Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Potential risks,prognostic indicators,and diagnostic and treatment modalities affecting survival in dogs with presumptive aspiration pneumonia: 125 cases (2005–2008)

Objective – To evaluate a clinical population of dogs diagnosed with presumptive aspiration pneumonia (AP) and determine diagnostic and treatment modalities contributing to survival. Design – Retrospective study. Setting – A university veterinary teaching hospital in an urban setting. Animals – One hundred and twenty‐five dogs with presumed AP treated from 2005 to 2008. Interventions – None. Measurements and Main Results – Dogs with presumptive AP identified by a review of medical records had an overall survival of 81.6% (102/125). Male large‐breed dogs (mean 24.9 kg; 82/125) were overrepresented and were more likely to develop AP in this study population. Recent anesthesia had been performed in 16% (20/125), and vomiting was reported in 64% (80/125). The most common radiographic findings were a predominantly alveolar pattern (187/272, [68.8%] total lung lobes) in the right middle lung lobe (80/115, [69.6%]). A mean of 2 lung lobes were involved radiographically, and the relationship between survival and the number of lung lobes affected was statistically significant (P=0.04). Neutrophilia with a left shift was common with no significant change on consecutive daily evaluations. The mean PaO₂ was 77.7 mm Hg (SD, 17.5 mm Hg) (range, 40.7–100 mm Hg) with a median alveolar‐arterial gradient of 41.1 mm Hg (range, 8.1–81.8 mm Hg). In this study population, 37.6% (47/125) of dogs had microbial cultures performed and of these, 76.6% (36/47) were positive for growth; Escherichia coli (38.8%), Mycoplasma spp. (21.3%), Pasturella spp. (19.1%), and Staphylococcus spp. (17%) were the most common isolates in either single or multiagent infections. No treatment modality was statistically associated with increased survival. Colloid therapy was a negative prognostic indicator. Conclusions – In this study the overall prognosis for AP was good. Patients with only 1 affected lung lobe appeared more likely to survive. Supportive treatment modalities are warranted for the hospitalized patient, although no individual treatment method was found to be clearly superior to others.     

Mitochondrial dysfunction influences apoptosis and autophagy in porcine parthenotes developing in vitro

Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H(2)O(2) to induce mitochondrial stress, examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 μM H(2)O(2) but was significantly decreased in the group supplemented with 500 μM H(2)O(2) (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 μM H(2)O(2). Furthermore, addition of H(2)O(2) (100 μM and 500 μM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real-time RT-PCR showed that H(2)O(2) significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H(2)O(2) had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1, in blastocysts. These results suggest that H(2)O(2) leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.     

Lipogenic gene expression in abdominal adipose and liver tissues of diet-induced overweight cats

The effect of overweight status on the expression of SREBP-1c and downstream lipogenic genes, such as ATP citrate lyase (ACL) and fatty acid synthase (FAS), in abdominal adipose and liver tissues was determined in cats using a diet-induced weight gain model. ACL and SREBP-1c mRNA expression was significantly reduced (～65% and 20%, respectively) in liver tissue, whereas FAS and SREBP-1c expression was significantly increased (～80% and 45%, respectively) in abdominal omental adipose tissue of overweight animals as compared to healthy animals. Additionally, ACL, FAS, and SREBP-1c expression was significantly reduced by ～50%, 75%, and 70%, respectively, in abdominal subcutaneous adipose tissue of overweight animals. Omental adipose tissue appeared to foster, whereas subcutaneous adipose and liver tissues appeared to defer lipid storage based on differences in SREBP-1c mRNA expression. Overall, reduced lipogenic gene mRNA expression patterns support the hypothesis that SREBP-1c expression is reduced in overweight and possibly obese cats, reflecting down-regulation of the lipogenic pathway to prevent further fat accumulation and weight gain.     

Molecular epidemiology of rabies virus circulating in South Korea, 1998-2010

We analyzed the nucleotide sequences of the G-L (glycoprotein-large protein) intergenic non-coding region of 33 strains of the rabies virus (RABV) isolated in South Korea in 1998-2010 and compared the sequences with those of previously reported non-Korean strains. The similarities of the nucleotide sequences of the G-L region among all Korean RABV isolates ranged from 97.1 to 100%. Based on the phylogenetic analysis of the G-L region, the Korean RABV isolates were classified into three distinct subgroups with high similarity and were most closely related to the non-Korean NeiMeng1025C isolate, which was isolated from a rabid raccoon dog in eastern China, suggesting that the Korean RABV isolates originate from a rabid raccoon dog in northeastern Asia. Our results indicated that G-L region, as a useful phylogenetic indicator, is equivalent to the nucleoprotein (N) or glycoprotein (G) gene for study of RABV molecular epidemiology and that the Korean RABV isolates showing a few substitutions in the G-L region are continuously circulating in South Korea.     

Kinematic gait analysis of the hind limb after tibial plateau levelling osteotomy and cranial tibial wedge osteotomy in ten dogs

This study identifies and compares the kinematic gait changes occurring in tibial plateau levelling osteotomy (TPLO) and cranial tibial wedge osteotomy (CTWO) limbs after transection of the cranial cruciate ligament (CrCL). Ten, healthy, adult beagle dogs were assigned to TPLO (five dogs) and CTWO (five dogs) groups. Hind limb kinematics were assessed, while dogs were trotted at speeds ranging from 2.0 to 2.3 m/s. The animals were evaluated preoperatively (prior to TPLO and CTWO surgery) and at both 8 and 12 weeks after surgery. Two-dimensional evaluation was synchronized to obtain the three-dimensional coordinates using the APAS motion analysis software. Gait patterns were assessed by measuring stifle, tibiotarsal joint angles and stifle joints angular velocity. Stifle and tibiotarsal joint functions were not affected by TPLO surgery, but stifle and tibiotarsal joint angles were changed, following CTWO surgery, compared with their preoperative values. The angular velocity patterns of CTWO were characterized by increased stifle joint extension velocity from the middle to end swing phase and decrease in the peak velocities (flexion) during swing phase. None of these changes was observed in the stance phase after the CTWO surgery. These kinematic results showed that dogs that underwent a CTWO procedure were more likely to have significantly hyperextended gait patterns of the swing phase postoperatively than the dogs that had a TPLO procedure for repair of a ruptured CrCL.     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.     

ULTRASONOGRAPHIC DIAGNOSIS—PYLOROGASTRIC INTUSSUSCEPTION IN A DOG

A 7-year-old female Maltese presented for evaluation of severe vomiting. A diagnosis of pylorogastric intussusception was made during ultrasonographic examination. The intussusception spontaneously underwent reduction by the following morning.     

The effect of active immunization against adrenocorticotropic hormone on cortisol, beta-endorphin, vocalization, and growth in pigs

Because the poor growth performance of intensively housed pigs is associated with increased circulating glucocorticoid concentrations, we investigated the effects of glucocorticoid suppression by inducing a humoral immune response to ACTH on physiological and production variables in growing pigs. Grower pigs (28.6 +/- 0.9 kg) were immunized with amino acids 1 through 24 of ACTH conjugated to ovalbumin and suspended in diethylaminoethyl (DEAE) dextran-adjuvant or adjuvant alone (control) on d 1, 28, and 56. The ACTH-specific antibody titers generated suppressed increases in cortisol concentrations on d 63 in response to an acute stressor (P = 0.002; control = 71 +/- 8.2 ng/mL; ACTH-immune = 43 +/- 4.9 ng/mL) without altering basal concentrations. Plasma beta-endorphin concentrations were also increased (P < 0.001) on d 63 (control = 18 +/- 2.1 ng/mL; ACTH-immune = 63 +/- 7.3 ng/mL), presumably because of a release from negative feedback on the expression of proopiomelanocortin in pituitary corticotropes. Immunization against ACTH did not alter ADG (P = 0.120; control = 1,077 +/- 25; ACTH-immune = 1,143 +/- 25 g) or ADFI (P = 0.64; control = 2,719 +/- 42; ACTH-immune = 2,749 +/- 42 g) and did not modify behavior (P = 0.681) assessed by measuring vocalization in response to acute restraint. In summary, suppression of stress-induced cortisol responses through ACTH immunization increased beta-endorphin concentrations, but it did not modify ADG, ADFI, or restraint vocalization score in growing pigs.