Protease-induced hyperactivation of canine spermatozoa associated with disappearance of lectin-binding glycoproteins on their surface

The relationship between the disappearance of glycoproteins from the surface of canine sperm and sperm capacitation was investigated in vitro. The protease (PR) concentration in flush fluids of the uterine horns and oviducts removed from 6 estrous, 5 diestrous, and 5 anestrous bitches was measured with a protease assay kit. Ejaculated sperm collected from 10 dogs were incubated for 4 hr in Eagle's MEM supplemented with 1 or 5 microg/ml PR, or to which no PR had been added (control). The glycoproteins on the surface of the sperm were stained with 4 different FITC-lectins (Con A, PHA-E, PNA, and WGA), and the percentages of hyperactivated (HA-) sperm and acrosome-reacted (AR-) sperm were evaluated. The mean PR concentration (5.95 microg/ml) in the flush fluid from the oviducts of the estrous bitches was significantly higher than in the fluid from their uterine horns (1.00 microg/ml; P<0.01). The PR concentrations of the flush fluids from the uterine horns and oviducts of both the diestrous and anestrous bitches were less than 0.05 microg/ml. Before incubation the acrosomal regions or entire heads of all sperm clearly stained with each FITC-lectin, but the percentages of sperm binding the 4 FITC-lectins decreased after incubation. The percentages of lectin-binding sperm in the MEM containing 5 microg/ml PR were significantly lower than in the control MEM (P<0.05 and 0.01). The mean percentages of motile sperm and HA-sperm after incubation in the MEM with PR were higher than in the control MEM, but there were no differences in the percentages of AR-sperm. The results indicate that HA-movement of sperm is induced by the disappearance of glycoproteins from the surface of canine sperm as a result of the action of PR in the oviductal fluid of estrous bitches.     

Aggregability and post-transfusion survival of canine platelets in stored whole blood

The effects of whole blood storage time on platelet aggregation and on post-transfusion platelet survival time were assessed in dogs. Citrate phosphate dextrose adenine-1 (CPDA-1) was used as a blood cell preservative. Storage time dependent decay of platelet aggregability was assessed. Platelet aggregation responses to collagen and ADP were maintained for at least 8 hr at room temperature. During blood storage, immunoglobulin became nonspecifically bound to platelets, suggesting the potential for immune destruction of platelets by the mononuclear phagocyte system after transfusion. To assess this assumption, the survival times of infused platelets, which were stored for 0 to 8 hr in whole blood, were measured. Post-transfusion survival of platelets was not affected by these storage times. These results suggest that canine platelets maintain viability when stored at room temperature for up to 8 hr in CPDA-1 treated whole blood intended for transfusion.     

Contraceptive effect of proligestone on spotted seals and crossbreeds of spotted seals and harbor seals

Spotted seals (Phoca largha) and related crossbreeds maintained at Kamogawa Sea World breed seasonally from the end of January to middle of March. For contraception in these animals, the effect of a single administration of a contraceptive synthetic luteal hormone for dogs, proligestone (PRG), was investigated. The animals tested were 10 seals aged 4-24 years old, and a total of 35 trials were performed over five years. PRG was administered in 23 trials during January, which was one month before the estimated estrus, and in 12 trials during December of the previous year, which was two months before the estimated estrus. The dose of PRG was 5 mg/kg in 32 trials and 10 mg/kg in 3 trials. The effect of the contraception was judged by the presence or absence of delivery. Among 23 animals treated in January, 2 animals treated with 5 mg/kg PRG became pregnant, but the contraception was successful in the other 21 animals. Contraception was successful in all 12 trials treated with 5 mg/kg PRG in December. Overall, contraception was successful in 94.3% (33/35). Therefore, a single administration of 5 mg/kg PRG in December may be an effective method of contraception for seals.     

Therapeutic effect of aromatase inhibitor in two azoospermic dogs with high plasma estradiol-17beta levels

Two azoospermic dogs with high plasma estradiol-17 beta (E(2)) levels were subcutaneously injected with an aromatase inhibitor (AI), 4-androstene-4-ol-3,17-dione, 2 mg every other day for 4 weeks. Before the AI treatment the plasma E(2) levels of the two dogs (21 and 22 pg/ml, respectively) were higher than those of 2 normal dogs (8.1 and 12.3 pg/ml), and they fell to 11-17 pg/ml between 1 and 4 weeks after the start of AI treatment. The plasma testosterone levels after the start of AI treatment had increased to 2.1-3.1 ng/ml. A small number of sperm were detected in the semen of the two dogs between 3 and 6 weeks after the start of AI treatment. These results indicate that the testicular function of infertile dogs with high plasma E(2) levels can be temporarily improved by AI therapy.     

Plasma concentration of estrone sulfate during pregnancy in different breeds of Japanese beef cattle

Plasma concentrations of estrone sulfate in different breeds of Japanese beef cattle and the relationship between those concentrations and feto-placental growth were examined in order to assess the possibility of monitoring abnormal growth of the fetus. Blood samples were obtained from cows from day 90 of gestation to parturition. The plasma concentration of estrone sulfate was measured by direct enzyme immunoassay. From day 180 of gestation, the mean concentration of estrone sulfate increased gradually and it was drastically elevated after day 240 of gestation with the maximum at day 285. Plasma concentrations of estrone sulfate on day 240 of gestation was significantly increased in F(1) cows (Holstein Friesian and Japanese Black) compared with those in other breeds of cow. From day 270 to 278 of gestation, estrone sulfate concentrations of Holstein Friesian cows inseminated by Holstein Friesian differed from those inseminated by Japanese Black. In the cow with retained placenta, the plasma concentration of estrone sulfate reached plateau at day 240 of gestation and did not increase thereafter. There was no significant relationship between estrone sulfate concentration and duration of gestation, calf birth weight, weight of placenta or viability of newborn calves. These results indicate that changes of plasma estrone sulfate concentration in Japanese beef cattle are very similar to those in Holstein dairy cattle. They also suggest that the plasma concentration of estrone sulfate is associated with the breed of pregnant cow and that its concentration is also affected by calf birth weight depending on the breed of bull. It seems possible to predict the incidence of retained placenta but not the calf birth weight and viability of newborn calves in Japanese beef cattle.     

Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin sample from a dog in Japan

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism.     

Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice

Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.     

Sequence comparison of the dopamine receptor D4 exon III repetitive region in several species of the order Carnivora

It was previously demonstrated that the dog dopamine receptor D4 (DRD4) gene is polymorphic in terms of the repeat number and/or order of 39- and 12-bp sequences located in the third exon. To examine whether or not the repetitive region is present in other species of the order Carnivora, the homologous region of DRD4 genes were sequenced in the gray wolf, raccoon dog, Asiatic black bear, common raccoon and domestic cat. In the family Canidae, the wolf had an identical sequence to that of the dog 447b allele, and a repetitive sequence similar to the dog DRD4 was also recognized in the raccoon dog. On the other hand, no obvious repeated structure was observed in the sequences of the bear, raccoon and cat.     

Influence of canine cumulus oophorus on homologous sperm motility

Sperm ejaculated by 8 beagle dogs and the cumuli oophori collected from 3 estrous beagle bitches were co-incubated, and penetration of the sperm into cumuli was observed to investigate the influence of cumuli on homologous sperm.The percentages of hyperactivated sperm and acrosome-reacted sperm were calculated after incubation with homogenized cumuli. The hyaluronic acid content of the incubated cumuli was measured, and hyperactivation and the acrosome reaction of the sperm were evaluated in medium containing hyaluronic acid. The mean percentage of hyperactivated sperm (33.0%) and number (3.0) of sperm that had penetrated a cumulus among sperm incubated for 7 hr were significantly higher than the values for sperm incubated for 0.5 hr (P<0.01). Almost all sperm that had penetrated the cumuli had intact acrosome, as though they were hyperactivated. The percentages of motile sperm (77.3%) and hyperactivated sperm (23.6%) after 2 hr incubation in the medium containing homogenized cumuli were significantly higher than in control medium (P<0.01), but there was no difference between cumulus and control media in the percentages of acrosome-reacted sperm. The hyaluronic acid content of a cumulus increased after 24 hr incubation. After 2 and 4 hr of incubation the percentages of hyperactivated sperm in the medium containing hyaluronic acid were significantly higher than in the control medium (P<0.01). These results suggest that canine hyperactivated sperm with intact acrosome can penetrate homologous cumuli and that the sperm are able to pass through the cumulus because the hyperactivated movement is maintained by hyaluronic acid secreted by the cumulus cells.