Effects of Para-Toluenesulfonamide on Canine Melanoma Xenotransplants in a BALB/c Nude Mouse Model.

The pharmacological pathway of para-toluenesulfonamide (PTS) restricts the kinase activity of the mammalian target of rapamycin, potentially leading to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical effect on tumorigenesis. We aimed to examine the antitumor effect of PTS or PTS combined with cisplatin on canine melanoma implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. The mice were randomly divided into four groups: control, cisplatin, PTS, and PTS combined with cisplatin. Mice treated with PTS or PTS combined with cisplatin had retarded tumor growth and increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase phosphorylation, decreased inflammatory cytokine levels, reduced inflammation-related factors, enhanced anti-inflammation-related factors, and inhibition of metastasis-related factors. Mice treated with PTS combined with cisplatin exhibited significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with those treated with cisplatin or PTS alone. PTS or PTS combined with cisplatin could retard canine melanoma growth and inhibit tumorigenesis. PTS and cisplatin were found to have an obvious synergistic tumor-inhibiting effect on canine melanoma. PTS alone and PTS combined with cisplatin may be antitumor agents for canine melanoma treatment.

WITHDRAWN: Correlation of SPINK1 gene polymorphisms in canine pancreatitis.

Ahead of Print article withdrawn by publisher.

Handmade trileaflet valve design and validation for patch-valved conduit reconstruction using generalized regression machine learning model.

Pulmonary valve diseases include the different degrees of aortic stenosis or congenital defects in children or adults. Valve repair or replacement surgery is commonly performed to relieve valvular dysfunction and improve the significant flow regurgitation in the aortic valve and the pulmonary valve. However, commercial valve stents and valved conduits are sometimes not available for children or patients with special conditions. The handmade trileaflet valve design has been used with different range of diameters for patch-valved conduit reconstruction. Thus, we propose a multiple regression model, as a generalized regression neural network (GRNN), to determine the optimal trileaflet parameters, including the width, length, and upper lower curved structure. Through computed tomography pulmonary angiography, while the diameter of the main pulmonary artery is determined, a leaflet template can be rapidly sketched and made. Using an experimental pulmonary circulation loop system, the efficacy of the valved conduit can be validated using the regurgitation fraction method. In contrast to commercial valve stents, experimental results indicate that the handmade trileaflet valve can also improve severe pulmonary regurgitations.

Benefits of Intraluminal Agarose Stents during End-to-End Intestinal Anastomosis in New Zealand White Rabbits.

In the present study, we evaluated the utility of an intraluminal agarose stent (IAS) for end-to-end intestinal anastomoses in rabbits. Female New Zealand white rabbits (n = 14) underwent conventional sutured anastomosis (CSA) with or without an IAS. IAS were used to maintain the luminal diameter for more rapid and accurate suturing, and then was squeezed transluminally to crush it into fragments, which passed through the intestines and were eliminated. The rabbits were euthanized on postoperative day 21. At necropsy, the anastomoses were assessed for adhesion formation, stenosis, and bursting pressure and were examined histologically for collagen content and blood vessel formation. Anastamosis surgery took less time in the IAS group (15.0 ± 2.6 min) than in the CSA-only group (30.1 ± 7.9 min). Only 1 postoperative death occurred (in the CSA group), and postmortem examination revealed evidence of anastomotic leakage. Adhesion formation and stenosis did not differ between groups, but bursting pressure, collagen content, and blood vessel formation were all significantly increased in the IAS group. IAS may decrease the operative time by maintaining a clear surgical field at the anastomotic site. In addition, the use of IAS promotes rapid healing and maintains the luminal diameter during end-to-end intestinal anastomosis.

Development of a sedation protocol using orally administered tiletamine-zolazepam-acepromazine in free-roaming dogs.

OBJECTIVE: To investigate the sedative effects in dogs of tiletamine-zolazepam-acepromazine (TZA) or ketamine-flunitrazepam (KF) administered orally and to evaluate the effectiveness of encapsulated TZA for capturing free-roaming dogs. STUDY DESIGN: Experimental study followed by a field trial. ANIMALS: Six research dogs and 27 free-roaming dogs. METHODS: In a pilot study, six research dogs were administered liquid TZA (20 mg kg-1 tiletamine-zolazepam and 2 mg kg-1 acepromazine) or liquid KF (50 mg kg-1 ketamine and 2 mg kg-1 flunitrazepam) orally: treatment 1, forcefully squirting liquid medication into the mouth; treatment 2, encapsulating liquid medication for administration in canned food; treatment 3, administering liquid medication mixed with gravy. Sedation was scored. A follow-up field trial attempted capture of 27 free-roaming dogs. RESULTS: In the pilot study, the median time (range) to lateral recumbency (% dogs) after TZA administration was: treatment 1, 47.5 (35-80) minutes (67%); treatment 2, 30 (15-65) minutes (83%); and treatment 3, 75 (45-110) minutes (100%). No dogs in KF treatment 2 or 3 achieved lateral recumbency. Based on these results, 20 free-roaming dogs were offered encapsulated TZA in canned food: TZ (20 mg kg-1) and acepromazine (2 mg kg-1). Of these, no further drugs to four dogs (one dog captured), 10 dogs were administered a second dose within 30 minutes (five dogs captured) and six dogs were administered TZ (5 mg kg-1) and xylazine (1.1-2.2 mg kg-1) intramuscularly by blow dart (six dogs captured). Seven dogs were initially offered twice the TZA dose (five dogs captured). In total, 63% free-roaming dogs were captured after administration of encapsulated TZA in canned food. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of encapsulated TZA in canned dog food can aid in the capture of free-roaming dogs, but additional drugs may be required. The sedation onset time and medication palatability influenced the capture rate.

An unusual case of thoracic ectopia cordis in a Toggenburg Goat and its three-dimensional images constructed with X-ray computed tomography.

A two-day-old female Toggenburg goat with thoracic ectopia cordis (EC) was diagnosed via radiography and computed tomography. The goat was born with EC, defects of the sternum and a supra-umbilical abdominal wall, but without the presence of Cantrell's syndrome. Necropsy and histopathological findings indicated the affected kid had malformation of the heart with an enlarged left ventricle. The findings showed the heart (9 x 5 x 5 cm) stayed outside the thorax, and was covered by a semitransparent membrane. This report is the first to describe a case of thoracic EC in a goat whose sternum was not developed fully and was not connected to the ribs. It is also the first paper to describe three-dimensional images of this condition constructed from computed tomography scans.

Effects of different cryoprotectants and freezing methods on post-thaw boar semen quality.

The current study aimed to investigate the effects of different concentrations of glycerol (0%, 1%, 2%, 3%, and 5%) and dimethylacetamide (DMA: 0%, 1%, 3%, and 5%) on post-sperm quality characteristics following semen freezing in dry ice (D) or liquid nitrogen (N). Semen was collected from Duroc boars and was allocated to 32 treatment groups for cryopreservation. Analysis of post-thaw semen quality and fertility after artificial insemination (AI) was used to examine the combinatorial effects of different treatments. The best scores for post-thaw sperm motility, sperm viability, and sperm acrosomal integrity were observed in semen frozen in: (a) dry ice in the presence of 5% glycerol and no DMA (16D-treatment); (b) dry ice in the presence of 3% glycerol and no DMA (9D-treatment); and (c) liquid nitrogen in the presence of 3% glycerol and 1% DMA (10N-treatment), with no significant difference observed among these three treatments. The farrowing rates after AI with post-thawed semen after 9D- and 10N-treatments were 33% and 50%, respectively. To summarize, the results of the present study indicated that the freezing extender containing 3% glycerol in combination with the straw-freezing method using dry ice produced the best post-thaw quality parameters of boar semen. Combinations of glycerol and DMA did not enhance the cryosurvival of boar spermatozoa.

Y chromosome analysis of dingoes and southeast asian village dogs suggests a neolithic continental expansion from Southeast Asia followed by multiple Austronesian dispersals.

Dogs originated more than 14,000 BP, but the location(s) where they first arose is uncertain. The earliest archeological evidence of ancient dogs was discovered in Europe and the Middle East, some 5-7 millennia before that from Southeast Asia. However, mitochondrial DNA analyses suggest that most modern dogs derive from Southeast Asia, which has fueled the controversial hypothesis that dog domestication originated in this region despite the lack of supporting archeological evidence. We propose and investigate with Y chromosomes an alternative hypothesis for the proximate origins of dogs from Southeast Asia--a massive Neolithic expansion of dogs from this region that largely replaced more primitive dogs to the west and north. Previous attempts to test matrilineal findings with independent patrilineal markers have lacked the necessary genealogical resolution and mutation rate estimates. Here, we used Y chromosome genotypes, composed of 29 single-nucleotide polymorphism (SNPs) and 5 single tandem repeats (STRs), from 338 Australian dingoes, New Guinea singing dogs, and village dogs from Island Southeast Asia, along with modern European breed dogs, to estimate the evolutionary mutation rates of Y chromosome STRs based on calibration to the independently known age of the dingo population. Dingoes exhibited a unique haplogroup characterized by a single distinguishing SNP mutation and 14 STR haplotypes. The age of the European haplogroup was estimated to be only 1.7 times older than that of the dingo population, suggesting an origin during the Neolithic rather than the Paleolithic (as predicted by the Southeast Asian origins hypothesis). We hypothesize that isolation of Neolithic dogs from wolves in Southeast Asia was a key step accelerating their phenotypic transformation, enhancing their value in trade and as cargo, and enabling them to rapidly expand and replace more primitive dogs to the West. Our findings also suggest that dingoes could have arrived in Australia directly from Taiwan, independently of later dispersals of dogs through Thailand to Island Southeast Asia.

Phylogenetic distinctiveness of Middle Eastern and Southeast Asian village dog Y chromosomes illuminates dog origins.

Modern genetic samples are commonly used to trace dog origins, which entails untested assumptions that village dogs reflect indigenous ancestry or that breed origins can be reliably traced to particular regions. We used high-resolution Y chromosome markers (SNP and STR) and mitochondrial DNA to analyze 495 village dogs/dingoes from the Middle East and Southeast Asia, along with 138 dogs from >35 modern breeds to 1) assess genetic divergence between Middle Eastern and Southeast Asian village dogs and their phylogenetic affinities to Australian dingoes and gray wolves (Canis lupus) and 2) compare the genetic affinities of modern breeds to regional indigenous village dog populations. The Y chromosome markers indicated that village dogs in the two regions corresponded to reciprocally monophyletic clades, reflecting several to many thousand years divergence, predating the Neolithic ages, and indicating long-indigenous roots to those regions. As expected, breeds of the Middle East and East Asia clustered within the respective regional village dog clade. Australian dingoes also clustered in the Southeast Asian clade. However, the European and American breeds clustered almost entirely within the Southeast Asian clade, even sharing many haplotypes, suggesting a substantial and recent influence of East Asian dogs in the creation of European breeds. Comparison to 818 published breed dog Y STR haplotypes confirmed this conclusion and indicated that some African breeds reflect another distinct patrilineal origin. The lower-resolution mtDNA marker consistently supported Y-chromosome results. Both marker types confirmed previous findings of higher genetic diversity in dogs from Southeast Asia than the Middle East. Our findings demonstrate the importance of village dogs as windows into the past and provide a reference against which ancient DNA can be used to further elucidate origins and spread of the domestic dog.

Detection of matrix metalloproteinase (MMP)-2 and MMP-9 in canine seminal plasma.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality.

Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan.

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our laboratory recently confirmed the first case of encephalitozoonosis in a rabbit in Taiwan; the prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being infected. This study surveys the seropositivity of E. cuniculi using carbon immunoassay (CIA) and enzyme-linked immunosorbent assay (ELISA). Serological examination of 171 rabbits using CIA and ELISA showed that 63.2% (108/171) and 67.8% (116/171) were seropositive against E. cuniculi, respectively. Thirteen of the 14 rabbits (92.9%) with neurological symptoms were seropositive. Except for gender, health status and location had a significant effect on E. cuniculi seropositivity (p<0.05). Adult rabbits aged older than 4 months exhibited significantly higher seropositivity for E. cuniculi than young rabbits (p<0.05). In conclusion, this study shows that E. cuniculi is present and widespread among healthy rabbits in Taiwan. Therefore, the fields of veterinary and human medicine in Taiwan should be aware of this zoonotic issue and the resulting public health concern of encephalitozoonosis.

Expression of progesterone receptor(s) during capacitation and incidence of acrosome reaction induced by progesterone and zona proteins in boar spermatozoa.

Sperm acrosome reaction (AR) is a prerequisite step for in vivo fertilization. In the vicinity of the oocyte, zona protein(s) (ZP) and progesterone (P4), a component of follicular fluid, are proven to be responsible for physiological AR induction. In the present study, a thorough analysis of the role of the progesterone receptor (PR) in this processing including in vitro physiological studies and biochemical isolation and characterization of the receptor protein was conducted. Following capacitation for 0, 2, 4 and 6h, pooled fertile boar semen samples (n=6) with >70% sperm motility were labeled with P4-BSA-FITC (100 microg/ml) to detect the activation of PR. Parallel sperm samples were treated with P4 (10 microg/ml) for 20 min to test AR inducing efficiency at different time points. To compare the ability of ZP and P4 to induce AR, spermatozoa capacitated in a modified medium supplemented with 1mg/ml heparin for 4h, were then treated with heat solubilized ZP (150 microg/ml), P4 (10 microg/ml) or ZP+P4 for 20 min. FITC-peanut agglutinin staining was applied to observe the disrupt acrosomal morphology. A purification protocol for crude boar sperm membrane proteins was developed based on ligand-receptor affinity chromatography procedures. The PR proteins were then identified by using mAb C262 raised against intracellular PR, combined with second antibody (SDS-PAGE, Western blotting). Their N-terminal amino acid sequence was determined. The amount of PR-activated spermatozoa was enhanced with time (onset: 27+/-5%, 2h: 41+/-4%, 4h: 49+/-3% and 6h: 52+/-4%, mean+/-S.E., n=6) as evidenced by increasing percentage of spermatozoa with completed cap fluorescent staining. In parallel sperm samples, percentages of AR induced by P4 were 9+/-2, 14+/-2, 18+/-2, and 24+/-2%, respectively. In solvent control at all time points, less than 10% spermatozoa had undergone AR. Capacitation for 4h or greater time periods resulted in optimal percentage of PR-activated and acrosome-reacted spermatozoa. After sperm incubation in heparin-medium, ZP+P4 treatment induced greater amounts of AR than either P4 or ZP alone (13+/-1% compared with 8+/-1 and 10+/-1%, P<0.01). Inducing capacity of P4 was comparable to that of ZP. The molecule weights of two apparent PR molecular masses were detected to be at Mr 74 kDa and Mr 63 kDa. N-terminal amino acid sequence of 74 kDa protein was XPXNIVLIFADXLXY, which had 78% homology to arylsulfatase A and 88% homology to 72 kDa protein from boar spermatozoa. The activation of PR is associated with the capacitating process and that appears to be required for P4-induced AR. P4 and ZP appear to be equally capable of independently inducing the AR but lack synergetic or additive effects in this induction process. Both might represent alternative pathways thus resulting in alternative systems for induction of the prerequisite acrosomal exocytosis (supported by NSC 90-2313-B-005-114; 91-2313-B-005-131).

Characterisation of the progesterone receptor on canine spermatozoa.

The present study was conducted to characterise and localise the progesterone receptor (PR) on canine spermatozoa. Using a progesterone-bovine serum albumin-fluorescein isothiocyanate conjugate (PBF) and different monoclonal antibodies (C262 and NCL-PGR against the steroid binding domain and N-terminus of intracellular PR, respectively, and h151 against the hinge domain of the intracellular oestrogen receptor), the PR was identified on the plasma membrane over the acrosomal region. Two proteins (54 kDa and 65 kDa) were detected by recognition of the three monoclonal antibodies using Western blotting. PBF labelling was observed in the majority of cauda epididymal spermatozoa (63 +/- 4%), but this labelling was markedly reduced (33 +/- 17%) after the addition of canine seminal plasma. Over a 7-h capacitation, the proportion of ejaculated spermatozoa exhibiting PBF labelling (indicating the presence of the PR) increased from 18 +/- 10% (onset) to 59 +/- 7% by 5 h, where it plateaued. Progesterone (P 4 ) induced the acrosome reaction (AR) in a dose-dependent manner (0, 0.1, 1 and 10 ug/mL P 4 corresponding to 10 +/- 5%, 16 +/- 9%, 23 +/- 7% and 30 +/- 7%). Pre-treatment of capacitated spermatozoa with canine seminal plasma reduced the incidence of the P 4 -induced AR (12 +/- 5%). In addition, treatment with the monoclonal antibodies significantly reduced the incidence of the P 4 -induced AR (10 microg/mL) in capacitated ejaculated spermatozoa from 19 +/- 6% to 11 +/- 4% (h151, 1 : 10) and 12 +/- 6% (C262, 1 : 10), respectively. A typical Scatchard plot revealed one binding with high affinity and low capacity, and another binding with low affinity and high capacity, suggesting at least two different characteristic PR. Taken together, these results demonstrate that P 4 induced the AR in a dose-dependent manner via functional transmembranal receptors in the acrosomal region of the canine sperm plasma membrane. The characteristics of this membrane receptor seem similar to those of other mammalian spermatozoa, and it shows structural homology to the intracellular PR.

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone.

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer.

This study investigates the efficacy of five extenders in contributing to the outcome of semen cryopreservation in Formosan Sika and Sambar deer. Pooled semen (n=4) of six males of each breed was used. In Sika deer, semen collection rate was 96% (23/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 0.5+/-0.4 ml, 77+/-6% and 1471.3+/-940.0 x 10(6) ml(-1), respectively. Post-thaw motility in respective extender was A: 66+/-16%; B: 71+/-2%; C: 73+/-6%; D: 9+/-4% and E: 26+/-12% (mean+/-S.D.). In extender C (74+/-14%) more viable spermatozoa were preserved than in the others (A: 64+/-10%; B: 48+/-11%; D: 41+/-16%; E: 47+/-6%; P<0.05). Acrosomal integrity was not influenced by extender composition. Post-thaw motility did not decrease during a 4-h incubation period, irrespective of the extender used (P>0.05). In Sambar deer, semen collection rate was 88% (21/24) over all electro-ejaculations. Volume, sperm motility and sperm concentration of fresh ejaculates was 1.3+/-0.5 ml, 82+/-4% and 379.1+/-252.2 x 10(6) ml(-1), respectively. Post-thaw motility was in respective extenders A: 69+/-2%; B: 74+/-6%; C: 73+/-2%; D: 13+/-6% and E: 31+/-20%. Extenders B and C were superior (P>0.05) with respect to sperm motility. Similarly, post-thaw viability in extenders A (70+/-7%), B (76+/-7%) and C (79+/-2%) was higher than that D (25+/-19%) and E (29+/-17%) (P<0.01). Sperm acrosomal integrity was better preserved in extenders B (86+/-4%) and C (83+/-4%) than in extenders A (54+/-13%), D (39+/-22%) and E (46+/-22%) (P<0.05). Post-thaw sperm longevity in extender A reduced from 69 to 16% during incubation (P<0.05) whereas only a slight decrease was observed in the other extenders after 4 h. In conclusion these data show that egg-yolk-Tris-Tes-glycerol based extender C containing Equex STM paste is optimal for freezing semen of Formosan Sika deer while egg-yolk-Tris-citric acid-glycerol based extender B containing Equex and extender C are superior in semen cryopreservation to others for Formosan Sambar deer.