increased LA for JT at mature stage. The α-diversity of bacterial communities of the silage fermentation with bloom was significantly lower than that without bloom. Bloom increased the abundance of  Silage, which is fermented mainly by lactic acid bacteria (LAB) existing on plant surface, is one of the important methods widely used in forage production. However, whether the cuticular wax of forage crop will influence its fermentation quality is still not clear.Cuticular waxes are mixtures of hydrophobic compounds covering the surface of land plants . These cSorghum bicolor Moench) is an important annual forage crop widely distributed all over the world   . These rNeolamarckia cadamba leaf silage by largely altering bacterial community  cultivars selected in this study were Yajin 2 (YJ) and Jintian (JT). The two cultivars differed in bloom content (YJ was bloom-depleted type and JT was bloom- enriched type). The seeds were surface sterilized in 10% H2O2 for 10 min, washed under tap water, and then were planted in Experiment Farm of Southwest University, located in Beibei district, Chongqing, China.The sweet sorghum  to determine the contents of crude protein (CP), ether extract (EE), crude fiber (CF), water soluble carbohydrate (WSC), and crude ash (Ash), according to the methods described by 2 flow rate 32.4 mL min–1; H2 flow rate 40.0 mL min–1; air flow rate 400.0 mL min–1; tail blowing 30 mL min–1; Split ratio 20:1. The ammonia nitrogen (NH3-N) was determined using the method described by –1; hydrogen flow rate 40.0 mL min–1; air flow rate 400.0 mL min–1; tail blowing 30 mL min–1; Split ratio 30:1.The fresh ensiled samples (20 g) were extracted with 180 mL distilled water at 4°C for 24 h, filtered through double-layer gauze and filter paper, and then the filtrates were stored in refrigerator at −20°C. The filtrate was used to determine pH, lactic acid, ammonia nitrogen and volatile fatty acids. pH was measured using a glass electrode pH meter. Lactic acids were analyzed using capillary gas chromatography . The detailed GC parameters were as follows: oven temperature 110°C; injection port temperature 250°C; detector temperature was 290°C; N1 under the master accession number of Bioproject PRJNA853257.The total genomic DNA was extracted from silage, and the high variation region of the bacterial 16S rRNA gene v3–v4 was obtained by 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR program was as follows: 95°C for 3 min, 27 cycles  with a final extension of 72°C for 10 min and 10°C until being halted. The PCR products were detected by 2% agarose gel electrophoresis, and the correct PCR products were identified and sequenced using Illumina PE300 sequencing platform . The original sequences were processed by Meiji Bio Cloud Platform. The RNA-Seq reads were deposited and available at the database Sequence Read Archive at NCBI2) = 3.1415 × diameter × height]. Then, the samples were extracted twice for 30 s with CHCl3 containing 5 μg of n-tetracosane as internal standard. The two extracts from each sample were then combined and filtered through glass wool, dried under N2, and then derivatized using pyridine  and N,O-bis(trimethylsilyl)trifluoroacetamide  at 70°C for 45 min. The excess reagents were dried under N2, and re-dissolved in 0.5 mL CHCl3 for GC analysis.The leaves and sheaths (third leaf from the top) and stems (between internode three and four) were sampled from healthy plants growing in fields at flowering and maturing stages, with three replicates for each cultivar. Wax extraction was based on the methods described by 2 was served as carrier gas. The temperature of the column membrane and FID detector was set at 300 and 320°C, respectively. The GC oven was held at 80°C for 10 min, heated at 5°C/min to 260°C, remained 10 min, then heated at 2°C/min to 290°C, and further heated at 5°C/min to 320°C, held for 10 min. Compounds were further detected with a GCMS-QP2010 Ultra Mass Spectrometric Detector  using HP-5 MS capillary column (30 m × 0.32 mm × 0.25 μm), and He as the carrier gas. Compounds were identified by comparing their mass spectra with published data and authentic standards. Wax quantification was based on the peak area of internal standard and presented as micrograms per unit leaf area.The GC analysis was carried out with FULI 9790II  using DM-5 capillary column (30 m × 0.32 mm × 0.25 μm) . NP < 0.05 according to the least significant difference test. The redundancy analysis (RDA) analysis was applied to analyze the relationship between fermentation qualities and microbial community at the phylum and genus level using Canoco 5. The significance of each variable was defined using Mantel tests (permutations = 999) and significance level was tested by Mantel R2 statistic and p-values. Spearman correlation heatmap was used to show the relationship between bacterial classifications and environmental variables .The presented data in figures and tables were the average of three biological replicates. Two-way ANOVA was applied to evaluate the influence of cultivars and bloom on fermentation qualities using SPSS software . Then, one-way ANOVA was applied to compare the difference in fermentation parameters between with bloom and without bloom treatments. Differences were regarded significance at –2 at flowering stage and from 0.63 to 1.11 μg cm–2 at maturity stage. Stem wax ranged from 5.09 to 9.82 μg cm–2 at flowering stage and from 9.21 to 16.80 μg cm–2 at maturing stage. Among the organs, sheath had the highest wax coverage at flowering stage, with sheath wax on and Jintian (JT) reaching 11.43 μg cm–2, followed by Yajin 2 (YJ) (8.82 μg cm–2). However, the wax coverage on sheath at maturing stage was lower than that at flowering stage, and even was lower than those on stem which increased from flowering stage to maturing stage. Using de-waxed cotton, the amounts of bloom collected from sorghum sheath reached 2.85 and 1.95 mg per 150 g plant for YJ, and 3.00 and 3.15 mg per 150 g plant for JT, at flowering and maturing stages, respectively. No significant difference of chemical profiles could be observed between sheath wax and bloom.The cuticular waxes on leaves, stems and sheaths of sorghums were analyzed separately. Overall, leaves were mainly predominated by fatty acids (averaged 26% across two cultivars and two stages), alkanes (31%), and primary alcohols (20%) . However–1, which was significantly lower than those in JT (200 g kg–1) at maturing stage, whereas the concentrations of EE in YJ was significantly higher those in JT at both stages. Compared with flowering stage, overall, the DM and WSC increased, whereas CP, CF and crude ash (Ash) reduced, at maturing stage.Besides their difference in wax depositions, the three sorghum cultivars also differed in their chemical properties related to feed qualities, particularly the concentrations of water-soluble carbohydrates (WSC) and EE . For exaAfter the sorghums were ensiled for 45 days, the chemical compositions were evaluated. Overall, the cultivar, sheath bloom and their interactions significantly influenced the chemical compositions of ensiled sorghums . For exaThe fermentation products were also influenced by cultivar, bloom and their interactions . The bloDifferences of bacterial communities in ensiled sorghum were revealed by the comparisons of diversity index . On averFirmicutes was the highest for sorghum silages irrespective of cultivars and growing stages, accounting for 96.95% (averaged across two cultivars) at flowering stage and 75.77% at maturing stage, followed by Proteobacterial accounting for 1.95% at flowering stage and 21.55% at maturing stage   . Compareng stage . Bloom aLactobacillus , the dominant microbe, for all cultivars at both stages, except for insignificant changes for JT at maturing stage. Bacillus was the second largest genus at flowering stage, the abundance (averaged 9.82%) of which was reduced by bloom for all cultivars. No Bacillus was detected at maturing stage. Weissella was the second largest genus at maturing stage (19.39%), the abundance of which was reduced by bloom from 33.22 to 27.53% for JT. The abundance of Weissella at flowering stage was all lower than 4%, which also was reduced by bloom for both cultivars. In addition, compared to flowering stage, there were more bacterial communities detected at the genus levels at maturing stage, such as Pediococcus, Kosakonia, and Rhodococcus.At the genus level, more obvious difference in microbial abundance was observed between ensiled sorghum with bloom and without bloom . Bloom iAs shown in the RDA biplots, a combination of variables explained 99.04 and 98.8% of the variance of the bacterial communities at the phylum level at flowering and maturing stage, respectively . The ParBacteroidota, and the relative abundances of Bacteroidota and Actinobacteriota were positively correlated with WSC, LA, and AN/TN, and negatively correlated with pH, PA, and BA, at flowering stage  for proteolysis  for YJ at flowering stage, whereas the reverse was true at maturing stage, and a higher AN/TN was observed at maturing stage than at flowering stage. There are varieties of factors influencing the hydrolysis of plant protein during ensiling process . The bloteolysis , therebyteolysis . Meanwhiteolysis . In thisGenerally, it is difficult to collect all waxes from sorghums using traditional method. Therefore, waxes from leaf, stem and intracuticular wax of sheath might all influence the effects of bloom on sorghum fermentation. However, our results clearly indicated that bloom would influence the fermentation qualities, depending upon the amounts and compositions of bloom, the wax profiles, and the nutrition characteristic of sorghums.Arabidopsis thaliana and Zea mays  and proteolytic enzyme (protease)  . The stuWeissella is beneficial bacteria to ensilage fermentation, which is strictly heterofermentative, producing a mixture of LA and AA by metabolizing WSC  under anaerobic conditions   . Sorghumlternata . Though tic acid .Lactobacillus come to dominate by consuming nutrients in silage, and it also dominated at the end of fermentation. This is consistent with many reports on silage bacterial community , but also decreased the bacterial Shannon diversity. The abundances of undesirable microbes were suppressed by the bloom, including PRJNA853257. Available at: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA853257&o=acc_s%3Aa.The data presented in this study are deposited in the NCBI’s Sequence Read Archive repository, accession number YG and WT designed the study. LL, YX, JZ, and HS participated in sample collection. WT performed the experiments and wrote the manuscript with the help of YC, LL, YX, and JZ. YG performed writing - review and edited final manuscript. All authors contributed to the article and approved the submitted version.


Neuroeconomics has been seldom used in investigating the impact of culture on international trade. This research proposes a scientific approach to investigate how cross-cultural differences contribute to the conceptualization of international trade patterns globally. International business relations are directly influenced by factors such as cultural variations which distinguish one foreign market from another. Therefore, the level of understanding these cultural differences is able to determine the success or not of business opportunities. In response to the scarcity of scientific investigation of cultural influence on international trade, the purpose of this study is to propose a neuroeconomic framework as a strategic instrument to elucidate the cross-cultural dimension of international commercial relations. Echoing this, our study uses cultural diversities and cognitive classifications established in literature to adopt a unique scientific tool for the conceptualization of international trade patterns across the world. This research establishes the cognitive mechanism of cross-cultural diversity, as a novel framework to conceptualize international trade patterns. By unveiling the cognitive process of cross-cultural diversity, this article provides an instrument to unlock trade barriers of individualism and collectivism across nations. The concept of economic integration among countries has long been demonstrated through classical economic theories and traditional frameworks of international organizations, whereby the role of cultural identity in the decision-making process had not been given a full attention . But recTo fill this gap, this study proposes a neuroscientific analysis of cultural effects on the cognitive mechanism in international trade decision-making process. For a global trade contest, we employed neuroeconomic techniques to investigate further the effect of cross-culture diversity in the conceptualization of international trade patterns, thus to respond to the question:1.How to conceptualize international trade patterns within a cross-cultural diversifies market?2.What framework can provide a central scientific interpretation for such cross-cultural diversity in the decision-making mechanism?We argue that for a complete understanding of the conceptualization of international trade patterns across nations/cultures, a neuroscientific examination of the cultural effect on the cognitive mechanism is imperative , 2005. TDefined as the exchange of capital, goods and services across territories, international trade is a complex process due to the numerous differences between patterns such as currency, government policies, judicial systems, laws, and markets influence trade . But to As in this context, cultural diversity creates a barrier to the transfer of knowledge, information, and competencies and ultimately the cost of doing business . AccordiAs well documented in literature, international trade decision-makings process has been totally attributed to market parameters based empirical research. However, the implication of cultural parameter pushes the discussion to take a broader dimension, where the decision-making process could be subjected to a cognitive scientific experimentation . ConsequAs established above, people’s perspective and reaction in international trade is directly influenced by their cultural background. In terms of the attitudes toward relations and business, literature has distinguished categorizations setting where cognitive mechanism is contrasted and compared to cultural identity . For insFurther classification of cultural backgrounds in typical negotiation situations could also be based on country differences. The Asian culture on the other side is characterized by a cultural and religious affiliation, with an adherence to tradition. The key to success is collectivism where, every stage of the negotiation process, the consent of the entire group is necessary. The Asian culture is a reserved culture, relationship-oriented, polychronic, and formal. When conducting negotiations, either successful or not, “saving face” is extremely important for the Asians. Relations between parties should be reserved in showing their feelings and emotions . The UniTo conclude, so far, there is no consensus on the patterns of international trading as they reflect different meanings and ways of doing business across cultures . From thUnderstanding the cognitive diversities across cultures, especially in the uncertain market, provides an assurance for successful results, as compared to a mere reliance on the classical economic parameters of market demand and supply. Indeed, an indebt examination of the neural interpretation of cultural diversities, and for this discussion very instrumental in bridging fundamental barriers of international trade . ObviousFounded on market parameters like demand and supply, the birth of classical economics was basically driven by rational and unemotional players with a focus on profit maximization. However, after a century-long detour, the recognition of psychological behaviors in the economic decisions making process opened up a new neoclassical era of economics traceable to In this discussion, we emphasize the impact of cross-cultural diversity on the conceptualization of international trade patterns. Obviously, cognitive difference across cultures does not facilitate a consensual trade pattern adapted for economic transactions across nations. Therefore, we propose a deeper analysis of the situation from a neuroscientific perspective, in order to analyze the functions of the brain in correlation with cultural identities and adapted business patterns. Moreover, we posit that, even with the trade barriers of individualism and collectivism across nations, from a neuroeconomics perspective, international trade patterns could be developed in correspondence with specific cultural identities . Based oEven though classical economic theories were instrumental in laying the foundations for the economic understanding, they became inadequate to explain broader determinants in the interpretation of the economic decision-making process. For instance, the implication of cultural diversity in the international trade procedures, need further expansion of the economic framework to include cognitive parameters . ConsequThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.GA designed and conducted the research and wrote the first draft of the manuscript. YW searched and analyzed the literature and wrote the main part of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This manuscript analyzed physicochemical and structural properties of 30 different types of sorghum starches based on their apparent amylose content (AAC). Current results confirmed that sorghum starch exhibited irregular spherical or polygonal granule shape with 14.5 μm average particle size. The AAC of sorghum starch ranged from 7.42 to 36.44% corresponding to relative crystallinities of 20.5 to 32.4%. The properties of enthalpy of gelatinization (ΔH), peak viscosity (PV), relative crystallinity (RC), degree of double helix (DD), degree of order (DO), and swelling power (SP) were negatively correlated with AAC, while the cool paste viscosity (CPV) and setback (SB) were positively correlated with AAC. Correlations analyzed was conducted on various physicochemical parameters. Using principal component analysis (PCA) with 20 variables, the difference between 30 different types of sorghum starch was displayed. Results of current study can be used to guide the selection and breeding of sorghum varieties and its application in food and non-food industries. Sorghum bicolor (L.) Moench] is a significant cereal crop which ranks the fifth largest cereal crop in the world after barley, corn, wheat and rice  and starch from other plant sources , 18. TheThirty varieties of sorghum were bred by the Chinese Academy of Sciences and were in the Academy’s sorghum database . Maize aThirty different kinds of sorghum were used to extract their starches following a previous method  with somThe moisture content of starch was determined using a halogen moisture analyzer  following the method of Li et al. . Sorghum2 solution was prepared by dissolving 0.5 g KI and 0.05 g I2 in 25 ml distilled water, and the KI-I2 solution was kept in dark at 4°C. Maize amylopectin and potato amylose were used to prepare starch standard curve with amylose content of 0, 20, 40, 60, 80, and 100%. Eight milliliters of urea-dimethyl sulfoxide solution (1:9 v/v) was added to 20 mg of starch, following by mixing using vortex for 2 min. The mixture was incubated in an 85°C water bath for 30 min and shaked every 5 min. The starch solution was cooled down to room temperature and fixed into a 25 ml volumetric flask. Aliquots (3 ml) of starch solution was mixed with 1 mL of KI-I2 solution to make 50 ml samples solution with distilled water. The sample was allowed to stand at 25°C for 15 min before measuring absorbance at 620 nm. The Amylose content was calculated based on the regression Equation 1. x is absorbance, y is the amylose content.The AAC was ascertained using the iodine binding-based method , 22 withScanning electron microscopy  was used to examine sorghum starch at a 5 kV accelerated voltage . The samg for 20 min after cooling down through room temperature water to 25°C. After transferring the supernatant to a petri dish, then supernatant was dried and weighed (W1). And the sediment was weighed and recorded as Ws. The following formulas were used to calculate WSI and SP:Swelling power  and water solubility index  were determined using a method modified from Tsai et al. . Starch –1 was deconvoluted using OMNIC 9.2 software to determine the absorbance of 1,047, 1,022, and 995 cm–1. Then, the values of 1,047/1,022 cm–1 (DO) and 995/1,022 cm–1 (DD) were calculated.The short-range ordered structures of different sorghum starches were evaluated by ATR-FTIR spectroscopy . The sample was set flat on the diamond ATR crystal’s surface in a homogeneous manner, the air background was gathered. The sample was subsequently scanned. ATR-FTIR spectroscopy in the range of 1,100–900 cmThe crystalline characteristics of sorghum starch were obtained using an X-ray diffractometer . The parameters were tested using 40kV and 30mA with Cu kα radiation. The samples, with a 0.03 step size, were measured between 5° and 40° . The relPasting properties of the samples were determined using a rapid visco-analyzer  based on to the method reported by Ambigaipalan et al. . The samTo), peak (Tp), conclusion (Tc) temperatures of gelatinization, and the enthalpy change (ΔH), were recorded.The melting curve of sorghum starch was determined using a NETZSCH DSC analyzer  based on to the method reported by Yang et al. . Starch p < 0.05, significant differences in the mean values were established. The correlation analysis between starch properties was determined by SPSS software V. 25. The principal component analysis (PCA) results were obtained in Origin 2021 V. 9.8 software.The mean and standard deviation were obtained by SPSS V. 25 software, and the significance of the results was evaluated by analysis of variance. At The moisture content of thirty sorghum starches ranged from 8.4 ± 0.3% in S19 to 13.5 ± 0.3% in S18 . Starch The average value of AAC (25.58%) was much lower than that of wheat starch (38.6%) , but higNo significant differences were observed in the SEM images of 30 different types of sorghum starches . AccordiAccording to r = −0.80, p < 0.001)  . Similar< 0.001)  and Li e< 0.001) .–1  of sorghum starch crystalline area, and the ratio of 995/1,022 cm–1 is relative to the double helix (DD). r = −0.701, p < 0.001) and DD , which indicated that the degree of order and double helix of the sorghum starch were influenced by AAC. High amylose content was associated with lower DD and DO. The present of amylose could disrupt granular crystalline ordering and stabilization  and DD was also positively correlated with SP . It showed that starch contained a high degree of order and double helix structure resulted in higher swelling power. The phenomenon corresponded to the findings of Du et al.  structures in starch have been reported to be sensitive to ATR-FTIR . The dec–1 , 51. Thiallinity . The ratlization . The paru et al.  and Zhonu et al. . The sweu et al. . DO and p < 0.05) between the thirty starches . Starch made up of an ordered crystalline region and an unordered amorphous region. Most of the noncrystalline and crystalline regions were made up of amylose and amylopectin, respectively  and DD . The degree of short-range order and double helix of sorghum starch suggested to be related to the degree of crystallinity, which is the same with previous studies  ranged from 7.87 ± 0.27 to 12.92 ± 0.42 J/g, 67.3 ± 0.4 to 75.8 ± 0.3°C, 73.5 ± 0.1 to 80.8 ± 0.1°C, 78.5 ± 0.2 to 85.7 ± 0.1°C, and 8.3 ± 0.3 to 12.4 ± 0.1°C, respectively. The thermal properties were similar with the literature reported by Singh et al.  plots can display the interrelationships between starch structural properties and the differences and similarities among diverse starches. , 70. TheAccording to To, Tp, Tc, and ΔH). The greater AAC existed in sorghum starch, resulting in the lower the PV and BD. In addition, high level of AAC in sorghum starch increased the HPV and SB. The RC, DO, and DD of starch were positively correlated with AAC. The thermal properties, pasting properties and swelling power have a strong correlation to RC, DO, and DD. Principal component analysis (PCA) was conducted for 20 variables, based on the differences between 30 types of sorghum starch. The PCA1 and the PCA2 were the two most significant components, which explained most of the variance. The results of this study can be utilized to direct the choice and breeding of sorghum cultivars as well as the application of sorghum starch.In summary, starches isolated from 30 varieties of sorghum exhibited significant differences in physicochemical properties. In this study, the morphological properties, apparent amylose content, thermal properties, swelling properties, crystalline structure and pasting properties of starch were synthetical investigated. The correlation analysis revealed that the thermal properties, pasting properties, crystalline properties, water solubility index and swelling power of sorghum starch were all related to the AAC. AAC prevented the expansion of starch granules and decreased the thermal characteristics (The original contributions presented in this study are included in the article/SY: investigation, methodology, and writing—original draft. ZaL and BW: supervision and project administration. TL, ZyL, and NZ: investigation and formal analysis. BC: conceptualization, resources, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.


In the present study, maize starch (MS), potato starch (PS), high-amylose maize starch (HAMS), and wheat starch (WS) were deep-fried in soybean oil that was continuously heated for 40 h under 180 °C. The thermodynamic and pasting properties of deep-fried starch samples were determined. The results suggested that starch–lipid complexes formed with the extension of frying oils’ usage; however, their number was not dependent on the frying oils’ life cycle. Importantly, the results of pasting properties revealed the following strength of intermolecular force in deep-fried starch samples: PS > MS > HAMS > WS. The results of XRD and FTIR analysis confirmed the formation of starch–lipid complexes during the deep-frying process. Furthermore, the results of the in vitro digestibility of deep-fried starch revealed that the formation of starch–lipid complexes inhibited the swelling of starch granules and prevented the entrance of amylase into the interior. Additionally, the results of the oxidation stability of deep-frying oil indicated that the formation of starch–lipid complexes did not alter the trend of lipid oxidation as an effect of the limited number of starch–lipid complexes. These results could have critical implications for the development of healthier deep-fried foods. Deep-frying is one of the most common cooking methods, during which foodstuff acquires a golden color, crisp texture, and pleasant flavor. However, oxidation, hydrolysis, and polymerization of triglycerides (TAGs) continue to occur during the deep-frying process . In partStarchy food is one of the most prevalent kinds of deep-fried foods. To assess the health value of deep-fried starchy food, starch–lipid complexes, which have been identified in deep-fried foods, have attracted the attention of researchers. Scientists found starch–lipid complexes in deep-fried foods such as instant noodles and batter . ResearcYang et al. illustrated that the increase of deep-frying temperature would lead to an increase of amylose content . They alStarch–lipid complexes are regarded as a new type of resistant starch . There iIn this paper, maize starch (MS), potato starch (PS), high-amylose maize starch (HAMS), and wheat starch (WS) were used for the preparation of nuggets with distilled water, and they were then deep-fried under real circumstances. Specifically, the starch samples were fried in soybean oil for 8 min under 180 °C, and the frying oil was fried continuously for 40 h. Starch–lipid complexes formed during the deep-frying process were detected. The thermodynamic properties and crystal structure of deep-fried substances were analyzed to determine the formation of starch–lipid complexes. The in vitro digestibility of deep-fried substances was investigated to illustrate the effect of starch types and deep-frying oils’ life cycle on the properties starch–lipid complexes. Furthermore, the effect of starch–lipid complexes’ formation on the oxidative stability of deep-frying oils was examined.WS was purchased from Qufeng Food Tech. Co., Ltd. ; HAMS was purchased from Ruitai Gaozhi Biotechnology Co., Ltd. ; MS, PS, potassium bromide, α-amylase, and anhydrous sodium acetate were all purchased from Macklin Biochemical Co., Ltd. ; soybean oil was purchased from Wilmar International Limited ; DNS chromogenic agent was provided by Solarbio Science & Technology Co., Ltd. ; α-Amyloglucosidase was purchased from BOSF Co., Ltd. .Starch and distilled water were mixed according to a certain mass ratio (6:5). The evenly mixed mixture of starch and water was then divided into a 2.5 cm × 2.5 cm × 1 cm mold to set. The mixture (30 g) was carefully removed and fried for 8 min in soybean oil (3000 g) under a temperature of 180 °C. The size of the fryer was 30 cm × 15 cm × 14 cm. The deep-fried oil was then kept at 180 °C for 12 min before another group of mixtures was processed. The soybean oil was kept at 180 °C for 8 h per day, and the deep-frying process lasted up to 5 d (40 h).The deep-fried starch samples were ground and defatted with petroleum. The samples were then sifted through 50 meshes.Amylose content was determined based on the method of Zhu et al. , with soThe thermodynamic properties of deep-fried starch samples were determined via differential scanning calorimetry (DSC), based on the method of Yang et al. , by makiThe viscosity of deep-fried starch samples was measured using a rapid viscosity analyzer (RVA). The specific operations were as follows: Deep-fried starch samples (3 g) were placed in a test canister. Distilled water (25 g) was then added to the canister to obtain a starch suspension. Before being loaded, the suspension was stirred several times with a plastic paddle to mix evenly. The test procedure was set as follows: The suspension was heated at 50 °C for 1 min, and it was then heated to 95 °C with a rate of 12.8 °C/min. Finally, the samples were cooled to 50 °C with a cooling rate of 12.8 °C/min and kept at 50 °C for 2 min.The wide-angle X-ray diffraction was performed, referring to the method of Chen et al. , with so−1, with a resolution of 4 cm−1 for 64 scans.The method of FTIR was modified by Wang et al. . The fin540nm of the solutions was recorded, and the glucose content was calculated (G0).The content of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in the deep-fried starch samples was analyzed according to the method described by Kang et al. . Specifi20) and 120 min (G120), respectively. The mixture was then centrifugated at 3000 r/min for 10 min, and the supernatant (0.5 mL) was added to a test tube with distilled water (1.5 mL). G0, G20, and G120 are called RDS, SDS, and RS, respectively.α-Amylase (1.0 g) and amyloglucosidase (20 μL) were added to the centrifuge tubes with the deep-fried starch samples and sodium acetate buffer, as mentioned above. The tubes were then shaken well and incubated under 37 °C. Enzymolysis solution (0.2 mL) was mixed with anhydrous ethanol (4.8 mL) at 20 min  of deep-frying oil were determined according to the official methods of the American Oil Chemists’ Society Cd 8b-90 and Cd 18–90, respectively. The total oxidation value (TOTOX) was calculated based on the equation:The peroxide value (POV) and p < 0.05). The SPSS package was used for all statistical analysis. The data graphs were plotted using Origin 2017.All analyses were performed in triplicate, and the results are presented as mean ± standard deviation (Mean ± SD). The statistical significance of the data was analyzed by one-way ANOVA and Tukey’s tests (As can be observed in As shown in TP = 68.82 °C) of MS disappeared, and new ones were found between 122.78 °C and 141.69 °C during the deep-frying process, indicating the gelatinization of starch and the formation of starch–lipid complexes [H) of deep-fried MS samples was significantly lower than that of native MS (9.95 J), suggesting the formation of starch–lipid complexes during the deep-frying process [H of amylose-lipid complexes is an indicator of the amount of complex and the degree of crystallinity in the starch, which is mostly related to the amount of complex [H of deep-fried MS samples at 32 h (6.61 J) was the highest among the samples where the oil had been heated from 8 h to 40 h. A previous study found that the structure of starch–lipid complexes with palmitic acid (C16:0), myristic acid (C14:0), and lauric acid (C12:0) was more ordered than that of complexes with stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) [The effect of the deep-frying process on the thermodynamic properties of starch samples is shown in omplexes . The melve MS 9.9 J, sugge process ,12,28. I complex ,28. Nota (C18:2) . Moreove (C18:2) . In addi (C18:2) . Soybean (C18:2) . Thus, mH of deep-fried PS samples was significantly lower than that of untreated PS (16.60 J), and the ΔH of deep-fried PS samples at 40 h (0.58 J) had the lowest value. The results suggested the formation of starch–lipid complexes during the deep-frying process. A similar trend was observed in HAMS and WS, where the gelatinization peak of native starch disappeared and new ones were found in deep-fried starch samples, indicating the formation of starch–lipid complexes during the deep-frying process.The gelatinization peak (63.31 °C) of native PS disappeared and new ones were found between 131.90 °C and 145.10 °C in deep-fried PS samples, indicating the melting of starch–lipid complexes. The ΔThe effect of the deep-frying procedure on the pasting properties of the four types of starch is presented in As in MS, a similar trend was observed in the viscosity for the other types of starch investigated in the present study. Notably, the peak viscosity of PS and HAMS decreased the most (more than 50%), and that of MS declined the least. Interestingly, the breakdown viscosity of PS decreased by more than 80% during the deep-frying process. However, there was no significant difference between the breakdown viscosity of native WS and that of deep-fried ones, indicating that the intermolecular force of deep-fried WS was weaker than the others. A significant increase in the final and setback viscosity of PS during the deep-frying process is presented in The diffraction patterns and relative crystallinity of four types of starch sample during the deep-frying process are displayed in As for PS, the diffraction peak at 15°, 20°, 22°, and 23° (B-type starch) disappeared, while the diffraction peak at 17° was presented either in native PS or deep-fried PS samples b. IntereThe diffraction peaks of HAMS, which contains a high level of B-type starch, disappeared, except for the peak at 17° during the deep-frying process c. Additi−1 (-OH) of all the starch samples investigated in the present study was significantly weakened during the deep-frying process, suggesting the ordered structure of starch granules was destroyed and the intramolecular or intermolecular hydrogen bonds were weakened. In addition, the intensity of -CH2 and -CH absorption bands (2931 cm−1) was weakened during the deep-frying process, as was that of the C-O-H absorption peak (1080 cm−1), indicating that the crystalline structure of starch particles was destroyed. The results were in line with previous studies [−1 and 2854 cm−1 [−1 was assigned to -C=O stretching vibration [−1 and 1746 cm−1 were observed in four types of deep-fried starch samples, where the bands indicated the vibrations of -C-H and -C=O in soybean oil, suggesting the presence of lipid in deep-fried starch samples. Therefore, the formation of starch–lipid complexes during the deep-frying process was confirmed. Although lipid was observed in native starch samples (The FTIR spectra of four types of deep-fried starch samples are displayed in  studies ,16,36. A854 cm−1 ,16,36. I samples , the lipThe contents of RDS, SDS, and RS of four types of starch samples are shown in As shown in p-AV, and TOTOX values are shown in POV, p-AV of the deep-frying oils, which was a crucial indicator of secondary oxidation product formation. Meanwhile, the lipid oxidation rate of deep-frying oil with MS and PS was much lower than that with WS and HAMS. As indicated in the present study, the intermolecular force of deep-fried PS and WS was the strongest and weakest, respectively. Overall, the strength of intermolecular force followed the following course: deep-fried PS > deep-fried MS > deep-fried HAMS > deep-fried WS. The results indicated that starch with a strong interaction could maintain the integrity of the swollen granules [Interestingly, the formation and destruction rate of hydroperoxides was different in various types of starch. The highest accumulation of hydroperoxides was observed in the deep-fried WS and HAMS system, indicating a higher lipid oxidation rate, and this was confirmed by the granules  and bounThis study investigated the effects of frying oils’ usage time on the formation of starch–lipid complexes in four types of starch, namely MS, PS, HAMS, and WS, as well as the effects of starch–lipid complexes on lipid oxidation. The thermodynamic properties, XRD, and FTIR analysis of deep-fried starch samples suggested that starch–lipid complexes formed during the deep-frying process. In addition, starch–lipid complexes formed with the extension of frying oils’ usage, while no correlation was observed between the amounts of starch–lipid complexes and frying oils’ life cycle. Furthermore, it was revealed that the interaction between starch and lipid relies more on the crystalline structure of native starch in comparison with the usage life of frying oil. Interestingly, the results of the pasting properties of deep-fried starch samples revealed that the strength of intermolecular force followed the following course: deep-fried PS > deep-fried MS > deep-fried HAMS > deep-fried WS. In vitro digestibility analysis showed a dramatical increase of RS content in deep-fried starch samples. However, the results of the present study suggest that the formation of starch–lipid complexes did not alter the trend of lipid oxidation during the deep-frying process, and this fact was attributed to the limited amounts of starch–lipid complexes.


Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents. Phaseolus vulgaris. L) as raw material. According to their findings, enzymatic digestion and heat treatment of starch boosted the production of RS. Das et al. [Starch is a vital carbohydrate for human health in nature. Based on its nutritional properties and release rate of glucose, starch can be classified into rapidly digestible starch, slowly digestible starch and resistant starch (RS) . Among ts et al.  focused s et al.  used beaThermostable α-amylase cleaves α-1,4 glucans into linear chains that can rapidly decrease the viscosity of gelatinous starch solution and promote the production of soluble dextrin and oligosaccharides . ExcessiResponse surface methodology (RSM) is a statistical method for solving multivariate factors. Specific data are obtained through the test, and a polynomial quadratic equation is used to fit a functional relationship between the variables and response. The fitted polynomial equation is analyzed to find the optimum process parameters. Wang et al.  developeIn this study, corn starch subjected to dual enzyme modification by using thermostable α-amylase and pullulanase was investigated using response surface methodology (RSM). Factors affecting enzymatic modifications were optimized by a single-factor test and RSM. Structural characteristics were assessed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and degree of polymerization (DP). This study aimed to optimize the dual enzyme modification conditions of corn starch to increase the content of RS and to reveal the structural differences between native and modified corn starch. The native corn starch with 27.9% amylose was obtained from a large supermarket . Its moisture content and protein content were 10.7% and ≤0.2%, respectively. Thermostable α-amylase (1400 U/mg) from porcine pancreas (type VI-B), pullulanase (1000 U/mL) and dinitrosalicylic acid (DNS) were purchased from Sigma–Aldrich Chemical Co. . A Megazyme Resistant Starch Assay Kit was purchased from Megazyme Int. Ireland Ltd., Co. . All other reagents were of analytical grade.Corn RS was prepared in accordance with the methods of Zhang et al.  with somThe RS content was measured using the Megazyme Resistant Starch Assay Kit based on the AACC-approved method 32-40 . SamplesIn order to determine the effects of different conditions on RS content, the following factors were carried out at various starch liquid concentrations , the amount of added thermostable α-amylase , the duration of enzymatic (thermostable α-amylase) hydrolysis  and the amount of added pullulanase . Then, followed the step in 1, X2, X3 and X4 are the concentration of starch liquid, the amount of added thermostable α-amylase, duration of enzymatic (thermostable α-amylase) hydrolysis and amount of added pullulanase, respectively; b0 is the interception coefficient; b1, b2, b3 and b4 are linear coefficients; b12, b13, b14, b23, b24 and b34 are interaction coefficients; and b11, b22, b33 and b44 are the quadratic coefficients. These coefficients represent the linear, quadratic and interaction effects of the variables, respectively. The fitted polynomial equation was calculated to visualize the relationship between the response and experimental levels of each factor and to deduce the optimum conditions [RSM is a collection of mathematical and statistical technique used to model and analyze problems in which a response of interest is influenced by several variables, and the objective of this technique is to optimize this response . Signifinditions . AnalysiThe average degree of polymerization (DP) of starch samples  was determined in accordance with the methods of Hizukuri et al.  with appThe morphologies of native and enzymatic-modified corn starch were observed using an SEM  at an accelerating voltage of 5.0 kV. Samples were fixed on an aluminum column and held in place with double-sided adhesive carbon tape before sputtering gold to a thickness of 50 nm.2 gas with a rate of 30 mL/min using an empty aluminum pan as a reference. The transition temperatures of gelatinization  and the enthalpy of gelatinization (ΔH) were analyzed using relevant software to characterize the thermal properties of starch samples. The thermal properties of starch samples were determined using a differential scanning calorimeter  based on the method of Gong et al.  with somp < 0.05).All the experiments were conducted in duplicate; the experimental data in this study were expressed as mean ± standard deviation. Data were analyzed with SPSS Statistics 20.0  and Design Expert 8.06. Analysis of variance (ANOVA) and Least Significant Difference (LSD) were used to determine significant differences (The effect of the starch liquid concentration on the RS content is listed in In The effect of the duration of enzymatic (thermostable α-amylase) hydrolysis on the RS content is shown in The effect of the amount of added pullulanase on the RS content is shown in 1 = 15%, X2 = 3 U/g, X3 = 35 min and X4 = 8 U/g. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The quadratic polynomial model of the RS content (Y) comprised the following variables: the concentration of starch liquid (X1), the amount of added thermostable α-amylase (X2), the duration of enzymatic hydrolysis (X3) and the amount of added pullulanase (X4).RSM results indicated that the RS content varied depending on the given conditions . The conp-values were used as a tool to check the significance of each coefficient, which in turn might indicate the pattern of the interactions between the variables. The smaller the p-value was, the more significant the corresponding coefficient would be. In the present study, the p-value of the regression model was less than 0.01 (32) followed by the quadratic term the amounts of added α-amylase (X22), quadratic term concentration of starch liquid(X12) and linear term the amounts of added α-amylase (X2). However, all other interaction terms were found insignificant (p > 0.05). The order of independent variables based on their main effects on increasing the RS content is X2 > X3 > X1 > X4, which indicates that the amounts of added α-amylase are the most important variable influencing the RS content, followed by the duration of enzymatic hydrolysis and concentration of starch liquid. The ANOVA results of the developed model and regression coefficients are presented in han 0.01 , confirmThe surface of the starch granules of RS was investigated through SEM. The surface of native starch granules was displayed as round or oval granular shapes without evidence of cracks a. Some cp < 0.05) were observed in T0, Tp, Tc and ΔH among the samples. Native corn starch showed a specific gelatinization pattern with a large gelatinization endotherm appearing at approximately 60 °C because of the melting of crystallites in native starch granules [0), peak (Tp) and conclusion temperature (Tc) and the transition enthalpy (ΔH) of treated samples increased significantly as compared with the native starch. Comparatively, the melting temperature of the dually-modified starches was higher than that of the single pullulanase-treated starch. The increase in ΔH can be explained in terms of increased ordering and stabilization of double helical structures through hydrogen bonds and other intermolecular forces [The thermal parameters and the enthalpy change (ΔH) are summarized in granules . The trar forces . Hence, r forces , who four forces .The combined enzymatic modification of corn starch with thermostable α-amylase and pullulanase resulted in a marked increase in RS. The amount of added thermostable α-amylase was the most important factor that influenced the RS content, and this factor was followed by the duration of enzymatic hydrolysis, concentration of starch liquid and the amount of added pullulanase. A maximum RS content of 10.75% was obtained using 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. This value was significantly higher than that of native and single enzyme-treated starch. SEM image, DSC and DP determination results demonstrated the remarkable effects of the dual enzyme modification pretreatment of corn RS.


Pseudohemiculter hainanensis  is a small Cyprinidae fish that has a wide distribution in China. In this study, we characterized the complete mitochondrial genome of P. hainanensis by the Illumina NovaSeq sequencing platform in Guangxi, China. The assembled mitogenome is 16,647 base pairs (bp) and consists of 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, and a control region (D-loop). Nucleotide composition of the complete mitogenome is 29.69% (A), 24.82% (T), 27.97% (C), and 17.52% (G), with an A + T bias of 54.51%. The maximum-likelihood tree based on 13 PCGs showed that Pseudohemiculter hainanensis formed an independent lineage and P. hainanensis was closer to T. houdemeri.The  Pseudohemiculter hainanensis  is a small Cyprinidae fish, that is widely distributed in the drainage basins of the Yuanjiang River, the Pearl River, Hainan Island, and the middle reaches of the Yangtze River in China. It is a  freshwater fish that lives in the upper middle layer of the water (Luo and Chen P. hainanensis was classified as least concern (LC) according to International Union for Conservation of Natures. No research on P. hainanensis has been documented till date; therefore, it is highly important to obtain the complete mitochondrial genome of P. hainanensis for further studies. This study obtained the complete mitochondrial genome of P. hainanensis, analyzed the structural features of the complete mitochondrial genome, and explored the phylogenetic relationships within Cultrinae to provide a basis for further studies on the genetic evolution and classification of P. hainanensis.The P. hainanensis was collected from the farmers market of Laibin, Guangxi Zhuang Autonomous Region, China  on 5 July 2021. The species was carefully distinguished as ‘Cypriniformes, Cyprinidae, Cultrinae, and Pseudohemiculter’ according to Culterinae. Fauna Sinica, Osteichthyes, Cypriniformes II book .Specimen of P. hainanensis captured in 2021 was dissected using a sterilized dissection tool, and 5 g of its dorsal muscle was taken and stored in a centrifuge tube at −80 °C in the refrigerator. Total genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method  server for functional annotation , 22 transfer RNA genes, two ribosomal RNA, and a control region (D-loop). Most PCGs of P. hainanensis were encoded on the N-strand, except for ND6 which was encoded on the J-strand. The overall base composition was 29.69% (A), 24.82% (T), 27.97% (C), and 17.52% (G), with an A + T bias of 54.51%. Twelve genes started with ATG while only COI started with GTG among the 13 PCGs. Eight genes shared the termination codon TAA including ND2, COI, COII, COIII, ATP6, ND4, ND4L, and Cytb, and the gene with TAG as the stop codon was ND1, ATP8, ND3, ND5, and ND6. The genome map is shown in The P. hainanensis and other 10 species from Cultrinae, phylogenetic trees were obtained using maximum-likelihood (ML) analyses based on the combined DNA sequences of 13 PCGs and corresponding amino acids (Ai et al. Danio rerio from Danioninae was selected as an outgroup. ML trees were based on 13 PCGs (P. hainanensis formed an independent lineage and P. hainanensis was closer to T. houdemeri.To investigate the phylogenetic relationships of  13 PCGs , while M 13 PCGs . The phyP. hainanensis were identical to most vertebrates (Boore P. hainanensis and Toxabramis houdemeri were in the same proximal branch, and P. hainanensis was differentiated earlier. Dai et al. (Pseudohemiculter, Toxabramis, and Hemiculter was small and the genetic relationship was close. All of the above showed that P. hainanensis and T. houdemeri may have similar genetic backgrounds and need further research.The gene arrangement pattern and translated orientation of es Boore . The phyi et al.  used theP. hainanensis formed an independent lineage and P. hainanensis was closer to T. houdemeri. This study can provide important basic information for species identification, geographic population division, and kinship identification of P. hainanensis in the freshwater waters of China.The results of this study showed that


The study was aimed to investigate the rules of postprandial changes in intestine digesta dry matter (DM) flow and amino acid digestibility of growing pigs fed low-protein (LP) diets made of different starch.Eight barrows (28.8±2.1 kg) with a T-cannula at the distal ileum were randomly allotted to an 8×3 Youden square design. Treatments included: waxy corn starch LP (WLP); corn starch LP (CLP) and pea starch LP (PLP). Diets were given at 08:00 and 20:00. Digesta samples were collected in six 2-h stages from 08:00 to 20:00.The Cr concentrations of ileal digesta increased and then decreased in WLP and CLP, while increased continuously in PLP as time passed after postprandial (p<0.05). Higher average Cr concentrations  and lower average DM flow  were observed in WLP and CLP, compared with PLP. The apparent ileal digestibility coefficient of most amino acids in WLP and CLP increased compared with that in PLP. No difference in lysine or methionine digestibility was observed. When digesta were collected in 2-h periods, the apparent ileal digestibility coefficient of amino acids did not change over time. When digesta was collected in 4-h periods from 16:00 to 20:00 and 6-h periods from 14:00 to 20:00 (p<0.05), WLP and CLP showed markedly higher amino acid digestibility than PLPHigh-amylose slowly digested starch can increase the DM flow at the distal ileum and reduce the apparent ileal digestibility coefficient of amino acids of pigs fed LP diets. Compared with waxy corn starch and corn starch, pea starch reduced the digestibility of amino acids in digesta collected after 4 h postprandial. Appropriate modulation of dietary starch profile can enhance nitrogen efficiency via optimizing the synchronization of energy and nitrogen supply in pigs ,2 and imFurthermore, the digestibility and digestion rate of nutrients are mainly decided by the rate of hydrolysis and digesta transit ,8. The hKim et al ,13 exploThe present experiment was approved by the China Agricultural University Animal Care and Use Committee . All pigs and experimental supplies were provided by the Fengning Swine Research Unit of China Agricultural University .Eight crossbred (Duroc×Landrace×Yorkshire) barrows (28.8 ±2.1 kg) fitted with a T-cannula at the terminal ileum were randomly allotted to 1 of 3 experimental diets in a 3-period Youden square design which resulted in 8 observations per dietary treatment . Dietaryad libitum. The temperature was maintained at 23°C±2°C. Humidity varied from 55% to 65% during the experiment.Daily feed allotment (about 4% of body weight) was divided into two equal meals given to each pig at 08:00 and 20:00 during the experimental period. All the pigs had access to fresh water Each experiment period consisted of 7 days (4 days for adaptation to diets and 3 days for sample collection). During sample collection period, ileal digesta samples were collected in six 2-h stages from 08:00 to 20:00 . A 300-mL plastic bag with 5 g chlortetracycline in it was attached to the cannula barrel using an elastic plastic rope. Bags were removed at least once every 30 min, and immediately stored at −20°C. Low-temperature storage and antibiotics supplementation were used to weaken microbial degradation of nutrients in digesta. After collection period, samples from each pig were mixed across the 3 collection days within collection stages. In summary, 6 digesta samples were collected from each pig in each 3-day collection period, and each sample representing a 2-h collection stage.N HCl for 24 h at 110°C before analysis for 15 amino acids with an Amino Acid Analyzer . The concentrations of methionine and cysteine were measured according to a common method. Briefly, samples were subjected to performic acid oxidation and hydrolyzed with 7.5-N HCl for 24 h at 110°C and determined using an Amino Acid Analyzer . Tryptophan concentrations in feed and digesta samples were determined using High Performance Liquid Chromatography  after NaOH hydrolysis for 22 h at 110°C. Chromium concentrations measurement was conducted using a Polarized Zeeman Atomic Absorption Spectrometer . Starch content in diet and ileal digesta was determined with commercial assay kits .Frozen digesta samples were placed at room temperature to thaw and then weighed. After adequate mixture, a subsample from each 2-h collection stage digesta sample was weighed, lyophilized, weighed, and finely ground. The analysis of feed and digesta samples was conducted in duplicate. The analysis of DM, crude protein, total calcium, and total phosphorus concentrations in feed and digesta samples was conducted according to the Association of Official Analytical Chemists procedures . SamplesThe ileal Cr flow and the ileal DM flow present the output of Cr and DM in ileal digesta, respectively. These result values were calculated for 4-, 6-, and 12-h collection stages on the basis of the following equation:Where sample (g) means the weight of the ileal digesta sample after freeze-drying.The ileal DM flows were calculated according to the following equation:diet means dietary Cr concentration (g/kg DM), and Crileal means Cr concentration in ileal digesta (g/kg DM).Where CrValues for apparent ileal digestibility coefficient of amino acids were calculated as previously described .Data were obtained with each 2-, 4-, 6-, and 12-h collection stages as one experimental unit. Experimental repeated measure data was analyzed using MIXED procedures of SAS version 9.4 . The differences were considered significant when p<0.05, and the differences were considered highly significant when p<0.001. The analysis results of analysis of variance method were used to describe the differences between treatment groups.Much research has reported the impact of starch structure modulation on pig husbandry, such as changing dietary glycemic properties, regulating growth performance and carcass traits ,19. HoweThe waxy corn starch, corn starch and pea starch used in this experiment are generally regarded as high-digestion rate, medium-digestion rate, and low-digestion rate starch, respectively . The staTo achieve the objective of the present study, a T-cannula was installed about 10 cm proximal to the ileo-blind junction by surgery for ileal digesta collection. In the experiment, when pigs were fed LP diets based on waxy corn starch (p<0.001) and corn starch (p<0.001), the Cr concentrations of ileal digesta collected in 2-h periods from 08:00 to 20:00 increased and then decreased . This obWhen pigs were fed WLP diet, the concentration of Cr in ileal digesta samples collected in postprandial 4 to 6 h, 6 to 8 h, and 8 to 10 h was higher than the concentration of Cr in ileal digesta samples collected in postprandial 0 to 2 h (p<0.05). Pigs fed CLP diet showed increased concentration of Cr in ileal digesta samples collected in postprandial 6 to 8 h and 8 to 10 h, compared with those collected in postprandial 0 to 2 h and 2 to 4 h (p<0.05). Chromium content in ileal digesta of the PLP fed pigs increased until 6 to 8 h after a meal (p< 0.05). There was no difference in Cr concentration in ileal digesta samples of the PLP fed pigs collected in postprandial 4 to 6 h, 6 to 8 h, 8 to 10 h, and 10 to 12 h. It is worth noting that whether pigs fed WLP, CLP, or PLP diets, the concentration of Cr in ileal digesta samples collected in 4-h period from postprandial 0 to 4 h was less than those collected in 4-h period from postprandial 4 to 8 h and from postprandial 8 to 12 h (p<0.05). When the collection time of digesta is divided into the first 6 h and the second, the concentration of Cr in the first 6 h is lower than that in the latter (p<0.001). This situation happened in all three treatment groups.In most sample collection periods, the concentration of Cr in pigs fed WLP and CLP diets was higher than that in pigs fed PLP diet in the corresponding collection period (p<0.05). These changes were mainly due to differences in DM flow because the DM flow had an opposite pattern compared with the concentration of Cr in the ileal digesta samples. During the whole 12 h of sample collection, the average concentration of Cr in ileal digesta and the average DM flow at the distal ileum were 0.78% and 181.1 g/kg DMI from pigs fed the WLP diet, 0.84% and 166.3 g/kg DMI from pigs fed CLP diet and 0.70% and 240.3 g/kg DMI from pigs fed PLP diet. Statistics proved that compared with WLP and CLP groups, PLP treatment group showed a decreased average Cr concentration of ileal digesta (p<0.05) and increased average DM flow at the distal ileum (p<0.001). This result suggested that the chemical components of ileal digesta depend on the pattern of dietary starch structure. Improved dietary amylose proportion induces increased DM flows through the foregut and decreased digesta Cr concentration. Previous research proved that the high dietary fiber content can increase the content of DM flows pass the small intestine but can decrease the concentration of Cr in the digesta and nutrient digestibility . TherefoThe data for the apparent ileal digestibility coefficient of amino acids further confirmed this hypothesis. The apparent ileal digestibility coefficient of threonine, tryptophan, leucine, valine, histidine, arginine, serine, glycine, aspartate, and total amino acids in pigs fed WLP and CLP diets was higher than that in pigs fed PLP diet p<0.001; . The appWhat draws our attention was that the apparent ileal digestibility coefficient of lysine or methionine was not different among treatments . These rThe apparent ileal digestibility coefficient of amino acid is described as the net disappearance of dietary amino acids from the digestive tract proximal to the distal ileum . The terWe show the apparent ileal digestibility coefficient of four key essential and non-essential amino acids based on the collection of ileal digesta during different time periods in Although the apparent ileal digestibility coefficient of amino acids in each treatment did not change significantly with time when digesta was collected in 2-h periods, which may be because of differences within the group. We still find that the digestibility of amino acids tended to increase over time or decrease after 10 h postprandial. These results are different from those of Kim et al  where thWhen comparing differences in amino acids digestibility of ileal digesta during different time periods between groups, the situation is similar to the comparison in Cr concentration. In general, essential (except lysine and methionine) and non-essential amino acids digestibility in WLP and CLP treatments was higher than that in PLP treatment during most time periods. When digesta was collected in 2-h periods and 4-h periods from postprandial 0 to 4 h, the apparent ileal digestibility coefficient of essential (except threonine) and non-essential amino acids between treatment groups did not differ. There was no difference in lysine and methionine digestibility between treatments, the only exception is that lysine digestibility in WLP and CLP treatments was higher than that in PLP treatment in ileal digesta samples collected during postprandial 8 to 10 h (p<0.001).When the digesta samples were collected in six 2-h periods, waxy corn starch (high amylopectin) and corn starch (medium amylopectin) induced the digesta DM flow to decrease and then increase, and pea starch (high amylose) caused the digesta DM flow to decrease and then stay on a plateau. Throughout digesta collection period, compared with waxy corn starch and corn starch, pea starch increased digesta DM flow and reduced the apparent ileal digestibility of dietary amino acids. Differences in amino acids digestibility between groups were mainly reflected in the digesta collected after 4 h postprandial.


CON); the CON dietary supplemented with 0.4 mg/kg selenium from selenized yeast (Se); the CON dietary supplemented with 5 mg/kg Cd + 50 mg/kg Pb +3 mg/kg Hg + 5 mg/kg Cr (HEM), and the HEM dietary supplemented with 0.4 mg/kg selenium from selenized yeast (HEM+Se). The dietary HEM significantly increased Cd, Pb, and Hg deposition in the egg yolk and ovary, and Cd and Hg deposition in the oviduct and in the follicular wall (p < 0.05). The HEM elevated Fe concentration in the egg yolk, ovary, and oviduct (p < 0.05). The HEM decreased Mn concentration in the egg yolk, Fe, Mn, and Zn concentrations in the egg white, Cu concentration in the ovary, Mg concentration in the oviduct, as well as Ca, Cu, Zn, and Mg concentrations in the follicular walls (p < 0.05). Dietary Se addition elevated Se concentration in the egg yolk, oviduct, and follicular walls and Mg concentration (p < 0.05) in the oviduct, whereas it reduced Fe concentration in the oviduct compared with the HEM-treated hens. Some positive or negative correlations among these elements were observed. Canonical Correlation Analysis showed that the concentrations of Pb and Hg in the egg yolk were positively correlated with those in the ovary. The concentration of Cd in the egg white was positively correlated with that in the oviduct. In summary, dietary Cd, Pb, Hg, and Cr in combination caused ion loss and deposition of HEM in reproductive organs of laying hens. Dietary Se addition at 0.4 mg/kg from selenized yeast alleviated the negative effects of HEM on Fe and Mg ion disorder in the oviduct and follicle wall of hens.The objective of this study was to explore the toxic effects of different heavy metals in combination with their deposition and ion homeostasis in the reproductive organs and eggs of laying hens, as well as the alleviating action of selenized yeast. A total of 160 Lohmann pink-shell laying hens (63-week-old) were randomly allocated into four treatments with 10 replicates of four hens each. The four dietary treatments were the corn-soybean meal basal dietary (control;  Heavy metal contamination in animal feed has received much attention in recent years due to intensive agricultural development. The cadmium (Cd), mercury (Hg), and lead (Pb) are considered contaminants in poultry feed or diet, which are a prior hazard to animal health because they can induce organ damage by oxidative stress, even at the low exposure levels , 2. ExceSelenium is an essential micronutrient for poultry , and canThe maximum permitted limits of Cd, Pb, Hg, and Cr are 0.5, 5, 0.1, and 5 mg/kg, respectively, in the complete poultry feed according to the guideline of China . Several2), lead nitrate (Pb(NO3)2), mercury bichloride (HgCl2), and chromium trichloride (CrCl3) were purchased from Kelong company of Chengdu . Selenized yeast (SY) which contains 0.2% selenium was provided by Chelota biotechnology Co. Ltd . The standard solution of Cd, Cr, Pb, Hg, Fe, Cu, Mn, Zn, Na, K, Mg, Se, and Ca were purchased from the center of the national standard. Laying hens were Lohmann pink-shell with similar production performance obtained from commercial layer farms .Cadmium chloride (CdClCON), the CON diet supplemented with 0.4 mg/kg selenium from selenized yeast (Se), the CON diet added with 5 mg/kg Cd, 50 mg/kg Pb, 3 mg/kg Hg, and 5 mg/kg Cr (HEM), and the HEM diet supplemented with 0.4 mg/kg Se (HEM+Se). The experiment lasted 12 weeks. The corn-soybean meal basal diet  was formulated to meet or exceed the requirement of laying hens  were randomly allocated into four treatments with 10 replicates of four hens. Two adjacent cages (two birds each cage) were used as one replicate unit. The four dietary treatments were as follows: the corn-soybean meal basal diet . Two birds were selected from each replicate and euthanized by cervical dislocation, and the samples of the ovary and oviduct were collected for the minerals analyses. And then, the follicular walls of the largest yellow follicles with a diameter ranging from 38 to 42 mm, also known as late preovulatory follicles prior to ovulation were also collected and stored at −20°C until the minerals analyses.3/HClO4  was added to the vessel and the digestion was carried out at 180°C using a heater . After digestion, the residual solution (about 0.2 ml) was dissolved with 0.5 % HNO3 solution in a certain volumetric flask to obtain the sample solution. This sample solution was directly used for measuring the concentrations of Cd, Cr, Pb, Hg, Fe, Cu, Mn, and Zn. For determining the concentrations of Na, K, Mg, and Ca, the lanthanum oxide  and cesium chloride  were added to the sample solution. The concentrations of the Fe, Cu, Mn, Zn, Cd, Pb, and Cr were determined using an atomic absorption spectrometer  according to the China National Standard GB/T 13885-2017  at 180°C using a heater , and the residual solution was dissolved with 4.73 M HCl containing 200 g/L (m/v) of potassium ferricyanide for Se analysis. The instrument detection limit of Se was 0.30 μg/kg. For quality control, a standard reference of wheat powder or bovine liver provided by the National Institute of Standards and Technology  was included in each batch of analysis to verify the determination validation. The R-squared values of standard curves for the different minerals were higher than 0.99. The recovery rates of Fe, Cu, Mn, Zn, Cd, Pb, Cr, Na, K, Mg, Ca, Hg, and Se were 90.1–99.5%, 93.8–102.3%, 90.9–103.4%, 95.3–105.4%, 91.8–98.4%, 93.9–97.4%, 96.9–100.3%, 97.3–104.1%, 93.8–102.4%, 94.5–102.6%, 96.3–105.4%, 97.9–103.7%, and 92.9–102.1%, respectively.Egg yolks and egg whites were lyophilized prior to analyses. Then, a 0.5 g sample of lyophilized egg yolk or egg white, or ovary, oviduct, or follicular wall on the wet-tissue basis was transferred into a polytetrafluoroethylene digestion vessel. Then, 10 ml of HNO885-2017 . The con885-2017 . The lan885-2017 . In brieThe PROC MIXED procedure of SAS software  was used to analyze the data about concentrations of Cd, Cr, Pb, Hg, Se, Fe, Cu, Mn, Zn, Na, K, Mg, and Ca in the egg yolk, egg white, ovary, oviduct, and follicular walls.The model is as follows:i is the fixed effect of treatment , εij is the random error associated with the observation. Each replicate was used as the experimental unit. The Tukey test was used for multiple population comparisons among the different treatments with letter groupings. The results are presented as the mean and standard error of the mean (SEM). Values of p < 0.05 were considered statistically significant. Correlations between elements were investigated using Pearson's correlation test.Y is an observation of the dependent variable, μ is the population mean for the variable, αr > 0.50 and p < 0.05 were considered as an indication of a strong correlation.Additionally, Canonical Correlation Analysis (CCA) was used to analyze correlations between heavy metals in the egg yolk and ovary, as well as egg white and oviduct, 1 and W1 were extracted in the two groups of variables, respectively. V1 and W1 were the linear combinations of the variables for the two variables group, respectively. The overall correlation between the two sets of data objects was reflected by using the relationship between two comprehensive variables  the concentrations of Cd, Pb, Hg, and Fe (p < 0.05) the concentration of Mn in the egg yolk compared to the CON. Decreased as down in p > 0.05). However, the HEM diet supplemented with SY increased (p < 0.05) the concentration of Se, did not significantly affect the concentrations of Cd, Pb, Hg, Fe, and Mn in the egg yolk (p > 0.05) compared to the HEM-treated group.Dietary HEM significantly increased  the concentrations of Fe, Mn, and Zn compared to the CON. Whereas the HEM diet supplemented with SY significantly reduced (p < 0.05) the accumulation of Ca in the egg white compared with the HEM treatment (p > 0.05).Dietary HEM significantly reduced (reatment . Dietaryp < 0.05) the concentrations of Cd, Hg, Pb, and Fe, but significantly reduced (p < 0.05) the concentration of Cu in the ovary compared to the CON (p > 0.05) on the concentrations of Cd, Pb, Hg, Fe, and Cu compared to the HEM-treated group. Dietary treatments did not significantly affect the concentrations of Cr, Se, Mn, Zn, K, Mg, and Ca in the ovary (p > 0.05).Dietary HEM significantly increased ( the CON . Howeverp < 0.05) the concentrations of Cd, Hg, and Fe compared to the CON, whereas it decreased (p < 0.05) the concentration of Mg in the oviduct compared to the CON (p < 0.05) the concentration of Fe and significantly increased the concentrations of Mg and Se (p < 0.05), and had no significant effect (p > 0.05) on the accumulations of Cd and Hg compared to the HEM-treated group. Dietary treatments also did not significantly affect the concentrations of Cu, Mn, Zn, Na, K, and Ca in the oviduct (p > 0.05).Dietary HEM significantly elevated ( the CON . The HEMp < 0.05) the accumulations of Cd and Hg, but reduced (p < 0.05) the concentrations of Cu, Zn, Ca, and Mg in the follicular walls compared to the CON (p < 0.05) the concentration of Mg, and had no effect (p > 0.05) on the concentrations of Cd, Hg, Cu, Zn, and Ca in the follicular walls compared to the HEM-treated group. Dietary treatments also did not significantly affect the concentrations of Pb, Cr, Fe, Mn, Na, and K in the follicular walls (p > 0.05).Dietary HEM increased ( the CON . The HEMp < 0.01), Hg (p < 0.05), and Fe (p < 0.05). Pb was positively correlated with Cd (p < 0.01) and negatively correlated with Mn (p < 0.01). Hg was positively correlated with Cd (p < 0.05) and Cr (p < 0.05). Se was negatively correlated with Mn (p < 0.05). Cu was positively correlated with Zn (p < 0.01) and Ca (p < 0.01). Zn was positively correlated with Cu (p < 0.01), K (p < 0.01), Mg (p < 0.01), and Ca (p < 0.01). K was positively correlated with Zn (p < 0.01) and Mg (p < 0.05). Mg was positively correlated with K (p < 0.05), Zn (p < 0.01), and Ca (p < 0.05).There were 13 significant correlations among the concentration of 13 elements in the egg yolk . The elep < 0.05) and Mg (p < 0.01). Pb was negatively correlated with Na (p < 0.05). Se was negatively correlated with Fe (p < 0.05). Mn was positively correlated with Zn (p < 0.05) and Mg (p < 0.05). K was positively correlated with Cu (p < 0.01), and Na (p < 0.01). Zn was positively correlated with Fe (p < 0.05) and Mn (p < 0.05). Mg was negatively correlated with Cd (p < 0.01) but positively correlated with Fe (p < 0.05) and Mn (p < 0.05).There were 10 significant correlations among the concentration of 13 elements in the egg white . Cd was p < 0.01) and Hg (p < 0.05) but negatively correlated with Cu (p < 0.05). Pb was positively correlated with Cd (p < 0.01), Hg (p < 0.05), and Fe (p < 0.05). Cr was correlated positively with Hg (p < 0.05), and negatively correlated with Mg (p < 0.05). Hg had a negative correlation with Cu (p < 0.05) and was positively correlated with Fe (p < 0.01), Cr (p < 0.05), Pb (p < 0.05), and Cd (p < 0.01). Cu was negatively correlated with Cd (p < 0.05) and Hg (p < 0.05), and was positively correlated with Ca (p < 0.05). Fe was positively correlated with Pb (p < 0.05) and Hg (p < 0.05), and negatively correlated with Zn (p < 0.05). K was positively correlated Mg (p < 0.05). Mg was positively correlated with K (p < 0.01), and negatively correlated with Cr (p < 0.05). Ca was positively correlated Cu (p < 0.05).There were 12 significant correlations among the concentration of 13 elements in the ovary . Cd was P < 0.01) and Na (P < 0.01), and was negatively correlated with Mg (p < 0.01). Pb was positively correlated with Ca (P < 0.05). Cr was positively correlated with Fe (p < 0.05). Hg was positively correlated with Cd (p < 0.01), Zn (p < 0.05), and Na (p < 0.05), and was negatively correlated with Mg (p < 0.05). Cu was positively correlated with Mn (p < 0.01) and Zn (p < 0.01). Fe was positively correlated (p < 0.05) with Cr (p < 0.05) and Na (p < 0.01), and was negatively correlated with Mg (p < 0.05). Mn was positively correlated with Cu (p < 0.01), Zn (p < 0.01), and Ca (p < 0.05). Zn was positively correlated with Hg (p < 0.05), Cu (p < 0.01), Mn (p < 0.01), and Na (p < 0.01). Na was negatively correlated with Mg (p < 0.01), and positively correlated with Cd, Hg, Fe, and Zn (p < 0.05).There were 16 significant correlations among the concentration of 13 elements in the oviduct . Cd was p < 0.01) and Hg (p < 0.05). Pb was positively correlated with Cd (p < 0.01), Hg (p < 0.01), and Fe (p < 0.05), but negatively correlated with Mn (p < 0.01). Hg was positively correlated with Fe (p < 0.05), Cd (p < 0.05), and Pb (p < 0.01). Se was negatively correlated with Mn (P < 0.05). Cu was positively correlated with Zn (p < 0.01) and Ca (p < 0.01). Zn was positively correlated with Cu (p < 0.01), K (p < 0.01), Mg (p < 0.01), and Ca (p < 0.01). K was positively correlated with Zn (p < 0.01) and Mg (p < 0.05). Mg was positively correlated (p < 0.01) with Zn (p < 0.05), K, and Ca (p < 0.05).There were 14 significant correlations among the concentration of 13 elements in the follicular walls . Cd was p = 0.03). The result indicated that there were positive correlations between the concentration of HEM in the ovary and in the egg yolk. Which one of HEM exists a positive correlation between the ovary and in the egg yolk? In order to find answers, the coefficient of X or Y in the first typical variables V1 and W1 regression equation would need to be judged. The composition of V1 and W1 is shown in p < 0.05) with those in the egg yolk.The first canonical correlation coefficient between the heavy metal concentration in the egg yolk and the ovary was 1.000  which rep = 0.006) (p < 0.05) with that in the oviduct  . The res oviduct .In this study, we found that the complete feed contaminated with the combination of relatively higher levels of Cd, Cr, Pb, and Hg increased the Pb, Cd, and Hg deposition in the egg yolk and ovary, as well as the accumulation of Cd and Hg in the oviduct and the follicular wall. These results suggested that the ovary was the target reproductive tissue of the Cd, Pb, and Hg, and the oviduct and follicular wall were the special target reproductive tissues of Cd and Hg in laying hens. In agreement, dietary contamination of Cd, Pb, or Hg individual increased its deposition in the egg yolk , 32, 33 2 for 90 days decreased the concentrations of Cu, Zn, and Ca, but increased Fe concentration in the ovary of hens aged 31 weeks. Qu et al. , and Cd may replace the essential metals Cu and Zn or share the MT with them .BW is employed by Chelota biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.