家兔胚胎细胞核移植与连续移植

将发育不同时期的兔胚和移核胚的卵裂球细胞核与去核的成熟卵母细胞共同组成移核胚,通过中间受体培养和移植实验检验胚胎的发育能力。结果表明,（１）兔囊胚之前各个时期的胚胎细胞核均可使移核胚发育到囊胚;（２）胚胎极化前后的卵裂球参与组成的移核胚发育到囊胚的比例无显著差异。但极化后 ６４细胞胚的卵裂球与去核卵母细胞的融合率低于极化前的 ８细胞胚胎卵裂球;（３）兔 １６细胞胚与去核的卵母细胞组成的移核胚可以发育到期,产仔率为 ３．１６％ ;（４）兔移核胚卵裂球用于连续核移植,其后 ２ 代均可发育成囊胚,其中第 １ 代移核胚与第 ２ 代移核胚发育率相似,但显著高于第 ３ 代移核胚;（５）兔移核胚和各代连续移核胚卵裂球与去核卵的融合率无显著差异。     

电激活对完全体外化牛细胞核移植的影响

牛卵母细胞在体外成熟２３～２４ｈ时去核，去核卵用８０Ｖ／ｍｍ４０μｓ两次电脉冲激活（Ⅰ组）或不激活（Ⅱ组），然后将体外受精、发育的８～３２细胞期胚胎的单个卵裂球注入卵周隙，用８０Ｖ／ｍｍ４０μｓ两次电脉冲诱导卵裂球与去核卵母细胞融合。Ⅰ组操作后存活率和融合率（８８．４％和５５．０％）显著低于Ⅱ组（９５．９％和６５．１％，Ｐ＜０．０５）。融合卵体外培养２４ｈ和５～８ｄ后，Ⅰ组核移植胚胎的卵裂率和桑椹／囊胚发育率（６３．０％和１５．２％）与Ⅱ组（５０．５％和７．８％）无显著差异，但Ⅰ组结果均好于Ⅱ组。将来自两组的２５枚桑椹和囊胚移植到１６头同期受体，在已检查过的８头受体中２头受体妊娠，其中１头于妊娠后４个多月流产，１头于妊娠期满后产出一雄性牛犊。研究结果表明：去核卵母细胞的电激活尽管对重组卵的存活与融合不利，但可改善核移植胚的卵裂和发育。本研究在我国首次获得牛细胞核移植的成功，证明用ＩＶＭ卵母细胞和ＩＶＦ胚胎进行完全体外化的牛细胞核移植是可行的。     

牛胚胎原代和继代细胞核移植结果比较

比较了原代和继代核移植的操作各环节以及核移植胚胎在体外发育能力上的差异。通过显微操作将体外受精发育而来的８～３２细胞期胚胎的单个卵裂球注入激活的去核卵母细胞的卵周隙内,并用８０Ｖ/mm、４０us２次电脉冲诱导卵裂球与去核卵母细胞融合,借此进行牛胚胎的原代核移 体外发育来的８～３２细胞期的原代核移植胚胎作为供体,用原代核移植相同的方法进行牛胚胎的继代移植。原代核移植的存活率和融合率（８７．３％和６８．５     

兔胚胎核移植操作条件的建立

本文对影响家兔胚胎细胞核移植的几个重要因素进行了研究。以不同的融合电场强度对重组胚进行处理，结果表明脉冲参数为１．６５ｋｖ／ｃｍ，脉宽为８５μｓ，脉冲数为１时，融合率达到７７．１％。显著高于其它场强的融合率。当场强增加至２．５０ｋｖ／ｃｍ时，多数重组胚发生溶解。融合液中的离子成份对重组胚的激活有重要影响。以０．３Ｍ甘露醇为融合液时，电激三次的激活率（５０％）显著高于一次（３６％）和二次（２２．２％Ｐ＜０．０５）。当加入１００μｎＣａ２＋时，电激三次后的激活率达到７１．４％，显著高于电激一次（４８％，Ｐ＜０．０１）和二次（６１．３％，Ｐ＜０．０５）。多次电激还可提高重组胚的发育率，当有Ｃａ２＋存在时，三次电激后的囊胚发育率为３１．４％。此外，随着卵母细胞卵龄的增加，重组胚的囊胚发育率下降，而融合胚的碎裂率则增高。当卵龄为ＨＣＧ后２１—２４小时，碎裂率高达５２％。对８－，１６－，３２－细胞期卵裂球的重组胚体外发育能力的研究表明，８－细胞期卵裂球的重组胚之囊胚发育率显著高于１６－及３２－细胞期卵裂的重组胚（３６．４％比２６％、２７．５％，Ｐ＜０．０５）。将部分３２－细胞期卵裂球的重组胚移入受体后，产出了３只核移植     

牛体外受精胚胎细胞核移植的实验研究

将采自肉联厂屠宰车间的牛卵巢中的卵母细胞置于ＴＣＭ－１９９＋１０％ＥＣＳ（０ｄ）＋ＦＳＨ（１万ＩＵ／Ｌ）＋ＬＨ（５ｍｇ／Ｌ）中培养成熟，用含０．３％透明质酸酶的消化液将卵母细胞周围的卵丘细胞去掉，并以７．５ｍｇ／Ｌ的ＣＢ液处理后，用微吸管吸去透明带内的第一极体及极体下面的部分卵母细胞质，然后将经体外受精并发育至８～１６细胞期胚胎的卵裂球注入去核卵母细胞的卵周隙中，再将移核胚置于电融合槽内进行融合（电场强度为１２００Ｖ／ｃｍ，持续时间为８０μｓ，１次脉冲刺激）。将融合的胚胎移入生长有单层贴壁颗粒细胞的发育培养液（ＴＣＭ－１９９＋１０％牛血清）中培养，观察移核胚的融合率及发育率，部分去核卵母细胞经染色后观察去核率。结果表明：（１）移核胚的融合率为８６．９％（５３／６１）；（２）发育至２～８细胞期的胚胎占培养的移核胚胎总数的２１．３％（１０／４７）；（３）卵母细胞的去核率为６８．２％（４５／６６）。     

牛胚胎体外生产技术的简化研究

利用屠宰牛卵巢分离的卵母细胞，以卵裂率和囊胚发育率为标准，对牛胚胎体外生产过程进行了简化试验。结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００￣２００枚／平皿）可代替加卵丘细胞的标准密度（５０枚／平皿）培养，其卵裂率（５７．６％比６２．５％）和囊胚发育率（２３．７％比２９．１％）没有显著差异（Ｐ〉０．０５），体外授精时间可从成熟培养后２２ｈ延长至２７ｈ，卵裂率和囊胚发育率均没有显著变化，卵母     

体内、外卵母细胞对猪体细胞克隆胚胎发育潜力的影响

为探讨猪体内、外成熟卵母细胞对核移植重组胚胎发育能力的影响,试验通过激素促排获得体内成熟卵母细胞和收集废弃卵巢获取体外成熟的卵母细胞,分别构建核移植重组胚,比较其卵裂率、囊胚率及胚胎移植受孕情况。结果显示,PGC+PMSG+HCG组的平均排卵数（27.8枚/头）显著高于PGC+HCG （12.5枚/头）、PMSG+HCG （13.7枚/头）及自然发情组（11.5枚/头）（P<0.05）,体内收集到的卵母细胞,可用于构建核移植重组胚的可用卵率均达到90%以上,与其他处理组差异不显著（P>0.05）,说明通过激素处理可获得更多的可用卵母细胞,而且卵母细胞的质量没有显著差异;以体内和体外成熟卵母细胞作为核移植受体构建的克隆胚胎,二者的胚胎融合率（80.31%和79.29%）和卵裂率（90.40%和86.51%）差异均不显著（P>0.05）,但来自体内成熟卵母细胞克隆的胚胎发育至囊胚期的比例显著升高（P<0.05）;将体内、外成熟卵母细胞构建的核移植重组胚分别移植代孕母猪,头平均移植30或60枚时,体内成熟卵母构建的克隆胚胎移植出生仔猪10头,而体外培养卵母细胞构建的克隆胚胎均未着床受孕,表明通过激素促排获得的卵母细胞质量更好,能显著提高克隆胚胎的囊胚率,减少胚胎移植数量,提高代孕母猪的怀孕率。     

水牛卵母细胞去核方法的比较

水牛卵母细胞体外成熟22h后，分别用盲吸法、点击法和纺锤体成像系统（Spindle—View System）去核。结果，三者的去核率分别为65．1％、81．8％和95．0％，差异极显著（P〈0．01）。以水牛胎儿成纤维细胞为供体，通过核移植构建的重构胚的融合率、分裂率和囊胚发育率，在上述3种方法之间均没有显著差异（P〉0．05）。认为点击法是一种较为简单、实用和有效的去核方法。     

牛胚胎体外生产技术的简化研究

利用屠宰牛卵巢分离的卵母细胞，以卵裂率和囊胚发育率为标准，对牛胚胎体外生产过程进行了简化试验。结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００～２００枚／平皿）可代替加卵丘细胞的标准密度（５０枚／平皿）培养，其卵裂率（５７．６％比６２．５％）和囊胚发育率（２３．７％比２９．１％）没有显著差异（Ｐ＞０．０５），体外授精时间可从成熟培养后２２ｈ延长至２７ｈ，卵裂率和囊胚发育率均没有显著变化，卵母细胞体外成熟后可以不经洗涤直接移入受精滴，原成熟培养皿内残留卵丘细胞再培养４８ｈ形成的单层细胞，可代替标准方法制作的单层小滴用于体外受精胚胎的共同培养，供胚胎发育培养的单层细胞使用多次不影响囊胚发育率，经简化的ＩＶＦ技术获得的胚胎非手术移入情期同步受体牛子宫角２～２．５个月后直检有５８．３％（７／１２）妊娠。我们认为，传统的牛胚胎体外生产技术经过简化可以提高生产效率，不会减少囊胚发育率和妊娠率，值得推广应用。     

供体细胞和去核卵母细胞的不同预激活对体细胞核移植胚胎体外发育的影响

研究用细胞质内注射法构建重组胚，比较了离子霉素和乙醇预激活供体细胞和去核卵母细胞对重组胚卵裂和体外发育的影响。结果表明：与未经处理的对照组相比，离子霉素预激活供体细胞对重组胚的卵裂率和囊胚发育率有所提高；而供体细胞和去核卵母细胞同时用离子霉素预激活，重组胚的卵裂率无明显提高，囊胚发育率下降；用离子霉素或乙醇预激活去核卵母细胞，对重组胚的卵裂和发育无明显影响。     

Phase I evaluation of CCNU (lomustine) in tumor-bearing cats

1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) is an alkylating agent in the nitrosourea subclass. A prospective evaluation of CCNU was done to determine the maximally tolerated dosage of CCNU in tumor-bearing cats. Response data were obtained when available. Twenty-five cats were treated with CCNU at a dosage of 50-60 mg/m3 body surface area. Complete hematologic data were available for 13 cats. Neutropenia was the acute dose-limiting toxicity. The median neutrophil count at the nadir was 1,000 cells/microL (mean, 2,433 cells/microL; range, 0-9,694 cells/microL). The time of neutrophil nadir was variable, occurring 7-28 days after treatment, and counts sometimes did not return to normal for up to 14 days after the nadir. Based on these findings, a 6-week dosing interval and weekly hematologic monitoring after the 1st treatment with CCNU are recommended. The nadir of the platelet count may occur 14-21 days after treatment. The median platelet count at the nadir was 43,500 cells/microL. No gastrointestinal, renal, or hepatic toxicities were observed after a single CCNU treatment, and additional studies to evaluate the potential for cumulative toxicity should be performed. Five cats with lymphoma and 1 cat with mast cell tumor had measurable responses to CCNU. Phase II studies to evaluate antitumor activity should be completed with a dosing regimen of 50-60 mg/m3 every 6 weeks.     

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.     

Prevalence of gastric lesions in racing Alaskan sled dogs

Human and equine athletes are reported to have a high prevalence of gastric disease, and anecdotal evidence suggests a similar phenomenon applies to racing sled dogs. To investigate the prevalence of gastric disease in racing sled dogs, we conducted 2 gastroscopy studies on dogs competing in the annual Iditarod Sled Dog Race. A pilot study of dogs that were either dropped from the 2000 Iditarod Sled Dog Race because of illness or that finished the race indicated that, approximately 5 days after competing, 10 of 28 dogs (35%) had endoscopic evidence of gastric ulceration, erosion, or hemorrhage. The next year, an endoscopic study of 73 dogs participating in the 2001 Iditarod race was performed in order to evaluate a larger population of dogs. Data from 70 of these dogs could be used; 34 (48.5%) had ulceration, erosion, gastric hemorrhage, or some combination of these findings. When this group of 70 dogs was compared retrospectively to a control group of 87 dogs presented to the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital, the Iditarod sled dogs had a significantly higher prevalence (P = .049) of gastric lesions. These findings suggest that, similar to athletes of other species, elite canine athletes have an increased prevalence of gastric disease compared to the canine population at large.     

The Epidemiology of Gastrointestinal Nematodes of Dairy Cattle in Central Kenya

The epidemiology of H. placei and of other gastrointestinal nematodes in yearling dairy cattle was examined on two farms in Kiambu District, central Kenya during each of 13 one-month periods from April 1993 to April 1994. On each farm, 32 newly weaned dairy calves were given a single dose of albendazole and then placed on experimental pastures. Twelve of the animals were designated for bi-monthly slaughter (n = 2) and analysis of worm population characteristics and 20 were designated for blood and faecal collection and for weighing. Two parasite-free tracer calves were grazed alongside the weaner calves each month throughout the study period and were also slaughtered for analysis of worm populations. Faecal egg counts, haematological and serum pepsinogen determinations, herbage larval counts, and animal live weight changes were recorded monthly. The study revealed that Haemonchus placei, Trichostrongylus axei, Cooperia spp. and Oesophagostomum radiatum were responsible for parasitic gastroenteritis and that H. placei was the predominant nematode present in the young cattle on both farms. Faecal egg counts from resident cattle and necropsy worm counts revealed that pasture larval levels were directly related to the amount of rainfall. The total worm burdens in the animals were highest during the rainy season (March–June and October–December) and lowest during the dry seasons (July–September and January–February). The very low recovery of immature larvae of H. placei from the tracer calves indicated that arrested development is not a feature of the life cycle of this parasite in central Kenya. The maintenance of the parasite population depended on continuous cycling of infection between the host and the pasture. The agroclimatic conditions of the study area were such that, in general, favourable weather conditions for the development and survival of the free-living stages of gastrointestinal nematodes existed all year round.     

Diversity of Ectoparasites in Sheep Flocks in Sa~o Paulo,Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of Sa~o Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the `Associaça~o Paulista de Criadores de Ovinos' (ASPACO; Sa~o Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.     

Evaluation of biohydrogenation rate of canola vs. soya bean seeds as unsaturated fatty acids sources for ruminants in situ

An experiment was conducted to study disappearance of C14 to C18 fatty acids, lag times and biohydrogenation (BH) rates of C18 fatty acids of ground soya bean and canola seeds in situ. Three ruminally fistulated Dallagh sheep were used to determine ruminal BH of unsaturated fatty acids (UFAs). Differences in the disappearance of fatty acids through the bags and lag times were observed between the oilseeds. We saw that the longer the incubation time of the oilseeds in the rumen, the lower the content of C18:2 and C18:3. Significantly higher lag times for both C18:2 and C18:3 were observed in ground canola compared to ground soya bean. BH rates of C18:2 and C18:3 fatty acids in soya bean were three times higher than those of canola. These results suggest that the fatty acid profile of fat source can affect the BH of UFAs by rumen micro‐organisms. So that UFAs of canola had higher ability to escape from ruminal BH. It seems that fatty acid profile of ruminant products is more affected by canola seed compared to soya bean seed.     

Serum biochemical values and mineral element contents of tissues in yaks

Serum biochemical values and the wet weight to dry weight ratios of tissues were determined in yaks in Shandan county of Gansu province. The liver, kidney, heart and muscle contents of seven elements in yaks were also analysed. Most serum biochemical values were similar to those of cattle, camels and sheep, but the calcium concentration was considerably above the normal range for other ruminants. The liver contained the highest concentrations of copper, manganese and cobalt and the kidney of selenium, iron and calcium.Abbreviations Alb albumin - ALT alanine aminotransferase - AST aspartate aminotransferase - BUN blood urea nitrogen - Chol cholesterol - Glob globulin - -GT -glutamyl transpeptidase - IP inorganic phosphorus - TP total protein     

Avian brood parasitism — a growing research area in behavioral ecology (part Ⅱ)

正We are pleased to publish the second special issue on avian brood parasitism and to be responsible guest editors for the two special issues of Chinese Birds (Vol. 3, No. 4, 2012 and Vol. 4, No. 1, 2013), entitled "Avian Brood Parasitism - A Growing Research Area in Behavioral Ecology". The first issue was published in December 2012. The goal of the two special issues is to publish accumulated knowledge and some of the recent developments in     

Internal Parasites and Haematological Values in Cattle Slaughtered in Buea Subdivision of Cameroon

Tropical Animal Health and Production -