Efficacy of evaluation of rooster sperm morphology using different staining methods

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin–nigrosin smears, which can determine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks reproduced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used to determine the efficacy of sperm morphology evaluation using four eosin–nigrosin staining methods (according to Blom, Bakst and Cecil, Morisson, Jaśkowski) and three examiners of different experience (high, medium, novice). There were significant (P ⩽ 0.01) differences in sperm morphology between Blom’s staining method and those of Bakst and Cecil, Morisson or Jaśkowski, irrespective of fowl breed and examiners experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa with normal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.     

Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols

Viability assessment is an important part of semen analysis, and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species-specific comparisons of viability stains. In raptor semen, similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2% [EB], eosin blue 2% with the addition of 3% sodium citrate [EB2], eosin blue–nigrosin 5% [EBN5], eosin yellow–nigrosin 5% [EYN5], eosin yellow–nigrosin 10% [EYN10], eosin blue–aniline blue [EBA], eosin yellow–aniline blue [EYA] and bromophenol blue–nigrosin [BBN] were evaluated in comparison with the fluorescence stain SYBR^® Green–propidium iodide [SYBR-PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centres, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability. Light microscopy using EB and using SYBR-PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strongly with EB only (r = .629; p < .001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.     

The advantages of using a combination of LDL and glutamine in comparison with TRIS egg yolk and Equex® STAMP extenders in the cryopreservation of canine semen

Twenty semen samples taken from 5 dogs were frozen in liquid nitrogen at ?196 °C in four different extenders: one control extender based on 20% egg yolk, 6% LDL alone (low density lipoproteins: the active cryoprotective principle in chicken egg yolk), 6% LDL combined with 20 mmol glutamine, and Equex® (a reference extender that we wish to compare with the LDL-glutamine combination). After thawing, spermatozoal motility was evaluated using a HAMILTON THORNE CERROS 12 image analyzer; the percentage of motile spermatozoa was 27.7% in the egg yolk extender (p < 0.05), 49.9% with 6% LDL alone (p > 0.05), 54.7% in the 6% LDL + 20 mmol glutamine extender, and 47.9% with Equex® (p > 0.05). The motility parameters (VAP, VCL, VSL and ALH) were also superior in the 6% LDL + 20 mmol glutamine extender in comparison with the other extenders.Finally, the spermatozoa were generally better protected during freezing with the 6% LDL + 20 mmol glutamine association than with the egg yolk, 6% LDL, or Equex extenders in terms of the flagellar plasma membrane (HOS test), DNA (Acridine orange test), and acrosome integrity (Spermac® test: no significant difference). The Equex® extender obtained the best results for the acrosome, followed by 6% LDL + 20 mmol glutamine (FITC-PSA test: p < 0.05 between each extender).     

Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows

In order to provide information that may help researchers to understand the main cause(s) of differences in bull fertility frequently observed in field trials, this study aimed to investigate conception rates as well as several in vitro sperm characteristics of different sires of unknown fertility utilized in a Timed-AI (TAI) program. Suckled Nelore cows submitted to the same TAI protocol were allocated into eight breeding groups of approximately 120 animals each. Frozen semen doses from three Angus bulls and three different batches from each bull were utilized. Approximately 100 doses from each batch were used in TAI. Sires, batches and AI technicians were equally distributed across breeding groups. Cows were examined for pregnancy diagnosis 40 d after TAI. For in vitro sperm analyses, the same thawing procedure was repeated in the laboratory to mimic field conditions. The following in vitro sperm characteristics were assessed: computerized motility, thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, morphology, morphometry and chromatin structure. No effect of breeding group, body condition score, AI technician and sire was observed. However, some significant differences among bulls were detected in laboratory analyses. Semen from sire presenting numerically lower (P > 0.05) pregnancy/AI also presented lower (P < 0.05) values in all sperm characteristics analyzed in thermal resistance test at 4 h (Total Motility, Progressive Motility, Average Path Velocity, Straight-Line Velocity, Curvilinear Velocity, Amplitude of Lateral Head Displacement, Beat Cross Frequency, Straightness, Linearity, and Percentage of Rapidly Moving Cells), higher (P < 0.05) Major and Total Defects in sperm morphological test, lower (P < 0.05) Length, Ellipticity and Fourier parameter (Fourier 0) in sperm morphometric analysis as well as higher (P < 0.05) chromatin heterogeneity. It was concluded that, although no bull effect was observed in the field experiment, the sire that presented numerically lower pregnancy/AI also presented lower semen quality according to the laboratory analyses performed.     

The effect of supplementation of flax-seed oil on interaction of Lactobacillus plantarum--Biocenol™ LP96 and Escherichia coli O8:K88ab:H9 in the gut of germ-free piglets

We investigated the influence of administration of flax-seed oil on interaction of Lactobacillus plantarum – Biocenol? LP96 and Escherichia coli O8:K88ab:H9 in the gut of germ-free piglets. When compared to animals supplemented with L. plantarum, the counts of lactobacilli in the jejunal and ileal mucosa and in the intestinal content were significantly higher in LMK group (p < 0.0001). Inter-groups comparison of the counts of E. coli K88 adhering to the jejunal and ileal mucosa revealed a significantly decrease in LMK animals (p < 0.001; p < 0.05).     

Gastrointestinal parasitism reduces the plasma availability of doramectin in lambs

A study was undertaken to investigate the effect of parasitism on plasma availability and pharmacokinetic behaviour of doramectin (DRM) in lambs. Fourteen parasitised grey face Suffolk lambs (26.9 ± 1.5 kg bodyweight) were selected for the study. Seven pairs of lambs were allocated to two groups to obtain an approximately even weight distribution. Group I (non-parasitised) was pre-treated with three repeated administrations of 5 mg/kg fenbendazole to maintain a parasite free condition. In group II (parasitised), the lambs did not receive any anthelmintic treatment. After the 85-day pre-treatment period, both groups of animals were treated with DRM by subcutaneous (SC) injection in the shoulder area at 200 μg/kg. Throughout the experimental period, both groups were maintained together under similar feeding and management conditions. Blood samples were collected by jugular venepuncture at different set times between 0.5 h and 60 days post-treatment. After plasma extraction and derivatisation, samples were analysed by high performance liquid chromatography (HPLC) with fluorescence detection. A computerised kinetic analysis was performed and the data were compared using the Student’s paired t test.The parent molecule was detected in plasma between 30 min and either day 20 (parasitised) or day 35 (non-parasitised) post-DRM treatment. The AUC values of the parasitised group (143.0 ± 18 ng d/mL) were significantly lower (P < 0.05) than those observed in the parasitically naïve animals (229.6 ± 21.7 ng d/mL). The mean residence time (MRT) in the parasitised group (3.4 ± 0.3 days) was significantly shorter (P < 0.05) than in the healthy group (6.6 ± 0.6 days). Study results have shown that parasitic disease, through alteration in the body condition, can produce significant changes in the plasma disposition of DRM when administered SC to parasitised lambs.     

Recurrence of Chlamydia suis infection in pigs after short-term antimicrobial treatment

The effect of short-term antimicrobial treatment on natural excretion of Chlamydia suis in rectal swabs and C. suis and Chlamydophila psittaci in nasal swabs was investigated in 47 clinically normal piglets by quantitative real-time PCR. Pigs were treated IM with 4 mg/kg enrofloxacin for 5 days (n = 22) or 2.5 mg/kg enrofloxacin for 3 days followed by 100 mg/mL tiamulin (n = 25). Antimicrobial treatment reduced the number of pigs positive for chlamydiae and the quantity of chlamydial DNA in positive swabs for a few days, but chlamydial excretion recurred in both groups. Short-term antimicrobial treatment at dosages recommended for treatment of other bacterial infections in pig herds was not effective in eliminating naturally occurring subclinical chlamydial infection in pigs.     

Pharmacokinetics and effects of cetirizine in horses with insect bite hypersensitivity

Horses with insect bite hypersensitivity (IBH) have difficulty in completely avoiding allergens, so effective treatment options are required. A randomised, placebo controlled and double blinded field study was conducted to determine the pharmacokinetics and efficacy in reducing dermatitis of the antihistamine cetirizine given orally at 0.4 mg/kg twice daily for 3 weeks. The influence of protection blankets and stabling were also investigated.The estimated maximum plasma concentration (C_max) and trough plasma concentration of cetirizine were 135 ng/mL and 18 ng/mL, respectively. There was no difference in dermatitis reduction between the treatment and placebo groups (P = 0.77). The findings indicated that cetirizine was of no apparent benefit in treating IBH at the dose rate tested. The use of blankets and stabling were shown to have favourable influence on the dermatitis (P < 0.05) and may be the preferred options to prevent this condition.     

Plasma atrial natriuretic peptide concentration in warmblood horses with heart valve regurgitations

ObjectivesThis study measured plasma atrial natriuretic peptide (ANP) concentration in horses with heart valve regurgitations (HVR) with and without atrial and ventricular dilatation.BackgroundIn humans and small animals, plasma ANP concentration is increased in heart disease and correlates with the severity of clinical signs and heart enlargement.Animals, materials and methodsTen healthy horses (control) and 36 horses with HVR were evaluated by auscultation, electrocardiography, echocardiography, and determination of plasma ANP.ResultsControl horses demonstrated mean plasma ANP concentration of 21 ± 5.4 pg/mL. Of the 36 horses with HVR, 17 horses possessed normal echocardiographic heart size (group 1), 10 horses had a left atrial dilatation (group 2) and 9 horses had both left atrial and ventricular dilatation (group 3). Mean plasma ANP concentration of groups 1, 2 and 3 was 20.1 ± 5.6 pg/mL, 22.9 ± 11.0 pg/mL and 27.6 ± 17.4 pg/mL, respectively. The plasma ANP concentrations of HVR and control horses were not significantly different. The highest ANP concentrations were observed in horses with atrial and ventricular dilatation. No correlation between left atrial or ventricular size, weight, or sex and the plasma ANP concentration was found.ConclusionsNo significant differences in plasma ANP concentration was observed between groups. Further study, especially in horses with clinical signs of heart failure is needed.     

Localised pyogranulomatous dermatitis due to Mycobacterium abscessus in a cat: a case report

A case of pyogranulomatous dermatitis, caused by Mycobacterium abscessus, an unusual opportunistic Mycobacterium spp., is described in a cat. Histopathological examination of the affected skin confirmed the diagnosis and Ziehl-Neelsen staining revealed acid-fast rods. A rapidly growing mycobacterium was found after culture on a Löwenstein–Jensen medium. Real-time polymerase chain reaction for the 16S rDNA (434 bp) sequence and the sequence of the rpoB gene (359 bp) revealed 99% and 100% matches, respectively, with M. abscessus. This is the first report of a feline infection caused by this organism in Europe.     

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca²⁺) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ?1 μM 2-APB significantly reduced ROS production, whereas 50 and 100 μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ?10 μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca²⁺ channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.     

Plasma concentrations of buprenorphine after epidural administration in conscious cats

Buprenorphine plasma concentrations were measured after administering buprenorphine (20 μg/kg) into the lumbosacral epidural space of conscious cats chronically instrumented with an epidural catheter. Blood was collected from a jugular vein before injection and 15, 30, 45 and 60 min and 2, 3, 4, 5, 6, 8, 12 and 24 h after administration. Plasma buprenorphine concentrations were measured using ELISA. Background concentration (before injection) was 1.27 ± 0.27 ng/mL (mean ± SD). Including background concentration, the mean peak plasma concentration was obtained 15 min after injection (5.82 ± 3.75 ng/mL), and ranged from 3.79 to 2.20 ng/mL (30 min–3 h), remaining between 1.93 and 1.77 ng/mL (4–12 h), and declined to 1.40 ± 0.62 ng/mL at 24 h. Elimination half-life was 58.8 ± 40.2 min and clearance 56.7 ± 21.5 mL/min. Results indicate early rapid systemic uptake of buprenorphine from epidural administration with plasma concentrations similar to using buccal or IM routes by 15 min postinjection.     

Microsphere immunoassay for the detection of cytokines in domestic cat (Felis catus) plasma: elevated IL-12/23 in acute feline immunodeficiency virus infections

We recently described the development and validation of a highly sensitive and specific microsphere immunoassay capable of simultaneously quantifying three domestic cat cytokines in tissue culture supernatant. Here we describe the modification of this assay to measure interferon gamma (IFNγ), interleukin (IL)-10 and IL-12/IL-23 p40 (IL-12/23) in domestic cat plasma, report values obtained from plasma collected after feline immunodeficiency virus (FIV) exposure, and compare plasma concentrations to blood cell mRNA expression. The validated quantitation limits of this assay are 31–1000 pg/ml for IFNγ, 63–2000 pg/ml for IL-10, and 20–625 pg/ml for IL-12/23. Plasma cytokine levels from domestic cats infected with pathogenic and/or apathogenic FIV were determined at 3–4 and 7–8 weeks post-infection. IL-12/23 was elevated (p < 0.05) during acute infection with both FIV strains in two similar studies, conducted five years apart in different feline cohorts (n = 44 total animals). IL-12/23 concentrations ranged from 377 to 1904 pg/ml in naïve cats and 552 to 3460 pg/ml in infected cats. In contrast, the majority of plasma samples had IFNγ and IL-10 concentrations below the lowest standard tested. The inability to consistently detect levels of IFNγ and IL-10 in plasma, despite the fact that mRNA changes were detected, suggests that these cytokines may be secreted and/or cleared in a more highly regulated manner than IL-12/23, or perhaps exert local effects under tighter peripheral constraints and/or at a lower effective concentration.     

Assessment of Different Sperm Quality Parameters to Predict in vitro Fertility of Bulls

Frozen‐thawed semen from six bulls with high (> 60%) and low (20–35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live‐dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin‐eosin (N‐E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N‐E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non‐invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development.     

Canine heartworm (Dirofilaria immitis) in Fresno and Madera Counties, California: prevalence differences between foothill and valley habitats

The aim of this study was to assess the prevalence of heartworm in domestic dogs in Madera and Fresno Counties, California, dependent on habitat and other host and environmental factors. Dogs were screened for presence of heartworm antigen using the PetChek^® ELISA on blood samples (N = 519) collected at seven sites during April-July 2009. Eighteen dogs were heartworm antigen positive. Pearson Chi-square analyses were conducted testing the presence of heartworm antigen against the following independent variables: elevation range, percentage of time spent outdoors during the day, percentage of time spent outdoors during the night, pet coat length, weight class, prevention status, and sex. Dogs that spent at least 50% of their time outdoors during the day were significantly more likely to have heartworm that those who spent less time outside (N = 519, df = 1, p = 0.031). Overall prevalence (3.47%) was lower than expected, with Madera County having 3.8% positive samples and Fresno County 3.5%; this prevalence is lower than in many previous studies. The effect of time spent outdoors on heartworm prevalence was similar to previous studies. The impact of elevation on infection, though not significant, requires further investigation, as does the prevalence and viability of larval stages in mosquitoes.     

Immunohistochemical detection of IgM and IgG in lung tissue of dogs with leptospiral pulmonary haemorrhage syndrome (LPHS)

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a severe form of leptospirosis. Pathogenic mechanisms are poorly understood. Lung tissues from 26 dogs with LPHS, 5 dogs with pulmonary haemorrhage due to other causes and 6 healthy lungs were labelled for IgG (n = 26), IgM (n = 25) and leptospiral antigens (n = 26). Three general staining patterns for IgG/IgM were observed in lungs of dogs with LPHS with most tissues showing more than one staining pattern: (1) alveolar septal wall staining, (2) staining favouring alveolar surfaces and (3) staining of intra-alveolar fluid. Healthy control lung showed no staining, whereas haemorrhagic lung from dogs not infected with Leptospira showed staining of intra-alveolar fluid and occasionally alveolar septa. Leptospiral antigens were not detected. We conclude that deposition of IgG/IgM is demonstrable in the majority of canine lungs with naturally occurring LPHS, similar to what has been described in other species. Our findings suggest involvement of the host humoral immunity in the pathogenesis of LPHS and provide further evidence to support the dog as a natural disease model for human LPHS.     

Radial strain and strain rate by two-dimensional speckle tracking echocardiography and the tissue velocity based technique in the dog

ObjectivesTwo-dimensional (2D) speckle tracking echocardiography (STE) is a new angle-independent ultrasound technique based on tracking of speckles within the myocardium on 2D grayscale images. The aims of this prospective study were as follows: (1) to assess the variability of left ventricular peak systolic radial strain (St) and strain rate (SR) in awake dogs using STE (Protocol 1); and (2) to quantify these variables in a healthy canine population and compare them with tissue Doppler imaging (TDI)-based St and SR values (Protocol 2).BackgroundSt and SR may be assessed using TDI, which is limited by angle dependency.Animals, materials and methodsThirty-six STE examinations were performed on 6 healthy dogs for Protocol 1 and 37 healthy dogs were recruited for Protocol 2. In both studies, STE measurements were obtained offline from the right parasternal short-axis view by the same trained observer using automatic frame-to-frame tracking of grayscale speckle patterns.ResultsAll within- and between-day coefficients of variation were <10% (Protocol 1). In Protocol 2, St (46.7 ± 12.2%) and SR (2.7 ± 0.6 s⁻¹) measured by STE were correlated with heart rate (p < 0.01), but not with the ratio of early mitral inflow velocity to early mitral annular velocity. There was a good correlation between STE and TDI for both St and SR values (p < 0.001).ConclusionsSTE is a repeatable and reproducible non-Doppler method for assessing radial St and SR. The combination of these indices with conventional echo-Doppler variables could provide a new approach for accurately quantifying canine systolic function.     

Effect of spironolactone on diuresis and urine sodium and potassium excretion in healthy dogs

ObjectivesTo document the diuretic effect of different oral doses of spironolactone (SP) in healthy dogs.BackgroundSP is currently mentioned as a diuretic agent in the dog. However, the recommended doses were empirically defined and their corresponding diuretic effect has never been documented in dogs.Animals, materials and methodsEight adult Beagle dogs were used for two separate 2 * 2 cross-over designs. In the first cross-over, 4 dogs received SP orally for 8 days at 1 and 2 mg/kg per day. In the second cross-over the 4 other dogs received SP similarly, but at 4 and 8 mg/kg per day. Dogs were weighed on the first and last day of each period. Plasma SP and canrenone (the main active metabolite of SP) were assayed by high performance liquid chromatography (HPLC). Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium and potassium were measured during the SP treatment.ResultsTwo hours after SP administration, SP was metabolized into canrenone. A significant 14 and 22% decrease in urine potassium excretion was observed at 1 and 2 mg/kg, respectively, but not at the two other dose levels. Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium were not significantly altered by the SP treatment regardless of dose.ConclusionsRepeated oral administration of SP at 1, 2, 4 or 8 mg/kg for 8 days had no effect on water and sodium diuresis in healthy dogs.     

The development of the pupillary light reflex and menace response in neonatal lambs and kids

The aim of this study was to investigate the development of the pupillary light reflex and menace response in neonatal lambs and goat kids. Thirty lambs and 33 kids were assessed daily from birth until the pupillary light reflex and menace response had become established. All animals had a controlled pupillary light reflex within 20 h of birth. Lambs and kids had developed menace responses by 8 ± 3 and 14 ± 2 days, respectively. The Mann–Whitney test revealed a significant difference (P < 0.001) in the ages at which lambs and kids developed a menace response. Male kids developed this response significantly (P = 0.006) later than females. There was no sex difference in the menace response in the lambs. Overall, the findings indicated that lambs develop a menace response earlier than kids, and female kids develop this response more rapidly than their male counterparts.     

Improvements of immune status, intestinal integrity and gain performance in the early-weaned calves parenterally supplemented with L-alanyl-L-glutamine dipeptide

Glutamine (Gln) is an important substrate for the innate immune cells including lymphocytes and macrophages. In this report, the effects of alanyl-glutamine dipeptide (Ala-Gln) on the naïve immune system, intestinal integrity and gain performance of early-weaned calves were investigated. Early-weaned Chinese Holstein calves were intravenously administered different dosages of Ala-Gln. The effects of Ala-Gln administration were ascertained by evaluating the blood for naïve T lymphocyte subpopulations, the concentrations of serum IgG, serum IgA and intestinal mucosal secretory IgA (s-IgA), the intestinal integrity, as well as the gain performance. Results demonstrated that intravenous administration of Ala-Gln dipeptide (1.01 g/kg × d^?1) for 7 days had a positive effect on gain performance, intestinal integrity and the immune system. Calves administered doses of Ala-Gln displayed an improvement in gain performance and health status concurrent with increases in blood CD2⁺ and CD4⁺ lymphocytes, the ratio of CD4⁺/CD8⁺, serum IgA and IgG, intestinal mucosal s-IgA while decreasing the occurrence of diarrhea. Moreover, we found that animals given the effective dose (1.01 g/kg × d^?1) of Ala-Gln resulted in improved immune status and intestinal integrity relative to those given a lower (0.49 g/kg × d^?1) or higher dose (1.99 g/kg × d^?1) of Ala-Gln. These findings suggest that maintaining a certain concentration of plasma and/or tissue glutamine in the early stages of weaning is an effective alternative approach for improvement of growth performance in early-wean calves.