Maíra Morales Brito

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.

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

ABSTRACT 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.

Effects of benign prostatic hyperplasia and finasteride therapy on prostatic blood flow in dogs

Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate accompanied by an increase in prostatic blood perfusion and vascularization. The most indicated treatment is to perform orchiectomy, however, medical treatment with finasteride can be an option for breeding dogs or elderly animals with a critical health status. In dogs, the influence of medical treatment on prostatic hemodynamics is still unknown. Therefore, this study aimed to evaluate the effects of benign prostatic hyperplasia and finasteride therapy on hemodynamic and vascular features of the canine prostate. For this purpose, twenty dogs of different breeds, body weights (10-30 kg) and ages (5-13 years) were used, assigned for: Healthy-non treated group (n = 5), BPH-non treated group (n = 5), Healthy-finasteride treated group (n = 5) and BPH-finasteride treated group (n = 5). Dogs that presented hematospermia and at least one general clinical sign (tenesmus, hematuria or dysuria) were presumptively diagnosed with BPH. Dogs were evaluated ultrasonographycally by B-mode and Doppler of the prostatic artery in a monthly interval (day 0, 30 and 60) in order to measure prostate volume (PV), expected prostate volume (EPV), prostate vascularization score (scored as minimum, intermediary and maximum) and prostatic artery blood flow parameters with the use of spectral and color Doppler ultrasound. It was possible to observe a decrease in prostate vascularization score between Day 0 (intermediary degree) and 60 (minimum degree) in finasteride treated dogs. Moreover, non-treated dogs had higher score of vascularization at Day 60 compared to animals treated with finasteride, regardless of BPH diagnosis. Healthy-non treated animals presented higher peak systolic:diastolic velocity (S/D) than BPH-non treated dogs. Furthermore, BPH-non treated dogs had lower S/D than BPH-finasteride treated dogs. In 30 and 60 days, no difference on PV was observed between BPH-finasteride treated group and Healthy-non treated group. At day 60, no difference between PV and EPV was observed for the BPH-finasteride treated group. In conclusion, finasteride treatment reduces simultaneously the volume, local vascularization and blood flow of the prostate, thus, being considered an effective and additional choice of therapy for BPH. Moreover, the course of therapy in dogs can be followed by analyzing changes in prostatic artery.

Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm

Abstract The present research aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs. Dogs were assigned into Young Group (n = 11) and Senile Group (n = 11), additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Males were evaluated for libido score and blood estrogen and testosterone assay. Sperm morphofunctional evaluations were performed based on Computer Assisted Sperm Analysis, morphology, mitochondrial activity, mitochondrial membrane potential, plasma and acrosomal membrane integrity, and DNA fragmentation. Sperm oxidative features were: protein oxidation, lipid peroxidation and production of advanced glycation end-products. Young dogs had higher libido score, sperm velocity average pathway, linearity of motility and mitochondrial activity index and lower percentage of major defects, total defects and proximal cytoplasmic droplet, despite the lack of difference between hormone profile of aged dogs. Fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs and to cryopreserved sperm. Cryopreserved semen of young dogs had higher acrosomal membrane integrity compared to the Senile Group. In conclusion, sperm of aged dogs have reduced quality, signaled by higher morphological defects, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury.

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

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.

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

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.

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

Characterization of the bitchs 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.

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37oC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.

Perfil de proteínas plasmáticas em cadelas gestantes e não gestantes

O estudo das interacoes orgânicas da gestacao e as mudancas fisiologicas que estao ocorrendo nesta fase sao de extrema importância para a avaliacao clinica da femea gestante ou para estabelecer o diagnostico de processos patologicos em andamento. O objetivo do presente estudo foi comparar o perfil das diversas proteinas sanguineas (fracoes proteicas do soro - albumina, a1, a2, b, g globulinas e proteina total) durante o periodo gestacional e no diestro em cadelas. Foram utilizadas 40 femeas caninas da raca Dogue Alemao, em idade variando entre 2 a 7 anos, clinicamente saudaveis. Os animais foram separados em dois grupos, denominados Grupo nao gestante (NG) e Grupo gestante (G), constituidos por 20 femeas em diestro e 20 femeas gestantes, respectivamente. Preconizou-se colheita de sangue semanalmente de cada animal durante 9 semanas. Nas femeas do grupo NG, as amostras foram colhidas a partir do inicio do diestro, ate a deteccao do inicio do anestro; no grupo G, as amostras foram colhidas do inicio do diestro ate o momento da paricao. A partir do soro sanguineo, foram determinadas as concentracoes de proteina total, albumina, a1, a2, b e g globulinas. Nao houve diferenca significativa quanto aos resultados de proteina serica total entre a 1a e 6a semanas de gestacao e diestro, havendo decrescimo gradual em ambos grupos. Ja ao final da gestacao (entre a 7a e 9a semanas), houve acrescimo significativo dos valores de proteina total, sugerindo acao anabolica. Os niveis de albumina sofreram queda da 1a a 9a semana, tanto no grupo gestante, como nao-gestante, com diferenca estatistica entre os dois grupos na 7a, 8a e 9a semanas. As concentracoes de α1 globulina nas femeas gestantes sofreram acrescimo significativo a partir da 2a semana, contudo, diferenca estatistica entre os grupos NG e G ocorreu somente na 8a e 9a semanas, coincidindo com a fase de preparacao a paricao. Houve aumento no perfil de α2 globulina entre a 2a e 3a semanas de gestacao, porem tais valores nao diferiram das femeas em diestro. Diferenca significativa de α2 globulina foi observada apenas durante a 4a, 5a, 6a, 8a e 9a semanas entre os dois grupos avaliados. As concentracoes sericas de β globulina nas cadelas nao-gestantes foi superior as das femeas gestantes durante a 2a, 4a e 7a semanas. Durante o primeiro terco da gestacao (1a a 3a semana), foi observado queda dos valores de γ globulina, coincidente ao periodo pre implantacional e durante a formacao dos sitios de implantacao. Entre a 8a e 9a semanas de gestacao, houve acrescimo significativo de γ globulina, possivelmente consequente ao aumento da producao de imunoglobulinas direcionadas a glândula mamaria, como constituinte do colostro. Em conclusao, as proteinas alteram-se de forma evidente durante o periodo de gestacao. Foi possivel inferir diferencas nas funcoes biologicas das proteinas sanguineas em cadelas gestantes e nao gestantes. As proteinas determinadas estao envolvidas com o estimulo inflamatorio durante a gestacao, alem dos mecanismos de regulacao hormonal e preparacao do organismo materno a lactacao.