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.

ContentsThe 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.

ContentsBenign 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.

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.

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.

No abstract

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.

ContentsCharacterization 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.