Successful production of blastocysts following ultrarapid vitrification with step-wise equilibriation of germinal vesicle-stage mouse oocytes

The purpose of this study was to evaluate the viability and subsequent developmental ability of murine germinal vesicle (GV) oocytes ultrarapidly vitrified after step-wise exposure to cryoprotectants (CPAs). Oocytes were transferred to a vitrification solution composed of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 M sucrose in a direct manner (non-preequilibrium) or in a step-wise manner (single-, two-, or ten-step preequilibrium). After ultrarapid vitrification and storage in liquid nitrogen, the oocytes were thawed, washed by diluting the CPAs in five steps, and then subjected to in vitro maturation, fertilization and culture. In the non-preequilibrium group, the rates of post-thawed oocytes surviving, maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 91.8, 87.1, 15.9 and 2.3%, respectively. In the single- and two-step groups, the corresponding rates were 97.0-98.2%, 92.2-95.0%, 22.0-29.4% and 8.8-15.6%, whereas in the ten-step group they were 98.2, 91.8, 38.6 and 22.8%, respectively. In the non-vitrified control group, the rates of oocytes maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 90.2, 75.2 and 51.5%, respectively. The present study shows that the ultrarapid vitrification of murine GV oocytes by a step-wise manner involving 10 steps preequilibrium may have an advantage in maintaining the viability and subsequent production of blastocysts.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

Evaluation of the Endophyte Enterobacter cloacae SM10 Isolated from Spinach Roots for Biological Control against Fusarium Wilt of Spinach

Six hundred sixty-three isolates of microorganisms, including fungi and bacteria, were collected from surface-sterilized roots of spinach (Spinacia oleracea L.) growing in commercial greenhouses in Kyoto Prefecture. These isolates were screened for their ability to control Fusarium wilt of spinach caused by Fusarium oxysporum f. sp. spinaciae. In primary screening, spinach seeds were treated with the isolates, sown in pots containing sterilized soil, and then challenge-inoculated with the pathogen. Nine bacteria were effective in reducing disease incidence. Subsequently, spinach seeds were treated with the selected isolates, then sown in an infested field and grown from June to July 1998. Four bacteria reduced disease incidence. One of these four, designated as SM10, significantly suppressed the disease. Based on bacteriological properties, SM10 was identified as a strain of Enterobacter cloacae. SM10 was observed within xylem vessels of spinach roots using light and immunoelectron microscopy, indicating E. cloacae SM10 was an endophytic bacterium of spinach. Received 4 July 2000/ Accepted in revised form 13 September 2000     

Lipopolysaccharide induces inflammatory cytokines in the pig amnion

Inflammatory mediators that are induced by gram-negative bacteria in the course of intrauterine infections threaten successful pregnancy. To compare the effect of two different routes of cytokine induction, bacterial lipopolysaccharide (LPS) was administered in vivo either into the cord vein or into the amniotic cavity of pig fetuses in the second half of gestation for 20 h and cytokines were detected in the amnion.Tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were induced in the amniotic epithelium after intra-amniotic but not after intra-venous administration of LPS. The presence of IL-8 was confirmed by RT-PCR. In contrast, transforming growth factor-beta1 (TGF-beta1) was expressed constitutively and was found in all samples of the amniotic epithelium.Amniotic fluid contained only minute levels of TNF-alpha. IL-8 levels in amniotic fluid increased after the treatment with LPS and the highest IL-8 levels were found in dead LPS-treated fetuses.     

No apoptotic cell death of erythroid cells of erythroblastic islands in bone marrow of healthy rats

A possibility of apoptotic cell death in erythropoietic regulation was examined by means of detailed light microscopical histoplanimetry, electron microscopy, the in situ nick-end labeling method, and an immunohistological method in the rat bone marrow. Serum erythropoietin concentrations were shown at normal levels. The erythroid series on a mature process presented several morphological features of apoptosis, i.e. the shrinkage of both nuclei and cytoplasm and the chromatin condensation. In the light microscopical histoplanimetry, however, morphological signs of final apoptotic cell death were never found in any erythroid cell within the erythroblastic islands. This finding was also supported by detailed ultrastructural observation: No erythroid cell bodies were trapped and degraded by the central macrophages of the erythroblastic islands, while the denucleated nuclei with small amount of cytoplasm of late erythroblasts were often trapped and degraded in the macrophages. Nuclear DNA fragmentation was not detected in any erythroblasts, but was detected in the lysosomes of the central macrophages. These findings suggest that erythropoiesis is regulated by other regulatory mechanisms than apoptotic cell death. An additional ultrastructural finding shows that the reticulocytes anchored to the central macrophages are transported into the peripheral blood circulation.     

Effect in sheep of dietary concentrate content on secretion of growth hormone, insulin and insulin-like growth factor-I after feeding

This study was designed to examine the effects of the proportion of concentrate in the diet on the secretion of growth hormone (GH), insulin and insulin‐like growth factor‐I (IGF‐I) secretion and the GH‐releasing hormone (GHRH)‐induced GH response in adult sheep fed once daily. Dietary treatments were roughage and concentrate at ratios of 100:0 (0% concentrate diet), 60:40 (40% concentrate diet), and 20:80 (80% concentrate diet) on a dry matter basis. Mean plasma concentrations of GH before daily feeding (10.00–14.00 hours) were 11.4 ± 0.4, 10.1 ± 0.5 and 7.5 ± 0.3 ng/mL on the 0, 40 and 80% concentrate diet treatments, respectively. A significant decrease in plasma GH concentration was observed after daily feeding of any of the dietary treatments and these decreased levels were maintained for 8 h (0%), 12 h (40%) and 12 h (80%), respectively (P < 0.05). Plasma IGF‐I concentrations were significantly decreased 8–12 h and 4–16 h after the end of feeding compared with the prefeeding level in the 40 and 80% concentrate diet treatments, respectively (P < 0.05). GHRH injection brought an abrupt increase in the plasma GH concentrations, reaching a peak 10 min after each injection, but, after the meal, the peak plasma GH values for animals fed 40% (P < 0.05) and 80% (P < 0.01) concentrate diet were lower than that for roughage fed animals. The concentrate content of a diet affects the anterior pituitary function of sheep resulting in reduced baseline concentrations of GH and prolonged GH reduction after feeding once daily.     

Laparoscopic cryptorchidectomy in standing bulls

Laparoscopic cryptorchidectomy without insufflation was applied in 10 standing bulls aged 3 to 15 months. Nine bulls were preoperatively pointed out intra-abdominal testes by computed tomography. Preoperative fasting for a minimum of 24 hr provided laparoscopic visualization of intra-abdominal area from the kidney to the inguinal region. Surgical procedure was interrupted by intra-abdominal fat and testis size. It took 0.6 to 1.5 hr in 4 animals weighing 98 to 139 kg, 0.8 to 2.8 hr in 4 animals weighing 170 to 187 kg, and 3 and 4 hr in 2 animals weighing 244 and 300 kg to complete the cryptorchidectomy. In conclusion, standing gasless laparoscopic cryptorchidectomy seems to be most suitable for bulls weighing from 100 to 180 kg.     

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province,South Africa

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved.     

Seasonal variations in and effect of incubation water temperature on vertebral number in naturally spawning chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>

Seasonal variations and reaction norms for vertebral number (V _N) in response to incubation water temperature were estimated in adult and juvenile naturally spawning chum salmon Oncorhynchus keta. The mean V _N of adults varied according to spawning time; the early-spawning population had higher V _N values than the late-spawning population. Moreover, the mean V _N values in the early-spawning population decreased with seasonal changes, whereas V _N values in the late-spawning population remained stable. Chum salmon embryos in three full-sib families were incubated at five different temperatures until hatching, and the V _N values of the resulting juveniles were analyzed. The V _N reaction norm to incubation water temperature showed a V-shaped curve that was lowest at an intermediate temperature. The mean V _N at the same incubation temperature varied among the three families. These results suggest that V _N values in chum salmon are influenced by genetic components and incubation water temperatures. V _N may be a useful parameter for estimating the environmental conditions during ontogenesis and the genetic background by detecting population changes.     

Analysis of the carbohydrates in an East African traditional beverage, togwa

In sub-Saharan Africa, togwa is a widely consumed beverage containing saccharified starch as a basis, being made from cereals and their malt flours. Togwa was prepared from maize flour and finger millet malt in a laboratory, and trace element and amino acid contents in the togwa were analyzed. The major constituents of togwa are carbohydrates, which are starch and starch hydrolysates that seem to be smoothly digested after ingestion. Carbohydrates in field and laboratory togwa were analyzed in detail by two different HPLC systems: normal phase chromatography and a gel permeation chromatography-multi angle laser light scattering system. Togwa was found to contain a very wide range of size distributions of molecules from glucose to starch that have molecular masses in the millions. It was concluded that togwa is an easily digestible and nutritious food.