Association between embryonic loss and damage to the zona pellucida by invasive micromanipulation during oviductal transfer of early-stage embryos in pigs

The aim of this study was to examine the impact of zona pellucida damage, which might arise during somatic cell nuclear transfer (SCNT), on the development and survival of transferred embryos. The zonae pellucidae of in vitro matured oocytes were either punctured with 8- to 10-microm square-ended nuclear injection pipettes and piezo pulses or slit with 35- to 40-microm enucleation pipettes. Intact oocytes were used as controls. These oocytes were electroactivated to induce parthenogenesis and transferred to the oviducts of estrus-synchronized recipient gilts. After 5 to 7 days, the recipient uteri were flushed to collect embryos, and embryonic development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-punctured, 129 zona-slitted and 57 intact embryos were transplanted into four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (64.3 to 79.1%). However, the zona-penetrated and incised embryos exhibited unstable development and survival compared with the controls; development and survival of the control embryos were 94.7 and 87.7%, whereas those of the zona-punctured embryos were 69.0 and 47.9% (P<0.01) and those of the zona-slit embryos were 64.7 and 50.0% (P<0.01). Cells with large foci that appeared to be macrophage giant cells were observed at the surface or inside the degenerated zona-damaged embryos. These results indicate that the recipient's immune response to damage to the zona pellucida may impair embryonic development after transplantation to the oviduct. This may be one of the factors causing the reduced efficiency of live progeny production by SCNT.     

Effects of acidic fog and ozone on the growth and physiological functions of Fagus crenata saplings

The effects of simulated acid fog (SAF) and ozone (O₃) stress on the growth and physiology of beech (Fagus crenata) saplings were investigated. Three-year-old beech saplings were exposed to SAFs of pH 3 and pH 5 (control) during May 2007 to July 2008. In each SAF treatment group, half of the saplings were exposed to 60 ppb of O₃ during September 2007 to July 2008. In comparison to the control saplings, those from the pH 3 treatment had lower total plant biomasses, epicuticular wax amounts, Ca²⁺ concentrations in their leaves, and lower starch concentrations in their leaves and roots. The effect of O₃ was significant only for the starch concentration in the roots, but the O₃ exposure also negatively affected the growth and physiology of beech saplings. Results show that acid fog exerts various severe effects, and that both chronic acid fog and O₃ exposure suppressed the physiological functions of beech saplings.     

Spatial variation in respiration from coarse woody debris in a temperate secondary broad-leaved forest in Japan

We measured the rates of respiration from snags and logs (“coarse woody debris”, CWD) of Japanese red pine (Pinus densiflora Sieb. et Zucc.) to examine the rate of decomposition and CO₂ efflux from these materials in a temperate secondary broad-leaved forest in Japan. At this site, a high quantity of CWD of P. densiflora had accumulated as a result of pine wilt disease during the 1970s. Respiration rates were measured using a dynamic closed chamber method combined with an infrared gas analyzer. We measured the respiration rate of 7 samples of snags and 10 samples of logs from August 2003 to January 2004. The responses of the respiration rates of snags (R_snag) and logs (R_log) to changing temperature were both exponential and the responses to water content were quadratic, and the same function could be used to estimate annual values of both R_snag and R_log. Intensive measurements of water contents of snags and logs showed a marked difference in water content. The mean water content of snags was 20% of log water content. This difference was likely responsible for the observed difference in annual R_snag and R_log. The decay rate constants estimated from the respiration rates measurement of snags and logs were 0.019 and 0.081 year⁻¹, respectively. Despite being lower than R_log, R_snag was a significant compartment of the CWD carbon budget at this site.     

Correlation between the proportion of stained eggs and the number of mites (Dermanyssus gallinae) monitored using a ‘non‐parallel board trap’

The poultry red mite (Dermanyssus gallinae) is a serious problem for the poultry industry worldwide. However, the relationship between the mite population and the damage that they cause is still unclear. In this study, the mite population in poultry houses was examined using an established trap method, and the risk of blood‐stained eggs caused by the mites was assessed. Traps were placed once a week outside the egg channels and/or on the floor in two poultry farms in Fukuoka Prefecture, Japan, from April 2012 to July 2014. The numbers of blood‐stained eggs and total eggs were counted at weekly intervals. The results showed that the number of mites increased from April to May, and reached a peak around the beginning of June when the average temperature and humidity were >24°C and 70–90%, respectively. In the segmented model, the correlation between the proportion of blood‐stained eggs and the number of mites or temperature was positive over a threshold. In conclusion, our established trap method is useful for monitoring mites and can be used to predict when poultry farms should be treated to prevent appearance of blood‐stained eggs.     

Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification,Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker

Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈–fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.     

Immunohistochemical analysis of the distribution of classical swine fever (CSF) viral antigen in boar-pig hybrids and pigs four weeks after infection

We detected the classical swine fever virus (CSFV) antigen in three boar-pig hybrids (hybrids) and three pigs. All animals were experimentally infected with CSFV strain JPN/27/2019 to optimize diagnostic sampling and risk assessment of virus dissemination. Two hybrids died 17- and 19-days post-inoculation (dpi). The other animals were euthanized at 28 dpi. The detection of CSFV antigen at 28 dpi in epithelial cells of the apocrine sweat and sebaceous glands in the skin, salivary glands, mucosal epithelial cells in the rectum, and epithelial cells in the kidney and urinary bladder, suggests that CSFV persists in these tissues and spreads via sweat, saliva, feces, and urine for at least 4 weeks. These findings reveal that hybrids and pigs represent a high risk of virus dissemination four weeks after infection with CSFV strain JPN/27/2019. Prominent CSFV antigens were also detected in hair follicles of the skin. These results suggest that postmortem sampling of animal skin may be effective for CSF diagnosis and can be used to develop a rapid and easy diagnostic method using hair follicles.     

Overexpression of CO2-responsive CCT protein,a key regulator of starch synthesis strikingly increases the glucose yield from rice straw for bioethanol production

Production of bioethanol from rice straw has attracted attention from the point of effective use of agricultural residue. Starch content is an important determinant for bioethanol production from rice straw. The overexpression of CO₂-responsive CCT protein (CRCT), which is the positive regulator of starch synthesis in vegetative organs, notably increased the starch content in rice straw. To produce the bioethanol from rice straw, the dilute acid pretreatment is a general pretreatment method. Importantly, the glucose yields in liquid hydrolyzate after dilute acid pretreatment was markedly increased in the CRCT overexpression lines compared with non-transgenic rice. In addition, the overexpression of CRCT enhanced the biomass production. In contrast, CRCT did not affect on the glucose yields from cellulose in acid-insoluble residue obtained after dilute acid pretreatment. With respect to byproduct in liquid hydrolyzate which inhibits the fermentation, the formic acid content was increased, whereas the furfural, 5-hydroxymethylfurfural and acetic acid contents were unchanged by the overexpression of CRCT. These results demonstrate that genetic engineering of CRCT is an effective method to increase the bioethanol production from rice straw.     

Production of a transgenic pig expressing human albumin and enhanced green fluorescent protein

We introduced a fusion gene of human albumin and enhanced green fluorescent protein (EGFP) into porcine oocytes using the sperm vector method, and produced a piglet that showed clear expression of GFP in the hooves and skin. PCR and Southern blotting analysis of genomic DNA extracted from the piglet's tissues, including the liver, showed that the tissues carried the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the tissues. The fact that human albumin gene was integrated and expressed in the liver of the transgenic pig opened a way for us to achieve our goal, which was the use of transgenic pigs for the bioartificial liver support system.     

Effect of mild restriction of food intake on the speed of racing Greyhounds

OBJECTIVE: To determine whether mild restriction of food intake affects clinicopathologic variables, body composition, and performance of dogs undertaking intense sprint exercise. ANIMALS: 9 trained healthy adult Greyhounds. PROCEDURE: Dogs were offered food free choice once daily for 9 weeks until body weight and food intake stabilized. Dogs were then randomly assigned to be fed either 85% or 100% of this quantity of food in a crossover study (duration of each diet treatment period, 9 weeks). Dogs raced a distance of 500 m twice weekly. Clinicopathologic variables were assessed before and 5 minutes after racing; food intake, weight, body composition, body condition score, and race times were compared at the end of each diet period. RESULTS: Compared with values associated with unrestricted access to food, there were significant decreases in mean body weight (by 6%) and median body condition score (from 3.75 to 3.5 on a 9-point scale) and the mean speed of the dogs was significantly faster (by 0.7 km/h) when food intake was restricted. Body composition and most clinicopathologic variables were unaffected by diet treatment, but dogs given restricted access to food had slightly fewer neutrophils, compared with values determined when food intake was unrestricted. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the common practice among Greyhound trainers of mildly restricting food intake of racing dogs to reduce body weight does improve sprint performance. A body condition score of approximately 3.5 on a 9-point scale is normal for a trained Greyhound in racing condition.     

Pharmacokinetic interactions of flunixin meglumine and enrofloxacin in dogs

OBJECTIVE: To examine pharmacokinetic interactions of flunixin meglumine and enrofloxacin in dogs following simultaneously administered SC injections of these drugs. ANIMALS: 10 Beagles (4 males and 6 females). PROCEDURE: All dogs underwent the following 3 drug administration protocols with a 4-week washout period between treatments: flunixin administration alone (1 mg/kg, SC); simultaneous administration of flunixin (1 mg/kg, SC) and enrofloxacin (5 mg/kg, SC); and enrofloxacin administration alone (5 mg/kg, SC). Blood samples were collected from the cephalic vein at 0.5, 0.75, 1, 1.5, 2, 3, 5, 8, 12, and 24 hours following SC injections, and pharmacokinetic parameters of flunixin and enrofloxacin were calculated from plasma drug concentrations. RESULTS: Significant increases in the area under the curve (32%) and in the elimination half-life (29%) and a significant decrease (23%) in the elimination rate constant from the central compartment of flunixin were found following coadministration with enrofloxacin, compared with administration of flunixin alone. A significant increase (50%) in the elimination half-life and a significant decrease (21%) in the maximum plasma drug concentration of enrofloxacin were found following coadministration with flunixin, compared with administration of enrofloxacin alone. CONCLUSIONS AND CLINICAL RELEVANCE: The observed decrease in drug clearances as a result of coadministration of flunixin and enrofloxacin indicates that these drugs interact during the elimination phase. Consequently, care should be taken during the concomitant use of flunixin and enrofloxacin in dogs to avoid adverse drug reactions.