Susceptibility of epididymal sperm against reactive oxygen species in dogs.

In order to add information to physiology of sperm maturation and help to underline future antioxidant supplementation treatment to epididymal sperm, the aim of this study was to evaluate susceptibility of caput, corpus and cauda epididymal sperm to different reactive oxygen species (ROS) in dogs. Epididymal sperm was separately collected from each segment (caput, corpus and cauda) and subjected to 4 different ROS-challenges: superoxide anion, hydrogen peroxide, hydroxyl radical, malondialdehyde (MDA) or maintained as control. After 30 min of incubation in each ROS, sperm was evaluated for sperm kinetics, plasma and acrossomal membrane integrity, mitochondrial activity and resistance to oxidative stress. Decreased total and progressive sperm motility and rapid velocity at epididymal corpus occurred after exposure to hydrogen peroxide, hydroxyl radical and MDA. However, for cauda epididymis, hydrogen peroxide and malondialdehyde promoted higher deleterious effect regarding sperm motility and velocity. Only at cauda epididymis MDA decreased sperm mitochondrial activity index and no kinetics alterations (motility or velocity) occurred after exposure to superoxide anion in corpus and cauda epididymis. In conclusion, corpus and cauda epididymal sperm are highly susceptible to deleterious effect of hydrogen peroxide, malondialdehyde and hydroxyl radical. In addition, epididymal canine sperm is relatively resistant to superoxide anion damage.

Reproductive and endocrinological effects of Benign Prostatic Hyperplasia and finasteride therapy in dogs.

Benign prostatic hyperplasia (BPH) is one of the most important reproductive disorders in aging dogs. Therapeutic measures include orchiectomy and pharmacological treatment, leading to reduction of prostate volume and clinical signs. One of the most common drugs used in BPH treatment is finasteride, but data regarding its possible side effects are scarce. Thus, the aim of this study was to evaluate the effects of BPH and short-term (2 months) finasteride therapy on clinical, endocrinological, and reproductive parameters in dogs. Dogs were allocated into four experimental groups: Non-affected (n = 5), BPH (n = 5), Non-Affected-Finasteride (n = 5) and BPH-Finasteride (n = 5) groups. Dogs were evaluated monthly during 2 months by a complete breeding soundness examination, B-mode ultrasound and Doppler ultrasonography of the testicular artery, hormonal profile (testosterone, estrogen and dihydrotestosterone) and oxidative profile of the prostatic fluid. After 2 months, dogs were gonadectomized and testicles were subjected to histologic analysis. Finasteride treatment reduced dihydrotestosterone concentrations, without negative influence on semen quality and also reverted testicular hemodynamics changes of BPH. On the other hand, BPH was accompanied by significant changes in testosterone and estrogen concentrations and semen quality, mainly related to sperm kinetics alterations. In conclusion, BPH dogs have important hormonal and sperm alterations, however, short-term finasteride treatment (2 months) was able to reduce overall effects of BPH, thus representing a method of therapy for BPH treatment.

Does finasteride treatment for benign prostatic hyperplasia influence sperm DNA integrity in dogs?

BACKGROUND: 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 (n = 5), BPH-Finasteride Group (n = 5), Non-BPH Finasteride-Treated Group (n = 5) and Non-BPH Untreated Group (n = 5). Sperm evaluation was performed monthly for 60 days after the start of finasteride therapy or BPH diagnosis (D0, D30 and D60). Sperm DNA integrity was analyzed through fragmentation susceptibility (toluidine blue staining and Sperm Chromatic Structure Assay - SCSA), direct evaluation of DNA fragmentation (Sperm Chromatin Dispersion Assay - SCDA) and sperm protamination (chromomycin A3). RESULTS: 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). CONCLUSION: In conclusion, benign prostatic hyperplasia causes post-testicular sperm DNA damage, albeit finasteride treatment itself does not directly influence sperm DNA integrity.

CONTEXTE: 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). RÉSULTATS: 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). CONCLUSIONS: 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.

Prostatic hyperplasia: Vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy.

As a consequence of a hormonal imbalance, Prostatic Hyperplasia (PH) is characterized by increased prostate volume, along with higher local angiogenesis and vascularization. Orchiectomy is the common treatment for dogs, however it is not an option for breeding animals. Thus, finasteride arises as the drug of choice for stud dogs. Therefore, the aim of this study was to evaluate the effects of orchiectomy or finasteride therapies on hormonal and vascular dynamics of PH dogs. Fifteen dogs, aged 6-13 years were assigned to: Untreated Group (dogs diagnosed with PH-n = 5), Finasteride treated group (PH dogs treated with finasteride-n = 5) and Orchiectomy treated group (PH dogs submitted to orchiectomy-n = 5). Evaluations were performed in a monthly interval (first day of treatment; after 30 and 60 days). Doppler ultrasonography was performed to measure prostatic volume, vascularization and hemodynamic profile of prostatic artery. Dihydrotestosterone, estrogen and testosterone concentrations were measured. At day 60, prostatic biopsy was performed for histological, immunohistochemical and qPCR analysis for VEGF-A expression. At day 60, vascularization score was higher in untreated compared to treated groups (finasteride and orchiectomy). Furthermore, VEGF-A expression was lower in the Orchiectomy Treated Group, but VEGF-A was immunohistochemically lower in both treated groups (finasteride and orchiectomy) compared to the Untreated Group. The efficiency of finasteride treatment in reducing clinical signs, prostate volume and vascularization appears to be similar to orchiectomy. In conclusion, both PH medical and surgical therapy lead to reduction in prostate dimension and VEGF-A expression and, consequently, lower local vascularization. However, orchiectomy promotes marked hormonal changes, which ultimately lead to prostate atrophy.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

A case trial study of the effect of ageing on fresh and post-thaw sperm in dogs.

The aim of this study was to compare fresh and post-thaw sperm morphofunctionality of young and old dogs of a case trial study. Young dogs had higher libido and fresh semen motility, vigour and plasmatic membrane integrity. Conversely, senile dogs had higher percentage of major sperm defects (proximal droplets). Young dogs presented higher post-thaw sperm acrosomal membrane integrity and lower percentage of sperm defects compared to senile dogs. In conclusion, senility has deleterious effect on fresh semen, causing high incidence of defected spermatozoa and plasma membrane lesion, decreasing sperm motility and vigour. Moreover, senile dogs are more susceptible to sperm cryodamage.

The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls.

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.

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress.

Taurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA + Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

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.

The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling.

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Impact of induced levels of specific free radicals and malondialdehyde on chicken semen quality and fertility.

Over the past decades, scientists endeavored to comprehend oxidative stress in poultry spermatozoa and its relationship with fertilizing ability, lipid peroxidation (LPO), free-radical scavenging systems, and antioxidant therapy. Although considerable progress has been made, further improvement is needed in understanding how specific reactive oxygen species (ROS) and malondialdehyde (MDA, a toxic byproduct of LPO) disrupt organelles in avian spermatozoon. Hence, this study examined functional changes in chicken spermatozoa after incubation with different ROS, and their implications for the fertility. First, semen samples from 14 roosters were individually diluted and aliquoted into five equal parts: control, superoxide anion, hydrogen peroxide (H2O2), hydroxyl radicals, and MDA. After incubation with these molecules, aliquots were analyzed for motility, plasma membrane and acrosome integrity, mitochondrial activity, and LPO and DNA damage. Hydrogen peroxide was more detrimental for sperm motility than hydroxyl radicals, whereas the superoxide anion and MDA exhibited no differences compared with controls. In turn, plasma membrane and acrosome integrity, mitochondrial activity, LPO and DNA integrity rates were only affected by hydroxyl radicals. Thereafter, semen aliquots were incubated under the same conditions and used for artificial insemination. In accordance to our in vitro observations, H2O2 and hydroxyl radicals sharply reduced egg fertility, whereas superoxide anion and MDA only induced slight declines. Thus, chicken sperm function was severely impaired by H2O2 and hydroxyl radicals, but their mechanisms of action seemingly comprise different pathways. Further analysis regarding susceptibility of spermatozoon organelles to specific radicals in other poultry will help us to understand the development of interspecific differences in scavenging systems and to outline more oriented antioxidant approaches.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Perfil de proteínas plasmáticas em cadelas gestantes e não gestantes / Plasmatic protein profile of pregnant and non-pregnant bitches

O estudo das interações orgânicas da gestação e as mudanças fisiológicas que estão ocorrendo nesta fase são de extrema importância para a avaliação clínica da fêmea gestante ou para estabelecer o diagnóstico de processos patológicos em andamento. O objetivo do presente estudo foi comparar o perfil das diversas proteínas sanguíneas (frações protéicas do soro - albumina, a1, a2, b, g globulinas e proteína total) durante o período gestacional e no diestro em cadelas. Foram utilizadas 40 fêmeas caninas da raça Dogue Alemão, em idade variando entre 2 a 7 anos, clinicamente saudáveis. Os animais foram separados em dois grupos, denominados Grupo não gestante (NG) e Grupo gestante (G), constituídos por 20 fêmeas em diestro e 20 fêmeas gestantes, respectivamente. Preconizou-se colheita de sangue semanalmente de cada animal durante 9 semanas. Nas fêmeas do grupo NG, as amostras foram colhidas a partir do início do diestro, até a detecção do início do anestro; no grupo G, as amostras foram colhidas do início do diestro até o momento da parição. A partir do soro sanguíneo, foram determinadas as concentrações de proteína total, albumina, a1, a2, b e g globulinas. Não houve diferença significativa quanto aos resultados de proteína sérica total entre a 1ª e 6ª semanas de gestação e diestro, havendo decréscimo gradual em ambos grupos. Já ao final da gestação (entre a 7ª e 9ª semanas), houve acréscimo significativo dos valores de proteína total, sugerindo ação anabólica. Os níveis de albumina sofreram queda da 1ª a 9ª semana, tanto no grupo gestante, como não-gestante, com diferença estatística entre os dois grupos na 7ª, 8ª e 9ª semanas. As concentrações de α1 globulina nas fêmeas gestantes sofreram acréscimo significativo a partir da 2ª semana, contudo, diferença estatística entre os grupos NG e G ocorreu somente na 8ª e 9ª semanas, coincidindo com a fase de preparação à parição. Houve aumento no perfil de α2 globulina entre a 2ª e 3ª semanas de gestação, porém tais valores não diferiram das fêmeas em diestro. Diferença significativa de α2 globulina foi observada apenas durante a 4ª, 5ª, 6ª, 8ª e 9ª semanas entre os dois grupos avaliados. As concentrações séricas de ß globulina nas cadelas não-gestantes foi superior às das fêmeas gestantes durante a 2ª, 4ª e 7ª semanas. Durante o primeiro terço da gestação (1ª a 3ª semana), foi observado queda dos valores de γ globulina, coincidente ao período pré implantacional e durante a formação dos sítios de implantação. Entre a 8ª e 9ª semanas de gestação, houve acréscimo significativo de γ globulina, possivelmente conseqüente ao aumento da produção de imunoglobulinas direcionadas à glândula mamária, como constituinte do colostro. Em conclusão, as proteínas alteram-se de forma evidente durante o período de gestação. Foi possível inferir diferenças nas funções biológicas das proteínas sanguíneas em cadelas gestantes e não gestantes. As proteínas determinadas estão envolvidas com o estímulo inflamatório durante a gestação, além dos mecanismos de regulação hormonal e preparação do organismo materno à lactação.(AU)

The study of the organic interaction and physiological adaptations during pregnancy is of utmost importance for clinical evaluation and diagnosis of pathological conditions of pregnant bitches. The aim of the present study was to compare the serum protein profile (total protein, albumin, a1, a2, b and g globulin) of pregnant and diestrous bitches. For this purpose, 40 healthy 2 to7-year-old Great Dane bitches were used. The bitches were allocated in two experimental groups: Non-pregnant group (NP; n=20) and pregnant group (P; n=20). From each female, blood was drawn weekly during 9 weeks, from the diestrous onset until the beginning of anestrus or parturition, respectively from NP and P groups. The concentration of total protein, albumin and a1, a2, b and g globulins were determined from serum samples. No statistical difference was found for total protein between 1st and 6th weeks of pregnancy or diestrus. In both groups, there was a progressive decline in total protein concentration. At the end of pregnancy (7th to 9th week), a significant increase in total protein was verified, suggesting an anabolic process. Albumin concentration decreased between the 1st and 9th week in both groups, however, more markedly in the P group (with significant difference between groups at 7th, 8th and 9th week). The levels of α1 globulin in pregnant bitches increased significantly from the 2nd week on. Statistical difference was observed between groups only at the 8th and 9th week, during which a preparatory phase for parturition occurs. A significant rise in α2 globulin was shown between the 2nd and 3rd week of gestation, however without difference from the NP group. There was significant difference for α2 globulin between groups at the 4th, 5th, 6th, 8th and 9th week. Serum concentration of b globulin in diestrous bitches was superior than pregnant bitches at the 2nd, 4th and 7th week. During early gestation (1st to 3rd week) there was a decrease in g globulin, simultaneously to the preimplantation period and formation of implantation sites. An increase in g globulin was shown between the 8th and 9th week in pregnant bitches, possibly due to the increase in immunoglobulin synthesis targeting the mammary gland to form the colostrum. In conclusion, markedly changes in protein profile occur during gestation. It was possible to state different biological function of blood proteins in pregnant and diestrous bitches. The determined proteins are enrolled in the inflammatory stimulus during gestation, as well as in the hormonal regulatory mechanisms and maternal preparatory process to lactation.(AU)