Postnatal development of hypoplastic thymus in semi-lethal dwarf pet/pet males

The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.     

Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.     

Blood biochemical values in Japanese Black breeding cows in Kagoshima Prefecture,Japan

To obtain blood biochemical basic data of Japanese Black breeding cows in Kagoshima Prefecture, Japan, blood samples were obtained from 857 clinically healthy multiparous cows on 71 farms. Cows were divided into three stages: the prepartum stage (between 20 and 80 days before parturition, n=290), lactation stage (between 20 and 80 days after parturition, n=283) and maintenance stage (between 120 and 200 days after parturition, n=284). The mean concentration of total protein and total cholesterol, and the mean activities of aspartate aminotransferase in the lactation stage tended to be higher than those in the prepartum and maintenance stages. The mean concentration of glucose in the prepartum stage tended to be lower than that in the lactation and the maintenance stages. The mean concentration of nonesterified fatty acids and beta-hydroxybutyrate in the prepartum stage tended to be higher than those in the lactation and maintenance stages. The mean concentration of serum retinol was approximately 30 µg/dl in all stages. These results suggest that the blood biochemical values of multiparous Japanese Black cows vary with breeding stages, and it is considered that the blood parameters obtained in this study are useful as indices for health management of Japanese Black breeding cows.     

Initial vegetation recovery following a blowdown of a conifer plantation in monsoonal East Asia: Impacts of legacy retention, salvaging, site preparation, and weeding

All or a part of a sequence of forest practices (i.e., salvage logging, site preparation, planting crop trees, and weeding) has been implemented after natural disturbances for the rapid re-establishment of tree cover. Forest policies in Japan have recently changed from monocultural planting of coniferous crop trees to planting native broadleaved trees to restore forests and nurture local biodiversity following large windthrows. However, the effects of this new practice on preserving biodiversity, as well as the effects of legacy retention, have never been verified in Asia. Thus, the objective of our research was to compare the effects of legacy retention with plantation after salvaging on the initial stage of vegetation recovery in a blowdown area, specifically focusing on plant species diversity, the occurrence of alien species, and the composition of plant species. Following the analysis of our results, we finally describe appropriate practices to alter disturbed coniferous plantations to bring the species composition closer to that of the original natural mixed forests.A control (A, legacy retention) and three experimental treatment sites (B, salvage logged, site prepared, and Quercus crispula seedlings planted; C, same as B, but weeded once during the summer; and D, residual rows that emerged after establishing sites for planting) were prepared, and quadrats were set. Eleven indicators of the ground condition and the number of vascular plant species, including ferns, were quantified, and the number and abundance of residual and newly colonized plants of the main woody species were estimated.Our main findings were as follows: (1) in unsalvaged sites and residual rows, the diversity of plant species was poor, but a variety of plant species compositions were observed due to the heterogeneous conditions of the ground and ample residual plants; (2) in the planting site, many species appeared, but little variety of the species composition was observed due to the homogeneous condition of the ground and the destruction of residual plants; (3) a large number of alien species emerged in broad, unvegetated areas; (4) the impact of site preparation overwhelmed the impact of salvage logging on the initial recovery of plant species; and (5) to restore a natural mixed forest, a combination of legacy retention and plantation after salvaging would be the most appropriate.     

In vitro and in vivo effects of recombinant bovine interferon-tau on bovine leukemia virus

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.     

Arrhythmogenic right ventricular cardiomyopathy coincided with the cardiac fibrosis in the inner muscle layer of the left ventricular wall in a boxer dog

A 7-year-old female boxer dog died suddenly without any clinical signs. It was suspected that the dog had arrhythmogenic right ventricular cardiomyopathy (ARVC) due to ventricular premature complexes and ventricular tachycardia at 3 years of age. The final diagnosis of ARVC was confirmed by histological characteristics, such as loss of cardiocytes and fibrofatty replacement, occurring in the right and left ventricular walls. In the cardiocytes, non-lipid vacuoles were observed. Cardiac fibrosis and intimal thickening of the small arteries occurred without fatty replacement in the inner muscle layer including the papillary muscles of the left ventricular wall. This paper describes the pathomorphological details of an ARVC case with coincidental cardiac fibrosis in the inner muscle layer of the left ventricular wall.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

Intestinal Tritrichomonas suis (=T. foetus) infection in Japanese cats

Tritrichomonas suis (=T. foetus) has recently been reported to be a causative agent of chronic large-bowel diarrhea in cats. While the disease was previously attributed to Pentatrichomonas hominis, the etiologic agent for feline trichomonal diarrhea was identified as T. suis. Although feline trichomonosis due to T. suis has been reported at prevalences ranging from 14 to 31% in Europe and the U.S., no reports of the pathogen have been published to date in Japan. In 2008, however, we encountered a case of feline trichomonosis at the Veterinary Teaching Hospital of Hokkaido University. The parasite was identified as T. suis by nested PCR amplification of partial internal transcribed spacer region 1 and 5.8S ribosomal RNA gene sequences with T. suis-specific primers and DNA sequencing of the amplified products. We then conducted surveys for feline trichomonosis in three different animal hospitals using either cultivation and/or PCR-based assays. The results revealed that 13 of 147 samples (8.8%) were positive for T. suis, and that 5 of the 13 infected cats, which ranged between 1 month and 7.5 years-old, showed chronic diarrhea. Seven of the infected cats were purebred and 6 were mixed breed. These findings suggested that feline trichomonosis is prevalent in Japan, and that T. suis may play a role as a causative agent of feline chronic diarrhea.     

Isolation of Araguspongine M,a New Stereoisomer of an Araguspongine/Xestospongin alkaloid,and Dopamine from the Marine Sponge Neopetrosia exigua Collected in Palau

A new stereoisomer of an araguspongine/xestospongin alkaloid, named araguspongine M (1), has been isolated together with 12 known compounds, araguspongines B (2) and D (3), dopamine, three galactosyl diacylglycerols, 24-methyl cholesterol, 5,6-dihydrocholesterol, β-sitosterol, and three 5α,8α-epidioxy sterols (11–13), from the marine sponge Neopetrosia exigua (formerly Xestospongia exigua) collected in Palau. The structure of 1 was assigned on the basis of its spectral data analysis. This is the first report on the isolation of dopamine from a marine sponge. This compound may be produced by an endosymbiotic Synechococcus-like cyanobacterium. Compounds 1–3 and 11–13 showed cytotoxicity against HL-60 at IC₅₀’s of 5.5, 5.5, 5.9, 22.4, 9.5, and 9.6 μM, respectively. The possible biosynthesis origin of the isolated metabolites is discussed.     

Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.