Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox

Semen manipulation and cryopreservation-thaw procedures may accelerate the generation of reactive oxygen species (ROS). Sperm exposure to large amounts of ROS has been shown to cause membrane lipid peroxidation and cellular injury to the sperm. The objective of this study was to overcome the ROS production in frozen-thawed ram semen by the addition of the antioxidants catalase or Trolox to semen following thawing. Frozen-thawed ram semen (100 x 10(6) sperm/straw) was supplemented with PBS (control group), 100 mu g/ml catalase, or 100 mu M Trolox/10(8) sperm (catalase and Trolox being dissolved in PBS) and incubated (37 degrees C) for 5 min. Under the experimental conditions used in this study, the catalase and Trolox antioxidants failed to protect the sperm from the spontaneous production of ROS. However, when lipid peroxidation was induced by iron (FeSO(4)), the addition of Trolox promoted a reduction (P < 0.05) in the formation of TBARS in the semen, compared to the control and catalase semen samples. The generation of TBARS and H(2)O(2) occurred in the extender alone, without the presence of sperm cells. In conclusion, the addition of Trolox to frozen-thawed ram semen could be beneficial as it decreases the production of TBARS when oxidative stress is induced. It is possible that a longer incubation period could lead to different results. The concentration of catalase also needs to be further evaluated. The extender could contribute to the oxidative stress of sperm, as it is a source of ROS during the cryopreservation of semen. (C) 2010 Elsevier B.V. All rights reserved.

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxidative metabolism of the neutrophils in sheep treated with sodium monensin and experimentally submitted to ruminal acidosis.

Ruminal acidosis is due to excessive ingestion of carbohydrates of rapid fermentation without previous adaptation of the microorganisms, causing severe metabolic disturbances to the animals. The objective of the present study was to assess the neutrophilic oxidative metabolism in sheep treated with sodium monensin in experimentally induced ruminal lactic acidosis. A total of 18 male sheep, half-bred (ideal x Merino), fistulated in the rumen, were used; nine of them received 33 mg/kg of the ionophore diet per day, for 30 days; the others were controls. The acidosis was induced by supplying 15g of sucrose/kg of body weight. The clinical evaluation and the rumen and blood samples were obtained before (0h) and at 6, 12, 24 and 48 hours post-induction. In both groups, all the animals presented clinical manifestations of ruminal lactic acidosis 6 hours after the induction. From this period on, a significant pH decrease (P<0.05) was observed in the ruminal fluid, which reached levels below 5. There were relevant differences (P<0.05) between the groups 12 hours after the induction, when the sheep treated with monensin had higher values than those of the control group. During this period, the oxidative metabolism of the neutrophils remained inhibited, and the reestablishment of this function only occurred in the sheep which received monensin. Blood pH, plasmatic glucose and the ionizable calcium suffered alterations within its levels. The seric cortisol concentration rose significantly (P<0.05) in both groups, although differences (P<0.05) between them were found at the end of the observation period. The treatment with monensin did not influence the oxidative metabolism of the neutrophils inhibited by the lactic acidosis; however, a faster recovery of this metabolism was verified in the animals treated with the ionophore.

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 +/- 6.05 mu mol/L) or 6 h (14.59 +/- 4.38 mu mol/L) and iron reached its lowest level at 96 h (3.08 +/- 1.18 mu mol/L) or 16 h (4.06 +/- 1.58 mu mol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of a portable clinical analyzer for the determination of blood gas partial pressures, electrolyte concentrations, and hematocrit in venous blood samples collected from cattle, horses, and sheep

Objective To compare results reported for blood gas partial pressures, electrolyte concentrations, and Hct in venous blood samples collected from cattle, horses, and sheep and analyzed by use of a portable clinical analyzer (PCA) and reference analyzer (RA).Animals-Clinically normal animals (24 cattle, 22 horses, and 22 sheep).Procedures-pH; Pco(2); Po(2); total carbon dioxide concentration; oxygen saturation; base excess; concentrations of HCO(3)(-), Na(+), K(+), and ionized calcium; Hct; and hemoglobin concentration were determined with a PCA. Results were compared with those obtained for the same blood sample with an RA. Bias (mean difference) and variability (95% confidence interval) were determined for all data reported. Data were also subjected to analyses by Deming regression and Pearson correlation.Results-Analysis of Bland-Altman plots revealed good agreement between results obtained with the PCA and those obtained with the RA for pH and total carbon dioxide concentration in cattle, K(+) concentration in horses and sheep, and base excess in horses. Except for Na(+) concentration and Hct in horses and sheep, correlation was good or excellent for most variables reported.Conclusions and Clinical Relevance-Data from blood gas and electrolyte analyses obtained by use of the PCA can be used to evaluate the health status of cattle, horses, and sheep. Furthermore, the handheld PCA device may have a great advantage over the RA device as a result of the ability to analyze blood samples on farms that may be located far from urban centers. (Am J Vet Res 2010;71:515-521)

Histopathology of the reproductive system of male sheep experimentally infected with Toxoplasma gondii

The aim of this study was to investigate the histopathological changes in reproductive system (testicles, epididymis, seminal vesicles, and prostate) of small male ruminants after Toxoplasma gondii infection. Eight sheep were inoculated with T. gondii: group I, four sheep (2.0 x 10(5) P-strain oocysts); group II, four sheep (1.0 x 10(6) RH-strain tachyzoites); and group III, two uninfected sheep maintained as control. Infection with T. gondii was confirmed by seroconversion (indirect fluorescent antibody test-IgG) in all the infected animals beginning on post-inoculation day (PID) 7. on PID 70, all the animals were euthanized and tissue samples (testicles, epididymis, seminal vesicles, and prostate) were collected and processed for histological analysis. The main changes detected were a focal mononuclear interstitial inflammatory infiltrate in the prostate and seminal vesicles; diffuse testicular degeneration associated with calcification foci and a multifocal mononuclear interstitial inflammatory infiltrate; and a mononuclear interstitial infiltrate and focal necrotic areas of the muscle fibers surrounding the seminal vesicles. The histopathological findings of this work, along with the detection of T. gondii in the examined parenchyma tissues (immunohistochemistry) and the results obtained by other authors examining different tissues, suggest that histological changes diagnosed in the reproductive system of rams infected with T. gondii are strongly suggestive of toxoplasmatic infection.

Effect of management practices on screw-worm among sheep in São Paulo State, Brazil

The screw-worm Cochliomyia hominivorax (Coquerel), whose larvae develop in wild and domestic animals as well as in man, is one of the major causes of myiasis in Brazil. Sheep raising is expanding in the state of São Paulo, where information about the infestation of screw-worm is necessary for appropriate control measures. The present data were obtained from questionnaires sent to all sheep breeders belonging to 'Associação Paulista de Criadores de Ovinos' - ASPACO (São Paulo State Association of Sheep Breeders). Response reliability was tested by random visits paid to 10.6% of the respondents. The results show that screw-worm is the most frequent ectoparasite in the flocks and is responsible for the greatest economic losses. Its occurrence is strongly associated with flock size, reaching 100% in hocks consisting of more than 500 animals. Infestation is higher in spring and summer and lowest in the fall. The number of cases increases in the presence of some management practices such as tail amputation. Furthermore, several other associations between the indicators surveyed were also found to be significant. The data obtained will permit the elaboration of strategies for the control of this parasitosis.

Diversity of ectoparasites in sheep flocks in São Paulo, Brazil

The occurrence of ectoparasites in sheep flocks is frequently reported but seldom quantified. Sheep production used to be a predominantly family activity in the state of São Paulo (Brazil), but it began to become a commercial activity in the past decade. Thus, information about the ectoparasites existing in sheep flocks has become necessary. The present data were obtained by means of questionnaires sent to all sheep breeders belonging to the 'Associação Paulista de Criadores de Ovinos' (ASPACO; São Paulo State Association of Sheep Breeders). Response reliability was tested by means of random visits paid to 10.6% of the respondents. Most of the properties (89.5%) reported the presence of one or more ectoparasites. Screw-worm (Cochliomyia hominivorax) was the most frequent ectoparasite (72.5%), followed by bot fly larvae (Dermatobia hominis, 45.0%), ticks (Amblyomma cajennense) and Boophilus microplus, 31.3%) and finally lice (Damalinia ovis, 13.8%). Combined infestations also occurred, the most common one being screw-worm with bot fly larvae (36.0%) followed by bot fly larvae with ticks (13.9%), screw-worm with ticks (9.3%), bot fly larvae with lice (6.9%), and ticks with lice (5.0%). The most common triple combination was screw-worm, bot fly larvae and ticks (12.8%). Breeds raised for meat or wool were attacked by bot fly larvae and ticks more often than other breeds. Lice were only absent from animals of indigenous breeds. The relationships among these ectoparasites are discussed in terms of sheep breeds, flock size, seasonality and the ectoparasitic combinations on the host.

Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep

This study was carried out to evaluate the relationship of abomasal inflammatory cells and parasite-specific immunoglobulin A (IgA) in mucus, with the resistance to Haemonchus contortus infection in three breeds of sheep naturally infected with gastrointestinal nematodes. The breeds were the native Santa Ines sheep, and the European Suffolk and Ile de France breeds. Mast cells, eosinophils and globule leucocytes were enumerated in abomasal mucosa. Eosinophils within the sub-mucosa also were counted separately. Histamine concentration was estimated in abomasal tissue samples. Enzyme-linked immunosorbent assay was carried out in mucus samples to determine the level of IgA anti-H. contortus third and fifth instar. There were no significant differences among group means of these variables (P > 0.05). The correlation coefficients between fecal egg counts (FEC) x mast cells (r = -0.490; P < 0.05) and FEC x eosinophils in sub-mucosa (r = -0.714; P < 0.01) was significant in the Santa Ines sheep. In the Ile de France group, the correlation coefficients between globule leucocytes x FEC (r = -0.879; P < 0.001) and histamine x worm burden (r = -0.833; P < 0.01) were also significant. In the Santa Ines and Ile de France sheep, correlation coefficients between IgA anti-L3 x worm burden and IgA anti-L3 x FEC were negative. In general, inflammatory cells and IgA-parasite-specific in abomasum were inversely associated with H. contortus worm burden and FEC indicating that they may impair parasite development or fecundity in the three breeds of sheep. However, similar mean values of inflammatory cells and IgA were found in the resistant (Santa Ines) and in the susceptible (Suffolk and Ile de France) breeds of sheep. The enumeration of cells by histological assessment does not provide information on their functional activity, which may be different among breeds. Thus, the effect of breed on the functional activity of these and other inflammatory cells is an important area for further study. (c) 2004 Elsevier B.V. All rights reserved.