Comparison of culturing Mycoplasma gallisepticum from fresh eggs and 18-day-old embryos

Twenty-four 70-week-old and sixteen 27-week-old white leghorn hens were challenged with R strain Mycoplasma gallisepticum (MG) by injection into the caudal thoracic air sac and infraorbital sinus. Eggs were collected daily and cultured within 7 days or incubated for 18 days. Vitelline membranes of eggs were cultured directly; in 18-day-old embryos, cultures were taken from the yolk sac, air sacs, and oral cavity. Culture of vitelline membrane of eggs within 2 days was compared with culture of eggs stored 10 days post oviposition. The first MG-positive egg was laid 2 days postinfection (PI). Hens continued to lay positive eggs to the end of the experiments. There was no significant difference in MG recovery between eggs cultured within 2 days and those cultured 10 days post oviposition. MG was isolated at a significantly higher rate from eggs than from 18-day-old embryos. MG was isolated at a higher rate from the yolk sac of 18-day-old embryos than from the air sacs or oral cavity of the same embryos.     

Development in culture of the chick embryo from cleavage to hatch

1. Early uterine embryos were obtained from hens by induced oviposition 7.5-8.0 h after the preceding egg was laid. They were cultured in vitro and then in recipient shells to hatch. As controls, embryos from freshly laid eggs were cultured in recipient shells to hatch. 2. For embryos cultured from uterine eggs, the hatch rate was 22.5%, and for embryos cultured from laid eggs, the hatch rate was 62.5%. 3. The weight of the chicks hatched from culture was about 60% of the weight of the preceding egg, or donor egg. Male and female chicks reached maturity and have produced viable offsprings. 4. The results show that it is possible to grow chick embryos in culture from the early cleavage stage (stage II) to hatch. They extend earlier findings on the culture of embryos from the blastoderm stage (Stage X) to hatch. The technique provides a basis for investigations on chick embryo cryopreservation.     

Development of porcine embryos in vitro: effects of culture medium and donor age.

We compared development of porcine embryos in three media and evaluated the effect of age of the donor on embryo development in vitro. In Exp. 1, embryos were collected from 35 postpubertal females on d 2 or 3 after onset of estrus. Embryos were cultured 144 h in Whitten's Medium (WM), North Carolina State University Medium-23 (NCSU-23), or Beltsville Embryo Culture Medium-3 (BECM-3) in 95% air: 5% CO2 at 39 degrees C. More (P < 0.01) embryos that were initially one cell or two cells developed to blastocysts when cultured in NCSU-23 (56%) and BECM-3 (43%) rather than in WM (7.5%). More (P < 0.01) embryos that were four cells at recovery developed to blastocysts in NCSU-23 (97%) than in BECM-3 (69%) or WM (69%). Blastocysts that developed from four-cell embryos cultured in BECM-3 had more (P < 0.01) nuclei than blastocysts that developed from four-cell embryos in the other two media. In Exp. 2, ovarian responses, fertilization rates, and in vitro embryo development in NCSU-23 and BECM-3 were compared for postpubertal (approximately 170-d-old) gilts vs gilts given exogenous gonadotropins at 102 d of age. Ovulation rate (P < 0.01), number of eggs recovered, and number of eggs fertilized per gilt (P < 0.001) were greater in the older gilts. The percentage of eggs fertilized, the number of unfertilized eggs, and the number of unclassifiable eggs were similar (P > 0.10) for both age groups. More (P < 0.10) blastocysts developed from embryos recovered from 170-d-old than from 102-d-old gilts, and more (P < 0.05) blastocysts developed in NCSU-23 than in BECM-3. Zona thicknesses and number of nuclei per embryo were similar (P > 0.10) for both ages. We conclude that embryos from prepubertal gilts do not have the same in vitro developmental potential as those from cyclic gilts. However, superior development of embryos in NCSU-23 from both 102-d-old and 170-d-old gilts indicates that media composition did not differentially affect embryos produced by younger vs older gilts.     

Effect of medium change on the development of in vitro matured and fertilized bovine oocytes cultured in medium containing amino acids

Bovine in vitro matured and fertilized oocytes were cultured for 153 hr in groups of 3 or 30 in 30 microl of modified synthetic oviduct fluid medium supplemented with amino acids. The concentration of ammonium in culture medium at 153 hr of culture was significantly decreased by medium change at 72 hr of culture. However, regardless of embryo density, medium change had no beneficial or detrimental effect on the development of bovine embryos. Increase in the development to blastocysts and production of ammonium were observed when embryos were cultured in groups of 30. These results indicated that the ammonium concentration detected in this culture system has a negligible effect on the development of bovine embryos to blastocysts.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

Inter-relationship between egg weight, parental age and embryonic development

A clear positive correlation was evident between egg weight at setting and embryo weight at 18 d of incubation for eggs laid at or after 32 weeks of age. The weight of embryos from the same size eggs were found to depend upon a curvilinear function of the age of parent flock, with the largest increase in embryo weight occurring between 28 and 32 weeks of age. Hatching weight was highly correlated with egg size at setting. Flock age had no effect on hatching weight from a particular sized egg. Within any of the five ages studied, chicks from smaller eggs tended to hatch slightly (but not significantly) earlier than those from larger eggs.     

活性氧(ROS)对小鼠早期胚胎胚细胞分裂的影响

在体外CZB培养的不同时期添加过氧化氢(2.1μmol/L),发育至囊胚阶段,制作胚胎标本,观察其囊胚发育率、胚细胞数和分裂相数。结果显示:0~12 h组和0~72 h组的胚胎发育率显著低于其他几组(P<0.05),对照组、12~24 h组、24~36 h组、36~48 h组之间均差异不显著(P>0.05);各组之间的胚细胞数和分裂指数均无显著差异。说明胚胎在体外培养时,2细胞期胚胎对外源性H2O2最敏感,也最容易发生发育阻滞。但是经H2O2处理之后,度过2细胞期发育阻滞的胚胎能在以后的发育中发生补偿性生长,其胚细胞数和分裂指数都和体外正常发育的胚胎没有差异。     

Production of quail-chick chimaeras by blastoderm cell transfer

1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.     

黄花棘豆生物碱对体外大鼠胚胎的发育毒性

应用大鼠全胚胎培养（ｗｈｏｌｅｅｍｂｒｙｏｃｕｌｔｕｒｅ，ＷＥＣ）方法，观察了黄花棘豆（Ｏｘｙｔｒｏｐｉｓｏｃｈｒｏｃｅｐｈａｌａ）主要有毒成分生物碱对体外胚胎生长发育、形态分化的影响。首先用醇类溶剂提取法从黄花棘豆中提取生物碱，薄板层析鉴定至少有４种生物碱成分。然后用体内同期胚胎对照及丝裂霉素Ｃ（ＭＭＣ）阳性对照确证了所建立的ＷＥＣ方法的可行性。在选定０．１％ＤＭＳＯ为该生物碱溶剂的基础上，分别在含有２５、５０、１００、２００ｍｇ／Ｌ生物碱的大鼠血清中培养９．５ｄ胚胎４８ｈ，结果，在５０ｍｇ／Ｌ以上可影响胚胎生长发育和器官分化，且呈剂量反应关系，主要表现为头长、颅臀长、体节数、蛋白质含量及１７项形态学总记分低于溶剂对照组，而且随剂量增加，胚胎畸形也增多，２００ｍｇ／Ｌ时畸形率为８０％，主要表现为小头、后脑水肿、无听泡、无视泡、心包积液、肢芽缺失、尾异常、翻转异常等。生物碱还可影响脏壁卵黄囊（ＶＹＳ）的血液循环，抑制其生长。结果提示，黄花棘豆生物碱对体外培养大鼠胚胎有直接发育毒性，ＶＹＳ功能障碍也是引起胚胎发育毒性的重要机制。     

OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响

为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。     

Experimental Infection of Mycoplasma ovipneumoniae in SPF Chicken Embryos

In order to evaluate the SPF chicken embryos as a candidate of the experimental model of Mycoplasma ovipneumoniae (Mo) infection,the 7-day-old SPF chicken embryos were infected with different concentrations of Mo(10⁸,10⁹ and 10¹⁰ ccu/mL) by injection of vitelline fluid and allantoic cavity.The mortality and Mo detection rate (PCR detection and isolation) of infected chicken embryos were employed to evaluate the optimal inoculation route,dose and sampling site.Three different isolates of Mo were submitted to injection of chicken embryos for pathogenicity comparison.The results showed that the optimal inoculation route and dose were vitelline fluid injection with 0.2 mL (10⁹ ccu/mL) Mo per embryo and the sampling site for detection was also vitelline fluid.Three Mo isolates revealed good infectivity and lethality to the chicken embryo.The chicken embryos mortality caused by FL3 strain (45%) and MoGH3-3 (40%) were both higher than that by A3 strain (25%),but with no significance (P>0.05).The detection rate of Mo from the chicken embryos in FL3 group (100%) was higher than that of MoGH3-3 strain(85%) and that of A3 strain (90%),but with no significance(P>0.05).This experiment preliminarily proved that the SPF chicken embryos could be infected and partially killed by Mo,and the virulence of different strains of Mo to the chicken embryos was different.The results provided the data for further establishment of the chicken embryo infection model of Mo,which would benefit the research on Mo pathogenicity and vaccine development.     

Pathogenesis of Brucella abortus in chicken embryos

Chicken embryos inoculated with Brucella abortus at 6, 10, and 12 days of incubation were examined by light and electron microscopy. B. abortus was identified by avidin-biotin immunoperoxidase and immunogold techniques. Death occurred from 2 to 5 days post-inoculation, depending on age of the embryo and route of inoculation. B. abortus was recovered from all infected eggs. Brucellae had spread throughout all tissues and localized preferentially within cells of mesodermal derivation. Organ distribution and degree of bacterial replication varied with age of the embryo at time of inoculation. In 6-day-old embryos, B. abortus localized preferentially in endoderm and mesoderm of yolk sac wall, extra- and intraembryonic serosal epithelia, and glomeruli of the mesonephros. In 10- and 12-day-old embryos, B. abortus spread to all tissues; renal glomeruli, liver, spleen, and heart were most severely infected. Intracellular B. abortus was within the rough endoplasmic reticulum of mesenchymal, mesothelial, yolk endodermal, and hepatic cells. In mononuclear phagocytes, endothelial cells, and granulocytes, bacteria were within membrane-bound vacuoles. Intracellular replication of B. abortus in embryonic tissues, especially yolk endoderm, closely resembled that in experimental infections of trophoblasts.     

Lipid Content of Non-cultured and Cultured Pig Embryo

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos . The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage . Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume – V(e), volume density of cytoplasm per unit volume of embryo – Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm – Vv(fat,c) and total volume of lipid droplets per whole embryo – V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).     

绵羊肺炎支原体人工感染SPF鸡胚的研究

为探索SPF鸡胚作为绵羊肺炎支原体实验室感染模型的可行性,本试验用不同浓度绵羊肺炎支原体（10⁸、10⁹、10¹⁰ ccu/mL）经由卵黄囊和尿囊腔两个部位接种7日龄SPF鸡胚,通过统计鸡胚死亡情况和不同鸡胚组织样品中绵羊肺炎支原体检测阳性率（PCR检测和支原体分离鉴定）,确定绵羊肺炎支原体鸡胚感染方式、感染剂量和最佳分离部位,再用3株不同来源的绵羊肺炎支原体分离株感染鸡胚,观察其对鸡胚的致病力。结果表明,绵羊肺炎支原体感染鸡胚最佳接种途径为卵黄囊接种,感染剂量为10⁹ ccu/mL、0.2 mL/只,最佳分离部位为卵黄液。3株支原体均能感染和致死鸡胚,并均能从卵黄液中分离到绵羊肺炎支原体,但对鸡胚的致病性存在一定差异：FL3株致鸡胚死亡率为45%,略高于MoGH3-3株（40%）,二者均高于A3株（25%）,但差异均不显著（P>0.05）;FL3株鸡胚检测阳率为100%,高于MoGH3-3株（85%）及A3株（90%）,但差异也不显著（P>0.05）。本试验初步确定了绵羊肺炎支原体可感染和致死SPF鸡胚,不同分离株对SPF鸡胚致病力有差异,表明SPF鸡胚可作为下一步建立绵羊肺炎支原体实验室感染模型的候选,为绵羊肺炎支原体致病性研究和疫苗研制奠定基础。     

Effect of time of the addition of glucose to a medium on the development of 1-cell stage mouse embryos

In the present study, 1‐cell stage mouse embryos were cultured with or without glucose, and their development to the blastocyst stage was compared. Embryos cultured in a glucose‐free medium had a higher percentage of development to the 8‐cell stage, and they had higher developmental speeds compared with those cultured in a glucose‐containing medium. The percentages of embryos that developed to the early blastocyst stage, blastocyst stage and expanded blastocyst stage were much lower than those developed in a glucose‐containing medium. This suggests that the culture of 1‐cell stage embryos in a glucose‐containing medium inhibits development at the 8‐cell stage, and that glucose is necessary for blastocyst formation. Previous reports indicate that, from the 1‐cell stage to the 4‐cell stage, glucose inhibits embryo development. In the present study, exposure of early embryos to a glucose‐free medium improved subsequent embryo development, and there was no difference in the percentage of development to the stages ranging from the 8‐cell stage to the expanded blastocyst stage between embryos cultured in a glucose‐free medium from the 1‐cell stage to the 2‐cell, 4‐cell and 8‐cell stage. This indicates that embryo development is improved when a 1‐cell stage embryo is exposed to a glucose‐free medium before and during the 2‐cell stage, and glucose only has an inhibitory effect on embryo development during conversion from the 1‐cell stage to the 2‐cell stage.     

Development of transferred xenogeneic vole embryos in mouse uteri

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

分离培养牦牛输卵管上皮细胞和卵泡颗粒细胞的目的是克服体外受精中早期胚胎发育阻断,建立有利于牦牛早期胚胎体外发育的共培养体系.采集牦牛输卵管上皮细胞进行原代及传代培养,同时从卵泡液中获取颗粒细胞进行原代培养,培养过程中观察2种细胞的生长方式和形态特点.输卵管上皮细胞贴壁生长时,呈多边形,且呈单层成簇生长.培养144 h～216 h,可形成细胞单层.颗粒细胞贴壁生长时,呈现聚集生长特性,贴壁细胞形状不规则,呈放射状.培养72 h～96 h,形成颗粒细胞单层.     

Twin embryos in mares. I: From ovulation to fixation

Recent findings on the origin and development of twins from ovulation (Day 0) to fixation (mean: Day 16) are reviewed. Available data show that almost all twins originate from multiple ovulations. Results of recent ultrasound studies indicate that the number of days between double ovulations does not affect the conception rate per ovum or embryo survival during the first 16 days after each ovulation. Embryo reduction is the natural elimination of excess embryos so that only one embryo enters the foetal stage. In two studies, embryo reduction before or on the day of fixation was not considered an important aspect of the natural correction of twins. Diameters and growth rates on Days 11 to 16 were similar between singletons and twins and the presence of two vesicles did not have a direct effect on their diameter other than that attributable to their age. Twin and singleton embryonic vesicles were mobile within the uterine lumen from the first day of detection (Days 9 to 11) to the day of fixation (mean: Day 16). In one study, the embryonic vesicles were in the uterine body for over 50 per cent of the time during Days 9 to 12 and thereafter were most often in the uterine horns. For twins of dissimilar size, the preference for uterine body versus horns appeared to be an independent function of each vesicle based on its age or size. After Day 12, the number of entries from the uterine body into the horns increased and the vesicles began a maximum mobility phase which continued until fixation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular fluid and cumulus cells synergistically improve mouse early embryo development in vitro

The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used independently, to improve preimplantation embryo quality in culture. This study was undertaken to evaluate the influence of a cumulus cell monolayer in human follicular fluid on mouse early embryo development in vitro. One-cell embryos were obtained from NMRI mice after superovulation with eCG and hCG. Cumulus cells were prepared from mouse egg-cumulus mass. These cells were separated from red blood cells using a Percoll gradient. Follicular fluid was collected from patients undergoing an IVF program during oocyte pick-up. The cumulus cell monolayer was prepared in follicular fluid (FC) and Ham's F10 (HC). Mouse one-cell embryos were cultured in FC, HC, Ham's F10 (HF) and follicular fluid (FF) for 120 h. Only 10.5% of embryos passed the two-cell block in HF. However, the proportions of embryos passing the two-cell block were 23.1%, 21.4% and 68.5% in FF, HC, and FC treatments, respectively; which were significantly different from HF (p<0.05). The differences between FC and the two other treatments were also significant (p<0.001). In FC, 33.7% of one-cell embryos continued to grow to the blastocyst stage whereas only 2.1% and 1.9% of one-cell embryos in FF and HC reached this stage and no embryos developed to blastocyst in HF. The proportion of blastocysts in FC was significantly higher than all other treatments (p<0.001). It can be concluded that follicular fluid and cumulus cells in monolayer form synergistically improve the early embryo culture condition.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.