Cortisol and behavioral responses to enrichment in military working dogs

Our objective was to compare the effects of two schedules of enrichment exposure, a distributed (high frequency) and regular regime versus a mass (low frequency) and irregular regime, on the cortisol and behavioral responses of 14 kennelled military working dogs. The enrichment experiences were exercise coupled with human contact. Both groups of dogs were exposed to approximately the same total amount of enrichment per week for a total of 7 weeks. The distributed and regular regime (DRR) furnished enrichment for 20-min intervals three to four times a day every weekday; the mass and irregular regime (MIR) randomly furnished enrichment for 16 or 24 continuous hours 1 day out of 5. Our data show that: (1) cortisol concentrations of DRR dogs significantly decreased between the first and the last week of observations; this was not the case in MIR dogs; (2) at the 7th week DRR dogs had lower cortisol concentrations than did MIR dogs; (3) all dogs in each group exhibited stereotypies. Our results suggest that MIR dogs underwent a decline in welfare throughout the experiment, whereas DRR dogs appeared to undergo a period of acute stress at the beginning of their training that resolved over 7 weeks. This study suggests that the welfare of kennelled dogs, particularly military working dogs, is affected among other factors by schedules of exercise and contact with people.     

Polymorphism analysis of tyrosine hydroxylase (TH) in military working dogs

Canine and human behavior are shaped by similar evolutionary processes, yet the identification of the behavioral phenotype is often difficult. A widely used method relies on breed stereotypes provided by experts such as dog trainers. To reveal a valid association between behavior and genetic factors, an association study of behavioral phenotyping and genotyping is essential. We screened for variable number of tandem repeat (VNTR) polymorphisms in intron 4 of the tyrosine hydroxylase gene in military working dogs (belonging to pass and fail groups based on the results of an in-training examination conducted by the drillmaster), which were scored based on possessiveness, audaciousness, concentration, and motor ability by qualification examination. We first characterized each genotype by sequencing, in which the 1/1 type consists of a single copy of a 36-bp sequence and the 2/2 type is a duplicated form of the 36-bp repeat unit. The 1/2 alleles showed a single nucleotide change as a heteroduplex, which generated a PCR product of similar size as that of the 1/1-182-bp. The military working dogs showed the 2/2 type of VNTR and heteroduplex. For the pass group, two dogs possessed 2/2 type (40 %), whereas three dogs were of the heteroduplex type (60 %). However, all members of fail group showed the 2/2 type (100 %). These data indicate that repeat polymorphisms with behavioral phenotyping can identify military working dogs that would pass or fail the in-training examination.     

Heritability of semen characteristics in dogs

Retrospective analysis was performed on semen collected from 24 dogs (parents: 14 Labrador retrievers and 10 Golden retrievers) aged between 16 and 28 months of age. The dogs were part of a large breeding programme but lived in the homes of volunteer families. The semen was subjected to a standardised examination procedure including assessment of: percentage normal motility, sperm concentration, total sperm output, percentage of live normal sperm, and total number of live normal sperm. Semen was subsequently collected from one son of each of the parents when the offspring were aged between 16 and 28 mo (offspring: 14 Labrador retrievers and 10 Golden retrievers), and was subjected to the same examination procedures conducted by the same technician. Examination of breeding records demonstrated that each of the 48 dogs achieved at least one pregnancy within a period of 3 months before to 3 months after the semen collection.There was a weak correlation between parents and offspring for each of the 5 semen parameters, although none of these were statistically significant. Narrow sense heritability measures were low for all parameters except for the heritability of high sperm motility (rN2 = 0.57) and the heritability of low total sperm output (rN2 = 0.57).It is plausible that breeding selection procedures may be useful in dog breeding programmes in an attempt to improve semen quality, although any impact upon fertility is yet to be proven.     

Learning and owner-stranger effects on interspecific communication in domestic dogs (Canis familiaris)

Domestic dogs are very successful at following human cues like gazing or pointing to find hidden food in an object choice task. They solve this kind of situation at their first attempts and from early stages of their development and perform better than wolves. Most of the authors proposed that these abilities are a domestication product, and independent from learning processes. There are few systematic studies on the effects of learning on dogs’ communicative skills. We aim to evaluate the effect of extinction and reversal learning procedures on the use of the pointing gesture in an object choice task. The results showed that dogs stopped following the pointing cue in the extinction and that they learned to choose the not pointed container in the reversal learning. Results suggest that instrumental learning plays an important role in interspecific communication mechanisms between humans and dogs. In both experiments for half of the subjects the pointer was the owner and for the rest was a stranger. A differential effect was found: extinction was slower but reversal learning was faster when the owner gave the cue. This data indicates that the relationship of the dog with the person who emits the cue influences performance.     

Seasonal study of the fungal biota of the fur of dogs

During a one year period, 944 dogs from the Municipal kennel of Barcelona were examined to detect animals with suspected dermatophytosis. Only a few animals (1.8%) presented skin lesions but none of them had dermatophytosis. A representative number of dogs without visible skin lesions (n=172), selected at random, were used to carry out a seasonal study of the mycobiota of their fur. Fifteen isolates belonging to the genera Microsporum and Trichophyton were isolated from 14 of the 172 (8.1%) dogs without lesions. The identity of these fungi was Microsporum gypseum (6/15), Trichophyton terrestre (4/15), M. canis (2/15), M. cookei (2/15) and Trichophyton ajelloi (1/15) (one strain each of M. gypseum and T. ajelloi were isolated from one dog). Species of Penicillium (% prevalence=89.5%), Alternaria (86.6%), Cladosporium (84.9%), Aspergillus (77.3%), Scopulariopsis (65.7%) and Chrysosporium (64.5%) were the most prevalent. No significant differences in the fungal biota were observed with respect to age, gender, hair length or between mixed and pure breed dogs. A large number of isolates, including species belonging to the genera Beauveria, Chrysosporium, Malbranchea and Scopulariopsis, that macroscopically and/or microscopically resemble dermatophytes and may be mistaken for them, produced a red color change in Dermatophyte Test Medium. No significant seasonal difference was detected among the isolates belonging to the the most frequently encountered genera, with the exception of Scopulariopsis (higher in summer and autumn) and Chrysosporium (higher in summer). Species from other genera, with lower occurrence also presented significant differences in their seasonal distribution. Arthrinium, Aureobasidium, Chaetomium and Phoma spp. presented maximum prevalence peaks in spring, Fusarium, Paecilomyces, Phoma and Rhizopus spp. in summer and Geotrichum and Mucor spp. in autumn. The Microsporum and Trichophyton species were more frequently isolated in summer.     

Spantide: failure to antagonize bombesin-induced stimulation of gastrin secretion in dogs

Spantide ([d-Arg1, d-Trp7,9, Leu11] substance P) was shown to function not only as a substance P receptor antagonist but also as a bombesin receptor antagonist. This study examined the effects of spantide on intravenous bombesin-induced stimulation of gastrin and acid secretion. Dogs were infused with spantide (1 or 10 nmol kg 1 hr 1) or saline and bombesin (60 pmol kg-1 hr-1), and the gastric acid and plasma gastrin responses were monitored. Spantide did not significantly modify gastrin or gastric acid secretion induced by bombesin. It is concluded that spantide may not be a useful bombesin antagonist for in vivo studies.     

Association of inflammatory markers elevation with aggressive behavior in domestic dogs

We tested the hypothesis that elevations of inflammatory markers such as C-reactive protein (CRP) and interleukin-6 (IL-6) could be associated with the presence of aggressive behavior in domestic dogs. Serum concentrations of CRP and IL-6 were determined by ELISA in eighteen adult male German Shepherd dogs showing no clinical signs but aggression. Eighteen healthy male dogs with a negative history of behavioral and neurological disorders were used as controls. Compared with normal dogs, those with aggression had significantly higher levels of CRP (P < 0.05) and IL-6 (P < 0.01) after adjustment for age, body weight, creatinine, blood urea nitrogen, total protein, total bilirubin and cholesterol. Our pilot data suggest for the first time that an activation of systemic inflammatory processes may contribute to the pathophysiology of aggression in domestic dogs. Further investigations are needed regarding the impact of our findings on treatment strategies.     

Update on the treatment and prevention of ocular thelaziosis (Thelazia callipaeda) in naturally infected dogs from Spain

This study examines the therapeutic and year-round prophylactic efficacy of different formulations used in dogs in three Spanish areas where canine thelaziosis is endemic. The study was conducted as a Good Clinical Practice, multicentre, randomised field study in privately owned outdoor dogs naturally infected with Thelazia callipaeda. The active pharmaceutical ingredients tested were: an oral formulation of milbemycin oxime 12.5 mg combined with praziquantel 125 mg (A), a subcutaneous sustained-release formulation of moxidectin 10 g (B), a moxidectin 2.5% weight/volume (w/v) spot-on formulation combined with imidacloprid 10% w/v (C), and an eye drop formulation (6 µg) of ivermectin 10 mg/ml diluted 10% in propylene glycol (D). Infected dogs were randomly allocated to treatment Groups A, B, C and D. Dogs testing negative for T. callipaeda inspection in two visits (Day 7/Day 14 and D30) were enrolled in the prophylaxis trial and reallocated to the corresponding study group (A, B, C or D). Treatment efficacy ranged from 70.4% recorded in Group A 1 week after treatment, to 100% recorded in Group C on Day 30 and in Group B on Day 60. Treatment was more efficacious in Group D (85.7% 1 week after treatment) than A, but was never 100% efficacious as in Groups B and C. Year-round prophylactic efficacy was 83.3% in Group A, 100% in Group B, 93.5% in Group C and 87.5% in Group D. In conclusion, products containing moxidectin were highly efficacious both in treating and preventing canine thelaziosis. Milbemycin also emerged as a good option. However, the off-label use of topical or subcutaneous ivermectin should be avoided due to possible adverse reactions such as pruritus, irritation or redness. In endemic areas, monthly prophylaxis to limit the spread of T. callipaeda to new areas across Europe and reduce zoonotic risks is essential.     

Age-dependent changes in uptake and recycling of ascorbic acid in erythrocytes of Beagle dogs

Uptake and recycling of ascorbic acid (AA) were studied in erythrocytes of 1- to 12-month-old Beagle dogs. At 1 month, both AA uptake and recycling capacity were high. Ascorbic acid entered erythrocytes mainly in the oxidized form with elevated activity of Glut 1 glucose transporter. However, this trait of erythrocytes was rapidly lost in the course of postnatal growth. At 3 months, ascorbic acid uptake and recycling capacity decreased to almost adult levels. Thereafter, AA was transported mainly in the reduced form, and its uptake and recycling capacity became one-third the levels of 1-month-old dogs. Postnatal anemia and recovery were indicated by changes in hemoglobin and packed cell volume levels at 1 and 3 months. Glutathione reductase (GR) activity was twice as high as in adults in 1-month-old dogs, allowing efficient reduction of oxidized ascorbic acid, which enters cells in large amounts due to elevated activity of the Glut 1 glucose transporter. One-month-old dogs need high levels of AA for antioxidant protection and skeletal development. The high AA recycling capacity of erythrocytes is considered to balance the expenditure of AA in young Beagle dogs.     

The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine (Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.     

Current status and applications of somatic cell nuclear transfer in dogs

Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications.     

浸提条件对小麦秸秆中化感物质检测结果的影响

通过对比不同浸提条件下小麦秸秆中化感物质的检测结果,发现浸提温度和时间对化感物质的最终结果有很大的影响,在室温条件下,化感物质的量随着浸提时间的延长而增长,但在50℃时,随着浸提时间的延长,化感物质的量反而降低。化感物质最大量在50℃,24h浸提条件下得到。在高温高压条件下提取到的化感物质的量多于多数条件下得到的量（除少于50℃,24h浸提条件下得到的量）。试验结果表明随着漫提温度升高,植物材料中的化感物质在水中的溶解速度加快,但是性质不稳定,容易分解变性。     

Release of VIP-like immunoreactivity in response to dopamine agonists, insulin hypoglycemia and feeding in conscious dogs

VIP levels were measured by radioimmunoassay in peripheral venous blood of conscious dogs. Bolus injections of the dopamine agonists apomorphine, 0.05 mg/kg, and bromocriptine, 0.2 mg/kg, were found to increase VIP levels from approximately 5 pmol/l to 150 pmol/l. The release responses were abolished by pretreatment with dopamine antagonists (haloperidol 0.1 mg/kg or halopemid 0.1 mg/kg) and by hexamethonium (1 mg/kg) a blocker of ganglionic transmission. Vagotomy did not inhibit the dopamine agonist induced output of VIP. Vagal activation by means of feeding or insulin hypoglycemia caused only minor rises of VIP levels (5-10 pmol/l). It is concluded that dopamine agonists stimulate the release of VIP from populations of neurons other than those affected by vagal and sympathetic activation. Possible sites of action for the VIP releasing effect exerted by dopamine agonists are discussed. Furthermore, it is suggested that some of the peripheral effects exerted by dopaminergic drugs are exerted via a previous release of VIP.     

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.     

Meteorological indicators for Amblyomma cajennense and population dynamics in the Tamaulipan Biotic Province in Texas

We studied the population dynamics of free‐living ticks in the Tamaulipan Biotic Province in south Texas from March, 2005 to November, 2008. We collected 70,873 ticks using carbon dioxide traps. Amblyomma cajennense represented 93.6% of the ticks identified. A. cajennense is distributed from northern Argentina to south Texas in the Tamaulipan Biotic Province. Emergence of larval A. cajennense ticks was observed two to five weeks after significant rain events (p<0.0001) and had a strong negative correlation with temperature (p<0.0001). More larvae were observed under humid conditions (p<0.05). Fewer larvae were observed during windy and warmer conditions (p<0.05). This observation indicates high sensitivity of larvae to desiccating conditions. Peaks in nymphal activity were observed after peaks of larval emergence. Activity of nymphs was negatively correlated with temperature (p<0.05). Adult activity was negatively correlated with humidity (p<0.05) and negatively correlated with total rain from three to six weeks prior to observation (p<0.05). Adult A. cajennense are particularly tolerant to drier conditions relative to other closely related ticks. Adult female activity was positively correlated with temperature (p<0.05). Peaks in rain activity and a summer behavioral diapause appear to be the dominant factors controlling emergence of larvae, and by extension, the life cycle of A. cajennense in the Tamaulipan Biotic Province.     

多因素对昆明小鼠胚胎干细胞原代集落形成和后期增殖的影响

目的：研究细胞因子LIF、EGF、bFGF和肝素钠,以及氧分压、温度等多因素对昆明系小鼠胚胎干细胞（KM-ESC）原代集落形成和后期增殖的影响。方法：本研究选择LIF、EGF、bFGF、肝素钠、5%O2,20%O2和37℃、39℃作为研究条件。并检测不同情况下小鼠胚胎干细胞原代集落形成和后期增殖情况。结果：LIF对KM-ESC原代集落形成和后期增殖具有显著促进作用,极显著高于EGF、bFGF和肝素钠组（P〈0.01）。温度对KM-ESC原代集落形成和增殖具有显著影响,39℃条件下,原代集落形成率、直径和后期增殖显著高于37℃（P〈0.05）;而氧分压对KM-ESC原代集落形成无显著作用（P〉0.05）,但是对原代集落直径和后期增殖有一定促进作用,20%O2组显著高于5%O2组（P〈0.05）。结论：LIF、EGF、bFGF、肝素钠、39℃、20%O2对小鼠胚胎干细胞原代集落形成和后期增殖具有显著促进作用。     

Toxoplasma gondii: Evidence for the transmission by semen in dogs

Ten male dogs were distributed into three experimental groups for infection with Toxoplasma gondii: GI - three dogs inoculated with 2.0 × 10⁵ P strais oocysts, GII - three dogs infected with 1.0 × 10⁶ RH strain tachyzoites, and GIII - four controls dogs. Several clinical parameters were evaluated. IFAT was performed to detect anti-T. gondii antibodies. Presence of the parasite in semen was evaluated by PCR and bioassay techniques. Tissue parasitism was examined using bioassays and immunohistochemistry in testicle and epididymis fragments collected after orchiectomy. In semen samples collected from these two groups, the presence of T. gondii was verified by bioassays and PCR. T. gondii was detected by immunohistochemistry in tissues (testicle and epididymis fragments) of all six experimentally infected dogs. The T. gondii-positive seminal samples were used in the artificial insemination (AI) of four female dogs free of toxoplasmic infection. Seven days after AI, all of the female dogs presented serologic conversion (IFAT). Fetal reabsorption occurred in two of the dogs, while the others sustained full-term gestation. Several T. gondii cysts were detected in the brains of four offspring. These results suggest that T. gondii can be sexually transmitted in domestic dogs.     

Overexpression of cathepsin B in gastric cancer identified by proteome analysis

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.