The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3’ and 5’ primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.     

The first case of feline sinonasal aspergillosis due to Aspergillus fischeri in Japan

Feline upper respiratory tract infection due to Aspergillus spp. is considered an emerging disease, with the number of reported cases continuing to rise. In this study, we report the first case of feline sinonasal aspergillosis caused by Aspergillus fischeri in Japan. The patient presented after 2 months of progressive facial deformity around the nose and nasal discharge. The isolate from this case was susceptible to itraconazole (ITZ), voriconazole and micafungin, but was resistant to amphotericine B. However, the infected cat died approximately 1 month after referral, despite treatment for 12 days ITZ administered orally at 10 mg/kg.     

Diagnostic imaging of intra-abdominal cyst in heifer using the computed tomography

A 10-month-old Japanese black heifer was diagnosed as having an intra-abdominal cyst using computed tomography (CT). Through a posterior ventral midline incision, the cyst was removed, and the heifer completely recovered after the surgery. CT scans enabled detection of the intra-abdominal cyst and measurements of the diameter of the cyst before the surgery.     

Persistence of uterine bacterial infection,and its associations with endometritis and ovarian function in postpartum dairy cows

We investigated the relationship between the persistence of uterine bacterial infections with cytologically determined endometritis and ovarian function in 65 postpartum Holstein cows. Vaginal mucus discharges were collected, and endometrial smear samples (n = 130) were collected for cytological and bacteriological examinations from the cows at weeks 5 and 7 postpartum (pp). Blood samples were collected at weeks 3, 5 and 7 pp to determine plasma progesterone concentrations to monitor ovarian activity. According to the bacteriological examination, cows were classified into four groups. The first group (n = 32; 49%) comprised cows negative for bacteria at weeks 5 and 7 pp. The second group (n = 11; 17%) comprised cows with bacterial infections at week 5 pp but that were clear of infection at week 7 pp. The third group (n = 12; 19%) comprised cows without bacteria at week 5 pp but that acquired an infection by week 7 pp. The fourth group (n = 10; 15%) comprised cows with bacterial infections at weeks 5 and 7 pp (persistence of infection). A positive correlation (P < 0.001) was noted between the severity of cytologically determined endometritis, purulent vaginal discharge and the persistence of infection. Cows with persistent infections had a significantly (P < 0.01) prolonged luteal phase compared with cows without infection. In conclusion, the prevalence of cytologically determined endometritis and prolonged luteal phase were significantly increased in cows with persistent infections.     

Production of Ban miniature pig embryos by in vitro fertilization: A comparative study with Landrace

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10⁶/mL) was lower (P < 0.01) compared with Landrace (4160 ± 42 × 10⁶/mL). However, sperm characteristics before and after freezing in Ban and Landrace were similar. The numbers of follicles with diameter larger than 2 mm per ovary in Ban females treated with equine chorionic gonadotropin and human chorionic gonadotropin (27.1 ± 1.3) were higher (P < 0.05) than those in Landrace (12.9 ± 2.0) and in non‐hormone stimulated Ban (no > 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone‐stimulated Ban and Landrace. The percentages of two‐cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non‐stimulated Ban (4.0 ± 3.8%) was lower (P < 0.05) than that in Landrace (15.3 ± 1.8%). In conclusion, an effective IVP system for good quality embryos in Ban, that is essential for genetic conservation of this breed, was established.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.     

Immobilization of Japanese black bears (Ursus thibetanus japonicus) with tiletamine hydrochloride and zolazepam hydrochloride

The effect of anesthetizing with a 1:1 combination of tiletamine hydrochloride and zolazepam hydrochloride (TZ) was evaluated in 75 Japanese black bears. TZ was administered to 43 captive and 11 wild, 8 captives and 13 hibernating captive bears at the doses of approximately 9.0 mg/kg (usual dosage), 18.0 mg/kg (high dosage) and 5.0 mg/kg (low dosage), respectively. Sufficient anesthetic effects were achieved in all bears, and rectal temperatures, heart rates and respiratory rates did not change significantly during an hour handling. Complete blood cell examinations showed no abnormal data. A combination of TZ would be an efficient and safe drug for chemical immobilization of Japanese black bears.