Evaluation of DNA fragmentation in dog sperm using the sperm chromatin dispersion test.

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.5% and 5%) in fourteen ejaculates from seven dogs and semen incubation with 0.3 M NaOH for 15 min at room temperature was assayed as a control for sperm DNA fragmentation. Data were analysed using a Mann-Whitney test and either Pearson's or Spearman's correlation. The selected ME concentration to use in the SCD test was 5%, as it produced the largest DNA dispersion halo while preserving the core nucleus structure. Four DNA halo patterns were identified as follows: large dispersion halos, medium halos, small halos and nuclei without halos. Semen incubated with NaOH showed 100% sperm without halos (damaged DNA). A significant positive correlation was observed between sperm with fragmented DNA and sperm with coiled tails. Thus, it was possible to adapt the SCD protocol to evaluate dog sperm DNA fragmentation in raw semen without using a commercial kit and establish incubation with NaOH as a DNA fragmentation control. Only coiled tails showed correlation with DNA fragmentation.

Detection of lipid peroxidation in dog spermatozoa with the fluorescent dye C11-BODIPY581/591.

C11-BODIPY581/591 is a fluorescent probe that has been successfully used to evaluate lipid peroxidation in different species, but it has not been completely studied in the dog. Thus, the aim of the present study was to assess lipid peroxidation of dog spermatozoa using C11-BODIPY581/591 and compare different positive controls of the technique. Twenty-four ejaculates were collected from 8 adult male dogs. Routine seminal characteristics were evaluated in raw semen. Lipid peroxidation evaluation was performed as described in other species. Samples were divided in three aliquots, exposed to UV radiation, incubated with hydrogen peroxide or left without treatment (control). Lipid peroxidation was significantly greater only in UV-exposed samples than in the control ones (91 ± 6% vs. 8.3 ± 3.5%, p Ë‚ .01). In conclusion, C11-BODIPY581/591 is useful to evaluate lipid peroxidation of dog spermatozoa and UV radiation is a good promoter of membrane oxidation, so irradiated samples can be used as a positive control of this technique.

Dog sperm DNA: Raw semen evaluation with Toluidine blue stain.

The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty-one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue-violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min) for any of the TB patterns. All DTT incubation times (2, 5 and 30 min) showed 100% sperm positive to TB. To conclude, it was possible to simplify the TB stain and determine the different patterns in canine spermatozoa. Also, DTT can be used both as a positive control for the stain and to evaluate individual susceptibility to decondensation in vitro.

Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.