Distribution of blastodermal cells transferred to chick embryos for chimera production using windowed eggs

1. To improve the production of chimeras, the distribution of donor blastodermal cells after transferring into recipient embryos was examined morphologically.

2. Donor blastodermal cells were distributed near the site of injection in the epiblast and in the subgerminal cavity and yolk. Some filled the hole made by the micropipette and were distributed outside the epiblast. Many were buried in yolk. In some cases, more donor blastodermal cells were located in the yolk than in the subgerminal cavity and some were located 800 μm below the under‐surface of the epiblast.

3. It is recommended that injection should be as shallow as possible to increase the proportion of chimeras produced, and that some means is needed to prevent blastodermal cells from escaping from the hole produced by injection.     

Clearance of Theileria sergenti-infected bovine red blood cells in severe combined immune deficiency mice

Clearance of Theileria sergenti-infected bovine red blood cells (Bo-RBCs) from the blood circulation of severe combined immune deficiency (SCID) mice was studied to help understand the mechanisms of anemia developing in cattle infected with T. sergenti. For the clearance test, Bo-RBC samples having 2%, 58%, and 76% parasitemia and, as a control, parasite-free Bo-RBCs were prepared in the Bo-RBC-SCID mouse model. The T. sergenti-infected Bo-RBCs and the uninfected control Bo-RBCs were separately labeled with two, green and red, fluorescent dyes, mixed together, and injected intravenously into SCID mice. The blood samples collected at various time points were observed under a fluorescent microscope, and the numbers of green and red fluorescing RBCs were counted differentially to determine the clearance rates of T. sergenti-infected and uninfected Bo-RBCs. This test clearly demonstrated that the Bo-RBC samples having higher parasitemias were cleared faster from the blood circulation of SCID mice. The results suggest that the intravascular clearance system in SCID mice may have a mechanism by which T. sergenti-parasitized and non-parasitized Bo-RBCs are recognized and cleared differentially.     

Comparative study of the effects of various cryoprotectants in preserving the morphology of frozen and thawed fowl spermatozoa

1. The effects of various cryoprotectants (glycerol, dimethyl‐sulphoxide (DMSO), methylformamide and ethylene glycol) of the same molarity on preserving the morphology of frozen and thawed fowl spermatozoa (especially midpiece and acrosome) were examined under the light (LM) and scanning electron microscope (SEM) to determine the most suitable.

2. Under LM, the mean respective increases in sperm deformity in semen diluted with ethylene glycol, methylformamide, glycerol and DMSO were 4·5, 4·9, 5·0 and 6·5 percentage points.

3. Under SEM, the mean respective increases in acrosomal deterioration in semen diluted with glycerol, ethylene glycol, DMSO and methylformamide were 6·6, 8·8, 13·1 and 31·2 percentage points.

4. From these results, it appears that glycerol is superior to ethylene glycol DMSO and methylformamide as a cryoprotectant.

     

Construction of monopartite geminivirus-based virus-induced gene silencing (VIGS) vectors using a two-component strategy

Journal of General Plant Pathology - Virus-induced gene silencing (VIGS) is a rapid and efficient tool to elucidate plant gene functions by inserting a target gene fragment into a viral genome and...     

Sarcoptes scabiei var. canis refractory to ivermectin treatment in two dogs

A 10-year-old castrated male Shih Tzu presented with severe generalized pruritus. Skin scrapings revealed the presence of Sarcoptes scabiei var. canis. A Yorkshire terrier in the same household simultaneously developed pruritus due to scabies. Both dogs were treated with 300 μg/kg ivermectin, at first orally and then subcutaneously at 14 day intervals. However, live mites were still found on day 35, and the skin condition deteriorated in both dogs. These findings suggested that the S. scabiei in these dogs was clinically refractory to ivermectin. The pruritus in both dogs rapidly and completely disappeared following topical fipronil administration. This appears to be the first report of canine scabies refractory to ivermectin treatment.     

Morphological observations on frozen and thawed Muscovy spermatozoa

1. Fresh Muscovy drake spermatozoa were examined using a scanning electron microscope (SEM). The average lengths of the segments were: acrosome 1·8 μm, nucleus 10‐9 μm, midpiece 3·6 μm and flagellum (exclusive of midpiece) 71 μm.

2. Under the light microscope, the incidence of abnormal spermatozoa in Muscovy semen subjected to freezing and thawing (almost all with crooked necks) was about 5% higher than that in diluted unfrozen semen.

3. In thawed semen, various abnormalities of the acrosome were observed under the SEM. It seemed that the most radical change was the complete separation of the acrosome from the apical part of the nucleus.

4. The incidence of abnormal acrosomes was increased more than 20% by freezing and thawing.

5. These results suggest that low fertility in thawed semen may be related to increases in the proportion of spermatozoa with crooked necks and acrosomal damage.

     

Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing

In order to investigate the factors causing crooked-necked spermatozoa (CNS) or those with abnormal acrosomes during freezing and thawing, fowl spermatozoa in NaCl or glucose solutions containing 92 ml glycerol/l were examined using a scanning electron microscope before and after freezing and thawing. The incidence of CNS in NaCl solution significantly increased after freezing and thawing, but not in glucose solution. The acrosomal damage caused by freezing and thawing was considerable in both solutions, and the incidence of damage in glucose solution was significantly higher than that in NaCl solution. In neither solution was there a significant difference between the incidence of acrosomal damage in CNS and in non-CNS. The ratios of incidences of abnormal acrosome after, versus before, freezing were higher in non-CNS than in CNS. It appears from these results that the factors during freezing and thawing which cause CNS may differ from those causing acrosomal damage.     

Prediction of enteric methane emission from beef cattle in Southeast Asia

We conducted a meta‐data analysis to develop prediction equations to estimate enteric methane (CH₄) emission from beef cattle in Southeast Asia. The dataset was obtained from 25 studies, which included 332 individual observations on nutrient intakes, digestibilities, and CH₄ emissions. Cattle were provided tropical forage or rice straw, with or without concentrates in individual pens equipped with indirect open‐circuit head hood apparatus. The simplest and best equation to predict daily CH₄ emission was CH₄ (g/day) = 22.71 (±1.008) × dry matter intake (DMI, kg/day) + 8.91 (±10.896) [R² = 0.77; root mean square error (RMSE) = 19.363 g/day]. The best equation to predict CH₄ energy as a proportion of gross energy intake (CH₄‐E/GEI, J/100 J) was obtained using DMI per body weight (DMIBW, kg/100 kg), content (g/100 g DM) of ether extract (EE) and crude protein (CP), and DM digestibility (DMD, g/100 g); CH₄‐E/GEI = ?0.782 (±0.2526) DMIBW ? 0.436 (±0.0548) EE ? 0.073 (±0.0218) CP + 0.049 (±0.0097) DMD + 8.654 (±0.6517) (R² = 0.39; RMSE = 1.3479 J/100 J GEI). It was indicated that CH₄ emissions from beef cattle in Southeast Asia are predictable using present developed models including simple indices.     

Inhibitory effects of Ecklonia cava extract on high glucose-induced hepatic stellate cell activation

Nonalcoholic steatohepatitis (NASH) is a disease closely associated with obesity and diabetes. A prevalence of type 2 diabetes and a high body mass index in cryptogenic cirrhosis may imply that obesity leads to cirrhosis. Here, we examined the effects of an extract of Ecklonia cava, a brown algae, on the activation of high glucose-induced hepatic stellate cells (HSCs), key players in hepatic fibrosis. Isolated HSCs were incubated with or without a high glucose concentration. Ecklonia cava extract (ECE) was added to the culture simultaneously with the high glucose. Treatment with high glucose stimulated expression of type I collagen and α-smooth muscle actin, which are markers of activation in HSCs, in a dose-dependent manner. The activation of high glucose-treated HSCs was suppressed by the ECE. An increase in the formation of intracellular reactive oxygen species (ROS) and a decrease in intracellular glutathione levels were observed soon after treatment with high glucose, and these changes were suppressed by the simultaneous addition of ECE. High glucose levels stimulated the secretion of bioactive transforming growth factor-β (TGF-β) from the cells, and the stimulation was also suppressed by treating the HSCs with ECE. These results suggest that the suppression of high glucose-induced HSC activation by ECE is mediated through the inhibition of ROS and/or GSH and the downregulation of TGF-β secretion. ECE is useful for preventing the development of diabetic liver fibrosis.