Simultaneous estimation of the pH of rumen and reticulum fluids of cows using a radio-transmission pH-measurement system

Circadian pH changes in the fluid of the rumen (bottom and middle) and reticulum were assessed simultaneously using wireless and wired radio-transmission pH-measurement systems in cows fed a control diet (C diet) or rumen-acidosis-inducing diet (RAI diet). The pH in the three sites decreased following the morning and evening feedings. In cows fed the C diet, the bottom-rumen and reticular pH reverted to the basal level by the next morning, while the middle-rumen pH did not recover completely, suggesting that active fermentation occurred in the middle of the rumen. The mean pH at 1 hr intervals was higher in the reticulum than at the bottom and in the middle of the rumen. The relatively stable reticular pH may result from dilution due to salivation. In cows fed the RAI diet, the bottom-rumen pH fell to approximately 5.2 after the evening feeding, but returned to the basal level by the next morning. In contrast, the middle-rumen pH did not return to the basal level (6.5) within 24 hr, presumably owing to continuous, vigorous fermentation. There were positive correlations between the pH at the bottom and in the middle of the rumen and at the bottom of the rumen and in the reticulum. These findings indicate that our radio-transmission pH-measurement system may be suitable tool for simultaneous measurement of pH in the rumen and reticulum fluid.     

Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.     

Passive immunoneutralization of endogenous inhibin increases ovulation rate in miniature Shiba goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.     

Glucoprivation-induced Fos expression in the hypothalamus and medulla oblongata in female rats

Glucoprivation induced by 2-deoxy-D-glucose (2DG) suppresses pulsatile luteinizing hormone (LH) secretion in female rats. The suppression is enhanced in the presence of estrogen. In the present study, 2DG-induced Fos expression was examined in the solitary tract nucleus (NTS), hypothalamic paraventricular nucleus (PVN), raphe obscurus nucleus (ROb) and raphe pallidus nucleus (RPa), which have been previously suggested to be involved in glucoprivation-induced suppression of LH secretion in female rats. Ovariectomized (OVX) or estrogen-primed ovariectomized (OVX+E(2)) rats were injected intravenously with 2DG (400 mg/kg BW). The brain was removed 1 h after the injection. The number of Fos-like-immunoreactive (Fos-li) cells in the PVN and NTS was significantly increased in OVX+E(2) rats compared with control groups, but did not show a significant increase in the OVX group. Few Fos-li cells were observed in the ROb and RPa in all groups. All of the Fos-li cells in the PVN and NTS were neurons because they had immunoreactivities to microtubule-associated protein 2. Some Fos-li cells (8.3%) had tyrosine hydroxylase-like immunoreactivities in the NTS in 2DG-treated OVX+E(2) rats. These results suggest that neurons in the PVN and NTS are involved in the estrogen-dependent neural cascade mediating glucoprivic suppression of LH secretion in female rats.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.     

Paraventricular alpha1- and alpha2-adrenergic receptors mediate hindbrain lipoprivation-induced suppression of luteinizing hormone pulses in female rats

Acute central lipoprivation suppresses pulsatile luteinizing hormone (LH) release and increases blood glucose levels through noradrenergic input to the hypothalamic paraventricular nucleus (PVN) in female rats. The present study was conducted to identify adrenergic receptor subtypes involved in central lipoprivation-induced suppression of pulsatile LH secretion and increases in plasma glucose levels in female rats. Acute hindbrain lipoprivation was produced by injection into the fourth cerebroventricle (4V) of 2-mercaptoacetate (MA), an inhibitor of fatty acid oxidation, in estradiol-implanted ovariectomized rats. Two min before MA injection, alpha1-, alpha2- or beta-adrenergic receptor antagonist was injected into the PVN. Injection of MA into the 4V suppresses pulsatile LH release in PVN vehicle-treated rats, whereas pretreatment of animals with injection of alpha1- or alpha2-adrenergic antagonist into the PVN blocked the effect of the 4V MA injection on LH pulses. beta-Adrenergic antagonist did not affect MA-induced suppression of LH pulses. The counter-regulatory increase in plasma glucose levels after 4V MA injection was also partially blocked by pretreatment with alpha1- and alpha2-adrenergic receptor antagonists. These results suggest that alpha1- and alpha2-adrenergic receptors in the PVN mediate hindbrain lipoprivation-induced suppression of LH release and counter-regulatory increases in plasma glucose levels in female rats.     

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.     

Arterial blood gas anomaly in canine hepatobiliary disease

Arterial blood gas analysis is an important diagnostic and monitoring tool for respiratory abnormalities. In human medicine, lung complications often occur as a result of liver disease. Although pulmonary complications of liver disease have not been reported in dogs, we have frequently encountered hypoxemia in dogs with liver disorders, especially extrahepatic biliary obstruction. In addition, respiratory disorders account for 20% of perioperative fatalities in dogs. Therefore, in this study, we evaluated the respiratory status in dogs with hepatobiliary disease by arterial blood gas analysis. PaO₂ and PaCO₂ were measured. Alveolar-arterial oxygen difference (AaDO₂), the indicator of gas exchange efficiency, was calculated. Compared to healthy dogs (control group), hepatobiliary disease dogs had significantly lower PaO₂ and higher AaDO₂. Hypoxemia (PaO₂ of ≤80 mmHg) was observed in 28/71 dogs with hepatobiliary disease. AaDO₂ was higher (≥30 mmHg) than the control group range (11.6 to 26.4 mmHg) in 32/71 hepatobiliary disease dogs. By classifying type of hepatobiliary disease, dogs with extrahepatic biliary obstruction and chronic hepatitis showed significantly lower PaO₂ and higher AaDO₂ than in a control group. Dogs with chronic hepatitis also had significantly lower PaCO₂. The present study shows that dogs with hepatobiliary disease have respiratory abnormalities more than healthy dogs. Preanesthetic or routine arterial blood gas analysis is likely beneficial to detect the respiratory abnormalities in dogs with hepatobiliary disease, especially extrahepatic biliary obstruction and chronic hepatitis.     

Genetic diversity of plums characterized by random amplified polymorphic DNA (RAPD) analysis

We investigated the genetic diversity of 42 plum varieties by RAPD analysis. Twenty primers discriminated all plum varieties excepting two synonymous pairs: 'Botankyou and ‘Kelsey’, and ‘Chairn’ and ‘Tragedy’. Two North American plums, ‘Beach Plum’ and ‘Glow’, were genetically distinct from the other plums by cluster analysis. Overlaps observed between the ‘European plum group’ and the ‘Japanese plum group’, were perhaps due to intercrossing. We could also discriminate ‘Sordum’ from 'Late Sordum and ‘Bansei Sordum’, although ‘Late Sordum’ and ‘Bansei Sordum’ are thought to be derived from bud mutants of ‘Sordum’. This revised version was published online in July 2006 with corrections to the Cover Date.