Made available in DSpace on 2016-08-17T17:11:04Z (GMT). No. of bitstreams: 1 DISSERTACAO_ARIEL DE ALMEIDA COELHO.pdf: 1092576 bytes, checksum: 5f74aa9cceb65b68fc6d8633937e2c89 (MD5) Previous issue date: 2014-03-20 / FUNDAÇÃO DE AMPARO À PESQUISA E AO DESENVOLVIMENTO CIENTIFICO E TECNOLÓGICO DO MARANHÃO / This study aimed to verify the effect of bovine somatotrophin (bST) on sexual development, semen and endocrine traits in peripubertal buffalo. A total of 20 male Murrah buffaloes (Bubalus bubalis) (14 months of age) were randomly distributed into two treatments (bST vs control). The body weight, body condition score, testicular measurements, semen traits and IGF-I levels were evaluated once a month for a year. The ejaculate was collected by electroejaculation and physical and morphological analyzes were proceeded. Data analysis was performed using the Statistical Analysis System for Windows, SAS®. There was no difference (P>0.05) between bST and Control treatments on body weight or body condition score. Testicular measurements also showed no differences (P>0.05) between treatments, but increases significantly in a similar trend. There was no positive effect of bST treatment on age at first appearance of spermatozoa in the ejaculate (P> 0.05), but the bST group showed a significant tendency to increase the number of animals exhibiting puberty at each breeding soundness evaluation (P = 0.054). Sperm motility, vigor and percentage of abnormalities showed no difference (P>0.05). There was no difference (P>0.05) between bST and Control treatments on IGF-I serum concentrations. In conclusion, the use of bST did not result in positive effects on sexual development, semen traits and endocrine profile of male buffaloes in peripubertal period. / Objetivou-se com este estudo verificar o efeito da somatotropina bovina (bST) sobre o desenvolvimento sexual, características seminais e hormonais em bubalinos no período peripuberal. Um total de 20 búfalos machos, não castrados, da raça Murrah (Bubalus bubalis) (12 meses de idade) foram distribuídos em dois tratamentos (bST vs controle) em um delineamento de blocos ao acaso com dez repetições. O peso corporal, escore de condição corporal, biometria testicular, características seminais e os níveis de IGF-I foram avaliados uma vez por mês durante oito meses. O ejaculado foi coletado por eletroejaculação e submetido a análise física e morfológica. A análise dos dados foi realizada utilizando o Statistical Analysis System for Windows, SAS®. Não houve diferença (P> 0,05) entre os tratamentos bST e Controle no peso e no escore de condição corporal. As mensurações testiculares também não apresentaram diferenças (P> 0,05) entre os tratamentos, mas aumentaram com a idade significativamente em uma tendência similar. Não houve efeito positivo do tratamento com bST sobre a idade ao aparecimento dos primeiros espermatozóides no ejaculado (P>0,05). O bST apresentou uma tendência significativa em antecipar a puberdade (P = 0,054). A motilidade, vigor e percentual de anormalidades espermáticas não apresentou diferença (P> 0,05) entre tratamentos. Não houve diferença (P> 0,05) entre os tratamentos bST e Controle nas concentrações séricas de IGF-I. Em conclusão, o uso de bST não resulta em efeitos positivos sobre o desenvolvimento ponderal e sexual em bubalinos machos no período peripuberal.

OBJETIVOS: Avaliar a eficácia de um meio de criopreservação sintético para sêmen humano contendo fosfolipídios e antioxidantes (ANTIOX-PL, Invitra/Supera Parque de Inovação e Tecnologia de Ribeirão Preto) em comparação com o meio convencional à base de gema de ovo (TEST-yolk buffer, Irvine Scientific), mensurada pelos parâmetros in vitro de motilidade progressiva dos espermatozoides e índice de fragmentação do DNA espermático. MÉTODOS: Ensaio clínico de não-inferioridade. Foram recrutados 63 homens (julho de 2015 a outubro de 2016), com idade entre 18 a 50 anos, amostra seminal com volume >= 1,5 mL, concentração de espermatozoides >= 15 x 106/mL e motilidade progressiva >= 32%. O sêmen foi dividido em duas alíquotas com volumes iguais, randomizadas aleatoriamente nos grupos estudo (ANTIOX-PC) e controle (TEST-yolk buffer) para receberem os meios de criopreservação. A avaliação dos desfechos foi cega para atribuição de grupo, verificados antes do congelamento e após o descongelamento. Os dados foram comparados pelo teste t pareado pelo SAS versão 9.3; ?=0,05. RESULTADOS: A motilidade progressiva (p=0.83) e o índice de fragmentação do DNA (p=0.32) analisados nas amostras de sêmen congeladas com o meio A (ANTIOXPL) não apresentaram diferença significativa em relação às que foram congeladas com o meio B (TEST-yolk buffer). A concentração (p=0.02), a concentração total (p=0.02), o percentual de espermatozoides I (p<0.01) e a vitalidade (p<0.01) foram superiores nas amostras congeladas com o meio B (TEST-yolk buffer). A motilidade não progressiva (p<0.01) e a motilidade total (p<0.01) foram superiores nas amostras congeladas com o meio A (ANTIOX-PC). E a morfologia (p=0.07) não apresentou diferença significativa entre os meios de criopreservação. CONCLUSÕES: Em relação aos desfechos primários analisados, motilidade progressiva dos espermatozoides e índice de fragmentação do DNA espermático, a nova formulação proposta neste projeto de pesquisa, ANTIOX-PC, mostrou-se não inferior ao meio convencional, TEST-yolk buffer. / OBJECTIVES: To evaluate the efficacy of a synthetic cryopreservation medium for human semen containing phospholipids and antioxidants (ANTIOX-PC, Invitra / Supera Parque de Inovação e Tecnologia de Ribeirão Preto) compared to conventional egg yolk medium (TEST-yolk buffer, Irvine Scientific), measured by the in vitro parameters of progressive sperm motility and sperm DNA fragmentation index. METHODS: Non-inferiority clinical trial. 63 men (July 2015 to October 2016), aged 18 to 50 years, seminal sample with volume >= 1,5 mL, spermatozoa >= 15 x 106/mL and progressive motility >= 32% were recruited. The semen was divided into two aliquots with equal volumes, randomly randomized in the study (ANTIOX-PC) and control (TEST-yolk buffer) groups to receive cryopreservation media. The evaluation of the outcomes was blind to group assignment, verified before freezing and after thawing. The data were compared by the t-test paired by SAS version 9.3; ? = 0.05. RESULTS: Progressive motility (p=0.83) and DNA fragmentation index (p=0.32) analyzed in the semen samples frozen with medium A (ANTIOX-PL) showed no significant difference in relation to those frozen with the medium B (TEST-yolk buffer). The concentration (p=0.02), the total concentration (p=0.02), the percentage of immobile spermatozoa (p<0.01) and vitality (p<0.01) were higher in the samples frozen with medium B TEST-yolk buffer). Nonprogressive motility (p<0.01) and total motility (p <0.01) were higher in samples frozen with medium A (ANTIOX-PC). And the morphology (p=0.07) showed no significant difference between the cryopreservation media. CONCLUSIONS: The new formulation proposed in this research project, ANTIOX-PC, was not inferior to the conventional medium, TEST-yolk buffer, in relation to the primary endpoints analyzed, sperm motile spermatozoa and sperm DNA fragmentation index.

Made available in DSpace on 2014-08-20T14:37:56Z (GMT). No. of bitstreams: 1 dissertacao_rafael_tonieto.pdf: 295673 bytes, checksum: cde6d39f839653a9d49de2dfbef8ef62 (MD5) Previous issue date: 2008-10-24 / The low pregnancy rates obtained with artificial insemination using frozen ram semen make its routine use unfeasible. As the cryopreservation process causes injuries in the sperm cells, the development of extenders that include state-of-the art cryoprotectant solutions is justified. This study tested the inclusion of low density lipoprotein (LDL) and trehalose in extenders for freezing ram semen, by evaluating parameters of post-thawing semen quality. In the first experiment, 3 treatments were compared: Tris including 20% egg yolk and 5% glycerol (T1); Tris including 10 mM trehalose (T2); and T1 including 50 mM trehalose (T3). Sperm motility did not differ across treatments (P > 0.05), but T2 presented higher proportion of sperm cells having membrane integrity (P < 0.05). In the second experiment, 4 treatments were compared: Tris including 20% egg yolk (T1); T1 including 5% glycerol (T2); T1 including 100 mM trehalose (T3); and t1 including 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2, T3 and T4 than for T1 (P < 0.05), but acrossome integrity did not differ among treatments (P > 0.05). In the third experiment, four treatments were compared: Tris including 20% egg yolk and 100 mM trehalose; and T1 with the replacement of egg yolk by LDL including 5% glycerol (T2); 100 mM trehalose (T3); and 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2 and T3 than for the other treatments (P < 0.05), but there was no further difference among the LDL treatments (P > 0.05). Acrossome integrity did not differ across treatments (P > 0.05). Therefore, the inclusion of LDL and trehalose as cryoprotectants in extenders for frozen ram semen was associated with improvement in post-thawing sperm motility and membrane integrity. / Em ovinos, as baixas taxas de prenhez obtidas com inseminação artificial com sêmen congelado inviabilizam o seu uso como rotina. Provocando o processo de criopreservação lesões nos espermatozóides, o desenvolvimento de diluentes que incluam crioprotetores de excelência se justifica. Este trabalho testou a inclusão da lipoproteína de baixa densidade (LDL) e da trealose em diluentes para congelamento de sêmen ovino, a partir da avaliação de parâmetros de qualidade seminal pós-descongelamento. No primeiro experimento, foram comparados 3 tratamentos: Tris com 20% de gema de ovo e 5% de glicerol (T1); Tris com 100 mM de trealose (T2); e T1 com 50 mM de trealose (T3). A motilidade não diferiu entre os tratamentos (P > 0,05), mas o T2 apresentou maior proporção de gametas com membranas íntegras (P < 0,05). No segundo experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo (T1); T1 com 5% de glicerol (T2); T1 com 100 mM de trealose (T3); e T1 com 100 mM de trealose e 5% de glicerol (T4). A motilidade e a integridade da membrana espermática do T2, T3 e T4 foram superiores a do T1 (P > 0,05), mas a integridade do acrossoma não diferiu (P > 0,05) em todos os tratamentos. No terceiro experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo e 110 mM de trealose (T1); Tris com 8% de LDL, incluindo 5% de glicerol (T2); Tris com 8% de LDL, incluindo 110 mM de trealose (T3); Tris com 8% de LDL, incluindo 110 mM de trealose e 5% de glicerol (T4). A motilidade dos espermatozóides para o T2 e para o T3 foram superiores aos demais tratamentos (P < 0,05), mas, com relação à integridade de membrana, não houve diferença entre os tratamentos com LDL (P > 0,05). A integridade do acrossoma não diferiu entre os tratamentos (P > 0,05). Portanto, o uso de LDL e trealose como crioprotetores foi associado com melhorias na motilidade e na integridade da membrana espermática, no sêmen ovino, após o descongelamento.

Made available in DSpace on 2014-08-20T14:38:49Z (GMT). No. of bitstreams: 1 tese_diones_bender_almeida.pdf: 702296 bytes, checksum: 79cdf77bf0f5d2b12aa8fc733442f59d (MD5) Previous issue date: 2012-02-27 / The Nile tilapia (Oreochromis niloticus) is one of the most significant species of the current fish farming. All knowledge related to the reproductive behavior of this species is important for defining some selection criteria as well as a better understanding of their reproductive characteristics. Therefore, this research aimed to evaluate the spermatic characteristics of the semen, together with the reproductive acting of females of three different strains of Nile tilapia (Supreme, Premium Aquabel and Chitralada). These evaluations are necessary before establishing genetic breeding program for the species, in order to determine what kind of selection criteria will be used. The collections of data related to the reproducers of both sexes were taken in the Aquabel Pisciculture Station, located in Rolândia/PR. Thirty males were used in the seminal evaluation and seventy five females in the reproductive evaluation of each strain. The reproduction of the females was observed for seven weeks through weekly collections. It was evaluated parameters as volume of eggs/spawing, number and weight of eggs/female, number of eggs/Kg of fish, hatching rates, percentage of females that had not spawned, porcentage of spawning/week, more than two spawning during the seven weeks, average of the spawnings in chronological order and the frequency of spawnings. Of the evaluated strains, the PA presented higher averages for all of the parameters morfometrics and for great majority of the appraised reproductive parameters. As the male influence is as important as for the female in the reproductive process, it was aimed to identify by certain variables, productive and reproductive indexes in the semen that could be selected. Among them it can be mentioned the pH, semen collection, velocity and type of movement, motility, concentration, spermatic viability and integrity of membrane, mitochondrial and DNA. Looking for to identify the line to gather the largest number of desirable characteristics for cultivation, SUP is highlighted by presenting better results for great majority of these parameters. All these variables, for both males and females, are fundamental because in the fertilization process it is necessary that they have good quality. After the choice of these variables it would be possible to identify, among the worked strains, the more appropriate ones for the implantation of a future genetic breeding program. / A tilápia do Nilo (Oreochromis niloticus) é uma das espécies mais significativas da piscicultura atual. Todo conhecimento relativo ao comportamento reprodutivo dessa espécie é importante para se definirem determinados critérios de seleção assim como uma melhor compreensão de suas características reprodutivas. Para isso, este projeto de pesquisa teve como objetivo avaliar as características espermáticas do sêmen, juntamente com o desempenho reprodutivo de fêmeas de três diferentes linhagens de tilápias do Nilo (Supreme, Premium Aquabel e Chitralada). Essas avaliações são necessárias antes de se estabelecer um programa de melhoramento genético para essa espécie, a fim de determinar que tipos de critérios de seleção serão utilizados. As coletas de dados relacionados aos reprodutores de ambos os sexos foram realizadas na Estação de Piscicultura Aquabel, localizada no município de Rolândia/PR. Foram utilizados 30 machos na avaliação seminal e 75 fêmeas na avaliação reprodutiva de cada linhagem. A reprodução das fêmeas foi realizada durante sete semanas através de coletas semanais, onde se avaliou parâmetros como volume de ovos/desova, número e peso de ovos/fêmea, número de ovos/Kg de peixe, taxa de eclosão, porcentagem de fêmeas que não desovaram, porcentagem de desovas/semana, mais de duas desovas durante as sete semanas, média das desovas em ordem cronológica e frequência de desovas. Das linhagens avaliadas a PA apresentou médias consideradas superiores para todos os parâmetros morfométricos e para grande maioria dos parâmetros reprodutivos avaliados. Como a influência do macho é tão importante quanto à da fêmea no processo reprodutivo, buscou-se identificar através de determinadas variáveis, índices produtivos e reprodutivos no sêmen, que pudessem ser selecionados. Dentre eles podemos citar o pH, coloração, velocidade e tipo de movimentação, motilidade, concentração, viabilidade espermática e integridades de membrana, mitocôndria e DNA. Buscando identificar a linhagem que reunisse o maior número de características desejáveis ao cultivo, a SUP se destacou por apresentar melhores resultados para grande maioria desses parâmetros. Todas essas variáveis, tanto nos machos como nas fêmeas, são fundamentais de serem estudadas porque no processo de fertilização é necessário que as mesmas apresentem boa qualidade. Somente após a escolha dessas variáveis é que podemos identificar, dentre as linhagens trabalhadas, aquela (s) mais adequada (s) para a implantação de um futuro programa de melhoramento genético.

Cryopreservation is used to freeze and store donor’s sperm, from men who are going throughcertain medical treatments such as chemotherapy or radiotherapy and from men withazoopermia or severe oligozoospermia. Stored sperm samples are used in artificialreproductive technologies. Formation of ice crystals is the biggest problem when freezingcells because of the risk severe damage to the cells. To guarantee optimal survival rates infrozen sperm samples, cryoprotectants which protect sperms from ice crystals formations, isadded.The main objective of the study was to compare two different freeze solutions and theirimpact on sperm survival in frozen sperm samples. In addition, an additional aim was todetermine if the amount of motile sperm changes when the samples is left for two hours inroom temperature.In this study, 31 samples were used. Each sample was divided into two groups. The firstgroup was mixed with a SpermFreeze Solution™ and the second group was mixed withSperm CryoProtectTM II. All samples were frozen using cooling vapor and stored in tankswith liquid nitrogen. The concentration of motile sperm was measured after thawing andpreparation. SpermFreeze SolutionTM showed significant better results in the number ofsperm who survived after freezing and thawing process Also, the results showed that werewas no significant difference on concentration of motile sperm in samples after being left atroom temperature for two hours. In summary, an improvement in the process of preparationrecommends to be performed to reduce the mechanical stress to ensure a greater quantity ofmotile sperm after the whole process.

Submitted in partial fulfillment of the requirements for the degree of Master of Health Sciences in Clinical Technology, Durban University of Technology, Durban, South Africa, 2017. / Introduction The prevalence of infertility is increasing at an alarming rate globally. Many couples are afflicted with infertility due to an array of diseases, trauma and psychological stresses. Renal disease is one such pathophysiological condition which is increasing amongst the younger age group. Often the progression of chronic renal disease leads to end stage renal failure that requires a renal transplantation. Post renal transplant, immunosuppressive agents are routinely prescribed to prevent allograft rejection. Immunosuppressive agents are potent drugs that can have deleterious side effects on semen parameters. However, the effects of the immunosuppressive agents on semen parameters in the literature are unclear and require further investigation. It is, therefore, important to assess the effects of immunosuppressive agents on semen, especially the three vital aspects of sperm concentration, motility and morphology which form the basis of male reproduction. Aims and Objectives of study This was a prospective observational study evaluating the effects of different immunosuppressive regimens on sperm parameters in post renal transplant male patients. The main aspects of semen parameters such as sperm concentration, motility and morphology that determine reproductive potential were assessed in the study patients and compared to the gold standard of semen analysis according to the World Health Organisation (WHO) reference values. Methodology Thirty-four renal transplant patients were recruited from the databases of both private nephrologists in the greater Durban area and the academic renal unit at Inkosi Albert Luthuli Central Hospital. Following bioethical approval and informed consent, patients were required to produce a semen sample by masturbation. A questionnaire documenting the patient’s lifestyle, aetiology of renal disease, transplant date and immunosuppressive duration and regimen were recorded. The semen samples were analysed comprehensively according to the protocol on semen analysis recommended by the WHO. This included the macroscopic investigation (volume, appearance, colour, viscosity, liquefaction time and pH) and microscopic evaluation (sperm concentration, total motility, morphology, IgG/IgA and vitality). Sperm concentration, total motility, morphology and vitality were examined and recorded in duplicate to strengthen the validity of the results. A biostatistician analysed the data and determined the statistical analysis. Descriptive statistics determined values of semen parameters in renal transplanted males and in each race demographic. The one sample t-test analysed the statistical significance between the mean study values and the WHO reference values. The effect of the immunosuppressive agent on semen parameters was determined using multiple linear regressions whilst ROC analysis determined the sensitivity and specificity of sperm concentration, total motility and morphology in predicting pregnancy from the patients that fathered children post renal transplant. Results The mean sperm concentration and morphology in the study patients were 14.0 mill/ml (95% Confidence Interval (CI) 10.2 – 17.7) and 3.3% (95% CI 2.7 – 3.9), respectively. Although values obtained were minimally lower than the WHO reference values, these results were within the 95% CI of the WHO guidelines. Motility evaluation revealed higher values of 43.2% (95% CI 36.6 – 49.7). In contrast, sperm vitality was considerably decreased, 47.5% (95% CI 40.6 – 54.4). All semen parameters exhibited no statistical significance (one sample t-test) when analysed against the WHO reference values except for sperm morphology, (p = 0.025; p< 0.05) which showed decreased morphology irrespective of immunosuppressive regimen. Semen volume 1.7 ml (95% CI 1.3 – 2.0) and pH 7.7 (95% CI 7.6 – 7.9) were both within the WHO guidelines. Descriptive statistics according to racial demographics showed no differences in semen values. An almost perfect linear relationship existed between total sperm motility and vitality (r = 0.967). Multiple linear regressions of duration and dosages of immunosuppressive drugs tacrolimus and mycophenolate mofetil, could not predict the effect of the immunosuppressive agents on sperm concentration, total motility and morphology. There was a significant difference in morphology between those with and without children post renal transplant. Those with children post renal transplant exhibited a higher morphology value, (p = 0.001; p< 0.05). Sensitivity and specificity analysis of the patients with children post renal transplant concluded that morphology is the most optimal indicator and predictor of pregnancy (AUC = 0.854). Tacrolimus was the common immunosuppressive agent used in the four patients that fathered children. This was more evident in patients that underwent therapy with Sirolimus followed by Cyclosporin A (CsA) and changed to Tacrolimus as the last immunosuppressive agent used for maintenance therapy. Conclusion The ability to procreate in renal transplanted males has become increasingly difficult and emotionally challenging. In this study sperm concentration and morphology of renal transplanted males exhibited parameters similar to the general fertile population. Total motility possessed a higher range of values in contrast to sperm vitality which showed a significant decrease from the WHO reference values. The effect of immunosuppressive treatment on semen parameters could not be clearly defined due to the number of immunosuppressive regimens that patients were subjected to intermittently resulting in small sample sizes within each immunosuppressive regimen grouping. The majority of patients underwent a triple maintenance therapy of tacrolimus, MMF and prednisone. The dosage and duration of these tacrolimus and MMF was inconclusive in determining a beneficial or detrimental relationship on semen parameters. Morphology was shown to be the most significant indicator in predicting pregnancy in patients that fathered children. Tacrolimus was a common immunosuppressive agent used in the majority of patients that fathered children. It may have protective effects on sperm parameters as shown in patients that fathered children. This was a study with a small sample size and further investigations are required in a larger cohort of patients to assess individualized effects of the different immunosuppressive agents on sperm parameters. / M

O uso de protocolos de diluição bitérmica está aumentando em centros de inseminação artificial e é necessário garantir que as doses de inseminação tenham a mesma qualidade quando comparado com protocolos de diluição isotérmica. Quatro ejaculados de cada 19 reprodutores PIC® foram coletados e distribuídos em split sample em três tratamentos: diluição bitérmica em duas etapas (T1), diluição isotérmica em duas etapas (T2) e diluição isotérmica em uma etapa (T3). A curva de temperatura para os três tratamentos foi feita utilizando um data logger com sensor de temperatura. As doses inseminantes foram preparadas utilizando o diluente BTS e armazenadas a 16ºC e usadas para avaliação dos parâmetros espermáticos através do Sistema CASA e avaliação de morfologia espermática, durante 120 horas. A temperatura das amostras de sêmen submetidas à diluição bitérmica em duas etapas alcançou 24,1ºC durante 120 minutos , enquanto que as amostras submetidas à diluição isotérmica em uma ou duas etapas alcançou 26,8ºC e 27,0ºC, respectivamente. A motilidade total, a progressiva e BCF foram influenciadas (P<0,05) pelo tempo de diluição, mas não pelo protocolo de diluição. A motilidade total e progressiva diminuiu com o tempo de armazenamento (91.0 ± 0.91 para 81.5 ± 1.08 % e 74.0 ± 2.48 para 60.4 ± 2.59% de 24h para 120h, para MOT e PROG, respectivamente) enquanto BCF diferiu entre 24 e 120h (28.6 ± 0.76 e 27.3 ± 0.79 Hz). As seguintes características de motilidade não foram afetadas pelo protocolo de diluição e pelo tempo de armazenamento: DAP, DCL, DSL, VAP, VCL, VSL, STR, LIN, WOB e ALH. Às 72 h de armazenamento, a morfologia espermática não diferiu entre os tratamentos (P>0.05), apresentando uma média geral de 9.2 ± 0.36 defeitos totais. Concluindo, a diluição bitérmica torna o processo de produção de doses inseminantes mais rápido, pois demoraram menos tempo para alcançar a temperatura próxima de armazenamento, sem comprometer a qualidade das doses inseminantes produzidas. / The use of bithermic dilution protocols is increasing in artificial insemination centers, being necessary to guarantee that the quality of insemination doses remain the same when compared to isothermic dilution protocols. Four ejaculates from each one of 19 crossbreed PIC® boars were collected and assigned, in a split sample design, in to three treatments: two step bithermic dilution (T1), two step isothermic dilution (T2) and one step isothermic dilution (T3). Temperature curve for the three treatments was recorded using a temperature sensor data logger. Semen doses prepared with BTS extender were stored at 16°C and were used to evaluate sperm parameters through CASA system and sperm morphology, during 120 h. The temperature in semen samples submitted to a two-step bithermic dilution reached 24.1ºC during 120 min, whereas one or two-step isothermic dilution samples reached 26.8ºC and 27.0ºC, respectively. Total motility, progressive motility and BCF were influenced (P<0.05) by the storage time but not by the dilution procedure. Total and progressive motility reduced throughout the storage time (91.0 ± 0.91 to 81.5 ± 1.08 % and 74.0 ± 2.48 to 60.4 ± 2.59% from 24h to 120h, for MOT and PROG respectively) whereas BCF differed between 24 and 120h (28.6 ± 0.76 and 27.3 ± 0.79 Hz). The following motility traits were neither affected by the dilution procedure nor by the time of storage: DAP, DCL, DSL, VAP, VCL, VSL, STR, LIN, WOB, and ALH. At 72 h of storage, sperm morphology was not different among treatments (P>0.05), showing an overall mean of 9.2 ± 0.36 total defects. In conclusion, the bithermic dilution makes the process of artificial insemination doses production faster by taking less time to reach a temperature close to that of storage, without impairing semen quality.

A capacidade de preservar a viabilidade do sêmen pelo emprego das biotecnologias, como refrigeração e criopreservação, oferece muitas vantagens a equideocultura. O estresse oxidativo é um dos principais entraves para estas biotecnicas e advêm do ataque de distintas espécies reativas de oxigênio (EROs). Assim, o objetivo do presente estudo foi verificar o impacto das diferentes EROs sobre os espermatozoides com e sem plasma seminal. Foram colhidos ejaculados de 13 garanhões Mangalarga Marchador de fertilidade conhecida. Os ejaculados foram divididos em duas frações (A e B). As frações foram centrifugadas (2200g/10min) e o pellet com os espermatozoides foi ressuspendido em solução salina fisiológica ou plasma seminal de modo que as duas frações apresentassem a mesma concentração final (sptz/mL). Ambas as frações (A e B) tiveram 4 alíquotas de 400&micro;L retiradas e submetidas a 3 sistemas distintos de produção de EROs (xantina + xantina oxidase que produz anion superóxido; peróxido de hidrogênio; ferro + vitamina C que produz radical hidroxil) e malondialdeído. Após a incubação, as amostras A e B foram avaliadas de acordo com os testes funcionais (coloração de eosina-nigrosina para membranas, fast-green/rosa bengala para acrossomos, coloração 3-3&#39;diaminobenzidina para atividade mitocondrial e SCSA&trade; para susceptibilidade a denaturação de cromatina) e avaliação do índice de peroxidação lipídica (TBARs).Todas as análises estatísticas foram realizadas pelo programa SAS System for Windows. A susceptibilidade do espermatozoide equino ao estresse oxidativo é variável de acordo com a espécie reativa de oxigênio empregada na incubação; as espécies mais deletérias no presente experimento foram a malondialdeído e principalmente o radical hidroxil o que já era esperado. Houve um efeito benéfico quanto a manutenção das funções celulares e status oxidativo em amostras cujo plasma seminal foi preservado. A proteção atribuída ao plasma seminal influenciou parâmetros como atividade mitocondrial, susceptibilidade a denaturação acida da cromatina e índice de peroxidação lipídica. O plasma seminal exerceu mais pronunciada proteção aos eventos desencadeados pela malondialdeído / Biotechnologies such as chilling and cryopreservation of equine semen have been largely used in horse breeding due to its importance on storage and propagation of genetic material. The oxidative stress is one of the main causes of poor results related to the fertility of processed stallion sperm. Oxidative injuries are mainly due to the attack of different reactive oxygen species (ROS). Thus, the aim of the present study was to evaluate the impact of distinct ROS attack on equine spermatozoa with and without seminal plasma. The collection of one ejaculate of thirteen Mangalarga fertile stallions were performed. Ejaculates were divided in two aliquots (A and B). These aliquots were centrifuged (600G /10 min) and pellets were resuspended in saline solution (0,9%) or seminal plasma certifying that each sample had the same concentration (sptz/mL). Both aliquots (A and B) were divided in four samples of 400 &micro;L each and incubated with four ROS generating systems (superoxide anion; hydrogen peroxide; hydroxil radical and malondialdehyde). After the 30 incubation, all the samples were evaluated for membrane integrity (eosin-nigrosin stain), acrosome integrity (simple stain of fast-green/ Bengal rose), 3-3&#39;diaminbenzidine for mitochondrial activity and SCSA&trade; for the chromatin suceptibility to the acid denaturation. Lipid peroxidation was accessed by TBARs assay. Statistical analysis was performed using SAS System for Windows. The susceptibility of equine sperm to the oxidative stress is different in each ROS generation system; but the most deleterious ROS were hydroxyl radical and malondialdehyde. A positive effect of seminal plasma on the sperm viability was observed which may be linked to its protective role. The protection occurred in mitochondria, chromatin and against lipid peroxidation. Higher beneficial effect of seminal plasma was observed in samples treated with malondialdehyde

OBJETIVOS: Avaliar a eficácia de um meio de criopreservação sintético para sêmen humano contendo fosfolipídios e antioxidantes (ANTIOX-PL, Invitra/Supera Parque de Inovação e Tecnologia de Ribeirão Preto) em comparação com o meio convencional à base de gema de ovo (TEST-yolk buffer, Irvine Scientific), mensurada pelos parâmetros in vitro de motilidade progressiva dos espermatozoides e índice de fragmentação do DNA espermático. MÉTODOS: Ensaio clínico de não-inferioridade. Foram recrutados 63 homens (julho de 2015 a outubro de 2016), com idade entre 18 a 50 anos, amostra seminal com volume >= 1,5 mL, concentração de espermatozoides >= 15 x 106/mL e motilidade progressiva >= 32%. O sêmen foi dividido em duas alíquotas com volumes iguais, randomizadas aleatoriamente nos grupos estudo (ANTIOX-PC) e controle (TEST-yolk buffer) para receberem os meios de criopreservação. A avaliação dos desfechos foi cega para atribuição de grupo, verificados antes do congelamento e após o descongelamento. Os dados foram comparados pelo teste t pareado pelo SAS versão 9.3; ?=0,05. RESULTADOS: A motilidade progressiva (p=0.83) e o índice de fragmentação do DNA (p=0.32) analisados nas amostras de sêmen congeladas com o meio A (ANTIOXPL) não apresentaram diferença significativa em relação às que foram congeladas com o meio B (TEST-yolk buffer). A concentração (p=0.02), a concentração total (p=0.02), o percentual de espermatozoides I (p<0.01) e a vitalidade (p<0.01) foram superiores nas amostras congeladas com o meio B (TEST-yolk buffer). A motilidade não progressiva (p<0.01) e a motilidade total (p<0.01) foram superiores nas amostras congeladas com o meio A (ANTIOX-PC). E a morfologia (p=0.07) não apresentou diferença significativa entre os meios de criopreservação. CONCLUSÕES: Em relação aos desfechos primários analisados, motilidade progressiva dos espermatozoides e índice de fragmentação do DNA espermático, a nova formulação proposta neste projeto de pesquisa, ANTIOX-PC, mostrou-se não inferior ao meio convencional, TEST-yolk buffer. / OBJECTIVES: To evaluate the efficacy of a synthetic cryopreservation medium for human semen containing phospholipids and antioxidants (ANTIOX-PC, Invitra / Supera Parque de Inovação e Tecnologia de Ribeirão Preto) compared to conventional egg yolk medium (TEST-yolk buffer, Irvine Scientific), measured by the in vitro parameters of progressive sperm motility and sperm DNA fragmentation index. METHODS: Non-inferiority clinical trial. 63 men (July 2015 to October 2016), aged 18 to 50 years, seminal sample with volume >= 1,5 mL, spermatozoa >= 15 x 106/mL and progressive motility >= 32% were recruited. The semen was divided into two aliquots with equal volumes, randomly randomized in the study (ANTIOX-PC) and control (TEST-yolk buffer) groups to receive cryopreservation media. The evaluation of the outcomes was blind to group assignment, verified before freezing and after thawing. The data were compared by the t-test paired by SAS version 9.3; ? = 0.05. RESULTS: Progressive motility (p=0.83) and DNA fragmentation index (p=0.32) analyzed in the semen samples frozen with medium A (ANTIOX-PL) showed no significant difference in relation to those frozen with the medium B (TEST-yolk buffer). The concentration (p=0.02), the total concentration (p=0.02), the percentage of immobile spermatozoa (p<0.01) and vitality (p<0.01) were higher in the samples frozen with medium B TEST-yolk buffer). Nonprogressive motility (p<0.01) and total motility (p <0.01) were higher in samples frozen with medium A (ANTIOX-PC). And the morphology (p=0.07) showed no significant difference between the cryopreservation media. CONCLUSIONS: The new formulation proposed in this research project, ANTIOX-PC, was not inferior to the conventional medium, TEST-yolk buffer, in relation to the primary endpoints analyzed, sperm motile spermatozoa and sperm DNA fragmentation index.

Thirty-six half-sibling Angus bulls were assigned one of three diets: 1) 60% corn-based (CON; S = 0.18%; n = 12); 2) 60% DDGS replacing corn (60DDGS; S = 0.55% DM; n = 12); 3) CON diet + equivalent sulfur of 60DDGS added as calcium sulfate (SULF; S = 0.54%; n = 12) to evaluate the effects of feeding diets containing DDGS or calcium sulfate on performance and semen characteristics. Bulls began the study at 9 months of age and gained 1.6 kg/day for 112 days. Treatment by day interactions (P < 0.05) were observed for glutathione peroxidase and trace mineral concentrations in seminal plasma. Effects of treatment (P < 0.05) were observed for semen kinematics and triiodothyronine in serum. Alterations observed when feeding 60% DDGS to developing bulls occurred in a manner that is not dependent on dietary sulfur; therefore, observed changes could be related to other components within DDGS.