利用夏季自然发情的云南黄牛进行奶牛胚胎移植效果研究

利用夏季自然发情的云南黄牛为受体,开展了奶牛体内冷冻胚胎移植.留用的114头受体牛中,本地黄牛59头,年龄在3～10岁,均为经产牛,最终移植27头,西杂牛56头(其中2岁以下的青年牛26头),移植29头(青年牛11头),两个品种受体利用率分别为45.8%和51.8%;移植后妊娠率分别为59.3%和55.2%;解冻的63枚胚胎移植给了56头受体牛,A级胚胎单独移植和B、C级胚胎搭配移植最终妊娠结果分别是58.5%(24/41)和53.3%(8/15);发情后第6 d和第7 d移植妊娠率分别为56%和58.1%.结果表明,1～10岁的西杂牛和3～10岁体型较大的本地黄牛均可作为受体移植奶牛胚胎;利用自然发情的云南黄牛做受体移植效果较为理想,是一种经济、方便、适合云南广大农村推广的技术途径;A级胚胎单独移植和B、C级搭配移植可获得较为理想的妊娠结果;处于发情后第6 d和第7 d的云南黄牛都可以作为桑囊期奶牛胚胎的移植受体.

杜泊羊冷冻胚胎移植的影响因素

下载PDF阅读器为进一步提高杜泊羊的冻胚移植成功率,于春秋两季同期处理湖羊和小尾寒羊受体共997只(次),同期发情782只(次),发情率达78.4%,其中湖羊春秋两季平均发情率为84.6%,显著高于小尾寒羊(73.9%)(P＜0.01),秋季两品种平均发情率83.3%,显著高于春季75.8%(P＜0.01);对431只发情受体羊进行移植,总妊娠率为58.5%(252/431),产羔率为58.0%(250/431).其中湖羊为受体的春秋两季平均妊娠率62.5%,高于小尾寒羊(54.7%);秋季两品种的平均妊娠率为64.8%,显著高于春季(52.5%).囊胚移植后的平均妊娠率为61.5%,显著高于桑椹胚(41.5%),其中A级囊胚为67.4%,显著高于B级囊胚(P＜0.05)和所有桑椹胚(P＜0.01);B级囊胚显著高于B级桑椹胚(P＜0.05),B级囊胚与A级桑椹胚间及A、B级桑椹胚间均无显著差异;C级囊胚和桑椹胚均未见妊娠.此外,受体羊卵巢黄体发育状况及移植技术熟练程度对妊娠率也有显著影响.

杜泊绵羊冷冻胚胎移植

介绍了从新西兰引进的冷冻杜泊羊胚胎解冻后移植到同期发情湖羊和小尾寒羊母羊受体的方法步骤,并分析、评价了胚胎移植的效果。结果表明:受体羊总平均同期发情率为78.4%,胚胎移植利用率达49.1%,妊娠率达59.4%,产羔率达58.0%,羔羊出生重为4.7 kg(公)和4.3 kg(母),羔羊1月龄和3月龄体重分别为14.9kg和32.4 kg;不同批次和不同品种受体间胚移效果亦存在不同程度的差异;4批次试验共移植冷冻胚胎475枚,产羔266只,现存活245只。该研究建立了可靠而有效的杜泊绵羊冷冻胚胎移植技术,有望在畜牧业科研和生产中广泛推广应用。

牛血清白蛋白对小鼠原核期胚胎玻璃化冷冻的影响

以小鼠原核期胚胎为对象, 以胚胎的存活率、卵裂率、囊胚率以及囊胚细胞数作为检测指标, 在M2 液的基础上添加8 种浓度(0 , 2 , 4 , 8 , 16 , 32 , 64 , 96 mg/ mL) 牛血清白蛋白(BSA) 配置防冻液, 探讨防 冻液和玻璃化冷冻后对胚胎发育的影响。BSA 防冻液对胚胎发育影响的实验结果表明, 8 个浓度组间以及与对 照组间胚胎的卵裂率、囊胚率以及囊胚细胞数无显著差异( P > 0105) , 说明在防冻液中加入一定浓度的BSA 对小鼠胚胎无毒性作用。防冻液经玻璃化冷冻后对胚胎发育影响的实验表明, 8 个浓度组间冷冻胚胎复苏后的 存活率、卵裂率、囊胚率及囊胚细胞数无显著差异( P > 0105) 。表明BSA 在这种防冻液中没有明显的保护作 用。从经济、实用、生物安全角度考虑, 不支持在玻璃化防冻液中添加BSA。

受体品种、体重及移植季节对杜泊羊胚胎妊娠率和新生羔羊体重的影响

胚胎移植可以提高家畜良种扩大的速度, 但移植成功率会受到很多因素的影响。用615～715 日龄 杜泊羊冷冻胚胎作为移植胚胎, 研究了季节和受体状况对杜泊羊胚胎移植妊娠率以及出生羔羊生长发育的影 响。在比较春秋两季236 枚冷冻胚胎的移植效果发现: 秋季移植的妊娠率(6816 %) 显著高于春季(5815 %) ; 春季和秋季移植后出生羔羊的体重, 初生时无显著差异; 但在30 日和60 日(断乳) 时, 春季移植的羔羊显著 高于秋季。比较不同品种的受体发现, 昭通绵羊受体的胚胎移植妊娠率最高, 湖羊次之, 小尾寒羊最低, 但受 体羊3 种品种间无显著差异; 较大受体羊( ≥40 kg) 生产的羔羊出生和30 d 的体重显著高于较小受体( < 40 kg) ; 但60 d 羔羊体重差异不显著, 说明受体体重会影响羔羊的出生体重, 但是对哺乳期的生长发育影响不大。 春季移植出生的羔羊, 其早期生长发育情况明显优于秋季。

影响杜泊羊冷冻胚胎移植成功率的因素

采用引进的杜泊羊冷冻胚胎, 以云南当地绵羊为受体进行胚胎移植。同期发情处理了158 只受体 羊, 同期发情率为82191 %; 对102 只进行了胚胎移植, 实际移植率为77186 % , 3 个情期内移植妊娠率达 7415 %; 出生68 只, 产羔率达6617 %。分析表明, 胚胎发育阶段及级别、卵巢黄体情况、以及胚胎移植技术 熟练程度直接影响胚胎移植成功率; 此外, 受体羊的处理程序及移植后的饲养管理、移植时机的把握、移植季 节以及胚胎冷冻及解冻方法也会影响杜泊羊移植妊娠率, 进而影响产羔率。

Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 muL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst. (C) 2000 by Elsevier Science Inc.

Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL, penicillin; 3) HECM-9 with 50 mug/mL streptomycin; 4) HECM-9 with 10 mug/mL,gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 mug/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay. (C) 2000 by Elsevier Science Inc.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

鸡胚背根神经节培养法测定神经生长因子(NGF) 活性的条件优化

目的　探索一套稳定可靠的鸡胚背根神经节测定N GF 活性的实验条件。　方法　通过观察培养 基、鸡血浆和鸡胚浸液的比例、背根神经节部位、培养时间等条件对N GF 刺激神经节生长的影响, 从而 建立一套该培养法检测N GF 活性的优化条件。　结果　以DM EM 为母液, 在含20% 鸡血浆和15% 鸡胚 浸液的培养基中, 利用伊莎褐鸡胚腰椎背根神经节, 在终浓度为30 ngöm l 的蛇毒N GF 的刺激下, 培养36h 可以得到较好的实验结果。　结论　该条件简单实用、分辨率高、重复性好。

CHARACTERISTICS OF FATTY ACID METHYL ESTERS (FAMEs) AND ENZYMATIC ACTIVITIES IN SEDIMENTS OF TWO EUTROPHIC LAKES

Enzymatic activities and fatty acid methyl esters (FAMEs) in the sediments of two eutrophic lakes in Wuhan city were investigated. The results showed phosphatase and dehydrogenase activities in the lotus zone and plant floating bed zone were significantly lower than those in other sites, and urease activity was the highest where microorganism agents were put in. Fatty acid group compositions indicated the predominance of aerobic bacteria in the surface sediments in shallow lakes. The ratios of FAMEs specific for bacteria and Gram-positive bacteria exibited significant differences between the two lakes. The results of trans to cis indicated that the microorganisms in Lake Yuehu could adapt themselves to environmental stress better. The enzymatic activities and FAMEs showed differences in different sites, indicating that ecological restoration measures and environmental conditions could affect lake sediment to some extent. But the monitoring, work would be done in series to exactly evaluate the effect of the remediation measures.

Molecular and expression characterization of two somatostatin genes in the Chinese sturgeon, Acipenser sinensis

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry. SMART cDNA was synthesized from the hypothalamus of Chinese sturgeon, and the full-length cDNAs of two somatostatin (SS) genes were cloned and sequenced. The first cDNA (AsSS1) encodes a 116-amino acid protein that contains the SS14 sequence at its C-terminal extremity. AsSS1 shows high identity to that of human and other vertebrates. The second cDNA (AsSS2) encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]-SS14 at its C-terminal extremity. Both the two SS mRNAs were expressed in brain and pituitary with different mRNA levels. But in peripheral tissues, AsSS2 was more widely distributed than AsSS1. High mRNA levels of AsSS2 were found in liver, kidney and heart, while low mRNA levels of AsSS2 were also detected in ovary. Throughout embryogenesis and early larval development only AsSS2 mRNAs were detected. Furthermore, in the hypothalamus of one to five year-old Chinese sturgeon, AsSS2 but not AsSS1 maintained stable expression. The mRNA distribution suggests that the Chinese sturgeon AsSS2 products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development. (C) 2009 Elsevier Inc. All rights reserved.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Protein Profiles in Zebrafish (Danio rerio) Embryos Exposed to Perfluorooctane Sulfonate

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and in wildlife, and it has the potential for developmental toxicity. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the proteins that are differentially expressed in zebrafish embryos exposed to 0.5 mg/l PFOS until 192 h postfertilization. Two-dimensional electrophoresis coupled with mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that 69 proteins showed altered expression in the treatment group compared to the control group with either increase or decrease in expression levels (more than twofold difference). Of the 69 spots corresponding to the proteins with altered expression, 38 were selected and subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 18 proteins were identified in this analysis. These proteins can be categorized into diverse functional classes such as detoxification, energy metabolism, lipid transport/steroid metabolic process, cell structure, signal transduction, and apoptosis. Overall, proteomic analysis using zebrafish embryos serves as an in vivo model in environmental risk assessment and provides insight into the molecular events in PFOS-induced developmental toxicity.

Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos

Hexabromocyclododecane (HBCD) is widely used as a brominated flame retardant, and has been detected in the aquatic environment, wild animals, and humans. However, details of the environmental health risk of HBCD are not well known. In this study, zebrafish embryos were used to assess the developmental toxicity of the chemical. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of HBCD (0, 0.05, 0.1, 0.5, and 1.0 mg L-1) until 96 h. Exposure to 0.1, 0.5, and 1.0 mg L-1 HBCD significantly increased the malformation rate and reduced survival in the 0.5 and 1.0 mg L-1 HBCD exposure groups. Acridine orange (AO) staining showed that HBCD exposure resulted in cell apoptosis. Reactive oxygen species (ROS) was significantly induced at exposures of 0.1, 0.5, and 1.0 mg L-1 HBCD. To test the apoptotic pathway, several genes related to cell apoptosis, such as p53, Puma, Apaf-1, caspase-9, and caspase-3, were examined using real-time PCR. The expression patterns of these genes were up-regulated to some extent. Two anti-apoptotic genes, Mdm2 (antagonist of p53) and Bcl-2 (inhibitor of Bax), were down-regulated, and the activity of capspase-9 and caspase-3 was significantly increased. The overall results demonstrate that waterborne HBCD is able to produce oxidative stress and induce apoptosis through the involvement of caspases in zebrafish embryos. The results also indicate that zebrafish embryos can serve as a reliable model for the developmental toxicity of HBCD. (C) 2009 Elsevier B.V. All rights reserved.