Tesi sul tema "Semen"

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Nas últimas décadas, muitos estudos têm sido realizados nas diferentes espécies com o intuito de se determinar os fatores envolvidos no processo da fragmentação do DNA espermático. A biologia molecular voltada à área da reprodução tem resultado em inúmeras técnicas para a avaliação da qualidade da cromatina e determinação da fragmentação do DNA espermático. Em trabalhos recentes, um novo teste denominado Halomax® tem se mostrado tão eficiente quanto a outros tradicionais para a avaliação da fragmentação do DNA espermático, apresentando como vantagens a sua praticidade e o fato não requerer o uso de equipamentos de alto custo. O presente experimento teve por objetivo comparar as raças Mangalarga Marchador (MM) e Quarto de Milha (QM) quanto à resistência à refrigeração, bem como utilizar o Teste Dispersão de Cromatina Espermática denominado Halomax® para avaliar a fragmentação da cromatina espermática do sêmen refrigerado dos garanhões dessas raças em duas temperaturas de armazenamento de sêmen refrigerado (5°C e 15°C). Os resultados deste trabalho demonstraram que não houve diferença entre as temperaturas de armazenamento (5°C e 15°C), mostran do que ambas são eficientes na manutenção da viabilidade espermática pelo período de 24 horas para ambas as raças estudadas. Além disso, foi observada uma característica espermática superior dos garanhões da raça Quarto de Milha quando comparada aos da raça Mangalarga Marchador, tanto nos parâmetros de velocidade espermática, avaliado pelo CASA, quanto com relação à fragmentação do DNA espermático, revelando uma maior sensibilidade dos animais da raça Mangalarga ao processo de refrigeração do sêmen
Many studies have been conducted in different species in order to determine the factors involved in the process of sperm DNA fragmentation. Molecular biology studies focused on the reproductive area has resulted in several techniques for assessing the quality of chromatin and sperm DNA fragmentation. Recently, a new test called HALOMAX ® has been shown to be as efficient as other traditional assessment of sperm DNA fragmentation, having the advantage of practicality and not require the use of expensive equipment. The aim of this study was to compare breeds Mangalarga and Quarter Horses for cooling resistance and use the Sperm Chromatin Dispersion Test called HALOMAX ® to evaluate the sperm chromatin fragmentation using two cooled semen storage systems (5°C and 15°C). Our results showed no difference between storage systems (5°C and 15°C), showing that both are effective in maintaining sperm viability for a period of 24 hours for both breeds studied. In addition, the Quarter Horse shown superiority in semen quality parameters assessed by CASA and sperm DNA fragmentation when compared with the Mangalarga, revealing higher sensitivity of the Mangalarga stallions for cooling semen

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Universidade Estadual Paulista (UNESP)
Objetivou-se estudar os efeitos do extrato de lipoproteínas de baixa densidade sobre o sêmen ovino durante a refrigeração. Vinte amostras de 5 carneiros foram refrigeradas por 24 e 48 horas na geladeira para refrigeração Minitube, nos meios diluentes Glicina Gema Leite, Glicina Gema purificada Leite e Glicina Extrato Leite e submetidas a teste de exaustão (37ºC/240 minutos) sendo avaliados in vitro por meio das análises da cinética espermática computadorizada, da morfologia e da integridade da membrana plasmáticas. Após 24 e 48 horas de refrigeração, os meios Glicina Gema purificada Leite e Glicina Extrato Leite apresentaram resultados superiores ao meio Glicina Gema Leite, após o teste de exaustão, para o parâmetro de integridade de membrana plasmática. Para a integridade de acrossomo o meio Glicina Gema purificada Leite foi superior (P<0,05) em relação ao meio Glicina Gema Leite durante o teste de exaustão. Nos demais parâmetros estudados de cinética espermática e morfologia (cauda dobrada), não houveram diferenças significativas (P>0,05) entre os meios. Entre os momentos, houve diferença significativa (P<0,05) em todos os meios durante o teste de exaustão
The objective was to study the effects of extract of low density lipoproteins on ovine semen during cooling. Twenty samples of five sheep were chilled for 24 and 48 hours in the refrigerator for cooling Minitube, in extenders Glycine Yolk Milk, Glycine purified Yolk Milk and Glycine Extract Milk and tested to exhaustion (37 ° C/240 min) were evaluated in vitro by means of computerized analysis of sperm kinetics, morphology and plasma membrane integrity. After 24 and 48 hours of refrigeration, the extenders Glycine purified Yolk Milk and Glycine Extract Milk showed better results than extender Glycine Yolk Milk, after the exhaustion test, for the parameter of membrane integrity. For the integrity of the acrosome through Glycine purified Yolk Milk was higher (P <0.05) than Glycine Yolk Milk during the exhaustion test. In other parameters of sperm kinetics and morphology, there were no significant differences (P> 0.05) among extenders. Between times, significant difference (P <0.05) in all extenders during the exhaustion test

Bibliography: leaves 267-292. This study aimed at finding better ways of storing ram semen at refrigerator or room temperature with particular reference to ingredients readily available in Indonesia, namely coconut extract and quail egg yolk. Coconut extract showed consistent advantages with regard to sperm motility and quail egg yolk was as effective as hen egg yolk. Investigations were extended to examine storage for subnormal semen such as would be produced during periods of heat stress. Motility was assessed visually and using a Hamilton Thorn semen evaluation apparatus.

L’enfocament que es proposa en aquesta tesi es basa en el fet que hi ha un buit d’informació sobre l’optimització de l’examen de la solidesa reproductiva juntament amb els protocols d’anàlisi de semen en els equids, tal com es va veure al capítol anterior. A l’ase, no es van definir protocols específics per a l’anàlisi de semen, només per a mostres de sementals on hi ha un acord general sobre el protocol a utilitzar. Quan es comprova el tracte reproductor, s’utilitza una ecografia en mode B com a mètode de diagnòstic fàcil i no invasiu. D’altra banda, es pot utilitzar una ecografia Doppler polsada per estudiar la perfusió testicular de sang. Els índexs de Doppler pulsat de l’artèria testicular i les mides ASG van mostrar una correlació positiva amb la qualitat del semen en sementals i diferents espècies. Malauradament, no es van fer estudis d’aquest tipus en ruc, cosa que constitueix el primer objectiu de la tesi. No obstant això, quan es tracta d’anàlisi de semen, la tecnologia CASA representa el millor mètode objectiu que s’utilitza avui en dia. Permet obtenir dades quantitatives fiables, fins i tot si és necessari definir protocols que assegurin la consistència i l’aplicació universal dels resultats. Tanmateix, aquesta estandardització mai s’ha fet seguint un punt de vista integrador, que compon el segon objectiu general. Hi ha tres aspectes principals a tenir en compte a l’hora d’optimitzar les anàlisis automatitzades de semen mitjançant la tecnologia CASA-Mot, a saber, el tipus, la profunditat de la cambra de comptatge, la dilució i la velocitat de fotogrames d’adquisició d’imatges. Els avenços tecnològics van permetre ara l’adquisició d’alta freqüència de captura i, al mateix temps, el procés d’aquestes imatges en segons. A més, es va definir l’estructura de les subpoblacions d’espermatozoides en marcs més alts i la distribució SP entre les races dels sementals. El tercer objectiu era col·laborar en l’ús i la millora de la nova tècnica Trumorph®, per a l’anàlisi de morfologia de l’esperma que permet l’anàlisi d’espermatozoides immobilitzats vius.
El enfoque defendido en esta tesis se basa en el hecho de que existe un vacío de información con respecto a la optimización del examen de solidez reproductiva junto con los protocolos de análisis de semen en équidos, como se vio en el capítulo anterior. En burro, no se definieron protocolos específicos para el análisis de semen, solo para muestras de sementales donde existe un acuerdo general sobre el protocolo a utilizar. Cuando se comprueba el tracto reproductivo, la ecografía en modo B se utiliza como un método de diagnóstico fácil y no invasivo. Por otro lado, la ecografía Doppler pulsado se puede utilizar para estudiar la perfusión sanguínea testicular. Los índices de Doppler pulsado de la arteria testicular y los tamaños de ASG mostraron una correlación positiva con la calidad del semen en sementales y diferentes especies. Lamentablemente, estos estudios no se realizaron en burro, lo que constituye el primer objetivo de la tesis. Sin embargo, cuando se trata de análisis de semen, la tecnología CASA representa el mejor método objetivo utilizado en la actualidad. Permite obtener datos cuantitativos confiables, incluso si es necesario para definir protocolos asegurando la consistencia y aplicación universal de los resultados. Sin embargo, esta estandarización nunca se ha hecho siguiendo un punto de vista integrador, que compone el segundo objetivo general. Hay tres aspectos principales a considerar al optimizar los análisis de semen automatizados mediante la tecnología CASA-Mot, a saber, el tipo, la profundidad de la cámara de recuento, la dilución y la velocidad de cuadros de la adquisición de imágenes. Los avances en tecnología permitieron ahora la adquisición de alta frecuencia de captura y al mismo tiempo el proceso de estas imágenes en segundos. Además, se definió la estructura de las subpoblaciones de espermatozoides en cuadros más altos y la distribución de SP entre las razas de sementales. El tercer objetivo fue colaborar en el uso y la mejora de la nueva técnica Trumorph®, para el análisis de la morfología espermática que permite el análisis de espermatozoides inmovilizados vivos.
The approach advocated in this thesis is based on the fact that there is a gap of information regarding optimization of breeding soundness examination along with semen analysis protocols in equids as seen in the previous chapter. In donkey, no specific protocols for semen analysis were defined, only for stallion samples where there is a general agreement about the protocol to use. When the reproductive tract is checked, B-mode ultrasonography is used as a non-invasive and easy diagnostic method. On the other hand, pulsed Doppler ultrasonography can be used to study testicular blood perfusion. Pulsed-Doppler indices of testicular artery and ASG sizes showed a positive correlation with semen quality in stallion and different species. Unfortunately, no such studies were done in donkey, which makes up the first objective of the thesis. However, when it’s about semen analysis, CASA technology represents the best objective method used today. It permits to obtain reliable quantitative data, even if it is needed to define protocols assuring the consistency and universal application of the results. However, this standardization has never been done following an integrative point of view, which composes the second general objective. There are three main aspects to consider when optimizing automated semen analyses by CASA-Mot technology, namely the type, depth of the counting chamber, the dilution, and the frame rate of image acquisition. The advances in technology allowed now the acquisition of high capture frequency and at the same time the process of these images in seconds. Also, was defined sperm subpopulations structure at higher frames and SP distribution between stallions’ breeds. The third objective was to collaborate in the use and the improvement of the new Trumorph® technique, for sperm morphology analysis which permits the analysis of alive immobilized sperm.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

Two successive ejaculates were collected from six mature Holstein bulls at 3 d intervals for 7 wks. Elevated testicular temperature was induced by complete coverage of the scrotum with insulated material for 48 h. Viability (motility and acrosome integrity) and morphological characteristics of sperm before and after thermal insult were examined. For assessment of results, collection days were grouped: days -6, -3, 0 = Period 1 (d 0 = day of testis coverage after semen collection on that day), days 3, 6, 9 = Period 2 , days 12, 15...39 = Period 3. Semen was cryopreserved on each day of collection until morphological abnormalities of sperm increased to >50%. Semen viability before and after freezing was lower in Period 3 than in Period 1 (P≤.01). These differences coincided with increased abnormal morphology. No differences in viability were observed between Period 1 and Period 2 for unfrozen semen. Once frozen, spermatozoa ejaculated during Period 2 were significantly different from Period 1 for both viability measurements, but only after 3 h incubation at 37°C (P≤.01). Mean percent pre-insult abnormal sperm level was 19.6 ± 5.7 and sperm morphology in Period 1 (pre-insult) did not differ from that in Period 2. Morphological change was first noted in Period 3 on d 12 and 15 (47.5 ± 27.4 and 65.0 ± 27.0 % abnormal sperm, respectively). Abnormal sperm peaked on d 21 (83.2 ± 22.8 %). Although bulls varied in degree and time of response post-insult, all bulls exhibited the same sequence of appearance for specific abnormalities. The sequence and peak means for these abnormalities observed over all bulls were as follows: decapitated sperm, d 15 (33.9 ± 28.8 %); diadem defect, d 18 (55.6 ± 25.8 %); pyriform heads and nuclear vacuoles (excluding diadems), d 21 (18.3 ± 17.6 and 20.8 ± 10.5 %, respectively); knobbed acrosomes, d 27 (11.6 ± 13.6 % ). Sperm morphology was followed through d 39, by which time all bulls were producing ≤50% abnormal cells (35.2 ± 8.0 %). We concluded that viability of epididymal/rete sperm was adversely affected by elevated testicular temperatures, as noted by lowered viability of cryopreserved semen, and that there is a sequence in appearance of abnormal cell types in repsponse to thermal insult of the testis.
Master of Science

The aim of the current study was to determine if frozen-thawed bull semen can be treated with the acidic buffer MES (2-[N- morpholino] ethanesulfonic acid) without any detrimental effect on the motility, plasma membrane, acrosomal membrane and longevity of sperm. Frozen bull semen was obtained from a local co-operative. The semen was frozen in 0.25 mL French straws at a concentration of 80 x 106 sperm cells per millilitre. Semen of two different batches from ten bulls of four different breeds was used in this study. Three frozen semen straws of each batch were thawed at 38° C for 25 seconds. The thawed semen was pooled and then split into two aliquots. The one aliquot was used as control, whilst the other was exposed to MES treatment. The motility, plasma membrane integrity, acrosomal membrane integrity and longevity of sperm were evaluated. The effect of MES on motility was minimal as only the percentage of aberrantly motile sperm increased two hours after treatment. Although no effect on the plasma membranes were observed, it can be assumed that some damage did occur due to the fact that the acrosomal membranes were affected significantly. No significant effect was found for longevity of sperm between the control and treated samples, but a significant effect was found for both the control and treated samples over time. Although the detrimental effects caused by MES treatment would render some sperm unable to fertilise an oocyte, it is likely that a sufficient portion of sperm would survive the treatment. It is probable that this treatment would also be effective in frozen-thawed buffalo semen. The following step would be to treat semen of footand-mouth disease positive bulls with MES to establish if treatment with MES will be effective in inactivating foot-and-mouth disease virus in semen of infected bulls. Copyright
Dissertation (MSc (Veterinary Science))--University of Pretoria, 2008.
Production Animal Studies
unrestricted

The object of this thesis was to determine the independent predictive value of semen parameters obtained from the conventional analysis of semen by the methods advocated by the World Health Organisation in 1987, and of the descriptive semen categories also advocated by the World Health Organisation in 1987. The laboratory error in assessing sperm motility, density and morphology was quantified, and minimised by examining the predictive value of semen analyses performed by one technician. It was determined whether it is important to employ stringent criteria for recruiting fertile controls, by comparing semen parameters obtained from 'fertile' men who were recruited by two different methods. The relationship between demographic and clinical female factors and the cumulative conception rate was studied. The relationship between semen parameters and fertility was studied by two different methods. Firstly 'fertile' controls were compared to men who had attended an infertility clinic who were grouped by details of their partners. Secondly the relationship between semen parameters and the cumulative conception rate was studied after having considered known female factors. The relationship between information obtained from the history and physical examination of the male partner and the cumulative conception rate was studied, and it was determined whether semen analysis furnished predictive information independent of these features. This study also attempted to determine how many semen samples should be examined for optimum predictive value.

Artificial insemination (AI) is a widely used part of the modern agricultural industry, with the number of animals inseminated globally being measured in the millions per anum. Crucial to the success of AI is that the sperm sample used is of a high Quality. Two factors which determine the quality of the sample are the number of sperm present and their motility. There are numerous methods used to analyse the quality of a sperm sample, but these are generally laboratory based, expensive and in need of a skilled operator to perform the analysis. It would, therefore be useful to have a simple and inexpensive system which could be used outside the laboratory, immediately prior to the insemination of the animal. Presented in this thesis is work developing a time of flight (ToF) technique which makes use of a quartz crystal microbalance (QCM), operating at 5 MHz, as the sensing element. Data is shown developing a device where a 50 μl sample of boar sperm is added to a liquid filled swim channel, which the sperm are allowed to self-propel down and attach to the surface of a QCM at the end. The attachment of the sperm to the surface causes a measurable frequency decrease in the QCM, aproximately 50 Hz. An average effective mass measurement was made using a QCM and gave a value of 8 ± 5 pg per sperm, which was used in conjunction with the frequency change to determine the number rate of sperm reaching the QCM. Additional data is presented to investigate the effect of environmental temperature on the ToF of the sperm, showing a decrease in ToF between 23 0C to 37 0C. The system was also used to investigate increasing the swim speed of the sperm by chemical means. A range of 20 μmol to 100 μmol of progesterone was added to the swim medium and the ToF was shown to decrease as a result. To further develop the system, large commercial electronics were replaced by smaller circuits built in-house. An oscillator circuit based on a Pierce oscillator was used to drive the QCM and a frequency counter circuit making use of a universal frequency to digital converter (UFDC-1) was used to measure the frequency of the QCM. ToF experiments were performed which showed these pieces of equipment to be effective for performing the analysis of sperm samples. The swim cell itself was also refined, resulting in a compact, modular design. Work was performed developing layer-guided, single-port acoustic resonators to replace the QCM as the sensing element in the sperm analysis device. A maximum mass sensitivity of 1110 Hzμg-1cm-2 was found for devices on a LiTaO3 substrate with a 6 μm guiding layer. While viscosity-density sensing experiments found a maximum sensitivity of 488 KHz Pa-1/2 kg1/2 for a 4 μm guiding layer.

Includes bibliographical references
This study aims to address the lack of data on the link between BMI and infertility in the South African population by describing the prevalence of male overweight and obesity in a group of men undergoing infertility investigation, as well as assessing any semen analysis abnormalities in these groups. It also aims to describe how well men can predict their BMI category and determine whether weight loss would be an acceptable part of infertility management in overweight or obese male partners. Beliefs surrounding healthy weight and fertility will also be addressed.

A theoretical study showed that Al can greatly affect the efficiency of sheep breeding schemes provided fertility is maintained at the highest levels. Factors that affect the survival of ram spermatozoa during preservation were studied. A pH range between 6 and 7 was well tolerated. The addition of 4% (v/v) glycerol to the diluted ram semen in Tris buffer lowered the motility and survival of spermatozoa during 5 hours of storage at 30'C. Following insemination of chilled ram semen, with and without glycerol in the diluent, lambing percentages of 59% and 73% respectively were obtained. Ram semen was frozen in 0.25 ml straws using various cooling combinations. The optimal procedure was found to be to cool rapidly from 5'C to -120'C at -20'C/min. When semen so treated was compared in a fertility trial with semen frozen by the pellet method of Evans and Maxwell (1987), lambing percentages of 14% and 18% respectively were obtained. Attempts were made to formulate a vitrifying diluent for ram semen. A method was developed for the assessment of semen in highly concentrated cryoprotective solutions. Semen tolerated 10% concentrations of each of glycerol, acetamide and propylene glycol applied together, but when concentrations were raised above this level sperm mortality was very high. A simple spectrophotometric procedure for the objective assessment of vigour of ram semen was developed and tested. Raffinose 66 mM in the freezing diluent improved the post-thawing revival rate of spermatozoa from 46% to 71%, and increased the post-thawing recovery of the swimmingup vigour (P< 0.01). Raffinose treatment reduced the ATP content of semen but did not reduce the rate of glucose oxidation by diluted spermatozoa at either the pre-freezing or post-thawing stages. Frozen storage of ram spermatozoa as pellets was best achieved using two volumes of Tris buffer diluent containing 18% (v/v) egg-yolk, 6% (v/v) glycerol and 66 mM raffinose to one volume of semen. The diluted semen was chilled to 5'C and frozen as 0.10 ml pellets on dry ice. For frozen storage of ram semen in 0.25 ml straws, best results were obtained when the Tris buffer diluent contained 18% (v/v) egg-yolk, 9% (v/v) glycerol and 66 mM raffmose, and cooling was at a rate of -30'C/min from 5'C to -120'C. Non-return rates were 21%, 20% and 31% for ewes inseminated with semen samples frozen as standard 0.20 ml pellets, as raffinose containing 0.10 ml pellets, and as raffinose containing 0.25 ml straws respectively. Of the in vitro tests, only the swim-up test was correlated with non-return rates (r=0.904, P< 0.1). Post-thawing survival of the spermatozoa was improved by the addition of raffinose which had no deleterious effect on fertility.

Thesis (Ing. Zootécnico)--Universidad Católica Boliviana San Pablo, Unidad Académica Campesina Tiahuanaco, Carrera Ingeniería Zootécnica, 2004.
Reproduced from copy at BYU's Benson Institute. Includes bibliographical references (leaves [53]-[56]).

Orientador: Paulo Augusto Neves
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Estudos prévios demonstraram que, em mamíferos, a interação entre os gametas, que antecede a fertilização propriamente dita, é mediada por uma glicoproteína, a D-manose. Os óvulos são ricos em D-manose e os espermatozóides apresentam, em sua superfície, receptores para D-manose, que, durante a interação, reconhecem os óvulos através de um mecanismo tipo chave-fechadura. Os espermatozóides, uma vez capacitados durante sua ascensão pelo trato genital feminino, interagem com os óvulos, sofrem a reação acrossômica e, a seguir, penetram a zona pelúcida. Inúmeros trabalhos procuraram estudar esta interação através de um teste de D-manose, porém não foram conclusivos quanto à metodologia nem quanto aos padrões de expressão dos receptores nos espermatozóides. O presente trabalho teve por objetivo padronizar o teste de D-manose no Laboratório de Reprodução Humana da Universidade Estadual de Campinas e avaliar o padrão de expressão de receptores de D-manose em espermatozóides de homens sabidamente férteis. No período de maio de 2001 a dezembro de 2002, 30 pacientes do programa de vasectomias da Universidade Estadual de Campinas foram selecionados e forneceram uma amostra de sêmen após período de abstinência sexual de três dias, após assinarem consentimento informado. Todas as amostras enquadraram-se dentro dos critérios de normalidade propostos pela Organização Mundial da Saúde. As amostras foram processadas de acordo com a técnica de swim-up e incubadas por um período de 20 horas para induzir a reação acrossômica. Foram colhidas alíquotas da amostra inicial, após processamento seminal por uma hora, e após incubação por 20 horas. As alíquotas seminais foram submetidas ao teste de D-manose, utilizando o corante Man-FITC-BSA, concomitantemente ao teste da reação acrossômica, utilizando o corante RITC - PSA. Para fins de análise, 3.000 espermatozóides foram analisados em cada momento, e os resultados das alíquotas foram comparados entre si. Os principais padrões de expressão dos receptores de D-manose em espermatozóides não reagidos acrossomicamente foram determinados. Os resultados revelaram que houve aumento significativo da proporção de espermatozóides reagidos acrossomicamente ao se comparar as alíquotas inicial e 20 horas e de uma e 20 horas, demonstrando que a incubação por 20 horas foi eficiente em provocar a reação acrossômica. Observou-se expressão de receptores de Dmanose em 18% dos espermatozóides na amostra inicial, em 21,5% na amostra incubada por uma hora e em 28% dos espermatozóides incubados por 20 horas. Ao se analisar especificamente os espermatozóides que expressaram receptores para D-manose, porém que se mantiveram com o acrossoma intacto, não houve diferença estatística entre os três momentos (zero hora, 1 hora e 20 horas): medianas de 4,0%, 5,5% e 5,0%, respectivamente. Os principais padrões de expressão dos receptores de D-manose em espermatozóides não reagidos acrossomicamente em homens férteis foram: padrão 1: marcação apenas da peça intermediária (25,1%), padrão 2: marcação do contorno da cabeça (12,2%), padrão 3: todo o corpo marcado (12,2%), padrão 4: somente região equatorial (7,6%) e padrão 5: cabeça toda marcada (6,3%) e outros padrões (36,6%). Uma vez estabelecido o comportamento dos receptores de D-manose em homens férteis, estudos comparativos com homens inférteis poderão ser propostos na tentativa de se estabelecer novos mecanismos fisiopatológicos da infertilidade masculina
Abstract: Several studies showed that, in mammals, gamete interaction is mediated by a glycoprotein, D-manose. The surface of the eggs is rich in D-manose, and the spermatozoa show, in their surface, receptors that interact with the female gamete in a key-lock manner. The sperm is capacitated during his ascension in the female tract, interacts with the egg, suffers acrosomal reaction and penetrates the zona pelucidae. Previous studies fail to establish the methodology of the Dmanose test, nor could determine usual patterns of expression of the receptors. The objectives of the present work are to standardize the D-manose test in normal fertile men and to determine the usual patterns of expression of the receptors at the Laboratory of Human Reproduction of the University of Campinas Medical School ¿ Brasil. The period at May-2001 to December-2002, thirty normal patients who presented for vasectomy provided a semen sample after 3 days of sexual abstinence. All samples were considered normal according to the criteria of World Health Organization (2001). All samples were submitted to swim-up procedure and further incubation for 20 hours, in order to induce acrosomal reaction. One aliquot of initial sample, after swim-up (1 hour incubation) and after 20 hours of incubation of all samples were collected and submitted concomitantly to D-manose test, using Man-FITC-BSA dye and to acrosomal reaction test, using RITC - PSA as dye. Three thousand sperms were analysed under epifluorescence microscope in every moment, and it was determined the main patterns of expression of D-manose receptors in spermatozoa without acrosomal reaction. The results showed that there was a significant increase of acrosomal reaction in sperm incubated for 20 hours, denoting that this period of incubation is adequate to induce acrosomal reaction. 18% of sperm expressed Dmanose receptors in initial samples, in 21,5% of sperm incubated for 1 hour and in 28% of sperm incubated for 20 hours. There was no significant statistical difference of the median percentage of expression of D-manose receptors in sperm without acrosomal reaction in the three moments (0, 1 and 20 hour incubation): 4.0, 5.5 and 5.0%, respectively. The most common patterns of expression of D-manose receptors in spermatozoa that not suffered acrosomal reaction were: pattern 1 ¿ intermediate piece (25.1%), pattern 2 ¿ around the head (12.2%), pattern 3 ¿ all body of sperm (12.2%), pattern 4 ¿ only equatorial region (7.6%), and pattern 5 ¿ all head (6.3%), other patterns (36.6%). Once established the usual patterns of expression of D-manose receptors of normal fertile donors, comparative studies can be proposed in order to evaluate infertile men and to disclose new physiopathological mechanisms of male infertility
Mestrado
Ciencias Biomedicas
Mestre em Tocoginecologia

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo do estudo foi comparar a efetividade de três diluidores empregados para criopreservação e refrigeração do sêmen bovino em relação aos padrões de motilidade, integridade de membrana plasmática e acrossomal, índice de peroxidação lipídica e fertilidade nos programas de inseminação artificial em tempo-fixo (IATF). No Trabalho científico número 1 foi comparado a viabilidade e fertilidade pós-descongelação proporcionada pelos diluidores Tris-frutose (TRIS, Controle) e Botu-Bov® (BB), ambos contendo 20% de gema de ovo como fonte de lipoproteínas, frente à diluição em Botu- Bov®-Lecitina de Soja (meio BB-L) apresentando 1% de lecitina em substituição ao produto de origem animal. No Trabalho 2 foram avaliados os mesmos diluentes quando utilizados para a refrigeração do sêmen bovino por 48 horas a 5°C. Já no Trabalho número 3 foi avaliada a taxa de concepção na inseminação artificial (C/IA) proporcionada pelo sêmen bovino refrigerado por 24 horas em meio Botu-Bov® em comparação ao sêmen convencionalmente criopreservado no mesmo diluidor. Os meios TRIS e BB a base de gema de ovo foram mais efetivos na manutenção da viabilidade espermática pósdescongelação, conferindo melhores resultados de C/IA (P<0,05) em relação ao meio BBL. No entanto, quando utilizado o sêmen na forma líquida e refrigerado (Trabalho número 2) foi observada uma maior proteção contra o estresse oxidativo proporcionado pelo diluidor a base de lecitina de soja, resultando em maior probabilidade de prenhez quando comparado às amostras refrigeradas em TRIS ou BB, alcançando índice de concepção similar ao obtido com o sêmen congelado. A utilização do sêmen bovino refrigerado por 24 horas levou ao aumento da C/IA de vacas submetidas a IATF quando comparado ao sêmen congelado em meio Botu-Bov®. Conclui-se que embora a lecitina de soja represente...
The aim of this study was to compare three different extenders used for cryopreservation of bovine semen, based on the results obtained during the cooling storage and post-thaw evaluation for motility patterns, integrity of plasmatic and acrossomal membranes, lipid peroxidation rate, as well as conception rate after fixed-time artificial insemination (FTAI). In Paper.1, the efficiency of Tris-Fructose extender (TRIS, control group), Botu-Bov® extender (BB), both containing 20% of egg yolk, and Botu-Bov®-Lecithin extender (BBL), which has 1% of soy lecithin instead of egg yolk, were compared in cryopreservation of bovine semen. In Paper.2, ejaculates from different bulls were cooled to 5°C for 48 hours using the same extenders of Paper.1. In Paper.3 the fertility trial was conducted either with frozen-thawed semen or cooled semen for 24 hours in the BB extender. The egg yolk extenders, TRIS and BB, demonstrated significant differences on the viability and the fertility of frozen-thawed bovine semen when compared to BB-L (P<0.05). However, the use of lecithin instead of egg yolk on semen extender resulted in a greater protection against oxidative stress; moreover, this extender improved the conception rates, reaching the results obtained in FTAI programs with frozen-thawed semen. The use of cooled bovine semen at 5°C for 24 hours improves the conception rate of Nelore cows submitted to FTAI. Although soy lecithin is an interesting alternative source of phospholipids in the elaboration of chemically defined extenders and decrease the risk of microbiological contamination, the egg yolk semen extenders are more effective in preserving the viability and fertility of frozen-thawed bovine semen. However, there was a higher production of free radicals in cooled semen with the use of egg yolk based extenders, resulting in lower conception rates when compared to frozen-thawed... (Complete abstract click electronic access below)

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Universidade Estadual Paulista (UNESP)
O presente estudo objetivou identificar o perfil eletroforético seminal, as características do espermograma e fertilidade in vitro, do sêmen de touros proveniente dos períodos pré e pós-indução do processo degenerativo testicular por insulação. Para isso, foram selecionados quatro animais da raça Nelore, com idade entre trinta e trinta e seis meses. Amostras de sêmen foram colhidas três dias antes (P0) da indução do processo degenerativo e, sete (P7), quatorze (P14) e vinte e um (P21) após a retirada do insulto testicular com cinco dias de duração. Para a análise dos dados foram considerados três experimentos: 1) efeito dos períodos experimentais (P0, P7, P14 e P21) sobre o espermograma e perfil eletroforético do plasma seminal e membrana plasmática dos espermatozóides; 2) efeito dos períodos experimentais e seleção espermática por gradiente de Percoll sobre as características seminais e 3) fertilidade in vitro e associação com características seminais oriundas do período pré e pósinsulto testicular. No experimento 1, os espermogramas foram avaliados por métodos convencionais, coloração de Fuelgen para estimativa do percentual de fragmentação nuclear (FN) e, perfil eletroforético do plasma seminal e espermatozóides por SDSPAGE a 12,5%...
The study was conducted to evaluate the seminal gel electrophoresis profile, spermogramme characteristics and in vitro fertility rate, of semen obtained before and after induction of the testicular degenerative process by scrotal insulation. Four Nelore bulls, between thirty and thirty-six months of age, were subjected to a five days scrotal insult. Semen samples were collected three days (P0) before scrotal insulation and seven (P7), fourteen (P14) and twenty-one (P21) days after the removal of the testicular insult. Three experiments were considered for data analysis: 1) effect of the experimental periods (P0, P7, P14 and P21) on the spermogramme and seminal plasma and plasmatic membrane of spermatozoa gel electrophoresis profile; 2) effect of the experimental periods and sperm selection by Percoll gradient on the seminal characteristics; and 3) in vitro fertility rate and association with seminal characteristics from periods before and after testicular insult. In the experiment 1, the spermogrammes were evaluated by conventional methods beside in the Fuelgen stain for nuclear fragmentation (NF) and proteins profile by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12,5%)...

The aim of this study was to determine the efficacy of vaccination in preventing LSDV excretion in semen and negative effects on semen quality. Lumpy skin disease (LSD) is caused by a virus in the genus Capripoxvirus of the family Poxviridae. The virus has been reported to be excreted in the semen of experimental infected nonvaccinated bulls. Nevertheless, vaccination has been the most widely used method to reduce and prevent the spread of the disease. This work was done to determine the efficacy of lumpy skin disease vaccination in preventing the excretion of lumpy skin disease virus (LSDV) in semen of experimentally infected vaccinated bulls. It also determined further the effect of vaccination and experimental infection on semen quality. Six serologically negative bulls 11-16 months of age were vaccinated with an attenuated Neethling strain of LSD vaccine, and a repeated dose of vaccine was given twenty one days later. These bulls were then experimentally infected by intravenous injection with a virulent field strain of LSDV (V248/93). Six unvaccinated bulls were similarly infected to act as controls. All animals were observed for clinical signs, blood and semen was collected and evaluated twice a week until day 40 post vaccination and every two days until day 28 post-infection when the trial was terminated. Serology was done using the serum neutralization test and viraemia was determined by virus isolation. Semen was examined by polymerase chain reaction (PCR) for the presence of virus. Semen evaluation was done visually and microscopically. Two of the unvaccinated controls developed severe LSD, two showed mild symptoms and two were asymptomatic. No clinical abnormalities were detected following vaccination, and clinical signs were limited to mild lymph node enlargement in four bulls following challenge of the vaccinated bulls. There was a significant difference (P<0.05) in semen quality after experimental infection of the unvaccinated bulls. In the vaccinated bulls, semen quality showed no significant difference (P>0.05) following vaccination and challenge. Three of the vaccinated bulls were serologically positive at the time of experimental infection and four at the end of the trial. Five unvaccinated bulls were found to be viraemic during the course of the trial. No vaccinated bulls were found to be viraemic at any stage. Four unvaccinated bulls excreted the virus in their semen during the course of the trial. Viral nucleic acid was not detected in any semen samples following vaccination or challenge in vaccinated bulls. This study provides evidence that vaccination against LSD prevented the excretion of viral particles in semen. It also illustrated that LSD vaccination prevented any effect on semen quality after experimental infection with virulent virus.
Dissertation (MSc (Production Animal Studies))--University of Pretoria, 2006.
Production Animal Studies
unrestricted

La vicuña (Vicugna vicugna) es una especie silvestre de Camélido Sudamericano (CSA). Está clasificada en Bajo Riesgo según la IUCN, no obstante, como continúa estando amenazada, se requiere estudios enfocados en su conservación. Una herramienta de conservación es la reproducción asistida, sin embargo, para hacer uso de ella, primero se requiere conocer la fisiología básica de la especie. En ese sentido, el presente estudio tuvo como objetivo desarrollar un protocolo de contención química y colección de semen en vicuñas mediante la técnica de electroeyaculación, así como caracterizar el eyaculado obtenido. Fueron utilizados 9 individuos machos adultos de V. vicugna, clínicamente sanos, pertenecientes al Parque Zoológico Huachipa (n igual 3), Lima; CIP Quimsachata-INIEA (n igual 4), Puno; y Zoológico Municipal Cerrito de La Libertad (n igual 2), Huancayo. La electroeyaculación se realizó bajo anestesia general, utilizando la combinación de ketamina (7,83 mg Kg-1), xilacina (1,20 mg Kg-1) y atropina (0,07 mg Kg-1) (n igual 19), además de midazolam (0,35 mg Kg-1) (n igual 9). La colección de semen (n igual 16) se llevó a cabo con un electroeyaculador que constaba de un transductor rectal de 2 cm de diámetro y 3 electrodos de cobre espaciados por 0,4 cm. El animal fue colocado en decúbito y se le introdujo el transductor rectal lubricado de 10 a 15 cm dentro del recto. El protocolo de electroeyaculación consistió de estímulos progresivos desde los 2 V hasta los 12 V. Fue colectado eyaculado en 15 de los procedimientos. Los valores seminales encontrados son los siguientes (x ± EE): volumen 0,85 ± 0,12 ml; pH 7,09 ± 0,16; motilidad espermática no progresiva 28,08 ± 3,56 %; concentración 166,29 ± 60,92 x 104 espermatozoides/ml y espermatozoides normales 62,77 ± 1,96 %. En cuanto al volumen testicular, el valor total encontrado fue de 22,95 ± 2,28 cm3, el cual no tiene correlación con el volumen seminal y la concentración espermática (r igual 0,06 y r igual 0,16; P menor 0,05). Se concluye que la colección de semen por electroeyaculación en vicuñas es factible, asimismo las características seminales observadas son similares a las otras especies de CSA.
The vicuna (Vicugna vicugna) is a wild species of South American Camelid (SAC). From the conservation point of view, is classified at Low Risk by the IUCN. However, it is still a threatened species so many studies for their conservation are required. The assisted reproduction is a conservation tool, however it is necessary to know first the basic physiology of the species. In such sense, the aim of this study was to develop a protocol for chemical immobilization and semen collection using the electroejaculation technique, as well as to characterize the ejaculate obtained. Nine adult males of vicuna, clinically healthy, located at the Huachipa Zoological Park, Lima (n same 3), Quimsachata Research Station, Puno (n same 4) and Zoo Cerrito de La Libertad, Huancayo (n same 2) were used. The electroejaculation procedure was carried out under general anesthesia. The combination of ketamine (7,83 mg Kg-1), xilacine (1,20 mg Kg-1) and atropine (0,07 mg Kg-1) (n same 19) were used, besides of midazolam (0,35 mg Kg-1) (n same 9). Semen collection (n same 16) was carried out with an electroejaculator with a 2 cm diameter probe with 3 ventral electrodes spaced about 0,4 cm. With the animal in recumbent position, the lubricated probe was inserted 10 to 15 cm into the rectum. Progressive electrical stimuli from 2 V to 12 V was applied. Fifteen ejaculates were collected. Seminal values of the ejaculates were as follow (x ± SE): volume 0,85 ± 0,12 ml, pH 7,09 ± 0,16, non progressive sperm motility 28,08 ± 3,56 %; sperm concentration 166,29 ± 60,92 x 104 spermartozoa/ml and sperm normal morphology 62,77 ± 1,96 %. In the case of testicular volume, the total value found was 22,95 ± 2,28 cm3, and do not show correlation with seminal volume and sperm concentration (r same 0,06 y r same 0,16; P less 0,05). These results demonstrate that it is possible to collect semen by electroejaculation and the vicuna´s seminal values are similar with the others (SAC).
Tesis

El semen de alpaca post-descongelación ha sido usado para inseminación artificial sin el éxito esperado, a pesar de usar diversos protocolos de criopreservación y criopreservantes para mantenerlo viable por un tiempo prolongado. Sin embargo, para elaborar un buen criopreservante es necesario conocer que parámetros bioquímicos del plasma seminal se ven afectados tras su descongelación. De allí que el objetivo del estudio fue determinar y comparar las características bioquímicas del plasma seminal de alpacas en fresco y post-descongelado. Se realizó en los laboratorios de Farmacología y Toxicología Veterinaria y Reproducción Animal de la FMV-UNMSM. Se realizó en 4 alpacas adultas, bajo las mismas condiciones de crianza. Se usó un electroeyaculador digital (Electrojac) y se colectó en tubos Falcón de 10 ml, se centrifugó a 3000 rpm por 30 minutos, para la separación del plasma seminal. Una parte se analizó en fresco y la otra parte se almacenó en nitrógeno líquido por 1 mes para su posterior descongelamiento y análisis bioquímico. Se hizo análisis cinético y fotocolorimétrico de niveles de glucosa, colesterol-total, colesterol-HDL, triglicéridos, proteínas-totales, albúmina, calcio, fosfatasa alcalina, ALT y γ-GT, usándose kits específicos (FAR, Italia), y un analizador bioquímico (SINOWA, China). Este procedimiento se realizó 1 vez por semana por 4 semanas. Las diferencias entre los valores bioquímicos en fresco y post-descongelado fueron evaluadas por T-Student pareado con un nivel de confianza del 95%. Sólo los valores de triglicéridos en plasma seminal de alpacas se modificaron debido al proceso de congelación/descongelación lo que se debería a la labilidad de estas moléculas a dichos procesos, los valores de triglicéridos (mg/dL) fueron 44.12±7.38 y 27.31±4.65 en fresco y postdescongelación, respectivamente. Se concluye que tras la descongelación del plasma seminal, únicamente los niveles de triglicéridos en plasma seminal se modifican, lo que supondría tomar en consideración este componente cuando se realice la formulación de criopreservantes.
Tesis

During the last half of the 20^th century, the world pork industry has achieved astonishing developments in pig breeding. Now swine farms are larger, ownership more concentrated, and farms have become more industrialized. Artificial insemination (AI) plays an important role in animal breeding increasing utilization of genetically superior sires. Currently boars selected for commercial use as AI sires are evaluated on grow-finish performance and carcass characteristics. The objectives of this study were to (A) estimate genetic correlations between production and semen traits in the boar; average daily gain (ADG), back fat thickness (BF) and muscle depth (MD) as production traits, and total sperm cells (TSC), total concentration (TC), volume collected (SV), number of extended doses (ND), and acceptance rate of ejaculates (AR) as semen traits; (B) to model the variances and covariances of total sperm cells (× 10⁹) over the active lifetime of AI boars; and (C) to compare multiple traits and random regression analyses applied to total sperm cells (TSC). Average heritability estimates were 0.39 for ADG, 0.32 for BF, 0.15 for MD, and repeatability estimates were 0.38 for SV, 0.37 for TSC, 0.09 for TC, 0.39 for ND, and 0.16 for AR. Semen traits showed negative genetic correlations with MD. Genetic correlations would indicate that current selection objectives are having a negative effect on semen traits. Therefore, current AI boar selection practices may be having a detrimental effect on semen production. In random regression analysis for total sperm cells, maximum log likelihood value was observed for sixth, fifth, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Best fit as determined by Akaike's Information Criterion was based on a model with sixth, fourth, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Best fit as determined by Schwarz Criterion was by fitting fourth, second, and seventh order polynomials for fixed, additive genetic and permanent environmental effects, respectively. Heritability estimates for total sperm cells ranged from 0.27 to 0.61 across age of boar classifications. Heritability for total sperm cells tended to increase with age of boar classification. The cyclic nature of heritability for total sperm cells that was observed over the active lifetime of boars may be due in part to number of observations across seasons limiting our ability to correct for seasonal effects on sperm production. In MTDFREML analysis, heritability estimates of 9, 12, 15, 18, 21, 24, and 27 months of age were, respectively, 0.28, 0.29, 0.26, 0.27, 0.30, 0.79, and 0.41. The results from MTDFREML seemed to be overestimated when compared to random regression. Therefore, random regression methods are the most appropriate to analyze semen traits as they are longitudinal data measured over the boars lifetime.

POOLPERM, PARIWAT.  Factors Influencing Semen Quality and Fertilityin Boars. (Under the direction of Drs.Glen W. Almond and William L. Flowers)

        The objectives of this researchwere 1) determine the influence of antibiotics on semen quality, 2) determinethe association among insulin-like growth factor I (IGF-I) in seminal plasma,semen quality, and subsequent fertility, and 3) retrospectively study theassociation among semen parameters and sow fertility.  In the firststudy, the effects of gentamicin (GM), amikacin (AM), neomycin sulfate(NM), and penicillin-streptomycin (PS) in semen extender on the percentagesof motile (MOT), morphologically normal sperm (MOR), and sperm with normalacrosome (NAR) were examined.  An in vitro penetration assay was conductedusing sperm cells on day 0 and day 5 of storage.  GM and NM groupsshowed higher (p<0.05) MOT than other groups after 5 days of storage. No differences in penetration rate were found among treatments; however,the penetration rate decreased (p<0.05) on day 5 of storage.
        In the second study, ejaculateswere collected and diluted in an extender (Vital?).  Gilts (n=113)and sows (n=375) were inseminated with the extended semen in homogenetic-homospermicregimens.  Farrowing rate (FR), total pigs born (TB) and born alive(TBA) were recorded.  IGF-I was determined in seminal plasma by radioimmunoassay. Concentration of IGF-I from 204 ejaculates was 95.38 ± 3.56 ng/ml (mean± SEM) and total amount of IGF-I/ejaculate was 23.50 ± 1.20 µg.  SeminalIGF-I differed (p<0.05) among genetic lines and had no effect (p>0.05)on MOT, MOR and NAR.  However, IGF-I was associated (p<0.05) withsemen volume, sperm concentration and total number of sperm/ejaculate. No association between IGF-I level in seminal plasma and fertility indiceswas found.
        The third study determinedthe associations among insemination parameters with subsequent fertilityof boar semen using data from the second study.  MOR was associated(p<0.05) with fertility parameters.  TB and TBA were associatedwith age of semen at the first insemination (SAGE) and number of spermper insemination dose (AIDOSE).  With stepwise regression analysis,it was evident that FR was associated with semen volume, MOR and SAGE. Meanwhile, TB and TBA were associated with SAGE, AIDOSE and total numberof spermatozoa/ejaculate.  In conclusion, the assessment of semencharacteristics may not necessarily delineate fertility between boars.

L’abeille domestique (Apis mellifera Linnaeus) joue un rôle crucial comme pollinisateur dans l’industrie de l’agriculture. Cependant, durant les dernières décennies, une mortalité des colonies d’abeilles a été observée partout à travers le monde. La conservation du sperme d’abeille est un outil efficace pour sauvegarder la diversité génétique. Sa conservation est possible à température pièce, mais la cryoconservation serait une meilleure méthode pour la conservation à long terme. Notre objectif général est de développer une méthode de cryoconservation de la semence d’abeille. L’hypothèse no.1 était que la cryoconservation de la semence d’abeille est plus efficace à long terme que les températures au-dessus de 0 °C. Nous avons évalué l’efficacité, basé sur la viabilité des spermatozoïdes, de deux températures de conservation: -196 °C et 16 °C. Après un an de conservation, la semence congelée avait une meilleure viabilité comparée à 16°C (76% ± 5% vs 0%; p < 0,05). Par la suite, la spermathèque des reines inséminées avec la semence cryoconservée a été évaluée par la migration des spermatozoïdes ainsi que la viabilité des spermatozoïdes. Il y avait beaucoup de variabilités dans nos résultats. Nous n’avons pas été en mesure de vérifier si l’ajout de la centrifugation après la conservation améliore la fertilité des reines après insémination. Toutefois, nos résultats confirment que la cryoconservation est une technique efficace pour conserver la semence d’abeille à long terme.
Honey bees (Apis mellifera Linnaeus) are critical players in the agricultural industry for food production as they account for the vast majority of insect pollination. In the last decades, however, there have been dramatic losses of honey bee colonies worldwide. Coupled with instrumental insemination, conservation of honey bee sperm is an effective strategy to protect the species and their genetic diversity. Sperm storage is possible at room temperature, but for many mammal species, cryopreservation is the preferred method for the long-term storage of gametes. However, cryopreservation of honey bee drone semen is not optimized. Our overall objective is to develop a method of drone semen cryopreservation, therefore, two experiments were conducted. Hypothesis #1 was that cryopreservation of drone semen is more effective for long-term storage than at above-freezing temperatures. We therefore compared the efficacy based on sperm viability, of two honey bee semen preservation temperatures: frozen (-196°C) and 16°C. After 1 year of storage, frozen sperm viability was higher than at 16°C (76% ± 5% vs. 0%; p < 0.05), showing that cryopreservation is necessary to conserve semen in vitro. However, the cryoprotectant used for drone sperm freezing, DMSO (dimethyl sulfoxide), is toxic to queens after instrumental insemination. Hypothesis #2, therefore, was that centrifugation of cryopreserved semen to remove DMSO prior to insemination improves queen fertility. Our results indicate that centrifuging semen does not affect sperm viability (78% ± 3% vs 75% ± 4% viable sperm; p > 0.05). After queen insemination, both spermathecae and brood production were evaluated, but the results varied greatly, possibly due to the undesirable mucus present in the semen. Therefore, we cannot yet confirm that centrifugation improves queen health after insemination. Nonetheless, our study confirms that cryopreservation of honey bee sperm is necessary and possible for long-term conservation.

Thesis (B.S.)--California Polytechnic State University, 2009.
Project advisor: Stan Henderson. Title from PDF title page; viewed on Jan. 21, 2010. Includes bibliographical references. Also available on microfiche.

Exosomes are cell-derived vesicles that circulate in bio-fluids and enclose cell-associated cargo, producing various consequences in intercellular communication and may contribute to microbial pathogenesis. Exosomes are similar in composition to enveloped viruses, making differentiation between exosomes and enveloped viruses difficult. Yet, exosomes and enveloped viruses may vary considerably in function. Exosomes produced from infected cells may incorporate viral material as they are simultaneously produced with viruses and depending upon the cargo, contribute to disease pathogenesis. Conversely, exosomes from uninfected cells may contribute to protection from infection. Exosomes from breast milk, vaginal fluid, and semen of healthy donors protect against HIV-1. The functional dichotomy of exosomes is unknown. Here, we focus on the function and physical qualities of exosomes found in semen (SE) and how these influence HIV-1. As semen is the major body fluid involved in HIV-1 transmission, exosomes from semen that regulate HIV-1 may contribute to the low incidence of HIV-1 sexual transmission in vivo. Previous studies indicate healthy donor derived SE but not blood exosomes (BE) inhibit HIV-1 in a donor-independent manner. The composition and function of exosomes depends on the status of the exosome-producing cell. Thus, donor characteristics that alter the condition of exosome-producing cells may alter the antiviral phenotype of SE. Illicit drug use enhances HIV-1 replication, negatively affects male fertility, and alters exosome biogenesis pathways. Thus, illicit drugs may alter SE physical, composition, and functional properties. Indeed, SE from donors with a history of illicit drugs were altered in composition which correlated with a diminished ability to inhibit HIV-1. Similarly, because exosomes derived from HIV-1 infected cell cultures promote infection, donor HIV status may contribute to a proviral phenotype of exosomes. Infectivity studies by HIV-infected ART-naïve SE revealed that SE from healthy donors and HIV-infected donors are inhibitory, but BE are not inhibitory. Therefore, the inhibitory phenotype of SE is conserved regardless of donor HIV status. Significantly, BE and SE from HIV-infected ART-suppressed donors not only inhibited HIV-1, but contained inhibitory levels of antiretroviral (ARV) medications, indicating that body-fluid derived exosomes may act as carriers of ARV drugs. Previous studies found that the antiviral mechanism of SE was targeted to multiple HIV-1 lifecycle steps; however, the mechanism of inhibition was unclear. Transcription specific analysis revealed that SE reduced HIV-transcription at multiple steps including association of transcription factors NF-kB and Pol II with the viral promoter, as well as transcription initiation and elongation. SE inhibited HIV-driven promoter activation and viral gene expression. Importantly, SE targeted inhibition of viral protein Tat transcriptional activities. Overall, these findings from donor characteristics that may alter the condition of the cellular source of SE demonstrate that SE antiviral factor(s) is highly conserved. Illicit drugs alter SE-associated factors and may reduce SE inhibitory activities. However, HIV status does not affect the antiviral function of SE. Significantly, donor-ARV medications are associated with SE and BE indicating a potential role of exosomes in drug delivery in vivo. Mechanistically, SE suppress HIV-1 by targeting host and viral transcription factors that could be therapeutically exploited for novel anti-HIV strategies. These data emphasize the need for additional studies on the composition and function of SE to harness the antiviral potential of SE inhibitory factors.

Handling of semen is a very important tool for a successful artificial insemination in both pig and horse farming. One of the main problems during liquid and frozen semen storage is the loss of fertilizing potential mainly due to a sudden reduction of temperature, an excessive production of reactive oxygen species and alterations in the antioxidant defense systems. Supplementation of sperm preparation with different antioxidants gave interesting and promising results and, during recent years, use of plant antioxidants has been gaining the attention of several research groups. Moreover, an increase in the fertilising ability of boar sperm has been demonstrated after photo-stimulation. On these bases, the objective of the present thesis was to assess whether: 1) Resveratrol (RESV) or Epigallocatechin-3-gallate (EGCG) supplementation of thawing boar semen extender is effective in influencing sperm quality parameters and in vitro fertilization ability; 2) EGCG and green tea extract polyphenols improve stallion semen parameters during cooling at 4°C; 3) Photo-stimulation improves frozen-thawed boar semen quality. The results obtained demonstrate that the addition of RESV or EGCG, alone or in combination, to thawed boar semen positively affects in vitro penetration rate while no positive effects were registered in cooled stallion semen up to 48 h storage; as a consequence, the addition of these substances in cooling medium for stallion sperm storage is not useful. Therefore, it is evident that the effects of antioxidants may vary depending on species as well as on the way semen is processed for preservation (cooled vs frozen-thawed). Moreover, photo-stimulation may be considered as a potential tool to increase the cryotolerance of poor freezability ejaculates (PFE) but more researches on the mechanisms underlying the detrimental effects on sperm motility are required; they may allow to understand whether such increase in cryotolerance has a significant impact upon the fertilizing ability of PFE.

O objetivo do presente estudo foi verificar a influência de diferentes pontos de risco de contaminação bacteriana durante a coleta do ejaculado suíno e seus efeitos sobre a qualidade da dose inseminante (DI). O experimento foi realizado em quatro centros de difusão genética (CDG), nos quais as coletas dos ejaculados foram observadas buscando possíveis pontos de risco de contaminação. Posteriormente, o sêmen in natura e 2 DIs, provenientes da coleta observada, foram avaliados no que se refere à quantificação bacteriana, morfologia e motilidade espermática e pH. Além do ejaculado, amostras de água e diluente foram avaliadas quanto ao número de unidades formadoras de colônia (UFC). Pêlos prepuciais compridos (>1,0 cm), a higiene da luva de coleta, líquido pingando pela mão do coletador para o interior do recipiente de coleta e a duração da coleta foram os 4, dentre os 12 fatores avaliados, que levaram a um aumento no percentual de ejaculados com valor superior a 220 UFC mL-1 de mesófilos aeróbios (P<0,05). Foi avaliado o efeito isolado ou associado de sete fatores (reprodutor sujo, óstio prepucial sujo, divertículo prepucial grande, pêlos prepuciais compridos, luva de coleta suja, pingos pela mão do coletador para dentro do recipiente de coleta e pênis escapou durante a coleta) que pudessem resultar diretamente na contaminação do ejaculado. Houve aumento significativo do número de ejaculados com contaminação superior a 220 UFC mL-1, a partir da associação de 2 fatores, quando comparados aos ejaculados obtidos em coletas sem nenhum fator predisponente para contaminação. Ao classificar as DIs conforme o grau de contaminação do diluente foram observadas redução na motilidade e no pH e aumento nas alterações de acrossoma das DIs, ao longo das 168 horas de armazenamento, no grupo cujo grau de contaminação do diluente foi superior a 14000 UFC mL-1 versus o grupo com contaminação inferior a 330 UFC mL-1. Doses inseminantes provenientes de ejaculados mais contaminados apresentaram maior grau de contaminação bacteriana. Aparentemente, quando o ejaculado é coletado com um protocolo de contaminação mínima, dificilmente seu grau de contaminação será capaz de produzir efeitos deletérios na qualidade da DI, exceto quando a origem da contaminação for proveniente de falhas higiênicas na linha de processamento das mesmas. A produção de DIs com alta qualidade do ponto de vista bacteriano, somente será possível com um rigoroso controle higiênico na linha de processamento, principalmente no que diz respeito à água e ao diluente, associados a um protocolo de contaminação mínima durante a coleta.
The aim of this study was to check the influence of different risk of factors for bacterial contamination during the collection of ejaculate and the effects in boar extended semen quality. The experiment was conducted in four boar studs, where semen collection was observed, searching for possible risk of factors for bacterial contamination. The ejaculate and two extended semen doses, deriving from the observed collection, were evaluated in regard to numbers of colony-forming units (CFU), sperm morphology and motility, and pH. Water and extender samples were also evaluated for CFU. Long preputial hair (>1 cm), the hygiene of the collection glove, liquid trickling from the hand of the technician into the semen container and the duration of the collection were the four, from twelve factors evaluated, that lead to an increase in the percentage of ejaculates with more than 220 CFU mL-1 of aerobic mesophiles (P<0.05). The isolated or combined effect of seven factors (bad hygiene of boars, dirty preputial ostium, large preputial diverticulum, long preputial hair, dirty collecting glove, liquid trickling from the hand of the technician into the semen container and escape penis during the collection), that could directly result in the contamination of ejaculates was evaluated. There was a significant increase in the number of ejaculates contaminated with more than 220 CFU mL-1 when two or more factors were associated, compared to ejaculates obtained from collections without any predisponent factors. When the extended semen doses were classified according to the degree of contamination of the extender, a decrease in motility and pH and an increase on acrosome alterations in extended semen, during 168 hours of storage, were verified in the group where the degree of contamination was higher than 14,000 CFU mL-1 versus the group with lower than 330 CFU mL-1. Extended semen derived from more contaminated ejaculates showed a higher degree of bacterial contamination. Apparently, when the ejaculate was collected with minimum contamination protocol, its degree of contamination will hardly be able to produce effects in the extended semen quality, unless when the source of contamination was hygienic failure in the processing. The production of semen extended with high quality in the bacterial point of view will only be possible with a strict hygienic control in the processing, mostly in respect to water and extender, associated with minimum contamination protocol during the collection.

A busca de estratégias eficientes para a manutenção de espécies em risco de extinção tem impulsionado a comunidade científica a pesquisar alternativas para a preservação de material genético, com intuito de formação de bancos de genoma e aplicação de biotécnicas reprodutivas. Este estudo teve por objetivo avaliar os efeitos de dois protocolos diferentes de resfriamento pré-congelamento para a criopreservação de sêmen de jaguatirica (n=3), gato-do-mato-pequeno (n=4) e gato doméstíco (n=15). Apenas seis amostras de sêmen de jaguatirica, sete de gato-do-mato-pequeno e treze amostras de gato doméstico apresentaram características qualitativas e quantitativas para serem submetidas aos dois métodos de resfriamento. Estas amostras foram processadas e diluídas em meio crioprotetor, com 4% de glicerol e divididas em duas alíquotas submetidas ao método rápido (30 min a 4°C) ou lento (= 2 horas a 4°C) de resfriamento e, em seguida congeladas. Após o descongelamento as amostras foram avaliadas para índice de motilidade, percentual de células com acrossoma intacto e de células viáveis. Ovócitos de gatas domésticas maturados in vitro foram utilizados para avaliar a capacidade de ligação espermática à zona pelúcida, em ensaios de ligação competitiva in vitro. Os espermatozóides criopreservados pelos dois métodos, diferenciados entre si pelo uso de corante vital, competiram pela ligação à zona pelúcida dos mesmos ovócitos, em iguais condições de tratamento. O índice médio da motilidade espermática, pós-congelamento, foi de aproximadamente 50% para todas as espécies nos dois métodos de resfriamento. O percentual de células com acrossoma intacto, para a jaguatirica, foi de aproximadamente 20% nos dois métodos de resfriamento enquanto o gato-do-mato-pequeno apresentou 30,8% de células com acrossoma intacto no método de resfriamento rápido e 33,4% no método lento. Para o gato doméstico, o percentual de células com acrossoma intacto foi de 33,4% e 32,7% nos métodos de resfriamento rápido e lento, respectivamente. O sêmen do gato-do-mato-pequeno sofreu a menor redução no percentual de células viáveis com 51,2% no método de resfriamento rápido e 44% no método lento. A jaguatirica apresentou os menores índices entre as três espécies com 24% de células viáveis no método rápido e 27,4% no método lento, e o gato doméstico apresentou 28,6% e 28,2% de células viáveis nos métodos rápido e lento, respectivamente. O número médio de espermatozóides ligados por ovócito foi maior (p < 0,05) para o resfriamento lento do sêmen de gato-do-mato-pequeno (5,8 +- 0,9) do que para o resfriamento rápido (2,7 +- 0,4). Já o resfriamento rápido proporcionou melhores resultados na taxa de ligação espermática para a jaguatirica (8,5 +- 1,3) (p < 0,01) e para gato doméstico (4,3 +-0,9) (p< 0,05), enquanto o método de resfriamento lento proporcionou 2,5 +- 0,3 espermatozóides de jaguatirica e 1,4 +- 0,2 espermatozóides de gato doméstico ligados por ovócito. As diferentes respostas entre as espécies podem ser devidas a características espermáticas espécie-específicas e individuais. Os resultados permitiram inferir que os protocolos de criopreservação devem ainda ser aperfeiçoados e adaptados à cada espécie e que o ensaio de ligação espermática competitiva foi eficiente na detecção de diferenças na função espermática entre tratamentos, o que não foi possível com as avaliações espermáticas de rotina

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Com a crescente disponibilidade das biotécnicas de reprodução animal, o comércio dos produtos envolvidos com essas práticas também está em expansão, oferecendo a possibilidade de melhoria dos índices zootécnicos às produções de bovinos. Porém deve-se levar em consideração que há riscos sanitários em práticas como a inseminação artificial caso não se realize o controle do material biológico utilizado. Dentre os agentes infecciosos que podem estar presentes no sêmen e passíveis de serem transmitidos por esse estão a leptospirose e a brucelose, enfermidades responsáveis por grandes perdas reprodutivas e econômicas na bovinocultura mundial. Este projeto teve como objetivos detectar molecularmente esses patógenos em amostras de sêmen bovino provenientes de centrais de comercialização brasileiras, utilizando um kit comercial para extração de DNA (“RTP Bacteria DNA Mini Kit” (Invitek®), aperfeiçoá-lo para a extração de DNA bacteriano a partir de sêmen e avaliar sua aplicabilidade à rotina laboratorial. O DNA bacteriano foi extraído e quantificado por eletroforese em gel de agarose. Pretendeu-se também realizar reação em cadeia da polimerase (PCR) utilizando os “primers” B4 e B5 para amplificação do DNA de Brucella spp. e os “primers” Lep 1 e Lep 2 para Leptospira spp. O kit de extração foi otimizado com sucesso, e todas as 96 amostras examinadas foram negativas para qualquer DNA bacteriano. Os resultados podem ser úteis para estabelecer alternativas de controle sanitário em touros doadores de sêmen e permitir o fornecimento de material genético livre de patógenos, aumentando o “status” sanitário da reprodução de bovinos no Brasil
With the increasing disponibility of animal reproduction biotechniques, trading of products involved with these activities is also in expansion offering improving possibilities in zootecnic indexes of bovine herds. However, considerations should be taken about sanitary risks in practices like artificial insemination if any control is applied to this biological material. Among infectious agents that could be present and transmitted by semen are leptospirosis and brucellosis, diseases that are responsible for numerous reproductive and economic losses in world’s cattle culture. The goals of this project were to molecularly detect these pathogens in bovine semen samples from Brazilian artificial insemination centers using a commercial kit for DNA extraction (RTP Bacteria DNA Mini Kit (Invitek®), to improve it for extracting bacterial DNA from semen and to analyze its applicability in laboratory routine. Bacterial DNA was extracted and quantified by agarose gel electrophoresis. We also intended to realize polymerase chain reaction (PCR) using primers B4 and B5 for amplification of Brucella spp. DNA and primers Lep 1 e Lep 2 for Leptospira spp. DNA. The extraction kit was successfully optimized and all of 96 examined samples were negative for any bacterial DNA. Results could be useful to establish alternative measures of sanitary control in semen donors and to allow the supplying of genetic material free of pathogens, increasing sanitary status of bovine reproduction in Brazil

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A infertilidade é definida pela inabilidade de engravidar após 12 meses ou mais de coito regular não protegido. O uso de polivitamínico e polimineral parece influenciar na qualidade seminal. Dados da literatura sobre o uso oral isolado ou combinado desses micronutrientes na melhoria dos parâmetros seminais e eventual fertilidade são controversos e escassos. Analisar os parâmetros seminais de indivíduos inférteis em uso de polivitamínico e polimineral e compará-los com indivíduos normais, comprovadamente férteis sem uso destas substâncias. Foram analisados os parâmetros Seminais de 57 casais inférteis acompanhados no ambulatório de esterilidade do Hospital das Clínicas da Faculdade de Medicina de Botucatu, no período de 2003 a 2007. Nos indivíduos inférteis a análise seminal foi realizada antes e com 90 dias de micronutrientes por via oral os quais foram comparados com 50 indivíduos saudáveis comprovadamente férteis sem uso destas substâncias. A avaliação do sêmen foi feita de acordo com os critérios da Organização Mundial de Saúde - OMS (1999) e morfologia de Kruger et al. (1986). A análise estatística foi feita utilizando os testes t de Student, Mann-Whitney e Wilcoxon, considerando um nível de significância de 5%. Os indivíduos inférteis e os comprovadamente férteis apresentaram similaridade quanto a idade (31,0±5,6 versus 30,3±6,5) (p=0,55) e ao tabagismo (29,8% versus 22,0) (p=0,36). Nos indivíduos inférteis, o uso desses micronutrientes aumentou significativamente a morfologia tanto pelos critérios estabelecidos pela OMS (18,3±9,6 para 22,6±11,8) (p=0,006) e por Kruger (6,9±4,1 para 9,1±5,2) (p=0,002). Verificou-se que os homens inférteis antes do uso de micronutrientes quando comparados aos férteis apresentavam significantemente uma menor concentração de espermatozóides/ml (68,0[37,8;101,2]...
Infertility is defined as the inability to become pregnant after 12 months or more of regular unprotected intercourse. The use of multivitamin/multimineral supplements seems to influence semen quality. Data on the isolated or combined use of these micronutrients to improve semen parameters and eventual fertility are controversial and scarce. To assess semen parameters in infertile individuals using multivitamin and multimineral supplements in comparison with healthy proven fertile individuals not using these substances. Semen parameters were evaluated in 57 infertile couples followed up in the Sterility Outpatient Clinic of Botucatu Medical School between 2003 and 2007. Semen analysis was performed before and after 90 days of oral micronutrient use in infertile individuals that were compared with 50 healthy proven fertile individuals not using these substances. Semen was evaluated according to the recommendations of the World Health Organization- WHO (1999) and the criteria described by Kruger et al. (1986). Statistical analysis was carried out using Student’s t test and the tests of Mann-Whitney and Wilcoxon with significance set at 5%. Infertile and proven fertile individuals showed similar age (31.0±5.6 versus 30.3±6.5) (p=0.55) and smoking status (29.8% versus 22.0) (p=0.36). In infertile individuals, the use of micronutrients significantly improved morphology according to the criteria of WHO (18.3±9.6 to 22.6±11.8) (p=0.006) and Kruger (6.9±4.1 to 9.1±5.2) (p=0.002). Before micronutrient use, infertile individuals compared with fertile males showed lower spermatozoa/ml concentration (68.0[37.8;101.2] versus 96.5[49.0;144.2]) and vitality (85,4±9,2 versus 89.6±6.9) and higher leukocyte count (600.0 [300.0;121.5] versus 350.0[100.0;675.0]). In infertile individuals using multivitamin and multimineral supplements, semen parameters... (Complete abstract click electronic access below)

The primary objective of this research was to study the effects of breed, age, season, and their interactions on semen morphological characteristics. The study was done on 329 bulls (271 Friesland and 58 Jersey) aged 12, 24, 36,48, 60, 72, 84, 96 and> 96 months. The collection of semen was carried out using the artificial vagina method in all four seasons of the year. Spermatozoa were screened for the percentages normal sperm, percentage and total major defects such as knobbed acrosome, pyriform, abnormal lose head, dag defects, nuclear vacuole, degenerative heads, mid-piece reflexes, percentages and total minor defects such as normal lose heads, distal droplets, curled end-piece, lose acrosome. Statistical analyses of the data were done using the general linear model (GLM) procedure of the Statistical Analyses System (SAS, 1999). The results of the study indicate that breed did not significantly affected the percentage normal sperm and percentage major sperm defects, but significantly affected the percentage minor defects (P = 0.01). The Least square means (LSM±SE) for the percentage normal sperm, major defects and minor defects in Friesland and Jersey bulls were 80.6 ±1.06%; versus 78.9±2.31 %; 14.8±0.90% versus 15.0± 2.62%, 5.1±0.43% versus 7.6±0.94%, respectively. The results obtained show that the prevalence of sperm defects that differed significantly between breeds was higher in Jersey bulls compared to Friesland bulls. The results of the study indicated the percentage of normal sperm to differ (P = 0.01) with season. The percentage of normal sperm during the summer, autumn, winter and spring, were 72.8±1.6%, 79.4±2.2%, 82.5±2.4% and 84.4±2.4% respectively. Season also affected the percentage of major defects (P = 0.01) and percentage of minor defects (P = 0.03). The results demonstrate that even though there was a higher variation in sperm morphology with season, better sperm morphology was recorded in spring and winter than summer and autumn. Results also indicate the percentage of normal sperm (P = 0.05) and major defects (P = 0.01) to be affected significantly by age. On the other hand, the percentage of minor defects did not differ significantly with age. Bulls of 36-48 months of age showed better semen quality than bulls older than 72 months and bulls younger than 36 months. The percentage of major defects, particularly the incidence of major defects such as knobbed acrosomes, pyriforms, dag defects and broken flagella were significantly affected by the interaction between age and breed (P = 0.05) and age and season (P = 0.05). There was an increase in the susceptibility to these sperm defects in Jersey bulls with an increase in age, while no variation was observed in Friesland bulls. With age and season combined, young bulls recorded poor semen morphology during winter, while old bulls showed poor morphology during summer. In conclusion, the study suggested that breed, age and season and their interactions are important sources of variation in sperm morphology. For a successful AI programme, semen collection should be done at the age of 36-48 months for both breeds. It is therefore recommended that age, breed and season should be given urgent attention in any bull management system employed in South Africa in order to obtain the best semen quality.
Dissertation (M Inst Agrar (Animal Production))--University of Pretoria, 2003.
Animal and Wildlife Sciences
unrestricted

Bromus sterilis L. (barren brome) spreads rapidly in many European regions. In the Czech Republic, its importance has increased dramatically over the past 10 years. Barren brome is reported as a problem weed in winter crops such as winter wheat, winter barley and oil seed rape, in vineyards and in other cultivated places. Barren brome has been becoming troublesome weed of winter cereals mainly in reduced soil tillage systems. The factors, that are important for its spreading and adaptability under different environmental conditions, are dormancy and germination. Optimal timing for seed germination varies with respect to natural conditions and it is determining for plant development. These traits are adapted to different conditions and habitats; therefore the dormancy and germination response patterns to those conditions vary significantly. Recently there are not many information available on germination behaviour, therefore this study was focused on seeds of barren brome and its characteristics, which were collected in different regions of the Czech Republic. Seeds were investigated under different temperatures, light regimes and water stress in a wide range of conditions. The following characteristics of seeds were studied the dormancy, the dynamics of germination, the temperature optimum, the age of seeds and dynamics of emergence from different depths and persistence in the soil profile under field conditions. Dormancy and germination are influenced by external conditions. The dormancy of seeds barren brome is very short or missing. The germination is influenced by light; the seeds germinated better under darkness than under light regime. The primary dormancy of B. sterilis was short and the seeds needed only three weeks for after-ripening. The seeds of B. sterilis showed broad ecological valence to hydrothermal factors germinating in the wide range of 3 to 35 °C. The temperature optimum is 5-23°C. The germination was only slightly influenced in an environment with low water potential. Germination was limited under water stress only at lower temperatures under 10°C. The response to light at various temperatures showed that seeds germinated better in darkness in all temperatures regimes, than in alternating light regime, especially at lower temperatures. The emergence declined significantly with burial depth (under 40 mm). The seeds were not able to survive in the soil seed bank for a longer time and fall seeds lost viability after 1 year burial in soil profile. These results may be of value for development of predictive models and understanding period when weed control may be most feasible.

Orientador: Mary Anne Heidi Dolder
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Borboletas e mariposas apresentam um dos casos mais evidentes de polimorfismo espermático, com a produção de dois tipos de espermatozóides: os apirenes e os eupirenes, que diferem em morfologia e função. Estudos ultra-estruturais descreveram a morfologia e organização de ambos os tipos de espermatozóides ao longo dos tratos reprodutores masculino e feminino da borboleta Euptoieta hegesia. Os espermatozóides apirenes extratesticulares adquirem membranas concêntricas externas provenientes do rearranjo da membrana plasmática. Já os eupirenes, adquirem um complexo envoltório que sofre modificações ao longo dos tratos reprodutores e parece ser parcialmente resultante do rearranjo dos apêndices laciniados. Foram usados também métodos citoquímicos, à saber: ácido fosfotúngstico-etanólico, ácido tânico, cuprolinic blue, vermelho de rutênio, tiosemicarbazida/proteinato de prata e lectinas. Apirenes e eupirenes apresentaram, diferencialmente, proteínas e carboidratos no lúmen dos microtúbulos e nos elementos de ligação do axonema, nas membranas e principalmente nas estruturas extra-celulares. Nos espermatozóides apirenes ainda foram detectados esses componentes no capuz anterior e nas regiões paracristalinas dos derivados mitocondriais. Nos espermatozóides eupirenes, os apêndices laciniados apresentaram, principalmente, componentes protéicos, enquanto os apêndices reticulados apresentaram carboidratos. Ambos os tipos de apêndices apresentaram organização paracristalina, formada por estruturas cilíndricas. Além disso, por meio da técnica de ácido tânico, foi verificada significativa similaridade entre os envoltórios apirenes e eupirenes, indicando uma possível origem comum. Com o uso de lectinas, os apêndices laciniados e os envoltórios apirenes e eupirenes apresentaram os mesmos glicoconjugados, sugerindo que os envoltórios se originaram do rearranjo destes apêndices. O método imunocitoquímico para detecção de tubulinas mostrou que os apêndices laciniados não são compostos microtubulares. No trato reprodutor feminino foram descritas as morfologias da espermateca e dos espermatozóides apirenes e eupirenes armazenados. O epitélio espermatecal, desconhecido na literatura, apresenta morfologia similar àquela encontrada em outras ordens de insetos
Abstract: Butterflies and moths present one of the most evident examples of sperm polymorphism, with the production of two types of spermatozoa: the apyrene and the eupyrene, that differ in functional and morphological characteristics. Ultrastructural studies were carried out to describe the morphology and organization of both sperm types along the male and female reproductive tracts of the butterfly Euptoieta hegesia. The extra-testicular apyrene spermatozoa acquire external concentric membranes as a result of the plasma membrane rearrangement. The eupyrene spermatozoa acquire a complex coat that is modified along the reproductive tracts and is apparently originates from the rearrangement of the lacinate appendages. Different cytochemical methods were applied: ethanol fosfotungstic acid, tannic acid, cuprolinic blue, ruthenium red, tiosemicarbazide/silver proteinate and lectins. Both sperm types presented differences in the proteins and carbohydrates found in the microtubule lumens and in the links binding the axoneme, in the cellular membranes and, principally, in the extracelullar structures. In apyrene sperm these components were detected in the anterior cap and in the paracrystalline cores of the mitochondrial derivatives. In the eupyrene sperm, the lacinate appendages were predominant1y protein in composition, while the reticular appendages seem to be composed principa11y of carbohydrates. Both appendage types presented paracrystalline organization, made up of small cylindrical structures. With the tannic acid technique, a significant similarity was verified between the coats of both sperm types, indicating a possible common origin. With t he lacinate technique, the lacinate appendages and the c oat of both sperm types were shown to contain the same glycoconjugates, suggesting that the coats are originated by reorganization of these appendages. The immunocytochemical method for tubulin detection demonstrated that the lacinate appendages are not microtubular structures. In the female reproductive tract, the morphology of the spermatheca was described as well as the apyrene and eupyrene spermatozoa, stored in this organ after mating. The morphology of the spermatheca epithelium, which had not been previously investigated for Lepidoptera, presented a somewhat similar organization to what is known for other insect
Doutorado
Biologia Celular
Doutor em Ciências Biológicas

Orientador: Mary Anne Heidi Dolder
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O termo "espermiocladística" defini a utilização de características estruturais e ultra-estruturais dos espermatozóides na composição de matrizes de caracteres para estudos filogenéticos. Estudos realizados, inclusive com insetos, têm fornecido dados consistentes e úteis para este fim, pelo menos para táxons superiores. Neste sentido, propusemos para este trabalho de tese, caracterizar detalhadamente a morfologia dos espermatozóides nas tribos Meliponini, Euglossini e Bombini, buscando identificar caracteres potenciais que pudessem contribuir para o entendimento das relações filogenéticas do grupo. Para tanto, utilizamos as metodologias usuais de (1) microscopia de luz, contraste de fase e fluorescência com DAPI e (2) Microscopia Eletrônica de Transmissão, convencional e citoquímica com ácido tânico e E-PTA. Ainda, comparamos nossos dados com o descrito na literatura para a tribo Apini, para que pudéssemos compor uma matriz consistente de caracteres. Nossas análises filogenéticas, embora incipientes, sugerem (Meliponini (Euglossini (Bombini + Apini))). Entretanto, acreditamos que nossos dados devam ser associados aos dados de morfologia somática, comportamento e biologia molecular, para que as análises filogenéticas futuras sejam mais conclusivas. Este trabalho originou 2 manuscritos em fase de submissão, um já aceito para publicação e um artigo já publicado: Zama, U.. Uno-Neto, J. & Dolder, H. 2001 . Ultrastructure of spermatozoa in Plebeia (Plebeia) droryana Friese (Hymenoptera: Apidae: Meliponina). Journal of Hymenoptera Research, 10 (2): 261-270
Abstract: The term "spermiocladistics" express the use of structural and ultrastructural charcteristics to compose character matrices for phylogenetic studies. Such studies, carried out for different animais, including insects, have been shown to be consistent and useful, at least for higher taxa. Therefore, we proposed, in this thesis, to carry out a detailed ultrastructural analysis of the spermatozoa of the tribes: Melliponini, Euglossini and Bombini, in order to identify characters that could potentially contribute to the understanding of phylogenetic relationships in this group. For this study, we employed the methods: (1) light microcopy using phase contrast, DAPI fluorescence and (2) transmission electron microscopy with conventional preparations and cytochemistry (EPT A). Our data were compared with those described in the literature for the Apini tribe, so as to compose a consistent character matrix. Phylogenetic analysis of our data suggested (Meliponini (Euglossini (Bombini + Apini»). However, we believe that our data should be considered together with the data obtained with research on somatic morphology, behavior and molecular biology, in an effort to develop more consistent results. This thesis includes two manuscripts that are being submitted for publication, one was accepted and one article already published: : Zama, U., Uno-Neto, J. & Dolder, H. 2001. Ultrastructure of spermatozoa in P/ebeia (P/ebeia) droryana Friese (Hymenoptera: Apidae: Meliponina). Journal of Hymenoptera Research, 10 (2): 261-270
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep[TM], or Cushion Fluid[TM]. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação para o Desenvolvimento da UNESP (FUNDUNESP)
O objetivo do presente estudo foi avaliar o efeito da adição da asolctina de soja ou da fosfatidilcolina de soja em diluente à base de gema de ovo sobre os parâmetros espermáticos e índices de fertilidade de sêmen congelado de garanhões. No Experimento I, dezenove ejaculados de três garanhões foram submetidos ao processo de criopreservação utilizando três diluentes de congelação: Botu-Crio® (BC), Botu-Crio® adicionado de asolctina de soja (BC+Ac) e Botu-Crio® adicionado de fosfatidilcolina de soja (BC+Fc), para posterior avaliação dos parâmetros de cinética espermática e integridade de membrana plasmática. No Experimento II, comparou-se a taxa de fertilidade referente aos três meios de congelação por meio de inseminação artificial das éguas com “pool” de espermatozoides dos três garanhões. Não houve diferença significativa no que se refere aos parâmetros espermáticos (P<0,05) de motilidade total (69,16±9,28; 64,63±11,05; 68,47±7,92), motilidade progressiva (25,00±5,43; 23,26±5,63; 26,16±5,50), integridade de membrana plasmática (39,37±8,82; 39,95±9,52; 41,63±9,24) e índice de concepção (46,7%, 13,3%, 37,5%) entre os meios BC, BC+Ac e BC+Fc, respectivamente. Entretanto, existiu uma tendência a maior (P>0,05) taxa de fertilidade do diluente Botu-Crio® quando comparado ao meio adicionado de asolctina de soja. Diante dos resultados encontrados, conclui-se que tanto a adição da asolctina de soja quanto da fosfatidilcolina de soja ao diluente Botu-Crio® apresenta a mesma eficácia que o diluente Botu-Crio® convencional na criopreservação de sêmen equino
The aim of this study was to evaluate the effect of addition of soybean asolectin and phosphatidylcholine in an egg yolk-based extender on sperm parameters and fertility rates of frozen stallion semen. In Experiment I, nineteen ejaculates from three stallions were submitted to cryopreservation process using three freezing extenders: Botu-Crio™ (BC), Botu-Crio™ with soybean asolectin (BC+Ac) and Botu-Crio™ with soybean phosphatidylcholine (BC+Fc), for further evaluation of kinetic parameters and sperm plasma membrane integrity. In Experiment II, the fertility rates from the three freezing extenders were compared through artificial insemination in mares using a sperm “pool” of three stallions. No significant differences were found on sperm parameters (P<0.05) of total motility (69.16±9.28; 64.63±11.05; 68.47±7.92); progressive motility (25.00±5.43; 23.26±5.63; 26.16±5.50); plasma membrane integrity (39.37±8.82; 39.95±9.52; 41.63±9.24) and fertility rates (46.7%, 13.3%, 37.5%), for the BC, BC+Ac and BC+Fc groups, respectively. However, the tendency toward higher (P>0.05) fertility rates in Botu-Crio™ (BC) freezing extender than the freezing extender containing soybean asolectin (BC + Ac). Based in these results, it is concluded that the both addition of soybean asolectin and phosphatidylcholine to Botu-Crio™ freezing extender has the same effectiveness as the conventional Botu-Crio™ used in the cryopreservation of equine semen

Se utilizarán eyaculados procedentes de machos de la línea R (línea de conejos seleccionada por velocidad de crecimiento durante el periodo de engorde)alojados en diferentes centros de inseminación artificial. Una vez recuperados los eyaculados se procederá a su valoración y una muestra de todos ellos será crioconservada. La calidad seminal será de nuevo valorada tras el proceso de congelación. Junto con los anàlisis seminales se utilizarán los datos de crecimiento y pedigree de los machos y de todos los animales de la línea R desde su fundación para estimar por un lado los parámetros genéticos de las variables relacionadas con la producción y calidad de dosis seminales en fresco y tras un proceso de crioconservación y la correlación genética existente entre las variables seminales anteriormente citadas y la velocidad de crecimiento. A su vez se estimará mediante un modelo recursivo la relación entre las variables seminales en fresco y tras la descongelación.
The general aim of this thesis was to study the genetic determinism for some traits related to artificial insemination (AI) dose production of fresh and frozen-thawed semen, in order to explore the interest and limitation of different strategies for their genetic improvement in a paternal line of rabbits selected for growth rate during the fattening period (28-63 days). In chapter 1, genetic parameters of sperm production traits are estimated as well as the genetic relationship with daily gain (DG). The heritabilities (h2) of the semen traits were 0.13±0.05, 0.08±0.04 and 0.07±0.03 for ejaculate volume (V), sperm concentration (CN) and sperm production (PROD) per ejaculate, respectively. A favourable and moderate genetic correlation was observed between V and DG (0.36±0.34). From this chapter it may be concluded that if a seminal trait is to be included as a selection objective, a useful one could be sperm production, as it is a trait in which both volume and concentration are included. Moreover, there is currently no evidence to suggest that selection for DG in rabbits will affect sperm production adversely. The aim of chapter 2 was to explore the genetic determinism of some sperm quality traits and their genetic relation with the selection criteria of the paternal rabbit line. The heritabilities (h2) of semen quality traits commonly evaluated in a classic spermiogram were 0.18, 0.19 and 0.12 for NAR (%, percentage of sperm with intact acrosome), ANR (%, percentage of sperm abnormalities) and MOT (%, percentage of total motile sperm cells) respectively. We also estimated the h2 of some motion CASA parameters 0.09, 0.11, 0.10, 0.11, 0.11 and 0.11 for VAP (µm/s; average path velocity), VSL (µm/s; straight-line velocity), VCL (µm/s; curvilinear velocity), LIN (%, linearity index), ALH (µm; amplitude of the lateral head displacement), STR (%, straightness). Genetic correlations between DG and semen traits showed a high HPD95% (interval of highest density of 95%). However there is some consistent evidence of the negativity of the genetic correlations of DG with NAR and MOT (-0.40 and -0.53, respectively). Chapter 3 aims to determine the repeatability and heritability of sperm head characteristics: width (W, ¿m), area (A, ¿m2),length (L, ¿m) and perimeter (P, ¿m), and explore the relationships between them and with the selection objective (DG). The results obtained showed that sperm head dimensions are heritable (ranged between 0.2 and 0.29). The genetic correlations between sperm traits were always high and positive (between 0.72 and 0.90), with the exception of L-W genetic correlation, which was moderate. Regarding the genetic correlations between DG and sperm head characteristics, the resulting means ranged from -0.09 for L-DG to -0.43 for W-DG, showing consistent evidence of the negativity of the genetic correlations. The environmental and male effects that could have an influence on sperm freezability are studied in Chapter 4. Six different traits were evaluated: sperm concentration (CONC, 106spermatozoa/mL), acrosome integrity in fresh (NAR, %) and frozen-thawed semen (Nar-FT, %), sperm motility in fresh (MOT, %) and frozen-thawed semen (Mot-FT, %) and the percentage of viable sperm in frozen-thawed semen (Live-FT, %). In addition, two synthetic traits were computed: the relative reduction of acrosome integrity (Rnar, %) and relative reduction of motility (Rmot, %) after the freezing-thawing process. A multiple-trait recursive model was used to analyse the relationships between the semen traits considered. For the fixed effects studied, the season had the highest impact on post-thaw semen characteristics. Results of the analysis of recursive coefficients showed that fresh semen concentration and motility influence the future freezability of the semen. All traits studied presented moderate repeatabilities, ranging from 0.11 to 0.38. These results provide conclusive evidence that sperm freezability in rabbits could be heritable. Regarding male correlations, there were large positive male correlations between fresh traits (rm=0.77-0.57), as well as between direct frozen-thawed traits (rm=0.72-1). Male effects on fresh and direct frozen-thawed traits were generally positively correlated. This correlation was moderate to high for MOT with all frozen-thawed traits (rm=0.41-0.74) and for Mot-FT and all fresh traits (rm=0.5-0.74); these results suggest that these traits could be genetically related. The final chapter of this thesis focused on estimating the heritability of semen freezability traits and estimating the genetic correlation between frozen-thawed sperm traits and the growth rate in a paternal rabbit line. Estimated heritabilities showed that frozen-thawed semen traits are heritable (ranged between 0.08 and 0.15). In the case of Live-FT, the estimated heritability is the highest and suggests the possibility of effective selection. After the study of genetic correlations, it seems that DG was negatively correlated with sperm freezability, but due to the high HPD95% no further conclusions could be drawn. More data should be included in order to obtain better accuracy for the estimates of these genetic correlations. If the results obtained in the present study were confirmed, it would imply that selection for DG could alter sperm cell membranes or seminal plasma composition, both components related to sperm cryoresistance.
Lavara García, R. (2013). Genetics of fresh and frozen-thawed semen traits and their relationship with growth rate in rabbits [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31657
TESIS

The chromosomal location of endocrine genes was established, and relationships between expression of specific endocrine genes and measures of testis function in normal and poor semen quality stallions was assessed. Consensus primer sequences for glucocorticoid receptor (GR) and luteinizing hormone receptor (LHR) were used to screen the CHORI-241 equine bacterial artificial chromosome (BAC) library. The identity of PCR-positive BAC clones was confirmed by sequencing. Verified BACs were mapped to horse metaphase chromosome spreads by fluorescence in situ hybridization (FISH). The BACs containing the GR and LHR were localized by FISH to ECA 14q16-q21 and ECA15q22-q23, respectively. In addition to FISH mapping, the 5000rad horse x hamster radiation hybrid (RH) panel was screened in duplicate. Two-point linkage analysis placed GR 0 cR from LEX047, while LHR was 36.67 cR from TKY011 on ECA14 and ECA15, respectively. Total testicular parenchymal weight, mean daily sperm production (DSP) per gram parenchyma and mean apoptotic rate (406.05 ± 24.33g vs. 180.01 ± 34.41g, 15.29 ± 0.87 vs. 10.24 ± 1.10, 6.70 ± 0.88 vs. 14.25 ± 1.11, respectively) differed (P<0.05) between normal (n=8) and poor semen quality (n=5) stallions. Also, plasma estradiol and inhibin concentrations were higher (P<0.05) in normal stallions than in poor semen quality stallions. Testicular expression of estrogen receptor beta (ER beta), βB inhibin, prolactin receptor (PRLR), growth hormone receptor (GHR) and insulin-like growth factor I receptor (IGF-IR) mRNAs were all lower (P<0.05) in poor semen quality stallions than in normal stallions. The BACs and primers developed in this study will facilitate future investigations of GR and LHR gene structure in the horse as well as providing a resource for physiological investigation of these two genes that are primary regulators of stress responsiveness and fertility. These data add important endocrine genes to the horse cytogenetic map. Also, important hormonal and gene expression changes have been identified in poor semen quality stallions for further investigation.

Thesis (MScMedSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The basic semen analysis plays a pivotal role in the diagnosis of male infertility and makes a significant contribution to the diagnostic process in andrology, gynecology and clinical urology. In 1902, the man considered to be ―the founding father of modern andrology‖ Edward Martin, proposed that an analysis of a semen sample should be incorporated into all infertility assessments. Following this suggestion in 1956, the scientist John MacLeod advanced the basic semen analysis from beyond a mere observation and introduced the importance of certain semen parameters such as morphology, motility and viscosity. The present day examination includes the analysis of certain established semen parameters, which can provide key information about the quality of a patient‘s semen and the functional competence of the spermatozoa. A semen analysis is also a valuable diagnostic tool in assessing possible disorders of the male genital tract and the secretory pattern of the male accessory sex glands. This information can help to determine the reproductive capacity of the male and can be used in conjunction with the partner to indicate the impact of male genital pathophysiology in the assessment of a couple‘s prospect for fertility. Patients attending the andrology laboratory at Tygerberg Academic Hospital for a semen analysis are referred based on primary, secondary or idiopathic infertility. Amongst these patients, an increase in semen viscosity has been observed over a period of time and created the need to assess the possible causes behind this trend. Despite viscosity being included in a routine spermiogram, it raises a considerable amount of concern as it is assessed semi-quantitatively. In the first part of this study, the possible correlation between seminal hyperviscosity and leukocytospermia was assessed. To achieve the most comprehensive assessment of viscosity, a new approach was used, which is a highly quantitative method to record viscosity in the international unit, centipoise (cP). The analysis of semen samples for possible leukocytospermia was approached by three methods the first of which was cytological. During this method granulocyte grading was performed on stained semen smears during the normal determination of morphology. The same approach was taken for the second method, whereby white blood cell concentrations were quantified with a leukocyte peroxidase test in the total sample group (n=200). Viscosity was compared between the samples classified as leukocytospermic positive or negative, according to the set reference values of the World Health Organisation (WHO). Correlation analysis between the two variables was also performed. In the biochemical approach of detecting leukocytospermia, an enzyme-linked immunoabsorbant assay (ELISA) was used to quantify the concentration of the extracellular polymorphonuclear (PMN) enzyme released from leukocytes. This test was performed on 124 randomly selected samples. All samples were fractionated before storage in liquid nitrogen, to allow for multiple assessments to be performed on each sample. The PMN elastase concentration was assessed against viscosity to investigate a possible correlation and relationship with the presence of leukocytospermia. All three methods of detecting possible infection showed a significantly positive relationship with increased viscosity in semen samples. The second approach in the study was to assess increased viscosity and leukocytospermia against parameters included in the spermiogram. An evaluation of hyperviscosity and its correlations to the various other semen parameters can allow for a detailed study into the effects that this anomaly may elicit. With the assessment of each of the sperm parameters against the leukocyte count and viscosity (cP), volume, concentration and morphology showed significance. To further the study, the third angle was to investigate a possible correlation between viscosity and the functional status of the male accessory sex glands. The biochemical approach of assessing the secretory patterns of the prostate and seminal vesicles against markers of infection can possibly further the understanding behind hyperviscous semen and leukocytospermia. Citric acid and fructose, secretory products of the prostate and seminal vesicles respectively, showed no significance when assessed against the leukocyte count and viscosity. However, this project was a pilot study and this approach offers an exciting avenue for further research. These research findings may provide a more comprehensive assessment of a man‘s fertility status. Seen in the context of patients attending the andrology laboratory of Tygerberg Academic Hospital, this is greatly needed as the majority of these patients cannot afford advanced assisted reproductive therapies. The introduction of a more accurate method of quantifying viscosity may possibly help to identify, diagnose and treat patients suffering from leukocytospermia in order to ultimately enhance their fertility potential.
AFRIKAANSE OPSOMMING: Die basiese semenanalise speel 'n belangrike rol in die diagnose van manlike infertiliteit en maak dus 'n betekenisvolle bydrae tot die diagnostiese proses in andrologie, ginekologie en kliniese urologie. In 1902 het Edward Martin, wat deur sommige navorsers as die vader van moderne andrologie beskou word, voorgestel dat 'n semenanalise deel moet vorm van alle infertiliteitsondersoeke. In 1956 het die wetenskaplike John MacLeod aanvoorwerk gedoen om die grondslag van 'n basiese semenanalise daar te stel, wat beteken het dat, in plaas van net 'n observasie studie te doen, 'n semenmonster kwantitatief analiseer moes word en dat parameters soos spermmorfologie, motiliteit en viskositeit as deel van die volledige analise gedoen moet word. Die hedendaagse analise sluit, behalwe die basiese semenparameters, ook inligting in oor die funksionele aspekte van spermatozoa. Die semenanalise is dus ook ‗n belangrike diagnostiese hulpmiddel om inligting rakende moontlike abnormaliteite in die manlike genitale traktus en die sekretoriese funksies van die manlike bykomstige geslagskliere te verskaf. Hierdie inligting kan help om 'n moontlike diagnose van die man se fertiliteitspotensiaal te maak. Terselftertyd kan dit ook tesame met die metgesel se reproduktiewe inligting meer lig werp op die impak van die man se genitale patofisiologie op die paartjie se fertilitetspotensiaal. Pasiënte wat die andrologielaboratorium van die Tygerberg Akademiese Hospitaal besoek word verwys op grond van primêre, sekondêre of idopatiese infertiliteit. Gedurende die laaste aantal jare is daar ‗n toename in voorkoms van verhoogde semenviskositeit onder hierdie groep pasiënte waargeneem. Dit het die behoefte laat ontstaan om die moontlike redes hiervoor te ondersoek. Ten spyte van die feit dat viskositeit deel vorm van die roetine semenanalise is dit tog kommerwekkend aangesien dit op 'n semi-kwantitatiewe manier bepaal word. In die eerste deel van hierdie studie is 'n moontlik korrelasie tussen seminale hiperviskositeit en leukositospermie ondersoek. Om die beste moontlike verwantskap te kon bepaal is 'n nuwe en hoogs kwantitatiewe metode gebruik om viskositeit in numeriese waardes volgens internasionale standaarde in centipoise (cP) te meet. Daar is van drie metodes gebruik gemaak om die teenwoordigheid van leukositospermie in 'n semenmonster te ondersoek. Die eerste metode was die sitologiese metode waar die teenwoordigheid van granulosiet op die gekleurde semensmeer tydens die standaard morfologie beoordeling bepaal word. Die tweede was deur middel van 'n leukosietperoksidase toets waarmee daar 'n kwantitatiewe telling gedoen kan word, soos teenwoordig in 'n voorbereide semenmonster. Hierdie twee bepalings is op die totale studiepopulasie van 200 pasiënte gedoen. Die viskositeit van monsters met of sonder die teenwoordigheid van leukositospermie, soos bepaal met die voorafgaande metodes en gebaseer op die WGO riglyne, is met mekaar vergelyk. Korrelasies is ook tussen hierdie twee veranderlikes en verskeie semenparameters van hierdie twee groepe gedoen. Die derde metode was 'n biochemiese ontleding met behulp van 'n ensiemgekoppeldeimmuunsorberende essai (ELISA) vir die bepaling van die ekstrasellulêre konsentrasie van polimorfonukleêre (PMN) elastase ensiem in die seminale plasma. Hierdie toets is op 124 lukraak gekose semenmonsters uitgevoer. Alle monsters is gefraksioneer voor berging in vloeibare stikstof om meervoudige analises van elke monster moontlik te maak. Die PMN elastase konsentrasies is vergelyk met die viskositeit van die semenmonsters vir 'n moontlike korrelasie en verwantskap met die teenwoordigheid van leukositospermie. Die resultate van al drie hierdie metodes, vir die moontlike bepaling van infeksie, het 'n betekenisvolle positiewe verwantskap met die toename in graad van viskositeit in semenmonsters aangetoon. Die tweede benadering van hierdie studie was om die viskositeitsgradering en die kwantitatiewe leukositopermie waardes te vergelyk met die semenparameters wat bepaal is tydens die semenanalise. Die doel van hierdie benadering was om enige verwantskap of effek van viskositeit asook die teenwoordigheid van witbloedselle op die semenparameters te ondersoek. Daar is betekenisvolle verwantskappe gevind tussen die viskositeitstatus van 'n semenmonster, die teenwoordigheid van witbloedselle en die semenparameters, soos motiliteit, morfologie en spermatosoa konsentrasie. Die derde benadering was om 'n ondersoek te doen na die moontlike verwantskap tussen viskositeit en die sekretoriese funksies van die manlike bykomstige geslagskliere, te wete die prostaat en seminale vesikula. Die biochemiese ondersoek na die sekresies van hierdie twee organe, naamlik fruktose en sitroensuur, is gedoen om te bepaal of die teenwoordigheid van infeksies van die manlike traktus, en waargeneem as leukositospermia, ook in verband gebring kan word met die viskositeitstatus van 'n semenmonster. Daar is geen verband gevind tussen die sekresies van hierdie twee kliere en die viskositeit van die semenmonsters nie. Aangesien hierdie deel van die studie net as 'n loodsprojek beskou is, is die biochemiese bepalings slegs op 'n beperkte aantal semenmonsters uitgevoer en kan hierdie tipe ondersoek as 'n moontlike verdere studie onderneem word. Hierdie navorsingsresultate kan lei tot ‗n meer omvattende assessering van mans se fertiliteitstatus. Dit is uiters noodsaaklik in die konteks van omstandighede van die pasiënte wat die andrologielaboratorium van die Tygerberg Akademiese Hospitaal besoek aangesien die meerderheid nie gevorderde in vitro behandeling kan bekostig nie. Die akkurate bepaling van 'n semenmonster se viskositeit kan dus moontlik waarde toevoeg tot die identifisering, diagnose en behandeling van pasiënte met leukositospermie om sodoende hulle fertiliteitspotensiaal te verbeter.

The aim of this diploma thesis is assessing the determination and evaluation of pathogens species spectrum in carrot (Daucus carota), parsley (Petroselinum crispum) and pepper (Capsicum annum) seeds. The microflora was determined in laboratory conditions. The surface disinfected seeds and non-disinfected seeds were cultivated on culture medium PDA and filter paper. The level of bacterial and yeast contamination was low. The major part of pathogenswere fungi pathogens. The most often found pathogens were species from Alternaria and Cladosporium genus. The species spectrum was not different between individual varieties of carrot, parsley and pepper. In most cases, non-disinfected seeds had significantly higher amount of pathogens than the disinfected seeds.