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Hair is one widely used alternative matrix for endocrine studies. Not only can it maintain hormone content during storage for long periods of time, but its collection also induces little to no stress. Noninvasive techniques have broadened the opportunities for endocrine research, particularly regarding wild animals. Despite its advantages, many sources of variation may affect the steroid concentration found in hair, such as body location harvested, fur color, reproductive status, and sex. Thus, domestic species, such as the dog, are an excellent and approachable model for understanding this variability. For such, we addressed diverse sources of variation in testosterone concentrations from 24 domestic dogs (Canis lupus familiaris) of the Poodle breed of various colors and neuter status, and from both sexes. The variation comprised the comparison between 2 different matrices (blood vs hair); 2 different extraction storage methods (refrigerator vs freezer); 3 body regions (head, torso, and limbs); 3 coat colors (black, brown, and white); different neuter status (intact vs castrated males) and, finally, sex. Our results showed no correlation between blood and hair testosterone concentrations. Additionally, we did not find differences related to the storage method, body region, or coat color. There were differences in concentration between males and females, but not between females and castrated males. We discuss hair testosterone levels exhibited reasonable stability, and we present practical applications for both domestic and wildlife animals.

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BACKGROUND: Some antioxidants have been used in semen extenders to reduce adverse effects caused by excessive reactive oxygen species (ROS) production. The study was carried out to assess the effect of quercetin (QC) antioxidant therapy on goat semen submitted to cryopreservation. OBJECTIVE: To evaluate the effect of quercetin incorporation in different phases of the cryopreservation process of goat spermatozoa. MATERIALS AND METHODS: Five ejaculates from each of four goats (n= 20) were collected and split into four groups: Control (G1), without QC; G2, 15 µM of QC added to semen before centrifugation; G3, 15 µM QC added to semen after centrifugation; G4, 15 µM QC added to semen before centrifugation and 15 µM of QC added to semen after centrifugation (total of 30 µM of QC); and cryopreserved. All semen samples were evaluated after thawing for sperm kinetics, plasma membrane integrity, and ROS levels. RESULTS: Although lower concentrations of ROS were associated with groups that received antioxidant supplementation (P=0.0213), linear and dose dependent (P<0.05) reductions of the total and progressive sperm motility, velocity and percentage of fast cells were related to the QC groups. Likewise, plasma membrane integrity was better preserved (P=0.0154) in the control group (35.5%) than in groups that received QC (G2=32.6%, G3=32.4% and G4=26.7%). CONCLUSION: Although quercetin was efficient at reducing the oxidative stress related to sperm cryopreservation, it exerted a deleterious dose-dependent effect on the kinetics and integrity of the frozen goat semen, contradicating its use in the tested concentrations.

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This study aimed to detect the most deleterious ROS for goat sperm and then supplemented the extender with a proper antioxidant. For this, 12 adult goats (aged 1-7) were used. Fresh samples were submitted to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and malondialdehyde (MDA-toxic product of lipid peroxidation). After experiment 1, sperms were cryopreserved in extenders supplemented to glutathione peroxidase (Control: 0 UI/mL; GPx1: 1 UI/mL; GPx5: 5 UI/mL, and GPx10: 10 UI/mL) and catalase (Control: 0 UI/mL; CAT60: 60 UI/mL; CAT120: 120 UI/mL, and CAT240: 240 UI/mL). Each sample was evaluated by motility, plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, assay of the sperm chromatin structure, mitochondrial activity (3,3-diaminobenzidine), and measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS]). It was possible to observe a mitochondrial dysfunction (DAB-Class IV) and low membrane integrity after hydrogen peroxide action. However, the high rates of TBARS were observed on hydroxyl radical. CAT240 presents the lower percentage of plasma membrane integrity. It was possible to attest that hydrogen peroxide and hydroxyl radical are the more harmful for goat sperm. Antioxidant therapy must be improving perhaps using combination between antioxidants.

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Embryo mobility occurs as a result of prostaglandin production by the embryo and endometrium, promoting uterine smooth muscle contractions, which propels the embryonic vesicle through the lumen. Non-steroidal anti-inflammatory drugs (NSAIDs), as flunixin meglumine, are routinely used in equine medicine and can alter the conceptus mobility if applied in early pregnancy, which may impair maternal recognition of pregnancy. The objective of this study was to evaluate and compare the effect of flunixin meglumine (FM; 1.1 mg/kg IV), firocoxib (FIRO; 0.2 mg/kg PO), and meloxicam (ML; 0.6 mg/kg, IV), on the embryo mobility. Thirty mares were divided into three groups (n = 10 per treatment). After the pregnancy diagnosis on day 12 after ovulation, the embryo mobility was evaluated by transrectal ultrasonography every 5 min for 1 h in order to visualize the location of the embryo. In all mares, three evaluations were performed: immediately before treatment (pre-treatment), after NSAID administration and 24 h after treatment. In group FM, embryo mobility decreased, from 5.8 ±â€¯0.3 movements/hour (m/h) to 2.3 ±â€¯0.5 m/h (p < 0.05) and, after 24 h the values were similar to the pre-treatment evaluation (5.9 ±â€¯0.2 m/h). Likewise, ML treatment caused a decrease of embryo movements, from 5.9 ±â€¯0.3 to 1.9 ±â€¯0.3 m/h (p < 0.05), 24 h after treatment values were 5.7 ±â€¯0.4 m/h. Treatment with FIRO did not interfere with embryo mobility (5.7 ±â€¯0.4; 5.8 ±â€¯0.3 and 5.6 ±â€¯0.3 embryo movements in the first, second and third evaluation, respectively). In conclusion, FIRO was the only NSAID that did not alter the embryo mobility and may be the safest NSAID for use in early pregnant mares.

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The ruminant diet is characterized by low lipid concentration, resulting from traditional diets composed by forage species. The use of agro industrial byproducts in animal feed may be interesting, once it reduces production costs and reduces environmental contamination. Among them, macadamia is known for interesting protein and carbohydrate contents; however, it is the amount of lipids that make it different. Fat supplementation can raise concentrations of blood cholesterol, a precursor metabolite of steroid hormones, which constitute biological membranes and possess specific and essential biological activities. The semen characteristics should be taken into account in the selection of the breeding herds, and the semen analysis makes it possible to evaluate the fertility of the sheep and allows obtaining important conclusions based on its results. The objective was to evaluate the seminal quality of Morada Nova sheep breed consuming diets supplemented with macadamia residue and protected fat. The experiment was carried out with 24 rams aged 18 or 30 months, distributed in four treatment groups: control (C), 50 g (MAC50) or 150 g (MAC150) of macadamia industrial byproduct; and 50 g of protected fat (Megalac®), added to the concentrate. Semen was collected at four intervals: before supplementation (day 0), 30, 60 and 75 days after the beginning of supplementation, and it was taken the measurements of volume, appearance, motility, vigour, turbulence, concentration and morphology. At days 60 and 75, semen was frozen for determination of plasma membrane integrity, acrosome integrity and mitochondrial activity after thawing. Analysis of variance was performed and the means were compared by the SNK test. In the analysis of fresh semen, a significant effect (p0.05). The inclusion of 50 or 150 g of macadamia residue or 50 g of Megalac in the...

A dieta dos ruminantes é caracterizada por baixa concentração de lipídeos, resultante de dietas tradicionais compostas por espécies forrageiras. A utilização de coprodutos agroindustriais na alimentação animal pode ser interessante, pois além de reduzir custos na produção, reduz a contaminação ambiental. Dentre eles a macadâmia é conhecida por teores interessantes de proteína e de carboidratos; entretanto é a quantidade de lipídeos que a torna diferenciada. A suplementação de gordura pode elevar as concentrações de colesterol sanguíneo, metabólito precursor dos hormônios esteroides, que constituem membranas biológicas e possuem atividades biológicas específicas e essenciais. As características seminais devem ser levadas em consideração na seleção dos reprodutores, sendo que a análise do sêmen possibilita avaliar a fertilidade do carneiro e permite obter importantes conclusões a partir dos seus resultados. O objetivo foi avaliar a qualidade seminal de carneiros Morada Nova consumindo dietas suplementadas com resíduo de macadâmia e gordura protegida. O experimento foi conduzido com 24 carneiros, com idade entre 18 e 30 meses, distribuídos em quatro grupos de tratamento: controle (C), 50 g (MAC50) ou 150 g (MAC150) de subproduto industrial da macadâmia; e 50 g de gordura protegida (Megalac®), adicionados ao concentrado. O sêmen foi coletado em quatro intervalos: antes da suplementação (dia 0), 30, 60 e 75 dias após o início da suplementação. O sêmen foi coletado para avaliação do volume, aparência, motilidade, vigor, turbilhonamento, concentração e morfologia. Nos dias 60 e 75, o sêmen foi congelado para determinação da integridade da membrana plasmática, integridade do acrossoma e atividade mitocondrial após o descongelamento. A análise de variância foi realizada e as médias comparadas pelo teste SNK. Na análise do sêmen fresco, foi observado efeito significativo (p<0,05) dos tratamentos na motilidade. Para...

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RESUMO

The ruminant diet is characterized by low lipid concentration, resulting from traditional diets composed by forage species. The use of agro industrial byproducts in animal feed may be interesting, once it reduces production costs and reduces environmental contamination. Among them, macadamia is known for interesting protein and carbohydrate contents; however, it is the amount of lipids that make it different. Fat supplementation can raise concentrations of blood cholesterol, a precursor metabolite of steroid hormones, which constitute biological membranes and possess specific and essential biological activities. The semen characteristics should be taken into account in the selection of the breeding herds, and the semen analysis makes it possible to evaluate the fertility of the sheep and allows obtaining important conclusions based on its results. The objective was to evaluate the seminal quality of Morada Nova sheep breed consuming diets supplemented with macadamia residue and protected fat. The experiment was carried out with 24 rams aged 18 or 30 months, distributed in four treatment groups: control (C), 50 g (MAC50) or 150 g (MAC150) of macadamia industrial byproduct; and 50 g of protected fat (Megalac®), added to the concentrate. Semen was collected at four intervals: before supplementation (day 0), 30, 60 and 75 days after the beginning of supplementation, and it was taken the measurements of volume, appearance, motility, vigour, turbulence, concentration and morphology. At days 60 and 75, semen was frozen for determination of plasma membrane integrity, acrosome integrity and mitochondrial activity after thawing. Analysis of variance was performed and the means were compared by the SNK test. In the analysis of fresh semen, a significant effect (p<0.05) of the treatments on motility was observed. For cryopreserved semen, there was no significate difference (p>0.05). The inclusion of 50 or 150 g of macadamia residue or 50 g of Megalac in the...(AU)

A dieta dos ruminantes é caracterizada por baixa concentração de lipídeos, resultante de dietas tradicionais compostas por espécies forrageiras. A utilização de coprodutos agroindustriais na alimentação animal pode ser interessante, pois além de reduzir custos na produção, reduz a contaminação ambiental. Dentre eles a macadâmia é conhecida por teores interessantes de proteína e de carboidratos; entretanto é a quantidade de lipídeos que a torna diferenciada. A suplementação de gordura pode elevar as concentrações de colesterol sanguíneo, metabólito precursor dos hormônios esteroides, que constituem membranas biológicas e possuem atividades biológicas específicas e essenciais. As características seminais devem ser levadas em consideração na seleção dos reprodutores, sendo que a análise do sêmen possibilita avaliar a fertilidade do carneiro e permite obter importantes conclusões a partir dos seus resultados. O objetivo foi avaliar a qualidade seminal de carneiros Morada Nova consumindo dietas suplementadas com resíduo de macadâmia e gordura protegida. O experimento foi conduzido com 24 carneiros, com idade entre 18 e 30 meses, distribuídos em quatro grupos de tratamento: controle (C), 50 g (MAC50) ou 150 g (MAC150) de subproduto industrial da macadâmia; e 50 g de gordura protegida (Megalac®), adicionados ao concentrado. O sêmen foi coletado em quatro intervalos: antes da suplementação (dia 0), 30, 60 e 75 dias após o início da suplementação. O sêmen foi coletado para avaliação do volume, aparência, motilidade, vigor, turbilhonamento, concentração e morfologia. Nos dias 60 e 75, o sêmen foi congelado para determinação da integridade da membrana plasmática, integridade do acrossoma e atividade mitocondrial após o descongelamento. A análise de variância foi realizada e as médias comparadas pelo teste SNK. Na análise do sêmen fresco, foi observado efeito significativo (p<0,05) dos tratamentos na motilidade. Para...(AU)

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The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

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The aim of the present study was to analyze seminal quality of young bulls subjected to different frequencies of gossypol supplementation. Forty-eight Nellore bulls, with 19 months of age and weighing 357.8 ±â€¯7.2 kg, were used in this study. Animals were fed with 10.5 kg of standard supplement containing free-gossypol from whole cottonseed (WCS) at the following frequency: 3x/week (G3x), 5x/week (G5x) or 7x/week (G7x - Control). Additionally, a negative control was provided, and the treated animals received only mineral supplement (MM) ad libtum. The experiment lasted for 84 days and semen was collected at the beginning and at the end for analysis and cryopreservation. Fresh semen was used for initial analysis and plasma membrane integrity and sperm morphology were also determined. General motility using computer assisted sperm analysis (CASA), plasma and acrosomal membranes integrity, mitochondrial activity, and induced oxidative stress were assessed in post-thawed semen. The study design was completely randomized. Parametric data were analyzed by ANOVA and non-parametric data by the Wilcoxon test, using the statistical program SAS. Level of significance was set at 5%. Supplementation with WCS, regardless the frequency, increased total (P = .009) and head (P = .005) defects in comparison to animals receiving only forage and mineral supplement. Infrequent supplementation, particularly 5 times in the week (G5X), increased head (P = .026) and midpiece (P = .014) abnormalities. Sperm motility in fresh semen was lower in animals that received daily supplementation than those supplemented on alternate days (P = .021). Additionally, animals supplemented daily showed lower percentage of spermatozoa with intact acrosome compared to those supplemented on alternate days (P = .005). Thus, regardless the frequency of supplementation, free-gossypol supplementation affects sperm quality. Although the amount of free gossypol supplied weekly was the same among treatments, daily supplementation compromised sperm kinetics, differently from infrequent supplementation that led to sperm defects developed during spermatogenesis.

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Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high-precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue-stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.

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Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post-mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer-assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3'3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.

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