Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

An assay for quantitative virulence in Rhynchosporium commune reveals an association between effector genotype and virulence

Detailed knowledge of the evolutionary genetics of virulence is needed to understand and predict host–pathogen dynamics. This study used a virulence assay based on digital image analysis and treated virulence as a quantitative rather than a binary trait. Such quantitative data may better reflect the genetic underpinning of virulence in many pathogen systems and provide better resolution in statistical investigations. A greenhouse experiment based on a common garden design was conducted to measure virulence (% of leaf area covered by lesions) of 126 genetically distinct isolates of the barley scald pathogen, Rhynchosporium commune, originating from nine field populations around the world. Virulence in this pathosystem was found to be a quantitative trait with a continuous distribution in all populations. By comparing population genetic differentiation for virulence and neutral microsatellite markers (i.e. a Q_ST/G_ST comparison), evidence that virulence is under stabilizing selection across populations was found. Heritability values were high and ranged from 0·52 to 0·96 with a mean heritability of 0·84. Virulence was positively correlated with spore production as predicted by the trade‐off theory of virulence evolution. Furthermore, an association analysis between virulence and sequence haplotypes of three known necrosis‐inducing effector genes (NIP1, NIP2 and NIP3) revealed a significant effect of NIP2 haplotypes and NIP1 deletions. Overall, the results support a quantitative model for virulence in the R. commune–barley pathosystem and very high evolutionary potential for this trait.     

Epidemiology of beet necrotic yellow vein virus in sugar beet at different initial inoculum levels in the presence or absence of irrigation

A field experiment was set up in 1988 to study the development of rhizomania disease of sugar beet at different inoculum levels of beet necrotic yellow vein virus (BNYVV) in soil. Five, tenfold different, inoculum levels were created by addition of the approximate amounts of 0, 0.5, 5, 50 and 500 kg infested soil per ha (the latter corresponding to 0.01% v/v calculated to the tillage layer). A drip irrigation treatment was applied to study the influence of soil moisture on disease. Susceptible sugar beet, cv. Regina, was grown for three consecutive years.In the first year, root symptoms were not observed, but BNYVV-infected plants were detected by ELISA in low numbers at all inoculum levels at harvest. After late drilling in 1989, high numbers of infected plants, up to 90–100% in plots with the highest inoculum level, were detected already in June. Root symptoms were also observed from June onwards. In both these years disease incidence increased in time and was significantly influenced by the initial inoculum level. In the third year, the whole field was heavily diseased, and only for the non-irrigated plots incidence differed for different initial inoculum levels. The expression of symptoms by BNYVV-infected plants was influenced by initial inoculum level, thus by the amount and timing of primary infection.Root weight at harvest was not affected, but sugar content decreased with increasing inoculum level already in 1988, leading to a reduction in sugar yield of 10% at the highest inoculum level. In 1989, both root weight and sugar content decreased progressively with increasing inoculum level, resulting in sugar yield reductions of 11–66% (down to approximately 3000 kg ha^–1) for low to high inoculum levels compared to the control. As the control plots became contaminated, all yields were low in 1990, still showing a decrease with increasing inoculum level in the non-irrigated plots, but an overall mean sugar yield of 3323 kg ha^–1 for the irrigated ones.Sodium and -amino nitrogen content of the root, additional quality parameters determining extractability of sucrose, showed an increase and decrease, respectively, with increasing initial inoculum level already in the first year. The relative differences in contents compared to those from the control were largest for Na content. A significant negative correlation was found between Na (mmol kg^–1 root) and sugar content (% of fresh weight); linear for 1988, exponential for 1989 and 1990.In spring 1989, the infestation of individual plots was assessed using a quantitative bioassay estimating most probable numbers (MPNs) of infective units of BNYVV per 100 g dry soil. The relationship between the MPns determined and root weight, sugar content and sugar yield at harvest could be described by Gompertz curves. The increase in disease incidence with increasing MPN in 1989 was adequately fitted with a logistic equation.     

Detection of early pregnancy factor in superovulated mice

Rosette inhibition tests for the detection of early pregnancy factor (EPF) were performed on naturally ovulated and superovulated mice from day 2 of pregnancy up to 4 days after parturition. In both groups of mice, the rosette inhibition titre (RIT) increased on day 2 of pregnancy, and persisted at high levels until day 15. Thereafter, the RITs of both groups of mice decreased to the non-pregnancy range. No significant differences of the mean RITs between these two groups were observed during the high RIT period. These results showed that the superovulatory treatment did not cause any changes or interference in the detection of EPF. In order to investigate the initial time of appearance of EPF in the maternal circulation in relation to the stage of fertilization, measurement of RIT and examination of the fertilization stage were carried out on superovulated mice 1 day after mating. The mean RIT of mice with pronucleus stage ova was significantly (p less than 0.01) higher than that of mice with sperm-penetrated ova. EPF was considered to appear in the maternal peripheral blood at the pronucleus stage.     

A serosurvey of Borna disease virus infection in wild rats by a capture ELISA

For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.     

Effects of supplemental vitamin E during the mature period on the reproduction performance of Taiwan Native Chicken cockerels

One-day-old Taiwan native male chicks were fed with maize-soybean rearing diets without supplemental vitamin E to 23 weeks of age. From 23 to 52 weeks of age, the cockerels (n = 90) were assigned at random to 5 dietary treatments and fed with maize-soybean diets supplemented with 0, 20, 40, 80 and 160 mg/kg of vitamin E (dl-alpha-tocopherol acetate). Pullets (225) of the same age were fed with standard diets throughout. They were artificially inseminated with one dose of 0.04 ml/bird intact and 5-fold diluted pooled semen at 31 to 43 weeks of age and at 49 weeks of age, respectively. The criteria evaluated included: semen quality, fertility and maximum and effective duration of fertility, blood characteristics, body and testes weight. Supplemental vitamin E did not affect cockerels' effective duration of fertility and percentage of fertility. However, when pullets were inseminated with diluted semen, supplementing 160 mg/kg vitamin E increased the maximum duration of fertility at 49 weeks of age. Cockerels receiving 40 to 160mg/kg supplements had higher sperm viability and motility after 39 weeks of age and those fed 80 mg/kg had higher sperm concentration at 39 weeks of age. Cockerels receiving supplements of more than 40 mg/kg vitamin E had higher body weight gain. Plasma cholesterol and testosterone were not affected by supplemental vitamin E. However, plasma luteinising hormone (LH) concentration was lower in cockerels fed 160 mg/kg. Lack of supplemental vitamin E over 39 weeks was associated with lower semen quality but did not reduce the proportion of fertile eggs laid by inseminated hens, perhaps because the insemination dose compensated for low sperm quality. We found that the maximum duration of fertility might be improved by supplementing 160 mg/kg vitamin E at 49 weeks of age.     

Effect of ensiling whole crop oat with lucerne in different ratios on fermentation quality,aerobic stability and in vitro digestibility on the Tibetan plateau

The objective of this study was to determine the effect of ensiling different ratios of whole crop oat to lucerne on fermentation quality, aerobic stability and in vitro digestibility of silage on the Tibetan plateau. Four experimental treatments were produced varying in the ratio of forages on a fresh matter (FM) basis: 1) 100% oat (control, dry matter (DM) content: 317 g/kg), 2) 90% oat + 10% lucerne (OL10, DM content: 316 g/kg), 3) 80% oat+ 20% lucerne (OL20, DM content: 317 g/kg) and 4) 70% oat+ 30% lucerne (OL30, DM content: 318 g/kg). All treatments were packed into laboratory‐scale silos and ensiled for 60 days and then subjected to an aerobic stability test for 15 days. Further, the four experimental treatments were incubated in vitro with buffered rumen fluid to study the nutrient digestibility. All silages were well preserved with low pH and NH₃‐N contents, and high lactic acid contents and V‐scores (evaluation of silage quality). Increasing the lucerne proportion increased (p < 0.05) crude protein (CP) content of silage, whereas neutral (NDF) and acid (ADF) detergent fibre contents were not affected. Under aerobic conditions, the control silage showed higher (p < 0.05) yeast counts (>10⁵ cfu/g FM) followed by OL10 silage, and OL10 silage improved aerobic stability for 74 h. OL20 and OL30 silages showed fewer (p < 0.05) yeasts (<10⁵ cfu/g FM) and markedly (p < 0.05) improved the aerobic stability (>360 h). After 48‐h incubation, OL30 silage increased (p < 0.05) in vitro dry matter digestibility (IVDMD) and neutral detergent fibre digestibility (IVNDFD) compared with the control silage. These results suggest that replacing oat with lucerne had no unfavourable effects on fermentation quality of silage, but improved CP content, aerobic stability IVDMD and IVNDFD. OL30 silage was the best among the three mixed silages.     

In vitro fertilization and cortical granule distribution of bovine oocytes having heterogeneous ooplasm with dark clusters.

In vitro maturation, fertilization and subsequent development of oocytes with homogeneous (category 1), or heterogeneous ooplasm (category 2) were investigated. No significant differences were observed in the nuclear maturation and total fertilization rates between the two categories. However, category 2 oocytes showed a higher normal fertilization rate due to their lower incidence of polyspermy as compared to category 1 oocytes. Electron microscopic study revealed that all category 2 oocytes had cortical granules lined up next to the plasma membrane, and that some category 1 oocytes still had small clusters of cortical granules after maturation. Although the proportion of cleaved zygotes was higher in category 2, the percentages of cleaved zygotes that developed to the blastocyst stage did not differ between the two categories. These results demonstrate that oocytes with heterogeneous ooplasm have a higher capacity for normal fertilization due to the reduction in polyspermy. This can be attributed to the normal distribution of cortical granules in category 2 oocytes after maturation.