Introduction of exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo

1. In order to introduce exogenous DNA into gonads of chick embryos, stage X blastoderms of freshly laid and unincubated eggs were transfected by lipofection and electroporation in vivo. 2. The introduced DNA, green fluorescence protein (GFP) gene, was efficiently expressed in the blastoderms incubated for 24 h (78.8%, 78/99). 3. The GFP gene was present in most of the embryonic bodies and extra-embryonic membranes died by d 10 of incubation, when analysed by polymerase chain reaction. On d 16 to 20 of incubation, the GFP gene was detected in 7.0 to 20.9% of embryos in the heart, liver, stomach and brain, but not in the sartorius muscle. For the gonads, the GFP gene was detected in 22.2% (6/27) of the testes and 6.3% (1/18) of the ovaries examined. 4. These results suggest that it is possible to introduce exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.     

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.     

The ostrich (Struthio camelus) blastoderm and embryo development following storage of eggs at various temperatures

1. The gross morphology of blastoderms in fresh unstored ostrich eggs and in eggs subjected to different regimen of storage and incubation was studied. Then the effects of storage duration of eggs (1, 2 and 3 weeks) and storage temperature (15, 20 and 25 degrees C) on blastoderm and embryo development were investigated.2. Only incubation following overnight storage at 18 degrees C advanced blastoderm development (1.5-fold increase in diameter) to a stage comparable to hypoblast. 3. Storage of eggs at 15 or 20 degrees C did not affect blastoderm stage and size whereas, at 25 degrees C, the blastoderm doubled in size and appeared to have advanced to a primitive streak stage. Embryo development was reduced after 2 weeks of storage regardless of the storage temperature. 4. After oviposition and during pre-incubation storage the ostrich blastoderm develops progressively over time in a temperature-dependent manner towards the hypoblast stage and beyond but the viability of the blastoderm and embryo development is seriously compromised by 2 weeks of storage.     

Effects of storage time on hatchability of artificially incubated Greater Rhea (Rhea americana) eggs

(1) A study was conducted to determine the effects of the length of the storage period on the hatchability of artificially incubated Greater Rhea eggs. Hatchability was evaluated in eggs gathered daily from a captive population and in eggs collected less frequently from a semi-captive population. (2) Eggs form both sites were either immediately incubated after being collected or were stored for 1 to 9 d prior to incubation. (3) The maximum number of days for which an egg could be stored without depressing hatchability (with respect to non-stored eggs) was longer in the eggs collected daily. (4) Eggs collected daily and stored for 4d or more showed total hatchability (28%) and fertile hatchability (43%) which was approximately 30% lower than non-stored eggs or eggs stored for 3 d or less. In the semi-captive population, the total and fertile hatchability of non-stored eggs and of eggs stored for one day were 40% greater than of eggs stored for 2 to 9 d (20 and 34%, respectively). (5) The period for which Greater Rhea eggs could be stored without depressing hatchability varied depending on the frequency of egg collection: non-daily egg collection reduces the possible period of storage.     

主动免疫血管活性肠肽（VIP）对家鸽繁殖性能影响的初探

将３０对青年鸽随机分为三组，第一组用血管活性肠肽（ＶＩＰ）类似物与牛血清蛋白（ＢＳＡ）的偶联物进行主动免疫ＶＩＰ，第二组注射与第一组等量的生理盐水，第三组不做任何处理。在试验的第一阶段，除第三组外，第一、二组鸽在产蛋后立即将书目是拿出，试验结果显示：第一组与第二组鸽月产蛋量和周抱窝数差异不显著。但第一、二组鸽产蛋量与第三组（自然饲养组）差异极显著。在试验的第二阶段（Ｂ处理过程）均不把蛋拿出，则三组     

Comparative Staging of Embryo Development in Chicken,Turkey, Duck,Goose, Guinea Fowl,and Japanese Quail Assessed from Five Hours After Fertilization Through Seventy-Two Hours of Incubation

Normal tables of chicken embryo development are used to define specific stages of morphogenetic progression from the first cleavage divisions through hatching. Although established for the turkey and Pekin duck, the application of the normal tables of chicken embryo development to other birds of commercial and research importance needs be examined. Chicken, turkey, Japanese quail, and Pekin duck blastoderms from oviductal eggs showed differences in the rate of development that were inversely correlated with egg size. Oviposited eggs from these and additional species (goose, Muscovy and mule ducks, and Guinea fowl) were examined after 24 to 72 h of storage and at 6-h intervals up to 72 h of incubation. There was variation in the developmental stages of the blastoderm at the time of oviposition between and within the species and strains examined. Although it is recognized that the temporal rate of development will differ between different species and strains, the external features of any embryo in any given stage will be nearly identical.     

滞育诱导温度和光照节律对家蚕sod基因和cat基因的表达的影响

家蚕(Bombyxmori)是卵滞育性鳞翅目模式昆虫,其滞育性受亲代卵孵化期(催青期)温度、光节律等环境因子诱导。为了研究家蚕滞育与温度和光照节律的关系,本文调查了温度、光照节律对家蚕sod基因和cat基因表达的影响。催青期中除了孵化期,25℃持续光照下(25LL)卵的sodmRNA均保持在较高水平,另外,sod基因在20℃光照黑暗(20LD)12h交替环境下的表达量要高于15℃持续黑暗(15DD)条件,且孵化后期这种现象更加明显。cat基因在25LL、20LD和15DD环境之间的表达量无差异,但是孵化后期(积温2160-3600℃.h),cat基因表达水平显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律比较敏感的结论相一致。此外,本文还调查了子代卵中sod基因和cat基因的表达情况。sod基因在人工活化卵(DTE)中的表达水平显著高于非浸酸的滞育性卵(DDE);卵龄24-192h,25LL子代卵逐渐进入滞育,sod基因表达量下调。低温黑暗条件会上调家蚕子代卵sod基因的表达水平。在初期的盐酸活化卵(DTE)中,cat表达量低于滞育卵(DDE),而后逐渐上升,直至接近滞育卵表达量,这表明盐酸可能会抑制cat基因表达。在恒温条件下,黑暗条件会促进子代卵上调cat表达。在温度光照协同作用下,20LD子代卵中cat表达量低于25LL和15DD。     

Factors related to shell deaths during artificial incubation of ostrich eggs

The ostrich industry experiences a high rate of embryonic mortalities during artificial incubation of eggs. Embryonic deaths were studied from data recorded on 37,740 fertile eggs incubated artificially during the 1998-2005 breeding seasons. Roughly 10,000 eggs that sustained embryonic mortalities were classified according to the stage and nature of death, i.e. before 21 days of incubation, after 21 days of incubation, deaths after pipping and rotten eggs. Although infection may have played a role in approximately 1300 rotten eggs, no detailed knowledge of the pathogens involved was available. The remainder of deaths could not be related to pathogens and the deaths were thus generally referred to as non-infectious. The overall level of embryonic mortality in all the eggs studied was 28.5 %. Overall embryonic mortality was affected by incubator, with higher levels (57.0 %) found in eggs incubated in an African Incubator and also in eggs that were transferred between incubators during incubation (38.1%). Overall embryonic mortality also increased in eggs produced by older females. Eggs produced in the autumn had the highest level of embryonic mortality at 53.6 %, whereas eggs produced in the winter had a marginally higher level of embryonic mortalities of 29.2 % compared with eggs produced during summer (27.4 %). Eggs produced by South African (SA) Black males crossed to Zimbabwean Blue females had high levels of embryonic losses of 45.7 %. The embryonic mortality of eggs produced by SA Blacks or Zimbabwean Blue breeding birds subjected to pure breeding was similar at approximately 33-34 %, but embryonic mortality was improved in eggs produced by Zimbabwean Blue males crossed to SA Black females (27 %). Embryonic mortality was increased in eggs that were set directly (32.0 %) or subjected to longer than 6 days of storage (43.5 %). Embryonic mortality was affected by year. The results that were obtained will assist in determining non-infectious factors that have a negative effect on hatching success. Steps can thus be taken to eliminate such factors that may compromise hatching success.     

Effect of short periods of high incubation temperature on hatchability and incidence of embryo pathology of turkey eggs

1. Turkey eggs were incubated at 38.0 degrees and 38.5 degrees C at different stages of embryo development and for periods of 3 to 25 d. Results were compared with control eggs incubated at 37.5 degrees C. The age of mortality, the incidence of malpositions and the incidence of morphological abnormalities were recorded from all unhatched eggs. 2. Eggs incubated at 38.5 degrees C for 5 or more days hatched significantly less well than eggs incubated at 37.5 degrees C. Eggs incubated at 38.5 degrees C for 3 d hatched worse than controls but not significantly so. Eggs incubated between 0 and 25 d and 7 and 12 d but not between 0 and 6, 13 and 18 and 19 and 25 d, at 38.0 degrees C had significantly lower hatchabilities than eggs incubated at 37.5 degrees C. 3. Lowest hatchabilities were obtained when the eggs were incubated at high temperatures between 7 to 12 d and 6 to 10 d, indicating that embryos at this stage of development are more likely to succumb to high temperature than at other ages. 4. Increases in embryo mortality due to overheating were seen in weeks 3 and 4 of incubation and at the pipping stage. This was observed even when the period of overheating occurred in weeks 1 and 2 of incubation. 5. Embryos malpositioned with their head in the small end of the egg were seen at a higher incidence when overheating in the 2nd or 3rd quarter of the incubation period.     

Length of the plateau and pipping stages of incubation affects the physiology and survival of turkeys.

1. The hypothesis tested was that accelerating the rate at which a turkey embryo passes through the Plateau and Pipping stages (incubation days 25 to 28) affects growth and embryonic mortality. 2. Eggs from 4 genetic lines were incubated similarly until 24 d of incubation. The eggs were divided at random, half incubated at 36.8 degrees C (CON) and the remaining half incubated at 37.3 degrees C (FAST). 3. The passage time for each developmental stage was recorded at 4 h intervals. Tissues were collected and measured for growth, and glycogen concentration at each developmental stage in response to the accelerated development. 4. The FAST treatment accelerated passage through the Plateau in some lines but not others, but time to pip was shortened in all genetic lines. Embryonic survival rate was affected differently by FAST. Slower growth in response to FAST enhanced survival. 5. In conclusion the rate at which genetically different embryos grow, mature and survive may involve choices among the 3 processes.     

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.     

Influence of the Laying Date on the Fertility and Hatchability of Red-Legged Partridge (Alectoris rufa) Eggs

In several countries, there is a well-developed market for Red-Legged partridge (Alectoris rufa) eggs for incubation. Although Red-Legged partridge eggs produced at game farms are sold with a guaranteed average hatchability, there is a marked seasonal variation in fertility and hatchability. Therefore, an average incubating hatchability value cannot be generalized across the whole breeding season. In this research, the influence that the laying date has upon the fertility and hatchability of incubated eggs and the hatchability of the fertile eggs incubated at a farm of Red-Legged partridge was analyzed. It was found that the laying date did indeed influence the fertility and hatchability of the incubated eggs. Fertility and hatchability were greater in the eggs set in the incubator between mid February and late March than those of the eggs set in late April and early May. Hatchability of fertile eggs was greater in the eggs set in the incubator in mid March and lower in the eggs set in late April. The higher values obtained during the middle of the laying period and the lower ones obtained at the end of the laying period cause the game farms’ need to inform their potential customers of the eggs’ expected hatchability as a function of their laying date.     

合成滞育激素对家蚕卵碳水化合物代谢的影响

为了进一步阐明家蚕滞育激素在诱导滞育卵发生以及对蚕卵滞育过程中碳水化合物代谢的调节作用 ,对低温 (16℃ )暗催青的二化性品种大造 ,在化蛹初期注射合成滞育激素 ,诱导其产下滞育卵 ,然后将这种滞育卵分别用 2 5℃和 5℃保护 ,调查其在滞育过程中糖元、海藻糖、山梨醇和甘油量的变化。结果发现 ,从化蛹第 3天开始 ,每天注射 15 0pmol/头合成滞育激素 ,连续 3次 ,可得到 70 %左右的滞育卵。这种滞育卵在产卵初期糖元的含量与自然滞育卵相似 ,但山梨醇的含量仅有自然滞育卵的 5 0 %左右。诱导滞育卵在 5℃中保护时 ,解除滞育的时间比自然滞育卵提前 ,孵化率比自然滞育卵低 2 0 %左右。未孵化卵大多在转青期死亡。由上述结果推测 ,这种诱导滞育卵在产卵初期糖元与山梨醇的代谢间存在某种障碍 ,从而影响山梨醇的合成 ,导致滞育的提前解除和胚胎发育异常。     

Effect of oxygen supplementation in the hatcher at high altitude on the incubation results of broiler eggs laid at low altitude

1. The object of this research was to investigate the effects of high altitude with supplementary oxygen during the last stage of incubation of broiler eggs laid at low altitude and incubated at low and high altitude. We analysed thyroid hormones and haematological variables. 2. The treatment groups were: low altitude (LA), high altitude with oxygen supplementation in the hatcher (HA-OX) and high altitude non-oxygen-supplemented (HA-NOX). 3. High altitude affected relative egg weight loss and early embryonic mortality. The hatchability of fertile eggs was lower at high than at low altitude. 4. Oxygen supplementation into the hatcher cabinet during the last stage of incubation decreased late embryonic mortality ratio (LEM(1)) and improved survival rates of embryos incubated at high altitude. 5. Eggs incubated at low altitude had a higher hatched chick weight and relative chick weight than those incubated at high altitude. Hatched chick weight and relative chick weight did not change with oxygen supplementation at high altitude. 6. High altitude caused an increase in plasma T(3) and T(4) concentrations as well as in the ratio of T(3):T(4) in embryos. High altitude newly hatched chicks showed a higher T(3):T(4) ratio than low altitude chicks; this ratio decreased with oxygen supplementation at high altitude. Altitude and oxygen supplementation did not affect the mean plasma T(4). 7. Newly-hatched chicks incubated at high altitude showed a higher plasma haematocrit (PCV) than the newly-hatched chicks from eggs incubated at low altitude. High altitude without supplementation increased haemoglobin (Hb), while oxygen supplementation returned the value to low altitude values.     

二化性家蚕滞育过程中谷胱甘肽S-转移酶活性的变化

二化性家蚕卵以25℃和15℃催青,可分别诱导成虫产下子代滞育和非滞育卵,而即时浸酸和5℃左右的低温处理可以分别阻止和解除胚胎滞育。利用1-氯-2,4-二硝基苯(CDNB)显色法测定了经上述处理后的胚胎滞育过程中谷胱甘肽S-转移酶(glutathione S-transferase,GST)的活性,结果表明:胚胎发育后期的蚕卵经25℃催青,卵中的GST活性显著高于15℃催青蚕卵;蚕卵即时浸酸未显著改变胚胎滞育发动阶段蚕卵中的GST活性;5℃低温处理显著提高了滞育卵中的GST活性,但是未能显著改变即时浸酸卵的GST活性。上述结果表明,蚕卵中的GST活性变化与二化性家蚕胚胎滞育诱导和解除密切相关。     

Implantation of chicken embryonic tissue and cells into unfertilised eggs

1. Chick embryo cells and halved embryos were successfully implanted into unfertilised eggs. Yolks containing implants were placed in recipient eggshells, covered by transparent vacuum-formed plastic cones and incubated for 72 h. 2. Dispersed cells were obtained from eggs expelled from the uterus or from eggs that had been laid. Implantation of these cells often resulted in aggregation and epithelial growth, in several cases with axial development. 3. Growth of implanted halved embryos of different ages was often observed, including one 10-somite embryo. Non-axial epithelia, sometimes with a central hole, a central fluid-filled cellular vesicle or a vesicle only, were also observed. 4. In another culture system, whole and halved embryos obtained from laid eggs were cultured on a vitelline membrane stretched across semi-solid egg albumen. During the 72 h incubation, axial development was observed only in whole embryos, while halved embryos grew either into epithelia containing fluid-filled cellular vesicles or into vesicles only. 5. It was found that daily drainage of the accumulating fluid from the embryo compartment encouraged axial development in halved embryos, and almost abolished vesicle formation. Holes were formed in half the embryos cultured on a vitelline membrane. 6. It appeared that physical and biological conditions could inflict serious malformations on the implants.     

二化性家蚕卵低温和常温催青的胚胎期蛋白质表达差异分析

家蚕二化性品种的滞育性受上代胚胎期环境条件调控,查找家蚕胚胎期滞育关联蛋白,可为最终阐明家蚕滞育的分子机制提供实验依据。以家蚕二化性品种秋丰的蚕卵为材料,分别在25℃常温和18℃低温条件下催青,提取胚胎不同发育时期蚕卵的易溶性和难溶性蛋白,采用蛋白质双向电泳(2-DE)和图像分析技术,研究蚕卵在胚胎不同发育时期的蛋白质差异表达谱。在易溶性和难溶性蛋白图谱中分别检测到5个和7个差异显著的蛋白点。对这些差异蛋白点进行质谱鉴定,有8个蛋白点得到可信的最佳匹配蛋白报告,共鉴定出卵特异蛋白、卵黄原蛋白、表皮蛋白和丙酮酸脱氢酶激酶4种蛋白。这些差异表达蛋白可能导致蚕卵胚胎中物质代谢和能量代谢的不同,据此推测低温和常温催青可能在蚕卵胚胎中诱导了不同的生理生化反应。     

Production of somatic chimera chicks by injection of bone marrow cells into recipient blastoderms

Several types of cells, including blastoderm cells, primordial germ cells, and embryonic germ cells were injected into early-stage recipient embryos to produce chimera avians and to gain insights into cell development. However, a limited number of studies of avian adult stem cells have also been conducted. This study is, to the best of our knowledge, the first to evaluate chicken bone marrow cells' (chBMC) ability to differentiate into multiple cell lineages and capability to generate chimera chicks. We induced random differentiation of chBMCs in vitro and injected immunologically selected pluripotent cells in chBMCs into the blastoderms of recipient eggs. The multipotency of BMCs from the barred Plymouth rock (BPR) was confirmed via AP staining, RT-PCR, immunocytochemistry, and FACS using specific markers, such as Oct-4 and SSEA-1, 3 and 4. Isolated chBMCs were found to be able to induce in vitro differentiation to multiple cell lineages. Approximately 5,000 chBMCs were injected into the blastoderms of white leghorn (WL) recipients and proved able to contribute to the generation of somatic chimera chicks with a frequency of 2.7% (2 of 73). Confirmation of chimerism in hatched chicks was achieved via PCR analysis using D-loop-specific primers of BPR and WL. Our study demonstrated the successful production of chimera chicks using chBMC. Therefore, we propose that the use of adult chBMCs may constitute a new possible approach to the production of chimera poultry, and may provide helpful studies in avian developmental biology.     

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.     

The role of preparation technique, culture media and incubation time for embryonation of Heterakis gallinarum eggs

The importance of preparation technique, culture media and incubation time in the embryonation of the infective egg stages of the intestinal nematode parasite Heterakis gallinarum was studied. Mature H. gallinarum worms were isolated from the caeca of infected chickens and separated by sex. In a first experiment intact female worms were kept for the development of their eggs in four different media (0.5% formalin, 2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) and incubated under constant temperature (20-22 degrees C) for 2, 4, 6 or 8 weeks. Afterwards the body of the worms were ruptured and the numbers of unembryonated and embryonated eggs were determined using a McMaster egg counting chamber, and the percentage of embryonated eggs was calculated. After 8 weeks of incubation in 0.5% formalin, 0.1 N sulphuric acid or 0.1% potassium dichromate 27.6%, 26.7% and 29.4% of the eggs, respectively, embryonated into third stage larvae (p > 0.05). In contrast, incubation in 2% formalin resulted in an embryonation of 18.6% only (p < 0.05). In a second experiment H. gallinarum eggs were directly harvested from worm uteri and cultivated afterwards in different media (2% formalin, 0.1 N sulphuric acid, 0.1% potassium dichromate) at 20 to 22 degrees C for 6 weeks. An incubation of isolated eggs in 2.0% formalin or 0.1% potassium dichromate during 6 weeks resulted in a significantly higher percentage of embryonation in comparison to the incubation of intact worms (first experiment). The results suggest that preparation technique, media and time of incubation has an essential influence on the development rate of H. gallinarum eggs.