堀 達也

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumors. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib, and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumor volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumor regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation. This article is protected by copyright. All rights reserved.

Tolerance towards fetal alloantigens in the maternal immune system is essential for maintaining pregnancy. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells characterized by their ability to suppress immune activity and maintain maternal-fetal immune tolerance. However, the mechanisms underlying MDSC induction have not been elucidated. Herein, we investigated the myeloid-derived suppressor cells (MDSCs) in the peripheral blood of pregnant canines and its induction mechanism. By analyzing the concentration of MDSCs in the peripheral blood of pregnant canines, elevation of MDSCs has been observed during pregnancy. In addition, MDSCs from pregnant canines inhibit T cell activation. These results suggest that the elevated MDSCs in canine pregnancy may contribute to reduces maternal immune activity. To clarify the cause of MDSCs elevation in canine pregnancy, we analyzed the relationship between pregnancy-related hormones (estradiol, progesterone, and relaxin) and MDSCs. Serum relaxin levels, but not estradiol and progesterone, were correlated with the ratio of monocyte MDSCs. Additionally, relaxin induced monocytic MDSCs as well as inhibited T cell activation in vitro. Therefore, relaxin contributes to the elevation of monocytic MDSCs in the peripheral blood of pregnant canines. Our findings highlight the novel role of relaxin in pregnancy and contribute to a better understanding of maternal-fetal immune tolerance.

In this study, cauda epididymal sperm were collected from Amur leopard cats with various causes of death as well as Tsushima leopard cats that underwent castration surgery, and sperm quality was compared with that in domestic cats. A sufficient number of sperm similar to those in domestic cats could be collected from the cauda epididymis of Amur leopard cats. However, in old leopard cats, no or very few cauda epididymal sperm were recovered, although there were no differences in sperm motility and sperm abnormality. There were no significant differences in sperm quality immediately after collection and after freezing-thawing of cauda epididymal sperm compared with corresponding estimates in domestic cats.

Oxidative stress owing to an imbalance between reactive oxygen species and antioxidants, such as coenzyme Q10 (CoQ10), is a major contributor to male infertility. We investigated the effects of the reduced form of CoQ10 (ubiquinol) supplementation on semen quality in dogs with poor semen quality. Three dogs received 100 mg of ubiquinol orally once daily for 12 weeks. Semen quality, serum testosterone, and seminal plasma superoxide dismutase (SOD) activity were examined at 2-week intervals from 2 weeks before ubiquinol supplementation to 4 weeks after the treatment. Ubiquinol improved sperm motility, reduced morphologically abnormal sperm, and increased seminal plasma SOD activity; however, it had no effect on testosterone level, semen volume, and sperm number. Ubiquinol supplementation could be used as a non-endocrine therapy for infertile dogs.

Contents In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris-fructose citrate extender, and stored at 4 degrees C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48hr of storage, after storage for 96hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p<.05, p<.01). Although sperm abnormality increased in all groups with increased storage time, the group without being sensitized to PF showed significantly higher sperm abnormality than did the groups with being sensitized to PF after storage for 24hr and more (p<.01). From these findings, we concluded that PF was necessary for the cooled preservation of the canine sperm because these sperms were protected from any effects of low temperatures by being sensitized to PF.

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.

A 5-year-old female domestic shorthair cat was presented with abdominal distension and serum biochemical evaluation indicated a high concentration of oestradiol (32.81 pg/ml). Exploratory laparotomy revealed a large cystic mass in the right ovary with cystic fluid containing a high level of oestradiol (18.80 pg/ml). The tumour was composed of immature neuroectodermal tissue, mature cartilage, smooth muscle, adipose tissue and aggregated, poorly differentiated mesenchymal cells. It contained cysts of various sizes that were lined by epithelium of different types. The basal layer of the lining epithelium was shown to express aromatase by immunohistochemistry. The findings suggest that this was a novel, malignant, oestrogen-secreting teratoma and that the aromatase-positive, neoplastic cells may have been the source of elevated levels of serum oestrogen. (C) 2016 Elsevier Ltd. All rights reserved.

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.

Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species.

We collected semen from a male Amur leopard cat using the transrectal electroejaculation method and investigated the semen qualities for about four years. In addition, the influence of the season on the spermatogenic function of the Amur leopard cat was investigated with regard to the semen qualities, testicular volume and serum testosterone level. As a result, we could collect semen with good sperm qualities that would be useable for artificial insemination. Some seasonality was noted in the testicular volume and serum testosterone level. We clarified that the semen qualities were favorable before and during the female breeding season compared with those after the breeding season.

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 alpha-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.

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.

Contents Human chorionic gonadotropin (hCG) is a glycoprotein used in the treatment of spermatogenic dysfunction. However, previous studies performed in dogs show that repeated administration of large doses of hCG produces antibodies against hCG. In this study, we examined the efficacy of low-dose injections of hCG in four male dogs with spermatogenic dysfunction and low plasma testosterone (T) levels. We administered 100IU hCG per animal, five times at 3-day intervals, and evaluated the changes in semen quality and plasma T levels. The total number of sperm in ejaculate, the percentage of progressively motile sperm and the plasma T levels had increased by 3-5weeks after the first injection of hCG in three of the four dogs, but were unchanged in the fourth dog. These findings indicate that temporary improvement of the semen quality of dogs with spermatogenic dysfunction and low plasma T levels is possible after five low-dose injections of hCG.

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.

The objective of the present study was to determine an optimum temperature and extender for short-term transport of canine ejaculated semen. There was no significant difference in the qualities of semen diluted with two kinds of extender, egg yolk Tris-citrate fructose (EYT-FC) or glucose (EYT-GC) extender, between the 2, 8 or 12 and the 4 degrees C control groups during storage for up to 48 hr, while the 16-24 degrees C groups showed decreased sperm motility during storage for 48 hr. However, the 2 degrees C group showed slightly lower sperm motility and slightly higher sperm abnormality than the 4 degrees C group. Therefore, we concluded that semen qualities can be maintained for up to 48 hr when canine semen samples are extended with EYT-FC or EYT-GC and stored at a temperature in the range of 4-12 degrees C.

© Medwell Journals, 2014. The purpose of this study is to reveal the effect of estrogen-inducing SOD activity on the endometrium of domestic rabbits. Researchers measured Superoxide Dismutase (SOD) activity to investigate the reason of occurrence of popular uterine adenocarcinoma in rabbits in estradiol-17β-stimulated endometrium epithelial cell culture. Then, we also examined the distribution of cells expressing Estrogen Receptor α (ERα)-like Immunoreactivites (ERα-like-IR) in the uterus prone to developing adenocarcinoma. The addition of estradiol-17β to the primary culture of endometrial epithelial cells obviously decreased SOD activity. Moreover, the areas of endometrium in which uterine glands were confined in control rabbits and notably larger in rabbits with glandular hyperplasia of the uterus compared in normal dog uterus that the occurrence of uterine adenocarcinoma is rare. ERα-like-IR cells were found in all proliferating glands in rabbits with hyperplasia.

Superoxide dismutase (SOD) has a key role in protecting1 cells from Reactive Oxygen Species (ROS). Cancer cells produce more ROS involved in proliferation and survival of cancer cells. We, therefore, investigated the percentage of castrated dogs with Prostatic Adenocarcinoma (PA) and the relation between the occurrence of PA in the dog and the prostatic SOD activity after castration. Between 2005 and 2012, diagnosis of Prostatic Adenocarcinoma (PA) was made in 24 dogs at the Veterinary Teaching Hospital of Nippon Veterinary and Life Science University. The percentage of castrated dogs in the 24 dogs with PA and the relation between the occurrence of PA in the dog and the prostatic superoxide dismutase (SOD) activity after castration were examined. Prostatic parenchyma specimens were collected from 6 healthy dogs just before castration and 6 months after castration to measure prostatic SOD activity with a SOD assay kit. Nineteen (79.2%) of the 24 dogs diagnosed with PA had been castrated at 1-6 (2.7±1.7) years of age. Their prostatic SOD activity was significantly lower after castration than before castration (p&lt 0.01). The results of this study indicated that castration could not prevent PA from occurring and the reduction of prostatic SOD activity after castration might lead to oncogenesis in PA of the dog. © 2014 Academic Journals Inc.

Purpose: Ejaculation in the male dog consists of three fractions. Observation of behavior and measurement of heart rate (HR), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were researched sequentially, and a fundamental examination of the features of sympathetic nerve activity during copulatory behavior induced by the hand method in the male dog was undertaken. Methods: We investigated the breeding capability of male dogs. HR, plasma NA level and plasma Ad levels were measured during ejaculation induced by the hand method. Results: HR was 125.8 ± 6.0 beats/min at rest, and peaked during mounting at 195.2 ± 8.2 beats/min. Moreover, HR at 3 min after the first fraction decreased to values similar to those at rest. Plasma NA and Ad concentrations during copulatory behavior induced by the hand method did not differ significantly from those at rest. However, although there was no significant difference, plasma NA concentration during ejaculation of the third fraction peaked at about 1.8 times the baseline value. Conclusions: In the male dog, excitation of sympathetic nerves of long duration during erection of the penis and ejaculation is questionable. However, inhibition of sympathetic nerves and activation of parasympathetic nerves is thought to occur during erection of the penis and ejaculation. © 2013 The Author(s).

In healthy male dogs, peripheral plasma testosterone (T), prostaglandin E-2 (PGE(2)) and seminal plasma PGE(2) levels were measured before, during and after ejaculation, and semen quality was examined after oral administration of PGE(2). Plasma T and PGE(2) levels did not change during these periods, but the seminal plasma PGE(2) level of combined the first and second fractions was significantly higher than those at 0-5 and 5-10 min after the start of ejaculation of the third fraction. Semen volume but not quality increased after PGE(2) administration. In conclusion, large amounts of PGE(2) are released from the prostate gland during the early part of ejaculation, and PGE(2) plays an essential role in secretion of seminal plasma.

Preservation of canine spermatozoa at 5°C is a helpful method for dog breeding management. However, in many species, oxidative damage due to generation of reactive oxygen species (ROS) and long term storage which specifically cause DNA damage, and lipid peroxidation during preservation techniques can cause a decline of sperm motility and fertility capacity. In this study, the effect of antioxidants catalase (CAT) and superoxide dismutase (SOD) on motility, viability and acrosomal integrity of chilled canine semen were investigated. Semen was collected by digital manipulation from 4 dogs (two ejaculates/dog). After removal of seminal plasma, spermatozoa were diluted in egg yolk Tris-fructose citrate solution (EYT-FC) with or without CAT, SOD or the combination of CAT and SOD at dosages of 100, 400 and 1,600 U/ml in each antioxidant. Diluted spermatozoa were kept at 5°C for 7 days. Sperm motility, viability and acrosomal integrity in the EYT-FC with CAT or/and SOD and without the antioxidants (control) were not significantly different at same point of time. At day 7, the percentage of mean compared between control and treatment groups for sperm motility were 65.6% and 65.0-66.9%, viability were 83.6% and 82.1-84.9% and acrosomal integrity were 87.2% and 85.9-88.5%, respectively. In conclusion, adding CAT and SOD or the combination of CAT and SOD in EYT-FC did not significantly improve the maintenance of motility, viability and acrosomal integrity during dog sperm storage at 5°C for 7 days.

Contents This study investigated the duration of the interval between the onset of vulval bleeding at pro-oestrus and ovulation estimated from the plasma progesterone concentration in a large number of beagle bitches. The influence and association of individual variation, ageing and duration of the oestrous cycle were also investigated. The mean time of ovulation after the onset of vulval bleeding was 11.1 +/- 0.2 days, but it widely ranged from 3 to 31 days. This timing was not influenced by age or duration of the oestrous cycle, and within-individual variation was small. As there has been no previous report in which the ovulation day was investigated by the age, these data may be very valuable.

Contents As a step towards elucidation of the timing and mechanism of the determination of the number of ovulated ova in dogs, we excised one ovary 2, 5 and 8 days after the beginning of vulval bleeding and examined whether the lost ovulation function, assessed by estimating the number of ovulated oocytes, would be compensated for by the remaining ovary. The number of ovulated ova was maintained by the remaining ovary in the group that underwent unilateral ovariectomy 2 days after the beginning of vulval bleeding. However, in the groups ovariectomized 5 or 8 days after the beginning of vulval bleeding, no compensation for the number of ova that would have been ovulated from the lost ovary was observed; ova were ovulated only from the follicles 3 mm or greater in diameter observed in the remaining ovary at unilateral ovariectomy. Thus, in dogs, the number of ovulated ova is considered to be determined within 5 days after the beginning of vulval bleeding.

A Beagle dog (3 years old) that ejaculated high percentages (mean +/- SE: 29.1 +/- 1.2%) of sperm with a knobbed acrosome abnormality and a low number of sperm and that also had a low plasma testosterone (T) level was given 10 subcutaneous injections of 1 mu g of gonadotropin-releasing hormone analogue (GnRH-A) at 3-day intervals. The plasma T level and number of sperm increased 12-14 weeks after the first injection. Although the percentages of sperm with knobbed acrosome abnormality did not change after the GnRH-A therapy, the number of sperm and percentage of actively motile sperm increased after the therapy, and a bitch gave birth to 5 healthy puppies after intravaginal artificial insemination with fresh semen collected 14 weeks after the first injection.

The fertility was compared between ejaculated and cauda epididymal sperm sensitized with prostatic fluid in dog after freeze-thawing using the fertility of ova from the contralateral ovary after injection (2 x 10(8) sperm) into dog uterus on the unilateral ovariectomized side, on the basis of the presence or absence of conception. No significant difference was observed in sperm quality after freeze-thawing between the two groups and conception rates were equivalent and low. Therefore, to achieve a high fertility by intrauterine insemination of canine frozen-thawed ejaculated and cauda epididymal sperm, intrauterine insemination on both sides is recommended, rather than insemination with a lot of sperm of the uterine horn on one side.

Glycoproteins (GPs) are known to be involved in the phenomenon of sperm maturation and capacitation. In the present study, we investigated the attachment of GPs on sperm cell membrane during the process of feline sperm maturation from testicular sperm to ejaculated sperm by using 8 FITC-labeled lectins. The results showed that 3 types of GPs were presented on testicular sperm and 7 on caput epididymal sperm. Corpus and cauda epididymal sperm and ejaculated sperm had GPs detected by 8 FITC-labeled lectins used in the present study. This study demonstrates the part of the characteristic of GPs that are present on the feline sperm cell membrane during the process of sperm maturation.

The mean post-thaw sperm motilities of feline frozen semen prepared with 1% OEP or 3 g/ml SLS as a cryoprotective agent, in addition to 7% glycerin, were 35.0 +/- 2.4 and 37.0 +/- 2.5%, respectively, showing no significant difference. On unilateral intrauterine insemination (UIUI) using these semen samples at a sperm number of 40 x 10(6), the conception rate was 70.0% (7/10) in the OEP group and 30% (3/10) in the SLS group, showing that the rate was higher in the OEP group, but the difference was not significant. It was suggested that sperm in frozen semen showing the above qualities were transferred to the contralateral uterine horn on UIUI.

We observed the influences of low-temperature storage of the feline epididymis on the epididymal semen qualities before and after cryopreservation to identify the optimal duration for low-temperature storage of the epididymis. After excision, the feline epididymis was stored at 4 degrees C for 0-72 hr and then subjected to epididymal sperm collection. When sperm from the refrigerated cauda epididymis were frozen and thawed, there was no significant difference in sperm motility between the 0- and 24-hr low-temperature storage groups, but sperm motility was significantly decreased in the 48-hr storage group. The above findings suggested that low-temperature storage of the epididymis until 24 hr is useful for frozen sperm collected from the feline cauda epididymis.

It has been shown that addition of the surfactant Orvus ES paste (OEP) and its main component sodium lauryl sulfate (SLS) to boar or dog semen before freezing improves post-thaw sperm motility and protects acrosome caps. In this study, we investigated the usefulness of the addition of OEP (0, 1, 2 and 4%) or SLS (0, 1, 2, 3 and 4 mg/ml) to cat ejaculates before freezing and their concentrations. Among the OEP addition groups, the 1% OEP group showed higher sperm motility than the other groups. Among the SLS addition groups, the 3 mg/m/ SLS group showed slightly higher sperm motility and viability than the other groups. Comparison between the 1% OEP and 3 mg/m/ SLS addition groups suggested a higher percentage of sperm with an acrosome cap in the 1% OEP group. The other sperm properties did not significantly differ between the 2 groups. These results indicate that addition of 1% OEP or 3 mg/m/ SLS is effective for freezing of cat ejaculated semen.

A Beagle with a low plasma testosterone (T) level and azoospermia was given 10 Subcutaneous injections of 1 mu g gonadotropin-releasing hormone analogue (GnRH-A) per head at intervals of 3 days (Experiment 1), and 6 months after the final injection was given, 15 Subcutaneous injections of 2 mu g GnRH-A were given at intervals of 2 days (Experiment 2). The plasma T level increased and peaked at 8 weeks after the first injection of GnRH-A in both Experiment 1 and Experiment 2. Motile sperm were detected in the semen collected 8 weeks and 7 weeks after the first injection in Experiment 1 and Experiment 2, respectively. The total number of sperm peaked 9 weeks after the first injection in both Experiment 1 (4.5 x 10(6)) and Experiment 2 (72.8 x 10(6)).

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.

Plasma progesterone (P(4)) concentrations are maintained in pregnant cats until parturition, but become low in pseudopregnant cats 40-45 days after infertile mating. This difference in P(4) concentrations is considered to be due to P(4) secretion by the placenta of pregnant cats. Therefore, to clarify these points, we performed ovariectomy (OVX) at various stages of pregnancy, examined the pregnancy status and measured LH and P(4) concentrations in peripheral, ovarian and uterine venous blood. After OVX, abortion occurred in 100% (5/5), 80% (4/5), 40% (2/5) and 60% (3/5) of Groups I (Day 35), II (Day 40), III (Day 45) and IV (Day 50) cats, respectively. In the remaining cats, normal delivery took place on days 63 - 69 [mean, 66.1 +/- 1.1 (SE)] of pregnancy. The time to abortion after OVX was 4 - 8 (mean, 5.6 +/- 0.8), 3 - 17 (mean, 8.0 +/- 3.6), 10 and 11, and 2 - 4 (mean, 3.0 +/- 0.7) days in Groups I, II, III and IV, respectively. The plasma P(4) concentrations were 1 - 2 ng/ml in all groups on the day after OVX, decreasing to less than 1 ng/ml from the 2nd day onwards. The concentrations of P(4) in ovarian venous blood at the time of OVX decreased with the stage of pregnancy, but were clearly higher than those in peripheral blood. The plasma P(4) concentrations in uterine venous blood were similar to those in peripheral blood. These results suggest that peripheral P(4) in pregnant cats is the result of P(4) secretion secreted only by the ovarian corpus luteum, not by the placenta, but indicate that either P(4) is not essential for the maintenance of pregnancy in cats from day 40 - 45 of pregnancy onwards, or that the placenta provides a local source of P(4) that does not appear in measurable amounts in the peripheral circulation.

Contents Female cats are known to be seasonal breeders and male cats annual breeders. Despite this, there are limited data on the influence of breeding season (BS) on hormone concentration and semen quality in the male cat. This study compared plasma concentrations of LH and testosterone ( T), and semen quality during the non-breeding season (NBS) and BS in five male cats subject to natural hours of daylight but a constant environmental temperature. Plasma LH and T concentrations were higher during the BS in 2/35 and 3/5 cats, respectively, although when comparing both hormones combined, values were higher during the BS than the NBS in all cats (p < 0.01). There were no significant differences in the percentage of abnormal sperm between the cats. Overall, semen quality was superior during the BS with larger semen volume in 2/5, sperm motility in 2/5 and sperm viability in 3/5 cats. Although there was a clear seasonal effect on hormone secretion and semen quality, during the NBS all cats were likely to have been fertile.

Although cats are induced ovulators, the relationship between the day of breeding, the number of matings and the likelihood of ovulation and conception have not been extensively investigated. In this experiment, cats were mated either once or three times on day 1 or day 5 of oestrus to study the incidence of the LH surge, ovulation and conception rates. The percentage ovulating and the conception rates after a single mating on day 1 of oestrus were 60% (6/10) and 33.3% (2/6), respectively, and for cats mated once on day 5 of oestrus were 83.3% (10/12) and 40% (4/10), respectively. When cats were mated three times on day 1 of oestrus, the ovulation rates and conception rates were 70% (7/10) and 85.7% (6/7), respectively, and for those mated three times on day 5 of oestrus were 100% (10/10) and 100% (10/10), respectively. The concentration of LH did not increase in non-ovulating cats, and cats that were mated three times had LH concentrations that were numerically higher than those that were mated once. Litter size was neither related to the day of mating nor to the number of matings. Although an increase in the number of matings on day 1 of oestrus produced a numerically larger LH surge, it did not increase the ovulation rate, suggesting that plasma oestradiol concentrations were not sufficiently elevated to induce a high pituitary response to mating stimulation. The conception rate after a single mating was low, suggesting that the number of sperm per mating was not sufficient. These results suggest that mating more than once in the middle of oestrus is required to improve ovulation rates and conception rates in cats.

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.

The effects of the time period between canine epididymis removal and cooling on post-thaw caudal epididymal sperm quality were investigated. Sperm recovered from the epididymis stored for 6 hr at 4 or 20 degrees C exhibited similar motility. However, when the epididymis was stored for 12 hr or longer at 20 degrees C, sperm motility was significantly lower than that at 4 degrees C (p<0.01). The post-thawed qualities of sperm recovered from the caudal epididymides that had been stored at 20 degrees C for 0 or 6 hr and then at 4 degrees C for 24 hr after removal were not significantly different. Therefore, leaving the canine epididymis at 20 degrees C for tip to 6 hr after its removal may have little effect on the post-thaw quality of recovered caudal epididymal sperm.

The proportions of Sertoli cell tumor (SCT), seminoma and Leydig cell tumor in 50 dogs with unilateral testicular tumors were 52%, 36% and 12%, respectively. The rate of occurrence of SCT in the cryptorchid testis was very high (71 %). The testicular superoxide dismutase (SOD) activity, testicular heat shock protein (HSP) 70 concentration and peripheral blood plasma inhibin (INH)-alpha concentration of 10 dogs with a unilateral cryptorchid testis and no testicular tumors, 10 dogs with SCT in a unilateral cryptorchid testis and 10 normal dogs, all aged 5-15 years, were measured in order to identify high risk factors for the occurrence of SCT in the canine cryptorchid testis. The mean SOD activity in cryptorchid testes and SCTs was significantly lower and higher, respectively, than in normal testes (both P < 0.01). The mean HSP 70 concentration in both cryptorchid testes and SCTs was significantly higher than in normal testes (both P < 0.01). The mean plasma INH-alpha concentration of the cryptorchid and SCT dogs was significantly lower and higher, respectively, than in normal dogs (P < 0.05 and 0.01, respectively). The low SOD activity in the cryptorchid testis, low blood plasma INH-alpha concentration of the cryptorchid dogs and high HSP 70 concentration in the SCTs may be related to the occurrence of SCT and tumor cell proliferation in canine cryptorchid testes.

The peripheral blood plasma testosterone (T) levels and superoxide dismutase (SOD) activity were measured in 5 azoospermic (AZ-) beagles. The mean values in the AZ-dogs were significantly lower than in 7 control beagles (P < 0.001). Subcutaneous injections of 1 mu g/kg GnRli analogue three times weekly in the AZ-dogs induced significant increases in mean T level and SOD activity (P < 0.05) and improvement in spermatogenesis. Thus, spermatogenic function in the dog appears to be maintained by T and normal SOD activity in the testis.

Plasma concentrations of progesterone (P-4) and prolactin (PRL) were measured in 35 bitches presented at veterinary clinics for symptoms of overt pseudopregnancy (PSP) between 50 and 95 days after the onset of proestrus. Results were compared to those from samples collected from 35 control bitches at comparable stages of the ovarian cycle (expressed as days after the onset of observed signs of proestrus). In the PSP bitches at 71.4 +/- 1.6 (mean +/- S.E.M.) days of the cycle, P-4 (1.5 +/- 0.2 ng/mL) was lower (P < 0.01) and PRL (16.0 +/- 1.9 ng/mL) was higher (P < 0.0 1), compared to P-4 (2.7 +/- 0.4 ng/mL) and PRL (2.9 +/- 0.6 ng/mL) in control bitches at 70.6 +/- 1.5 days of the cycle. Low P-4 was not a prerequisite for elevated PRL. Although elevated (>= 10 ng/mL) PRL (20.9 +/- 2.0 ng/mL) occurred more often with low (< 2 ng/mL) P-4 (20 of 24 cases) it also occurred with P4 above 3 ng/mL in two affected bitches and in two control bitches. Whether the occurrence of relatively low PRL concentrations (< 4 ng/mL) in samples obtained from 4 of the 35 pseudopregnant bitches reflected variable and often elevated PRL secretion or increased sensitivity to PRL in the absence of elevated prolactin in those animals was not determined. We inferred that elevated plasma PRL was often involved in the etiology of overt PSP; furthermore, a premature decline in circulating P-4 concentrations may be a factor in some instances. (c) 2007 Elsevier Inc. All rights reserved.

We measured the blood plasma testosterone (T) levels and superoxide dismutase (SOD) and catalase activities in the seminal plasma of the ejaculates of 5 normal (2-5 years old) and 5 asthenozoospermic (AZ-) (3-5 years old) Beagles. Sperm ejaculated by AZ-dogs was incubated for 3 hr in Eagle's MEM only (controls) or Eagle's MEM containing 100 units/ml of SOD or catalase. Sperm motility was examined during incubation. The mean (SE) plasma T level of the AZ-dogs (1.2 +/- 0.2 ng/ml) was significantly lower than in the normal dogs (2.5 +/- 0.2 ng/ml) (P < 0.005). The mean (+/- SE) seminal plasma SOD and catalase activities (18.8 +/- 1.9 and 0.5 +/- 0.1 unit/g protein, respectively) were significantly lower in the AZ-dogs than in the normal dog (43.3 +/- 2.5 and 2.2 +/- 0.4 unit/g protein, respectively) (P < 0.001 and 0.01, respectively). The motility of sperm incubated in Eagle's MEM containing SOD or catalase was significantly higher than that of control sperm incubated in only Eagle's MEM after 2 or 3 hr of incubation (P < 0.05). The results of this study indicate that poor T secretion by the testes and low antioxidant enzyme activities are related to AZ in the dog.

Ultrasonography-guided transabdominal needle aspiration was carried out to remove 3 to 14 ml of purulent matter from the cavities of prostatic abscesses in 6 dogs, and the same volume of tea tree oil was injected into the cavities to treat the abscesses. The same treatment was repeated 3 weeks later in 4 dogs, and subsequent disappearance of the purulent matter in the cavities and a marked reduction in the volume of the cavities were observed. These findings indicate that the treatment of prostatic abscesses by aspiration of the purulent matter and injection of tea tree oil into the cavities is very effective in dogs.

The effects of day of mating and litter size on gestation length in dogs were studied in 36 beagle bitches (age 2-10 yr). The day that plasma progesterone concentrations exceeded 2 ng/mL was considered the day of ovulation; dogs were randomly assigned to be bred once, 1-5 days after the estimated day of ovulation. The interval from mating to parturition was negatively correlated with the number of days from estimated ovulation to mating (P < 0.01). Gestation length (interval from ovulation to parturition) was almost constant at 63.9 +/- 0.2 days (mean +/- S.E.M.), with no significant relationship between the number of fetuses and the duration of gestation. (c) 2006 Elsevier Inc. All rights reserved.

Semen quality was determined in a sexually mature male Giant Panda, electroejaculated 13 times during a 5-year interval, before, during and after estrus of a female Giant Panda housed nearby. Testis volume and plasma testosterone concentrations were also measured. Mean testis volumes were 1223.0 +/- 64.7(S.E.M.) cm(3) (before estrus), 1213.2 +/- 218.2 cm(3) (during estrus), and 1360.2 +/- 160.4 cm(3) (after estrus). Compared to before and during estrus in the female, testis volume decreased 70 days after estrus and there was no projectile ejaculation. The mean semen volume and sperm count were 2.2 +/- 0.7 mL and 8.3 +/- 3.1 x 10(8) before estrus, 2.4 +/- 0.9 mL and 5.7 +/- 0.9 x 10(8) during estrus, and 1.3 +/- 0.3 mL and 8.1 +/- 1.7 x 10(8) after estrus, respectively. The semen volume, sperm count, and testis volume markedly differed from 90 days before estrus until 66 days after estrus, whereas no marked differences in sperm motility, sperm viability, and proportion of morphologically abnormal spermatozoa were observed. Plasma testosterone concentrations were elevated both before and during estrus (0.62 +/- 0.23 ng/mL and 0.95 ng/mL), but decreased substantially after estrus (0.20 +/- 0.0 ng/mL). We inferred that spermatogenesis was active in this male panda from approximately 3 months before estrus to 2 months after estrus in the adjacent female. (c) 2006 Elsevier Inc. All rights reserved.

We investigated the use of a single treatment of estradiol benzoate (E2B) to prevent pregnancy in mismated dogs, including effects of dose, side effects, and mechanism of action. We used 74 female beagles aged 1-10 year. A single treatment of E2B (0.025, 0.05, 0. 10, or 0.20 mg/kg; 10 dogs/dose) given 2 days after mating (5 days after ovulation) resulted in pregnancy rates of 70, 50, 10, and 0%, respectively, whereas pregnancy rate was 100% in 5 untreated (control) dogs. There were no side effects in dogs given E2B, regardless of dose. To clarify the mechanism of prevention of pregnancy, we investigated embryonic descent in the uterine tube and embryonic degeneration after treatment with 0.025 or 0.20 mg/kg of E2B; treatment delayed embryonic descent in the uterine tube and increased the incidence of embryonic degeneration. In conclusion, a single treatment of 0.020 mg/kg of E2B given 2 days after mating effectively and safely prevented pregnancy in dogs. (c) 2006 Elsevier Inc. All rights reserved.

The success rate of in vitro fertilization (IVF) in dogs is low and only early embryos have been obtained. In the present study, we investigated the use of intrauterine transfer of early-stage canine embryos to obtain pups. Twenty-three female dogs, in which the date of ovulation (based on plasma progesterone concentrations) differed by +/- 1 day, were used (10 donors and 13 recipients). The uterine tube was extirpated under general anesthesia 1-4 days after mating (5-7 days after ovulation), and descending perfusion was done to collect embryos. Embryos were examined and transferred into the uterine horn of a recipient, ipsilateral to the ovary with the most corpora lutea. Pregnancy was established in one of eight bitches that received early embryos (zygote to 4-cell embryos); she received two zygotes and one 2-cell embryo and delivered two puppies. Although intrauterine transfer of early embryos (zygote to 4-cell embryos) was difficult, pregnancy was achieved, suggesting that uterine tube transfer is appropriate for these early-stage embryos. (c) 2006 Elsevier Inc. All rights reserved.

The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN2) vapor and the height from LN2, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN2 surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN2. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: 4 to -22 degrees C/min) and 10 cm/15 min groups (cooling rate: -6 to -10 degrees C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9 degrees C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN2 to reduce the temperature at a slow cooling rate of about -10 degrees C/min, followed by plunging into LN2 after sensitization for 10-15 min, provides good semen qualities after thawing.

The addition of Orvus ES paste (OEP) to extender may be essential for preparing frozen dog semen. The major ingredient of OEP is sodium lauryl sulfate (SLS). In this study, we compared the effect of SLS on frozen dog semen with that of OEP. There were no significant differences between the 2-mg/ml SLS group and OEP group concerning sperm motility, viability and the percentage of viable sperm with intact acrosomes after freeze-thawing. These results suggest that the effectiveness of frozen dog semen extender containing 2 mg/ml of SLS is similar effective to that demonstrated for OEP.

An intrauterine insemination technique using a fiberscope was investigated in me giant panda. A septal wall cm from the vulva, the external urethral orifice was present in the ventral region, and the entrance (pseudocervix) to the vagina was present in the dorsal region. The uterovaginal region protruded in the dorsal region about 15 cm from the pseudocervix. The external uterine orifice was present in the uterovaginal region, revealing that intrauterine insemination can be easily performed. This technique may greatly contribute to artificial reproduction of the giant panda.