The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199 + FCS with various concentrations (0-20 mu-M) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 mu-M nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 mu-M nocodazole. Exposure to 10 mu-M nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 mu-M nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% O2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 mu-M nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.

Health and ruminal variables were intensively measured during adaptation to grain-based diets in 6 beef cattle with fistulated rumens. The cows had been maintained on prairie grass hay-supplemented diets, and were converted to a grain-based finishing ration by feeding each successive diet (diets 1-4, respectively) for a period of 7 days. Each cow was evaluated and samples were obtained 3 times each day for the first 5 days that each diet was fed. Health variables monitored were rectal temperature, pulse, respiratory and rumen motility rates, fecal consistency, demeanor, blood pH, and blood glucose and L(+) lactate concentrations. Ruminal variables monitored were pH and glucose, DL-lactate, and volatile fatty acid concentrations of rumen contents. Data were analyzed by use of a multivariate ANOVA. We determined that most of the health variables were within reference rang limits throughout the adaptation period; however, analysis of pulse and respiratory rates indicated that diets 2 and 4 were stressful. Although blood pH continually decreased during feeding of the 4 diets (7.38 to 7.30), blood L(+) lactate and glucose concentrations had large increases only within diet 4. The pH of ruminal contents decreased progressively from 6.8 to 5.3. Rumen glucose concentration was low (< 1 micromole/ml), except with diet 4 in which values were 8 times higher than for other diets. By the end of the study, the ruminal contents of all animals were acidic (pH < 5.5), and, on the basis of higher than background amounts of ruminal glucose and DL-lactate, it was determined that rumen microbial equilibrium had not yet been achieved. Analysis of results of this study suggested that ruminal imbalance could be evaluated by monitoring pulse and respiratory rates, blood pH, and blood glucose concentrations. Assessment of the rumen alone could be accomplished by monitoring the variables of rumen pH, rumen glucose, and DL-lactate concentrations.

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 mu-M nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used-for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. IN experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres no treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres oat 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P<0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P<0.05) and development to the blastocyst stage (P<0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.