Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).     

Characterisation of endobacterial communities in ectomycorrhizas by DNA- and RNA-based molecular methods

The diversity of endobacteria associated with ectomycorrhizas of Suillus variegatus and Tomentellopsis submollis, in two Corsican pine (Pinus nigra) stands was analysed by cultivation-dependent and cultivation-independent molecular methods. Denaturing gradient gel electrophoresis (DGGE) analysis revealed the cultivable endobacterial communities associated with S. variegatus were similar within the same stand. The most abundant cultivable bacterial species belonged to the genera Pseudomonas and Burkholderia. Cultivation-independent molecular analysis indicated that the structure of the endobacterial communities in ectomycorrhizas was consistent across all samples regardless of ECM fungal species or the pine stand from which the samples were collected. However, comparison between rDNA- and rRNA-derived DGGE gels showed that metabolically active endobacterial species were not always detected in rDNA-based profiles. Clone libraries constructed from rRNA molecules indicated that Pseudomonas and Burkholderia spp. were metabolically active bacteria. As some of the most abundant cultivable bacteria, including Bacillus/Paenibacillus spp., were not detected in cultivation-independent DGGE profiles, a combination of cultivation-dependent and -independent approaches provided a more complete assessment of the diversity of endobacteria associated with ectomycorrhizas.     

Dietary calcium/phosphorus ratio influences the efficacy of microbial phytase on growth,mineral digestibility and vertebral mineralization in juvenile tiger puffer,Takifugu rubripes

A 2 × 3 factorial feeding trial was conducted to determine effects of dietary Ca/P ratio and dietary microbial phytase on growth, mineral digestibility and vertebral mineralization in tiger puffer. The treatments consisted of three levels of Ca/P ratios (0.5, 1.0 and 1.5) combined either with phytase (2000 FTU kg^?1 diet) or without supplementation, respectively. The Ca/P ratios were achieved by supplementing calcium at 0, 6 and 12 g kg^?1 combined with the same level of inorganic P at 5 g kg^?1. After a 50‐day feeding trial, puffer fish fed the diet at low Ca/P ratio (0.5) together with phytase had significantly higher growth rate and feed intake (FI) than other groups. Both dietary Ca/P ratio and phytase supplement were independent effects on plasma minerals and alkaline phosphatase. Interactive effect between both dietary treatments was observed on P and Zn contents in vertebrae and whole body. P and Zn digestibilities tended to increase with increased Ca/P ratio from 0.5 to 1.0, especially when phytase was supplemented. In conclusion, fish fed a diet with highest Ca/P ratio (1.5) showed the poorest growth performance and nutrients utilization. Dietary Ca/P ratio of 0.5 (without Ca supplement) with 2000 FTU phytase per kg would be the optimum combination in the diet of tiger puffer.     

Developmental Competence of Frozen-thawed Blastocysts from Fair-quality Bovine Embryos Cultured with β-Mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

Influence of food availability,plant productivity,and indigenous forest use on ranging behavior of the endangered samango monkey (Cercopithecus albogularis schwarzi), in the Soutpansberg Mountains,South Africa

Understanding the determinants of ranging patterns in species susceptible to habitat fragmentation is fundamental for assessing their long‐term adaptability to an increasingly human‐dominated landscape. The aim of this study was to determine and compare the influence of ground‐based food availability, remotely sensed plant productivity, and indigenous forest use on the ranging patterns of the endangered samango monkey (Cercopithecus albogularis schwarzi). We collected monthly ranging data on two habituated samango monkey groups, from February 2012 to December 2016, from our field site in the Soutpansberg Mountains, South Africa. We used linear mixed models to explore how food availability, plant productivity, and indigenous forest use influenced monthly ranging patterns, while controlling for group size, number of sample days and day length. We found that as more areas of high plant productivity (derived from remotely sensed EVI) were incorporated into the ranging area, both total and core monthly ranging areas decreased. In addition, both total ranging area and mean monthly daily path length decreased as more indigenous forest was incorporated into the ranging area. However, we found no effect of either ground‐based food availability or remotely sensed plant productivity on ranging patterns. Our findings demonstrate the behavioral flexibility in samango monkey ranging, as samangos can utilize matrix habitat during periods of low productivity but are ultimately dependent on access to indigenous forest patches. In addition, we highlight the potential of using remotely sensed areas of high plant productivity to predict ranging patterns in a small ranging, forest‐dwelling guenon, over ground‐based estimates of food availability.     

A plastidic glucose-6-phosphate dehydrogenase is responsible for hypersensitive response cell death and reactive oxygen species production

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.