Lipid peroxidation assays in canine fresh semen

Lipid peroxidation (LP) is a potential cause of infertility in males of numerous species. Levels in which the spermatozoa loses mobility in vitro is correlated with the rate of LP that it suffers. The objective of this work was to determine the fatty acid composition and analyze the sensitivity to LP (ascorbate–Fe++ dependent) in spermatozoa obtained from different samples of canine fresh semen. LP was evaluated using chemiluminescence (CL) (cpm/mg of protein) and fatty acid (FA) profile by means of gas chromatography. The saturated FA content found in the analyzed spermatozoa was approximately 40%, whereas the total unsaturated FA content was approximately 55% with a prevalence of docosapentaenoic acid (C22:5 n6). When the control and ascorbate–Fe++ dependent samples were compared, it was observed a significant increase in the light emission (CL). Consequently, significant decrease in the percentage of the polyunsaturated fatty acids (PUFA) was obtained, being more affected: C18: 2 n6, C20: 4 n6, C22: 4 n6, C22: 5 n6 and C22: 6 n3. The unsaturation index used to evaluate the alterations generated during the LP was correlated with the data mentioned before. Our results indicate that dog semen contains great amounts of PUFA, which were vulnerable to the LP. The alteration in the PUFA composition could be the common base of different degenerative processes

Antioxidant effect of lutein protects against oxidative damage to porcine spermatozoa

<p Boar sperm is especially susceptible to peroxidative damage generated by reactive oxygen species (ROS). Chemiluminescence was initiated by incubating porcine semen in an in vitro ascorbate-Fe++ system, a technique that allows the evaluation of oxidative stress in these cells. Lutein is known for its antioxidant effects, chemically it is a dihydric derivative of α-carotene and belongs to the group of xanthophylls. The main objective of this study was to investigate the antioxidant effect of lutein on boar spermatozoa. The effect of lutein was analyzed by two methods: 1) by adding lutein: the sperm samples were placed in an in vitro ascorbate- Fe++ system, during 120 min at 37°C adding increasing amounts of lutein (50, 150 and 250 μg) per mg of protein and 2) by incubation with lutein in an in vitro ascorbate-Fe+2 system, for 120 min at 37°C, using spermatozoa obtained from porcine semen samples previously incubated with lutein (0.15 and 0.25 mg/ml) during 24 h at 15°C. In both methods a control group (without lutein) was used. Peroxidation was measured by chemiluminescence using a liquid scintillation counter, the light emission being quantified in cpm (counts per minute). Analyzing the effect of lutein by the two methods, it was observed that the total amount of cpm/mg of protein originated by chemiluminescence was lower in samples obtained from the lutein group than in the control group (without lutein). Total chemiluminescence (cpm total) was lower in samples obtained from the lutein group than in the control group (without lutein), with a significance of p<0.005. Percent inhibition of peroxidation was not concentration dependent. These results would demonstrate that lutein could act as an antioxidant that would protect the membranes of the sperm from oxidative damage.p>Boar sperm is especially susceptible to peroxidative damage generated by reactive oxygen species (ROS). Chemiluminescence was initiated by incubating porcine semen in an in vitro ascorbate-Fe++ system, a technique that allows the evaluation of oxidative stress in these cells. Lutein is known for its antioxidant effects, chemically it is a dihydric derivative of α-carotene and belongs to the group of xanthophylls. The main objective of this study was to investigate the antioxidant effect of lutein on boar spermatozoa. The effect of lutein was analyzed by two methods: 1) by adding lutein: the sperm samples were placed in an in vitro ascorbate-Fe++ system, during 120 min at 37°C adding increasing amounts of lutein (50, 150 and 250 μg) per mg of protein and 2) by incubation with lutein in an in vitro ascorbate-Fe+2 system, for 120 min at 37°C, using spermatozoa obtained from porcine semen samples previously incubated with lutein (0.15 and 0.25 mg/ml) during 24 h at 15°C. In both methods a control group (without lutein) was used. Peroxidation was measured by chemiluminescence using a liquid scintillation counter, the light emission being quantified in cpm (counts per minute). Analyzing the effect of lutein by the two methods, it was observed that the total amount of cpm/mg of protein originated by chemiluminescence was lower in samples obtained from the lutein group than in the control group (without lutein). Total chemiluminescence (cpm total) was lower in samples obtained from the lutein group than in the control group (without lutein), with a significance of p<0.005. Percent inhibition of peroxidation was not concentration dependent. These results would demonstrate that lutein could act as an antioxidant that would protect the membranes of the sperm from oxidative damage

Susceptibility to peroxidation and fatty acid composition of fresh boar semen obtained from different hog farms

Objectives of this study were to determine the fatty acid composition and to analyze the sensitivity to lipid peroxidation of different boar fresh semen samples from two herds, H1 and H2. Lipid peroxidation was evaluated using chemilu­minescence (cpm/mg of protein) and fatty acid profile by means of gas chromatography. The saturated fatty acid content found in the analyzed spermatozoa was approximately 43% in H1 and 33% in H2, whereas the total unsaturated fatty acid content was 47% in H1 and 59% in H2. When control and ascorbate-Fe++ dependent samples were compared, it was observed a significant increase in light emission. Consequently, significant decrease in the percentage of the polyunsaturated fatty acids was determined, being more affected: C22: 5 n6 and C22: 6 n3 in both herds, whereas C20: 4 n6 and C22: 4 n6 only in H2. The great amounts of polyun­saturated fatty acids found in H2 samples could be related to the loss of acrosomal integrity. Our results indicate that boar semen contains great amounts of polyunsaturated fatty acid in its composition, which were vulnerable to the lipid peroxidation

Comparison of dogs treated for primary immune-mediated hemolytic anemia in Tuscany, Italy and Texas, USA

This retrospective study compared clinical characteristics between dogs treated for IMHA by veterinary teaching hospitals in Tuscany, Italy and Texas, USA between 2010 and 2018

Evaluation of Clinico-Pathological Alterations Including Some Leukocyte Ratios and Survival Rate in Dogs with IMHA Transfused and Not Transfused: A Retrospective Study

Immune-mediated hemolytic anaemia (IMHA) is a common hematological disorder in dogs. It can be primary or secondary and it is characterized by anti-RBC antibodies production. IMHA requires a detailed diagnostic pathway as well as a complex therapeutic approach that can include blood transfusion. Unfortunately, IMHA presents a high mortality rate, especially within 15 days after onset. This retrospective study evaluated: a) the clinical and clinico-pathological alterations that influenced the choice to perform a blood transfusion in an IMHA patient; b) if blood transfusion could be an additional therapeutic approach; c) application of leukocyte ratios in the prognosis. Sixty-seven cases of IMHA, both primary and secondary, admitted to the Veterinary Teaching Hospital between May 2010 and July 2018, were included. Signalment, history, clinical signs, clinico-pathological parameters and survival rate were collected. Patients were divided in two groups: 44 patients (IMHAnt) treated with immunosuppressive therapy alone (primary n=36, secondary n=8) and 23 patients (IMHAt), which received also a blood transfusion (primarily packed RBC) (primary n=16, secondary n=6). For all collected parameters, both groups were statistically compared. The IMHAt patients compared to IMHAnt patients (un-regarding to primary or secondary cause) presented: worse marks according to Tokyo Score System (TSS) (Chi Squared, p=0.003); a lower erythrocyte count (T-test, p=0.039), hemoglobin concentration (T-test, p=0.029) and platelet count (Mann-Withney, M-W, p=0.008); a higher value of band neutrophils (M-W, p=0.022), band neutrophil to lymphocyte ratio (M-W, p=0.005), (band neutrophil/neutrophil) to lymphocyte ratio (M-W, p=0.006) and a lower value of lymphocyte to monocyte ratio (M-W, p=0.013); a higher value of C-reactive protein (M-W, p=0.011) and activated partial thromboplastin time (M-W, p=0.014); and a lower survival rate at day 120 (Kaplan-Meyer, logrank, p=0.004) and not at 7, 15 and 30 days. Blood transfusions were performed based on the severity of clinical and clinico-pathological signs. IMHAt patients showed a more severe disease (according to TSS), a greater acute inflammatory condition and more coagulative defects. The high death rate among IMHAt patients at 120 days was related to their critical condition, which is probably why the desired benefit of blood transfusion wasn’t reached. However, a link between blood transfusions and the related worse clinical signs in IMHAt patients could not be ruled-out. Finally, the leukocyte ratios in dogs affected by IMHA were assessed for the first time so far and they were proven to be useful markers of acute inflammation and could have a prognostic value

Tel-eVax: a genetic vaccine targeting telomerase for treatment of canine lymphoma

Background: we have recently shown that Tel-eVax, a genetic vaccine targeting dog telomerase (dTERT) and based on Adenovirus (Ad)/DNA Electro-Gene-Transfer (DNA–EGT) technology can induce strong immune response and increase overall survival (OS) of dogs affected by multicentric Diffuse Large B cell Lymphoma (DLBCL) when combined to COP therapy in a double-arm study. Here, we have utilized a clinically validated device for veterinary electroporation called Vet-ePorator , based on Cliniporator technology currently utilized and approved in Europe for electro- chemotherapy applications and adapted to electrogenetransfer (EGT). Methods: 17 dogs affected by DLBCL were vaccinated using two Ad vector injections (Prime phase) followed by TM DNA–EGT (Boost phase) by means of a Vet-ePorator device and treated in the same time with a 27-week Madison Wisconsin CHOP protocol. The immune response was measured by ELISA assays using pool of peptides. Results: No significant adverse effects were observed. The OS of vaccine/CHOP animals was 64.5 weeks, in line with the previous study. Dogs developed antibodies against the immunizing antigen. Conclusions: Tel-eVax in combination with CHOP is safe and immunogenic in lymphoma canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and hold promise for the treatment of dogs affected by other cancer types. More importantly, our findings may translate to human clinical trials and represent new strategies for cancer treatment

Faecal microbiota in dogs with multicentric lymphoma

Malignant lymphoma B-cell type is the most common canine haematopoietic malignancy. Changes in intestinal microbiota have been implicated in few types of cancer in humans. The aim of this prospective and case-control study was to determine differences in faecal microbiota between healthy control dogs and dogs with multicentric lymphoma. Twelve dogs affected by multicentric, B-cell, stage III-IV lymphoma, and 21 healthy dogs were enrolled in the study. For each dog, faecal samples were analysed by Illumina sequencing of 16S rRNA genes and quantitative PCR (qPCR) for selected bacterial groups. Alpha diversity was significant lower in lymphoma dogs. Principal coordinate analysis plots showed different microbial clustering (P = .001) and linear discriminant analysis effect size revealed 28 differentially abundant bacterial groups in lymphoma and control dogs. The qPCR analysis showed significant lower abundance of Faecalibacterium spp. (q < .001), Fusobacterium spp. (q = .032), and Turicibacter spp. (q = .043) in dogs with lymphoma compared with control dogs. On the contrary, Streptococcus spp. was significantly higher in dogs with lymphoma (q = .041). The dysbiosis index was significantly higher (P < .0001) in dogs with lymphoma. In conclusion, both sequencing and qPCR analyses provided a global overview of faecal microbial communities and showed significant differences in the microbial communities of dogs presenting with multicentric lymphoma compared with healthy control dogs.dog

Hematological parameters in subadult female dolphins (Grampus griseus)

The purpose of this study is to identify CBC Reference Intervals on Risso’s dolphins. There is no database of blood parameters in cetacean species and the correct evaluation is fundamental to interpret any pathological variations. In three years, 818 blood samples were collected from 9 pre-pubertal females hosted under human care during clinical routine checks. Blood was obtained from periarterial venous rete in ventral side of the flukes and then placed in EDTA tubes. The Complete Blood Count (CBC) parameters were considered and are going to be reported as mean ± sd (MIN; MAX). Studies on CBC parameters for dolphins are rare and obsolete. This is the first study carried out on a large number of samples of Risso's dolphins, which differ in feeding habits and in some ethological aspects from the other genera of the family. This is a preliminary research for future studies related to the difference for sex and age