Different prostaglandin F2α secretion in response to oxytocin injection between pregnant and non‐pregnant cows: effect of the day of oxytocin challenge test for determining the difference

The present study was conducted to determine the difference in plasma prostaglandin F₂α metabolite concentrations following oxytocin (OT) challenge between pregnant and non‐pregnant cows. Experiment 1: cows were subjected to the OT challenge test on days 12, 14 or 16 (day of estrus = day 0) with or without prior insemination and plasma 13,14‐dihydro‐15‐keto prostaglandin F₂α (PGFM) concentrations were measured from ?30 to 180 min after OT injection. On day 16, the increment of plasma PGFM concentrations in response to OT injection was significantly smaller in pregnant than that in cyclic cows. On days 12 and 14, there was little OT‐induced PGFM secretion and no difference in PGFM increase between the pregnant and cyclic cows. Experiment 2: cows were inseminated on day 0 and subjected to the OT challenge test on day 16. Cows were classified into non‐pregnant/early embryonic death (NP/EED), late embryonic death (LED) and pregnant (PREG) groups. The increment of PGFM concentrations in response to OT injection was less in both PREG and LED groups than that in the NP/EED group. In conclusion, plasma PGFM secretion induced by OT is suggested as the base of pregnancy diagnosis prior to returning estrus in cows.     

Molecular screening of canine GM1 gangliosidosis using blood smear specimens after prolonged storage: detection of carriers among shiba dogs in northern Japan

Molecular screening of GM1 gangliosidosis in Shiba dogs was carried out in northern Japan using blood smear specimens after prolonged storage. Of 125 specimens obtained from 3 veterinary teaching hospitals for this screening, 68 specimens (54%) were adequate for direct amplification in a polymerase chain reaction (PCR)-based DNA test, and the percentage of adequacy was different at each hospital (34%, 73%, and 100%), suggesting that the amount of blood on the smear and the storage condition of specimens may affect adequacy. Of the 68 dogs examined, 2 dogs (2.9%) were heterozygous carriers for this disease and the other dogs were all genotypically normal. The results suggest blood smear specimens can be useful for PCR testing after prolonged storage provided specimens contain a generous amount of blood and have been adequately stored. The study also suggests that GM1 gangliosidosis may be widely prevalent in the Shiba dog population in northern Japan.     

A new method of measuring the transepidermal water loss (TEWL) of dog skin

Human skin barrier function is evaluated by measuring transepidermal water loss (TEWL). However, this conventional method has not been applied to assess canine skin barrier function because the equipment is not suitable for dogs due to the effects of air turbulence resulting from movement of the subject and vapor from the subject's hair coat. The TEWL analyzer CC-01 was developed as a closed-chamber method device; this means that instead of using the open-chamber method, it has a ventilated chamber that uses dry air. TEWL values measured by CC-01 show less variability than those measured by the conventional method. An ambient temperature of 20-26 degrees C is optimal for measurement with the CC-01, and humidity affects the length of measurement but not the values. The CC-01 may be more reliable for measurement of TEWL than the conventional methods and may give new insights in the evaluation of skin barrier function in dogs.     

Anti-anxiety effect of ovary lipid extracted from Skipjack tuna (Katsuwonus pelamis) in rats

Using an elevated plus-maze test, we evaluated anxiety level in rats given ovary lipid extracted from Skipjack tuna (Katsuwonus pelamis; OLS). The percentage of open time was significantly higher in rats given OLS than in rats in the control group, but lower than in rats given diazepam (1.0 mg/kg body weight). Based on this fact and findings about other indicators, this study showed that OLS does not have as fast-acting and strong an anti-anxiety effect as diazepam but that continuous ingestion of OLS causes an anti-anxiety effect in animals.     

Evaluation of serum amyloid A and surfactant protein D in sera for identification of the clinical condition of horses with bacterial pneumonia

In the present study, the concentrations of serum amyloid A and surfactant protein D in sera were measured to evaluate them for identification of the clinical condition of horses with bacterial pneumonia. The study utilized 185 clinically healthy control thoroughbreds and 9 thoroughbreds for experimental infectious study with S. zooepidemicus. Blood samples were collected from the 185 healthy control thoroughbreds. The 9 thoroughbreds were experimentally infected S. zooepidemicus using an endoscopic injection to a lung lobe and were then observed of clinical conditions. Blood samples were collected before inoculation and on the 1-15th, 22nd, and 29th days after inoculation (follow-up group). The levels of SAA and SP-D in the healthy control thoroughbreds were very low. In the follow-up group, the levels of SAA and SP-D changed in parallel with the horses' clinical condition. The pyrexia observed after bacterial inoculation faded by the 11th day, and the changes in SAA and SP-D occurred simultaneous to disappearance of the clinical signs. Measurement of SAA and SP-D proved useful for monitoring the clinical condition of the horses with bacterial pneumonia. Changes in the SP-D value were preceded by changes in the SAA value. Since the changes in SP-D occurred approximately simultaneous to the changes in the horses' clinical signs, we believe that they reflect the condition of the alveolar membranes. We conclude that measurement of SAA and SP-D in sera is useful for identification of the clinical condition of horses with bacterial pneumonia.     

Development of a programmable piggyback syringe pump and four-times-a-day injection regimen for superovulation in non-lactating Holstein cows

The objectives of the present study were to develop a programmable piggyback syringe pump for bovine superovulation and to evaluate the effects of a four-times-a-day injection regimen using the pump. Non-lactating Holstein cows were treated with a total of 30 armour units of porcine FSH by injection four times a day with the pump (study, n = 9) or injection twice a day manually (control, n = 9) for four consecutive days from D10 of the estrous cycle. The pump-driven program successfully induced superovulation in all cows tested. The numbers of small (3– < 5 mm in diameter) and large (≥ 10 mm in diameter) follicles were greater in the study group on D11-13 and D14, respectively. There were fewer unovulated follicles detected on D21 (7 days after estrus) in the study group than in the control group (1.2 ± 0.4 and 3.2 ± 0.6, respectively).     

Development of cleaved amplified polymorphic sequence (CAPS)-based markers for identification of sweetpotato cultivars

To develop a simple and reliable method to identify sweetpotato cultivars, we designed cleaved amplified polymorphic sequence (CAPS)-based markers and used them to perform genotyping of Japanese sweetpotato cultivars. In order to screen the CAPS-based markers, 13 primer pairs were designed from the exon sequences of 11 sweetpotato genes to amplify fragments containing an intron. By digesting the amplified products with 8 restriction enzymes having different recognition sites, a total of 27 polymorphic marker fragments were obtained. Genotyping of 60 Japanese sweetpotato cultivars using these markers suggested that the markers can effectively distinguish sweetpotato cultivars. Among the genes used for primer design, the gene encoding the dihydroflavonol 4-reductase (DFR) showed the largest degree of polymorphism. To our knowledge, this is the first report on the development of CAPS-based markers in sweetpotato.     

Sequence homogeneity of the ψserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regions of ‘Candidatus Liberibacter asiaticus’ isolates around Okinawa Main Island in Japan

In Japan and Southeast Asia, ‘Candidatus Liberibacter asiaticus’ (Las) is the dominant causal agent of citrus greening (huanglongbing) disease. Using PCR techniques, we determined the 11168-nucleotide sequence of the ψserA-trmU-tufB-secE-nusG-rplKAJL-rpoB gene cluster and the flanking regions for 51 Japanese, four Taiwanese, four Indonesian, and three Vietnamese isolates of Las. The sequence is identical in 62 isolates collected from Japan, Taiwan, Indonesia, and Vietnam, except for nucleotide substitutions at 11 positions. Some Las isolates from Sakishima Islands near Taiwan had unique nucleotide mutations, but all Las isolates around Okinawa Main Island were homologous. On the basis of the pattern of single nucleotide polymorphisms (SNPs) of the 11 nucleotide substitutions, the 62 Las isolates from Japan, Taiwan, Indonesia, and Vietnam could be divided into 12 pattern groups, and the 51 Japanese isolates consisted of six patterns. The results suggested that one unique genetic group is dominant around Okinawa Main Island, whereas several different are commonly distributed around islands near Taiwan.     

Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.