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.

Oxidative stress and acid-base balance during the transition period of neonatal Holstein calves submitted to different calving times and obstetric assistance.

During the neonatal period, constant adaptations to the extrauterine environment are required. These adaptations are made impossible or hindered when unexpected obstetrical events take place. This study aimed to identify clinical changes of vitality, metabolic and acid-base balance, and oxidative stress of newborn calves born to assisted dams that experienced different durations of calving. A prospective and observational study was conducted with 44 Holstein calves allocated to 3 groups according to the duration of calving: 2 h (n = 16), 2-4 h (n = 16), and >4 h (n = 12), and to 2 subgroups: spontaneous calving (n = 22) and assisted calving (n = 22). We observed that obstetric assistance was beneficial when calving lasted more than 4 h, because 100% of assisted calves had a high Apgar score (≥7) at 5 min, similar to that of spontaneously born calves after 2 h of calving. Rectal temperature of all calves remained within normal values, albeit with a reduction at 20 min of life. Calves born after a calving time >4 h had the highest rectal temperatures, especially those that required obstetrical assistance. There was a progressive increase in blood oxygenation with time, but calves in the >4 h group showed the highest partial pressure of carbon dioxide at birth, whereas calves in the 2 h and 2-4 h groups had lower values. A decrease in partial pressure of CO2 was observed at 120 min in the 2-4 h and >4 h groups. In the 2-4 h group, calves with obstetrical assistance had higher oxidative stress (measured as thiobarbituric acid reactive substances, TBARS) at 120 min of life compared with that at birth. In spontaneously born calves, TBARS increased only after 240 min. In the >4 h group, TBARS increased progressively with time, regardless of obstetrical subgroup. Calves born spontaneously within the 2 h group had higher TBARS values at birth than calves in the >4 h group with obstetrical assistance. Calves in the >4 h group showed higher levels of glutathione peroxidase (an antioxidant enzyme) at birth than those in the remaining groups. Glutathione peroxidase decreased at 120 min in the >4 h group and at 240 min for the 2 h and 2-4 h groups. In conclusion, obstetric assistance did not improve neonatal vitality and blood oxygenation when calving lasted less than 2 h. Calves born after 2 h of calving showed decreased vitality, hypercapnia, hypoxemia, and increased antioxidant status (glutathione peroxidase).

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.

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.

Exogenous surfactant replacement immediately at birth as preventive therapy for lung prematurity in neonatal lambs.

Surfactant treatment is a manner to reduce alveolar superficial tension and increase pulmonary compliance in premature neonates. Thus, we aimed to analyze the effect of exogenous surfactant treatment in combination with manual ventilation for preterm lambs. We used 15 ewes and their lambs (n = 16), prematurely born at 135 days. At birth, lambs were submitted to orotracheal intubation attached to a handheld resuscitation device and randomly allocated to: Control Group (n = 5; only manual ventilation), Single Surfactant Group (n = 5; manual ventilation coupled by intratracheal administration of 100 mg/kg surfactant) and Double Surfactant Group (n = 6; surfactant volume was divided into two doses (50 mg/kg + 50 mg/kg) administrated at birth and 30 min thereafter). A complete physical exam, arterial gas analysis, blood glucose, urea and creatinine concentration and chest radiographic assessment were performed at fixed times. All lambs had decreased body temperature until 20 min after birth. However, control and double surfactant groups reached a thermic plateau after 30 min. Regardless of the time-point, control lambs had higher heart rate in comparison to treated neonates, including bradycardia in Single Surfactant Group. Single instillation led to lower oxygenation degree, compared to the Double Surfactant Group, suggesting that surfactant treatment was not able to adequately spread within the alveoli. Lambs treated with surfactant had severe impairment of aerobic activity, leading to anaerobic metabolism. All groups had hypercapnia, which can be explained by inadequate respiratory pattern and pulmonary opacity (89% of the lambs had severe or moderate lung content). In conclusion, exogenous surfactant therapy in association with manual ventilation is ineffective in reverting pulmonary immaturity of the preterm lamb, leading to less vitality, hypoxemia, delayed pulmonary clearance and high mortality rate.

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus).

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such 'labour-intensive technology'.

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.

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.

Influence of the duration of calving and obstetric assistance on the placental retention index in Holstein dairy cows.

The aim of this study is to evaluate the influence of duration of calving and obstetric assistance on retained placenta incidence of high milk production Holstein cows. Experimental groups were determined according to the duration of the expulsive phase of calving: 2 h (n = 16), 2-4 h (n = 16) and >4 h (n = 12), and additionally allocated in two sub-groups: spontaneous calving (n = 22) and intervention calving (n = 22). Diagnosis of retained placenta was considered with a threshold of 8 h after the expulsive phase. Cows without obstetric intervention, with labor duration of 2 h, presented reduced time needed for placental release, in comparison to those with obstetric assistance. In the 2-4 h group and >4 h, there was no statistical difference. The 2 h and 2-4 h groups with intervention and the spontaneous >4 h group were considered retained placenta groups. On the other hand, performing obstetric intervention when calving period was superior to 4 h nulled the occurrence of retention of fetal membranes. As a conclusion, obstetric assistance predisposes placental retention to calving with <2 h of duration in dairy cows. Conversely, when calving is more than 4 h, performing fetal extraction has a beneficial influence on preventing retained placenta.

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs.

BACKGROUND: During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis (lipids were extracted, transmethylated and analyzed by chromatography). RESULTS: Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids. CONCLUSIONS: These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.

Acute-phase protein profile during gestation and diestrous: proposal for an early pregnancy test in bitches.

Early pregnancy diagnosis in bitches has special relevance for adequate medical assistance to assure normal gestation, diagnosis of abortion or embryonic resorption, undesirable pregnancy interruption and dog owners wishing to assure medical assistance during parturition and to program participation of females in dog shows. The aim of this study was to verify acute-phase protein profile variation as a consequence of generalized inflammatory reaction due to embryonic endometrial invasion and use alterations as a method for early pregnancy diagnosis. Also the relationship between hormonal status and acute-phase proteins concentrations was assessed. Weekly serum samples were collected from 20 non-pregnant (NP) bitches and 20 pregnant females (P) to determine levels of fibrinogen, haptoglobin, ceruloplasmin, seromucoid, glycoprotein, alpha(2) globulin, progesterone and estradiol-17beta. No correlation was found between the implantation sites formed (number of pups born) and the hepatic stimulus for the acute-phase protein production. The conclusions are that acute-phase proteins can be used as an early pregnancy test for bitches from the 3rd week of gestation (14th-21st day post LH peak) for haptoglobin assay (values above 112.42 mg/dl of HbCN binding capacity), from the 4th to the 6th week (21st-42nd day post LH peak) for ceruloplasmin (values above 12.76+/-5.29 U/l), from the 4th week (21st-28th day post LH peak) for glycoprotein (values above 13.67%) and from the 4th week of gestation (21st-28th day post LH peak) for alpha(2) globulin (values above 0.61 g/dl). Fibrinogen and seromucoid increased in the P group from the 5th to the 6th week, respectively, thus not being suitable as parameters for an early pregnancy diagnosis. Relationship between ceruloplasmin and estradiol-17beta and seromucoid and progesterone were verified. For the acute-phase protein test it is important to verify bitches' healthy condition and to assure the precise mating dates to avoid false-positive and -negative results, respectively.

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)