Eiichi Kawakami

Effects of canine oviduct epithelial cells on movement and capacitation of homologous spermatozoa in vitro

In this study, the interaction between canine sperms and oviduct epithelial cells (OECs) was examined in vitro. The oviducts of eight bitches in the follicular (F-) phase and six bitches in the luteal (L-) phase were removed under halothane inhalation anesthesia. The entire oviduct was opened longitudinally, and the oviductal epithelium of bitches in the F- and L-phases was scraped with a scalpel into tissue culture medium (Eagles MEM) containing 10% estrous bitch serum and 10% diestrous bitch serum, respectively. The OEC collected were preincubated for 24h and then coincubated with ejaculated canine sperms at 38 degrees C under an atmosphere of 5% CO2 in air. The percentages of sperms exhibiting active tail movement (% TM), hyperactivated sperms (% HA), and acrosome-reacted sperms (% AR) were investigated until 72 h after the start of coincubation. The percentage of sperms labeled with fluoresceinated Ca indicator (% Ca) was evaluated to assess the influx of Ca into sperms cytoplasm during capacitation. Canine sperms attached to both ciliated OEC and non-ciliated OEC. All of the mean % TM of the OEC-binding sperms in the F-OEC and L-OEC media after 24, 48, and 72 h of coincubation were significantly higher than the values of the freely swimming sperms (P< or =0.01). Conversely, the mean % AR and % Ca of the OEC-binding sperms were significantly lower (P<0.01). All of the mean % HA and % AR of the freely swimming sperms in the F-OEC medium after 24, 48, and 72 h of coincubation were significantly higher than the values of the sperms in the L-OEC medium (P< or =0.01). These results indicate that attachment of canine sperms to the OEC prolongs their viability and motility arid inhibits Ca influx into the sperms and sperm capacitation. These phenomena may be responsible for maintaining the active movement and the fertile life of canine sperms in homologous oviducts.

Prolonged Duration of Fertility of Dog Ova

The fertile period for natural mating in dogs extends from before ovulation until day 5 post ovulation (PO) and involves a delay in oocyte maturation until 2-3 days PO and viability of secondary oocytes for 48-60 h or more. Spermatozoa do not enter the uterus after vaginal insemination in late oestrus. Cervical closure appears to occur on average 5 days PO, but conception may occur following intrauterine artificial insemination (IUAI) up to 8 days PO. Therefore, the present study was conducted to clarify the duration of fertility of canine ova. Using IUAI at 6, 7, 8 and 9 days PO (n = 5 bitches each) conception rates were 100%, 71.4%, 37.5% and 0%, respectively, with an average litter resorption rate of 30.8%, and with mean litter sizes and times to delivery PO being 4.3 +/- 1.6 and 64.3 +/- 0.3 days, 4.0 +/- 1.4 and 66.3 +/- 0.4 days, and 2.5 and 68 days for IUAI at 6, 7 and 8 days, respectively. The high pregnancy rates with IUAI at 6 and 7 days PO confirm that many canine oocytes are fertile at 4-5 days after maturation. The high rate of resorption was presumably because of aging of ova or asynchrony between embryonic development and the intrauterine environment.

Effects of medium containing heparin and theophylline on capacitation and metabolic enzyme activities of ejaculated spermatozoa from dogs with asthenozoospermia

The percentages of motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR) of four beagle dogs with asthenozoospermia (AS) and five normal beagle dogs were determined during 7 h of incubation. The metabolic enzyme activities of the sperm was examined after 0 and 4 h of incubation. The sperm were incubated in canine capacitation medium (CCM) and CCM containing either 20 microg ml(-1) heparin (HE), 10 microg ml(-1) theophylline (TH) or 20 microg ml(-1) HE + 10 microg ml(-1) TH in glass tubes at 38 degrees C under 5% CO2 in air. The %HA and %AR were determined by counting the sperm exhibiting star-spin like movement and by the triple stain technique. The spermatozoa in HE + TH CCM were homogenized and centrifuged, and the metabolic activities of hexokinase, fructokinase, glucose-6-phosphodehydrogenase (G6PD), and pyruvate kinase in the sperm cytosol in the supernatant was measured with a spectrophotometer. The mean %MO and %HA values of both AS and normal dogs in the four types of CCM were highest in HE + TH CCM, with a mean %HA in HE + TH CCM of 78 +/- 5% (S.E.) after 7 h of incubation. However, there was little difference in %AR among the four types of CCM. The mean activities of the four enzymes in the sperm of AS dogs before incubation was significantly lower than in the sperm of normal dogs (P < 0.05, 0.01). However, after 4 h of incubation the activities of all enzymes in the sperm of both AS and normal dogs was clearly higher in HE + TH CCM than in the control CCM. These findings indicate that HE and TH in the medium are effective inactivating metabolic enzymes, maintaining longer sperm motility, and efficiently inducing HA even of the sperm of AS dogs.

Changes in plasma luteinizing hormone, testosterone and estradiol-17β levels and semen quality after injections of gonadotropin releasing hormone agonist and human chorionic gonadotropin in three dogs with oligozoospermia and two dogs with azoospermia

Plasma luteinizing hormone (LH) and testosterone (T) levels in three normal male Beagles increased markedly, the LH levels peaking at 30 or 45 min and the T levels at 45 or 60 min respectively, after a subcutaneous injection of 1 microgram kg-1 gonadotropin releasing hormone agonist (GnRH-A). Two Collies and a Great dane diagnosed as oligozoospermic and two Shetland sheep dogs diagnosed as azoospermic by evaluation of semen quality were treated with 1 microgram kg-1 GnRH-A after blood collection. Their plasma levels of LH, T and estradiol-17 beta (E2) were measured by radioimmunoassay for the purpose of investigating the effect of hormone therapy on spermatogenic dysfunction and the mechanism on improvement of semen quality. The semen quality of one of the Collies had improved 4 weeks after the GnRH-A treatment. The dog was treated with GnRH-A again and mated with a bitch 4 days later. The bitch gave birth to five puppies. The other dogs, whose semen quality had not improved, were treated with an intramuscular injection of 500 or 1000 IU human chorionic gonadotrphin (hCG) per animal. Since the semen quality of the other Collie and the Great Dane improved temporarily 2 and 4 weeks, respectively, after hCG treatment, the former was mated with a bitch 5 days later. The bitch gave birth to a litter of seven puppies. These hormone treatments, however, had no effect on the azoospermia in the two Shetland sheep dogs. Although the mean plasma LH and T levels in the dogs with oligozoospermia had been low, their LH levels gradually increased after hormone treatment. There were no marked changes in plasma T or E2 levels. These findings indicate that the semen quality of dogs with oligozoospermia can be temporarily improved between 2 and 4 weeks after a single injection of GnRH-A or hCG and the fertility of the dogs restored by the injection.

Effect of Catalase and Superoxide Dismutase on Motility, Viability and Acrosomal Integrity of Frozen-Thawed Cat Spermatozoa

The aim of this study was to evaluate the effect of antioxidant catalase (CAT) and superoxide dismutase (SOD) in semen extender on motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa. Semen was collected by using an artificial vagina from five domestic cats (two ejaculates/cat). Spermatozoa were diluted in egg yolk Ttris-fructose citrate solution (EYT-FC) without glycerol and cooled at 4 degrees C for 1 h, then diluted further with EYT-FC with glycerol (7% final concentration) and 400 IU/ml of CAT (treatment 1) or SOD (treatment 2) or without antioxidants (control). Before freezing using a styrofoam box, diluted spermatozoa filled in 0.25-ml straws were equilibrated for 1 h at 4 degrees C. After thawing, spermatozoa were assessed for motility, viability and acrosomal integrity. Cryopreservation significantly impaired sperm motility, viability and acrosomal integrity (p < 0.05). However, motility, viability and acrosomal integrity of frozen-thawed cat spermatozoa in the EYT-FC with CAT, SOD and without the antioxidants were not significantly different. The average percentages of spermatozoa motility after thawing compared between control, treatment 1 and treatment 2 group were 43.5 +/- 3.2, 42 +/- 4.1 and 38 +/- 4.5; for viability: 44.8 +/- 3.5, 50.6 +/- 5.7 and 47.1 +/- 4.1 and for acrosomal integrity: 45 +/- 3.5, 44.9 +/- 3.4 and 44.4 +/- 3.3, respectively. In conclusion, adding CAT and SOD to EYT-FC did not improve motility, viability and acrosomal integrity in cryopreserved cat spermatozoa.

Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma

Four dogs with poor semen quality, low seminal plasma superoxide dismutase (SOD) activity and low blood plasma testosterone (T) levels were orally administered one vitamin E tablet containing 50 mg α-tocopheryl acetate per dog daily for 4 weeks. The mean values of semen quality were temporarily improved after the start of vitamin E treatment and the values of 4, and 5 weeks after that were significantly different from those before the treatment (P<0.05–0.001). The mean blood plasma T and seminal plasma SOD activity values slightly increased in the 4 dogs after the treatment. The results of the present study indicate that poor semen quality in dogs with low seminal plasma SOD can be improved by vitamin E treatment.

Superoxide dismutase activity in the oviductal and uterine fluid of the bitch and the effects of the enzyme on viability, motility and hyperactivation of canine sperm in vitro.

ABSTRACT Superoxide dismutase (SOD) activity in flushings from oviducts and uterine horns of 8 anestrous, 5 estrous and 7 diestrous bitches was measured. SOD activity in oviductal fluid in estrous bitches was significantly higher than that in anestrous and diestrous bitches (P<0.01). SOD activity in uterine fluid of diestrous bitches was, however, significantly higher than that in anestrous and estrous bitches (P<0.01). Additionally, sperm collected from normal dogs were incubated in MEM and in MEM containing SOD (SOD-MEM) for 24 hr. The percentages of sperm with viability, motility and hyperactivation in SOD-MEM were higher than those in MEM. SOD produced in oviduct and uterus may be able to maintain or improve sperm quality and fertility in the dog.

MicroRNA expression profiling in canine prostate cancer

Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.

Influence of different methods of collection from the canine epididymides on post-thaw caudal epididymal sperm quality

Canine epididymal sperm was collected from the cauda epididymis using 2 different methods (flushing and mincing) to compare the qualities (the percentage of progressively motile, viable, morphologically abnormal, immature and intact acrosomes) before and after freezing and thawing. No significant difference was noted in the quality of the cauda epididymal sperm immediately after collection and after freezing-thawing between the collection methods, although the mean levels of sperm quality with the flushing method were slightly better than that of the mincing method. The flushing method is simple and free of blood contamination, although the vas deferens was too small to be perfused in only 1 dog, and our results suggest that the flushing method is preferable to the mincing method for collecting sperm from the canine cauda epididymis.

Intrauterine insemination with fresh semen in Amur leopard cat (Pionailurus bengalensis eutilura) during non-breeding season

Equine and human chorionic gonadotropins were administered to two female Amur leopard cats to induce estrus and ovulation during non-breeding season. Fresh semen collected from male cats was surgically inseminated into the uterine horn of the females. In one animal, two fetal sacs without heartbeats were observed on abdominal ultrasonography 31 days after insemination, which indicated that embryo death had occurred. In the other animal, fetal heartbeats were detected in two fetal sacs 29 days after insemination, which confirmed as pregnancy. This animal delivered two newborns 68 days after insemination; the one of the kittens was assumed to be stillbirth, and the other grew normally. In this study, we successfully obtained a kitten from an Amur leopard cat by artificial breeding for the first time in Japan.