牛肉中孕酮的气相色谱/燃烧炉/同位素比质谱检测

建立了牛肉中孕酮的气相色谱/燃烧炉/同位素比质谱(GC/C/IRMS)检测方法。牛肉样品用乙腈振荡和超声辅助提取,经Na Cl脱水,有机相离心和旋转蒸发后以Zn Cl2脱脂,然后用LC-C18、LC-Si、LC-NH2固相萃取柱净化,过滤液经半制备液相色谱(Pre-HPLC)的C18柱纯化,最后分析物以GC/C/IRMS系统分析。牛肉中加标外源性孕酮δ13C值为-30.64±0.24‰(n=6),牛肉内源性孕酮δ13C值为-25.70±0.13‰(n=6),单因素方差分析(ANOVA,p值=2.23×10-140.05)显示,内源性孕酮和外源性孕酮的δ13C值存在显著差异性,且牛肉中加标外源性孕酮δ13C值与孕酮标准溶液的δ13C值无差异性。同时,经模拟实验可知,实际样品中内外源性孕酮混合物的δ13C值与外源性孕酮的δ13C值具有同源性。结果表明,本方法特异性和准确性好,GC/C/IRMS是鉴别激素来源的有效工具,该方法填补了国内鉴别激素来源技术空白。     

气相色谱-燃烧炉-同位素比质谱法测定奶粉中 孕酮δ13C值

目的基于高效液相色谱纯化分离,应用气相色谱-燃烧炉-同位素比质谱法(gas chromatography-combustion-isotope ratio mass spectrometry,GC-C-IRMS)建立奶粉中测定孕酮激素碳同位素比值的方法,以气相色谱-质谱法提供定量支持。方法样品经水溶解和乙腈提取,经乙腈饱和的正己烷除脂肪和过C18固相萃取柱净化,经高效液相色谱纯化、浓缩后进行GC-C-IRMS检测。结果外源性孕酮δ~(13)C平均值为-32.297‰,内源性孕酮δ~(13)C平均值为-21.387‰,方法回收率为52.9%,检出限为22.7μg/kg,相对标准偏差小于5%(n=6)。结论内源性和外源性孕酮δ~(13)C值有显著性差异,本方法可以区分内外源性孕酮。但由于方法局限性,只能检测孕酮大于22.7μg/kg的样品。     

青梅酒中乙醇稳定碳同位素的测定

建立了气相色谱-稳定同位素比值质谱法测定青梅酒中乙醇的稳定碳同位素比值(δ~(13)C)。青梅酒与丙酮混合后,用GC-IRMS法测定乙醇的δ~(13)C值。结果表明,该方法能快速准确的测定青梅酒中乙醇的碳稳定同位素比值,准确率和精确度均满足测定需求。通过测定4个品牌青梅酒中乙醇的δ~(13)C值,品牌A、B、C和D青梅酒中乙醇δ~(13)C值分别为-13.87‰~-13.56‰、-14.30‰~-13.60‰、-14.29‰~-13.86‰和-14.09‰~-13.52‰。表明本方法前处理简单、测定结果准确,可用于青梅酒的产地溯源。     

液相色谱/元素分析仪-同位素质谱法鉴定蜂蜜 掺假的方法改进

目的改进液相色谱/元素分析仪-同位素质谱法鉴别蜂蜜掺假的方法。方法对现有欧盟标准方法优化液相色谱条件,结合元素分析仪-同位素质谱法,将二糖分离为麦芽糖、蔗糖,提出一个新的参数—麦芽糖、蔗糖δ~(13)C值之差δ~(13)C_(M-S)。结果根据本研究检测113个国内外不同来源纯正蜂蜜样本的数据,提出纯正蜂蜜δ~(13)C值新要求:蜂蜜蛋白质与蜂蜜同位素差值δ~(13)C_(P-H)大于-0.97‰;果糖、葡萄糖δ~(13)C值之差δ~(13)C_(F-G)在-0.60‰至0.56‰范围内;麦芽糖、蔗糖δ~(13)C值之差δ~(13)C_(M-S)在-0.73‰至0.98‰范围内;各个组分δ~(13)C最大差值δ~(13)C_(max)小于2.05‰;根据上述4个参数来确认蜂蜜是否掺假。在日常检测和市场销售的160个样品中,原方法阳性检出率为16.2%,而新方法阳性检出率达21.9%。结论本研究提升了蜂蜜掺假检测能力,此方法的建立更好、更精确打击掺假的同时,也维护消费者权益。     

气相色谱-燃烧-同位素比质谱法测定鱿鱼中甲醛稳定碳同位素比值

建立了一种气相色谱-燃烧-同位素比质谱法(GC/C/IRMS)测定鱿鱼中甲醛(FA)稳定碳同位素比值的方法。鱿鱼样品经水充分提取后,高速离心除油脂,上清液与2,4-二硝基苯肼在酸性、60℃条件下发生液相衍生化反应1 h,衍生液经正己烷萃取,氮吹干后复溶即可进行稳定碳同位素测试。二氯甲烷、甲醇、正己烷、乙腈均可作为衍生产物甲醛2,4二硝基苯腙的同位素测试的溶剂。甲醛的衍生化反应未发生碳同位素分馏,由质量平衡方程计算得到的衍生产物甲醛2,4二硝基苯腙的δ¹³C值与实测值差值的标准偏差为0.215‰。分析9个市售不同产地鱿鱼中甲醛的含量为0.341~39.132 mg/kg,内脏部分含量较高。甲醛含量较高的2、3、4、5号样品δ¹³C值差异高达23.653‰。水产品中甲醛稳定碳同位素指纹特征可作为其来源解析的特征性指标。     

元素分析/液相色谱-碳同位素比值质谱法在纯正 苹果汁掺假鉴别中的应用

目的建立一种鉴别检测苹果汁掺假情况的元素分析/液相色谱-同位素比值质谱法(elemental analysis/liquid chromatography-isotope ratio mass spectrometry,EA/LC-IRMS)。方法通过对不同产区不同种类118个纯正苹果汁的糖、有机酸、果糖、葡萄糖、二糖、寡糖碳同位素比值(δ~(13)C值)的测定,建立纯正苹果汁同位素数据库进而提出纯正苹果汁应满足的δ~(13)C值要求。结果有机酸和糖差值Δδ~(13)CO-S在-1.38‰至1.09‰范围内,果糖和葡萄糖差值Δδ~(13)CF-G在-0.70‰至0.57‰范围内,而各组分最大差值Δδ~(13)Cmax4.36‰。对市售105个苹果汁进行检测,采用本方法检出32个阳性样品,而采用本实验室的糖浆标志物法仅检出12个阳性样品。结论本方法大大提高了苹果汁的掺假鉴别,有很大的实际应用潜力。     

利用GC-C-IRMS技术追溯食醋的来源与制作工艺

利用同位素技术测定食品中的δ13C值已经成为食品质量检验的一种重要手段。该实验中,通过有机溶剂稀释与气相色谱-燃烧-同位素比值质谱联用(GC-C-IRMS)方法来测定3种工业用冰醋酸、2种食用醋酸以及14种商品化食醋中的醋酸的δ13C值,发现食用醋酸的δ13C值在-11.57‰~-20.66‰之间,而工业冰醋酸的δ13C值在-24.45‰~-29.14‰之间;镇江香醋中的醋酸δ13C值在-22‰~-25‰之间,山西陈醋的δ13C值在-13‰~20‰之间,白醋的δ13C值在-14‰~-25‰之间。因此GC-C-IRMS技术不但可以区分工业冰醋酸和食用醋酸,而且可以区别部分不同来源和不同酿造工艺的食醋。     

基于食醋中乙醇的δ13C值对其真伪鉴别的应用初探

文章新建了液相色谱联用稳定同位素比率质谱(LC-IRMS)对食醋中乙醇δ13C值进行测定的方法。简述了仪器的运行过程,通过对色谱柱的选择,从出峰时间的角度考察了其他组分(糖、酸)对乙醇δ13C值测定的影响,使用蔗糖标准物质标定了高纯CO2参考气的δ13C值并考察了线性范围下的测定重复性。对16批次代表性样品进行测定,分析食醋、食醋沉淀物、食醋中乙醇三者间δ13C值的关系,乙醇的δ13C值基本在-28‰~-31‰,与其余二者的δ13C值范围有所差别,低于食醋的δ13C值,说明了在转化过程中发生了一部分的同位素损失;对市场上常见的多种酒精的δ13C值使用该分析方法进行测定,发现工业酒精、玉米食用酒精、淀粉酒精δ13C值在-10‰~-17‰,大米食用酒精在-29.1‰±1.2‰,高粱食用酒精在-17.6‰±1.3‰,其中仅有大米食用酒精的δ13C值与食醋中乙醇的δ13C值区间重合,其他均能轻易分辨出,通过测定食醋中乙醇的δ13C值或可起到真伪鉴别的作用。     

δD,δ~(18)O和δ~(13)C同位素比率质谱法在鲜榨苹果汁鉴伪中的应用

为识别苹果汁中糖和水的掺假,采用同位素比率质谱法(isotope ratio mass spectrometry,IRMS)对不同品种的鲜榨苹果汁和掺假果汁的δD,δ~(18)O和δ~(13)C值进行分析确定。结果表明,鲜榨苹果汁水的δD,δ~(18)O值均显著高于外源水掺假的果汁,且掺假果汁水的δD与δ~(18)O值呈线性关系,同时δD、δ~(18)O值随掺水量增加而降低;δ~(13)C值显示鲜榨苹果汁中糖组分含量分别为:果糖-25.64‰～-26.83‰,葡萄糖-25.01‰～-26.36‰,二糖-22.41‰～-23.24‰;果糖、葡萄糖、二糖占总糖的百分比范围分别48.84%～52.39%,14.34%～28.85%,10.47%～18.78%,未检出寡糖,掺假果汁二糖的δ~(13)C值不在上述范围。说明鲜榨果汁与掺假果汁的碳氢氧稳定同位素比率存在着差异,可进一步探索建立数据库以用于市售鲜榨苹果汁掺假的判别,为IRMS在果汁鉴伪中的应用提供试验依据。     

基于87Sr/86Sr和δ13C对进口大麦进行产地溯源

目的 建立基于87Sr/86Sr和δ13C稳定同位素比质谱法(stable isotope ratio mass spectrometry, IRMS)的进口大麦产地溯源技术。方法 样品干燥粉碎后, 经硝酸消解, 锶特效树脂净化后利用热电离质谱法(thermal ionization mass spectrometry, TIMS)分析检测87Sr/86Sr; 样品用锡囊包裹后, 利用元素分析-稳定同位素比质谱法分析检测δ13C; 利用SPSS 25.0软件对进口大麦的87Sr/86Sr和δ13C进行正态性验证、方差分析、事后多重比较分析和判别分析。结果 不同进口国大麦中的87Sr/86Sr和δ13C具有显著性差异, 经判别分析, 进口自澳大利亚、法国和美国的大麦可以达到100%正确判别率, 整体正确判别率达到86.2%, 若将美国和加拿大进口的大麦归类为北美洲进口大麦, 则可实现进口自北美、法国和澳大利亚大麦的100%正确判别率。结论 利用87Sr/86Sr和δ13C可以对不同进口国大麦进行产地溯源分析。     

高效液相色谱- 串联质谱法测定猪肉中10 种类固醇类激素残留

建立猪肉中类固醇类激素残留高效液相色谱- 串联质谱(LC-MS/MS)分析方法。样品经甲醇提取,固相萃取柱净化后采用LC-MS/MS 进行检测分析;并对样品前处理条件和质谱参数进行研究。结果表明：这些激素物质的线性范围均为0.5～100μg/kg;低、中、高3 种加标水平的加标回收率为71%～89%;最低检出限为0.2～2μg/kg。本方法快速简便、灵敏度高、选择性好、测定结果令人满意,可在各食品检测机构推广应用。     

Simultaneous determination of 30 hormones illegally added to anti-ageing functional foods using UPLC-MS/MS coupled with SPE clean-up

A novel analytical method employing solid-phase extraction (SPE) coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 30 hormones in anti-ageing functional foods (capsules, powders and tablets). The analytes were extracted with acetic acid–acetonitrile (1–99 v/v), methanol and acetone, respectively. The extract was purified using a combined column, followed by analyte detection with electrospray ionisation in positive- or negative-ion modes. The results indicated that the 30 compounds had good linear correlations in the range of 1–1000 μg kg^–1, and the correlation coefficients were above 0.99. The limits of detection (LOD) and limits of quantification (LOQ) were 0.03–2 and 0.1–5 μg kg^–1, respectively. The average recovery of 30 compounds at the three spiked levels varied from 74.7% to 124.1%, and the relative standard deviation (RSD) was 2.4–15.0%. This method was applied to the analysis of hormones in 14 real samples of which seven hormones (such as estrone, dienestrol) were detected in four samples, but the remainder of the hormones were not detected. The developed method is sensitive, efficient, reliable and applicable to real samples.     

动物性食品中环境激素对人体的影响及其防治

文中分别介绍了环境激素的定义、激素与环境激素间的关系,以及动物性食品中所包含的环境激素种类,通过分析动物性食品中环境激素的污染途径及其对人体的影响,提出了控制措施以增强人们对其危害的重视,并减少环境激素对人体损害,有利于控制环境激素在动物性食品中的含量。     

超高效液相色谱串联质谱检测动物组织中卡马西平及其代谢物的残留量

卡马西平为治疗癫痫和三叉神经痛等精神疾病药物,但其具有刺激抗利尿激素释放,可引起水钠潴留副作用。水钠潴留可造成组织水肿,体重异常,为养殖环节非法注水提供了保水抗利尿的可能,因此检测动物组织中卡马西平对打击非法注水意义重大。本文采用超高效液相色谱串联质谱建立动物组织中卡马西平及其代谢物环氧卡马西平残留检测方法。样品经过0.2%甲酸乙腈提取、Oasis PRi ME HLB柱净化后,经Acquity UPLC BEH C18(100 mm×2.1 mm,1.7μm)分离,以甲醇和0.1%甲酸水溶液为流动相进行梯度洗脱,正离子MRM信号采集模式,两种药物能在8 min内分离完好,在1.0~50.0μg/L浓度范围内,线性良好,相关系数均在0.999以上;方法定量限均为1.0μg/kg,通过1.0、10、20μg/kg三个浓度的加标回收实验表明,回收率为72.3%~106.8%,RSD%值为1.43%~10.3%。该方法为防范动物产品中非法药物添加和注水等违法行为提供了一定的技术参考。     

蜂王浆对雌性大鼠生长发育的影响

目的：探讨高剂量蜂王浆对雌性大鼠生长发育的影响。方法：实验组大鼠按3 g/（200 g·d）（以体质量计,下同）灌胃新鲜的蜂王浆溶液,对照组按3 mL/（200 g·d）灌胃生理盐水,4 周后,停止灌胃,继续饲养2 周。每两周解剖一批大鼠,称质量计算心、肝、脾、肺、肾、肾上腺、子宫、卵巢脏器系数,用放射免疫技术检测血清雌二醇（estradiol,E2）、促卵泡素（follicle stimulating hormone,FSH）、促黄体素（luteinizing hormone,LH）、孕激素（progestrone,P）含量,实时定量聚合酶链式反应（real-timepolymerase chain reaction,real-time PCR）检测卵巢和子宫中雌激素受体α（estrogen receptor α,ERα）、雌激素受体β（estrogen receptor β,ERβ）、促卵泡素受体（follicle stimulating hormone receptor,FSHR）、促黄体素受体（luteinizing hormone recepter,LHR）、孕激素受体（progestrone receptor,PR）mRNA表达量。结果：灌胃2 周,实验组大鼠体质量、主要脏器系数、血清主要性激素含量及其卵巢和子宫中受体表达量与对照组相比均无显著差异;灌胃4 周,实验组大鼠体质量显著低于对照组,实验组血清E2显著高于对照组,实验组卵巢PR mRNA表达量显著高于对照组,实验组子宫中ERα、ERβ、PR mRNA表达量显著高于对照组。实验组与对照组脏器系数无显著差异;停止灌胃2 周后,实验组大鼠体质量、主要脏器系数、血清主要性激素含量与对照组相比均无显著差异,实验组卵巢中FSHR、ERβ、PR mRNA表达量显著高于对照组,实验组子宫中ERα、ERβ、FSHR和PR mRNA表达量显著高于对照组。结论：高剂量的蜂王浆在一定程度上影响雌性大鼠的生长发育。     

半胱胺对鲤鱼生长及血液生化指标的影响

在鲤鱼基础饵料中分别添加0、200、400、600和800 mg/kg的半胱胺,经过42 d的饲养试验,研究不同水平的半胱胺对鲤鱼生长、相关激素水平、血清生化指标的影响.结果表明:①与对照组相比,鲤鱼饵料中添加200、400、600 mg/kg半胱胺均不同程度提高了鲤鱼的生长性能,400 mg/kg半胱胺显著提高了鲤鱼的相对增重率(P＜0.05),并降低了鲤鱼的饵料系数(P＞0.05).②与对照组相比,400 mg/kg半胱胺显著提高了鲤鱼血液中GH水平(P＜0.05),T3水平也高于其他各组(P＞0.05).③400 mg/kg半胱胺组的碱性磷酸酶活性、血糖和总蛋白质水平均高于其他各组,其中总蛋白质水平显著高于对照组(P＜0.05).分析认为,本试验鲤鱼饵料中半胱胺适宜的添加量为400 mg/kg.     

Polymorphism in the bovine growth hormone gene affects endocrine release in dairy calves

The objective was to test whether calves with the Leu/Leu genotype release more growth hormone (GH) than calves with Leu/Val and Val/Val genotypes. Danish Holstein (n = 286), Danish Red (n = 68), and Danish Jersey (n = 61) calves were genotyped for the Leu/Val polymorphism in the GH gene and assessed for GH release following inducement by the growth hormone releasing hormone (GHRH). Three GH traits were assessed for each calf: BASELINE, PEAK, and RATE. BASELINE and PEAK are the mean concentration of GH in blood sampled before and after GHRH inducement. RATE is the disappearance rate of GH in blood sampled after GHRH inducement. Danish Jersey calves with Leu/Leu genotype had a higher PEAK and RATE than calves with the Val/Val genotype, whereas the Leu/Val genotype had an intermediate response. The contribution of the Leu/Val polymorphism to the total genetic variation of the BASELINE, PEAK, and RATE traits was 5, 30, and 27%, respectively. By contrast, the amount of GH released by the Danish Holstein and Danish Red calves was not influenced by their GH genotype. Further studies involving calves with all three genotypes are required to further elucidate whether this polymorphism has a functional role or whether it works through a linked-gene effect specific to certain cattle breeds.     

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation,follicle dynamics,and circulating follicle-stimulating hormone in lactating dairy cows

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 μg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (～18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5–11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.     

Intravaginal instillation of gonadotropin-releasing hormone analogues with an absorption enhancer induced a surge of luteinizing hormone in lactating dairy cows

Our objectives were to evaluate circulating LH concentrations after intravaginal (IVG) instillation of GnRH analogs in lactating dairy cows. In 2 experiments, lactating Holstein cows (experiment 1: n = 32; experiment 2: n = 47) received the experimental treatments 48 h after the first of 2 PGF_2α treatments given 12 h apart and 7 d after a modified Ovsynch protocol (GnRH at ?7 d, PGF_2α at ?24 h, PGF_2α at ?56 h, GnRH at 0 h). In experiment 1, cows were stratified by parity and randomly allocated to receive the following treatments: 2 mL of saline IVG (SAL, n = 6), 100 µg of gonadorelin (Gon) i.m. (G100-IM, n = 5), and 100 (G100, n = 7), 500 (G500, n = 8), or 1,000 µg of Gon IVG (G1000, n = 7). In experiment 2, treatments were SAL (n = 8), G100-IM (n = 8), G1000 (n = 7), 1,000 µg of Gon plus 10% citric acid (CA) IVG (G1000CA, n = 8), 80 µg of buserelin IVG (B80, n = 8), and 80 µg of buserelin plus 10% CA IVG (B80CA, n = 8). In both experiments, blood was collected every 15 min from ?15 min to 4 h, and every 30 min from 4 to 6 h after treatment. Data for area under the curve (AUC), mean LH concentrations, and time to maximum LH concentration were analyzed by ANOVA with (mean LH only) or without repeated measures using PROC MIXED of SAS (version 9.4, SAS Institute Inc., Cary, NC). The proportion of cows with a surge of LH was evaluated with Fisher's exact test using PROC FREQ of SAS. In both experiments, LH concentrations were affected by treatment, time, and the treatment by time interaction. In experiment 1, the AUC for LH and maximum LH concentration were greatest for the G100-IM treatment and were greater for the G1000 than for the SAL and G500 treatments. The proportion of cows with an observed surge of LH was 100 and 0% for cows that received Gon i.m. and IVG, respectively. In experiment 2, the AUC and maximum LH concentrations were greater for the G100-IM, G1000CA, and B80CA treatments than for the other IVG treatments. The proportion of cows with a surge of LH differed by treatment (SAL = 0%, G100-IM = 100%, G1000 = 14%, G1000CA = 88%, B80 = 13%, and B80CA = 100%). For the treatments with a surge of LH, time to maximum concentration of LH was the shortest for the G100-IM treatment, intermediate for the G1000CA treatment, and the longest for cows in the B80CA treatment. In conclusion, Gon (up to 1,000 µg) absorption through intact vaginal epithelium after a single IVG instillation was insufficient to elicit a surge of LH of normal magnitude. Conversely, IVG instillation of 1,000 µg of Gon and 80 µg of buserelin with the addition of citric acid as absorption enhancer resulted in a surge of LH of similar characteristics than that induced after i.m. injection of 100 µg of Gon.     

Hormonal and lactational responses to growth hormone-releasing hormone treatment in lactating Japanese Black cows

Ten multiparous lactating Japanese Black cows (beef breed) were used to evaluate the effects of bovine growth hormone-releasing hormone (GHRH) analog on milk yield and profiles of plasma hormones and metabolites. The cows received 2 consecutive 21-d treatments (a daily s.c. injection of 3-mg GHRH analog or saline) in a 2 (group) x 2 (period) Latin square crossover design. The 5 cows in group A received GHRH analog during period 1 (from d 22 to 42 postpartum) and saline during period 2 (from d 57 to 77 postpartum), and those in group B received saline and GHRH analog during periods 1 and 2, respectively. Mean milk yield decreased in saline treated compared with that during the 1-wk period before treatment 7.4 and 19.1% during periods 1 (group B) and 2 (group A), respectively. Treatment with GHRH analog increased milk yield 17.4% (period 1, group A) and 6.3% (period 2, group B). Treatment with GHRH analog induced higher basal plasma concentrations of growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin, and glucose compared with saline-treated cows. In glucose challenge, the GHRH analog-treated beef cows had greater insulin secretion than the saline-treated beef cows. In insulin challenge, however, there were no significant differences in the areas surrounded by hypothetical lines of basal glucose concentrations and glucose response curves between GHRH analog- and saline-treated cows. These results demonstrate that GHRH analog treatment facilitates endogenous GH secretion in lactating Japanese Black cows, leading to increases in milk yield and plasma concentrations of IGF-1, insulin, and glucose.