鸡胚发育过程中胚胎液中几种游离氨基酸的动态变化

本实验旨在研究鸡胚发育过程中尿囊液和羊水中天冬氨酸、谷氨酸、精氨酸和亮氨酸浓度的动态变化。选用72枚6胚龄的三黄鸡鸡胚,按重量相近原则分为6组,每组12个重复,分别在孵化第7、8、11、13、14、18天采集尿囊液和羊水,并利用高效液相色谱法分析其氨基酸浓度。结果表明:尿囊液中,除8胚龄外,其他各胚龄精氨酸浓度均高于另外3种氨基酸(P<0.05),13胚龄精氨酸浓度显著低于其他胚龄(P<0.05),而18胚龄显则著高于其他胚龄(P<0.05)。羊水中,各胚龄精氨酸浓度均高于其他3种氨基酸;除11胚龄外,13胚龄精氨酸浓度显著高于其他胚龄(P<0.05);7胚龄天门冬氨酸浓度显著高于13、14、18胚龄(P<0.05);但18胚龄谷氨酸及亮氨酸浓度则显著升高(P<0.05)。以上结果说明,在尿囊液及羊水中,所测4种氨基酸浓度变化不尽相同,但总体精氨酸含量最丰富,这可能与鸡胚发育的营养与代谢有关。     

相对湿度对孵化失重、胚胎成活率及尿囊液量的影响

通过孵化试验,采用随机区组设计对120个种蛋,分别在相对湿度60%、65%、70%的条件下孵化12 d,研究了不同相对湿度对孵化失重、胚胎成活率及尿囊液量的影响。结果表明,随着相对湿度的升高,种蛋失重及失重率逐渐降低,获得尿囊液量逐渐升高,其中相对湿度为70%的孵化组12日龄失重率最低,获得单位胚重生成的尿囊液量最多,而不同湿度对胚胎成活率几乎没有影响。     

蛋形指数对蛋壳强度、孵化失重、羊水及尿囊液量影响的研究

通过孵化试验，研究了蛋形指数对蛋壳强度、孵化失重、羊水及尿囊液量的影响。结果表明，蛋形指数与蛋壳强度呈正相关，相关系数为0.30；蛋形指数在0.800~0.819时，蛋壳强度最大，大于和小于这个范围均呈下降趋势；蛋形指数在0.780~0.799时，蛋的失重最大，羊水和尿囊液的总量最少，大于和小于这个范围蛋的失重逐渐减小，而羊水和尿囊液的总量则逐渐增多。     

蛋重对孵化失重、羊水及尿囊液量、雏鸡初生重影响的研究

通过孵化实验,研究了蛋重对孵化失重、羊水及尿囊液量、雏鸡初生重的影响.结果表明,蛋越重,失重越大,羊水及尿囊液的量越多,雏鸡初生重也越大;蛋重在60.0～64.9 g范围时,失重率最大;蛋重与雏鸡初生重呈强正相关,相关系数为0.868.     

新城疫病毒LaSota株在鸡胚中的繁殖动态

用新城疫病毒（ＮＤＶ）ＬａＳｏｔａ株尿囊腔接种１０日龄非免疫鸡胚，以血凝（ＨＡ）试验检测接种后２４、４８、７２、９６，１２０、１４４和１６８小时所收集的鸡胚液（尿囊液和羊水）、胚体、尿囊膜的ＨＡ价。结果表明，在上述时间内，胚体、尿囊膜的含毒量很低，无收获价值；病毒在鸡胚液中自接种后２４小时繁殖呈线性上升，至１２０小时，胚液中含毒量最高，以后则下降，故此时是收获的最佳时间。     

禽流感H9亚型SD696株疫苗生产工艺改进试验

分别采用10日龄和12日龄SPF鸡胚增殖禽流感H9亚型SD696株病毒,并改变后孵化温度,将死亡高峰期、最终死亡率、平均单胚收获量、尿囊液血凝价HA、病毒含量EID50进行对比,结果表明用12日龄SPF鸡胚比用10日龄SPF鸡胚增殖禽流感H9亚型SD696株病毒,死亡高峰期推迟12-24 h,最终死亡率、尿囊液的HA和EID50无差异,平均单胚收获量提高了2-3mL。35℃培养比37℃培养鸡胚死亡高峰期延迟、最终死亡率降低、平均单胚收获量提高、尿囊液的血凝价HA及病毒含量EID50偏高。选择12日龄SPF鸡胚,35℃培养温度为生产工艺,既提高疫苗产量,降低成本,又能够提高产品的质量。     

鸡传染性支气管炎H120、W93株活疫苗生产工艺的改进

分别以鸡传染性支气管炎病毒(In-fectiousbronchitisvirus,IBV)H120株、W93株不同病毒量接种10、12日龄SPF鸡胚,并改变后孵化温度生产抗原液,接种后不同时间收获胚液,根据鸡胚的早死率以及胚液的收获量、病毒滴度,选择最佳的生产工艺。结果表明,IBVH120株、W93株以102.7EID50/0.1mL的病毒量接种12日龄鸡胚尿囊腔,在34.5℃孵育35h,可较为显著地降低早死胚率,提高产毒量,且抗原液病毒滴度符合疫苗制备规程的要求。     

孵化时种蛋不同放置方式对胚胎后期发育及孵化率的影响

采用迪卡父母代所产种蛋５００枚，随机分成２组，每组各２５０枚，分别平放和坚放孵化，孵化条件正常。试验分别在孵化和１１，１３，１５，１７，１９天随机抽取两组正常胚蛋各１０枚进行观测，结果表明：平放有利于胚体从蛋壳，蛋黄，蛋白中吸收营养物质；平放可提高受精蛋孵化率近５个百分点，但差异不显著；不论平放竖放，１３胚龄左右尿囊液量最大，且平放组显著高于竖放组。     

孵化温湿度对鸡胚尿囊液的影响

在疫苗生产中常用鸡胚培养毒株.鸡胚中尿囊液量的多少直接影响着疫苗生产的数量和经济效益.生产实践早已证明,胚胎对湿度的适应范围比较广,不像温度那么严格,湿度有良好的导热作用,可以使孵化初期的胚胎受热良好,有利胚胎后期生理散热及胚胎发育.现有研究报道,关于孵化的湿度研究较多,却未见关于获得最大量尿囊液的湿度的研究,而实际生产中,尿囊液量的多少直接影响着疫苗的产量和经济效益.因此本试验的目的是研究湿度、温度两个因素对鸡胚孵化过程中(0～11d)尿囊液量的影响.     

减蛋综合症病毒在鸭胚中增殖规律的研究

通过在鸭胚上增殖规律的研究，证实了鸡减蛋综合症病毒在尿囊腔接种１０日龄鸭胚，接种后２４ｈ以前病毒未增殖，２４ｈ后才开始增殖，１２０ｈ ，病毒增殖到高峰，以后随时间延长下跌；同时还表明，鸡减蛋综合症病毒在尿囊膜、尿囊液、羊水中含量较高，病毒纯化及其他研究提供了依据。     

鸽新城疫四元材料蜂胶佐剂灭活苗免疫期试验研究

采用四批四元材料混合病毒（鸡胚尿囊液、羊水、尿囊膜、羊膜）制备的鸽新城疫（No）蜂胶佐剂灭活疫苗（0701、0702、0703、0704）分别免疫30日龄非免疫健康鸽,不定期采血,连续检测HI抗体的动态变化,这四批疫苗免疫后5d均可检测出抗体,14d达到最高峰（平均为11．31log2）,6个月的平均HI抗体滴度均在6．4log2以上。因此,该苗对鸽的ND免疫保护期至少为6个月。     

Effect of in ovo vaccine delivery route on herpesvirus of turkeys/SB-1 efficacy and viremia

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.     

Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.     

Distribution of chicken cathepsins B and L,cystatin and ovalbumin in extra-embryonic fluids during embryogenesis

1. Concentrations of chicken cathepsin B, cathepsin L, cystatin and ovalbumin were determined in the allantoic fluid, amniotic fluid and extracts of chorioallantoic membranes during days 6 to 12 of embryogenesis.

2. Similar trends for cystatin and ovalbumin were observed in the allantoic fluid with maximum concentrations of cystatin on day 7 (12?±?4?µg/ml) and ovalbumin on day 8 (～19?±?2·5?µg/ml) of embryonic development. The highest concentrations of cathepsin B was found on day 7 and of cathepsin L on day 10, but were significantly lower than those of cystatin and ovalbumin.

3. In the allantoic fluid, especially on day 7, considerable proportions of cystatin and ovalbumin were phosphorylated and contained phosphorylated serine.

4. Concentrations of cathepsin B and L, cystatin and ovalbumin in the amniotic fluid were variable but were comparable to those in allantoic fluid.     

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.     

Metabolomic Profile of Allantoic and Amniotic Fluid in Late-term Gestational Mares Characterized by 1H-nuclear Magnetic Resonance Spectroscopy

The amniotic and allantoic fluid compartments in the mare serve essential roles throughout pregnancy and parturition. Although the global metabolomic profile of amniotic fluid in women has been extensively characterized, current data for equine fetal fluids are limited. Therefore, the goal of this study was to characterize the global metabolomic profile of equine allantoic and amniotic fluid through nuclear magnetic resonance spectroscopy. Fetal fluids were collected between 270 and 295 days of gestation from 12 pregnancies through ultrasound-guided transabdominal puncture. A total of 24 samples (n = 10 allantoic fluid; n = 9 amniotic fluid; n = 5 admixed fluid) were analyzed by one-dimensional proton (¹H) and two-dimensional (¹H-¹³ C) nuclear magnetic resonance spectroscopy. Metabolites were integrated and compared between fluid types using a Kruskal–Wallis test at P < .05 significance. A total of 28 distinct metabolites were found in allantoic and admixed fluid, whereas 23 metabolites were identified in amniotic fluid. Allantoic fluid contained significant elevations (P < .05) in the metabolites betaine, creatine, creatinine, citrate, histidine, nitrophenol, tryptophan, π-methylhistidine, and unknown metabolite #1 compared with amniotic fluid, whereas amniotic fluid contained statistically increased concentrations of the metabolite lactate compared with allantoic fluid (P = .003).     

Biochemical Profile of Amniotic and Allantoic Fluid During Different Gestational Phases in Mares

Fetal fluids have different vital functions that sustain both pregnancy and normal parturition. The biochemical composition of amniotic fluid during gestation is not well established; thus the purpose of the present study was to determine the biochemical profile of both amniotic and allantoic fluids from mares during initial, mid, and latter third phases of pregnancy. Samples were collected after slaughter, using allantocentesis and amniocentesis. Sixty samples of fetal fluids were analyzed. Alkaline phosphatase (AP), glucose, total protein (TP), urea, creatinine, Ca, chloride (Cl), Na, and K concentrations were measured using commercially available kits. The AP concentration in amniotic fluid was higher than that in allantoic fluid during the three gestational phases (P < .05). There were no differences between glucose mean values of allantoic and those of amniotic fluids (P < .05). However, glucose values were higher in the allantoic fluid in the last trimester of pregnancy. TP was higher in the amniotic fluid than in allantoic fluid (P < .05). Urea values varied among the phases; however, there were no differences between the amniotic and allantoic fluid values (P > .05). Creatinine values were higher in allantoic fluid (P < .05). Na and Cl concentrations were higher in amniotic fluid (P < .05). However, Ca and K concentrations were higher in the allantoic fluid.     

Abnormalities of foetal fluids in sheep: two case reports

SUMMARY Two cases of abnormal accumulation of foetal fluids in sheep are described. In the first case, a ewe, at 64 days of pregnancy, had 2 sacs of fluid, one at each of the tips of the pregnant and non-pregnant horns. Neither sac contained a foetus. The composition of the fluids differed from those of the amniotic and allantoic fluids associated with a single foetus. The composition resembled that of foetal plasma. We conclude that the 2 sacs were amniotic sacs from conceptuses that had been resorbed, and that at this early (0.4) stage of pregnancy, amniotic fluid can accumulate in the absence of a viable foetus.
The second case was a rare case of hydrallantois in a ewe at 100 days of pregnancy. The volume of allantoic fluid (1800 mL) was more than 10 times normal. The composition was normal, except for high chloride and calcium concentrations and a very low creatinine concentration. The foetus and the volume and composition of the amniotic fluid were normal. The hydrallantois might have occurred as a result of some alteration in function of the allantoic membrane, perhaps as a result of altered hormonal status of the ewe.     

Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 μg) was used for in‐solution tryptic digestion, and mass spectrometry analysis (nano‐LC‐MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty‐one proteins were found in the amniotic fluid, and twenty‐one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha‐1‐antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha‐2‐macroglobulin and alpha‐2‐HS‐glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha‐2‐macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.