Marcelo Demarchi Goissis

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.

Elevated progesterone concentrations enhance prostaglandin F2α synthesis in dairy cows.

The objective was to evaluate the influence of varying plasma progesterone (P(4)) concentrations throughout the luteal phase in dairy cows on PGF(2alpha) production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha); PGFM) following treatment with estradiol-17beta (E(2)) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n=9), received 20mg LH (n=7), or had ovarian follicles larger than 6mm aspirated (n=8). In Experiment 2, cows on Day 6 were untreated (n=9) or received 5000 IU hCG (n=10). In Experiments 1 and 2, all cows received 3mg E(2) on Day 17, and blood samples were collected every 30 min from 2h before to 10h after E(2). Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n=4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n=7) received the alternate sequence. Blood was collected daily for measurement of P(4) 4h after concentrate feeding. On Day 17, blood was collected from 1h before to 1h after a 100 IU OT injection. In Experiment 1, both plasma P(4) and release of PGF(2alpha) were similar between LH-treated and control cows (P>0.10). In Experiment 2, plasma P(4) was elevated to a greater extent on Day 17 in cows treated with hCG (P<0.05) and plasma PGFM was also greater in hCG-treated animals (treatment x time interaction; P<0.05). In Experiment 3, there was a group x period interaction (P<0.01) for plasma P(4), indicating that less concentrate feeding was associated with greater plasma P(4). Release of PGF(2alpha) in response to OT was greater for cows receiving less concentrate (group x period interaction; P<0.05). In conclusion, dairy cows with more elevated blood P(4) concentrations released more PGF(2alpha) in response to E(2) or OT.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

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.

Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro‐produced early‐ and late‐dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo‐produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast‐ and slow‐dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast‐ and slow‐dividing embryo groups, whereas blastocysts from the slow‐dividing group were more similar to in vivo‐produced blastocysts. Gene ontology enrichment analysis showed that the slow‐dividing and in vivo‐produced blastocysts shared biological processes related to groups of up‐ or down‐regulated genes when compared to the fast‐dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro‐produced bovine embryos. Embryos from fast‐ and slow‐dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post‐in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage‐stage and morulae groups, but was higher in the blastocysts of the slow‐dividing group. On the other hand, blastocysts of the fast‐dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324–336, 2016.

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.

Effect of age on expression of spermatogonial markers in bovine testis and isolated cells

Spermatogonial stem cells (SSC) are the most undifferentiated germ cell present in adult male testes and, it is responsible to maintain the spermatogenesis. Age has a negative effect over stem cell, but the aging effect on SSC is not elucidated for bovine. The present study aim to evaluate the effect of age on the expression of undifferentiated spermatogonial markers in testis and in enriched testicular cells from prepubertal calves and adult bulls. In this matter, testicular parenchyma from calves (3-5 months) (n=5) and bulls with 3 years of age (n=5) were minced and, isolated cells were obtained after two enzymatic digestions. Differential platting was performed for two hours onto BSA coated dish. Cell viability was assessed by Trypan Blue solution exclusion method and testicular cells enriched for SSC was evaluated by expression of specific molecular markers by qRT-PCR (POU5F1, GDNF, CXCR4, UCHL1, ST3GAL, SELP, ICAM1 and ITGA6) and flow cytometry (GFRA1, CXCR4 and ITGA6). CXCR4 and UCHL1 expression was evaluated in fixated testes by immunohistochemistry. We observed that age just affected the expression of selective genes [SELP (Fold Change=5.61; p=0.0023) and UCHL1 (Fold Change=4.98; p=0.0127)]. By flow cytometry, age affected only the proportion of ITGA6+ cells (P<0.001), which was higher in prepubertal calves when compared to adult bulls. In situ, we observed an effect of age on the number of UCHL1+ (p=0.0006) and CXCR4+ (p=0.0139) cells per seminiferous tubule. At conclusion, age affects gene expression and the population of cells expressing specific spermatogonial markers in the bovine testis.

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.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.

Sensibilidade e especificidade do exame eletrocardiográfico na detecção de sobrecargas atriais e/ou ventriculares em gatos da raça Persa com cardiomiopatia hipertrófica

Hypertrophic cardiomyopathy (HCM) is the most common feline heart disease and is characterized by increased cardiac mass with a hypertrophied and not dilated left ventricle. The echocardiography is the best noninvasive diagnostic tool for the differentiation of cardiomyopathies and is considered the gold standard for detection of ventricular hypertrophy present in HCM. Electrocardiographic changes are also common in animals with HCM and the electrocardiogram (ECG) is quick, easy and highly available screening test for the detection of ventricular hypertrophy in humans. In cats, few studies have been conducted regarding the sensitivity and specificity of ECG in detecting ventricular hypertrophy. With the intention of evaluating the use of ECG as a screening tool for diagnosis of HCM in cats, Persian cats (n=82) were evaluated by echocardiographic and electrocardiographic examinations. Animals with blocks and/or conduction disturbances were excluded from statistical analysis (n=22). Subsequently the animals included were classified as normal (n=38), suspicious (n=6) and affected by HCM (n=16). Statistical differences were observed in the P-wave amplitude in DII and R-wave amplitude in DII, CV6LL and CV6LU, with higher values in animals with HCM. Velocities and pressure gradient of aortic flow, left atrial diameter (LA) and LA/Ao ratio were higher in cats with HCM. Among the animals with ECG changes suggestive of left atrial enlargement (n=7), only two actually had LA enlargement on echocardiography, and among animals with left atrial enlargement on echocardiogram (n=7), only two had ECG changes suggestive of overload AE (40,4% of sensibility and 90,9% of specificity). Among the animals with ECG changes suggestive of left ventricular hypertrophy (n=6), five actually had ventricular hypertrophy on echocardiography, and among animals with HCM by echocardiography (n=16), only five showed electrocardiographic abnormalities suggestive of LV hypertrophy (31,25% of sensibility and 97,72% of specificity). We observed a positive correlation between diastolic thickness of the interventricular septum and/or left ventricular free wall and R-wave amplitude in DII and CV6LU. The electrocardiogram is quick and easy to perform, has good specificity in detecting ventricular hypertrophy in cats, however, has low sensitivity, with large numbers of false negative animals. Thus, the ECG assists in the diagnosis, but does not replace echocardiography in confirming ventricular hypertrophy.