Adriano Felipe Perez Siqueira

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome.

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.

System for evaluation of oxidative stress on in-vitro-produced bovine embryos

This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.

Modeling the Photocatalytic Process of Variation in Chemical Oxygen Demand via Stochastic Differential Equations

Several papers in the literature on Advanced Oxidation Processes (AOPs) confirm the process as a viable alter- native for the treatment of a variety of industrial effluents. In many of these works, modeling the variations of Chemical Oxygen Demand (COD) as a function of different experimental conditions was performed by techniques such as Design of Experiments, Artificial Neural Networks and Multivariate Analysis. These techniques require both a large number of parameters and a large quantity of experimental data for a systematic study of the model parameters as a function of ex- perimental conditions. On the other hand, the study of Stochastic Differential Equations (SDE) is presently well devel- oped with several practical applications noted in the literature. This paper presents a new approach in studying the varia- tions of COD in AOPs via SDE. Specifically, two effluents, from the manufacture of paints and textiles were studied by combined treatment of the photo-Fenton process and catalytic ozonization.

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

Catalytic Ozonation Using Fe 2+ in the Treatment of Dairy Effluent in a Semi-Batch Process with Recycle

This work describes the treatment of dairy industry effluent using catalytic ozonation with Fe2+ as its catalyst in a semi-batch process with recycle. A fractional factorial design 24-1 was used with a reduction percentage of total organic carbon (TOCred) as response. Optimal conditions were obtained by the reaction time of 30 min, ozonator power of 35 W, O2 flow rate of 0.125 L min-1, Fe2+ concentration of 1.0 g L-1 and pH 4.0 for a 2 L raw effluent. TOCred of 64.03% represents a decrease in concentration from 473.0 to 170.1 mg L-1, with an estimate cost of US

Quantificação do interferon-tau durante o reconhecimento materno da gestação em fêmeas bovinas Bos taurus indicus

0.03 L-1effluent. The treatment performed was not sufficient to discharge it directly on surface water; however, significant reduction of TOCred, among physical and chemical characteristics makes a better product to be proceeded into a biological treatment.

Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing

Durante o periodo critico do reconhecimento materno, compreendido entre o 15o e 19o dias da gestacao, o concepto deve sintetizar competentemente moleculas capazes de bloquear a sintese de prostaglandina F2α (PGF2α) e a luteolise. Em bovinos, a principal macromolecula proteica envolvida em tal bloqueio e o interferon-tau (IFN-τ). Durante o periodo critico, falhas neste reconhecimento determinam a mortalidade embrionaria em ate 40% das femeas inseminadas. Informacoes sobre o IFN-τ em animais Bos taurus indicus, ainda sao restritas. Este estudo objetivou uma avaliacao quantitativa do IFN-τ durante o periodo critico do reconhecimento materno, em lavados uterinos obtidos por sonda de Foley (dias 14, 16 e 18 posestro) ou post-mortem (dia 18 pos-estro). Para tanto, foram utilizadas femeas multiparas azebuadas (Bos taurus indicus), ciclicas ou prenhes, nos dias 14, 16 e 18 pos-estro. Para a obtencao dos lavados, os uteros foram infundidos com solucao de Ringer Simples. Os lavados foram concentrados por ultra-filtracao e liofilizados. As macromoleculas proteicas foram separadas por Eletroforese Unidimensional SDSPAGE, em gel com 15% de poliacrilamida. A quantificacao do IFN-τ nos lavados uterinos foi realizada por Western-Blotting e densitometria. Tanto nos lavados obtidos por sonda de Foley quanto nos post-mortem foi possivel observar bandas de proteinas que apresentaram reacao cruzada com os anticorpos utilizados no Western-Blotting. O IFN-τ foi detectado apenas nos lavados uterinos post-mortem de vacas prenhes (P<0,05). A densidade optica nao foi afetada pelo dia do periodo critico, estado (ciclico ou prenhe) ou interacao dia x estado. Nos lavados post-mortem nao houve efeito de peso do concepto ou concentracao de progesterona plasmatica no dia do lavado na densidade da banda proteica referente ao IFN-τ . Concluiu-se que a deteccao e quantificacao do IFN-τ no ambiente uterino de vacas azebuadas, nestas condicoes experimentais, e possivel apenas em lavados uterinos obtidos post-mortem.

Ureaplasma diversum and Its Membrane-Associated Lipoproteins Activate Inflammatory Genes Through the NF-κB Pathway via Toll-Like Receptor 4

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production.