Investigation of the Role of Androstenedione-19-oic Acid in the Presence of 19-Norandrostenedione in Intact Male Horse Plasma Using Liquid Chromatography–Tandem Mass Spectrometry

A liquid chromatography–tandem mass spectrometry method was developed to confirm the presence of androstenedione-19-oic acid in intact male equine plasma and to show the source of 19-norandrostenedione in equine plasma. Androstenedione-19-oic acid was recovered from acidified plasma by liquid–liquid extraction using methyl tert-butyl ether and separated on an Ace 5 C₈ column. A triple quadrupole mass spectrometer was used to detect the analytes in negative electrospray ionization mode. Limits of detection, quantification, and confirmation of the method were 0.1, 0.5, and 1.0 ng/mL, respectively. The linear dynamic range of quantification was 0.5–50 ng/mL. The presence of androstenedione-19-oic acid was confirmed in all plasma samples obtained from intact male horses but not those from gelded and female horses; the average concentration was 3.1 ± 1.6 ng/mL, suggesting androstenedione-19-oic acid is an endogenous compound only in intact male horse plasma samples. The conversion of androstenedione-19-oic acid to 19-norandrostenedione in equine plasma was demonstrated by spiking androstenedione-19-oic acid into blank plasma and monitoring the generation of 19-norandrostenedione and its increase in concentration during storage. Results indicated that androstenedione-19-oic acid was readily converted into 19-norandrostenedione; the higher the storage temperature, the faster the conversion. The conversion was not affected by the types of plasma samples collected from gelded and female horses or by anticoagulants used in blood collection to harvest plasma. Compared with other matrices such as water, methanol, and phosphate-buffered saline, the conversion of androstenedione-19-oic to 19-norandrostenedione in equine plasma was faster, suggesting that there is an unknown factor(s) in equine plasma that enhances the conversion.     

颈静脉灌注不同模式的氨基酸混合物对泌乳奶牛乳蛋白合成的影响

本试验旨在比较颈静脉灌注酪蛋白模式和理想模式的氨基酸混合物对泌乳中期荷斯坦奶牛乳产量、乳成分以及乳腺对氨基酸摄取利用的影响。选择8头泌乳中期[泌乳天数:(82±11)d]荷斯坦奶牛作为试验动物,试验采用随机区组设计,将试验牛随机分为2组,分别颈静脉灌注160g/d酪蛋白模式(Casein组)和理想模式的氨基酸混合物(R组)。2个试验组分别以各自灌注前作为空白对照组(C1组为Casein组的空白对照组,C2组为R组的空白对照组)。预试期14d,灌注期5d。试验采用全混合日粮(TMR)饲喂,以玉米、豆粕、棉籽粕、玉米青贮、苜蓿干草和羊草为主要原料,参照NRC(2001)奶牛饲养标准配制。结果表明:灌注酪蛋白模式的氨基酸混合物后,乳蛋白产量和含量较灌注前呈上升趋势(乳蛋白产量上升7.14%,P=0.078;乳蛋白含量上升3.27%,P=0.072);并且,奶牛动脉血浆中异亮氨酸(Ile)、亮氨酸(Leu)、赖氨酸(Lys)和组氨酸(His)的浓度较灌注前有不同程度的上升(Ile的浓度提高31.5%,P=0.097;Leu的浓度提高65.9%,P=0.041;Lys的浓度提高36.9%,P=0.088;His的浓度提高40.1%,P=0.010),而苏氨酸(Thr)、缬氨酸(Val)、蛋氨酸(Met)、苯丙氨酸(Phe)和精氨酸(Arg)的浓度在数值上虽较灌注前高但无显著差异(P0.05)。灌注酪蛋白模式的氨基酸混合物后,奶牛乳腺对天冬氨酸(Asp)和半胱氨酸(Cys)的摄取率显著升高(Asp的摄取率提高95.2%,P=0.031;Cys的摄取率提高49.6%,P=0.031),而奶牛乳腺对甘氨酸(Gly)的摄取率显著降低(降低158.3%,P=0.041)。灌注理想模式的氨基酸混合物后,乳蛋白含量比灌注前有上升趋势(提高5.78%,P=0.064),而乳脂产量显著低于灌注前(降低8.57%,P=0.015);并且,奶牛动脉血浆中Arg的浓度有上升趋势(提高18.0%,P=0.093),而酪氨酸(Tyr)的浓度呈下降趋势(降低47.8%,P=0.074)。灌注理想模式的氨基酸混合物后,奶牛乳腺对谷氨酸(Glu)、Cys和Ile的摄取率显著上升(Glu的摄取率提高118.7%,P=0.015;Cys的摄取率提高77.4%,P=0.032;Ile的摄取率提高46.0%,P=0.012),而奶牛乳腺对Ser的摄取率呈下降趋势(降低56.2%,P=0.052)。灌注氨基酸混合物后,Casein组乳脂产量增量显著高于R组(P=0.012),且Casein组的乳产量增量(P=0.095)和乳糖产量增量(P=0.091)较R组有升高的趋势,而2组间其他指标增量无显著差异(P0.05)。由此得出,在本试验条件下,颈静脉灌注酪蛋白模式和理想模式的氨基酸混合物均可提高泌乳奶牛的乳蛋白含量,而灌注酪蛋白模式氨基酸混合物同时可以促进乳蛋白产量的升高,因此,颈静脉灌注酪蛋白模式氨基酸混合物的效果优于灌注理想模式氨基酸混合物。     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

无氮日粮纤维水平对猪内源氨基酸测值的影响

以醋酸纤维素为纤维源配制成粗纤维水平分别为２．０％、５．０％、８．０％的３种无氮日粮,采用３头回—直肠吻合猪,分３期试验,通过３×３拉丁方设计研究在等进食量（７５０ｇ／ｄ）条件下日粮的纤维水平对回—直肠吻合猪消化道内源氨基酸测值的影响。结果表明：除缬氨酸、异亮氨酸和胱氨酸外,无氮日粮的粗纤维水平对回肠末端食糜干物质中其他内源氨基酸含量（％）无显著影响（Ｐ＞０．０５）。而对回肠末端内源氨基酸的总排泄量影响显著（Ｐ＜０．０５）或极显著（Ｐ＜０．０１）。相关分析表明,粗纤维水平与各种内源氨基酸的排泄总量之间存在显著（Ｐ＜０．０５）或极显著（Ｐ＜０．０１）的强相关关系,随无氮日粮中粗纤维水平的升高,各种内源氨基酸排泄量呈直线增加。     

不同氧化方法对羽毛粉中胱氨酸生成磺基丙氨酸效果的影响

采用均匀设计方法研究了过甲酸法、过乙酸法氧化羽毛粉中胱氨酸为磺基丙氨酸的效果，结果表明，两种方法的效果无显著差异（Ｐ＞０．０５）。从实用经济角度来考虑，以使用过乙酸氧化法为宜。使用此法的最适条件为：过乙酸用量（Ｘ１）０．１ｍＬ／ｇ羽毛粉，过乙酸组成（Ｘ２）（过氧化氢∶乙酸）１∶８；过乙酸的浓度（Ｘ３）９０％；氧化温度１１０℃（Ｘ４）；氧化时间（Ｘ５）８ｈ。在此条件下，磺基丙氨酸最佳得率为８．０２％。氧化条件（Ｘ）与生成磺基丙氨酸（Ｙ）之间的回归方程：ｙ＝０．９４２２＋０．０６８６Ｘ１＋０．５７８７Ｘ２＋０．００４９Ｘ３＋０．００５２Ｘ４＋０．２９５９Ｘ５。     

Ascorbic acid ameliorates dysregulated folliculogenesis induced by mono-(2-ethylhexyl)phthalate in neonatal mouse ovaries via reducing ovarian oxidative stress

Phthalates, including di-(2-ethylhexyl)phthalate (DEHP), are common industrial chemicals in the environment. Recent evidence indicates that DEHP and its active metabolite mono-(2-ethylhexyl)phthalate (MEHP) negatively modulate reproductive functions and induce reactive oxygen species. Ascorbic acid (AA) is a dietary requirement for primates, and it acts as a potent free radical scavenger to protect tissues against oxidative stress. In this study, to investigate the toxic effects of MEHP on the follicle development and the beneficial role of AA, neonatal mouse ovaries were treated with different concentrations of MEHP with or without AA for 6 days. Then, the follicle constitution and oxidative status were compared in different groups. Results showed MEHP accelerated primordial follicle recruitment by increasing the percentage of primary and secondary follicles and decreasing the percentage of primordial follicles in the ovaries. Moreover, MEHP-induced ovarian oxidative stress by significantly increasing malondialdehyde (MDA) concentration and the expression of GSS and SOD1. When ovaries were co-administrated with MEHP and AA, follicle constitution was normalized, and the oxidative status was significantly decreased. These results suggested that AA ameliorated MEHP-induced ovarian oxidative stress and follicular dysregulation, which attested the clinical significance of AA for ovary protection in the case of MEHP exposure.     

Fatty acid composition of intramuscular fat from Nellore steers fed dry or high moisture corn and calcium salts of fatty acids

The purpose of this work was to evaluate the fatty acid composition of the Longissimus muscle from carcasses of Nellore steers fed diets with calcium salts of fatty acids (CSFA) and high moisture corn. Forty eight steers were fed during 70 days four diets containing dry corn (DC), high moisture corn (HM), dry corn plus CSFA (DC-CSFA) or high moisture corn plus CSFA (HM-CSFA). Fatty acid composition of the Longissimus muscle was determined by gas chromatography. Corn type had no effect on the ether extract percentage and in the content of the majority of the fatty acids, although steers fed HMC showed higher levels of polyunsaturated fatty acids and polyunsaturated/saturated ratio. Feeding CSFA increased ether extract percentage but had no effect on total of saturated, unsaturated and saturated: unsaturated ratio. Both high moisture corn and calcium salts of fatty acids increased CLA (cis9, trans11) and total CLA concentrations in intramuscular fat.     

Concentration of conjugated linoleic acid in grazing sheep and goat milk fat

The conjugated linoleic acid (CLA) content of grazing sheep and goat milk fat, throughout their lactation period, was examined. Six sheep and six goat representative farms were selected at random and milk samples were taken at monthly intervals for fatty acids profile determination. Sheep and goat nutrition was based on natural grazing and on supplementary feeding during the winter months. From April onwards, grazing native pastures was the only source of feed for sheep and goats. The University farm whose sheep are kept indoors all year round without any grazing, was also used as reference. Fifteen individual milk samples were also taken in April from a sheep and goat farm respectively, in order to see the variability of CLA inside the farm. The results showed that: a. the CLA content of grazing sheep and goat milk fat increased significantly in April–May (early growth stage of grass) and then declined while that of indoors kept sheep was more or less constant during the same period, b. the isomers cis-9, trans-11 and trans-10, cis-12 of CLA were found in grazing sheep milk fat, while in indoors kept sheep and goats' milk fat only the cis-9, trans-11 isomer was found, c. the CLA content of sheep milk fat was much higher than that of goats, d. a negative correlation between sheep milk fat and CLA content was found and e. there was considerable variation in milk fat CLA content between sheep and goat farms and inside the farms.