Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

Mycotic aneurysm caused by Graphium species in a dog

A 10-year-old castrated male mixed-breed dog exhibited vomiting, lethargy and anorexia. An abdominal ultrasound examination revealed a focal dilation of the abdominal aorta. The dog died 2 days after examination, and necropsy revealed a ruptured aneurysm at the abdominal aorta between the kidneys. Histological examination revealed severe granulomatous and necrotizing pan-arteritis with fungal organisms. Graphium species was identified through DNA sequence analysis of the PCR product from formalin-fixed paraffin-embedded samples. To our knowledge, this is the first report of aortic aneurysm caused by Graphium species in a dog.     

Gastrointestinal motility modulation by stress is associated with reduced smooth muscle contraction through specific transient receptor potential channel

Excessive stress response causes disability in social life. There are many diseases caused by stress, such as gastrointestinal motility disorders, depression, eating disorders, and cardiovascular diseases. Transient receptor potential (TRP) channels underlie non-selective cation currents and are downstream effectors of G protein-coupled receptors. Ca²⁺ influx is important for smooth muscle contraction, which is responsible for gastrointestinal motility. Little is known about the possible involvement of TRP channels in the gastrointestinal motility disorders due to stress. The purpose of this study was to measure the changes in gastrointestinal motility caused by stress and to elucidate the mechanism of these changes. The stress model used the water immersion restraint stress. Gastrointestinal motility, especially the ileum, was recorded responses to electric field stimulation (EFS) by isometric transducer. EFS-induced contraction was significantly reduced in the ileum of stressed mouse. Even under the conditions treated with atropine, EFS-induced contraction was significantly reduced in the ileum of stressed mouse. In addition, carbachol-induced, neurokinin A-induced, and substance P-induced contractions were all significantly reduced in the ileum of stressed mouse. Furthermore, the expression of TRPC3 was decreased in the ileum of stressed mouse. These results suggest that the gastrointestinal motility disorders due to stress is associated with specific non-selective cation channel.     

Atrazine intoxication in cattle,confirmed by gas chromatography–mass spectrometry

Ten of 40 cows died within 48 h of gaining access to a barn in which various chemicals were stored. Some of the surviving cows exhibited drooling, muscle tremors, and agitation. Postmortem examinations of 2 cows were performed in the field, and revealed nonspecific, moderate-to-severe pulmonary congestion. Liver and rumen contents, each from a different cow, were analyzed using a qualitative, multi-residue GC-MS method validated for the detection of pesticides and other chemical analytes. Using this method, extracts from the liver and rumen content samples were compared to atrazine (neat standard) and matrix-matched, control samples fortified with atrazine. GC-MS analysis detected atrazine at 215 m/z (NIST match >97%) with a retention time of ~13 min in liver and rumen content samples from our case. Detection of atrazine in the samples from the cows in this herd, combined with the clinical history, indicate that atrazine toxicity was the likely cause of clinical signs and death observed in this herd.     

Time-course comparison of pulmonary inflammation induced by intratracheal instillation of four different nickel oxide nanoparticles in male Fischer rats

Occupational exposure to nickel oxide (NiO) is an important cause of respiratory tract cancer. Toxicity is known to be associated with the dissociated component, i.e. nickel (II) ions. To address the relationship between physicochemical properties, including solubility in artificial lysosomal fluid, of NiO and time-course changes in the pulmonary response, we conducted an intratracheal instillation study in male Fischer rats using four different well-characterized NiO products, US3352 (NiO A), NovaWireNi01 (NiO B), I small particle (NiO C), and 637130 (NiO D). The NiOs were suspended in purified water and instilled once intratracheally into male F344 rats (12 weeks old) at 0 (vehicle control), 0.67, 2, and 6 mg/kg body weight. The animals were euthanized on days 3, 28, or 91 after instillation, and blood analysis, bronchoalveolar lavage fluid (BALF) testing, and histopathological examination were performed. The most soluble product, NiO B, caused the most severe systemic toxicity, leading to a high mortality rate, but the response was transient and surviving animals recovered. The second-most-soluble material, NiO D, and the third, NiO A, caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, NiO C induced barely detectable inflammation in the BALF examination, and no marked changes were noted on histopathology. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility.     

Cardiorespiratory and anesthetic effects produced by the combination of butorphanol,medetomidine and alfaxalone administered intramuscularly in Beagle dogs

This study evaluated anesthesia quality, degree of analgesia and cardiorespiratory parameters after intramuscular (IM) injection of a combination of butorphanol (0.1 mg/kg), medetomidine (10 µg/kg) and alfaxalone (1.5 mg/kg) in ten healthy adult Beagle dogs. Rectal temperature (T), heart rate (HR), respiratory rate (f_R), arterial pressure, arterial blood gases and M-mode echocardiographic left ventricular (LV) indices were measured before drug administration and every 10 min thereafter until extubation. Mean duration of anesthesia, recovery and analgesia were 89 ± 17, 6 ± 1 and 80 ± 12 min. HR, f_R, partial pressure of arterial CO₂ and O₂, arterial pressure, and LV contractility were significantly altered during anesthesia. IM administration of the drug combination provided acceptable anesthesia, but produced substantial cardiorespiratory suppression.     

Right ventricular systolic and diastolic function assessed by two-dimensional speckle tracking echocardiography in dogs with myxomatous mitral valve disease

Pulmonary hypertension (PH) is a common comorbidity in dogs with myxomatous mitral valve disease (MMVD), and can induce various changes in the right heart, such as right ventricular (RV) hypertrophy, dilatation, and dysfunction. We hypothesized that RV function, not only systolic function but also diastolic function, could be worsened with PH progression. We aimed to compare RV systolic and diastolic function in dogs with MMVD. Twenty healthy dogs and sixty-eight dogs with MMVD were enrolled. Dogs with MMVD were classified into the probability of PH. Two-dimensional and Doppler echocardiographic indices for right heart and two-dimensional speckle tracking echocardiography indices were measured. The morphological indicators of the right heart were significantly higher only in the high probability of PH group. The RV strain, early-diastolic and systolic strain rates were significantly lower in the high probability of PH group than those in the low and intermediate probability of PH groups. Multivariate analysis showed that increased RV internal dimension normalized by body weight and RV myocardial performance index were significantly associated with the presence of right-sided congestive heart failure. Speckle tracking echocardiography-derived RV systolic and diastolic function were activated in the low and intermediate probability of PH groups. However, dogs with high probability of PH showed RV myocardial dysfunction and dilatation. Increased RV myocardial performance index and end-diastolic RV internal dimension normalized by body weight were significantly associated with the presence of right-sided congestive heart failure in dogs with MMVD.     

Spontaneous Rhabdomyosarcoma in a Common Marmoset ( Callithrix jacchus )

The common marmoset (Callithrix jacchus) is now widely used in various research fields, including toxicology. However, information about the background pathology of this species is scarce. Here, we report a case of rhabdomyosarcoma that spontaneously occurred in a common marmoset. A 44-month-old male common marmoset was euthanized due to bilateral hind limb paralysis. At necropsy, a 2×2×5-cm intramuscular mass was observed in the lower right back. Histologically, the mass was mainly composed of interlacing bundles of spindle-shaped tumor cells. Immunohistochemically, the tumor cells were positive for myogenin, desmin, vimentin and alpha-smooth muscle actin. Ultrastructurally, the tumor cells contained bundles of myofilaments with Z-band-like structures. Thus, the tumor was diagnosed as a rhabdomyosarcoma. To our knowledge, this is the first report of spontaneous rhabdomyosarcoma that was definitely diagnosed in the common marmoset.     

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS–based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.     

Protectivity conferred by immunization with intranasal recombinant outer membrane protein H from Pasteurella multocida serovar A:1 in chickens

Recombinant outer membrane protein H (rOmpH) is a potential fowl cholera vaccine candidate. The present study was aimed at developing rOmpH formulations for intranasal administration. The rOmpH was purified and formulated with either Escherichia coli enterotoxin B (LTB) or CpG oligodeoxynucleotides (ODN) as an adjuvant. Antibody responses in chickens intranasally immunized with rOmpH in combination with 2 different adjuvants were significantly increased (P<0.05) post immunization. Chicken survival rates showed that rOmpH formulated with ODN and LTB elicited 90% and 70% protection, respectively. Our findings indicated that rOmpH formulated with ODN elicited protection better than that formulated with LTB. Therefore, the vaccines formulations in the present study can be considered new intranasal vaccine formulations for fowl cholera in chickens.