Background

Leptin is a polypeptide hormone produced mainly by adipose cells[1],being also expressed by other tissues such as skeletal muscle[2],mammary gland[3],and others[4].The main physiological function of leptin is energy homeostasis by controlling feed intake by inhibiting hunger[5-7].It acts on a specific receptor(LEPR)in the brain,most specific to the hypothalamus in ventromedial,dorsomedial,and arcuate nuclei[8].Although the physiological role of leptin is to control food intake,obese individuals generally present greater circulating levels of the hormone due to their greater percentage of body fat[9].Obese people usually present resistance to leptin by alterations in the LEPR signaling and/or diminished crossing into the blood brain barrier[10].

Fat metabolism and deposition is important for livestock,affecting the quality of the products(beef,milk,etc)and providing energy reserves for reproduction and lactation[11].Soon after the discovery of the obese gene in 1994[1],its product,leptin,was identified for potential applications in animal production[12].Indeed,research has been done on different aspects of leptin in livestock,including the use of its polymorphisms in animal breeding programs[13-17]and the use of circulating plasma leptin as a predictor of body composition[18,19].

In sheep,plasma concentration of leptin is more related to variation in body fatness(35%)than to nutritional status(17%)[20].Corroborating this result,leptin levels in beef cattle(Bos taurus)were positively associated with adipose cell size[21],body fatness[22],and with the 12thrib and rump fat thickness,explaining 16.8%of the variation in the 12thrib fat thickness[19].In the same work,the authors[19]showed the circulating leptin was positively associated with residual feed intake(RFI),a measure of feed efficiency,however,they explained very little of the variation in RFI(＜3.2%　of the variance).A moderate association(r=0.31)of leptin with RFI was also showed by Richardson et al.[23]in Angus steers.

另外，高科技企业技术团队的高薪支出税费的抵扣也是企业的另一个关注点。以中国排名前几的技术有限公司为例，2017-2018年华为技术团队个人年薪30-60万不等，百度技术团队个人年薪35-60万不等，阿里巴巴技术团队个人年薪40-60万不等，腾讯技术团队个人年薪25-48万不等，其他职位最高年薪20万元以下。将各企业技术团队个人年薪折中与其他职位最高年薪作比较，由图3可以看出企业技术团队的高年薪支出成为企业迫切希望纳入进项税抵扣的一个关注点。

In Bos indicus,the majority of beef cattle grown in Brazil,Leptin　genepolymorphismswereassociated mostly with growth and carcass traits[17,24,25],but no study was found on the circulating plasma leptin and/or muscle gene expression association with feed efficiency.Thus,the aim of this work was to characterize the circulating plasma leptin and its muscle gene expression in young Nellore bulls with divergent residual feed intake.

Methods

Animals and experimental design

1.4.2 粪污处理设施 可根据养殖规模配套建设干粪处理房和堆积发酵池，或使用干粪处理机加工生产有机肥料，同时还应配备沼气等粪污处理设施。

A:从描述来看，并没有什么好让你担心的。孩子喜欢和比自己大的人交朋友，这很正常。他的社会圈子还小，就只认识这位小姐姐，于是特别关注。你可以创造适当的机会让他们多接触，同时也让他有机会交同龄朋友，以平常心看待就是了。

In this study,it was shown that circulating plasma leptin and muscle gene expression were greater in high feed efficiency group(LRFI)than in low feed efficiency group(HRFI)of Nellore bulls.In addition,feed efficient animals showed less fat deposition,probably a consequence of significant less DMI.In a previous work from the group,it was demonstrated that LRFI animals have less circulating plasma cholesterol at the end of the feeding trial[26].Since the main role of leptin is regulate food intake in different species[29-31],these results support the physiological role of this hormone in Nellore cattle,since leptin is controlling DMI,plasma cholesterol,and fat deposition.Supporting this concept,less efficient animals have less muscle gene expression and circulating leptin which increases DMI,plasmatic cholesterol,and fat deposition.

Plasma samples were collected at 56 d of feeding trial and at slaughter(80 d)by jugular venipuncture using vacutainer tubes containing sodium heparin as anticoagulant(BD Vacutainer Plus,BD,Brazil).All blood samples were centrifuged at 3,500×g for 15 min at 4 °C,and plasma was collected and stored at-20°C until assayed for leptin.The concentration was determined in duplicate 100 μL aliquots of plasma samples,using the leptin RIA kit(Multi-species leptin RIA kit,Cat.#XL-85 K,Millipore,St.Charles,MO,USA)and following the manufacturer’s instructions.Intra and interassay CVs for the leptin assay were less than 10%as described by Delavaud et al.[20].

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

Animals were slaughtered on d 80 at the Experimental slaughterhouse of University of São Paulo,after fasting from feed for 16 h.Immediately after slaughter,samples of the medial portion　from the right Longissimus lumborum muscle(between the 12thand 13thribs)were taken from 10 animals of each RFI group.The samples were collected and immediately immersed in RNA stabilization solution　(RNAholder-　Bioagency,São Paulo/SP,Brazil).The sample were maintained overnight at 4 °C and then stored at-80 °C until RNA extraction.

A feeding trial of 98 Nellore bulls(16 to 20 months old and 376±29 kg BW)was conducted at the Faculdade de Zootecnia e Engenharia de Alimentos,Universidade de São Paulo(FZEA/USP),Pirassununga,SP,Brazil.All the details regarding animals,traits,and diet can be found in Alexandre et al.[26].Briefly,the data collection period consisted of 70 d preceeded by 21 d of adaptation to diet and environment.Animals were weighed in the beginning,the end,and every 14 d of the experimental period.Additionally,daily dry matter intake(DMI)for each animal was measured by weighting the orts every day.Residual feed intake(RFI)was calculated[27],and two groups were formed:low RFI(LRFI)and high RFI(HRFI),each composed of 20 extreme animals.

Gene expression of Leptin

现代医院管理制度包括3个层面的逻辑架构[4]：宏观层面的政府治理机制、中观层面的法人治理机制和微观层面的医院内部管理制度。

Total RNA was extracted from 300 mg of powdered tissue samples using Trizol reagent following the manufacturer’s protocol(Invitrogen,Carlsbad,CA,USA).After quantification in a spectrophotometer,1 μg of total RNA was treated with DNAse(Invitrogen,Carlsbad,CA,USA)and reverse transcribed into cDNA using Go Script™Reverse Transcription System kit(Promega Corporation,Madison,WI,USA).Real time-PCR was performed on a CFX ConnectTM　Real-TimePCR　detectionsystem　(Bio-Rad,Hercules,CA,USA),using SYBR Green RT-PCR kit(Applied Biosystems,Foster City,CA,USA)with the following cycle parameters:95°C for 3 min and 40 cycles at 95 °C for 10 s and 60 °C for 30 s.Primers utilized for PCR were leptin(F)5’GGGCACGTCAGCATCTATTA3’and leptin(R)5’CCTGTCTGCTGTTATGGTCTTA3’,and for the endogenous control,the ribosomal 18S(F)5’CCTGC GGCTTAATTTGACTC3’and 18S(R)5’AACTAAGAACGGCCATGCAC3’were used.The amplification efficiency was 0.90 to 0.99 for leptin and 18S,respectively.All reactions were performed in triplicate,and the method of Livak and Schimttgen was used for gene expression analysis[28].

(5)成槽作业时，抓斗出入导墙口时要轻提慢放，防止泥浆动荡。抓斗上升时，及时补充合格泥浆，抓斗上升速度与泥浆补充速度相适应，并保持泥浆液面在导墙顶面以下300 mm以内，避免出现槽内泥浆下降过快而产生塌孔现象。

Statistical analysis

All analyses were performed using GraphPad Prism version 6.0 for Mac(GraphPad Sotware,La Jolla California,USA).The phenotypic measures assessed in the LRFI and HRFI groups were first tested for Gaussian distribution by the Shapiro-Wilk test,and later,they were tested for difference between the means of the groups by Student’s　t-testfor　normaldistributed　data　and Mann-Whitney-Wilcoxon　test　for　nonparametric data.　The　correlation　between　the　two　leptin measures was performed with the Pearson test.A two-way ANOVA followed by the Fisher post-test was used for leptin plasmatic concentration on d 56 and d 80.Statistically significant results were considered when P＜0.05.

Results

Characterization of feed efficiency groups

Phenotypic traits(related to body weight,feed efficiency,muscle and fat deposition,liver weight,and cascass yeld),are already published and no significant difference of sire or age on RFI was found[26].Animals of LRFI and HRFI groups showed no difference in body weight(initial,final,or average daily gain)and rib eye area(initial,final,and gain).On the other hand,DMI and RFI were different between groups,wherein LRFI animals presented less DMI[26].In addition,LRFI animals presented less fat deposition in both back fat and rump fat and less pelvic and kidney fat relative weight in comparison with HRFI.Thus,LRFI animals eat less and are leaner than HRFI animals[26].

Circulating plasma leptin and muscle gene expression

The correlation between those two measures was moderate to strong and significant(r=0.5817 and P=0.0071).In this experiment,leptin concentration in plasma increased over time in both groups(Fig.1a),accounting for 21.3%of the total variation and was considered significant(P＜0.0001).In addition,leptin concentration was different between groups(P=0.0049).However,no interaction between time and groups was noted though(P=0.513).

(4)制作布局。制作时要注意画面中对象的数量、布局、色彩搭配等，还有按钮的位置、样式等，如直接使用故事中的卡通形象作为按钮，让它说话或运动。

Leptin gene expression was performed in muscle samples collected at slaughter.LRFI animals showed greater expression of leptin(P=0.0022)compared to HRFI animals,reflecting the increased leptin plasma concentration of animals at slaughter(Fig.1b).

Discussion

Plasmatic leptin quantification

Nonetheless,in other experiments on cattle,leptin concentration was positively associated with DMI,body fat,and RFI([19,23,32].In these cases,leptin seems to be related to body fat mass but not with the control of food intake.Interestingly,this phenotype resembles the resistance to leptin in obese humans where even elevated levels of leptin produced by greater percentage of body fat failed to control hunger and modulated body fat[33].In accordance with the hypothesis,a recently published work from Foote et al.[34]suggested that beef cattle on a high grain finishing ration could become slightly leptin resistant.

Fig.1　Circulating plasma leptin and muscle gene expression in young Nellore bulls divergently selected for feed efficiency.a Circulating plasma leptin were evaluated on d 50 and at the end of the experiment on d 80.b Leptin gene expression in muscle was performed at the end of the experiment on d 80.*notes statistical difference(P＜0.05)

The opposing results observed in the current study and others suggest the hypothesis that animals in this experiment are not in an “obese”phenotype,being instead where leptin reflects fat body mass.This could be explained by the conjunction of:(1)having used young bulls while others used steers[18,19,23,34]or heifers[34,35]and/or;(2)Nellore is an indicine breed and most experiments on cattle are performed in taurine breeds.The first possibility is probably the most relevant since the effect of sex on fat deposition has long been described.Total rate of fat deposition relative to muscle is similar for heifers and steers but significantly lower in bulls[36].These authors concluded that differences in fattening patterns among sexes result from a combination of fattening at a lighter weight of carcass muscle in heifers than steers and in steers than bulls,in addition to a more rapid rate of fat deposition relative to muscle in heifersand steers compared to bulls.In addition,the effect of sex is well known to be related to feed efficiency,where bulls are more efficient than steers or heifers[37,38].In humans,females have greater circulating leptin as compared to males,even after the correction for differences in body fat mass[39].In vitro experiments with human adypocytes in primary culture showed that both testosterone,and its biologically active metabolite dihydrotestosterone inhibited leptin secretion up to 62%,supporting that sex is clearly a relevant factor on leptin metabolism[39].

We would also like to thank Michael James Stablein of the University of Illinois Urbana-Champaign for his translation services and review of this work.

Therefore,it is plausible that the increased muscle gene expression of leptin and circulating leptin levels in feed efficient young Nellore bulls in comparison to less efficient animals,although,others demonstrated the opposite.This result also calls attention for the use of leptin as a possible predictor for body fat mass in cattle,especially if considered for use in Nellore cattle.

Conclusion

To the best of our knowledge,this work demonstrated for the first time that leptin plays its physiological role in young Nellore bulls,rather its pathological role,controlling food intake because feed efficient animals have more circulating leptin and lower residual feed intake.

Abbreviations

ADG:Average daily gain;BWf:Final body weight;BWg:Body weight gain;BWi:Initial body weight;DMI:Dry matter intake;HRFI:Low feed efficient;RBg:Rib eye gain;REAf:Final rib eye area;REAi:Initial rib eye area;RFI:Residual feed intake;RIA:Radioimmunoassay;LEPR:Leptin receptor;LRFI:High feed efficient

2.通过各种渠道提高自身学历层次。学校应该为教师提供到其他高校或者到国外进修的机会。应用型本科院校的商务英语教师学历多为本科学历，无论从专业发展的角度还是从提升自身能力的角度，教师都应继续考取本专业的硕士和博士，扩大自己的视野，不仅要掌握扎实的语言知识，还应掌握较强的商务实践能力。只有这样才能有效保证教学质量，培养社会真正需要的应用型商务人才。应用型本科院校可以根据本学校实际，选拔优秀青年教师到全国一流特色大学进行进修；利用假期为教师提供短期的商务培训；为教师创造出国进修以及出国提高学历的机会，学习国外的先进教育经验和理念。

Acknowledgements

One should also consider the possible effect of genetic differences between Nellore and taurine breeds for the observed effect in this experiment.Indicine cattle are known to be more adapted to tropical climate but with less growth performance than taurine breeds.Corroborating this idea,Marcondes et al.[40]demonstrated the dry matter intake and performance of steers were higher in Nellore crossbreds(Nellore-Simmental and Nellore-Red Angus)than that of pure Nellore,however,no difference in feed efficiency was noted between groups.Paschal and colleagues[41]studying the postweaning and feedlot growth and carcass characteristics of five indicine breeds and one taurine(Angus)showed that Zebu crosses(including Nellore)grew faster postweaning and were heavier and taller than Angus crosses.However,the Angus crosswasmoredesirablein　marblingscoreand quality grade.Beef products derived from Nellore are recognized by the international market as very lean meat due to lack of intramuscular fat[42].The same authors demonstrated the difference in intramuscular fat content in skeletal muscle of Nellore and Angus cattle is due to a slightly enhanced muscle adipogenesis in Angus cattle.Altogether,these results support the concept that Nellore have diferent growth curves in comparison to taurine breeds taking more time for fat deposition.

Slaughter and tissue sample collection

This work was financially supported by “Fundação de Amparo à Pesquisa do Estado de São Paulo”(FAPESP 2010/05650-5;2014/02493-7,2014/07566-2).

所谓的“专业”在具体的实务情境当中有不同的体现，在很多人看来，一线社工尚未显示出自身不可替代的专业性，但是确实在不断走向专业化的路途中。而在真正实现专业化的前期阶段，外界就会形成对社工职业的初步认知，而对社工职业来说，其最看中的是所持有的价值理念并不容易凸显出来的时候，就很容易给外界一种不专业的形象。即目前社会公众对于社工职业的认知和社工的专业化是同时进行的，如何形成对于社工职业的专业认知是每一位一线社工共同去面对的。

Availability of data and materials

脑梗死为临床常见的脑血管疾病，病死率及致残率高。经手术治疗后，患者多会出现不同程度的后遗症，如头昏、高血压、便秘等。常规西医护理方法，以给药、健康宣教及病情监测为主，患者后遗症发生率较高。有研究指出，将中医护理手段应用到脑梗死后遗症患者的护理中，有助于促进病情康复，提高患者的生活质量。为改善预后，本文于本院2016年6月—2018年6月收治的脑梗死后遗症患者中，随机选取78例作为样本，阐述了脑梗死后遗症患者的中医护理方法，并观察了护理效果。

Authors’contributions

PAA,CMB,SLS,PRL,and HF designed the experimental study;LFMM and CMB performed the leptin circulating analysis;PAA,MHS,LFMM,and HF conducted the in vivo experiment and sample collection;ET helped LFMM on gene expression analysis;LFMM,PAA,FJN,and HF drafted the manuscript.All authors read,made critical revisions,and approved the final manuscript for submission.

(2)提高乘客乘坐舒适性。CFRP的阻尼是普通金属的10～100倍，其高阻尼特性可有效降低结构的振动，改善乘客的乘坐舒适性。表2为CFRP与金属材料的导热性能对比。由表2可以看出CFRP具有良好的隔热性能，其不但可以提高车体的隔热性能，还能减少车辆隔热材料的使用量。CFRP还具有热膨胀系数小、尺寸稳定性较好、抗磁、绝缘、隔音等多种优良性能，以上性能均能提高车辆舒适度。

Ethics approval and consent to participate

All animal protocols were approved by the Institutional Animal Care and Use Committee of Faculdade de Zootecnia e Engenharia de Alimentos,Universidade de São Paulo(FZEA-USP-protocol number 14.1.636.74.1).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

从检测结果显示，本次监测仅在郊区农贸市场查出淡水鱼类华支睾吸虫囊蚴阳性，分析其原因市区农贸市场和超市的淡水鱼类一般来源于人工养殖鱼塘，郊区河塘分布，野生小鱼较多，在郊区农贸市场贩售的淡水鱼类很有可能捕捉于河塘，且居民有煎炸小鱼作为特色菜肴的进食习惯。再有本次检出阳性的淡水鱼类均为麦穗鱼和鳑鲏鱼，与浙江省一些地区研究结果相符［18-19］。此类小鱼体积较小、野生，生活在浅水区，易接触栖息于水域边和水草上的淡水螺逸出的尾蚴而感染，而一般大型鱼种多活动在水域下层，接触尾蚴而感染的机会较小［20］，故此类小鱼多在当地河塘捕捉后出售于就近农贸市场，超市并无供应。

Author details

1Departmento de Zootecnia,Universidade Federal dos Vales do Jequitinhonha e Mucuri,Diamantina,MG 39100-000,Brazil.2Present adress:Faculdade de Ciências Agrárias e Veterinárias,Universidade Estadual Paulista,Jaboticabal,SP 14884-900,Brazil.3Departamento de Medicina Veterinária,Faculdade de Zootecnia e Engenharia de Alimentos,Universidade de São Paulo,Av.Duque de Caxias Norte n°225,Pirassununga 13635-900,SP,Brazil.4Departmento de Zootecnia e Desenvolvimento Agrossocioambiental Sustentável,Faculdade de Veterinária,Universidade Federal Fluminense,Niteroi,RJ 24230-340,Brazil.5Departamento de Zootecnia,Faculdade de Zootecnia e Engenharia de Alimentos,Universidade de São Paulo,Pirassununga 13635-900,SP,Brazil.

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