Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

The aim of this study was to compare measurement of spermatozoal membrane status using computer‐assisted spermatozoal quantification (CASQ) and eosin‐nigrosin (EN) staining with manual counting after CFDA/PI staining. Analysis was performed on both fresh and thawed cryopreserved canine semen. Membrane‐disrupted spermatozoa (MDS) were counted using CASQ (n = 311) in an untreated sample and a completely membrane‐disrupted sample, and the percentage of membrane‐intact spermatozoa (MIS) calculated: (Total count ? Untreated sample count) ÷ Total count × 100. Spermatozoa were stained with a one‐step EN stain (n = 501), and then, at least 100 spermatozoa were manually examined under ×1,000 magnification and classified as MDS (stained with eosin) or MIS (non‐stained). Spermatozoa from the same samples were also stained with CFDA/PI, and then, at least 200 spermatozoa were manually examined under ×1,000 magnification and classified as MIS (completely stained by CFDA) or MDS. The percentage of progressively motile spermatozoa (PMS) was determined by both computer‐assisted semen analysis (CASA) and subjective methodologies, and the data were subsequently analysed to measure the agreement between the CASQ and EN methods with the CFDA/PI technique using Bland–Altman methodology. Pearson's correlation was measured between the MIS and PMS percentage samples and correlation coefficients compared. The mean MIS percentage was lower for CASQ and higher for EN than in CFDA/PI for all comparisons. The agreement of MIS percentage between CASQ and CFDA/PI was ?20.2% to 32.0%, and between EN and CFDA/PI was ?32.9% to 14.9%. In all methods, the MIS and PMS percentages were correlated (p < .001). Measurement of CFDA/PI appeared to be the most reliable and accurate method of determining MIS percentage in dogs. Further investigation is required to determine whether the CASQ technique can be improved. Eosin‐nigrosin staining also appeared to be unreliable at MIS <80% and overestimated the MIS percentage.     

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

The aim of this study was to develop and validate a novel, computer‐assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 28) with a haemocytometer using light microscopy, CASQ and computer‐assisted semen analysis (CASA; MMC sperm), following three independent dilutions. The limits of agreement between the haemocytometer and CASQ were ?13.1% to 13.8% and ?27.0% to 28.6% between the haemocytometer and CASA. The precision CVs (limits of agreement) were 5.7% (?7.8% to 8.9%) for the haemocytometer, 6.2% (?8.8% to 12.3%) for CASQ and 10.8% (?16.0% to 19.5%) for CASA. In Experiment B, spermatozoa were manually counted (n = 42) with the haemocytometer under fluorescent illumination using the CASQ sample. The limits of agreement between the CASQ and the haemocytometer were satisfactory (?4.6% to 4.6%) and the precision CVs (limits of agreement) were 6.2% (?9.0% to 11.4%) for the haemocytometer and 4.4% (?5.8% to 8.6%) for CASQ. The CASQ method was then clinically applied to compare the haemocytometer (light and fluorescent methods) with CASQ and CASA. Outlying data were removed. These studies demonstrated that CASQ was reliable and that the MMC sperm CASA was unreliable as methods for determining spermatozoal concentration in canine semen. Computer‐assisted spermatozoal quantification was also determined to be more precise than manual counting with the haemocytometer. Using the clinical protocol, the agreement between the haemocytometer and CASQ method was acceptable, but it was worse than in the experiments where duplicate samples and a larger volume of semen were analysed. The CASQ method may be a useful method to measure the membrane status of canine spermatozoa; however, further investigation is required. Counting spermatozoa using fluorescent microscopy and the haemocytometer may improve the efficiency of counting and the accuracy of the method.     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Protein characterization using electrophoresis and immunofixation; a case‐based review of dogs and cats

Protein electrophoresis and immunotyping can be a useful adjunct to the standard biochemical techniques for characterizing serum and urine proteins. This paper reviews currently available and commonly used methods for diagnostic protein electrophoresis, including both agarose gel and capillary zone electrophoretic techniques and total protein assessments. Immunofixation and immunosubtraction methods for identification of immunoglobulin location and class are also presented. Practical application of quality assurance and quality control strategies in compliance with American Society of Veterinary Clinical Pathology (ASVCP) best practices are discussed. Commonly encountered serum and urine electrophoretic diagnostic patterns, including electrophoretically normal, acute‐phase protein responses, polyclonal gammopathies, restricted polyclonal/oligoclonal gammopathies, paraproteinemias (monoclonal or biclonal gammopathies), and Bence‐Jones proteinurias are also reviewed using relevant case material. Cases in which immunofixation electrophoresis are particularly useful are highlighted, and methodologies to more accurately quantify serum monoclonal proteins (M‐proteins), monitoring tests commonly used in human medicine, are discussed.     

Patient characteristics influencing the variability of distributed parameter‐based models in DCE‐CT kinetic analysis

Kinetic parameter variability may be sensitive to kinetic model choice, kinetic model implementation or patient‐specific effects. The purpose of this study was to assess their impact on the variability of dynamic contrast‐enhanced computed tomography (DCE‐CT) kinetic parameters. A total of 11 canine patients with sinonasal tumours received high signal‐to‐noise ratio, test‐double retest DCE‐CT scans. The variability for three distributed parameter (DP)‐based models was assessed by analysis of variance. Mixed‐effects modelling evaluated patient‐specific effects. Inter‐model variability (CV_inter) was comparable to or lower than intra‐model variability (CV_intra) for blood flow (CV_inter:[4–28%], CV_intra:[28–31%]), fractional vascular volume (CV_inter:[3–17%], CV_intra:[16–19%]) and permeability‐surface area product (CV_inter:[5–12%], CV_intra:[14–15%]). The kinetic models were significantly (P<0.05) impacted by patient characteristics for patient size, area underneath the curve of the artery and of the tumour. In conclusion, DP‐based models demonstrated good agreement with similar differences between models and scans. However, high variability in the kinetic parameters and their sensitivity to patient size may limit certain quantitative applications.     

Optimization and physicochemical properties of nutritional protein isolate from pork liver with ultrasound‐assisted alkaline extraction

The aim of this study was to investigate the optimal conditions of ultrasound‐assisted alkaline extraction (UAAE) on pork liver protein isolate (UPLPI) and its physico‐chemical properties. Response surface methodology was used to determine the optimal conditions for UAAE, which were at ultrasonic power 265 W, ultrasonic time 42 min, NaOH concentration 0.80%, temperature 50°C, and solvent/raw material ratio 70. The extraction yield and efficiency of UPLPI were significantly improved over the conventional alkaline extraction (PLPI). The results of amino acid composition showed that UAAE could increase serine (36.5 g/kg), arginine (38.1 g/kg), alanine (37.5 g/kg), proline (48.7 g/kg), phenylalanine (55.6 g/kg) and lysine (47.2 g/kg) elution amount. The changes in fourier transform infrared spectra indicated unfolding and destruction of the protein structure in UPLPI. The differential scanning calorimetry analysis presented UPLPI with a slightly lower onset and peak denaturation temperature over PLPI. Surface hydrophobicity increased and the microstructures presented larger and more pores of UPLPI, therefore, it had better in vitro digestibility than PLPI. Therefore, UPLPI might have a potential application prospect in the food field due to its changes on molecular structure as well as on the microstructure of protein by UAAE.     

Triple‐phased mesenteric CT angiography using a test bolus technique for evaluation of the mesenteric vasculature and small intestinal wall contrast enhancement in dogs

Computed tomography angiography is widely used for the assessment of various mesenteric vascular and bowel diseases in humans. However, there are only few studies that describe CT angiography application to mesenteric vessels in dogs. In this prospective, experimental, exploratory study, the mesenteric vasculature and enhancement pattern of the intestinal wall were evaluated on triple‐phase CT angiography, and improvement of the visibility of vasculature was assessed on multiplanar reformation, maximum intensity projection, and volume rendering technique. After test bolus scanning at the level of the cranial mesenteric artery arising from the aorta, mesenteric CT angiography was performed in 10 healthy, male, Beagle dogs. Scan delay was set based on time‐to‐attenuation curves, drawn by placing the regions of interest over the aorta, intestinal wall, and cranial mesenteric vein. Visualization and enhancement of mesenteric arteries and veins were evaluated with multiplanar reformation, maximum intensity projection, and volume rendering techniques. The degree of intestinal wall enhancement was assessed on the transverse images in precontrast, arterial, intestinal, and venous phases. Pure arterial images were obtained in the arterial phase. Venous phase images allowed good portal vascular mapping. All CT angiography images were of high quality, allowing for excellent visualization of the anatomy of mesenteric vasculature including the small branches, particularly on maximum intensity projection and volume rendering technique. Distinct contrast enhancement of the intestinal wall was observed in both intestinal and venous phases. Findings indicated that this technique is feasible for the evaluation of mesenteric circulation in dogs.     

Assessment of tumor enhancement by contrast‐enhanced CT in solid tumor‐bearing dogs treated with toceranib phosphate

In humans, contrast‐enhanced CT (CECT) has been used to indirectly assess the antiangiogenic effects demonstrated by a number of tyrosine kinase inhibitors. This retrospective, cross‐sectional study aimed to quantitatively evaluate changes in tumor contrast‐enhancement (CE) using CECT in solid tumor‐bearing dogs treated with toceranib phosphate (TOC). The changes in tumor size and CE were measured using the Hounsfield unit (HU) scale in CECT images before TOC treatment and between 30 and 90 days after initiating the treatment. Among the 36 dogs treated with TOC, eight (22.2%) showed a partial response, 22 (61.1%) showed stable disease, and six (16.7%) showed progressive disease. Thirty (83.3%) of 36 dogs showed a decrease in tumor CE (median: ?20%, range: ?1% to ?48%) after initiating the treatment. The results indicated that tumor CE and size changes were observed in tumor‐bearing dogs that were treated with TOC; however, tumor CE was not significantly correlated with tumor regression. We suggest that these results could serve as pilot data to evaluate the antiangiogenic effects associated with TOC.     

Three‐dimensional CT observation of position and movability of the scapula in the horse using carcasses of Falabella

The three‐dimensionally real position and movement of the scapula in the lateral side of the ribcage could not be clarified in the horse, since the body size of the horse is too large to apply the CT scanning and image analysis methods. In this study, therefore, we examined the position and the movability of scapula using a carcass of the Falabella which is one of the smallest breeds of the horse. The whole skeletal system in thoracic part of the Falabella could be three‐dimensionally observed by CT scanning method. The three‐dimensional images show that the scapula cranially slides and the ventral part of the scapula dorso‐cranially rotates, when the shoulder joint moves to the most cranial position as simulation. The three‐dimensional rotation angle was approximately 10 degrees. As a result of comparative osteology of the scapula between Falabella and the large draft horse, the infraspinous fossa was caudally enlarged in the larger draft horse, whereas the Falabella had narrower infraspinous fossa. We suggest that it may be due to the adaptational morphological change in the scapula bearing various body weights among breeds. The three‐dimensional CT image analysis and the simulation in carcass contribute to the analysis of the bone movements of the horse during walking and running locomotion as well as the motion analysis from external functional‐morphological data.     

Effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars,analysed with a CASA‐Mot system

Motility is the most widely used indicator of sperm quality. Computer‐Assisted Semen Analysis (CASA) allows the objective evaluation of sperm motility parameters. CASA technology is a common tool to predict semen doses in farm animal reproduction. The kinds of video cameras used until now for image acquisition have presented limited frame rates (FR), which have a negative influence on the quality of the obtained data. The aim of the present work was to define the optimal frame rate for a correct evaluation of boar sperm motility and its subpopulation structure. Eighteen ejaculates from nine mature boars of the Pietrain breed were used. Using the ISAS^®v1 CASA‐Mot system, with a video camera working up to 200 Hz, six FRs (25, 50, 75, 100, 150 and 200 fps) were compared. ISAS^®D4C20 counting chambers, warmed to 37°C, were used. FR affected all the kinematic parameters, with curvilinear velocity (VCL) and BCF the most sensitive ones. All the parameters showed differences among animals. Non‐linear correlation showed the asymptotic level for VCL at 212 fps, being the highest FR for all the parameters. For future studies based just on progressive motility, almost 100 fps FR for 0.5 s must be used, while when kinematics must be considered, almost 212 fps for one‐second should be analysed. Three principal components were obtained (velocity, progressivity and oscillation), being similar at 50 and 200 fps. Cells were grouped in four subpopulations but with different kinematic and cellular distribution at both FRs.     

Evaluation of platelet function in dogs with cardiac disease using the PFA‐100 platelet function analyzer

Background: Cardiac disease has the potential to alter platelet function in dogs. Evaluation of platelet function using the PFA‐100 analyzer in dogs of multiple breeds and with a broad range of cardiac conditions would help clarify the effect of cardiac disease on platelets. Objectives: The objective of this study was to assess differences in closure time (CT) in dogs with cardiac disease associated with murmurs, when compared with that of healthy dogs. Methods: Thirty‐nine dogs with cardiac murmurs and turbulent blood flow as determined echocardiographically were included in the study. The dogs represented 23 different breeds. Dogs with murmurs were further divided into those with atrioventricular valvular insufficiency (n=23) and subaortic stenosis (n=9). Fifty‐eight clinically healthy dogs were used as controls. CTs were determined in duplicate on a PFA‐100 analyzer using collagen/ADP cartridges. Results: Compared with CTs in the control group (mean±SD, 57.6±5.9 seconds; median, 56.5 seconds; reference interval, 48.0–77.0 seconds), dogs with valvular insufficiency (mean±SD, 81.9±26.3 seconds; median, 78.0 seconds; range, 52.5–187 seconds), subaortic stenosis (71.4±16.5 seconds; median, 66.0 seconds; range, 51.5–95.0 seconds), and all dogs with murmurs combined (79.6±24.1 seconds; median, 74.0 seconds; range, 48.0–187 seconds) had significantly prolonged CTs (P<.01). Conclusions: The PFA‐100 analyzer is useful in detecting platelet function defects in dogs with cardiac murmurs, most notably those caused by mitral and/or tricuspid valvular insufficiency or subaortic stenosis. The form of turbulent blood flow does not appear to be an important factor in platelet hypofunction in these forms of cardiac disease.     

Tolerability and pharmacokinetic profile of a novel benzene‐poly‐carboxylic acids complex with cis‐diammineplatinum (II) dichloride in dogs with malignant mammary tumours

The pharmacokinetic profile, tolerability and efficacy of benzene‐poly‐carboxylic acids complex with cis‐diammineplatinum (II) dichloride (BP‐C1) were studied in dogs with mammary cancer. A three‐level response surface pathway designed trial was performed on seven dogs. At each level BP‐C1 was administered subcutaneously daily for 7 days followed by a 7‐day rest period in a dose escalating manner. Adverse events according to VCOG‐CTCAE, performance status and tumour progression were recorded. The pharmacokinetic profile followed a two‐compartment model with rapid absorption, short distribution, and a slow elimination phase. The overall elimination half‐life was 125 h. The maximum tolerated dose of BP‐C1 was estimated to be above 0.46 mg kg^?1. A significant reduction in VCOG‐CTCAE toxicity which correlated negatively with increasing dose was found. The dogs' general performance status remained unchanged. No decrease in total tumour burden was found, although temporary tumour reduction was seen in some target tumours.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Computed tomographic‐lymphography as a complementary technique for lymph node staging in dogs with malignant tumors of various sites

Locoregional lymph nodes are routinely examined in order to define the spatial extent of neoplastic disease. As draining patterns of certain tumor types can be divergent from expected anatomical distribution, it is critical to sample the lymph nodes truly representing the draining area. The aim of this bicenter prospective pilot study was to describe the technique of computed tomographic (CT)‐lymphography for primary draining lymph node mapping in tumor staging in dogs. Forty‐five dogs with macro‐ or microscopic tumors in specified localizations were evaluated. Depending on body weight, 0.8–2 ml contrast agent (iohexol) was injected into four quadrants around the tumor, and CT‐images were obtained at 1, 3, 6, 9, and 12 minutes post‐injection. Attenuation of chosen regions of interest (Hounsfield units (HU)) and patterns of enhancement were assessed for 284 lymph nodes in the precontrast study with median HUs of 31.1 (Interquartile range (IQR) = 18.4) and for 275 in the intravenous postcontrast study with 104.3 HU (IQR = 31.2) (paired Wilcoxon test, P < 0.001). In the CT‐lymphography study, 45 primary draining lymph nodes with a significantly higher median HU value of 348.5 (IQR = 591.4) (one‐sample t‐test, P < 0.001) were identified. Primary draining lymph nodes were found to be clearly visible after 1–3 minutes after local injection, often concurrent with a good visibility of the lymphatic vessel system. The herein described technique of peritumorally injected CT‐contrast agent followed by subsequent CT‐lymphography for primary draining lymph node mapping works well in a majority of cases in all investigated sites and warrants further validation for different tumor entities.