Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

Effects of supplementation with cellooligosaccharides on growth performance of weaned calves on pasture

We conducted two feeding experiments to evaluate the effects of cellooligosaccharide (CE) supplementation on growth performance in grazing beef calves. Calf sex and age and duration of the experimental period differed between the experiments. Experiment 1 (10 weeks) used 5.6‐month‐old castrated males; Experiment 2 (13 weeks) used 3.9‐month‐old females. Eight Japanese Black calves were assigned to either a control group (CON) or an experimental group (CE) fed CE at a rate of 10 g/day mixed with concentrate. Calves were stocked in fields in which Kentucky bluegrass was dominant. In both experiments, average daily gain tended to be greater in CE than in CON, especially in late stages of experiments, but no significant differences in body weight changes were observed. In Experiment 2, final heart girth was larger in CE than in CON calves. We monitored rumen microbial community composition in Experiment 2 and found increases in fibrolytic bacteria and methanogenic archaea in CE calves, but the overall microbial composition did not differ between the groups. Our results suggest that supplementation with CE may positively affect growth performance in weaned calves on pasture, but longer‐term CE supplementation may be required to exhibit the effect.     

Influence of subsoiling on the yield of sugar-beets

Root growth and yield of sugar-beets were studied on two Belgian silt loam soils and one alluvial clay soil after subsoiling to a depth of 60 cm. The removal of the plough sole layer, characterized by penetration resistances > 3 MPa, resulted in an increase of the root quantity and a higher availability of water. As a consequence, a mean yield increase of 5 tons/ha for sugar-beets was found on the subsoiled plots in drier years. The yield increase was accompanied by a higher uptake of nutritive elements.     

Neuromuscular and vascular hamartoma of the small intestine in an F344 rat

An intestinal mass was found in the border area of the jejunum and ileum of a 110-week-old male F344 rat. Histopathologically, the mass protruded into the lumen and was covered with intestinal epithelium, exhibiting a normal architecture. The lesion was located in the submucosa and consisted of loose connective tissue, smooth muscle, scattered ganglion cells, and blood vessels of various sizes. Although these components showed an irregular and disordered structure, no cellular atypia, increased proliferation activity, or invasive growth to adjacent tissues were detected. Immunohistochemical analyses revealed that smooth muscle, ganglion, and endothelial cells were positive for α-smooth muscle actin and vimentin, S-100, and CD34 and von Willebrand factor, respectively, indicating maturation of these cells. Thus, the mass was diagnosed as a neuromuscular and vascular hamartoma of the small intestine. To the best of our knowledge, this is the first report of this type of lesion in rodents.