Uterine haemodynamic, vascularization and blood pressure changes along the oestrous cycle in bitches.

Characterization of the bitch's reproductive physiology is of utmost importance for the development of new reproductive techniques and the diagnosis of reproductive diseases. In this respect, uterine B-mode ultrasonography has been employed in several studies; however, the focus on haemodynamic changes along the oestrous cycle is yet to be described. Thus, the aim of this study was to characterize haemodynamic changes (uterine vascularization, systemic arterial blood pressure and heart rate) throughout the oestrous cycle in bitches. For this purpose, ten Golden Retriever bitches were evaluated during an entire oestrous cycle, twice during each stage of the cycle. Uterine artery blood flow, velocity wave forms, haemodynamic parameters and vascularization were analysed by Doppler ultrasonography. Furthermore, uterine diameter, arterial blood pressure and heart rate were measured. Uterine artery pulsatility index at early prooestrus was significantly lower in comparison with early oestrus, mid- and late anoestrus. Uterine artery resistance index was higher at early oestrus when compared to late oestrus and uterine diameter was significantly higher during late prooestrus. Furthermore, mean arterial blood pressure was lower and heart rate was higher during late prooestrus in comparison with the other oestrous cycle stages. In conclusion, haemodynamic changes in the uterine artery, uterine diameter, systemic blood pressure and heart rate occur during the canine oestrous cycle. Specifically, there is an increase in uterine artery perfusion, uterine diameter and mean arterial blood pressure during prooestrus, while uterine blood flow diminishes during oestrus and anoestrus.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients.

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

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

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

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

Effect of reduced glutathione (GSH) in canine sperm cryopreservation: In vitro and in vivo evaluation.

The aim of this study was to compare the in vitro and in vivo efficiency of different concentrations (0, 10 and 20 mM) of reduced glutathione supplemented to the extender for canine semen cryopreservation. Six normospermic dogs were used and each ejaculate was divided in 3 experimental groups, according to GSH concentration (GSH-0, GSH-10 and GSH-20 Groups). After thawing, samples were evaluated by sperm motility by computer-assisted sperm analysis (CASA), flow cytometric evaluation of plasma and acrosome membrane integrity, mitochondrial membrane potential and activity, chromatin susceptibility to acid-induced denaturation, and measurement of spontaneous and induced production of thiobarbituric acid reactive substances (TBARS). In vivo tests were carried out with GSH-0 and GSH-10 groups, for which six bitches were inseminated with semen cryopreserved in extender without GSH or containing 10 mM GSH. Intrauterine insemination was performed by cervical catheterization on the 5th and 6th days after the LH surge, detected by serum progesterone and LH assays. In the CASA evaluation, GSH-20 group had the lowest total and progressive motility and lower percentage of sperm with rapid and slow speed. Groups treated with glutathione showed lower percentage of acrosome damage, but higher percentage of plasma membrane injury. GSH-20 group had higher percentage of sperm with low mitochondrial activity and higher concentration of induced TBARS. Both groups (GSH-0 and GSH-10) had positive pregnancies. In conclusion, 20 mM GSH supplementation to canine cryopreservation extender promoted sperm damage, especially to mitochondrial activity. However, addition of 10 mM GSH resulted in acrosome protection, preserving fertility rate.

The oxidative stress, antioxidant profile and acid-base status in preterm and term canine neonates.

During the initiation of neonatal pulmonary respiration, there is an exponential increase in reactive oxygen species that must be scavenged by antioxidant defences. However, neonate and preterm newborns are known to possess immature antioxidant mechanisms to neutralize these toxic effects. The purposes of this study were to compare the development of antioxidant system between preterm and term canine neonates and to evaluate the magnitude of acid-base balance during the initial 4 h of life. A prospective study was conducted involving 18 neonatal puppies assigned to Term Group (63 days of gestation; n = 5), Preterm-57 Group (57 days of gestation; n = 8) and Preterm-55 Group (55 days of gestation; n = 5). Neonates were physically examined through Apgar score and venous haemogasometry within 5 min, 2 and 4 h after birth. No difference on amniotic fluid and serum superoxide dismutase (SOD), glutathione peroxidase (GPx) and the marker of oxidative stress (thiobarbituric acid reactive substances; TBARS) was verified. Irrespective of prematurity, all neonates presented low vitality, hypothermia, acidosis, hypoxaemia and hypercapnia at birth. However, term puppies clinically evolved more rapidly than preterm newborns. During the course of the study, premature neonates presented more severe complications, such as prolonged hypoxaemia and even death. In conclusion, premature puppies have no signs of immature enzymatic mechanisms for controlling oxidative stress, although SOD and GPx may participate in achieving acid-base balance. Aside from initial unremarkable symptoms, premature puppies should be carefully followed up, as they are at high risk of succumbing to odds of prematurity.

Sperm maturation in dogs: sperm profile and enzymatic antioxidant status in ejaculated and epididymal spermatozoa.

Spermatozoa become more susceptible to the attack of reactive oxygen species during maturation. To avoid oxidative damage, the epididymis must provide the necessary antioxidant protection. The aim of this study was to compare the canine sperm profile and the enzymatic antioxidant status of the ejaculated fractions and samples collected from the different segments of the epididymis (caput, corpus and cauda). Five adult dogs were used, and after 1-3 weeks, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy and computer-assisted motility analysis: sperm plasma membrane permeability and the activity of the antioxidant enzymes catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the caput and corpus showed lower values for most of the motility variables evaluated, indicating different levels of immaturity. Catalase activity was observed only in ejaculated samples. Conversely, GPx activity was higher in the cauda epididymidis. Correlations were found between SOD and GPx and SOD and sperm motility in the epididymal cauda and corpus, highlighting the importance of the enzymes for the protection of spermatozoa during the transit along the epididymis.

Semen analysis of Golden Retriever healthy dogs and those affected by muscular dystrophy.

This study aimed to characterise canine flow cytometry semen analysis, as well as seminal reactive oxygen species dosage using the Golden Retriever breed as model of study. Moreover, we searched for the influence of muscular dystrophy in Golden Retriever dogs on semen parameters. Thirty-seven semen samples were obtained from healthy Golden Retrievers (n = 15) and from muscular dystrophy affected dogs (n = 22). Sperm-rich fractions were analysed by standardised breeding soundness examination in addition to the assay of fluorescence assisted cell sorting for acrosome integrity, mitochondrial activity and DNA fragmentation. Volume of ejaculate, per cent of motile spermatozoa and vigour were similar between groups; there were no differences in the per cent of minor and major defects. Integrity of acrosomal membrane, mitochondrial potential and sperm DNA fragmentation had no significant differences between groups either. Animals from control group had higher concentration of spontaneous seminal oxidative species in comparison with affected animals. Dogs affected by dystrophy had seminal parameters similar to those observed in healthy dogs except for the lower concentration of oxidative species. Future studies aiming to establish reference values for canine seminal parameters should be considered preferably with distinction of breeds.

Prenatal and neonatal adaptations with a focus on the respiratory system.

Among the modifications that occur during the neonatal period, pulmonary development is the most critical. The neonate's lungs must be able to perform adequate gas exchange, which was previously accomplished by the placenta. Neonatal respiratory distress syndrome is defined as insufficient surfactant production or pulmonary structural immaturity and is specifically relevant to preterm newborns. Prenatal maternal betamethasone treatment of bitches at 55 days of gestation leads to structural changes in the neonatal lung parenchyma and consequently an improvement in the preterm neonatal respiratory condition, but not to an increase in pulmonary surfactant production. Parturition represents an important challenge to neonatal adaptation, as the uterine and abdominal contractions during labour provoke intermittent hypoxia. Immediately after birth, puppies present venous mixed acidosis (low blood pH and high dioxide carbon saturation) and low but satisfactory Apgar scores. Thus, the combination of physiological hypoxia during birth and the initial effort of filling the pulmonary alveoli with oxygen results in anaerobiosis. As a neonatal adaptation follow-up, the Apgar analysis indicates a tachypnoea response after 1 h of life, which leads to a shift in the blood acid-base status to metabolic acidosis. One hour is sufficient for canine neonates to achieve an ideal Apgar score; however, a haemogasometric imbalance persists. Dystocia promotes a long-lasting bradycardia effect, slows down Apgar score progression and aggravates metabolic acidosis and stress. The latest data reinforce the need to accurately intervene during canine parturition and offer adequate medical treatment to puppies that underwent a pathological labour.

Cortisol profile and clinical evaluation of canine neonates exposed antenatally to maternal corticosteroid treatment.

The effects of glucocorticoids on both foetal canine lung and endogenous serum cortisol concentration have not been clearly delineated. Therefore, we aimed to investigate whether maternal corticosteroid treatment can alter maternal and neonatal cortisol profile and improve neonatal vitality. We allocated six bitches of different breeds and their neonates into two groups: control group (CONT)--maternal administration of saline solution at 55 days post-ovulation (n = 3); and betamethasone group (BETA)--administration of a single dose of 0.5 mg/kg betamethasone (Celestone Soluspan(®) ) at 55 days post-ovulation (n = 3). Caesarean sections were scheduled for day 63 after ovulation. However, BETA group dams showed precocious signs of labour, and c-sections were performed at 58 days post-ovulation. Maternal and neonatal evaluations were performed periodically between betamethasone administration and birth, respectively. Neonates from both groups presented unsatisfactory (<5) Apgar score at birth. However, in spite of an earlier improvement on vitality found on CONT group and the premature delivery on BETA group, both groups showed acceptable Apgar score 120 min after birth. Neonatal cortisol concentrations were higher on CONT group compared to BETA group at birth. In addition, a gradual decrease on maternal cortisol concentrations was observed in the BETA group from treatment until parturition. These findings suggest that despite the down-regulation on the hypothalamic-pituitary-adrenal axis and the induction of premature delivery, betamethasone treatment was able to provide similar vitality when compared to the untreated neonates born at term.

Perinatal cortisol and blood glucose concentrations in bitches and neonatal puppies: effects of mode of whelping.

The decision on how and when to assist whelping is crucial for the survival rate of puppies and health status of the dam. However, medical or surgical therapy in dystocia can impact both maternal and neonatal stress and glucose response differently. This study aims to compare perinatal cortisol and glucose among different modes of delivery in bitches and neonates. We analyzed 50 puppies derived from 27 healthy bitches. According to the condition at birth, bitches and their puppies were allocated into either a Eutocia Group (vaginal birth with no whelping assistance), Fetal Dystocia Group (whelping assistance with fetal manipulation), Maternal Dystocia Group (whelping assistance because of partial uterine inertia corrected by oxytocin administration), or Cesarean Section Group (fetal or maternal dystocia bitches subjected to C-section). Maternal blood cortisol and glucose concentrations were analyzed during the perinatal period (prepartum, intrapartum, postpartum, and 1 h after postpartum). Neonatal blood samples were collected within 5 min and 1 h after birth for assessment of cortisol and glucose. Maternal dystocia bitches had higher cortisol concentrations at postpartum than the Fetal Dystocia Group. At 1 h postpartum, the Cesarean Section Group had higher cortisol concentrations compared with fetal dystocia bitches. The Eutocia Group presented increased cortisol concentrations at intrapartum and postpartum, whereas fetal dystocia bitches had higher intrapartum cortisol concentrations than at 1 h postpartum. The Maternal Dystocia Group presented higher postpartum cortisol concentrations than at prepartum and 1 h postpartum. Maternal glucose had a progressive increase throughout peripartum and was higher during postpartum and at 1 h postpartum. C-section bitches had the highest blood glucose concentration. Neonatal cortisol concentrations at birth were higher than 1 h after birth. Fetal dystocia puppies had higher cortisol concentrations, whereas caesarian section puppies had lower cortisol levels. Fetal dystocia and C-section puppies had higher glucose concentrations than the Eutocia Group. In conclusion, maternal dystocia leads to high cortisol concentrations in bitches immediately postpartum, whereas only fetal dystocia causes increased neonatal cortisol concentrations. Moreover, fetal dystocia and C-section are hyperglycemic obstetrical conditions for neonatal puppies; on the other hand, only C-section causes hyperglycemia in bitches.

Endocrine, prostatic vascular, and proapoptotic changes in dogs with benign prostatic hyperplasia treated medically or surgically.

Benign prostatic hyperplasia (BPH) is a disorder related to hormone imbalance, local angiogenesis, and prostate growth, which can be treated surgically (orchiectomy) or medically (most commonly with finasteride). However, finasteride therapy is not completely established in dogs regarding local action and posology. This study aimed to evaluate the effect of different doses of finasteride and orchiectomy on hormonal profile, prostate apoptosis, blood flow, and biometry in dogs with BPH. Dogs were assigned to the following groups: untreated, 0.1 mg, 0.2 mg, and 0.5 mg/kg/d of finasteride and orchiectomy. All dogs were assessed monthly: day 0 (before treatment), day 30, and day 60 and subjected to prostate B-mode and Doppler ultrasonography and hormonal analysis (testosterone and dihydrotestosterone). After 60 d, prostatic biopsy was performed for histology and immunohistochemical evaluation for apoptosis (caspase-3). On day 60, percentage reduction of prostatic volume was greater in orchiectomized dogs than that in finasteride groups, which, conversely, was greater than untreated dogs. On day 60, 0.2-mg finasteride, 0.5-mg finasteride, and orchiectomy groups had higher prostatic blood flow than 0.1-mg finasteride and untreated groups. In addition, both 0.5-mg finasteride and orchiectomy groups had an increase in prostate artery resistance. Orchiectomy significantly decreased androgen concentrations at 30 d onward, differing from the remaining groups. The orchiectomy group had lower caspase-3 immunostaining, however, not different from untreated and 0.5-mg finasteride. In conclusion, 0.5 mg/kg finasteride promoted more effective prostate apoptosis and hemodynamic effects among medical treatments, whereas orchiectomy caused prostate atrophy and sharp endocrine changes in dogs with BPH.

Morphometric and functional pulmonary changes of premature neonatal puppies after antenatal corticoid therapy.

Among prematurity complications, the most important disorder is structural immaturity and inadequate production of pulmonary surfactant. Betamethasone is the drug of choice to artificially improve pulmonary capacity, thus we aimed to verify the effect of prenatal maternal treatment on lung development of premature puppies. Pregnant bitches were allocated in Term Group (n = 7), Preterm-Treated Group (interrupted pregnancies with maternal administration of betamethasone; n = 7), Preterm-Control Group (untreated interrupted pregnancies; n = 7), Extremely-Preterm Group (interrupted pregnancies at 55d; n = 6). Puppies were subjected to chest radiographic at birth, morphometric description of pulmonary structures and immunohistochemical analysis of surfactant protein B, proliferating cell nuclear antigen and cytokeratin were performed. In Preterm-Treated Group it was possible to more clearly identify cardiac silhouette and lung parenchyma by X-Ray. Saccular formation was higher in Preterm Groups, while Term Group had higher subsaccular development. Lung septation was higher in Treated and Term Groups. Term Group had higher number of cells marked for SP-B, whereas higher proliferation was observed in Extreme-Preterm and Preterm-Control Groups. Preterm Treated and Term Groups had higher tissue differentiation. In conclusion, antenatal maternal corticotherapy in dogs acted by increasing lung morphology and development of areas of gas exchange, regulate metabolism of pulmonary fluids rather than stimulate surfactant production.

Peripartum haemodynamic status of bitches with normal birth or dystocia.

There has been limited investigation of parturition in the bitch and there is little information published on clinical and obstetrical examination other than opinion and anecdote. While there are substantial data on haemodynamic and vascular changes during normal parturition in humans, little is known about the physiological events in the dog. This study was aimed at maternal haemodynamic changes occurring during normal parturition and to investigate how these were modified in bitches with dystocia (DYST) treated either medically or via assisted delivery and caesarean operation. Three groups of 10 bitches were investigated; those with normal parturition, those with DYST corrected by manipulative assistance or caesarean operation and those with uterine inertia treated by oxytocin administration. Heart rate, systolic and diastolic blood pressure, electrocardiogram and blood glucose concentration were measured pre-partum, intra-partum, immediately after parturition and 1 h later. Heart rate was high at all times throughout the study and the majority of bitches had normal sinus rhythm. Blood pressure was generally within the normal range, and although systolic and diastolic blood pressure was highest during the intra-partum period and sometimes during the immediate post-partum period, there were no significant differences between groups. All bitches had blood glucose concentrations within the normal range throughout the study although pre-partum concentrations were statistically lower than many of the other time periods. The study provides useful physiological data that will facilitate monitoring and clinical management of bitches throughout normal parturition and DYST.

Pulmonary maturation in canine foetuses from early pregnancy to parturition.

Development of the foetal respiratory system includes both pulmonary growth and maturation. In human medicine, a higher incidence of respiratory distress is reported in newborn males. This study aimed to identify different phases of canine foetal lung maturation throughout pregnancy, to determine the stage of pregnancy in which surfactant production begins and to compare pulmonary development of male and female foetuses. Pregnant bitches (34) were subjected to elective ovariohysterectomy and allocated into four groups, according to the stage of pregnancy: 30-40 days of pregnancy (n = 10), 41-50 days (n = 10), 51-60 days (n = 10) and bitches in the first stage of parturition (n = 4). Foetal lungs were histologically processed and evaluated by optical microscopy. The pseudoglandular phase was identified between the 35th day and 46th day of gestation; the onset of canalicular and saccular periods was observed, respectively, from the 48th day and 60th day of pregnancy. Lungs from foetuses at term were in the saccular phase; thus, the development into the alveolar period occurs in the neonatal period. The histological analyses revealed that respiratory tract development is centrifugal, from upper to lower airways. Therefore, it is possible to identify distinct development periods in different portions of the same organ. In conclusion, the saccular phase of lung development begins around 57 and 60 days of pregnancy, the period in which surfactant production is believed to occur. Male and female foetuses present similar pulmonary development from early pregnancy until parturition.

Neonatal clinical evaluation, blood gas and radiographic assessment after normal birth, vaginal dystocia or caesarean section in dogs.

This study aimed to standardize signs and diagnostic criteria of respiratory function in newborn puppies delivered normally or after dystocia and caesarean operation. A total of 48 neonates were allocated into groups: eutocia (n = 20), dystocia (n = 8), caesarean (c)-section (n = 20). Neonatal health was assessed using the Apgar score and body temperature was determined at 0, 5 and 60 min after delivery. Venous blood gases (pO(2) and SO(2)) was measured immediately and 60 min after delivery, and a thoracic radiograph was made between 0 and 5 min of life. The c-section group had significantly lower Apgar scores at birth and 5 min. Hypothermia was present at 5 min in the eutocia and c-section groups, and at 60 min in all groups. The eutocia group had an irregular respiratory pattern in 78% of puppies at birth, 27.7% at 5 min and 21% at 60 min compared with 87.5%, 62.5% and 12.5% of the pups in the dystocia group where there was irregular respiratory rhythm, moderate to intense respiratory sounds with agonic episodes. The c-section group had respiratory alterations in 70%, 45% and 16% of puppies at 0, 5 and 60 min, respectively. Radiographic abnormalities were present in 17% of the pups in the eutocia group, 25% of the pups in the dystocia group and 30% of the pups in the c-section group, respectively. The c-section group had significantly lower SO(2) values at 60 min than at birth. All puppies had hypoxaemia, but a significant decrease was observed in the c-section group. Newborn puppies had tissue hypoxia and irregular respiratory pattern at birth. Caesarean-section puppies had lower vitality; however, all developed satisfactory Apgar scores at 5 min of life, regardless of the obstetric condition.

Acid-base changes in canine neonates following normal birth or dystocia.

There are limited data concerning blood gas parameters in neonatal dogs. Knowledge of the normal physiology may facilitate effective therapeutic intervention and potentially reduce neonatal mortality. This study examined acid-base parameters in pups born at normal parturition (n = 27) compared with those born after obstetrical assistance or caesarean operation (n = 13) and those born following oxytocin (OXY) administration for treatment of uterine inertia (n = 11). Pups were subjected to an objective scoring method of neonatal health adapted from use in humans (the Apgar score) at birth and again at 5 and 60 min after birth. Venous blood samples were collected at 5 and 60 min after birth for evaluation of blood gas parameters. At birth, all pups had low Apgar scores and a mixed acidosis. The base excess was lowest for pups delivered after OXY administration. The Apgar score improved for all pups after 5 min of birth and there was an improvement in carbon dioxide tension, base excess and venous blood pH at 1 h, although in all pups a metabolic acidosis persisted. These data provide an important insight into neonatal physiology and the variability of blood gas parameters in pups born at normal and abnormal parturition and provide the basis for clinical decision making following dystocia.

Oestrogen and progesterone receptor gene expression in canine oocytes and cumulus cells throughout the oestrous cycle.

The aim of this research was to analyze oestrogen receptor-alpha (ERalpha), ERbeta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells' total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ERbeta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ERbeta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ERalpha and ERbeta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ERalpha was expressed throughout the oestrous cycle.

Influence of ascorbic acid and glutathione antioxidants on frozen-thawed canine semen.

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 microM and AA 250 microM. Initial sperm motility was significantly higher in sperm diluted with AA 50 microM; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

Evaluation of efficacy and safety of zinc gluconate associated with dimethyl sulphoxide for sexually mature canine males chemical neutering.

The aim of this research was to evaluate the efficacy of zinc gluconate associated with dimethyl sulphoxide (DMSO) for chemical neutering in canine males. Fifteen sexually mature male dogs were divided in two groups, named control and treated. An injection was administered to both testicles, at a concentration of 26.2 mg zinc gluconate per ml and 0.5% DMSO in the treated group (11 dogs). The control group was given injections of saline solution (four dogs). Clinical examination and blood collection for a haemogram were done both before and after drug injection. There were 12 spermograms performed to analyse sperm motility, sperm vigour, ejaculate volume, testicle size, pathology and sperm concentrations. Libido was also measured. An ultrasound examination and histopathology were performed at the end of the experiment. Dogs' libido after chemical injection was reduced by over 50%. The spermogram analysis showed final mean results of 14.54% for sperm motility, 0.72 of sperm vigour and 37,150 per million spermatozoa per millilitre, values considered below the necessary levels at which fertilization can occur. Ultrasound and histopathology analyses of testicles for the treated group revealed more intense injuries when compared with the control group, with compromised testicular parenchyma and a decrease of germ cell number leading to total atrophy, indicating that the treatment reduced the fertilizing potential of male dogs, promoting a possible subfertile status.