Mielorradiculopatia esquistossomótica: relato de caso em paciente pediátrico / Schistosomal Myeloradiculopathy: case report in a pediatric patient

A esquistossomose é uma endemia parasitária típica das Américas, Ásia e África. A Mielorradiculopatia Esquistossomótica surge como uma evolução severa da infecção por esquistossomose e, apesar de muito comum, sua prevalência em áreas endêmicas vem sendo subestimada. Objetivo: relatar caso de Mielorradiculopatia Esquistossomótica ocorrido em paciente pediátrico. Metodologia: estudo descritivo do tipo Relato de Caso retrospectivo, submetido e aprovado pelo Comitê de Ética em Pesquisa do Centro Universitário CESMAC, CAAE: 28835220.0.0000.0039, N.º do Parecer: 3.898.292. Relato de caso: paciente do sexo masculino, previamente hígido, 11 anos, iniciou quadro com história álgica aguda em membros inferiores que piorava no período da noite acompanhada de relato de febre. Quadro clínico evoluiu com lombalgia, disúria, oligúria, posterior anúria e formação de globo vesical. Evoluiu, também, com paresia de membros inferiores. A investigação realizou-se com Exame Parasitológico de Fezes positivo para esquistossomose, além de Ressonância Magnética de coluna lombo-sacra que corroboraram com a hipótese diagnóstica. Instituiu-se tratamento com Albendazol, Praziquantel e pulsoterapia com Metilprednisolona durante internação. Paciente teve alta hospitalar com melhora de quadro neurológico, em uso de prednisona 40 mg/dia. Conclusão: a MRE constitui a forma mais grave dentre as manifestações ectópicas da esquistossomose. A dificuldade do reconhecimento do quadro clínico e a limitação no acesso aos métodos complementares diagnósticos contribuem para o subdiagnóstico da enfermidade, acarretando sequelas graves para os portadores da doença e ocultando sua importância epidemiológica principalmente em pacientes pediátricos e jovens. (AU)

Schistosomiasis is a parasitic endemic typical of the Americas, Asia and Africa. Schistosomal Myeloradiculopathy is a severe evolution of schistosomiasis infection and, although very common, the prevalence in endemic areas has been underestimated. Objective: to report Schistosomal Myeloradiculopathy case in a pediatric patient. Methodology: descriptive study of the type Case Report retrospective, submitted and approved by the Research Ethics Committee of the CESMAC University Center, CAAE: 28835220.0.0000.0039, Opinion N.º: 3.898.292. Case report: a previously healthy 11-year-old boy, started with a history of acute pain in lower limbs that worsened during the night accompanied of fever. Evolved with low back pain, dysuria, oliguria, subsequent anuria, vesical globe formation and lower limbs paresis. The investigation resulted in positive stool examination for schistosomiasis and magnetic resonance imaging of lumbosacral spine that corroborated the diagnostic hypothesis. The treatment included Albendazol, Praziquantel and pulsetherapy with Methylprednisolone during hospitalization. The patient was discharged from the hospital with improved neurological status, using prednisone 40 mg/day. Conclusion: Schistosomal Myeloradiculopathy is the most severe form of the ectopic manifestations of schistosomiasis. The difficulty in recognizing the clinical condition and the limitation of access to complementary diagnostic methods contributes to the underdiagnosis of the disease, causing severe sequels for patients with disease and hiding its epidemiological importance, especially in pediatric and young patients. (AU)

Uncovering sperm metabolome to discover biomarkers for bull fertility.

BACKGROUND: Subfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported. RESULTS: A total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, ß-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored. CONCLUSIONS: This is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility.

Major seminal plasma proteome of rabbits and associations with sperm quality.

The present study was conducted to describe the major seminal plasma proteome of rabbits and potential associations between seminal proteins and semen criteria. Semen samples were collected from 18 New Zealand adult rabbits, and seminal plasma proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Sperm motility, vigor, concentration, morphology and membrane sperm viability were evaluated. Rabbits ejaculated 364 ±â€¯70 million sperm/ml, with 81 ±â€¯6.1% motile cells, 3.8 ±â€¯0.2 vigor and 66.7 ±â€¯2.5% sperm with normal morphology. Based on the viability and acrosome integrity assay, there were 65.8 ±â€¯2.5% live sperm with intact acrosome and most spermatozoa had both intact acrosome and functional membrane. On average, 2-D gels of rabbit seminal plasma had 232 ±â€¯69.5 spots, as determined by PDQuest software (Bio Rad, USA). Mass spectrometry allowed the identification of 137 different proteins. The most abundant proteins in rabbit seminal plasma were hemoglobin subunit zeta-like, annexins, lipocalin, FAM115 protein and albumin. The intensity of the spots associated with these five proteins represented 71.5% of the intensity of all spots detected in the master gel. Multiple regression models were estimated using sperm traits as dependent variables and seminal plasma proteins as independent ones. Also, sperm motility had positive association with beta-nerve growth factor and cysteine-rich secretory protein 1-like and a negative one with galectin-1. The percentage of rabbit sperm with intact membrane was related to seminal plasma protein FAM115 complex and tropomyosin. Then, the population of morphologically normal sperm in rabbit semen was positively linked to carcinoembryonic antigen-related cell adhesion molecule 6-like and down regulated by seminal plasma isocitrate dehydrogenase. Based on another regression model, the variation in the percentage of live sperm with intact acrosome was partially explained by the amount of leukocyte elastase inhibitor and the peptidyl-prolyl cis-trans isomerase A in the rabbit seminal fluid. The current study reports the identification of 137 proteins of rabbit seminal plasma. Major proteins of seminal secretion relate primarily to prevention of damages caused by lipid peroxide radicals and oxidative stress, membrane functionality, transport of lipids to the sperm membrane and temperature regulation. Moreover, finding seminal plasma proteins as indicators of semen parameters will improve assisted reproductive technologies.

Proteomic landscape of seminal plasma associated with dairy bull fertility.

Male fertility is the ability of sperm to fertilize the egg and sustain embryo development. Several factors determine the fertilizing capacity of mammalian sperm, including those intrinsic to sperm and components of the seminal plasma. The present study analyzed the seminal fluid proteome of Bos taurus and potential associations between proteins and fertility scores. Mass spectrometry coupled with nano HPLC allowed the identification of 1,159 proteins in the dairy bull seminal plasma. There were 50 and 29 seminal proteins more abundant in high (HF) low fertility (LF) bulls, respectively. Based on multivariate analysis, C-type natriuretic peptide, TIMP-2, BSP5 and sulfhydryl oxidase indicated relationship with HF bulls. Clusterin, tissue factor pathway inhibitor 2, galectin-3-binding protein and 5'-nucleotidase were associated with LF bulls. Abundance of NAD(P)(+)-arginine ADP-ribosyltransferase, prosaposin and transmembrane protein 2 proteins had the highest positive correlations with fertility ranking. Quantities of vitamin D-binding protein, nucleotide exchange factor SIL1 and galectin-3-binding protein showed the highest negative correlations with fertility ranking. A fertility ranking score was calculated and the relationship with these proteins was significant (Spearman's rho = 0.94). The present findings represent a major and novel contribution to the study of bovine seminal proteins. Indicators of fertility can be used to improve reproductive biotechnologies.

Acetylation and methylation of sperm histone 3 lysine 27 (H3K27ac and H3K27me3) are associated with bull fertility.

Epigenetic modifications in histones are crucial for proper sperm physiology, egg activation and reproductive development of males. The objectives of this study were to determine the conservation and interactomes of histone three (H3) and ascertain the expression dynamics of acetylated and methylated H3 lysine 27 (H3K27ac and H3K27me3) in spermatozoa from Holstein bulls with different fertility. Methods in immunocytochemistry and flow cytometry were used to evaluate the expression dynamics of H3K27ac and H3K27me3 in spermatozoa from 10 bulls with different in vivo fertility. Computational biology methods including Clustal Omega and Cytoscape were performed to determine the evolutionary conservation and interactome of H3. The post-translational modifications (PTM) of H3 (H3K27ac and H3K27me3) had different spatiotemporal dynamics in the sperm head. Intensities of methylation were higher than those of acetylation and inversely correlated between the two fertility groups (p = .0032). The interacting proteins of H3 are involved in critical subcellular processes such as regulation of methylation, nucleosome assembly, regulation of DNA replication and chromatin assembly. These results are significant because they help advance fundamental science and biotechnology of mammalian reproduction.

Proteomic analysis of seminal plasma from locally-adapted "Curraleiro Pé-Duro bulls" (Bos taurus): identifying biomarkers involved in sperm physiology in endangered animals for conservation of biodiversity.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

Anethole improves the in vitro development of isolated caprine secondary follicles.

The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 µg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM+), defined as the control treatment, α-MEM+ supplemented with ascorbic acid at a concentration of 100 µg/mL (AA), or α-MEM+ supplemented with three different concentrations of anethole (30, 300, 2000 µg/mL) for a period of 18 days. Treatments were named as α-MEM+, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 µm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM+ and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.

Proteomic analysis of seminal plasma and sperm cells and their associations with semen freezability in Guzerat bulls.

The objective of the present study was to describe the relationship of seminal plasma and total sperm cell proteins with the semen freezability parameters of Guzerat bulls. Thirteen bulls were subjected to breeding soundness evaluation. Semen samples were collected, cryopreserved, and then post-thawing sperm kinetics were assessed, where high ( = 7) and low ( = 6) freezability groups were defined. Seminal plasma and total sperm proteins from the 2 groups were separated by 2-dimensional SDS-PAGE, and spots were identified by mass spectrometry. Semen parameters post-cryopreservation were as follows in the high and low freezability groups, respectively: mean total motility, 52.4 ± 20.5 and 13.7 ± 3.9; percentage of normal sperm, 89.0 ± 2.6 and 64.7 ± 14.0; and reactivity of hypo-osmotic swelling test, 38.9 ± 4.7 and 13.6 ± 3.7. Three seminal plasma proteins (osteopontin-K, DNase γ precursor, and DNASE1L3) and 6 proteins from sperm cells (acrosome formation-associated factor isoform 2, annexin A1, disintegrin and metalloproteinase domain-containing protein 2, dihydrolipoyl dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase) were highly expressed ( < 0.05) in the high freezability group. Another 6 seminal plasma proteins (acrosin inhibitor 1, glutathione peroxidase 3, metalloproteinase inhibitor 2, ephrin-A1, annexin A1, and platelet-activating factor acetylhydrolase) were significantly higher ( < 0.05) in the low freezability group. We described the associations of seminal plasma and sperm cell proteins with post-thawing sperm viability of Guzerat bulls raised in a typical semiarid environment. Such associations indicate that specific seminal plasma proteins more abundant in bulls of low semen freezability may be a response to an early oxidative stress that is not detected by conventional prefreezing semen evaluation. Moreover, specific sperm proteins were more associated with good freezability. The results presented here can serve as guidelines for future research aiming to develop better extenders and/or to improve bull semen selection for cryopreservation.

Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro.

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 µg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 µg/mL BSP1 (77.8 ± 3.1%), or 20 µg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 µg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 µg/mL BSP1 (22.4 ± 2.9%) and 40 µg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 µg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 µg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 µg/mL BSP1 (35.6 ± 2.5%) and 40 µg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma.

Seminal plasma proteins and their relationship with percentage of morphologically normal sperm in 2-year-old Brahman (Bos indicus) bulls.

The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (p<0.05). A multiple regression analyses of these SPPs, using three models; non-parametric Tree Model, Generalized Additive Model, and a step-wise selection method were conducted, and 6 SPPs could be used to predict PNS24; four SPPs had positive and two had negative association with PNS24. Together these spots explained 35% of the phenotypic variation in PNS24. Using mass spectrometry (MALDI-ToF and TripleToF-MS) the SPPs with positive relationship contained mainly apolipoprotein A-I (1310), protein DJ-1 and glutathione peroxidase 3 (2308), phosphoglycerate kinase 1 (6402) and apolipoprotein A-I and secretoglobin family 1D member (8008). The SPPs inversely associated with PNS24 were clusterin/seminal plasma protein A3 (1411) and epididymal secretory protein E1 (8108). This is the first comprehensive report on the association between seminal plasma protein composition in Bos indicus Brahman bulls and sperm morphology.

Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively.

Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles.

Aquaporins (AQPs) are a well-conserved family of small (approximately 30 kDa) membrane channel proteins that facilitate rapid movement of fluids and have a unique tissue-specific pattern of expression. These proteins have been found in the female reproductive systems of humans, rats, and mice. However, the expression and cellular localization of AQPs have not extensively been studied in the female reproductive system of sheep. Therefore, this study aimed to evaluate, by real-time polymerase chain reaction and immunohistochemistry respectively, the levels of messenger RNA and the immunolocalization of AQP3, AQP7, and AQP9 in large isolated ovine secondary follicles over a period of IVC. Our analysis revealed that AQP3 and AQP9 were present predominately in follicles that exhibited antrum formation, suggesting a crucial role of these AQPs in the formation of the antrum. Interestingly, AQP7 was only expressed in follicles that had not formed an antrum by Day 12 of culture. In conclusion, the presence of protein channels (AQP3 and AQP9) seems to be essential for the formation of the antrum in isolated ovine secondary follicles cultured in vitro and thus plays an important role during folliculogenesis in this species.

Growth, testis size, spermatogenesis, semen parameters and seminal plasma and sperm membrane protein profile during the reproductive development of male goats supplemented with de-oiled castor cake.

The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots (n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS-PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires.

Meat quality assessment from young goats fed for long periods with castor de-oiled cake.

Diet can influence both the qualitative and quantitative traits of ruminant meat. This study evaluated the effects of castor de-oiled cake on the meat of mixed-breed male goat kids. After 165days of diet treatment, no alterations (p>0.05) were observed in the in vivo performance, anatomic components, dissection and proximate composition of the Longissimus dorsi muscle, as well as in the color and pH of the carcasses. However, diet had an effect (p<0.05) on energy metabolites, fatty acid profile, and expression of certain proteins of the Longissimus dorsi muscle. To conclude, this study showed that the establishment of castor de-oiled cake diet for a long period to goats led to alterations in meat quality, without compromising its consumption qualities.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production.

OBJECTIVE: Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS: Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS: GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS: Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.

Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries.

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-ß were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-ß (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-ß were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats.

Seminal plasma proteome of electroejaculated Bos indicus bulls.

The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4 ± 2.3 and 64 ± 3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70 kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 d-isomerase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase 1. In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization.