Soroprevalência, isolamento e diagnóstico molecular para leptospirose em bovinos de fazendas de cria e de centrais de receptoras de embrião da região de Bauru- SP: Diagnóstico de leptospirose bovina em Bauru - SP

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Perfil lipídico da membrana citoplasmática de espermatozoides in natura e criopreservados de jumentos e cavalos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

AÇÃO REGULATÓRIA DO GnRH NO DESENVOLVIMENTO EMBRIONÁRIO PRECOCE E VITRIFICAÇÃO DE EMBRIÕES SUÍNOS PELO MÉTODO DE MICROGOTA (ROLE OF GnRH DURING EARLY EMBRYONIC DEVELOPMENT AND DEVELOPMENT OF SWINE EMBRYOS FOLLOWING VITRIFICATION BY MICRODROPLET METHOD)

The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB- 75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento (“hot warm”) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

COMPARATIVE MORPHOLOGICAL STUDY OF SOME TELLINIDAE FROM THAILAND (BIVALVIA: TELLINOIDEA)

A comparative study of seven species of Tellinidae, collected at Kungkrabaen Bay, Gulf of Thailand, is presented. The species are Serratina capsoides (Lamarck, 1818), Moerella cf. Miens (Deshayes, 1854), Cadella cf. semen (Hanley, 1845), Pinguitellina cf. pinguis (Hanley, 1844), Elpidollina sp., and Tellinides timorensis (Lamarck, 1818) of the subfamily Tellininae, and Macomona sp. of the subfamily Macominae. With the objective of performing the study in a comparative and testable scenario, a phylogenetic analysis was carried out based on 43 morphological characters (94 states). A single cladogram was obtained as follows: (Serratina capsoides (Tellinides timorensis (Pinguitellina cf. pinguis (Cadella cf. semen (Moerella cf. Miens (Elpidollina sp. Macomona sp.)))))). A semelid and a solenid were operationally analyzed as part of the ingroup; polarisation was based on a plicatulid. In this analysis, Tellinoidea and Tellinidae were monophyletic, supported by 19 and 9 synapomorphies, respectively. Tellininae was paraphyletic. Good and significant morphological differences were found for all taxa, including at the species level. Some structures are reported for the first time, such as pseudogills, insertion of the pedal protractor dividing the anterior adductor muscle, and a circular muscle surrounding part of the anterior adductor muscle.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

Heritability Estimate and Genetic Correlations of Reproductive Features in Nellore Bulls, Offspring of Super Precocious, Precocious and Normal Cows Under Extensive Farming Conditions

The present work aimed to estimate heritability and genetic correlations of reproductive features of Nellore bulls, offspring of mothers classified as superprecocious (M1), precocious (M2) and normal (M3). Twenty one thousand hundred and eighty-six animals with average age of 21.29 months were used, evaluated through the breeding soundness evaluation from 1999 to 2008. The breeding soundness features included physical semen evaluation (progressive sperm motility and sperm vigour), semen morphology (major, minor and total sperm defects), scrotal circumference (SC), testicular volume (TV) and SC at 18 months of age (SC18). The components of variance, heritability and genetic correlations for and between the features were estimated simultaneously by restricted maximum likelihood, with the use of the vce software system vs 6. The heritability estimates were high for SC18, SC and TV (0.43, 0.63 and 0.54; 0.45, 0.45 and 0.44; 0.42, 0.45 and 0.41, respectively for the categories of mothers M1, M2 and M3) and low for physical and morphological semen aspects. The genetic correlations between SC18 and SC were high, as well as between these variables with TV. High and positive genetic correlations were recorded among SC18, SC and TV with the physical aspects of the semen, although no favourable association was verified with the morphological aspects, for the three categories of mothers. It can be concluded that the mothers sexual precocity did not affect the heritability of their offspring reproduction features.

Genetic correlations and heritability estimates of reproductive traits of Nellore bulls

Estimates of phenotypic, genetics and residual variances for reproductive traits in 5903 Nellore bulls were obtained. The experimental model used was multiple trait derivative-free restricted maximum likelihood. The values obtained for heritability were 0.24 +/- 0.05 for scrotal circumference at 450 days of age and 0.37 +/- 0.05 at 21 months for age at the time of the breeding soundness evaluation; 0.24 +/- 0.05 and 0.26 +/- 0.05 for left and right testicle length; 0.29 +/- 0.05 and 0.31 +/- 0.05 for left and right testicle width; 0.12 +/- 0.04 for testicle format; 0.33 +/- 0.06 for testicle volume; 0.11 +/- 0.03 for gross motility; 0.08 +/- 0.03 for individual motility and 0.05 +/- 0.02 for spermatic vigor; 0.20 +/- 0.04, 0.03 +/- 0.02 and 0.19 +/- 0.04 for larger defects, smaller defects and total defects, respectively. The values for heritability for testicular biometric characteristics were moderate to high while the seminal characteristics, presented low values. Genetic correlations between scrotal circumference with all the reproductive traits were favorable, suggesting the scrotal circumference as a feature of choice in the selection of bulls.

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Rapid morphological changes in populations of hybrids between Africanized and European honey bees

African honey bees, introduced to Brazil in 1956, rapidly dominated the previously introduced European subspecies. To better understand how hybridization between these different types of bees proceeded, we made geometric morphometric analyses of the wing venation patterns of specimens resulting from crosses made between Africanized honey bees (predominantly Apis mellifera scutellata) and Italian honey bees (A. mellifera ligustica) from 1965 to 1967, at the beginning of the Africanization process, in an apiary about 150 km from the original introduction site. Two virgin queens reared from an Italian parental were instrumentally inseminated with semen from drones from an Africanized parental. Six F-1 queens from one of these colonies were open mated with Africanized drones. Resultant F-1 drones were backcrossed to 50 Italian and 50 Africanized parental queens. Five backcross workers were collected from each of eight randomly selected colonies of each type of backcross (N = 5 bees x 8 colonies x 2 types of backcrosses). The F-1 progeny (40 workers and 30 drones) was found to be morphologically closer to the Africanized than to the European parental (N = 20 drones and 40 workers, each); Mahalanobis square distances = 21.6 versus 25.8, respectively, for the workers, and 39.9 versus 46.4, respectively, for the drones. The worker progenies of the backcrosses (N = 40, each) were placed between the respective parental and the F-1 progeny, although closer to the Africanized than to the Italian parentals (Mahalanobis square distance = 6.2 versus 12.1, respectively). Consequently, the most common crosses at the beginning of the Africanization process would have generated individuals more similar to Africanized than to Italian bees. This adds a genetic explanation for the rapid changes in the populational morphometric profile in recently colonized areas. Africanized alleles of wing venation pattern genes are apparently dominant and epistatic.

Gonadal function in male patients with ankylosing spondylitis

Objective: To assess reproductive function in male ankylosing spondylitis (AS) patients in comparison to healthy controls. Methods: Twenty AS patients were compared to 24 healthy male subjects with regard to demographic data, urological examination, testicular ultrasound (US), semen analysis, anti-sperm antibodies, and hormone profile. Exclusion criteria were present use of sulfasalazine or methotrexate, and ever use of biological/cytotoxic agents. Disease activity of AS was evaluated by clinical and laboratory assessments. Results: Demographic data were similar in AS and controls (p = 0.175). Varicocele was found significantly more frequently in AS patients than in controls (40% vs. 8%, p = 0.027). Semen analysis revealed no significant differences in sperm quality between AS patients and controls (p > 0.05). By contrast, the median of normal sperm forms was significantly lower in AS patients with vs. those without varicocele [13.5 (range 2-27) vs. 22 (range 10-32.5)%, p = 0.049] whereas no difference in sperm morphology was observed comparing AS patients and controls without varicocele (p = 0.670). Comparison of AS patients with and without varicocele showed that anti-sperm antibodies, hormones, inflammatory markers, and disease activity scores did not contribute to the impaired sperm morphology observed in AS patients with varicocele. Conclusions: An increased frequency of varicocele was found in AS patients associated with sperm abnormalities but independent of therapy, anti-sperm antibodies, hormonal alterations, or disease parameters. Investigation for varicocele should be routine in AS patients with fertility problems.

Grade 3 Varicocele in Fertile Men: A Different Entity

Purpose: Although varicocele size has an inverse relationship with baseline semen parameters and a direct relationship with seminal reactive oxygen species in infertile patients, to our knowledge the effect of varicocele grade in fertile men is unknown. We evaluated the impact of varicocele grade on seminal parameters, testicular size and seminal reactive oxygen species in fertile men. Materials and Methods: We prospectively evaluated 194 men from July 2004 to April 2010. Of the men 156 were fertile and classified by presence of varicocele. A total of 38 infertile patients with varicocele as the only identifiable cause of infertility comprised the control group. Physical examination, semen parameters and seminal reactive oxygen species were compared between the groups. Results: Of 156 fertile men 43 (24.3%) had clinical varicocele, which was grade 1 to 3 in 22, 11 and 10, respectively. The remaining 113 men (72.7%) had no varicocele. Infertile men had smaller testes, decreased semen parameters and higher seminal reactive oxygen species than the fertile groups. Testicular size, reactive oxygen species and semen parameters did not differ between fertile men with vs without varicocele. Fertile men with varicocele grade 3 had higher seminal reactive oxygen species than those with lower grade varicocele. As varicocele grade increased, seminal reactive oxygen species increased and sperm concentration decreased. Conclusions: Although fertile men have more efficient defense mechanisms to protect against the consequences of varicocele on testicular function, these mechanisms may not be sufficient in those with varicocele grade 3. Further research is needed to clarify whether they are at increased risk for future infertility.

Assessment of field fertility and several in vitro sperm characteristics following the use of different Angus sires in a timed-AI program with suckled Nelore cows

In order to provide information that may help researchers to understand the main cause(s) of differences in bull fertility frequently observed in field trials, this study aimed to investigate conception rates as well as several in vitro sperm characteristics of different sires of unknown fertility utilized in a Timed-AI (TAI) program. Suckled Nelore cows submitted to the same TAI protocol were allocated into eight breeding groups of approximately 120 animals each. Frozen semen doses from three Angus bulls and three different batches from each bull were utilized. Approximately 100 doses from each batch were used in TAI. Sires, batches and AI technicians were equally distributed across breeding groups. Cows were examined for pregnancy diagnosis 40 d after TAI. For in vitro sperm analyses, the same thawing procedure was repeated in the laboratory to mimic field conditions. The following in vitro sperm characteristics were assessed: computerized motility, thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, morphology, morphometry and chromatin structure. No effect of breeding group, body condition score, AI technician and sire was observed. However, some significant differences among bulls were detected in laboratory analyses. Semen from sire presenting numerically lower (P > 0.05) pregnancy/AI also presented lower (P < 0.05) values in all sperm characteristics analyzed in thermal resistance test at 4 h (Total Motility, Progressive Motility, Average Path Velocity, Straight-Line Velocity, Curvilinear Velocity, Amplitude of Lateral Head Displacement, Beat Cross Frequency, Straightness, Linearity, and Percentage of Rapidly Moving Cells), higher (P < 0.05) Major and Total Defects in sperm morphological test, lower (P < 0.05) Length, Ellipticity and Fourier parameter (Fourier 0) in sperm morphometric analysis as well as higher (P < 0.05) chromatin heterogeneity. It was concluded that, although no bull effect was observed in the field experiment, the sire that presented numerically lower pregnancy/AI also presented lower semen quality according to the laboratory analyses performed. (C) 2012 Elsevier B.V. All rights reserved.

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT cooling rate of -0.55 degrees C min-1 and freezing rate of -19.1 degrees C min-1) and automated (AT cooling rate of -0.23 degrees C min-1 and freezing rate of -15 degrees C min-1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fishers test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 +/- 1.41% and 30.50 +/- 1.06%, with ethylene glycol was 21.17 +/- 1.66% and 21.67 +/- 1.13% and with dimethyl formamide was 8.33 +/- 0.65% and 9.17 +/- 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 +/- 1.49% (CT) and 15.83 +/- 1.26% (AT) to glycerol, 9.20 +/- 1.31% (CT) and 9.92 +/- 1.29% (AT) to ethylene glycol 4.65 +/- 0.93% (CT) and 5.17 +/- 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Evaluation of Sperm DNA Peroxidation in Fertile and Subfertile Dogs

Contents Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n=5); subfertile group (G2): dogs with low sperm count (<20x106sptz/ml) and/or more than 30% of total sperm pathology (n=6). Both groups received 500mg/day of vitamin C and 500mg/day of vitamin E for 60days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2'-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500mg/day of vitamin C and 500mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.