Assisted reproductive techniques for canines: preservation of genetic material in domestic dogs.

Assisted reproductive techniques (ARTs), such as artificial insemination, in vitro fertilization, and cryopreservation of gametes/zygotes, have been developed to improve breeding and reproduction of livestock, and for the treatment of human infertility. Their widespread use has contributed to improvements in human health and welfare. However, in dogs, only artificial insemination using frozen semen is readily available as an ART to improve breeding and control genetic diversity. A recent priority in sperm cryopreservation is the development of alternatives to egg yolk, which is widely used as a component of the sperm extender. Egg yolk can vary in composition among batches and is prone to contamination by animal pathogens. The latter can be a problem for international exchange of cryopreserved semen. Low-density lipoprotein and skim milk are promising candidates for use as extenders, to ensure fertility after artificial insemination. Although not tested for its effects on fertility following artificial insemination, polyvinyl alcohol may also be a useful alternative to egg yolk as an extender. The development of cryopreservation techniques for canine embryos lags behind that for other mammals, including humans. However, given the success of non-surgical embryo transfer in 2011, studies have sought to refine this approach for practical use. Research on sperm cryopreservation has yielded satisfactory results. However, investigation of other approaches, such as cryopreservation of oocytes and gonadal tissues, remains insufficient. Techniques for the efficient induction of estrus may aid in the development of successful canine ARTs.

Evidence for existence of insulin-like factor 3 (INSL3) hormone-receptor system in the ovarian corpus luteum and extra-ovarian reproductive organs during pregnancy in goats.

Insulin-like factor 3 (INSL3), initially described as a male hormone, is expressed in female reproductive organs during the estrous cycle and pregnancy but its function has not yet been established. This study explores the function of INSL3 in pregnant Saanen goats by characterizing the expression dynamics of INSL3 and its receptor, relaxin family peptide receptor 2 (RXFP2) and by demonstrating specific INSL3 binding in reproductive organs, using molecular and immunological approaches and ligand-receptor interaction assays. We demonstrate that the corpus luteum (CL) acts as both a source and target of INSL3 in pregnant goats, while extra-ovarian reproductive organs serve as additional INSL3 targets. The expression of INSL3 and RXFP2 in the CL reached maximum levels in middle pregnancy, followed by a decrease in late pregnancy; in contrast, RXFP2 expression levels in extra-ovarian reproductive organs were higher in the mammary glands but lower in the uterus, cervix and placenta and did not significantly change during pregnancy. The functional RXFP2 enabling INSL3 to bind was identified as an ~ 85 kDa protein in both the CL and mammary glands and localized in large and small luteal cells in the CL and in tubuloalveolar and ductal epithelial cells in the mammary glands. Additionally, INSL3 also bound to multiple cell types expressing RXFP2 in the uterus, cervix and placenta in a hormone-specific and saturable manner. These results provide evidence that an active intra- and extra-ovarian INSL3 hormone-receptor system operates during pregnancy in goats.

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility, free thiol content and seasonality.

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.

Cryopreservation of canine spermatozoa using a skim milk-based extender and a short equilibration time.

Although useful spermatozoa cryopreservation techniques have been established, long-term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)-based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p < .05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p < .05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen-thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM-based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used.

Gene expression analyses of the small intestine of pigs in the ex-evacuation zone of the Fukushima Daiichi Nuclear Power Plant.

BACKGROUND: After the accident at the Fukushima Daiichi Nuclear Power Plant, radioactive contaminants were released over a widespread area. Monitoring the biological effects of radiation exposure in animals in the ex-evacuation zone should be continued to understand the health effects of radiation exposure in humans. The present study aimed to clarify the effects of radiation by investigating whether there is any alteration in the morphology and gene expressions of immune molecules in the intestine of pigs and inobuta (wild boar and domestic pig hybrid) in the ex-evacuation zone in 2012. Gene expression analysis was performed in small intestine samples from pigs, which were collected from January to February 2012, in the ex-evacuation zone. Pigs lived freely in this zone, and their small intestine was considered to be affected by the dietary intake of radioactive contaminants. RESULTS: Several genes were selected by microarray analysis for further investigation using real-time polymerase chain reaction. IFN-γ, which is an important inflammatory cytokine, and TLR3, which is a pattern recognize receptor for innate immune system genes, were highly elevated in these pigs. The expressions of the genes of these proteins were associated with the radiation level in the muscles. We also examined the alteration of gene expressions in wild boars 5 years after the disaster. The expression of IFN-γ and TLR3 remained high, and that of Cyclin G1, which is important in the cell cycle, was elevated. CONCLUSIONS: We demonstrated that some changes in gene expression occurred in the small intestine of animals in the ex-evacuation zone after radiation. It is difficult to conclude that these alterations are caused by only artificial radionuclides from the Fukushima Daiichi Nuclear Power Plant. However, the animals in the ex-evacuation zone might have experienced some changes owing to radioactive materials, including contaminated soil, small animals, and insects. We need to continue monitoring the effects of long-term radiation exposure in living things.

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome.

Haploinsufficiency of the voltage-gated sodium channel NaV1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of NaV1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the NaV1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.

Synthesis and biological evaluation of novel imidazol-1-ylacetic acid derivatives as non-brain penetrant bombesin receptor subtype-3 (BRS-3) agonists.

Novel compounds based on 1a were synthesized with the focus of obtaining agonists acting upon peripheral BRS-3. To identify potent anti-obesity compounds without adverse effects on the central nervous system (CNS), a carboxylic acid moiety and a labile carboxylic ester with an antedrug functionality were introduced. Through the extensive synthetic exploration and the pharmacokinetic studies of intravenous administration in mice, the ester 2b was selected owing to its most suitable pharmacological profile. In the evaluation of food intake suppression in C57BL/6N mice, 2b showed significant in vivo efficacy and no clear adverse effects on blood pressure change in dogs administered the compound by intravenous infusion.

Synthesis and biological evaluation of novel chiral diazepine derivatives as bombesin receptor subtype-3 (BRS-3) agonists incorporating an antedrug approach.

Novel compounds based on the lead BRS-3 agonists from our HTS compounds 2a and 2b have been synthesized with the focus on obtaining peripheral BRS-3 agonists. To identify potent anti-obesity compounds without adverse effects on the central nerve system, a labile carboxylic ester with an antedrug functionality was introduced onto the terminal position. Through the extensive synthetic exploration and the pharmacokinetic studies of oral administration in mice, the phenol ester 17c was selected due to the most suitable pharmacological profile. In the evaluation of food intake suppression in B6 mice, 17c showed significant in vivo efficacy and no clear adverse effect on heart rate and blood pressure change in dog iv infusion. Our study paved the way for development of anti-diabetes and obesity drugs with a safer profile.

A randomized, open-label trial of edoxaban in Japanese patients with severe renal impairment undergoing lower-limb orthopedic surgery.

BACKGROUND: Edoxaban is an oral, direct, factor Xa inhibitor approved in Japan for thromboembolic prophylaxis after lower-limb orthopedic surgery (LLOS), but contraindicated in patients with severe renal impairment (SRI; creatinine clearance [CLCR] ≥15 to <30 mL/min). METHODS: This open-label study compared the safety of edoxaban 15 mg once daily in Japanese patients with SRI to that of edoxaban 30 mg in patients with mild renal impairment (MiRI; CLCR ≥50 to ≤80 mL/min; N = 30) undergoing LLOS. Patients with CLCR ≥20 to <30 mL/min were randomized to receive edoxaban 15 mg (N = 22) or subcutaneous fondaparinux 1.5 mg once daily (N = 21). All patients with CLCR ≥15 to <20 mL/min received edoxaban 15 mg (N = 7). Treatment was administered for 11 to 14 days. RESULTS: Major or clinically relevant non-major bleeding occurred in 6.7%, 3.4%, and 5.0% of patients in the MiRI edoxaban 30-mg, SRI edoxaban 15-mg, and SRI fondaparinux groups, respectively; there were no major bleeding events. No thromboembolic events occurred. At all time points assessed, edoxaban plasma concentrations and changes in coagulation biomarkers were similar between the SRI and MiRI groups. CONCLUSIONS: These results suggest edoxaban 15 mg once daily is well tolerated in Japanese patients with SRI undergoing LLOS. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01857583.

Transcriptional wiring for establishing cell lineage specification at the blastocyst stage in cattle.

Mice and cattle use distinct pathways for the first cell segregation into inner cell mass (ICM) and trophectoderm (TE) lineages at the blastocyst stage. However, limited knowledge is available regarding the reliable transcriptional networks that orchestrate the complex developmental processes at this stage in nonrodent species. In order to elucidate the site-dominant transcriptomic properties of bovine blastocysts, we separated cell samples into the ICM and TE using both mechanical and chemical methods and performed in silico prescreening for candidate genes that were site-dominantly expressed in bovine blastocysts. We further performed quantitative real-time PCR and in situ hybridization using the site-specific cell samples. As a result, we identified seven ICM-dominant genes and five TE-dominant genes not found in earlier studies. Our findings provide novel insights into the mechanism of cell-fate specification in the pre-implantation bovine embryo.

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

Cryopreservation of canine embryos.

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

Effect of graft site and gonadotrophin treatment on follicular development of canine ovarian grafts transplanted to NOD-SCID mice.

PURPOSE: To investigate the effect of graft site and gonadotrophins administration on the number and survival rate of follicles of canine ovarian grafts transplanted to NOD-severe combined immune deficiency (SCID) mice. METHODS: Fresh ovarian cortex slices obtained from immature bitches were grafted subcutaneously (SC), under kidney capsule (KC) or into ovarian bursa (OB) in NOD-SCID mice. Two months after surgery, the mice allocated into non-treated and treated gonadotrophins groups that injected with porcine follicle stimulating hormone during 7 days and human chorionic gonadotrophin 48 h later. Ovarian grafts were collected after 10 h of last injection and processed for histology. RESULTS: The number of transitional and preantral follicles under KC and into OB was significantly higher in gonadotrophins-treated mice than those who received saline. Furthermore, the survival rates of primary, transitional and preantral follicles under KC and into OB grafts were significantly higher than those placed SC in the treated gonadotrophins group, and in the non-treated gonadotrophins group; the proportion of primary and preantral follicle survival was significantly higher under KC and into OB than SC grafts. CONCLUSIONS: In canine ovarian xenografting, administration of gonadotrophin could be effective for improvement of survival of transplanted ovary. Furthermore, the grafting into OB appeared to be better than grafting under KC, which in turn is better than SC.

Effect of bovine cumulus-oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes.

PURPOSE: To investigate the ability of medium conditioned with bovine cumulus-oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries. METHODS: Cumulus-oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO2, 5% O2 and 90% N2. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO2, 98% air. RESULTS: The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate. CONCLUSIONS: The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.

Prediction of postoperative ambulatory status 1 year after hip fracture surgery.

UNLABELLED: Hirose J, Ide J, Yakushiji T, Abe Y, Nishida K, Maeda S, Anraku Y, Usuku K, Mizuta H. Prediction of postoperative ambulatory status 1 year after hip fracture surgery. OBJECTIVES: To assess the validity of Estimation of Physiologic Ability and Surgical Stress (E-PASS) for predicting the postoperative risk and ambulatory status long-term follow-up after hip fracture surgery and to establish an algorithm for predicting their ambulatory status. DESIGN: Cohort study. SETTING: Twelve hospitals belonging to the regional network for hip fracture in Japan. PARTICIPANTS: The study population was composed of 421 patients; 268 underwent surgery between April 2004 and March 2006 (group A), and 153 were treated surgically between April 2006 and March 2007 (group B). All were operated at 3 surgical hospitals and, subsequently, transferred to 9 rehabilitation centers. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: We evaluated various factors, including their E-PASS scores to determine whether there was a correlation with the patients' mortality rate and their ability to walk at discharge and 1 year after surgery (group A). Using multiple regression analysis, we then developed algorithms to predict the ability of elderly patients to walk after hip fracture surgery. We applied the algorithms to group B patients and compared their actual and predicted ambulatory status. RESULTS: In group A patients, the postoperative walking ability and mortality rate were highly correlated with their E-PASS scores and dementia status. In group B, our algorithms exhibited good correlations between the predicted and actual walking ability at both time points (rho=0.6, P<.001). CONCLUSIONS: In candidates for hip fracture surgery, the E-PASS scores exhibited a good correlation with the patients' functional and survival prognoses, and the algorithm including E-PASS scores and dementia status can accurately estimate the ambulatory status at discharge and 1 year after surgery.

Vitrification of canine cumulus-oocyte complexes in DAP213 with a cryotop holder.

Purpose: The effects of the cryoprotectant and the container (holder) used for the vitrification of canine germinal vesicle stage oocytes were examined to improve the cryopreservation method for canine oocytes and embryos. Methods: Canine cumulus-oocyte complexes (COCs) were collected from ovaries, and were vitrified with E30S (30% ethylene glycol and 0.5 M sucrose) or DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol) solution held by a cryotube or cryotop sheets. After warming, the oocytes were stained with propidium iodide for the assessment of their plasma membrane integrity. Results: In all the vitrification groups, more than 65% of the vitrified oocytes displayed a normal morphology (E30S-top, 65.6%; DAP-tube, 67.3%; DAP-top, 80.0%). However, when assessed by propidium iodide staining, the viability of oocytes in the DAP-top group (43.6%) was higher than that in the E30S-top group (21.3%, P < 0.05). Furthermore, the viability of the oocytes in the DAP-top group (43.6%) was higher than that in the DAP-tube group (4.1%, P < 0.05). Conclusions: These results suggest that a combination of DAP213 as the cryoprotectant and a cryotop sheet as the holder improved viability after the vitrification of canine oocytes at the germinal vesicle stage.

Development of Small-Diameter Elastin-Silk Fibroin Vascular Grafts.

Globally, increasing mortality from cardiovascular disease has become a problem in recent years. Vascular replacement has been used as a treatment for these diseases, but with blood vessels <6 mm in diameter, existing vascular grafts made of synthetic polymers can be occluded by thrombus formation or intimal hyperplasia. Therefore, the development of new artificial vascular grafts is desirable. In this study, we developed an elastin (EL)-silk fibroin (SF) double-raschel knitted vascular graft 1.5 mm in diameter. Water-soluble EL was prepared from insoluble EL by hydrolysis with oxalic acid. Compared to SF, EL was less likely to adhere to platelets, while vascular endothelial cells were three times more likely to adhere. SF artificial blood vessels densely packed with porous EL were fabricated, and these prevented the leakage of blood from the graft during implantation, while the migration of cells after implantation was promoted. Several kinds of 13C solid-state NMR spectra were observed with the EL-SF grafts in dry and hydrated states. It was noted that the EL molecules in the graft had very high mobility in the hydrated state. The EL-SF grafts were implanted into the abdominal aorta of rats to evaluate their patency and remodeling ability. No adverse reactions, such as bleeding at the time of implantation or disconnection of the sutured ends, were observed in the implanted grafts, and all were patent at the time of extraction. In addition, vascular endothelial cells were present on the graft's luminal surface 2 weeks after implantation. Therefore, we conclude that EL-SF artificial vascular grafts may be useful where small-diameter grafts are required.

Discovery of DS-1971a, a Potent, Selective NaV1.7 Inhibitor.

A highly potent, selective NaV1.7 inhibitor, DS-1971a, has been discovered. Exploration of the left-hand phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane derivatives with high NaV1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral neuropathic pain, with a favorable safety profile.

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes.

PURPOSE: The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa. METHODS: After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated. RESULTS: The rates of oocyte activation were comparable (90.6-100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P < 0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa. CONCLUSIONS: These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.

Individual fertility differences in the frozen-thawed spermatozoa among semen donors in the Labrador Retriever.

Purpose: We aimed to validate and determine the possible application of transcervical insemination of frozen semen for improved breeding in guide dogs for the blind in Japan. Methods: From February 2004 to March 2007, a total of 53 Labrador Retriever bitches, used for the breeding of guide dogs for the blind, were transcervically inseminated with frozen-thawed semen from 13 males by means of a cystoscope. Results: The overall whelping and pregnancy rate with the frozen semen was 42%. Pregnancy rates ranged widely from 0 to 100% depending on the semen donor male. Of 13 males, 6 males exhibited severely poor fertility (less than 20% pregnancy rate) and 3 males exhibited high fertility (over 70% pregnancy rate) on artificial insemination. However, the spermatozoa motility after thawing was not significantly different among these dogs. In addition, heterospermic insemination revealed the optimal timing for transcervical insemination with frozen-thawed semen to be by day 6 after the LH surge. Conclusions: Although transcervical insemination of frozen-thawed semen is effective for breeding of guide dogs for the blind, some modification of freeze-thawing procedures might be required to overcome individual fertility differences in the frozen-thawed spermatozoa among semen donor dogs,. In addition, the motility of spermatozoa after thawing might not be an appropriate indicator of the relative fertility of frozen-thawed spermatozoa in dogs.