Production of recombinant goldfish gonadotropins by baculovirus in silkworm larvae

Recombinant gonadotropins (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm larvae. Hemolymph containing recombinant FSH (rFSH) or LH (rLH) was collected from silkworm larvae, and its biological activity was examined in vivo using male goldfish and female bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish, whereas only rLH induced ovulation in female bitterling. These results suggest, that biologically active goldfish recombinant GTH could be applied for the induction of gonadal development in aquaculture fishes as a substitute of pituitary extract, if a method of large scale production is established.     

Transgenic Nile tilapia (Oreochromis niloticus) over-expressing growth hormone show reduced ammonia excretion

The growth of aquaculture has negatively affected the environment due to the high levels of nitrogen excreted by farmed fish. Here we propose that modifying the nitrogen metabolism of the fish themselves using transgenic technology might solve the pollution problem. Growth hormone (GH) is known to increase protein retention and absorption, and is thought to reduce ammonia excretion. Thus, we produced transgenic Nile tilapia (Oreochromis niloticus) that over-expressed the GH gene throughout their bodies. Our findings showed that the food-conversion efficiency of the transgenic fish was 35% higher than that of their non-transgenic siblings. The rearing period required for the transgenic fish to reach a body weight of 20 g was about 75% of that required for non-transgenic fish that were fed the same type and quantity of food. The total amount of ammonium-nitrogen excreted by the transgenic fish was about 69% of that excreted by the wild-type fish over their lifetime. These results suggest that our transgenic approach has the potential to reduce the amount of nitrogen pollution caused by farmed fish. This strategy is a promising option for making aquaculture more ‘eco-friendly’.     

Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain

A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of isolate Tokachi09 showed identical size (1341 and 4092 nucleotides, 446 and 1363 amino acids, respectively) and high similarity (98.0% and 99.0% at the nucleotides, 97.3% and 99.0% at the amino acids, respectively) to those of strain NC99. However, the isolate had a 185-nucleotide deletion from four bases after the 3'-terminal end of the spike gene, resulting in the absence of the open reading frame predicted to encode a 4.7-kDa nonstructural protein in strain NC99. These results suggest that the 4.7-kDa nonstructural protein is not essential for viral replication, at least in cell culture, and that the Japanese strain probably originated from a different lineage to the North American strain. This is the first equine coronavirus to be isolated from adult horses with pyrogenic and enteric disease.     

Degree of polymerization of inulin-type fructans differentially affects number of lactic acid bacteria, intestinal immune functions, and immunoglobulin A secretion in the rat cecum

This study examined the role of degree of polymerization (DP) of inulin-fructans in modulating the interaction between lactic acid bacteria and IgA cecal secretion. Rats were fed a control diet or a diet containing one of the fructans with different DP. Consuming fructans increased the cecal IgA concentrations in the order DP4 > DP8 > DP16. Cecal lactobacilli counts were higher in DP4, DP8, and DP16, whereas bifidobacteria were higher in DP8, DP16, and DP23. Cecal IgA concentrations were correlated with cecal lactobacilli counts (P < 0.01). DP4, DP8, and DP16, but not DP23, significantly increased IgA-producing plasma cells in the cecal mucosa. IFN-γ and IL-10 production in the cecal CD4(+) T cells was enhanced solely in DP4. The results show that fructans with lower DP enhance cecal IgA secretion and increase the plasma cells and suggest that the increased lactobacilli may contribute to the stimulation of cecal IgA secretion.     

The Dnmt3b splice variant is specifically expressed in in vitro-manipulated blastocysts and their derivative ES cells

Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos.     

Cerebral vascular hamartoma with thrombosis in a dog

A cerebral vascular hamartoma was identified in the frontal lobe, striatum and thalamus of the right side of the brain of a male, 7-year-old Shih Tzu. Histologically, the lesion consisted of thin-walled vessels, which showed various sizes and occasionally contained fibrin thrombi. These vascular walls were composed of a single layer of fibromuscular tissue lined by flat endothelium with various amount of collagen, but devoid of large coat of smooth muscles and elastic tissue. Immunohistochemically, the lining endothelial cells were positive for von Willebrand Factor antibody. Neuropil between the vessels was stained with Klüver-Barrera stain, and positive for synaptophysin and GFAP antibodies. Based on these findings, the lesion was diagnosed as vascular hamartoma, which might resemble venous malformation in humans.     

Formation of an Antral Follicle–like Structure of Bovine Cumulus–Oocyte Complexes Embedded Individually or in Groups in Collagen Gels

Culture techniques of antral follicle–like structure (AFLS) derived from cumulus–oocyte complexes (COCs) might provide important insights into follicular development and oocyte maturation. This study was undertaken to investigate the effects of embedding bovine COCs individually (one COC) or in groups (4–5 COCs) in collagen gels on the formation of AFLS and the meiotic status of oocytes. The observations of AFLS formation were performed every second day for 14 days. The AFLS was formed at Day 2 or 4 after the start of culture (Day = 0), irrespective of the culture methods. The mean diameters of AFLS during Days 4–14 using the individual culture method were significantly higher (p < 0.05) than those using the group culture method. However, the AFLS formation rate in the individual culture method was significantly lower compared to that in the group culture method (26.1% vs 62.7%, p < 0.01). Almost all oocytes had undergone the germinal vesicle breakdown stage, irrespective of the culture method or AFLS formation. In conclusion, comparison with the individual culture method revealed that the mean diameters of AFLS in the group culture method were smaller, but more COCs formed AFLS. The group culture method might be useful for evaluating the various hypotheses of follicular formation and interfollicular communication. However, improvement of the group culture system is necessary to prevent the meiotic resumption of oocytes, because the AFLS formation is dependent on the cumulus/granulosa cells surrounding oocytes.