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Benign prostatic hyperplasia (BPH) is one of the most common reproductive disorders in both male dogs and men. Finasteride, a synthetic inhibitor of the enzyme 5α-reductase, is widely used as medical treatment. Although sperm can be affected by both BPH and finasteride treatment, the direct influence on DNA integrity remains unclear. Thus, the aim of this study was to verify the direct effect of BPH and/or finasteride treatment on DNA integrity of dog spermatozoa. A 2 × 2 factorial experiment was designed with 20 male dogs assigned to 4 experimental groups: BPH Group (Sperm DNA integrity was not affected by finasteride treatment. However, BPH dogs had higher susceptibility to sperm DNA acid denaturation (SCSA) compared to dogs not presenting BPH, as well as lower percentage of sperm with DNA integrity (toluidine blue staining).In conclusion, benign prostatic hyperplasia causes post-testicular sperm DNA damage, albeit finasteride treatment itself does not directly influence sperm DNA integrity.L’hyperplasie bénigne de la prostate (HBP) est l’un des troubles de la reproduction les plus courants chez le chien et chez l’homme. Le finastéride, un inhibiteur synthétique de l’enzyme 5α-réductase, est largement utilisé comme traitement médical. Bien que le sperme puisse être affecté à la fois par l’HBP et par le traitement avec le finastéride, l’influence directe sur l’intégrité de l’ADN reste peu claire.Le but de cette étude était ainsi de vérifier l’effet direct de l’HBP et/ou du traitement par finastéride sur l’intégrité de l’ADN des spermatozoïdes de chien. Dans la présente étude, 20 chiens mâles ont été randomisés selon un plan factoriel en 2x2 à l’un des 4 groupes expérimentaux suivants : Groupe HBP (n=5), Groupe HBP-Finastéride (n=5), Groupe non-HBP traité par Finastéride (n=5), et Groupe non-HBP non traité (n=5). L’analyse Le sperme a été réalisée mensuellement pendant 60 jours (J0, J30 et J60) soit après le début du traitement par finastéride ou à partir du diagnostic de HBP. L’intégrité de l’ADN des spermatozoïdes a été analysée par l’évaluation de la susceptibilité à la fragmentation (coloration au bleu de toluidine ; détermination de la structure de la chromatine des spermatozoïdes - SCSA), par l’évaluation directe de la fragmentation de l’ADN des spermatozoïdes (détermination de la dispersion de la chromatine des spermatozoïdes - SCDA) et par l’évaluation de la protamination des spermatozoïdes (chromomycine A3).L’intégrité de l’ADN des spermatozoïdes n’a pas été affectée par le traitement par finastéride. Cependant, les chiens avec HBP ont une susceptibilité plus élevée à la dénaturation acide de l’ADN des spermatozoïdes (SCSA) par comparaison aux chiens ne présentant pas d’HBP, ainsi qu’un pourcentage plus bas de spermatozoïdes avec intégrité de l’ADN (coloration au bleu de toluidine).L’hyperplasie bénigne de la prostate induit des altérations de l’ADN des spermatozoïdes, alors que le traitement par finastéride n’influence pas directement par lui-même l’intégrité de l’ADN des spermatozoïdes.

In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.

SummaryAlthough bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

Wild African Lion Panthera leo populations are decreasing due to inbreeding and reduced genetic variability. Thus, the use of assisted reproduction in the species could one day become essential. Before this is possible, however, studies need to be conducted on the basic reproductive traits of the species, especially those regarding sperm cells. This study aimed to analyze the semen of African Lions in field conditions. We included seven captive African Lions in our study. The animals were chemically restrained and electro-ejaculated. Twenty sperm samples were selected and analyzed for sperm motility and progressive motility, sperm motility index, and sperm morphology. In addition, the samples were analyzed for membrane and acrosome integrity (hypoosmotic swelling test and fast green/rose Bengal dyes, respectively) and assessed for cytochemical activity of the mitochondria. We found that sperm motility rate was 75.25%±2.03, progressive motility rate was 3.25%±0.10, and sperm motility index was 70.12%±1.71. We found morphologic abnormalities roughly at the expected rate with 34.61%±7.22 of the sperm cells having an intact plasma membrane and acrosome integrity of 92.27%±2.73; high mitochondrial activity was 54.26±4.88% and absence of mitochondrial activity was 2.72±0.68% in the sperm cells. These findings show that conventional tests for sperm motility and sperm morphology bring about the expected results for lions according scientific literature. Though a hypoosmotic swelling test may be performed using different concentrations, it might lead to a higher number of sperm cells with membrane damage. Fast green/rose Bengal stain and 3’3 diaminobenzidine assay, however, can be used in sperm analysis of lions in field conditions.

Contents 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.

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P

A predição do desempenho de touros na produção in vitro de embriões (PIVE), pela avaliação seminal, não está plenamente estabelecida. Com este estudo, objetivou-se caracterizar as diferenças nos atributos espermáticos de touros com baixo e alto desempenho, baseado nas taxas de clivagem e de blastocisto, visando estabelecer métodos preditivos de maior eficiência na PIVE. A partir do histórico de três anos da empresa In Vitro Brasil®, foram selecionados touros que haviam resultado nas menores e maiores taxas de clivagem e de blastocisto, estabelecendo-se 4 grupos experimentais: baixa taxa de clivagem (BC, n=5), alta taxa de clivagem (AC, n=5), baixa taxa de blastocisto (BB, n=5) e alta taxa de blastocisto (AB, n=5). Foram adquiridas palhetas de sêmen congelado dos touros de cada grupo, nas quais foram avaliados, antes (Pré-Percoll®) e após centrifugação em gradiente de Percoll® (Pós-Percoll®), os atributos espermáticos: cinética, em sistema CASA; integridade de membranas plasmática e acrossomal (FITC-PSA/PI), potencial de membrana mitocondrial (JC-1), status oxidativo (DHE, CellROXTM green/PI, MitoSox red), apoptose (FITC-FMK-VAD/PI) e DNA (SCSA), por citometria de fluxo; morfologia; atividade mitocondrial (DAB); capacitação (CTC); teste funcional e TBARS induzido. Diferenças entre grupos (BC vs. AC e BB vs. AB) foram estimadas pelo teste t. Para todas as análises estatísticas foi considerado nível de significância de 5%. Na ausência de interações significativas, o efeito dos grupos foi analisado ao fundir os momentos Pré e Pós-Percoll®. Os grupos AC e AB apresentaram valores superiores de VAP, VCL, ALH e DNC e valores inferiores de BCF, STR, LIN e WOB, cinética indicativa de hiperativação. Adicionalmente, estes grupos apresentaram indícios de menor ocorrência de estresse oxidativo, evidenciados pelo percentual de espermatozoides com membrana lesada e estresse oxidativo (CellROXTM green/PI) no grupo AC e nível de DHE no grupo AB. Para os demais atributos estudados, não se obteve diferença entre os grupos. Por meio de análise de regressão logística multivariada, constatou-se que a taxa de clivagem sofre efeito de: VCL, BCF e percentual de espermatozoides com integridade de membrana e sem estresse oxidativo; já a taxa de blastocisto, sofre efeito de: defeitos menores, VSL, DABIII e DHE, os quais foram inclusos nos respectivos modelos preditivos. As taxas obtidas com o modelo preditivo foram comparadas às taxas reais por análise de correlação de Pearson, que resultaram em intensidade moderada tanto para clivagem (r=0,56) quanto para blastocisto (r=0,44). Subpopulações espermáticas com diferentes perfis de cinética em CASA, foram determinadas em três etapas estatísticas segundo Núñez-Martínez et al. (2006). Foram detectadas 4 subpopulações: 1-Rápidos e progressivos; 2- Hiperativados; 3- Lentos e sinuosos; 4- Lentos e progressivos. Touros com alto desempenho (AC e AB) apresentaram maior percentual de subpopulação 2, Pré-Percoll®, e das subpopulações 1 e 2, Pós-Percoll®. Com isto, demonstra-se ser possível diferenciar touros de baixo e alto desempenho na PIVE, pela avaliação dos atributos espermáticos, com o estabelecimento de modelos preditivos para as taxas de clivagem e de blastocisto. Ademais, o estudo das subpopulações espermáticas constitui-se no método mais efetivo para a predição do desempenho in vitro. The prediction of bull performance on the in vitro production (IVP) of bovine embryos, by the seminal evaluation, is not fully established. Thus, the aim of this study was to characterize the differences in the sperm features of bulls with low and high performance, based on cleavage and blastocyst rates, in order to establish predictive methods for a greater efficiency in the IVP. Based on the results obtained in three years from the company In Vitro Brasil®, bulls were selected by the lowest and highest cleavage and blastocyst rates, establishing 4 experimental groups: low cleavage rate (BC, n=5), high cleavage rate (AC, n=5), low blastocyst rate (BB, n=5) and high blastocyst rate (AB, n=5). Frozen semen were obtained from the bulls of each group, in which were evaluated, before (Pre-Percoll®) and after Percoll® gradient centrifugation (Post-Percoll®), the sperm features: kinetics, in CASA; integrity of plasma and acrosomal membranes (FITC-PSA/PI), mitochondrial membrane potential (JC-1), oxidative status (DHE, CellROXTM green/PI, MitoSox red), apoptosis (FITC-FMK-VAD/PI) and DNA (SCSA), by flow cytometry; morphology; mitochondrial activity (DAB); capacitation (CTC); functional test and induced TBARS. Differences between groups (BC vs. AC and BB vs. AB) were estimated by t test. A significance level of 5% was considered for all statistical analysis. In the absence of significant interactions, the effect of the groups was analyzed by merging the moments Pre and Post-Percoll®. The AC and AB groups presented higher values of VAP, VCL, ALH and DNC and lower values of BCF, STR, LIN and WOB, kinetics indicative of hyperactivation. In addition, these groups showed less evidence of oxidative stress, observed by the percentage of spermatozoa with damaged membrane and oxidative stress (CellROXTM green/PI) in the AC group and level of DHE in the AB group. For the others sperm features, there was no difference between groups. By multivariate logistic regression analysis, it was verified that the cleavage rate is affected by: VSL, BCF and percentage of spermatozoa with membrane integrity and without oxidative stress; and the blastocyst rate, is affected by: minor defects, VSL, DABIII and DHE, which were included in the respective predictive models. The rates obtained with the predictive model were compared to the real rates by Pearson correlation analysis, which resulted in moderate intensity for both cleavage (r=0.56) and blastocyst (r=0.44). Spermatic subpopulations with different kinetic profiles at CASA were determined in three statistical steps according to Núñez-Martínez et al. (2006). Four subpopulations were detected: 1-Fast and progressive; 2- Hyperactivated; 3- Slow and sinuous; 4- Slow and progressive. Bulls with high performance (AC and AB) presented higher percentage of subpopulation 2, Pre-Percoll®, and subpopulations 1 and 2, Post-Percoll®. In summary, it is possible to differentiate low and high-performance bulls at IVP by evaluating the sperm features, with the establishment of predictive models for the cleavage and blastocyst rates. Furthermore, the study of sperm subpopulations is the most effective method for predicting in vitro performance.

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p

SummaryThe cryopreservation of epididymal sperm is an important technique that allows genetic material to be preserved, even post mortem. However, cryopreservation leads to increased oxidative stress and impaired sperm viability. Polyunsaturated fatty acid (PUFA) supplementation may improve certain sperm characteristics, but it also makes sperm more susceptible to oxidative stress, therefore adding antioxidants that counteract oxidative stress has become an option. In this context, this study aimed to evaluate the effect of the interaction between docosahexaenoic acid (DHA) and antioxidants on the quality after the cryopreservation of epididymal bull sperm. Twenty epididymides were collected after slaughter, and epididymal sperm was cryopreserved with bovine extender supplemented with docosahexaenoic acid (DHA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). We verified an improvement in motility in the group that was treated only with DHA 5 µM and a concentration-dependent effect on susceptibility to lipid peroxidation that was associated with DHA concentration (1 µM, 5 µM or 10 µM). Moreover, treatment with DHA (5 µM) and SOD (20 IU/ml) resulted in higher sperm motility. Thus, the association between DHA (5 µM) and SOD (20 IU/ml) appears to be an option for increased epididymal sperm features in bulls.

Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.

Contents 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.

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ± 19.16 ng/mL; Bad cooler: 159.72 ± 15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ± 18.33%; Bad cooler: 18.33 ± 1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ± 18.22 ng/mL; Bad cooler: 131.92 ± 12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ± 6.72 ng/mL; Medium-high: 140.45 ± 11.70 ng/mL; Medium-low: 202.57 ± 16.30 ng/mL and Low: 231.02 ± 32.35 ng/mL; p 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma.

The aim of this study was to determine the pattern of follicular development in ewes following ablation of all follicles associated with ovarian superstimulatory treatment. After confirmation of reproductive cyclicity using ultrasonography and progesterone levels, five adult ewes of undetermined breed received a vaginal pessary containing 60mg of medroxyprogesterone, maintained for 10 days, with the application of 100μg cloprostenol on the fourth day. On the tenth day, 300UI equine chorionic gonadotropin (eCG) was applied together with 80mg FSH, and all visible follicles were aspirated by laparoscopic ovum pick-up (LOPU). Following LOPU, ovarian structures were assessed using sonography every 8 hours for a period of 64 hours, and the number and diameter of small (4.5mm) follicles were recorded. The average diameter of small follicles reduced from 5.6±1.5mm initially to 0.8±1.3mm by 56 hours. Population size of the medium-sized follicles during the observation period followed a parabolic distribution wherein the theoretical maximum size emerged at 34.6 hours with no difference at (P>0.05) 24 (4.6±1.5) and 48 hours (4.8±2.3). It was concluded that in ewes subjected to ovarian superstimulation immediately following LOPU, follicular development is characterized by a new wave of follicle growth with a predominance of medium-sized follicles (2.5-4.5mm) between 24 and 48 hours, and a theoretical population maximum occured at 34.6 hours. RESUMO: O objetivo deste estudo foi determinar o padrão do desenvolvimento folicular em ovelhas após a ablação de todos os folículos, associado a tratamento superestimulatório ovariano. Após confirmação da ciclicidade reprodutiva por avaliação ultrassonográfica e dosagem de progesterona, cinco ovelhas adultas, sem raça definida, ao início do tratamento, receberam pessário vaginal contendo 60mg de acetato de medroxiprogesterona. Após quatro dias, foi administrado 100µg cloprostenol sódico e, no décimo dia, realizou-se a remoção do pessário e aspiração de todos os folículos ovarianos visíveis, guiada por laparoscopia, além da administração de 300UI eCG associada a 80mg FSH. Ao final do tratamento, a cada 8 horas, foi realizada avaliação ultrassonográfica das estruturas ovarianas por um período de 64 horas, onde o número e o diâmetro de folículos pequenos (4,5mm) foram contabilizados. O número de folículos pequenos, que inicialmente apresentava média 5,6±1,5, foi reduzido para 0,8±1,3 a partir de 56 horas, folículos grandes somente foram vistos a partir de 32 horas (0,2±0,4); a população de folículos médios, durante o período de observação, apresentou uma distribuição parabólica cujo vértice, considerado o ponto teórico de maior concentração, foi às 34,6 horas, não havendo diferença (P>0,05) nessa população de folículos, entre 24 e 48 horas (4,6±1,5 vs 4,8±2,3). Conclui-se que, em ovelhas submetidas à superestimulação ovariana iniciada imediatamente após remoção de todos os folículos visíveis à laparoscopia, o desenvolvimento folicular é caracterizado pela emergência de nova onda de crescimento, com predomínio de folículos médios (2,5 a 4,5mm) entre 24 e 48 horas, sendo o ponto teórico de maior ocorrência às 34,6 horas.

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.

Made available in DSpace on 2019-10-06T16:16:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Escherichia coli is the most common contaminant of the bovine uterus in the first few weeks postpartum, and one of the most important pathogens involved in uterine infections. This bacterium is characterised by diverse virulence factors (VF) however, the profile of E. coli VF in physiologic postpartum uterine contamination is not well established. Therefore, the present study aimed to investigate the prevalence of intrauterine bacteria, a set of virulence factors and phylogroups of E. coli isolates, leukogram and uterine cytology in 75 Holstein cows at 24 h (Sampling time 1) and 14 days (Sampling time 2) postpartum. Escherichia coli, α-haemolytic Streptococcus, Trueperella pyogenes, and Pasteurella multocida were the most common microorganisms isolated in pure culture, whereas E. coli and Clostridium perfringens Type A, E. coli and α-haemolytic Streptococcus, and E. coli and Proteus mirabilis were the most frequent microorganisms in mixed colonies at both studied sampling times. In the 59 E. coli isolates after 24 h (n = 35) and 14 days (n = 24) postpartum, the genes detected for VF were fimH, iucD/aer, kps, hlyA, usp, vt1 and vt2. Most E. coli strains detected in both moments of study belonged to Commensal phylogenetic groups A and B1, whereas Pathogenic groups D and B2 were identified at 24 h and 14 days postpartum respectively. All cows presented suitable immune response against the presence of bacteria in uterine lumen, observed by leukocytosis, neutrophilia, lymphocytosis and monocytosis at leukogram and a high number of polymorphonuclear leukocytes in uterine cytology, in both studied moments. Thus, a complex diversity of aerobic and anaerobic bacteria is involved in uterine contamination in the early postpartum of cows, besides the predominance of E. coli. Moreover, the genes fimH, iucD/aer, hlyA, kps, usp, vt1 and vt2 play a key role in the virulence of E. coli in this period. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Doutor Walter Mauricio Correa Street Department of Genetics Evolution and Bioagents Institute of Biology University of Campinas, 255 Monteiro Lobato Street, Postal Address 6109 Department of Veterinary Clinics School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Doutor Walter Mauricio Correa Street Department of Veterinary Hygiene and Public Health School of Veterinary Medicine and Animal Sciences (FMVZ) São Paulo State University (UNESP) Prof. Doutor Walter Mauricio Correa Street Rubião Junior Postal Address 560 Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Doutor Walter Mauricio Correa Street Department of Veterinary Clinics School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Doutor Walter Mauricio Correa Street Department of Veterinary Hygiene and Public Health School of Veterinary Medicine and Animal Sciences (FMVZ) São Paulo State University (UNESP) Prof. Doutor Walter Mauricio Correa Street Rubião Junior Postal Address 560

It is reported a case of necrotizing mastitis by Staphylococcus aureus in goat whose signs were right ceiling with alteration on coloration, tenderness, consistency and bloody discharge. Supportive treatment with parenteral fluid therapy and antibiotics were performed prior to surgery to remove the affected hemigland. Additionally, postoperative topical therapy with hydrogen peroxide, tincture of iodine 2% and copper sulfate was conducted at different times. The treatment applied and the emergency care of the animal in this case were effective for improvement of the clinical case and were extremely important to maintaining the animal´s life.

Summary The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica–silane and colloidal silica–polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica–silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal–silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal–PVP. In conclusion, sperm selection in colloidal silica–silane and colloidal silica–PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica–silane is the preferred method because its effectiveness and lower cost.

Made available in DSpace on 2016-04-01T18:44:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:44Z : No. of bitstreams: 1 ISSN1828-051X-2010-09-64-344-347.pdf: 588640 bytes, checksum: 85e53369e1a536a4cc29c392f771c89c (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The aim of this study was to evaluate the response of prepubertal ewe lambs to exogenous administration of either medroxyprogesterone acetate (MAP) or long-acting progesterone (LAP) together with biostimulation. Two Pool Dorset adult males and 75 mixed-breed prepubertal ewe lambs (average of 179 days-old and 30.0 kg) were used. The females were randomly assigned to three different groups. In the first group the females were submitted to the insertion of intravaginal sponges containing MAP (60 mg) for 12 days and were then biostimulated for eight weeks. In the second group the females were submitted to a single injection of LAP (225 mg) and then to biostimulation for eight weeks. In the last group, the females were only submitted to biostimulation for eight weeks. Animals were considered cyclic when plasma progesterone (P4) concentration exceeded 1.0 ng/mL in at least one of two consecutive blood samples taken within a 7-day interval in three distinct experimental moments. After treatments 93.3% of the females disregarding their group started their cyclicity and most of them (92.0%), continued to be cyclic after 63 days of either MAP or LAP together with biostimulation under both male and female effect. We conclude that prepubertal ewe lambs when submitted to protocols of either MAP or LAP followed by biostimulation result in puberty at the 7 month of age. It can be deducted that some ewe lambs submitted to the administration of either MAP or LAP together with biostimulation promoted a multiplier effect upon the other young females that were then stimulated to start cyclicity. Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil

Frozen and thawed ovine semen undergo morphological and functional changes that prevent or decrease the efficiency of fertilization. Sperm selection methods seek to improve the quality and viability of the fertilizing materials. Four sperm selection methods were employed, using two silica colloidal solutions coated with silane (silica colloidal-silane) or by polyvinylpyrrolidone (silica colloidal-PVP), and varying the volume of colloidal solution. Sperm kinematic and sperm recovery were evaluated by means of CASA. The protocols using silica colloidal-silane showed higher total motility (TM), progressive motility (PM) and percentage of rapid sperm (%RAP) compared to the methods employing silica colloidal-PVP and to the samples prior to sperm selection. The silica colloidal-PVP had greater sperm recovery compared to the silica colloidal-silane. Only the method using 4mL of silica colloidal-PVP was not efficient in selecting samples with better quality compared to the samples analyzed prior to sperm selection. The methods using lower volumes of colloidal solution did not differ from those using higher volumes and the best results were shown by the method with 1mL silica colloidal-silane. The results found in the study indicated greater efficiency of the silica colloidal-silane solution for sperm selection of thawed ovine semen when compared to selection using silica colloidal-PVP. The method using 1mL of silica colloidal-silane was equally efficient to the method with higher volume, presenting itself as an alternative to process samples with lower sperm concentration. RESUMO O sêmen ovino congelado e descongelado sofre alterações morfofuncionais que impossibilitam ou diminuem a eficiência na fecundação. Os métodos de seleção espermática visam melhorar a qualidade e a viabilidade do material fecundante. Foram utilizados quatro métodos de seleção espermática utilizando duas soluções de sílica coloidal revestida por silano (sílica coloidal-silano) ou por polivinilpirrolidona (sílica coloidal-PVP), variando o volume de solução coloidal. Foram testadas a cinética espermática no CASA e a recuperação espermática. Os protocolos utilizando sílica coloidal-silano apresentaram maior motilidade total (MT), motilidade progressiva (MP) e porcentagem de espermatozoides rápidos (% RAP) quando comparados aos métodos utilizando a sílica coloidal-PVP e às amostras antes da seleção espermática. A sílica coloidal-PVP teve maior recuperação espermática quando comparada à sílica coloidal-silano. Somente o método utilizando 4mL de sílica coloidal-PVP não foi eficiente na seleção de amostras com melhor qualidade quando comparado às amostras analisadas antes da seleção espermática. Os métodos utilizando menores volumes de solução coloidal não diferiram dos métodos de maior volume, sendo a sílica coloidal-silano com 1mL o método que apresentou os melhores resultados. Como conclusão, os resultados encontrados no trabalho apontaram a maior eficiência da sílica coloidal-silano em selecionar sêmen ovino congelado e descongelado quando comparado à seleção em sílica coloidal-PVP. O método utilizando 1mL de sílica coloidal-silano foi igualmente eficiente ao método com maior volume, sendo uma alternativa para processar amostras com baixa concentração espermática.