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.     

Effects of the dietary protein to energy ratio on growth,feed utilization and body composition in Macrobrachium nipponense

Oriental river prawn (Macrobrachium nipponense) has been widely cultured in Asian countries. However, its nutritional studies are very limited. In the present 8‐week study, we investigated the effects of dietary protein to energy ratio (P/E ratio) on the growth, feed utilization and body composition in juvenile M. nipponense (initial weight 0.302 ±0.03 g). Two‐factor experiment was designed and nine semi‐purified diets were formulated to contain three lipid levels (20, 80 and 140 g kg^?1) and three protein levels (330, 380 and 430 g kg^?1), producing P/E ratios from 16.5 to 23.4 mg KJ^?1 protein. The results indicated that the growth, survival rate and protein efficiency were dose dependently improved by the increased dietary lipid, but not dietary protein content. Increased dietary lipid content and/or protein content increased lipid accumulation in whole body, hepatopancreas and muscle, but did not change the feed intake and hepatopancreas weight. In conclusion, our present study indicated that M. nipponense is a species with relatively high‐energy requirement. It could utilize dietary lipid content up to 140 g kg^?1, while the dietary protein with more than 330 g kg^?1 would not promote growth and protein efficiency. Taken together, 330 g kg^?1 dietary protein and 140 g kg^?1 dietary lipid level with P/E ratio 16.49 could be optimum for M. nipponense.     

Effect of dietary betaine levels on growth performance and hepatic intermediary metabolism of GIFT strain of Nile tilapia Oreochromis niloticus reared in freshwater

An 8‐week feeding experiment was conducted to determine the effect of dietary betaine levels on the growth performance and hepatic intermediary metabolism of genetically improved farmed tilapia (GIFT) strain of Nile tilapia Oreochromis niloticus (mean initial body weight: 78.3 ± 1.3 g, means ± SD). Six practical diets were formulated with the incorporation of betaine at the levels of 0 (control), 5, 10, 15, 20 and 25 g kg⁻¹. Survival showed no significant differences among the treatments (P > 0.05). The highest and lowest weight gain (WG) and specific growth rate (SGR) were observed for fish fed the diets containing 5 and 0 g kg⁻¹ (control) betaine, respectively. Feed intake showed similar trend with WG and SGR. In contrast, feed conversion ratio was the lowest when dietary betaine level was 5 g kg⁻¹. In general, dietary betaine supplementation showed no significant effect on hepatic composition of tilapia. Condition factor and viscerosomatic index tended to increase with increasing dietary betaine levels from 0 to 5 g kg⁻¹ and then decline when dietary betaine levels further increased from 5 to 25 g kg⁻¹. In contrast, hepatosomatic index declined with increasing dietary betaine levels (P < 0.05). Dietary betaine levels significantly influenced several hepatic enzymatic activities, including succinate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, lipoprotein lipase and hepatic lipase, suggesting that dietary betaine addition had significant effects on nutrient metabolism in the liver. Based on the second‐order polynomial regression analysis of WG, 12.5 g kg⁻¹ of dietary betaine level seemed optimal for genetically improved farmed tilapia strain of O. niloticus.     

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 quantitative real‐time PCR assay for detection of Colletotrichum lindemuthianum in navy bean seeds

Bean anthracnose is a seedborne disease of common bean (Phaseolus vulgaris) caused by the fungal pathogen Colletotrichum lindemuthianum. Using seed that did not test positive for the pathogen has been proven to be an effective strategy for bean anthracnose control. To quantify the extent of anthracnose seed infection, a real‐time PCR‐based diagnostic assay was developed for detecting C. lindemuthianum in seeds of the commercial bean class navy bean. The ribosomal DNA (rDNA) region consisting of part of the18S rDNA, 5^.8S rDNA, internal transcribed spacers (ITS) 1, 2 and part of the 28S rDNA of seven races of C. lindemuthianum, 21 isolates of Colletotrichum species and nine other bean pathogens were sequenced with the universal primer set ITS5/ITS4. Based on the aligned sequence matrix, one primer set and a probe were designed for a SYBR Green dye assay and a TaqMan MGB (minor groove binder) assay. The primer set was demonstrated to be specific for C. lindemuthianum and showed a high sensitivity for the target pathogen. The detection limit of both assays was 5 fg of C. lindemuthianum genomic DNA. To explore the correlation between the lesion area and the DNA amount of C. lindemuthianum in bean seed, seeds of the navy bean cultivar Navigator with lesions of different sizes, as well as symptomless seeds, were used in both real‐time PCR assays.