Hair as an alternative noninvasive matrix: sources of variation in testosterone levels.

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.

Paradoxical Effect of Quercetin Antioxidant on Goat Sperm Parameters After Cryopreservation.

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.

Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats.

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.

The effect of flunixin meglumine, firocoxib and meloxicam on the uterine mobility of equine embryos.

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.

Effect of sheep diets with macadamia by-product and protected fat on the quality of fresh and frozen semen / Efeito de dietas de ovinos com coproduto de macadâmia e gordura protegida na qualidade de sêmen fresco e congelado

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...

Effect of sheep diets with macadamia by-product and protected fat on the quality of fresh and frozen semen / Efeito de dietas de ovinos com coproduto de macadâmia e gordura protegida na qualidade de sêmen fresco e congelado

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)

Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes.

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.

Free gossypol supplementation frequency and reproductive toxicity in young bulls.

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.

A fast, low-cost and efficient method for the diagnosis of sperm DNA fragmentation in several species.

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.

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours).

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.

The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)

The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)

Effect of Prostaglandin F2 at the beginning of the ovulation synchronization protocol in dairy Bos indicus, Bos taurus and Bos indicus x Bos taurus heifers / Efeito da Prostaglandina F2 no início do protocolo de sincronização da ovulação em novilhas leiteiras Bos indicus, Bos taurus e Bos indicus x Bos taurus

The objective of the present study was to evaluate the effect of treatment with prosta-glandin F2α (PGF2α) at the beginning of the protocol for ovulation synchronization on follicular dy-namics in Bos indicus (Gyr; n=11), Bos taurus (Holstein Black and White, HBW; n=10), and crossbred animals (Gyr x HBW; n=12). On a random day of the estrous cycles (day 0, D0), the animals received 2.0 mg estradiol benzoate (EB) intramuscularly plus an intravaginal progesterone (P4) device, which was maintained for 8 days (day 8, D8). Half the heifers of each group received a dose of 25 mg PGF2αintramuscularly at the time of insertion of the intravaginal P4 device. When the intravaginal device was removed (D8), all animals received another dose of 25 mg PGF2α intramuscularly, followed by intramuscular injection of 1.0 mg EB 24 h later (day 9, D9). Ultrasonographic evaluations were per-formed at intervals of 24 hours from D0 to D8 and at intervals of 12 hours from removal of the P4 device to 96 hours thereafter. Samples were collected on days 0, 3, 6, 8, 10 and 22 for the measurement of P4. The mean maximum diameter of the dominant follicle (DF) was smaller (P=0.01) in Gyr heifers (10.0 ± 0.8 mm) than in Gyr x HBW (13.0 ± 0.6 mm) or HBW (12.5 ± 0.8 mm). Furthermore, treatment with PGF2α on D0 increased (P=0.02) the maximum diameter of DF (12.9 ± 0.5 vs. 10.9 ± 0.7 mm)...

O objetivo do presente estudo foi avaliar o efeito do tratamento com prostaglandina F2α (PGF2α)no início do protocolo de sincronização da ovulação, sobre a dinâmica folicular de novilhas Bos indicus (Gir; n=11), Bos taurus (Holandesa Preto e Branco - HPB; n=10) e cruzadas (Gir x HPB; n=12). Em dia aleatório do ciclo estral (dia 0 - D0), os animais receberam 2,0 mg de benzoato de es-tradiol (BE) via intramuscular, mais um dispositivo intravaginal de progesterona (P4) que foi man-tido por oito dias (dia 8 – D8). Metade das novilhas de cada grupo recebeu uma dose de 25 mg de PGF2α via intramuscular no momento da inserção do dispositivo intravaginal de P4. Na retirada do dispositivo intravaginal (D8), todos os animais receberam outra dose de 25 mg de PGF2α via intra-muscular e 24 h após (dia 9 - D9) receberam 1,0 mg de BE via intramuscular. Foram realizadas ava-liações ultrassonográficas com intervalos de 24 horas do D0 até o D8 e com intervalos de 12 horas da retirada do dispositivo de P4 até 96 horas. Para a dosagem de P4, foram colhidas amostras nos dias 0; 3; 6; 8; 10 e 22. A média do diâmetro máximo do folículo dominante (FD) foi menor (P=0,01) nas novilhas Gir (10,0 ± 0,8 mm) do que nas cruzadas Gir x HPB (13,0 ± 0,6 mm) ou HPB (12,5 ± 0,8 mm). Além disso, o tratamento com PGF2α no D0 aumentou (P=0,02) o diâmetro máximo do FD (12,9 ± 0,5 mm vs. 10,9 ± 0,7 mm) nos três grupamentos genéticos avaliados. A taxa de crescimento do FD foi menor (P=0,008) nas novilhas Gir (0,8 ± 0,1 mm/dia), do que nas novilhas cruzadas (1,3 ± 0,1 mm/dia) ou HPB (1,2 ± 0,1 mm/dia)...

Effect of Prostaglandin F2 at the beginning of the ovulation synchronization protocol in dairy Bos indicus, Bos taurus and Bos indicus x Bos taurus heifers / Efeito da Prostaglandina F2 no início do protocolo de sincronização da ovulação em novilhas leiteiras Bos indicus, Bos taurus e Bos indicus x Bos taurus

The objective of the present study was to evaluate the effect of treatment with prosta-glandin F2α (PGF2α) at the beginning of the protocol for ovulation synchronization on follicular dy-namics in Bos indicus (Gyr; n=11), Bos taurus (Holstein Black and White, HBW; n=10), and crossbred animals (Gyr x HBW; n=12). On a random day of the estrous cycles (day 0, D0), the animals received 2.0 mg estradiol benzoate (EB) intramuscularly plus an intravaginal progesterone (P4) device, which was maintained for 8 days (day 8, D8). Half the heifers of each group received a dose of 25 mg PGF2αintramuscularly at the time of insertion of the intravaginal P4 device. When the intravaginal device was removed (D8), all animals received another dose of 25 mg PGF2α intramuscularly, followed by intramuscular injection of 1.0 mg EB 24 h later (day 9, D9). Ultrasonographic evaluations were per-formed at intervals of 24 hours from D0 to D8 and at intervals of 12 hours from removal of the P4 device to 96 hours thereafter. Samples were collected on days 0, 3, 6, 8, 10 and 22 for the measurement of P4. The mean maximum diameter of the dominant follicle (DF) was smaller (P=0.01) in Gyr heifers (10.0 ± 0.8 mm) than in Gyr x HBW (13.0 ± 0.6 mm) or HBW (12.5 ± 0.8 mm). Furthermore, treatment with PGF2α on D0 increased (P=0.02) the maximum diameter of DF (12.9 ± 0.5 vs. 10.9 ± 0.7 mm)...(AU)

O objetivo do presente estudo foi avaliar o efeito do tratamento com prostaglandina F2α (PGF2α)no início do protocolo de sincronização da ovulação, sobre a dinâmica folicular de novilhas Bos indicus (Gir; n=11), Bos taurus (Holandesa Preto e Branco - HPB; n=10) e cruzadas (Gir x HPB; n=12). Em dia aleatório do ciclo estral (dia 0 - D0), os animais receberam 2,0 mg de benzoato de es-tradiol (BE) via intramuscular, mais um dispositivo intravaginal de progesterona (P4) que foi man-tido por oito dias (dia 8 D8). Metade das novilhas de cada grupo recebeu uma dose de 25 mg de PGF2α via intramuscular no momento da inserção do dispositivo intravaginal de P4. Na retirada do dispositivo intravaginal (D8), todos os animais receberam outra dose de 25 mg de PGF2α via intra-muscular e 24 h após (dia 9 - D9) receberam 1,0 mg de BE via intramuscular. Foram realizadas ava-liações ultrassonográficas com intervalos de 24 horas do D0 até o D8 e com intervalos de 12 horas da retirada do dispositivo de P4 até 96 horas. Para a dosagem de P4, foram colhidas amostras nos dias 0; 3; 6; 8; 10 e 22. A média do diâmetro máximo do folículo dominante (FD) foi menor (P=0,01) nas novilhas Gir (10,0 ± 0,8 mm) do que nas cruzadas Gir x HPB (13,0 ± 0,6 mm) ou HPB (12,5 ± 0,8 mm). Além disso, o tratamento com PGF2α no D0 aumentou (P=0,02) o diâmetro máximo do FD (12,9 ± 0,5 mm vs. 10,9 ± 0,7 mm) nos três grupamentos genéticos avaliados. A taxa de crescimento do FD foi menor (P=0,008) nas novilhas Gir (0,8 ± 0,1 mm/dia), do que nas novilhas cruzadas (1,3 ± 0,1 mm/dia) ou HPB (1,2 ± 0,1 mm/dia)...(AU)

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation.

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer-assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.

Cytoplasmic droplet acting as a mitochondrial modulator during sperm maturation in dogs.

Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18-24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Effect of different semen extenders for the storage of chilled sperm in Tigrina (Leopardus tigrinus).

To enhance the conservation of endangered populations, the present study aimed to evaluate whether Tigrinas (Leopardus tigrinus) sperm could be conserved under refrigeration for short periods while maintaining sufficient quality for use in assisted-reproductive techniques (i.e., cryopreservation, in vitro fertilization). For this purpose, semen samples from 15 Tigrinas individuals were submitted to conventional and functional tests after different cooling periods (4 °C; 0, 12, and 24 hours postcooling), using TCM 199 (TCM), Ham's F10 (HAM), Ham's F10 with bovine serum albumin (HBSA), and Tris-citrate egg yolk (TEYC) extenders. In a second step, semen cooled using TEYC was supplemented with reduced glutathione (GSH) at different concentrations (0, 0.5, 1.0, and 1.5 mM). TEYC yielded superior results compared with TCM, HAM, and HBSA even after 24 hours of cooling in regard to the sperm motility index (SMI-TEYC: 50.2 ± 1.7%), high mitochondrial activity (TEYC: 51.4 ± 1.9%), plasma membrane integrity (TEYC: 53 ± 2.1%), and DNA integrity (TEYC: 56.3 ± 2.9%). In regard to the concentration of thiobarbituric-acid-reactive substances (TBARS), TEYC (1900.1 ± 341.4 ng/106 spermatozoa) showed higher levels compared with the other extenders (HAM: 638.7 ± 121.6 ng/106 spermatozoa; HBSA: 468.7 ± 95.6 ng/106 spermatozoa; TCM: 169.6 ± 31.6 ng/106 spermatozoa). However, GSH therapy had no effect. In conclusion, the TEYC extender may be useful in maintaining sperm parameters of Tigrinas for up to 24 hours at 4 °C. Furthermore, these results allow the transport of this material at a minimum quality to be further used for artificial insemination, in vitro fertilization, and the development of semen cryopreservation protocols.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility.

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm.

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post-mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty-six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2-3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer-assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre-freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post-thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.