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.

Addition of alanine and taurine blocked killing of lymphocytes caused by culture at 45 C. The optimal concentration for thermoprotection was achieved at 12.5 mM for L-alanine and 5 mM for taurine. Both D and L forms of alanine provided thermoprotection. The effect of these agents was not simply to increase osmolarity of the culture medium, because NaCl did not provide thermoprotection at comparable concentrations. Alanine and taurine were each tested at concentration of 50 mM for ability to block heat shock-induced killing and developmental retardation of 8- to 16-cell mouse embryos. Both agents enhanced embryo development after exposure to high temperature, though development remained less than that for embryos not exposed to high temperature. In one experiment, for example, 81% of embryos cultured at 38 C advanced in development during culture vs 0% at 42 C, 15% at 42 C with alanine, and 32% at 42 C with taurine. The beneficial effect of alanine at high temperature may have been partly attributable to effects independent of thermoprotection, because development of embryos cultured at 38 C was also improved by alanine.

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.

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p less than 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p less than 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

Transabdominal ultrasonography has been popularly used in animal reproduction for the assessment of pregnancy status and foetal viability where as transvaginal method for pregnancy diagnosis is rarely used for pregnancy diagnosis in goats. The study was conducted in 74 Attappady Black goats from Government Goat Farm, Attappady and Livestock Research Station, Thiruvizhamkunnu to evaluate the accuracy of trans-vaginal methods and to identify the fetal characteristics from day 20 to 75. Transvaginal ultrasonographic examination was performed using endocavity transducer with frequency of 6.5 to 8 MHz. Observations were made in all the pregnant does at three different stages of pregnancy i.e., 20-35 days, 35-55 days and 55-75 days. Does were diagnosed as pregnant from the observation of gestational sac, embryo or foetus, embryonic or foetal heartbeat, placentomes or foetal skeleton. Sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy along gestation period were 98.03, 92.3, 96.15, 96 and 95.62%, respectively. It is concluded from the present study that transvaginal ultrasonography can be used for herd pregnancy diagnosis at early stages from day 20 to 45 of pregnancy diagnosis in goats.