Daniel de Souza Ramos Angrimani

Pontos críticos no manejo pré-abate de frango de corte: jejum, captura, carregamento, transporte e tempo de espera no abatedouro

Today the brazilian poultry industry sets Brazil as the third largest producer of chicken meat. The poultry production sector is extremely organized in all its segments, from birth to slaughter poultry. One of the most laborious is the pre-slaughter, which ranges from the feed withdrawal to the lairage time at the plant. Due to this fact this research highlights the most critical points in the pre-slaughter period, as: feed withdrawal, capture, catching, transport and lairage time in the plant.

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.

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

BackgroundDuring 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).ResultsCaput 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.ConclusionsThese findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.

Susceptibility of Stallion Spermatozoa to Different Oxidative Challenges: Role of Seminal Plasma

Abstract Seminal cryopreservation provides several advantages in horse breeding. However, improvement on post‐thaw sperm survival is still necessary. One of the main factors known to impair post‐thaw quality of stallion’s sperm is the oxidative damage caused by reactive oxygen species (ROS). In this context, the aim of this study was evaluated the effects of ROS on stallion sperm and the possible influence of seminal plasma (SP). Toward this aim, 13 ejaculates from adult stallions (n = 13) were divided into two aliquots which were centrifuged (600g/10 minutes), and the SP was removed and reserved. Pellets were suspended in physiological saline solution (A) or SP (B). Each of these solutions was divided into four aliquots and subjected to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical [OH−]) and the toxic by‐product of lipid peroxidation (malondialdehyde [MDA]). Samples were then evaluated for the susceptibility to lipid peroxidation (thiobarbituric acid reactive substances) plasma membrane integrity (eosin–nigrosin), acrosome integrity (Fast Green/rose bengal), mitochondrial activity (3′3 diaminobenzidine), and DNA integrity. Samples incubated in the presence of SP were highly impaired by OH− with regard to motility, plasma membrane integrity, mitochondrial activity, and DNA integrity. On the other hand, in the absence of SP, MDA was highly deleterious, especially with regard to motility, plasma membrane integrity, and mitochondrial activity. Thus, these results indicate that SP may have an important role on the protection of stallion sperm against the damages caused by MDA, an important product of lipid peroxidation. HighlightsSeminal plasma (SP) has a protective effect against oxidative damages.Hydroxyl radical in the presence of SP is the most deleterious reactive oxygen species (ROS) to equine spermatozoa.Malondialdehyde is more deleterious than ROS to equine spermatozoa in the absence of SP.Hydroxyl radical is extremely deleterious to equine sperm DNA.Malondialdehyde impaired membrane integrity and mitochondrial activity.

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.

Cystic endometrial hyperplasia–pyometra syndrome in bitches: identification of hemodynamic, inflammatory, and cell proliferation changes

Abstract Cystic endometrial hyperplasia (CEH)—pyometra syndrome is one of the most common diseases of noncastrated female dogs. However, determination of etiological mechanisms and differential diagnosis of CEH—pyometra syndrome are undefined. The aim of this study is to compare immunohistochemically the expression of cyclooxygenase-2 (COX-2) inflammatory mediator, Ki-67 antigen proliferation marker, vascular endothelial growth factor (VEGF-A) angiogenesis mediator and its FLT-1 and KDR receptors, and correlate with Doppler velocimetry of uterine artery and endometrial vascularization in bitches with CEH—pyometra syndrome. Bitches were allocated into CEH-mucometra Group (n = 13), Pyometra Group (n = 11), and Control Group (n = 8). Pyometra Group presented cytoplasmatic staining intensity for COX-2, VEGF-A, and FLT-1 and KDR receptors in luminal epithelium cells significantly higher compared to CEH-mucometra and Control groups. For the glandular epithelium, Pyometra Group had higher immunostaining score for VEGF-A and its receptors (FLT-1 and KDR). Hemodynamic indexes showed negative correlation with VEGF-A and its receptors as well as with COX-2. On the other hand, uterine vascularization score showed positive correlation in relation to immunostaining of COX-2, VEGF-A, and receptors in the endometrium luminal epithelium. In conclusion, uterus of bitches with CEH—pyometra syndrome show inflammatory process characterized by COX-2 expression, resulting in greater expression of proliferative Ki-67 marker as tissue response against the infectious agent. Furthermore, the increased VEGF-A expression and its receptors in CEH—pyometra reflect the increased blood flow and lower vascular resistance. Therefore, canine pyometra is characterized by an inflammatory, proliferative, and vascular disorder. Summary Sentence Canine CEH—pyometra syndrome is characterized by an inflammatory, proliferative and vascular disorder, with higher uterine blood flow and vascularization.

Carnosine as malondialdehyde scavenger in stallion seminal plasma and its role in sperm function and oxidative status

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ± 19.16 ng/mL; Bad cooler: 159.72 ± 15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ± 18.33%; Bad cooler: 18.33 ± 1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ± 18.22 ng/mL; Bad cooler: 131.92 ± 12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ± 6.72 ng/mL; Medium-high: 140.45 ± 11.70 ng/mL; Medium-low: 202.57 ± 16.30 ng/mL and Low: 231.02 ± 32.35 ng/mL; p < 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma.

Induced sperm oxidative stress in dogs: Susceptibility against different reactive oxygen species and protective role of seminal plasma

Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.

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.

Hemodynamics of the uterine and umbilical arteries during the perinatal period in ewes

Examining feto-maternal blood flow is extremely important for accurate prenatal control and predicting adverse conditions during puerperium. We aimed to analyze blood flow of uterine artery (UA) and umbilical artery (UMA) from and subsequent to mid-gestation, lambing and postpartum of 15 ewes, subjected to lambing induction with aglepristone. Hemodynamic variables, diameter and blood flow of UA and UMA were determined by Doppler and B-mode ultrasonography at 60, 90 and 120 days of pregnancy, at 12 h before, 12 h after first aglepristone injection and 12 h after induction and days 1, 3, 5, 7, 15 and 30 after lambing. The UMA peak systolic:diastolic velocity (S/D), resistance index (RI) and pulsatility index (PI) decreased from mid-pregnancy until 120 d, whereas peak systolic velocity (PSV), end diastolic velocity (EDV) and time average maximum velocity (TAMAX) increased towards 120 d. The UMA RI additionally decreased at 12 h after lambing induction. The UMA diameter and volume increased from mid-gestation until 12 h before lambing induction. UA PSV and TAMAX increased from 60 until 90 days of pregnancy, while EDV, PSV and TAMAX decreased soon after parturition, with a further reduction at 7 days. The UA diameter and volume decreased during postpartum, stabilizing by 15 days after parturition. In conclusion, pregnancy is marked by progressive increase in uterine and umbilical blood flow, while lambing acutely diminishes umbilical vascular resistance, suggesting lack of progesterone influence on vasodilation. Conversely, puerperium is characterized by reduction in uterine blood supply mostly within the first 2 weeks of postpartum in sheep.