Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

A Simple and Rapid PCR‐Based Method for Ostrich Sexing Using Micro Amounts of DNA

The aim of the present study is to identify ostrich sex by using polymerase chain reaction (PCR) on micro amounts of DNA from blood, bloodstain and feathers. Sixteen male and 18 female ostriches were used as test individuals. Genomic DNA as a template was extracted by the Chelex method. Ostrsex‐P1 and P2 primers were designed to perform PCR amplification on the template. PCR products were checked using agarose gel electrophoresis with ethidium bromide staining and ostrich sex was determined directly by the bands shown on the gel. The results demonstrate that ostrich sex can be determined by the extraction of DNA from as little as 0.0125 μl blood using Chelex, whereby the use of large amounts of organic solvents such as phenol and chloroform are unnecessary. In addition, it is possible to identify ostrich sex using micro amounts of DNA extracted from bloodstains and/or feathers. The use of feathers particularly avoids unwanted sampling problems such as the difficulty of collecting ostrich blood, the stress to the ostrich caused by bleeding, and the demand for a lot of manpower for ostrich restraint.     

Serum biomarker profiling in cancer studies: a question of standardisation?

Companion animals are exposed to similar environmental conditions and carcinogens as humans. In some animal cancers, there also appears to be the same genetic changes associated as in humans. However, little work has been carried out in cancer biomarker identification in animals. The recent dramatic advances in molecular medicine, genomics, proteomics and translational research will allow biomarker identification, which may provide the best strategies for veterinarians and clinicians to combat disease by early diagnosis and administration of effective treatments. Proteomics may have important applications in cancer diagnosis, prognosis and predictive clinical outcome that could directly change clinical practice by affecting critical elemen‐ts of care and management. This review summarizes the advances in proteomics that has propelled us to this exciting age of clinical proteomics, and highlights the future work that is required for this to become a reality. In this review, we will discuss the available proteomic technologies and their limitations, and highlight the key areas of research and how they have been used to discover cancer biomarkers. The principles described here are equally applicable to human and animal disease, but implementation of ‘omic’ technologies requires stringent guidelines for collection of clinical material, the application of analytical techniques and interpretation of the data.     

Comparative study of plasma leptin concentration between solid ruminal and liquid abomasal feeding in weaned adult sheep

This experiment was conducted to investigate the difference between ruminal (solid feed, SF) and abomasal (liquid feed, LF) feeding on the plasma leptin concentration in sheep. The experiment consisted of 2 weeks to adapt the animals to SF, 4 weeks of feeding on SF, 2 weeks adaptation to LF, 8 weeks of feeding on LF, 2 weeks of adaptation to SF, and 4 weeks of feeding on SF. The LF directory flowed into the abomasums of sheep by bottle feeding. Plasma leptin concentration before morning feeding was almost constant in the SF periods, whereas it showed between‐day variations when measured during the LF periods. Mean plasma leptin levels were higher for LF (7.77 ± 0.76 ng/mL; mean ± SE) than for SF periods (3.95 ± 0.16 ng/mL; mean ± SE). Although plasma leptin concentration did not show any change after feeding in the SF and LF periods, plasma insulin and glucose levels increased within 15 min after liquid abomasal feeding, but not after solid ruminal feeding. The high plasma leptin concentration during the LF periods in weaned sheep could be due to change of digestible energy intake and changes in plasma insulin and glucose levels accompanying the changes in digestive processes and nutrient supply.     

Measurement of C‐peptide concentrations and responses to somatostatin,glucose infusion,and insulin resistance in horses

Reasons for performing study: Hyperinsulinaemia is detected in horses with insulin resistance (IR) and has previously been attributed to increased pancreatic insulin secretion. Connecting peptide (C‐peptide) can be measured to assess pancreatic function because it is secreted in equimolar amounts with insulin and does not undergo hepatic clearance. Hypothesis: A human double antibody radioimmunoassay (RIA) detects C‐peptide in equine serum and concentrations would reflect responses to different stimuli and conditions. Methods: A validation procedure was performed to assess the RIA. Six mature mares were selected and somatostatin administered i.v. as a primed continuous rate infusion, followed by 50 nmol human C‐peptide i.v. Insulin and C‐peptide concentrations were measured in horses (n = 6) undergoing an insulin‐modified frequently sampled i.v. glucose tolerance test, and in horses with insulin resistance (n = 10) or normal insulin sensitivity (n = 20). Results: A human RIA was validated for use with equine sera. Endogenous C‐peptide secretion was suppressed by somatostatin and median (range) clearance rate was 0.83 (0.15–1.61) ml/min/kg bwt. Mean ± s.d. C‐peptide‐to‐insulin ratio significantly (P = 0.004) decreased during the glucose tolerance test from 3.60 ± 1.95 prior to infusion to 1.03 ± 0.18 during the first 20 min following dextrose administration. Median C‐peptide and insulin concentrations were 1.5‐ and 9.5‐fold higher, respectively in horses with IR, compared with healthy horses. Conclusions: Endogenous C‐peptide secretion decreases in response to somatostatin and increases after dextrose infusion. Results suggest that relative insulin clearance decreases as pancreatic secretion increases in response to dextrose infusion. Hyperinsulinaemia in insulin resistant horses may be associated with both increased insulin secretion and decreased insulin clearance. Potential relevance: Both C‐peptide and insulin concentrations should be measured to assess pancreatic secretion and insulin clearance in horses.     

Risk Factors for the Development of Hypokalemia in Neonatal Diarrheic Calves

Background

Neonatal diarrheic calves have a clear negative potassium balance because of intestinal losses and decreased milk intake but in the presence of acidemia, they usually show normokalemic or hyperkalemic plasma concentrations.

Objectives

To assess whether marked hypokalemia occurs in response to the correction of acidemia and dehydration and to identify factors that are associated with this condition.

Animals

Eighty‐three calves with a clinical diagnosis of neonatal diarrhea.

Methods

Prospective cohort study. Calves were treated according to a clinical protocol using an oral electrolyte solution and commercially available packages of 8.4% sodium bicarbonate, 0.9% saline and 40% dextrose infusion solutions.

Results

The proportion of hypokalemic calves after 24 hours of treatment (19.3%) was twice as great as it was on admission to the hospital. Plasma K⁺ after 24 hours of treatment was not significantly correlated to venous blood pH values at the same time but positively correlated to venous blood pH values on admission (r = 0.51, P < .001). Base excess on admission (Odds ratio [OR] = 0.81, 95% confidence interval [CI] = 0.70–0.94), duration of diarrhea (OR = 1.37, 95% CI = 1.05–1.80), milk intake during hospitalization (OR = 0.54, 95% CI = 0.37–0.79) and plasma sodium concentrations after 24 hours (OR = 1.12, 95% CI = 1.01–1.25) were identified to be independently associated (P < .05) with a hypokalemic state after 24 hours of treatment.

Conclusions and Clinical Importance

Findings of this study suggest that marked depletion of body potassium stores is evident in diarrheic calves that suffered from marked metabolic acidosis, have a low milk intake and a long history of diarrhea.     

Effect of Chronic Administration of Phenobarbital,or Bromide,on Pharmacokinetics of Levetiracetam in Dogs with Epilepsy

Background

Levetiracetam (LEV) is a common add‐on antiepileptic drug (AED) in dogs with refractory seizures. Concurrent phenobarbital administration alters the disposition of LEV in healthy dogs.

Hypothesis/Objectives

To evaluate the pharmacokinetics of LEV in dogs with epilepsy when administered concurrently with conventional AEDs.

Animals

Eighteen client‐owned dogs on maintenance treatment with LEV and phenobarbital (PB group, n = 6), LEV and bromide (BR group, n = 6) or LEV, phenobarbital and bromide (PB–BR group, n = 6).

Methods

Prospective pharmacokinetic study. Blood samples were collected at 0, 1, 2, 4, and 6 hours after LEV administration. Plasma LEV concentrations were determined by high‐pressure liquid chromatography. To account for dose differences among dogs, LEV concentrations were normalized to the mean study dose (26.4 mg/kg). Pharmacokinetic analysis was performed on adjusted concentrations, using a noncompartmental method, and area‐under‐the‐curve (AUC) calculated to the last measured time point.

Results

Compared to the PB and PB–BR groups, the BR group had significantly higher peak concentration (C _max) (73.4 ± 24.0 versus 37.5 ± 13.7 and 26.5 ± 8.96 μg/mL, respectively, P < .001) and AUC (329 ± 114 versus 140 ± 64.7 and 98.7 ± 42.2 h*μg/mL, respectively, P < .001), and significantly lower clearance (CL/F) (71.8 ± 22.1 versus 187 ± 81.9 and 269 ± 127 mL/h/kg, respectively, P = .028).

Conclusions and Clinical Importance

Concurrent administration of PB alone or in combination with bromide increases LEV clearance in epileptic dogs compared to concurrent administration of bromide alone. Dosage increases might be indicated when utilizing LEV as add‐on treatment with phenobarbital in dogs.