In a Modular High Temperature Gas Reactor (MHGTR), the Pressurized Conduction Cooldown (PCC) event is an accident scenario in which there is a loss of forced convection of the coolant through the system but the pressure boundary remains intact. When the PCC event occurs, natural circulation onsets which results in a flow and temperature reversal, resulting in hot helium flowing into the upper plenum which could imping upon the upper plenum head. The High Temperature Test Facility (HTTF), which is being designed and constructed at Oregon State University, is a scaled thermal test facility of the MHTGR which is being built for both code validation purposes and to examine specific flow phenomena of the gas reactor. This study is being performed to determine how the HTTF simulates the same conditions as the MHTGR under this scenario through the use of scaling parameters and computational fluid dynamics. / Graduation date: 2013

The need for energy is growing in the world and the market of nuclear power is now once more expanding. Some issues of the current light-water reactors can be solved by the next generation of nuclear power, Generation IV, where sodium-cooled reactors are one of the candidates. Phénix was a French prototype sodium-cooled reactor, which is seen as a success. Although it did encounter an earlier unexperienced phenomenon, A.U.R.N., in which a negative reactivity transient followed by an oscillating behavior forced an automatic emergency shutdown of the reactor. This phenomenon lead to a lot of downtime of the reactor and is still unsolved. However, the most probable cause of the transients is radial movements of the core, referred to as core-flowering. This study has investigated the available documentation of the A.U.R.N. events. A simplified model of core-flowering was also created in order to simulate how radial expansion affects the reactivity of a sodium-cooled core. Serpent, which is a Monte-Carlo based simulation code, was chosen as calculation tool. Furthermore, a model of the Phénix core was successfully created and partly validated. The model of the core has a k_eff = 1.00298 and a neutron flux of (8.43+-0.02)!10^15 neutrons/cm^2 at normal state. The result obtained from the simulations shows that an expansion of the core radius decreases the reactivity. A linear approximation of the result gave the relation: change in k_eff/core extension = - 60 pcm/mm. This value corresponds remarkably well to the around - 60 pcm/mm that was obtained from the dedicated core-flowering experiments in Phénix made by the CEA. Core-flowering can recreate similar signals to those registered during the A.U.R.N. events, though the absence of trace of core movements in Phénix speaks against this. However, if core-flowering is the sought answer, it can be avoided by design. The equipment that registered the A.U.R.N. events have proved to be insensitive to noise. Though, the high amplitude of the transients and their rapidness have made some researcher believe that the events are a combination of interference in the equipment of Phénix and a mechanical phenomenon. Regardless, the origin of A.U.R.N. seems to be bound to some specific parameter of Phénix due to the fact that the transients only have occurred in this reactor. A safety analysis made by an expert committee, appointed by CEA, showed that the A.U.R.N. events are not a threat to the safety of Phénix. However, the origin of these negative transients has to be found before any construction of a commercial size sodium-cooled fast reactor can begin. Thus, further research is needed.

Thesis (MScEng (Mechanical and Mechatronic Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: This study is aimed at the development of an efficient and reliable method of evaluating the performance of an air-cooled steam condenser (ACSC) under windy conditions, using computational fluid dynamics (CFD). A two-step modelling approach is employed as a result of computational limitations. The numerical ACSC model developed in this study makes use of the pressure jump fan model, amongst other approximations, in an attempt to minimize the computational expense of the performance evaluation. The accuracy of the numerical model is verified through a comparison of the numerical results to test data collected during full scale tests carried out on an operational ACSC. Good correlation is achieved between the numerical results and test data. Further verification is carried out through a comparison to previous numerical work. Satisfactory convergence is achieved for the most part and the few discrepancies in the results are explained. The effect of wind on ACSC performance at El Dorado Power Plant (Nevada, USA) is investigated and it is found that reduced fan performance due to distorted flow at the inlet of the upstream fans is the primary contributor to the reduction in performance associated with increased wind speed in this case. An attempt is subsequently made to identify effective wind effect mitigation measures. To this end the effects of wind screens, solid walkways and increasing the fan power are investigated. It is found that the installation of an appropriate wind screen configuration provides a useful means of reducing the negative effects of wind on ACSC performance and an improved wind screen configuration is suggested for El Dorado. Solid walkways are also shown to be beneficial to ACSC performance under windy conditions. It is further found that ACSC performance increases with walkway width but that the installation of excessively wide walkways is not justifiable. Finally, increasing the fan power during periods of unfavourable ambient conditions is shown to have limited benefit in this case. The model developed in this study has the potential to allow for the evaluation of large ACSC installations and provides a reliable platform from which further investigations into improving ACSC performance under windy conditions can be carried out. / AFRIKAANSE OPSOMMING: Hierdie studie is daarop gemik om die ontwikkeling van 'n geskikte en betroubare metode van evaluering van die verrigting van ’n lugverkoelde stoom-kondensator (air-cooled steam condenser, ACSC) onder winderige toestande, met behulp van numeriese vloei-dinamika. ’n Twee-stap modelleringsbenadering is aangewend as gevolg van rekenaar beperkings. Die numeriese ACSC-model wat in hierdie studie ontwikkel is, maak gebruik van die druksprong waaier model, asook ander benaderings, in ’n poging om die berekeningskoste van die verrigting-evaluering te verminder. Die akkuraatheid van die numeriese model is bevestig deur middel van ’n vergelyking van die numeriese resultate met toetsdata ingesamel tydens die volskaal toetse uitgevoer op ’n operasionele ACSC. Goeie korrelasie is bereik tussen die numeriese resultate en toetsdata. Verdere bevestiging is uitgevoer deur middel van ’n vergelyking met vorige numeriese werk. Bevredigende konvergensie is in die algemeen bereik en die paar verskille in die resultate word verduidelik. Die effek van wind op ACSC verrigting by El Dorado Power Plant (Nevada, VSA) is ondersoek, en daar is bevind dat verlaagde waaierverrigting, as gevolg van vervormde vloei by die inlaat van die stroomop waaiers, die primêre bydraer is tot die afname in ACSC werkverrigting geassosieer met verhoogde windsnelheid in hierdie geval. ’n Poging word dan aangewend om effektiewe wind-effek velagingsmaatreëls te identifiseer. Windskerms, soliede wandelvlakke en die verhoging van die waaierkrag word gevolglik ondersoek. Daar is bevind dat die installasie van ’n toepaslike windskerm-opset ’n nuttige middel tot ’n vermindering van die negatiewe effekte van wind op ACSC verrigting bied, en ’n verbeterde windskerm opset is voorgestel vir El Dorado. Soliede wandelvlakke word ook aanbeveel as voordelig vir ACSC verrigting onder winderige toestande. Dit is verder bevind dat die ACSC prestasie verhoog met wandelvlak breedte, maar dat die installasie van ’n te ruim wandelvlak nie regverdigbaar is nie. Ten slotte, word bewys dat die verhoging van die waaierkrag tydens periodes van ongunstige omgewingsomstandighede ’n beperkte voordeel in hierdie geval het. Die model wat ontwikkel is in hierdie studie het die potensiaal om voorsiening te maak vir die evaluering van groot ACSC- installasies en bied ’n betroubare platform vanwaar verdere ondersoeke tot die verbetering van ACSC verrigting onder winderige toestande uitgevoer kan word.

The behavior of gas-solid reactors known as compact-fixed and moving beds, is analyzed from a theoretical viewpoint. For a compact fixed-bed the solution of the energy balance equations is obtained for the cases of a uniform temperature inside the solid pellets (i.e., the Biot number is zero) and for the case in which there are temperature gradients within the pellets (Bi > 0). For short contact times, beds with Bi > 0 have gas- and solid- temperatures which are greater than the temperatures within beds with Bi = 0. For long times, the situation is reversed. For a compact-moving bed the solution of the mass balance equations is obtained for the cases of a feed-solid with constant concentration and a feed solid with an oscillating concentration. In both cases the steady states obtained are unique, and internal recycling is observed only for a feed-solid with an oscillating concentration. Recycling is that situation when the concentration of the solid falls below that of the gas for a bed in which the feed-solid is greater than the feed-gas. This occurred when the period of oscillation was smaller than the residence time of the solid provided that the residence time of the solid was not very short (i.e., provided that B(,s) > 0.1). For both types of beds there is an equivalence between mass transfer and energy transfer so that the solutions can be interchanged with suitable definitions of dimensionless variables.

Pressurizer modeling for predicting the dynamic pressure of the PIUS system is presented. The transient behavior of this model for the PIUS system was investigated. The validity of this model for the PIUS system is limited to transients that are neither too large nor too long in duration. For example, the model is not capable of describing events following a complete loss of liquid for the pressurizer. However, the model can be used for qualitative prediction of the PIUS system behavior for a wide variety of severe transients. A review of pressurizer modeling indicates that the neglecting of the change in the internal energy of the subcooled water during transients is an acceptable assumption. The inherently safe feature of the PIUS system was confirmed through the self-shutdown of the reactor or, in some cases, through reactor power reduction as a result of the ingress of the pool boric acid solution into the primary system. This dynamic model was constructed of three major components: (1) The primary loop, (2) The secondary loop, and (3) The natural convection loop through the pool. A lumped parameter model, uniform heat transfer, and point kinetics have been the main approximations in this model. Other approximations are mentioned during the modeling of each component of the model. The dynamic model was simulated using the DARE-P continuous system simulation language which was developed in the Electrical Engineering Department at the University of Arizona.

O objetivo do presente estudo foi avaliar a viabilidade da refrigeração do sêmen bovino comparada com a criopreservação. Este estudo foi realizado em dois experimentos. No primeiro experimento foram comparados os efeitos in vitro do sêmen refrigerado em três meios diluidores comerciais a 5° C por até 48 horas, e criopreservado em dois meios. Para este experimento foram utilizados ejaculados de 10 touros da raça Nelore. Cada ejaculado foi dividido em três alíquotas, sendo diluídas nos meios Botubov®, Steridyl® e Bovidyl®. Após a diluição o sêmen foi envasado em palhetas e refrigerado nos três diluidores e criopreservado utilizando somente os meios Botubov® e Steridyl®. A refrigeração do sêmen foi realizada a 5° C por até 48 horas em sistema passivo de refrigeração BotuFlex® e a criopreservação realizada em sistema automatizado TK 4.000®. O sêmen foi avaliado nos tempos 0, 24, 36 e 48 horas após a refrigeração e após a descongelação, para cada diluidor. Foram realizadas análises dos padrões de cinética espermática pelo sistema computadorizado de análise espermática (CASA, programa SCA - Sperm Class Analyser), integridade das membranas plasmática e acrossomal, potencial de membrana mitocondrial e estresse oxidativo por sondas fluorescentes, sob microscopia de epifluorescência e morfologia espermática por microspcopia de contraste de interferência diferencial (DIC). Notou-se efeito de tempo de refrigeração para os três diluidores para de 0 para 24h, se mantendo semelhante até 48 h. Os diluiores Botubov® e Steridyl® preservaram as características espermáticas de forma semelhante até 48 horas de refrigeração diferindo apenas na variável de velocidade curvilianear; no entanto, ambos foram superiores ao diluidor Bovidyl®, para as variáveis, motilidade total, motilidade progressiva, células rápidas, velocidade curvilinear, velocidade progressiva, velocidade do trajeto, retilinearidade, integridade da membrana plasmática, alto potencial de mitocondrial e espermatozoides com membranas plasmática e acrossomal íntegras e alto potencial mitocondrial. O segundo experimento foi realizado, baseado nos resultados do primeiro experimento, para avaliar os efeitos da refrigeração e da criopreservação do sêmen sobre a fertilidade in vivo. Foram utilizados ejaculados de três touros das raças Brangus, Braford e Angus, com idade entre 5 e 7 anos. O sêmen foi colhido por meio de vagina artificial, o ejaculado foi dividido em três alíquotas, sendo duas alíquotas para refrigeração e uma para criopreservação. Para a refrigeração o sêmen foi diluído nas concentrações de 15x106 (R15) e 30x106 (R30) espermatozoides/palheta e para a criopreservação diluído na concentração de 30x106 espermatozoides/palheta (CRIO). Para todas as diluições foi utilizado o meio Botubov® e o sêmen armazenado em palhetas de 0,5 mL. A refrigeração foi realizada a temperatura de 5° C por 48 horas em sistema passivo de refrigeração BotuFlex® e a criopreservação em sistema automatizado TK®. Foram sincronizadas 552 vacas da raça Brangus em programas de inseminação artificial em tempo fixo. Os resultados da taxa de prenhez para os grupos de vacas inseminadas foram de 49,4% para R15, 43,38% para R30 e 47,59% para o sêmen criopreservado. Pode-se concluir que a refrigeração do sêmen a 5° C por 48 horas resulta em taxa de prenhez semelhante às obtidas com o sêmen criopreservado, sendo que esses resultados indicam que a refrigeração por até 48 horas pode ser uma opção de uso para IATF. / The objective of the present study was to evaluate the viability of cooling bovine semen compared to cryopreservation. This study was carried out in two experiments. In the first experiment, the in vitro effects of cooled semen were compared in three commercial extenders at 5° C for up to 48 hours, and cryopreserved in two extender. For this experiment were used ejaculates of 10 Nellore bulls. Each ejaculate was divided into three aliquots, being diluted in the Botubov®, Steridyl® and Bovidyl® extenders. After dilution, the semen was cooled into three extenders and cryopreserved using the Botubov® and Steridyl®. Semen refrigeration was performed at 5° C for up to 48 hours in BotuFlex® passive refrigeration system and cryopreservation performed in TK 3.000® automated system. Semen was evaluated at 0, 24, 36 and 48 hours after refrigeration and after thawing, for each diluent. The sperm kinetics of the spermatic kinetics (CASA, SCA - Sperm Class Analyzer), plasma and acrosomal membrane integrity, mitochondrial membrane potential and oxidative stress by fluorescent probes were analyzed under epifluorescence microscopy and sperm morphology By Differential Interference Contrast Microscopy (DIC). The Botubov® and Steridyl® diluents were very similar after 48 hours of cooling differing significantly only in the Curvilianear Velocity (VCL) 106.04 m/s and 124.56 m/s. The Bovidyl® diluent yielded results significantly lower than the 48 hours of refrigeration for the variables: total motility (MT), progressive motility (MPRO), fast cells (RAP), curvilinear velocity (VCL), progressive velocity (AP), plasma membrane integrity (PI), high mitochondrial potential (AP), and spermatozoa with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIA). The second experiment was carried out, based on the results of the first experiment I, to evaluate the effects of cooled and cryopreservation of semen on in vivo fertility. We used ejaculates of three bulls of the Brangus, Braford and Angus breeds of a IA center, aged between 5 and 7 years. The semen was collected by artificial vagina, the ejaculate was divided into three aliquots, two aliquots for refrigeration and one for cryopreservation. For cooling, the semen was diluted in the concentrations of 15x106 (R15) and 30x106 (R30) spermatozoa/straw, for cryopreservation diluted in the concentration of 30x106 spermatozoa / vane (CRIO). For all dilutions, the Botubov® medium and the semen stored in 0.5 mL vial were used. Refrigeration was carried out at a temperature of 5° C for 48 hours in BotuFlex® passive refrigeration system and cryopreservation in an automated TK® system. 552 Brangus cows were synchronized in fixed-time artificial insemination programs. The results of the pregnancy rate for the groups of inseminated cows were 49.4% for R15, 43.38% for R30 and 47.59% for cryopreserved semen. It can be concluded that the cooling of the semen at 5° C for 48 hours results in pregnancy rate similar to those obtained with cryopreserved semen, and these results indicate that refrigeration for up to 48 hours may be an option of use for FTAI.

Diversos fatores respondem por variações na viabilidade do sêmen eqüino e na sua qualidade espermática; em humanos, reportam-se correlações entre fatores endócrinos e parâmetros seminais, mas em eqüinos poucos estudos contribuem para esta correlação. Para se investigar eventuais correlações entre hormônios sexuais e tireoideanos com as características seminais físicas e morfológicas, além de longevidade seminal, foram mensuradas as concentrações plasmáticas de estradiol, testosterona, T3 e T4 de garanhões e analisadas tais características espermáticas no momento da colheita seminal e após 24h, 48h e 72h de armazenamento a 5oC. 05 garanhões férteis em idade reprodutiva foram submetidos à colheita de sêmen (2 vezes/semana) e sangue (1 vez/semana, a cada 6 horas) durante 6 semanas. Estradiol, testosterona, T3 e T4 foram analisados por RIE. Após a colheita, o sêmen foi analisado e resfriado a 5oC, com análises subseqüentes após 24h, 48h e 72h de armazenamento. O sêmen foi avaliado quanto às características seminais físicas e morfológicas, além de integridade de membrana. Significativas (p<0.05) diferenças foram observadas entre os animais quanto às concentrações hormonais; o estradiol mostrou as maiores variações entre indivíduos, embora todos os resultados tenham se mostrado compatíveis aos valores de referência da literatura. Logo após a colheita, não se observou diferenças significativas (p<0.05) para vigor e motilidade total/progressiva, em exceção ao garanhão 4, que apresentou significativa menor motilidade progressiva inicial. Após 24h, 48h e 72h de resfriamento, diferenças significativas (p<0.05) foram observadas entre os garanhões; os menores índices de vigor/motilidade progressiva foram observados nos garanhões 1 e 4. Diferenças significativas (p<0.05) também foram observadas entre os animais para as características espermáticas morfológicas e integridade de membrana. Análises de covariância e correlações não demonstraram correspondência entre concentrações hormonais, longevidade espermática e características seminais físicas/morfológicas, tendo \"tempo de resfriamento\" e \"indivíduos\" como fatores. Correlação positiva foi observada entre motilidade progressiva/vigor e espermatozóides vivos com acrossomo intacto (r=0.70 e r=0.71 respectivamente); em analogia, correlação negativa foi encontrada entre motilidade progressiva/vigor e espermatozóides com acrossomo intacto (r=-0.70 e r=-0.72 respectivamente), indicando que, vivos ou mortos, apresentar acrossomo intacto é fator crítico para a viabilidade espermática mesmo após diferentes tempos de armazenamento em resfriamento. Os resultados mostram que a longevidade do sêmen eqüino provavelmente não é diretamente afetada por razões hormonais primárias; o mesmo pode ser considerado para morfologia espermática. Por outro lado, a longevidade do sêmen eqüino parece ser diretamente influenciada pela integridade de membrana espermática e eventualmente fatores bioquímicos, metabólicos e enzimáticos. / Several factors respond for variations of equine semen viability and sperm quality; in humans, scientific reports try to correlate endocrine issues and sperm parameters. In horses, few studies contribute to this correlation. In order to investigate eventual correlations between sexual and thyroid hormones with physical and morphologic characteristics and also longevity of equine semen,were measured plasma concentrations of estradiol, testosterone, T3 and T4 of stallions and analyzed physical and morphologic characteristics and membrane integrity of spermatozoa at the moment of collection and after 24, 48 and 72 hours of storage at 5oC. Five fertile stallions in reproductive age had semen (twice a week) and blood (once a week, every 6 hours) collected during 6 weeks. Estradiol, testosterone, T3 and T4 were analyzed by RIA. After collection, semen was analyzed and cooled at 5oC, with further analyses after 24, 48 and 72 hours of storage. Semen analysis consisted in physical, morphologic and embrane integrity characteristics. Significant (p<0.05) differences were observed among stallions when compared for plasma hormonal concentrations; estradiol showed the greater variations between individuals, despite all results were compatible to reference values in literature. Just after collection, no significant differences (p<0.05) were observed for vigor and total/progressive motility, in exception to stallion 4, that presented a significant lower initial progressive motility. After 24, 48 and 72 hours of storage, significant differences (p<0.05) were observed among stallions; lowest values of vigor/progressive motility were observed for stallions 1 and 4. Significant differences (p<0.05) were also observed among stallions for morphologic characteristics of sperm and membrane integrity. Analyses of covariance and correlations showed no correspondence among hormonal plasma concentrations, sperm longevity and physical/morphologic characteristics, having time of storage and individuals as factors. Meanwhile, a positive correlation was observed among progressive motility/vigor and live sperm with intact acrosome (r=0.70 and r=0.71 respectively); in analogy, a negative correlation was found among progressive motility/vigor and dead sperm with intact acrosome (r=-0.70 and r=-0.72 respectively), indicating that neither being live or dead, but having acrosome membrane intact is a critical factor for spermatozoa\'s viability even after different times of storage at cooling temperatures. Results show that longevity of equine semen is probably not directly affected by primary hormonal reasons; the same can be considered for sperm morphology. Otherwise, longevity of equine semen seems to be directly influenced by sperm membrane integrity and eventually by biochemical, metabolic and enzymatic factors.

O sêmen eqüino é muito utilizado na forma refrigerada, porém, para alguns garanhões, a longevidade espermática é baixa. Durante a refrigeração o espermatozóide pode ser afetado por vários fatores, que podem causar a perda da integridade da membrana plasmática, danos no acrossomo e disfunções mitocondriais, resultando em perda irreversível de motilidade e capacidade fecundante. A composição e concentração do plasma seminal podem causar efeitos deletérios ao espermatozóide, sendo a centrifugação do sêmen uma alternativa para minimizar estes efeitos. Este experimento foi realizado para verificar o efeito da centrifugação nas características de movimento, integridade e peroxidação lipídica das membranas do espermatozóide eqüino refrigerado a 5°C por até 72h. Foram realizadas cinco colheitas de sêmen de quatro garanhões. O sêmen foi avaliado quanto ao volume e concentração, e em seguida dividido em dois tratamentos, sem centrifugação (S) e centrifugado (C) (300g/10min). Ambos foram diluídos na concentração final de 30 x106 eptz/mL em diluidor a base de leite desnatado e então refrigerados. Foram analisadas as características do movimento espermático (CASA), morfologia espermática (DIC), integridade das membranas plasmática e acrossomal (PI/FITC-PSA), potencial de membrana mitocondrial (JC-1) e peroxidação lipídica das membranas (C11-BODIPY581/591), sendo estas três últimas análises por citometria de fluxo. As análises foram realizadas no tempo 0 e 24, 48 e 72 horas após a refrigeração. O delineamento experimental foi em blocos generalizados adicionado do fator medidas repetidas no tempo. Os dados foram analisados pelo programa SAS, submetidos à análise de variância e às interações determinadas pelo teste de Greenhouse-Geisser ao nível de 5% de significância. O espermatozóide do sêmen centrifugado apresentou valores médios maiores de motilidade total e progressiva, ALH, STR e LIN (P<0,05), porém os valores das velocidades e BCF foram menores (P<0,05). Houve efeito do tempo nas características de movimento espermático (P<0,05), exceto para STR. Não houve efeito da centrifugação e nem do tempo quanto às características morfológicas dos espermatozóides refrigerados, esta só diferiu entre os garanhões (P<0,05). Por sua vez, a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial espermáticas foram maiores nos sêmen centrifugado (P<0,05) e suas porcentagens decresceram com o tempo (P<0,05). A peroxidação lipídica da membrana do espermatozóide refrigerado aumentou somente nas primeiras 24h (P<0,05) e não foi influenciada pela centrifugação. Conclui-se que a centrifugação e a retirada do plasma seminal melhoram a qualidade do sêmen refrigerado eqüino, principalmente em garanhões com baixa longevidade, sendo esta influenciada pelas características iniciais da morfologia espermática. Ainda, tempo de refrigeração induz a um declínio da qualidade seminal. E, por fim, a peroxidação lipídica da membrana do espermatozóide parece não ser a principal causa de alterações da integridade das membranas e do potencial de membrana mitocondrial durante a refrigeração do sêmen eqüino. / Equine cooled semen is frequently used, although the longevity for some stallions is low. During the cooling process the spermatozoon can be affected by several factors that can cause loss of plasma membrane integrity, acrosomal damage and mitochondrial dysfunctions, resulting in irreversible loss of motility and fertilizing capacity. The composition and concentration of seminal plasma could cause harmful effects to the sperm, being semen centrifugation an alternative to minimize these effects. This experiment was designed to verify the effects of the centrifugation in motion characteristics, membranes integrity and lipid peroxidation of cooled equine sperm to 5°C until 72 hours. Five ejaculates from four stallions were collected. The semen was evaluated for volume and concentration, then split in two treatments, no centrifugation (N) and with centrifugation (W) (300g/10min). Both were diluted in the final concentration 30 x106 spermatozoa/mL in a skin milk extender and then cooled. The following analyses were performed: the sperm motion characteristics (CASA), morphology (DIC), plasma and acrosomal membranes integrity (PI/FITC-PSA), mitochondrial membrane potential (JC-1) and membrane lipid peroxidation (C11-BODIPY581/591), the last three analyses were assessed in flow citometry. The analyses were performed in time 0 and 24, 48 and 72 hours after cooling. The experimental design was random blocks with repeated-measures. The data were analyzed by the SAS program, submitted to analysis of variance and the interactions by the test of Greenhouse-Geisser at the level of 5% of significance. The values were larger in W semen for total and progressive motility, ALH, STR and LIN (P<0,05), however the values of the three types of speed and BCF were smaller (P<0,05). There was effect of time in the motion characteristics of sperm (P<0,05), except for STR. There was not any effect of centrifugation and time for the morphologic characteristics of cooled spermatozoa, that only differed among stallions (P<0,05). On the other hand, the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane were larger in W semen (P<0,05) and their percentages decreased within time (P<0,05). The membrane lipidic peroxidation of the cooled sperm only increased in the first 24 hours (P<0,05) and it was not influenced by centrifugation. In conclusion, the centrifugation and withdrawal of the seminal plasma improve semen quality of the cooled equine sperm, mainly in stallions with low longevity, which is influenced by the initials characteristics of sperm morphology. Moreover, time of cooling induces a decline of seminal quality. Finally, the membrane lipidic peroxidation of the cooled sperm seems not to be the main cause of alterations in the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane during the cooling of equine semen.

O objetivo do presente estudo foi avaliar a viabilidade da refrigeração do sêmen bovino comparada com a criopreservação. Este estudo foi realizado em dois experimentos. No primeiro experimento foram comparados os efeitos in vitro do sêmen refrigerado em três meios diluidores comerciais a 5&deg; C por até 48 horas, e criopreservado em dois meios. Para este experimento foram utilizados ejaculados de 10 touros da raça Nelore. Cada ejaculado foi dividido em três alíquotas, sendo diluídas nos meios Botubov&reg;, Steridyl&reg; e Bovidyl&reg;. Após a diluição o sêmen foi envasado em palhetas e refrigerado nos três diluidores e criopreservado utilizando somente os meios Botubov&reg; e Steridyl&reg;. A refrigeração do sêmen foi realizada a 5&deg; C por até 48 horas em sistema passivo de refrigeração BotuFlex&reg; e a criopreservação realizada em sistema automatizado TK 4.000&reg;. O sêmen foi avaliado nos tempos 0, 24, 36 e 48 horas após a refrigeração e após a descongelação, para cada diluidor. Foram realizadas análises dos padrões de cinética espermática pelo sistema computadorizado de análise espermática (CASA, programa SCA - Sperm Class Analyser), integridade das membranas plasmática e acrossomal, potencial de membrana mitocondrial e estresse oxidativo por sondas fluorescentes, sob microscopia de epifluorescência e morfologia espermática por microspcopia de contraste de interferência diferencial (DIC). Notou-se efeito de tempo de refrigeração para os três diluidores para de 0 para 24h, se mantendo semelhante até 48 h. Os diluiores Botubov&reg; e Steridyl&reg; preservaram as características espermáticas de forma semelhante até 48 horas de refrigeração diferindo apenas na variável de velocidade curvilianear; no entanto, ambos foram superiores ao diluidor Bovidyl&reg;, para as variáveis, motilidade total, motilidade progressiva, células rápidas, velocidade curvilinear, velocidade progressiva, velocidade do trajeto, retilinearidade, integridade da membrana plasmática, alto potencial de mitocondrial e espermatozoides com membranas plasmática e acrossomal íntegras e alto potencial mitocondrial. O segundo experimento foi realizado, baseado nos resultados do primeiro experimento, para avaliar os efeitos da refrigeração e da criopreservação do sêmen sobre a fertilidade in vivo. Foram utilizados ejaculados de três touros das raças Brangus, Braford e Angus, com idade entre 5 e 7 anos. O sêmen foi colhido por meio de vagina artificial, o ejaculado foi dividido em três alíquotas, sendo duas alíquotas para refrigeração e uma para criopreservação. Para a refrigeração o sêmen foi diluído nas concentrações de 15x106 (R15) e 30x106 (R30) espermatozoides/palheta e para a criopreservação diluído na concentração de 30x106 espermatozoides/palheta (CRIO). Para todas as diluições foi utilizado o meio Botubov&reg; e o sêmen armazenado em palhetas de 0,5 mL. A refrigeração foi realizada a temperatura de 5&deg; C por 48 horas em sistema passivo de refrigeração BotuFlex&reg; e a criopreservação em sistema automatizado TK&reg;. Foram sincronizadas 552 vacas da raça Brangus em programas de inseminação artificial em tempo fixo. Os resultados da taxa de prenhez para os grupos de vacas inseminadas foram de 49,4% para R15, 43,38% para R30 e 47,59% para o sêmen criopreservado. Pode-se concluir que a refrigeração do sêmen a 5&deg; C por 48 horas resulta em taxa de prenhez semelhante às obtidas com o sêmen criopreservado, sendo que esses resultados indicam que a refrigeração por até 48 horas pode ser uma opção de uso para IATF. / The objective of the present study was to evaluate the viability of cooling bovine semen compared to cryopreservation. This study was carried out in two experiments. In the first experiment, the in vitro effects of cooled semen were compared in three commercial extenders at 5&deg; C for up to 48 hours, and cryopreserved in two extender. For this experiment were used ejaculates of 10 Nellore bulls. Each ejaculate was divided into three aliquots, being diluted in the Botubov&reg;, Steridyl&reg; and Bovidyl&reg; extenders. After dilution, the semen was cooled into three extenders and cryopreserved using the Botubov&reg; and Steridyl&reg;. Semen refrigeration was performed at 5&deg; C for up to 48 hours in BotuFlex&reg; passive refrigeration system and cryopreservation performed in TK 3.000&reg; automated system. Semen was evaluated at 0, 24, 36 and 48 hours after refrigeration and after thawing, for each diluent. The sperm kinetics of the spermatic kinetics (CASA, SCA - Sperm Class Analyzer), plasma and acrosomal membrane integrity, mitochondrial membrane potential and oxidative stress by fluorescent probes were analyzed under epifluorescence microscopy and sperm morphology By Differential Interference Contrast Microscopy (DIC). The Botubov&reg; and Steridyl&reg; diluents were very similar after 48 hours of cooling differing significantly only in the Curvilianear Velocity (VCL) 106.04 m/s and 124.56 m/s. The Bovidyl&reg; diluent yielded results significantly lower than the 48 hours of refrigeration for the variables: total motility (MT), progressive motility (MPRO), fast cells (RAP), curvilinear velocity (VCL), progressive velocity (AP), plasma membrane integrity (PI), high mitochondrial potential (AP), and spermatozoa with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIA). The second experiment was carried out, based on the results of the first experiment I, to evaluate the effects of cooled and cryopreservation of semen on in vivo fertility. We used ejaculates of three bulls of the Brangus, Braford and Angus breeds of a IA center, aged between 5 and 7 years. The semen was collected by artificial vagina, the ejaculate was divided into three aliquots, two aliquots for refrigeration and one for cryopreservation. For cooling, the semen was diluted in the concentrations of 15x106 (R15) and 30x106 (R30) spermatozoa/straw, for cryopreservation diluted in the concentration of 30x106 spermatozoa / vane (CRIO). For all dilutions, the Botubov&reg; medium and the semen stored in 0.5 mL vial were used. Refrigeration was carried out at a temperature of 5&deg; C for 48 hours in BotuFlex&reg; passive refrigeration system and cryopreservation in an automated TK&reg; system. 552 Brangus cows were synchronized in fixed-time artificial insemination programs. The results of the pregnancy rate for the groups of inseminated cows were 49.4% for R15, 43.38% for R30 and 47.59% for cryopreserved semen. It can be concluded that the cooling of the semen at 5&deg; C for 48 hours results in pregnancy rate similar to those obtained with cryopreserved semen, and these results indicate that refrigeration for up to 48 hours may be an option of use for FTAI.

O sêmen eqüino é muito utilizado na forma refrigerada, porém, para alguns garanhões, a longevidade espermática é baixa. Durante a refrigeração o espermatozóide pode ser afetado por vários fatores, que podem causar a perda da integridade da membrana plasmática, danos no acrossomo e disfunções mitocondriais, resultando em perda irreversível de motilidade e capacidade fecundante. A composição e concentração do plasma seminal podem causar efeitos deletérios ao espermatozóide, sendo a centrifugação do sêmen uma alternativa para minimizar estes efeitos. Este experimento foi realizado para verificar o efeito da centrifugação nas características de movimento, integridade e peroxidação lipídica das membranas do espermatozóide eqüino refrigerado a 5°C por até 72h. Foram realizadas cinco colheitas de sêmen de quatro garanhões. O sêmen foi avaliado quanto ao volume e concentração, e em seguida dividido em dois tratamentos, sem centrifugação (S) e centrifugado (C) (300g/10min). Ambos foram diluídos na concentração final de 30 x106 eptz/mL em diluidor a base de leite desnatado e então refrigerados. Foram analisadas as características do movimento espermático (CASA), morfologia espermática (DIC), integridade das membranas plasmática e acrossomal (PI/FITC-PSA), potencial de membrana mitocondrial (JC-1) e peroxidação lipídica das membranas (C11-BODIPY581/591), sendo estas três últimas análises por citometria de fluxo. As análises foram realizadas no tempo 0 e 24, 48 e 72 horas após a refrigeração. O delineamento experimental foi em blocos generalizados adicionado do fator medidas repetidas no tempo. Os dados foram analisados pelo programa SAS, submetidos à análise de variância e às interações determinadas pelo teste de Greenhouse-Geisser ao nível de 5% de significância. O espermatozóide do sêmen centrifugado apresentou valores médios maiores de motilidade total e progressiva, ALH, STR e LIN (P<0,05), porém os valores das velocidades e BCF foram menores (P<0,05). Houve efeito do tempo nas características de movimento espermático (P<0,05), exceto para STR. Não houve efeito da centrifugação e nem do tempo quanto às características morfológicas dos espermatozóides refrigerados, esta só diferiu entre os garanhões (P<0,05). Por sua vez, a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial espermáticas foram maiores nos sêmen centrifugado (P<0,05) e suas porcentagens decresceram com o tempo (P<0,05). A peroxidação lipídica da membrana do espermatozóide refrigerado aumentou somente nas primeiras 24h (P<0,05) e não foi influenciada pela centrifugação. Conclui-se que a centrifugação e a retirada do plasma seminal melhoram a qualidade do sêmen refrigerado eqüino, principalmente em garanhões com baixa longevidade, sendo esta influenciada pelas características iniciais da morfologia espermática. Ainda, tempo de refrigeração induz a um declínio da qualidade seminal. E, por fim, a peroxidação lipídica da membrana do espermatozóide parece não ser a principal causa de alterações da integridade das membranas e do potencial de membrana mitocondrial durante a refrigeração do sêmen eqüino. / Equine cooled semen is frequently used, although the longevity for some stallions is low. During the cooling process the spermatozoon can be affected by several factors that can cause loss of plasma membrane integrity, acrosomal damage and mitochondrial dysfunctions, resulting in irreversible loss of motility and fertilizing capacity. The composition and concentration of seminal plasma could cause harmful effects to the sperm, being semen centrifugation an alternative to minimize these effects. This experiment was designed to verify the effects of the centrifugation in motion characteristics, membranes integrity and lipid peroxidation of cooled equine sperm to 5°C until 72 hours. Five ejaculates from four stallions were collected. The semen was evaluated for volume and concentration, then split in two treatments, no centrifugation (N) and with centrifugation (W) (300g/10min). Both were diluted in the final concentration 30 x106 spermatozoa/mL in a skin milk extender and then cooled. The following analyses were performed: the sperm motion characteristics (CASA), morphology (DIC), plasma and acrosomal membranes integrity (PI/FITC-PSA), mitochondrial membrane potential (JC-1) and membrane lipid peroxidation (C11-BODIPY581/591), the last three analyses were assessed in flow citometry. The analyses were performed in time 0 and 24, 48 and 72 hours after cooling. The experimental design was random blocks with repeated-measures. The data were analyzed by the SAS program, submitted to analysis of variance and the interactions by the test of Greenhouse-Geisser at the level of 5% of significance. The values were larger in W semen for total and progressive motility, ALH, STR and LIN (P<0,05), however the values of the three types of speed and BCF were smaller (P<0,05). There was effect of time in the motion characteristics of sperm (P<0,05), except for STR. There was not any effect of centrifugation and time for the morphologic characteristics of cooled spermatozoa, that only differed among stallions (P<0,05). On the other hand, the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane were larger in W semen (P<0,05) and their percentages decreased within time (P<0,05). The membrane lipidic peroxidation of the cooled sperm only increased in the first 24 hours (P<0,05) and it was not influenced by centrifugation. In conclusion, the centrifugation and withdrawal of the seminal plasma improve semen quality of the cooled equine sperm, mainly in stallions with low longevity, which is influenced by the initials characteristics of sperm morphology. Moreover, time of cooling induces a decline of seminal quality. Finally, the membrane lipidic peroxidation of the cooled sperm seems not to be the main cause of alterations in the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane during the cooling of equine semen.