Absorption and metabolism of estrogens from the stomach and duodenum of pigs

To determine the absorption and metabolism of 17β-estradiol (E₂) by the stomach and liver of the pig, crystalline E₂ was placed in the stomach of prepubertal gilts. Blood samples were subsequently obtained from the hepatic portal and jugular veins and plasma was assayed for E₂, estrone (E₁), 17β-estradiol-glucuronide (E₂G), estrone-glucuronide (E₁G) and estrone-sulfate (E₁S). Concentrations of E₂, E₁, E₂G and E₁S rose in the hepatic portal vein within five min and remained elevated for several hr. Concentration of E₂ represented only 6% of the total estrogen detected in the hepatic portal vein during the sampling period, indicating that most of the E₂ was converted or conjugated prior to entering the hepatic portal vein. The metabolism of E₂ presumably occurred in the stomach mucosa because food had been withheld for 26 hr before infusion of E₂. Concentrations of E₂G, E₁G and E₁S, but not E₂ and E₁, rose in the jugular vein and remained elevated for several hr. The lack of a rise in E₂ and E₁ in the jugular vein indicates that the E₂ and E₁ from the hepatic portal vein were completely converted and/or removed by the liver. Most of E₂ was converted to E₁ and then to E₁G. The infusion of bile containing normal estrogens from pregnant gilts into the duodenum of prepubertal gilts resulted in a peak of E₁G and E₂G in the hepatic portal and jugular veins within a few minutes. This was followed in about 180 min by a second sustained rise. The first peak was essentially abolished by extracting E₁ and E₂ from the bile before infusion. The second peak failed to occur in gilts given antibiotics orally to reduce gut bacteria before infusion of bile.     

Concentrations of free steroids in the jugular and hepatic portal veins of pigs after ingestion of testosterone, estrogen, or progesterone or transplantation of ovaries to the intestine.

Estrogen, progesterone or testosterone were administered orally to ovariectomized gilts fitted with indwelling catheters. Blood samples were taken from the jugular and hepatic portal veins at intervals varying from a few minutes to daily over periods that varied from 1 d to several months. Concentrations of free steroids rose dramatically in the hepatic portal vein within a few min of feeding steroids and remained high for 3-8 hr before declining to base levels. Concentrations in the jugular vein sometimes rose very slightly for the same period. At 48 hr after administration the concentrations remained at baseline in the hepatic portal vein, but rose several-fold in the jugular and remained elevated for several days. Autotransplantation of the ovaries to the intestine resulted in very large ovaries consisting of many large, heavily luteinized cystic follicles. Concentrations of progesterone and estrogen in the hepatic portal vein were very high, but were low in the jugular vein. The gut wall allows for passage of some orally administered free steroids to the liver via the hepatic portal vein. The free steroids are essentially metabolized before they reach the jugular vein, but can be recirculated via the enterohepatic route.     

Effect of a nonsteroidal aromatase inhibitor on in vitro and in vivo secretion of estradiol and on the estrous cycle in ewes.

Three experiments were performed to study effects of decreased concentrations of estradiol-17β (E₂) on lifespan and function of ensuing ovine corpora lutea (CL). In experiment 1, 52 follicles were collected from 10 ewes and placed into individual culture with 0 or .01 μCi ³H-androstenedione (10 ng; ³H-A) and 0, 10⁻¹¹, 10⁻⁹, 10⁻⁷, or 10⁻⁵ M of a nonsteroidal aromatase inhibitor, CGS16949A (CGS). Concentrations of E₂ secreted into the medium, and synthesis of estrogens as estimated by formation of ³H-water from ³H-A were decreased by 10⁻⁵ and 10⁻⁷ (P<.01), but not 10⁻⁹ or 10⁻¹¹ M CGS. In experiment 2, luteolysis was induced in 24 ewes by injection of PGF₂ on days 5 to 10 of the estrous cycle (0 hr). Ewes received 0, 0.5, 1.0, 2.0 or 4.0 mg CGS per kg BW i.v. at −12, 0, 12 and 24 hr, and an ovulatory dose of hCG at 36 hr. Jugular (P<.001) and vena caval (P<.001) concentrations of E₂ were decreased by CGS at all doses tested for 8 to 10 hr, but had returned to levels similar to control ewes by the time of the next injection. Concentrations of E₂ around the time of the LH surge were similar in control and treated ewes. During the subsequent luteal phase, concentrations of progesterone (P₄) were similar in control and treated ewes. Thus, transient decreases in E₂ during the follicular phase were not deleterious to the subsequent luteal phase. In experiment 3, luteolysis was induced in 18 ewes by injection of PGF₂ on days 6 or 7 (0 hr) of the estrous cycle. Ewes received 0 or 1 mg CGS per kg BW i.v. every 8 hr from 0 to 40 hr. Ovulation was induced with hCG at 36 hr. CGS reduced jugular (P<.001) and vena caval (P<.001) concentrations of E₂, prevented an endogenous surge of LH (P<.05) and increased (P<.001) concentrations of FSH. All ewes had ovulated a marked follicle by 72 hr, but onset of the luteal phase, as assessed by concentrations of P₄, was delayed (P<.01) in ewes receiving CGS. Delayed luteal phases were not solely attributable to the presence of new CL or to luteinization of follicular cysts. When data were aligned according to the day ewes were observed in estrus, profiles of P₄ did not differ with treatment. Therefore, normal luteal function ensued following estrus whether or not ewes re-ovulated. In conclusion, decreased secretion of E₂ by the preovulatory follicle was not involved in the ontogeny of CL of short lifespan or subnormal function. Instead, adequate production of E₂ or precisely timed E₂ secretion may be required during follicular development for subsequent functional luteinization.     

In vitro release of progesterone and estrone by the porcine placenta throughout gestation

In vitro release of progesterone (P₄) and estrone (E₁) by the porcine placenta was characterized at 12 days of gestation between d 20 and 110. Placental P₄ rose linearly from d 25 to 40, plateaued between d 40 and 50, continued to increase to a peak concentration at d 100, then decreased sharply to d 110. Placental E₁ decreased abruptly from d 30 to a nadir at d 40, then increased continuously from d 70 to peak values at d 110. This biphasic pattern for E₁ mimicked the pattern observed in allantoic fluid and maternal plasma pools. Only trace amounts of testosterone (T) were measured at d 20, 50, 60, and 100, suggesting rapid aromatization of C₁₉ steroids to estrogens. The results of this in vitro study indicate that the porcine placenta collected throughout gestation can release large quantities of P₄, although the lack of an increase in systemic P₄ suggests that under in vivo conditions P₄ is utilized and/or metabolized within the uterus.     

Circulating concentrations of 17-estradiol influence pattern of LH in circulation of cows

The objective of the research was to determine the relationship between circulating 17β-estradiol (E2) and secretion of luteinizing hormone (LH) in cows. A second objective was to determine if response to E₂ was influenced by interval between ovariectomy and the start of E₂ treatment. Thirty-one nulliparous cows 3 yr of age were randomly assigned to a 2 × 4 factorial arrangement of treatments. Sixteen cows were ovariectomized at 18 mo of age (long term), and the other 15 cows were ovariectomized at 36 mo of age (short term). At the time of ovariectomy of cows in the short term group, 11 cows in the short term group and 12 cows in the long term group were implanted subcutaneously with 1, 2 or 4 polydimethylsiloxane capsules containing E₂. The other eight cows served as non-implanted controls (n=4-short term, n=4-long term). All cows were fitted with jugular vein catheters on day 29 of treatment, and on day 30 blood samples were collected at 12-min intervals for 6 hr. At the end of 6 hr, luteinizing hormone-releasing hormone (LHRH) was administered and blood sampling continued at 12-min intervals for an additional hour. Serum was analyzed for LH and E₂. Variables of LH secretion analyzed were mean concentration, frequency of pulses, amplitude of pulses and maximum concentration after LHRH. There were no significant interactions for any of the variables of LH among cows ovariectomized for the long and short term. There was a significant linear increase in mean concentration of LH with increased circulating concentration of E₂. Frequency of LH pulses was not affected by circulating concentration of E₂. As circulating concentration of E₂ increased, amplitude of LH pulses increased and response to LHRH increased - resulting in an increase in mean LH. Interval from time of ovariectomy to the start of E₂ treatment only had a minor influence on mean concentration of LH and profile of LH concentrations in circulation.     

Effects of estradiol on secretion of LH, hypothalamic function and testicular development in bull calves

Two experiments were conducted in order to determine the effects of estradiol (E₂) on the development of the hypothalamic-pituitary-testicular axis in bull calves. In experiment 1, calves were assigned randomly to one of the following groups: 1) intact, 2) intact E₂-treated, 3) castrated, or 4) castrated E₂-treated. Treatments began when the calves were 7.5 wk of age and continued for 16.5 wk. Samples of blood were collected once a week from 3 to 14 wk of age and every 10 min for 6 hr at 8, 12 and 16 wk of age. Concentrations of E₂ in plasma decreased between 3 and 4 wk of age and were further reduced by castration. Maximum concentrations of E₂ (24.3 pg/ml) were observed 72 hr after insertion of E₂ implants, however, plasma E₂ stablized at 5–9 pg/ml by 2 wk after insertion of implants. Treatment with E₂ eliminated the pulsatile secretion of LH in intact and castrated calves and retarded testicular growth. In experiment 2, calves were assigned to a control (n=4) or E₂-treated (n=6) group. Implants of E₂ were inserted at 7.5 wk of age. At 24 wk of age, calves were bled and then sacrificed to collect hypothalamic and pituitary tissues. Age-related changes in testicular weight and secretion of LH were blocked by E₂. Neither the morphology nor the intensity of immunostaining of GnRH nerve cell bodies in the preoptic area (POA) were affected by E₂. However, the density of GnRH fibers and beads in the stalk median eminence (SME), and concentrations of pituitary GnRH receptors were greater (P<.01) in E₂-treated compared to control calves. In addition, concentrations of norepinephrine (NE) in the SME were lower in E₂-treated calves when compared to controls. Based on these observations, it is concluded that administration of E₂ at 7.5 wk of age causes profound alterations in hypothalamic function including, changes in metabolism of NE and suppression of GnRH release.     

胸腺上皮细胞中雌激素诱导lncRNA的鉴定分析及表达载体构建

为研究胸腺上皮细胞（thymic epithelial cells,TECs）中雌激素（estradiol,E₂）对长链非编码RNA（long non-coding RNA,lncRNA）表达的调节作用,本研究首先培养小鼠胸腺髓质上皮细胞系1（medullary thymic epithelial cell line 1,MTEC1）,经50 nmol/L E₂作用24 h后,观察对细胞表型变化的影响,并用CCK-8试剂盒检测细胞活力;提取细胞总RNA,运用实时荧光定量PCR技术验证E₂对lncRNA-2410006H16Rik表达的调节作用;最后运用RT-PCR技术扩增其目的基因,构建pEGFP-N1-lncRNA-2410006H16Rik重组过表达载体。结果显示,50 nmol/L E₂能够明显抑制MTEC1的增殖,且相较于对照组细胞,50 nmol/L E₂处理组细胞的D_{450 nm}值极显著降低（P<0.01）,表明其细胞活力极显著下降。实时荧光定量PCR结果显示,在E₂作用下,lncRNA-2410006H16Rik在MTEC1细胞中的表达极显著上调（P<0.01）,约是对照组的2倍,与高通量测序结果一致。经RT-PCR、双酶切及测序结果分析显示,试验成功构建pEGFP-N1-lncRNA-2410006H16Rik表达载体。结果表明,TECs中lncRNA-2410006H16Rik的表达与E₂作用密切相关,为后续在细胞水平上进一步验证lncRNA-2410006H16Rik的调节功能奠定了基础。     

Effects of Estrogen on COX-1 and COX-2 Expression in Bovine Oviduct Smooth Muscle

The aim of this study was to clarify the effects of estrogen (E₂) on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) gene expression in the cow oviduct tissue in vitro.Quantitative Real-time PCR and Western blotting were used to analyze the mRNA and protein expression of COX-1 and COX-2 at different time points after different concentrations of E₂ treatment.The results indicated that the relative expression of COX-1 and COX-2 mRNA reached the highest levels with 10^-11 mol/L E₂ treatment,the relative expression of COX-1 mRNA reached the highest level at 8 h after treatment;The relative expression of COX-2 mRNA reached the highest level at 2 h after treatment.The relative expression of COX-1 protein increased significantly at the range from 8 to 48 h after E₂ treatment.However,we did not detect COX-2 protein expression.     

雌激素对奶牛输卵管组织COX-1和COX-2表达的影响

试验旨在阐明雌激素(E₂)对体外培养的奶牛输卵管组织中环氧合酶-1(cyclooxygenase-1,COX-1)与环氧合酶-2(cyclooxygenase-2,COX-2)基因表达的影响。分离培养奶牛输卵管组织,用不同浓度的E₂作用于奶牛输卵管组织,采用实时荧光定量PCR与Western blotting检测COX-1与COX-2 mRNA和蛋白的表达量。结果表明,E₂作用浓度为10^-11 mol/L时奶牛输卵管组织中的COX-1与COX-2 mRNA相对表达量达到高峰;E₂作用时间为8 h时COX-1 mRNA的相对表达量达到高峰,E₂作用时间为2 h时COX-2 mRNA的相对表达量达到高峰;浓度为10^-11 mol/L的E₂作用不同时间后,COX-1蛋白的相对表达量在8~48 h显著升高;没有检测到COX-2蛋白的表达。     

CYP19A1调控内源性雌激素合成促进牦牛COCs细胞自噬和早期发育能力

旨在探索牦牛卵母细胞成熟过程中细胞色素P450芳香化酶(cytochrome P450arom, CYP19A1)对内源性雌激素(17β-estradiol, E₂)分泌、卵母细胞自噬和后续胚胎发育能力的影响。本研究在牦牛卵丘卵母细胞复合体(cumulus-oocyte complexes, COCs)体外成熟过程中,分别用等体积生理盐水、最佳浓度E₂(10^-7 mol·L^-1)、CYP19A1诱导剂黄曲霉毒素B1(aflatoxin B1, AFB1)、CYP19A1抑制剂双酚A(bisphenol A, BPA)处理,实时荧光定量PCR(real-time quantitative PCR, qRT-PCR)、Western blot和免疫荧光技术检测各组成熟COCs中CYP19A1表达水平,酶联免疫吸附方法(enzyme-linked immunosorbent assay, ELISA)检测诱导和抑制CYP19A1处理组牦牛成熟COCs培养液中E₂水平;分析不同处理组C...     

雌二醇单克隆抗体制备及间接竞争ELISA检测方法的建立

为建立敏感、特异、快速的雌二醇（estradiol,E₂）残留免疫检测方法,本研究利用雌二醇人工抗原（E₂-BSA）免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术制备特异性雌二醇单克隆抗体,利用高效价、高特异性单克隆抗体建立间接竞争ELISA（icELISA）方法。结果显示,试验成功筛选获得一株稳定分泌抗雌二醇抗体的杂交瘤细胞株（3H3）,抗体效价可达1∶320 000,利用3H3腹水抗体优化间接竞争icELISA反应条件,检测抗体的敏感度,IC₅₀为1.636 ng/mL,IC₂₀~IC₈₀线性范围为0.202~13.281 ng/mL。雌二醇腹水抗体与雌三醇、乙炔雌二醇的交叉反应率分别达0.31%和0.25%,而与雌酮、戊酸雌二醇、苯甲酸雌二醇、炔雌醚、己烯雌酚、壬基酚的交叉反应率均<0.1%。结果表明,利用本研究制备的单克隆抗体建立雌二醇间接竞争ELISA检测方法,可满足食品中雌二醇残留高灵敏度的检测要求。     

Preparation of Monoclonal Antibody and Establishment of Indirect Competitive ELISA of Estradiol

In order to establish a sensitive,specific and rapid method for the detection of estradiol (E₂) residues, an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on monoclonal antibody (MAb) for E₂ was developed. BALB/c mice were immunized by E₂-BSA and cell fusion technology was employed to screen hybridoma cell lines. One hybridoma cell line (3H3) was isolated, which produced monoclonal antibody that could binding E₂. Under the optimized conditions, the icELISA based on 3H3 for E₂ showed a half maximum inhibition concentration (IC₅₀) values of 1.636 ng/mL and detection ranges of 0.202 to 13.281 ng/mL with cross-reactivities for estriol and ethinyloestradiol of 0.31% and 0.25%, respectively, and negligible cross-reactivities with other E₂ analogs including estrone, estradiol valerate, estradiol benzoate, quinestrol, diethylstilbestrol and nonylphenol. The results demonstrated that the developed method could meet the requirements of high sensitivity detection of E₂ residue in food samples.     

Opioid and 17β-estradiol regulation of LH and FSH secretion during sexual maturation in heifers

The objective of the present study was to examine the involvement of opioid neuropeptides and E₂ in regulating circulating concentrations of gonadotropins during sexual maturation in the bovine female. Prepubertal (immature) and postpubertal (mature) bovine females were used. Mean concentrations of luteinizing hormone (LH) and follicle- stimulating hormone (FSH) in circulation before and after administration of naloxone were determined in ovariectomized heifers administered E₂ and ovariectomized heifers not administered E₂. A linear decline (P<0.01) in opioid suppression of LH and FSH occurred during the experimental period in immature heifers receiving E₂. This decline in opioid suppression of LH and FSH occurred during the same period of time that intact control heifers were initiating estrous cycles at puberty. Little change of opioid suppression of LH and FSH occurred during the experimental period in immature heifers not receiving E₂ and mature heifers receiving E₂. Our research indicates that opioid neuropeptides and E₂ act together to regulate LH and FSH secretion during sexual maturation in the bovine female.     

水牛发情周期生殖激素变化规律及唾液结晶的分析

试验对水牛发情周期血清和唾液中雌二醇(E_2)和孕酮(P_4)的浓度变化规律、水牛唾液结晶与卵泡发育变化分别进行了分析研究,为进一步探讨水牛发情规律、指导生产提供依据。采用酶联免疫分析法(ELISA)测定发情母水牛血清和唾液中E_2和P_4的浓度变化,并对血清和唾液的激素变化规律进行相关性分析。结果表明,水牛血清和唾液中的E_2和P_4呈波动性变化。发情前期,唾液中P_4浓度一直维持在6.50～7.10 ng/mL,发情第13天达到11.09 ng/mL,随后快速下降。唾液中E_2浓度在发情第3～5天出现一个峰值178.53 pg/mL,在第14～17天唾液中E_2浓度显著升高,出现第二个峰值179.10 pg/mL。母水牛唾液中E_2和P_4浓度的变化趋势与其在血清中的变化趋势基本一致,均呈显著相关(P<0.05);唾液中E_2与P_4浓度呈极显著相关(P<0.01)。水牛发情当天唾液结晶呈现明显的蕨类作物形状且分维值显著低于其他时间点(P<0.05)。水牛发情周期唾液结晶图形的变化与卵巢卵泡发育基本同步,可作为监测水牛发情及预测排卵的可靠指标之一。     

Metabolic profiles and bile acid extraction rate in the liver of cows with fasting-induced hepatic lipidosis

This study was designed to monitor lipid profile in the portal and hepatic blood of cows with fasting-induced hepatic lipidosis, and to compare the results with those in the jugular blood. The work was also carried out to investigate bile acid (BA) in these vessels, and further to investigate BA extraction rate in the liver. Five cows were equipped with catheters in the portal, hepatic and jugular veins (day 0), fasted for 4 days (day 1-day 4) and then refed (day 5-day 11). Before morning feeding, blood was sampled before, during and after fasting from the catheterized vessels. In the portal blood, the concentration of non-esterified fatty acids (NEFA) showed a progressive increase and at day 5 there was an approximate twofold rise. Increased NEFA concentrations were also found similarly in the other two veins. At day 5, beta-hydroxybutyrate (BHBA) in the portal, hepatic and jugular blood rose to 197, 190 and 186% of the pre-fasting value, respectively. However, the concentrations of NEFA and BHBA in the three veins gradually returned to pre-fasting concentration during the refeeding period. Compared with the pre-fasting value at day 0, the content of liver triglyceride (TG) increased significantly at day 5 (P < 0.01). In the liver, the hepatic extraction rate of BA dropped from 3.1 times pre-fasting to 2.2 times during fasting. There were no significant differences in the concentrations of glucose, TG, total cholesterol, cholesterol esters, free cholesterol and phospholipids. The results of the current study show that metabolic alterations occur in the portal, hepatic and jugular veins during induction of hepatic lipidosis in cows, and mostly metabolites, with exception of BA concentration, run parallel. The decreased BA extraction rate in the liver of fasted cows was considered to reflect hepatic cell impairment caused by TG accumulation. Hopefully, the findings, at least in part, contribute to the explanation of the pathophysiology of hepatic lipidosis in dairy cows.     

EVALUATION OF EXPERIMENTALLY INDUCED CANINE HEPATIC CIRRHOSIS USING DUPLEX DOPPLER ULTRASOUND

Portal blood flow was measured with duplex Doppler ultrasound in ten normal dogs and in ten dogs with hepatic cirrhosis induced by common bile duct ligation 4 weeks previously. Mean portal blood flow velocity in the 10 dogs with experimentally induced hepatic cirrhosis was markedly reduced (9.2 ± 1.70 cm/sec vs. normal 18.1 ± 7.6 cm/sec, p < 0.01). Mean portal blood flow was also significantly decreased compared to normal (17.2 ± 4.9 cc/min/kg versus normal 31.06 ± 9.1 cc/min/kg, p < 0.01) while portal vein diameter remained unchanged. The dogs with induced hepatic cirrhosis developed extensive extrahepatic portosystemic shunting that was confirmed at necropsy. It was concluded that decreased portal velocity and portal flow which resulted from hepatic cirrhosis was detectable noninvasively with Doppler ultrasound.     

Effect of Human Menopausal Gonadotropin,17β-estradiol and Epidermal Growth Factor on the Quality of in vitro Maturation Bovine Oocytes

For optimizing in vitro maturation system of bovine oocytes,we firstly examined the influence of four different hormonal regimes(FSH+LH,HMG,FSH+LH+E₂ and HMG+E₂) on oocyte maturation rates.Then we studied the effects of epidermal growth factor (EGF) in the above defined medium on bovine oocyte maturation,in vitro development and quality of parthenogenetic embryos.The cell apoptotic index of parthenogenetic blastocysts was detected by TUNEL.No significant difference was observed in maturation rates in four groups supplemented with different hormones.However,human menopausal gonadotropin (HMG) provided steady maturation results in replicates.Maturation of oocytes was promoted by supplementation with 17β-estradiol (E₂).Combination of HMG and E₂ gave rise to steady and efficient mature results.The presence of EGF at 30 ng/mL concentration significantly increased maturation rate and blastocyst rate and reduced apoptotic cells in parthenogenetic blastocysts.Therefore,the optimal oocyte maturation solution could be supplemented with 0.075 IU/mL HMG,1 μg/mL E₂ and 30 ng/mL EGF.     

Study on Colloidal Gold Immunochromatographic Assay for Rapid Detection of Estradiol in Milk

In order to develop a sensitive and specific method for the detection of estradiol residues (E₂) in milk, a colloidal gold immunochromatographic method was established. Colloidal gold particles were prepared by trisodium citrate reduction method and labeled with rabbit polyclonal antibodies (PcAb) by physical adsorption method. After optimization of reaction conditions such as the amount of coating antigen and labeled antibody to assemble test strip, the sensitivity, specificity, repeatability and accelerated preservation of the test strip were determined. The estrogen analogue cross reaction and milk matrix interference reaction were also measured. The results showed that the optimized concentration of E₂-OVA antigen was 2 mg/mL and the optimized antibody concentration was 20 μg/mL, visual detection limits of E₂ was 10 μg/L in PBS within 10 min. The cross reaction rate of this method with estriol was 40%, there were no negligible cross-reactivities with other estrogen compounds including estradiol valerate, estradiol benzoate, estrone, diethylstilbestrol, quinestrol, ethinyloestradiol and nonylphenol. Milk samples only needed two times diluted before analysis, and the results could judge by naked eye after 10 min with cut-off of 20 μg/L. The results demonstrated that the developed method was suitable for rapid on-site screening of E₂ residues in milk samples.     

牛奶中雌二醇胶体金试纸条快速检测技术研究

为了建立快速、敏感、特异的雌二醇（E₂）残留免疫检测方法,本研究应用胶体金免疫层析技术,研究制备一种快速检测牛奶中E₂的方法。采用柠檬酸三钠还原法制备胶体金颗粒,采用物理吸附法将E₂兔多克隆抗体偶联至胶体金,经过优化抗原包被量和多克隆抗体标记量等反应条件组装成检测试纸条,测定检测试纸的灵敏度、特异性、重复性和加速保存等参数,并对E₂类似物交叉反应和牛奶基质干扰反应进行了测定。结果表明,试纸条最优包被抗原浓度为2 mg/mL,抗体浓度为20 μg/mL。采用消线法判定结果,检测时间10 min,PBS缓冲液中E₂检测限为10 μg/L,该方法与雌三醇的交叉反应率为40%,与戊酸雌二醇、苯甲酸雌二醇、雌酮、己烯雌酚、炔雌醚、乙炔雌二醇、壬基酚等类似物均无可见交叉反应,牛奶样品经2倍稀释消除基质干扰直接用于检测,该方法在牛奶样品中的判定值为20 μg/L。本研究建立的E₂胶体金试纸条使用简单方便,适合现场快速检测牛奶中E₂残留。     

人尿促性腺激素、雌二醇及表皮生长因子对牛卵母细胞体外成熟质量的影响

试验旨在研究不同激素配比及表皮生长因子(EGF)浓度对牛卵母细胞体外成熟及卵母细胞质量的影响。将随机分组的卵丘-卵母细胞复合体于添加FSH+LH、HMG、FSH+LH+E₂、HMG+E₂ 4种不同激素组合配比的成熟基础液中培养,对比其体外成熟率,比较了EGF对牛卵母细胞体外成熟率和孤雌胚胎体外发育的影响,并采用TUNEL法检测添加不同浓度EGF的牛孤雌激活囊胚细胞凋亡情况。结果表明,添加HMG的成熟试验结果稳定,E₂对牛卵母细胞成熟有一定的促进作用,HMG+E₂联合使用可以得到高效稳定的成熟结果;在此基础上,在成熟液中添加30 ng/mL EGF对牛卵母细胞的成熟质量、胚胎发育及降低胚胎细胞凋亡都有明显的促进作用。因此,在体外成熟培养液中添加0.075 IU/mL HMG、1 μg/mL E₂和30 ng/mL EGF对牛卵母细胞的成熟和质量较为有益。