Mechanical comparison of materials used for extra-capsular stabilisation of the stifle joint in dogs

Objective The mechanical properties of three materials (No. 2 polypropylene, No. 5 polybutilate-coated multifilament polyester and 18, 27 and 36 kg test monofilament nylon leader material) commonly used for extra-capsular stabilisation of the stifle in dogs with cranial cruciate ligament insufficiency were determined. The ability of No. 5 polybutilate-coated multifilament polyester and 36 kg test monofilament nylon leader material, when placed as extra-capsular sutures, to mitigate cranial drawer was evaluated in hindlimbs of cadavers. Design An in vitro mechanical study. Animals Seven pairs of hindlimbs harvested from adult greyhound dogs recently euthanased for other reasons. Procedure Samples of each material, including samples of 27 kg test leader material that had been sterilised by one of three methods (ethylene oxide, one or five cycles in an autoclave), were loaded to determine tensile and stress relaxation properties. The effect of cyclic loading on a No. 5 polybutilate-coated multifilament polyester and 36 kg test leader material was also determined. Using the harvested hindlimbs, cranial drawer was measured before and after transection of the cranial cruciate ligament and on the first and twelfth cycle following extra-capsular stabilisation with either No. 5 polybu-tilate-coated multifilament suture or 36 kg test leader material. Results Leader material was found to have the most suitable mechanical characteristics for use as extracapsular stabilisation of the cranial cruciate ligament deficient stifle. Of the sterilisation methods, ethylene oxide was found to have the least detrimental effects on the handling and material characteristics of the leader material. Stifles stabilised with 36 kg test leader material had significantly less drawer than those stabilised with No. 5 polybutilate-coated multifilament polyester suture. Clinical implications Monofilament nylon leader material would appear to have suitable mechanical properties for extra-capsular stabilisation of the cranial cruciate ligament deficient stifle. If possible the material should be sterilised using ethylene oxide.     

A Chemoautotrophically Based Cave Ecosystem

Microbial mats discovered in a ground-water ecosystem in southern Romania contain chemoautotrophic bacteria that fix inorganic carbon, using hydrogen sulfide as an energy source. Analysis of stable carbon and nitrogen isotopes showed that this chemoautotrophic production is the food base for 48 species of cave-adapted terrestrial and aquatic invertebrates, 33 of which are endemic to this ecosystem. This is the only cave ecosystem known to be supported by in situ autotrophic production, and it contains the only terrestrial community known to be chemoautotrophically based.     

Evaluation of flow cytometric and automated methods for detection of activated platelets in dogs with inflammatory disease

OBJECTIVE: To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease. ANIMALS: 20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease. Procedures-Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets. Results-12 dogs with inflammatory disease had P-selectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet surface-associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease.     

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.     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane‐bound G‐coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out‐of‐season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen‐2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out‐of‐season breeding. GG and CC genotypes were having a higher prevalence in the studied population.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.