The role of altered uterine-embryo synchrony on conceptus growth in the pig

This study was conducted to determine whether inducing an embryo-uterine asynchrony during the preimplantation period would alter fetal and(or) placental size at term. Yorkshire gilts (n = 24) were checked twice daily for estrus and bred to a Yorkshire boar 24 h after the first exhibition of estrus. Embryos (1 to 4 cells) were flushed from the oviducts of each donor gilt on d 2.5 of gestation and transferred in equal numbers to the oviducts of a recipient gilt on d 1.5, 2.5, or 3.5 of the estrous cycle. Gilts were slaughtered on d 112 of gestation (calculated on the age of the conceptus) and fetal and placental weight, placental surface area, and implantation site lengths were determined. Although litter sizes were similar (9.1+/-0.9), conceptuses transferred to d 3.5 recipients became heavier fetuses (1.44+/-0.05 vs 1.23+/-0.04 kg, P < 0.001), with larger placental surface areas (1,793+/-60 vs 1,459+/-43 cm2, P < 0.01), and longer implantation sites (32.1+/-1.5 vs 24.9+/-0.6 cm, P < 0.001) than those transferred to recipients on d 2.5. These data demonstrate that oviductal transfer of embryos into a reproductive tract that is more advanced by as little as 24 h can result in alterations in placental growth and function during gestation.     

Successful direct transfer of a deep frozen-thawed equine embryo

Embryos were flushed on day 7 after ovulation from two mares, and frozen using a conventional slow freezing procedure in phosphate buffered (PBS) saline supplemented with 10% FCS, 1.5 mol/L ethylene glycol and 0.25 mol/L sucrose. One of the two embryos was thawed after 10 months of storage in liquid nitrogen and transferred directly (without dilution of the cryoprotectant and quality examination) to a synchronized recipient. This transfer resulted in the birth of a live female foal. To our knowledge, this is the first live foal born after direct transfer of a frozen-thawed equine embryo.     

精子介导法制备GHRH转基因猪的研究

以eGFP为报告基因,生长激素释放激素(GHRH)为目的基因,利用精子介导的基因转移方法,将构建慢病毒载体质粒LV-EGFP-GHRH直接和猪精子孵育,再通过体外人工受精建立GHRH转基因猪模型.经PCR检 测,52头后代中发现7头阳性个体,转基因效率为7.42%.本研究探索了应用精子介导的转基因方法将GHRH转移到动物体内,为猪的转基因育种提供了新的依据.     

Growth of the conceptus in alpacas

OBJECTIVE: To determine growth of the alpaca conceptus during early gestation, using ultrasonography. ANIMALS: 40 pregnant alpacas (20 multiparous, 20 nulliparous). PROCEDURE: Transrectal ultrasonography with a 5-MHz probe was performed 3 times weekly from days 7 through 45 after copulation (day of copulation = day 0). Blood samples were collected for progesterone assay on days of ultrasonography. RESULTS: Forty embryonic vesicles and embryos were identified and measured. Embryonic vesicles were first detected on day 12 after copulation and appeared spherical. Cumulative percentage of embryonic vesicles detected was 50, 82.5, and 100% on days 12, 14, and 16, respectively. Embryos were first detected by day 22 and heartbeats by day 24. Similarly, cumulative percentage of embryos detected was 17.5, 82.5 and 100% for 22, 24, and 26 days after copulation, respectively. Embryonic vesicles occupied both uterine horns beginning on day 19. A positive correlation existed between length and width of embryonic vesicles. Size of embryonic vesicles and embryos and progesterone concentrations did not differ between multiparous and nulliparous females. Five sets of twins were detected from days 21 through 33 after copulation, but the conceptus identified in the right uterine horn became nondetectable between days 28 and 33. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasonography was useful in detecting the earliest appearance of alpaca embryonic vesicles and determining the changes in growth from days 12 through 45 after copulation. Embryos were first detected by day 22. Transrectal ultrasonography was not harmful to animals and did not provoke embryonic death in this study.     

Growth and development of the ovine conceptus

This study was designed to characterize development of the ovine conceptus throughout gestation to establish the temporal relationships in metabolites, electrolytes, fluid volumes within the placenta, and hormonal changes with fetal growth. Length and weight of placentae, weight of cotyledons, and uterine weight increased between d 25 and 80 of gestation in advance of increases in fetal growth between d 80 and 140 of gestation. Allantoic fluid volumes changed (P < 0.01) between d 25 (21 mL) and 40 (91 mL), decreased to d 70 (32 mL), and then increased to d 140 (438 mL). Concentrations and total amounts of proteins in allantoic fluid were reduced between d 25 and 50, but total protein increased (P < 0.01) from d 40 (63 mg) to d 140 (2,991 mg). Concentrations of fructose in allantoic fluid varied between 2 and 6 mg/mL throughout gestation, but total fructose increased (P < 0.01) between d 25 (46 mg) and d 120 (679 mg). Concentrations of glucose ranged from 0.1 to 0.3 mg/mL, and total glucose increased (P < 0.05) from d 25 (3 mg) to d 140 (63 mg) of gestation. Amniotic fluid volume increased (P < 0.01) between d 30 and 140. Concentrations of estrogens in allantoic fluid, maternal uterine artery, and uterine vein increased (P < 0.01) with advancing pregnancy, and concentrations of progesterone in allantoic fluid (P < 0.07) and plasma (P < 0.05) were affected by day of gestation. Concentrations of glucose were greater (P < 0.05) in uterine artery than uterine vein, but concentrations of electrolytes and osmolarity of plasma were not affected by day of gestation. Increases in weights of fetal organs were proportional to increases in fetal weight during gestation. Results of the present study of conceptus growth and development highlight areas of needed research and provide benchmarks for comparisons when evaluating effects of various treatments, environmental conditions, and epigenetics on successful outcomes of pregnancy in sheep.     

T-cell tolerance to the developing equine conceptus

One of the most intriguing and dramatic examples of immunological tolerance is displayed by the mammalian foetal-placental unit, which thrives as a semi-allograft in the mother's uterus during pregnancy. The success of the so-called foetal allograft stands in stark contrast to the failure of most tissue and organ grafts to survive without genetic matching of donor and recipient or drastic immunosuppression of the recipient's immune system. Experiments conducted over the past 60 years have revealed multiple mechanisms that enable the conceptus to avoid immunological detection or destruction. Many of these mechanisms are directed towards evading immune-mediated damage by maternal T lymphocytes, and they can be grouped into three classes: (i) downregulation of major histocompatibility complex (MHC) gene expression in placental trophoblast cells; (ii) local and systemic alterations in maternal immune reactivity; and (iii) innate defence mechanisms of the trophoblast cells that comprise the barrier between foetal and maternal tissues. The redundancy in these protective mechanisms helps ensure the transmission of life from generation to generation and provides a rich field of study of ways in which functional immunological tolerance can be manifest. The variation in placental forms and function among mammalian species present opportunities to discover and understand novel tolerogenic mechanisms that may have broad application in biology, medicine and animal husbandry. This review focuses on the evidence obtained from studies of pregnancy in the mare that support the case for selective T-cell tolerance to the mammalian conceptus.     

Inhibitory effect of the bovine conceptus on lymphocyte stimulation

Experiments were conducted to determine whether the preimplantation bovine conceptus produces an immunosuppressant as measured by the inhibition of lectin-induced incorporation of thymidine into lymphocytes. The results of the experiments clearly indicated that media from cultured 18-d-old embryos inhibited lectin-induced incorporation of thymidine into bovine lymphocytes. The inhibitory substance was not produced by heat-killed cells from the conceptus, was not toxic to lymphocytes, was not lectin-specific, but did act in a partially reversible manner. The inhibitor had no apparent effect on porcine lymphocytes, a moderate effect on ovine lymphocytes and a potent effect on bovine lymphocytes. The substance appeared to have a molecular weight of between 500 and 10,000 and was ether insoluble and heat stable. The possible chemical nature of the inhibitor and its potential effect on survival of the preimplantation conceptus are discussed.     

Impacts on conceptus survival in a commercial swine herd

An estimated 30 to 40% of potential piglets are lost before farrowing in U.S. or European pig breeds. Because these studies were conducted in limited numbers of university research herds, we decided to characterize the timing, pattern, and extent of conceptus loss in a commercial swine herd in Iowa (Pig Improvement Company; Camborough Line). Sows (parities 2 to 14) were slaughtered on d 25 (n = 83), 36 (n = 78), or 44 (n = 83) of gestation. These days coincide with periods before, during, and after uterine capacity becomes limiting to conceptus survival. At slaughter, numbers of corpora lutea (CL) and uterine horn length were determined, and conceptuses were removed and evaluated. Uterine horn length and CL number did not differ among these days of gestation, averaging 217 cm and 26.6 CL, respectively. In contrast, numbers of conceptuses decreased (P < 0.05) from 15.8 on d 25 to 12.9 on d 36, then remained relatively constant through d 44 (12.1). Thus, conceptus survival averaged 60.2% on d 25, 50.1% on d 36, and 46.3% on d 44, based on numbers of CL present. There was a positive correlation (P < 0.001; r = 0.50) between numbers of viable conceptuses on d 25 and ovulation rate, but this association was completely lost by d 36 (P > 0.10) when uterine capacity becomes limiting. In agreement with this premise, uterine horn length and conceptus number were not associated on d 25 but exhibited positive correlations (P < 0.05) on d 36 (r = 0.36) and d 44 (r = 0.40). On all 3 d examined, the numbers of viable conceptuses were not associated with fetal weight but were negatively correlated (P < 0.05) with placental weight. Compared with the commonly reported values for ovulation rate and percentage conceptus loss in university research herds, values from these production animals were extremely high. Data suggest that throughout this period, larger litters were associated with conceptuses exhibiting small placentae. These data lend support to the concept that increased placental efficiency (fetal weight/placental weight) may contribute to increased litter size in the pig.     

鸡和猪脾转移因子活性组分的提取及免疫活性研究

采用Sephadex G-25柱层析法分离提取鸡、猪脾转移因子(Transfer Factor,TF)各组分。利用环磷酰胺建立小鼠免疫抑制模型,通过MTT法比较鸡脾TF各组分及TF原液对免疫抑制小鼠和正常小鼠的淋巴细胞转化的作用,同时比较了不同来源的鸡脾和猪脾TF各组分及原液对正常小鼠淋巴细胞转化的作用。结果表明鸡脾和猪脾TF均由三大组分构成,其中鸡脾TF三大组分及原液均对免疫抑制小鼠的外周血淋巴细胞转化具有明显的免疫增强作用,与对照组相比均表现差异显著(P<0.01),其中组分Ⅲ的淋巴细胞转化最佳,组分Ⅰ与组分Ⅱ接近,但均优于原液。而对正常小鼠的外周血淋巴细胞转化却表现不同的结果,其中组分Ⅰ和原液具有显著的免疫增强作用(P<0.05),组分Ⅱ作用不显著,组分Ⅲ则起到极显著的抑制作用(P<0.01)。而不同来源的猪脾和鸡脾TF各组分及原液对正常小鼠的淋巴细胞均表现出一致的免疫活性。     

猪脾转移因子对小鼠吞噬细胞吞噬功能的影响

成年健康小鼠分别口服或注射不同剂量的转移因子 ( Transfer factor,TF) ,第 8d检测小鼠外周血中性粒细胞对葡萄球菌的吞噬率 ,第 1 1 d检测小鼠腹腔巨噬细胞对鸡红细胞的吞噬率。结果表明 ,注射 TF的 、 、 组小鼠中性粒细胞吞噬率分别为( 38.5 6± 3.82 0 ,( 4 4.6 4± 2 .48) ,( 5 1 .0 7±3.84) ,而对照组的相应值为 ( 2 9.81± 2 .5 3) ;注射 TF的 、 、 组小鼠巨噬细胞吞噬率分别为 ( 5 8.0 4± 2 .1 9) ,( 6 7.8± 3.38) ,( 75 .82± 1 .73) ,对照组为 ( 4 6 .1 6± 1 .6 7) ;口服 TF的 、 、 组小鼠中性粒细胞吞噬率分别为 ( 4 0 .6 4± 4.85 ) ,( 5 3.45±5 .39) ,( 5 1 .0 7± 3.84) ,对照组为 ( 2 9.81±2 .5 3) ;口服 TF的 、 、 组小鼠巨噬细胞吞噬率分别为 ( 5 7.71± 2 .80 ) ,( 6 7.8±3.38) ,( 80 .39± 2 .30 ) ,对照组为 ( 4 6 .1 6±2 .6 8)。注射、口服 TF组小鼠脾脏重量与对照组比较 ,无明显差异 ( P >0 .0 5 )     

Select nutrients and their effects on conceptus development in mammals

The dialogue between the mammalian conceptus(embryo/fetus and associated membranes) involves signaling for pregnancy recognition and maintenance of pregnancy during the critical peri-implantation period of pregnancy when the stage is set for implantation and placentation that precedes fetal development. Uterine epithelial cells secrete and/or transport a wide range of molecules, including nutrients,collectively referred to as histotroph that are transported into the fetal-placental vascular system to support growth and development of the conceptus. The availability of uterine-derived histotroph has long-term consequences for the health and well-being of the fetus and the prevention of adult onset of metabolic diseases. Histotroph includes numerous amino acids, but arginine plays a particularly important role as a source of nitric oxide and polyamines required for fetal-placental development in rodents, swine and humans through mechanisms that remain to be fully elucidated. Mechanisms whereby arginine regulates expression of genes via the mechanistic target of rapamycin cell signaling pathways critical to conceptus development, implantation and placentation are discussed in detail in this review.     

单胚胎RT-qPCR对猪体细胞核移植囊胚全能性的分析

为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。     

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.