调控家禽肌肉和肝脏代谢的胰岛素信号

本文综述了胰岛素通过受体对鸡肝脏和肌肉的一般代谢和基因表达的调控作用。与哺乳动物相比,家禽血液循环中具有相似的胰岛素浓度,但仍然可以维持高血糖水平,这可能是家禽对外源性胰岛素敏感性低的结果。为了确定这种低敏感性是否是家禽与哺乳动物之间胰岛素受体信号差异的结果,家禽组织中的胰岛素受体已经得到了验证,并在肝脏和肌肉组织发现了两种胰岛素受体底物(胰岛素受体底物-1和结构域蛋白C)。与哺乳动物相比,目前我们对家禽胰岛素信号传导的认识还不全面。当考虑到参与胰岛素级联反应的成分异构体数量时,这一点尤其明显,许多异构体可能尚未在家禽中得到验证。尽管现有数据存在一些不足,但鸡肝脏中的胰岛素信号与哺乳动物相似,但肌肉与哺乳动物不同。在腿部肌肉中,鸡与哺乳动物在胰岛素信号级联的早期阶段不同,其中,鸡的磷脂酰肌醇-3激酶(PI3K)活性大约是大鼠的30倍,这种肌肉PI3K的高活性可能过度刺激鸡的反馈抑制通路,从而使鸡肌肉对胰岛素不敏感。     

The impact of the chromium supplementation on insulin signalling pathway in different tissues and milk yield in dairy cows

Thirty days before expected time of parturition, 20 Holstein cows were divided into ?Cr and +Cr groups. From day 25 before parturition (BP ) up to day 30 after parturition (AP ), +Cr cows received 10 mg of Cr (chromium‐enriched yeast) daily. Muscle and adipose tissue samples were taken at days ?30, ?10, +7 and +10 related to parturition, when body condition score (BCS ) was also determined. Hepatic tissue samples were taken at days ?10 and +7. Tissue samples were used for determination of the insulin signalling pathway protein expressions. Intravenous glucose tolerance test (IVGTT ) was performed at days ?28, ?7, +10 and +30. Milk yield was recorded during first 14 weeks AP . Milk composition was obtained at days 7 and 28 AP . At day 10 BP , protein content of β ‐subunit of insulin receptor (IR β ) was significantly higher (p ? 0.05) in muscle, and phosphorylation of insulin receptor substrate 1 at serine 307 (pIRS ‐1 Ser³⁰⁷) was significantly lower (p ? 0.05) in hepatic tissue of +Cr group. After parturition, pIRS ‐1 Ser³⁰⁷ was significantly lower in muscle tissue at days 7 and 28 (p ? 0.05 and p ? 0.001, respectively), while phosphorylation of Akt at serine 473 (pA kt Ser⁴⁷³) was significantly higher (p ? 0.01) in hepatic tissue at day 7 AP in +Cr group. Chromium had opposite effect on insulin kinetics during IVGTT s obtained BP and AP . Insulin secretion was significantly reduced at day 7 BP and significantly enhanced at day 10 AP , when NEFA concentration was also significantly increased. Milk yield and ECM value were depressed in +Cr group. DMI and BCS were significantly enhanced in +Cr group at day 7 BP . In conclusion, chromium modulates insulin signalling pathway in dairy cows, but targeted signalling molecules are different in antepartal then post‐partal period, probably due to duration of exposure to chromium and different energy status between those periods.     

成纤维细胞生长因子信号通路在小鼠牙齿发育中的表达及其作用

小鼠牙齿发育是一个外胚层来源的上皮和神经嵴来源的间充质相互作用的过程.在这个相互作用过程中,成纤维细胞生长因子(FGFs)信号通路在牙齿发生位置的确定,牙齿发育的起始、细胞的增殖分化和牙齿的形态建成等方面都发挥着重要作用.该家族由23个配体(Fgf1-23)组成,通过4种特异性受体(FGFR1-4)来发挥作用.本文综述FGFs信号通路的配体和受体在小鼠牙齿发育中具体的表达模式及其作用.     

Hypertriglyceridaemic insulin-resistant obese dog model: effects of high-fat diet depending on age

In humans, obesity is closely associated with insulin resistance (IR) and dyslipidaemia. The purpose of this study was to explore the effect of age on metabolic disturbances related to obesity in dogs ( n  = 25). Three age-groups of dogs (puppies, young adults and mature adults) were overfed to induce obesity, and body composition, insulin sensitivity index (I_IS) (euglycaemic–hyperinsulinaemic glucose clamp) and plasma lipids were measured. Fat mass was similar in the three obese groups (30 ± 1% in puppies, 34 ± 1% in young adults and 39 ± 1% in mature adults). In mature adults, body weight (BW) increased (+45%, p < 0.001) and I_IS decreased (−60%, p < 0.001) over 22 weeks. In young adults, BW gain was similar but slower (60 weeks) and I_IS decreased to a lesser extent (−49%, p < 0.001). Overfed puppies weighed 30% more (p < 0.01) than normally-fed control puppies, but there was no change in I_IS. Unlike young and mature adults, obese puppies did not exhibit significant changes in triglycerides (TG) and free fatty acid concentrations. In conclusion, as in humans, obese dogs develop IR that is associated with high TG levels; however, younger animals may be better able to balance energy needs with energy consumption.     

WNT信号通路的研究进展

Wnt(wingless-type MMTV integration site family members)信号通路与细胞的发育分化密切相关,对正常和肿瘤细胞生长都至关重要,因其启动蛋白为Wnt蛋白而得名。本文综述了Wnt蛋白家族、Wnt信号通路、Wnt信号通路与疾病的关系以及WNT信号通路的研究进展,并对家族中一些成员的研究进行了展望。     

雏鸡烟曲霉感染NOD1信号通路关键分子mRNA水平的变化

为明确烟曲霉菌感染雏鸡后肺组织内核苷酸结合寡聚化结构域1(nucleotide-binding oligomerisation domain1,NOD1)信号通路中关键分子mRNA水平的变化情况,取10日龄雏鸡40只,随机分为2组。感染组(Aspergillus fuigatus,Af)雏鸡滴鼻烟曲霉菌孢子悬液,50μL/只;对照组雏鸡滴鼻等量生理盐水。分别于感染后12、24、48 h处死雏鸡。无菌取肺组织,菌落计数法测定肺组织中烟曲霉菌的负荷;实时定量PCR方法检测肺组织中NOD1、Rip2、NF-kBp65 mRNA的表达情况。结果表明,Af组0 h未见烟曲霉菌生长,其他各时间点均有霉菌生长,且霉菌负荷量逐渐增加。对照组未见烟曲霉菌生长。在烟曲霉菌感染后24、48 h肺组织中NOD1和NFkBp65 mRNA的表达水平显著高于对照组(P<0.05)。感染后12、24 h肺组织中Rip25 mRNA的表达水平显著高于对照组(P<0.05)。由此说明,NOD1信号通路在烟曲霉菌感染后被激活,其在抗烟曲霉菌感染免疫中必定扮演重要作用,详细机制有待进一步研究。     

Effects of a supplement containing chromium and magnesium on morphometric measurements, resting glucose, insulin concentrations and insulin sensitivity in laminitic obese horses

Reasons for performing study: Obesity and insulin resistance are risk factors for laminitis in equids and supplements containing chromium and magnesium might improve insulin sensitivity. Hypothesis: A supplement containing chromium, magnesium and other nutraceuticals would alter morphometric measurements, blood variables, and insulin sensitivity in laminitic obese horses. Methods: Twelve previously laminitic obese (body condition score ≥ 7/9) horses were randomly allocated to treatment (n = 6) and control (n = 6) groups and 2 obese horses with clinical laminitis were included in the treatment group. Treated animals received 56 g supplement with 0.25 kg oats once daily for 16 weeks. The supplement contained chromium (5 mg/day as yeast), magnesium (8.8 g/day as oxide/proteinate), and other nutraceuticals. Insulin‐modified frequently sampled i.v. glucose tolerance tests were performed with hay provided at 0, 8 and 16 weeks, and insulin sensitivity was estimated by minimal model analysis. Physical measurements were collected at the same points. Horses were not exercised. Results: Hyperinsulinaemia (>30 µu/ml) was detected in 12 of 14 horses prior to treatment. Glucose and insulin data from one mare with clinical laminitis were excluded because of persistent pain. Mean ± s.d. insulin sensitivity was 0.64 ± 0.62 × 10^?4 l/min/mu prior to treatment for the remaining 13 horses. Time and treatment × time effects were not significant for any of the variables examined, with the exception of resting insulin concentrations, which significantly increased over time (P = 0.018). Health status remained the same. Conclusions: The supplement containing chromium and magnesium evaluated in this study did not alter morphometric measurements, blood variables, resting insulin concentrations or insulin sensitivity in laminitic obese horses. Potential relevance: Additional research is required to determine the appropriate use of chromium and magnesium supplements in horses.     

Involvement of the Ca2+ signaling pathway in osteoprotegerin inhibition of osteoclast differentiation and maturation

The purpose of this study was to determine whether the Ca²⁺ signaling pathway is involved in the ability of osteoprotegerin (OPG) to inhibit osteoclast differentiation and maturation. RAW264.7 cells were incubated with macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) to stimulate osteoclastogenesis and then treated with different concentrations of OPG, an inhibitor of osteoclast differentiation. The intracellular Ca²⁺ concentration [Ca²⁺]_i and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in the different treatment groups were measured by flow cytometry and Western blotting, respectively. The results confirmed that M-CSF + RANKL significantly increased [Ca²⁺]_i and CaMKII phosphorylation in osteoclasts (p < 0.01), and that these effects were subsequently decreased by OPG treatment. Exposure to specific inhibitors of the Ca²⁺ signaling pathway revealed that these changes varied between the different OPG treatment groups. Findings from the present study indicated that the Ca²⁺ signaling pathway is involved in both the regulation of osteoclastogenesis as well as inhibition of osteoclast differentiation and activation by OPG.     

乳酸菌发酵饲料对SPF鸡免疫功能的影响

本试验旨在研究乳酸菌发酵饲料对SPF鸡免疫功能和磷脂酰肌醇三激酶/蛋白激酶B(PI3K/Akt)信号通路的影响。选取14日龄无特定病原体(SPF)雏鸡120羽,随机分为4组,每组5个重复,每个重复6羽。对照组饲喂基础饲料,发酵饲料组饲喂乳酸菌发酵饲料,基础阻断剂组为基础饲料加PI3K阻断剂组,乳酸菌阻断剂组为乳酸菌发酵饲料加PI3K阻断剂。试验期21 d。结果表明:发酵饲料组与对照组、乳酸菌阻断剂组与基础阻断剂组比较,乳酸菌发酵饲料可降低雏鸡耗料增重比(P<0.05),提高日增重、T淋巴细胞转化率、B淋巴细胞转化率、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)、干扰素-α(IFN-α)、免疫球蛋白M(IgM)、免疫球蛋白A(IgA)和免疫球蛋白G(IgG)含量(P<0.05),提高雏鸡新城疫抗体(NDV-Ab)水平(P<0.05),提高雏鸡脾脏中PI3K、Akt mRNA和蛋白表达量(P<0.05);基础阻断剂组与对照组、乳酸菌阻断剂组与发酵饲料组比较,PI3K阻断剂可以提高雏鸡耗料增重比(P<0.05),降低日增重、T淋巴细胞转化率、B淋巴细胞转化率、IL-2、IL-6、IFN-α、IgM、IgA和IgG含量(P<0.05),降低雏鸡NDV-Ab水平(P<0.05),降低雏鸡脾脏中PI3K、Akt mRNA和蛋白表达量(P<0.05)。由此可见,乳酸菌发酵饲料可能是通过激活PI3K/Akt信号通路来提高SPF雏鸡的免疫功能。     

枯草芽孢杆菌RZ001对乳鼠肠道发育、肠道菌群和Wnt信号通路相关基因表达的影响

本试验旨在研究枯草芽孢杆菌RZ001的早期定植对乳鼠早期生长、肠道发育、肠道菌群和Wnt信号通路相关基因表达的影响.选择4窝数量相同的新出生的C57BL/6小鼠乳鼠,分为2个组(对照组和试验组),每组2个重复.从出生后第2天开始,试验组乳鼠连续14 d灌胃10μL枯草芽孢杆菌RZ001(1×107 CFU/d),对照组...     

补充牛磺酸对高脂饮食C57BL/6J小鼠糖脂代谢的影响

【目的】通过建立高脂动物模型,探究牛磺酸对肥胖小鼠糖脂代谢的影响。【方法】将20只5周龄SPF级C57BL/6J雄性小鼠随机分为4组：空白组、模型组、3%牛磺酸组和5%牛磺酸组,每组5只,试验期为15周。空白组小鼠饲喂对照饲粮,模型组饲喂高脂饲粮,其余两组在模型组饲粮的基础上分别添加3%、5%的牛磺酸。试验结束前2周分别进行口服葡萄糖耐受实验（OGTT）和胰岛素耐受实验（ITT）。试验结束后乙醚麻醉眼球取血,采用颈椎脱臼法处死小鼠,采集肝脏、肾脏、脾脏、附睾脂肪组织并称重,计算脏器/脂肪系数。测定血清中各项生化指标及瘦素（LEP）、脂联素（ADPN）含量,以及肝脏中甘油三酯（TG）、总胆固醇（TC）的含量,采用油红O染色和苏木精-伊红（HE）染色分别观察肝脏脂滴分布和脂肪组织形态变化。【结果】①与空白组相比,模型组小鼠体重极显著增加（P<0.01）,血糖（GLU）及血脂指标出现异常,葡萄糖耐受和胰岛素耐受的曲线下面积极显著升高（P<0.01）,符合肥胖模型的特征。②与模型组相比,添加牛磺酸后小鼠总增重、肝脏系数和脂肪系数均极显著降低（P<0.01）;血清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）活性及低密度脂蛋白（LDL）含量极显著升高（P<0.01）,GLU含量显著下降（P<0.05）;肝脏中TG和TC含量极显著下降（P<0.01）,ADPN含量极显著升高（P<0.01）;葡萄糖耐受和胰岛素耐受的曲线下面积均极显著降低（P<0.01）;肝脏中的脂滴数量减少,脂肪细胞的细胞截面积极显著减小（P<0.01）,细胞数量极显著增多（P<0.01）,脂肪细胞分布较均匀。【结论】牛磺酸可通过调节高脂饮食诱导的肥胖小鼠糖脂代谢水平,改善其肝脏及脂肪的组织病理形态,可能对肝脏具有保护作用。     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

抗脂肪细胞膜蛋白抗体对试验性肥胖大鼠5种激素水平的影响

为了验证抗脂肪细胞膜蛋白抗体减少动物脂肪沉积的作用效果,并对该抗体用于人类肥胖的治疗提供理论依据和试验资料,本试验分别对4组试验性肥胖大鼠注射0、0.1、0.5、1.0mg/mL剂量的抗脂肪细胞膜蛋白抗体,并检测4周内大鼠血清中5种激素含量及变化。结果显示,试验组血清中胰岛素(INS)含量低于对照组,Ⅰ组与Ⅳ组4周内均差异显著(P0.05);试验组与对照组、胰岛素样生长因子1(IGF-1)含量无显著差异(P0.05);注射抗体后,试验组生长激素(GH)含量显著增高,且Ⅰ、Ⅲ组4周内均差异显著(P0.05);第1周内,试验组三碘甲状腺原氨酸(T3)含量与对照组无显著差异(P0.05),后3周内Ⅰ组和Ⅳ组差异显著(P0.05);第1、3、4周内,Ⅰ组和Ⅳ组四碘甲状腺原氨酸(T4)含量差异显著(P0.05),第2周内无显著差异,Ⅱ组和Ⅲ组差异均不显著(P0.05)。此外,不同组间的差异显著性随注射剂量不同而不同。     

胰岛素对雏鸡心肌发育及其超微结构的影响

选取16只体重相近的健康雏鸡,随机分为两组。试验组每天定时腹腔注射生理剂量胰岛素,对照组腹腔注射等剂量的生理盐水。连续注射10d后称重,剖检取样并制片,通过分析心体比和透射电镜观察雏鸡心肌的超微结构变化,探讨胰岛素对雏鸡心肌发育及其超微结构的影响。结果表明,与对照组相比,试验组雏鸡的体重、心脏重和心体比没有显著变化,但其心肌的线粒体数量和横截面积明显增加。提示生理剂量胰岛素有促进心肌细胞代谢的作用。     

BMP4诱导的Smad9在小鼠卵泡发育中的作用及其机制的初步研究

本研究旨在探讨Smad9在小鼠卵泡发育中的作用,为卵泡生长发育及其调控机制的研究提供新思路。将6周龄的雌性昆明小鼠随机分成3组,分别腹腔注射生理盐水、骨形态发生蛋白-4（BMP4）和Smad9抑制剂（LDN-193189）,依次作为对照组、BMP4组和LDN组。48 h后收集卵巢制作石蜡切片,HE染色后观察卵泡发育情况并对各级卵泡进行计数;采用Western blotting和实时荧光定量PCR技术检测Smad9蛋白和mRNA水平。同时通过ELISA试验测定各组小鼠血清中雌二醇（E₂）、孕酮（P₄）、黄体生成素（LH）、卵泡刺激素（FSH）和芳香化酶等的浓度,并检测了CYP19a1、LHR、PRLR和FSHR基因的表达情况,进一步探究Smad9可能的作用机制。结果显示,与对照组相比,BMP4组Smad9蛋白和mRNA水平较高,而LDN组较低;BMP4组有腔卵泡显著增加（P<0.05）,而LDN组显著减少（P<0.05）。此外,ELISA试验结果显示,BMP4组血清E₂、FSH和芳香化酶含量增加,而P₄和LH含量降低;LDN组中E₂和芳香化酶的含量降低。实时荧光定量PCR结果显示,BMP4组FSHR和CYP19a1基因的mRNA水平升高,而LHR和PRLR基因的mRNA水平降低;LDN组PRLR基因的mRNA水平升高,而CYP19a1基因的mRNA水平降低。由以上试验结果可以得出,BMP4诱导的Smad9能促进小鼠有腔卵泡的发育,而其可能主要通过影响E₂、PRL和芳香化酶等的产生而起作用。     

钼对F1代雌性小鼠雌激素的影响

本试验主要探讨金属元素钼(Mo)对F1代雌性小鼠雌激素的影响。试验选择健康昆明系ICR F1雌性小白鼠20只,体重30~40 g,适应5 d后,随机分为4组。基础日粮相同,试验组分别在其饮水中加入100、200和400 mg/L钼酸钠,对照组给予正常饮水。喂养7周后,测量其体重并记录。采用眼球摘除采血法采集血液制备血清,测定小鼠的肝脏、肾脏、脾脏,计算肝脏、肾脏、脾脏的脏器指数。采用发光免疫技术测定小鼠血清雌二醇水平。     

牛卵泡抑制素抗体对小白鼠和大白鼠卵巢与子宫的作用

从用牛卵泡抑制素α－亚基片段免疫过的健康鸡所产蛋的卵黄液中提取抗体。得粗提物：然后用不同剂量的粗提物被动免疫２０只性成熟小白鼠和２０只发情周期大白鼠。并以生理盐水处理作为对照。结果发现,小白鼠用０．３ｍｌ抗体处理后,卵巢与子宫增重显著高于对照组（Ｐ〈０．０５）。大白鼠用０．６ｍｌ抗体处理后,卵泡平均数显著高于对照组（Ｐ〈０．０５）。这些结果表明,牛卵泡抑制素抗体被动免疫对小白鼠和大白鼠的子宫和卵泡     

Effects of dietary resveratrol supplementation during gestation and lactation of sows on milk composition of sows and fat metabolism of sucking piglets

The purpose of this article was to investigate the effects of dietary resveratrol supplementation during gestation and lactation of sows on the milk composition of sows and the fat metabolism of sucking piglets. Forty sows were allotted to two experimental treatment groups that included the following: (a) control sows (CON treatment, n = 20) fed with a corn–soybean meal control diet and (b) treatment sows (RES treatment, n = 20) fed with a control diet with addition of 300 mg/kg resveratrol. The results showed that the content of lactose in the colostrum was increased (p < 0.05) and the content of fat in 21‐day milk was increased (p < 0.05) by dietary resveratrol supplementation. In the RES treatment group, the concentrations of high‐density lipoprotein‐cholesterol (HDL‐C), low‐density lipoprotein‐cholesterol (LDL‐C), lipase activity and insulin (INS) in plasma of sucking piglets were increased (p < 0.05). In the adipose tissue, the enzyme activities of hormone‐sensitive lipase (HSL), acetyl‐CoA carboxylase (ACC) and lipoprotein lipase (LPL) increased significantly by RES treatment (p < 0.05), and the mRNA levels of acetyl coenzyme A‐alpha (ACCα), LPL, fatty acid transport protein (FATP1) and CCAAT–enhancer‐binding protein gene (C/EBPα) were higher in the RES treatment group (p < 0.05). In conclusion, resveratrol supplementation on gestational and lactating sows improved the content of lactose in the colostrum and the content of fat in milk at day 21 of lactation. In addition, resveratrol supplementation on sows increased HDL and LDL in the plasma of piglets. In piglet adipose tissue, the enzyme activity and mRNA level related to lipolysis, fatty acid uptake from circulating triacylglycerols and lipogenesis are partially improved by resveratrol supplementation on sows. These aspects affect fat metabolism in piglets.     

Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses

Reasons for performing study: Insulin resistance (IR) is a risk factor for pasture‐associated laminitis in equids and alimentary carbohydrate overload may trigger laminitis. Whether glucose metabolism responses to carbohydrate overload are more pronounced in insulin‐resistant horses requires further study. Hypothesis: Horses pretreated with endotoxin to alter insulin sensitivity differ significantly in their glucose and insulin responses to carbohydrate overload. Methods: Horses (n = 24) were divided into 3 groups. A lipopolysaccharide (LPS; n = 8) group that received endotoxin as an 8 h 7.5 ng/kg bwt/h i.v. continuous rate infusion, an oligofructose (OF; n = 8) group that received an infusion of saline followed by 5 g/kg bwt OF via nasogastric intubation, and a LPS/OF (n = 8) group that received LPS followed 16 h later by OF. Glucose and insulin dynamics were evaluated at ‐24 h and 48 h using the frequently sampled i.v. glucose tolerance test and minimal model analysis. Physical examinations and haematology were performed and the severity of laminitis assessed. Results: Horses receiving LPS developed leucopenia and both LPS and OF induced clinical signs consistent with systemic inflammation. Insulin sensitivity significantly decreased (P<0.001) over time, but responses did not differ significantly among groups. Time (P<0.001) and treatment × time (P = 0.038) effects were detected for the acute insulin response to glucose, with mean values significantly increasing in LPS and LPS/OF groups, but not the OF group. Five horses in the LPS/OF group developed clinical laminitis compared with 0 and 2 horses in the LPS and OF groups, respectively. Conclusions: Endotoxaemia and carbohydrate overload reduce insulin sensitivity in horses. Endotoxin pretreatment does not affect the alterations in glucose metabolism induced by carbohydrate overload. Potential relevance: Insulin sensitivity decreases after carbohydrate overload in horses, which may be relevant to the development of pasture‐associated laminitis.