No description available.

The objectives of this study were to assess growth and reproductive performance of boars fed a diet supplemented with organic selenium (Se). Crossbred boars received one of three treatments: I. basal diet with no supplemental Se, II. basal diet supplemented with 0.3 ppm organic Se (Sel-Plex), and, III. basal diet supplemented with 0.3 ppm sodium selenite. Nursery (n = 13 pens/treatment) boar performance was not affected (P > 0.1) by diet and only grow-finish (n = 11 pens/treatment) G:F was greater (P < 0.06) for Sel-Plex (0.378) compared with selenite (0.368) or control (0.363) boars. At 15-mo of age semen was collected from boars (n = 10/treatment) over 5-d. Semen quality declined over time, but the negative impact day had on sperm motility was less pronounced with Sel-Plex boars. Effects of treatment x day were detected for progressively motile (P = 0.02) and rapidly moving (P = 0.03) spermatozoa, sperm path velocity (VAP; P = 0.05), and average velocity (VSL; P = 0.05). At 17-mo of age, semen was collected from boars (n = 10/treatment), extended and stored over 10-d. Although semen quality decreased over time, sperm from Sel-Plex boars resisted the negative effects of day on sperm motility and pH. Effects of treatment x day were detected for percent motile spermatozoa (P < 0.01), static spermatozoa (P < 0.01), VAP (P = 0.06), amplitude of head displacement (ALH; P = 0.02), straightness (P = 0.01), and pH (P < 0.01). At 23-mo of age, semen was collected (day 0) from boars (n = 6/treatment), extended, stored and evaluated at d 1 and 8 using in vitro fertilization. Dietary Se treatment failed to affect (P < 0.05) in vitro fertilizing rates of boars. In summary, dietary supplementation with Sel-Plex enhanced G:F in grow/finish boars. Dietary Sel-Plex supplementation may decrease the effects that stressors, such as intensive semen collection or semen storage, have on boar sperm characteristics such as sperm motility. The mechanisms for these responses remain to be elucidated. / Ph. D.

This study was conducted to: (a) determine the optimum extender system for two extenders that will maintain the highest level of cellular integrity when stored at either 5C or 15C for a minimum of 72 hr; (b) evaluate the fertilizing capacity of stored spermatozoa using the optimum extender system; (c) critically analyze enzymatic and morphological changes associated with storage and aging of boar spermatozoa; and (d) characterize properties of boar spermatozoa important to fertilization. The least-squares means for the percentage of normal apical ridge acrosomes were significantly affected by ejaculate within boar (P< .001), boar x extender (P< .05), boar x storage temperature (P< .01), extender x storage temperature (P<.01), extender x cooling rate (P< .05) and antibiotic x storage temperature (P< .01). The regression equations of storage time x treatment effect were reported. The least-squares means for acrosin activity were significantly affected by ejaculate within boar (P<.001), ejaculate within boar x antibiotic (P< .05), ejaculate within boar x extender (P<. 001), ejaculate within boar x storage temperature and extender x storage temperature (P< .05). The regression of storage time x storage temperature was reported. The least-squares means for progressive forward motility were significantly affected by extender (P< .05), antibiotic (P< .05), ejaculate within boar (P< .001), antibiotic x extender (P< .05) and extender x storage temperature (P< .01). The regressions of storage time x treatment effects were reported. The least-squares means for vibrational and/or rotational motility were affected by ejaculate within boar (P< .001), ejaculate within boar x extender (P<.001), ejaculate within boar x cooling rate (P<.05), antibiotic x extender and extender x storage temperature (P< .01). The optimum extender system consisted of Purdue extender containing penicillin/streptomycin and cooled to a 15C storage temperature in 4 hours. This extender system maintained a 70% minimum fertilization rate for 84 hours. / Ph. D.

There is an increased demand for artificial insemination and shipping canine semen in clinical practice. However, we need to process the semen samples using centrifugation and dilution with extenders to help preserve the breeding dose and semen quality. Our objective was to determine a clinically relevant range of centrifugation and concentration parameters for processing canine semen. In the first experiment, we hypothesized that higher g force and longer treatment improves sperm recovery rates yet causes greater decline in semen parameters over a 48-hour cooling period. Our study design used the raw semen evaluations which served as each dog's own control. Sperm RR (%) was calculated post-centrifugation, and sperm viability (%, Nucleocounter® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (NM%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 (T2) and 48 hours (T3) after cooling. Sperm losses were minimal and similar for all treatment groups (median >98%, P≥0.062). Spermatozoa viability was not different between centrifugation groups at any time point (P≥0.38) but declined significantly during cooling (T1 vs. T2/T3, P≤0.001). Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 (P≤0.02). In conclusion, our study showed that centrifugation within a range of 400g-900g for 5-10 minutes is appropriate for processing canine semen. In the second phase, we compared different sperm concentrations for cooled canine semen storage and hypothesized that lower concentrations would result in better semen quality. Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 x106 sperm/ml (C200), then serially diluted to 100, 50, and 25 x106 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24h, then centrifuged and re-extended. Parameters were assessed in raw semen (T0), post-extension (T1), after 24h of cooling (T2), and after processing at 24h (T3). Cooling resulted in significant declines in STM and NM for all groups, and in decreased PMI for CON and C25-50. After cooling (at T2), PMI was significantly lower for C25 compared to all groups and higher for CON compared to C25-100 (p≤0.038). For the motility parameters and NM, C25 performed worse than all or most of the other groups. Comparing CON at T3 with C25-200 at T2, PMI, STM and NM for CON were significantly lower than C25-200, C200, and C100-200, respectively. In conclusion, our results show that cooling canine semen for 24h at 200 x106 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on highest PMI. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24h of cooled storage. / Master of Science / We need to process canine semen using centrifugation and dilution for cooled shipments or cryopreservation. This is due to the increased demand for shipping canine semen for artificial insemination. Our goal was to define an acceptable range of centrifugation and concentration parameters (gravitational (g) force and time and sperm/ml) without severe negative impact on semen quality. In the first experiment, we hypothesized that higher g force (900g vs. 400g or 720g) and longer treatment (10 min. vs. 5 min.) improves sperm recovery rates yet causes greater decline in semen parameters over a 48-hour cooling period. Initial raw semen evaluations served as each dog's own control. Sperm recovery rates post-centrifugation were similar between treatment groups. Sperm viability, motility and morphology were not different between centrifugation treatment groups but declined over time. In conclusion, our range of 400-900g for 5-10 minutes centrifugation provides clinically viable semen quality after up to 48 hours of cooled storage in dogs. In the second phase, we compared different sperm concentrations for cooled canine semen storage and hypothesized that lower concentrations would result in better semen quality. Individual ejaculates were divided into a control aliquot (CON) extended 1:3 vol:vol with a commercial extender. The remaining sample was centrifuged and extended to 200 x106 sperm/ml (C200), then serially diluted to 100, 50, and 25 x106 sperm/ml concentrations (C100-C25). Aliquots were cooled for 24h, then centrifuged and re-extended. Cooling resulted in significant declines in subjective total motility and normal morphology (NM, %) for all groups, and in decreased viability for CON and C25-50. After cooling, viability of the sperm cells was significantly lower for C25 compared to all other groups, and higher for CON compared to C25-100 (P≤0.038). For motility parameters and NM, C25 performed worse than all or most of the other groups. In conclusion, our results show that cooling canine semen for 24h at 200 x106 sperm/ml final concentration after processing or extending 1:3 vol:vol without centrifugation is preferred based on highest plasma membrane integrity (PMI) or sperm cell viability. If volume restrictions apply, processing raw semen and extending to the desired volume with higher sperm concentrations at the collection facility is superior to centrifugation and volume adjustment after 24h of cooled storage.

Profiles for testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA), and progesterone (P) in bovine semen were established for a group of four post-pubertal bulls by depleting the bulls' extra-gonadal sperm reserves (EGR) on three occasions at two week intervals. Each depletion consisted of 14 consecutive ejaculates combined into five pools to reduce variation and to provide adequate volume for steroid radioimmunoassay. Concentration of T and A in semen decreased significantly during depletion and reached basal concentrations. Basal concentrations were regarded as those amounts continually being produced and were considered to have been reached if there were no significant decreases in these concentrations for two or more successive pools. Testosterone concentration decreased from a mean (±SE) of 1.09 (±.17) ng/ml for the first two ejaculates (pool 1) to .26 (±.04) for ejaculates 11 to 14 (pool 5). Decreases in DHEA and P with depletion of EGR were not significant. Only T concentration was similar for pools 4 and 5, but repeatability was dependent on EGR depletion. A difference of approximately .4 ng T/ml was required to establish a significant difference among bulls in this study. Seminal T concentration decrease was not accompanied by a corresponding decrease in seminal fructose concentration nor were any other semen quality characteristics related to seminal T concentration. / M.S.

Tesis por compendio / Los objetivos generales de esta tesis fueron desarrollar nuevos diluyentes de inseminación artificial (IA) suplementados con un análogo de GnRH y caracterizar el proteoma del semen de conejo. En el capítulo I, se evaluó la inclusión de un cóctel de inhibidores de proteasas en el diluyente de inseminación (DI) para evitar parte de la actividad proteasa del plasma seminal de conejo. La calidad seminal y la fertilidad no se vieron afectadas por el cóctel. Sin embargo, la prolificidad fue significativamente menor en el grupo experimental en comparación con los grupos de control positivo y negativo (8,2±0,22 vs. 9,3±0,23 y 9,2±0,26 gazapos por parto, respectivamente). De este capítulo, se puede concluir que la adición de una amplia variedad de inhibidores de proteasas en el diluyente de semen de conejo afecta negativamente la tasa de prolificidad y que en el futuro sería aconsejable probar inhibidores específicos de aminopeptidasas (AMIs). En el capítulo II, suplementamos el DI con AMIs (bestatina y EDTA), y estudiamos su efecto sobre la calidad seminal y el rendimiento reproductivo. La inclusión de AMIs no afectó la calidad seminal, ni la fertilidad (85,3 vs. 88,6%), ni la prolificidad (10,12 vs. 10,51 gazapos por parto) en comparación con el grupo control. Por lo tanto, concluimos que los AMIs se pueden utilizar en los DIs de conejo para inhibir parte de la actividad aminopeptidasa del plasma seminal (PS). En el capítulo III, probamos nuevos DIs de conejo que contenían AMIs con o sin nanopartículas de quitosano (CS)-sulfato de dextrano (DS) que atrapan el análogo de GnRH. Se estudiaron los siguientes diluyentes: C4 (4 µg de buserelina/coneja en medio control (MC): tris-ácido cítrico-glucosa suplementado con AMIs), C5 (5 µg de buserelina/coneja en MC), Q4 (4 µg de buserelina/coneja en nanopartículas CS-DS en MC) y grupo Q5 (5 µg de buserelina/coneja en nanopartículas CS-DS en MC). La fertilidad fue significativamente menor en el grupo C4 en comparación con los grupos C5, Q5 y Q4 (0,7 frente a 0,85, 0,85 y 0,82, respectivamente). Por el contrario, la prolificidad fue similar en los cuatro grupos experimentales. Por lo tanto, las nanopartículas de CS-DS como transportador de acetato de buserelina permiten reducir la concentración de la hormona en diluyentes con AMIs sin afectar la fertilidad ni la prolificidad. Por ello, la nanoencapsulación parece ser un sistema prometedor para proteger el análogo de GnRH en los diluyentes de IA de conejos. Por otro lado, el objetivo de los últimos tres capítulos fue caracterizar las proteínas del semen de conejo. En los capítulos IV y V, se estudiaron las proteínas del PS de conejo. Las muestras de semen se recuperaron utilizando 6 machos de cada línea genética (A y R) y seleccionando una muestra heteroespérmica del comienzo, del medio y del final de cada estación y de cada línea (24 muestras en total). En el capítulo IV, utilizamos la técnica de electroforesis en gel de poliacrilamida 1D. Siete bandas proteicas fueron significativamente diferentes entre las líneas genéticas y tres bandas entre las estaciones. En el capítulo V, se sometió el PS a nano LC-MS/MS y se identificaron y cuantificaron 402 proteínas. 23 proteínas se expresaron diferencialmente entre genotipos. Con respecto al efecto de la estación en el proteoma del PS de conejo, los resultados mostraron que no hubo un patrón claro de variación de proteína a lo largo del año. En el capítulo VI, se caracterizaron las proteínas del espermatozoide de conejo. Se recuperaron 6 muestras espermáticas utilizando 5 machos de cada genotipo y se sometieron a nano LC-MS/MS, identificándose y cuantificándose 487 proteínas. 40 proteínas se expresaron diferencialmente entre genotipos. En conclusión, los resultados de los tres últimos capítulos evidencian que el genotipo está relacionado con una abundancia específica de proteínas del PS y del espermatozoide. Finalmente, se creó la / The general objectives of this thesis were to develop new artificial insemination (AI) extenders supplemented with a GnRH analogue and to characterise the proteomic profile of rabbit semen. In chapter I, the inclusion of a protease inhibitors cocktail in the insemination extender (IE) to avoid part of the rabbit seminal plasma protease activity was evaluated. Seminal quality and fertility rate were not affected by the cocktail, having similar values between experimental and control groups. However, prolificacy rate was significantly lower in experimental group compared to positive and negative control groups (8.2 ±0.22 vs. 9.3 ±0.23 and 9.2 ±0.26 total born per litter, respectively). From this chapter, it may be concluded that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate and it in the future it would be advisable to test specific aminopeptidase inhibitors (AMIs). Therefore, in chapter II, we supplemented the IE with AMIs (bestatin and EDTA), and we studied their effect on rabbit seminal quality and reproductive performance. Seminal quality was not affected by AMIs. Regarding reproductive performance, the inclusion of AMIs, did not affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery) in comparison with control group. Thus, we concluded that AMIs can be used in rabbit IEs to inhibit part of the seminal plasma aminopeptidase activity. In chapter III, we test new rabbit IEs containing AMIs with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. The following experimental extenders were studied: C4 (4 µg buserelin/doe in control medium (CM): Tris-citric acid-glucose supplemented with AMIs), C5 (5 µg of buserelin/doe in CM), Q4 (4 µg of buserelin/doe into CS-DS nanoparticles in CM) and Q5 group (5 µg of busereline/doe into CS-DS nanoparticles in CM). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 vs. 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied. Thus, CS-DS nanoparticles as carrier for buserelin acetate allow reducing the hormone's concentration in extenders supplemented with AMIs without affecting the fertility and prolificacy of rabbit females. Therefore, nanoencapsulation seems to be a promising system to protect the GnRH analogue in rabbit AI extenders. On the other hand, the aim of the last three chapters was to characterize rabbit semen proteins. In chapters IV and V, rabbit seminal plasma (SP) proteins were studied. Semen samples were recovered using 6 males from each genetic line (A and R). For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment (24 pools in total). In chapter IV, we used a 1D polyacrylamide gel electrophoresis approach. Seven protein bands were significantly different between genetic lines and three protein bands were significantly different between seasons. In chapter V, SP was subjected nano LC-MS/MS and 402 proteins were identified and quantified. Twenty-three proteins were differentially expressed between genotypes. Regarding the effect of season on rabbit SP proteome, results showed that there was no clear pattern of protein variation throughout the year. The results obtained in both chapters evidence that genotype is related to a specific abundance of SP proteins. In chapter VI, rabbit sperm proteins were characterised. Six samples were recovered during two months using 5 males from each genotype. Sperm proteins were subjected to nano LC-MS/MS and 487 proteins were identified and quantified. Forty proteins were differentially expressed between genotypes. In conclusion, rabbit sperm proteins showed that genotype has also a huge impact on their abundance. Finally, with these data, the first publicly accessible database of rabbit semen proteome was c / Els objectius generals d'aquesta tesi van ser desenvolupar nous diluents d'inseminació artificial (IA) suplementats amb un anàleg de GnRH i caracteritzar el proteoma del semen de conill. En el capítol I, es va avaluar la inclusió d'un còctel d'inhibidors de proteases en el diluent d'inseminació (DI) per evitar part de l'activitat proteasa del plasma seminal de conill. La qualitat seminal i la fertilitat no es van veure afectades pel còctel. No obstant això, la prolificitat va ser significativament menor en el grup experimental en comparació amb els grups de control positiu i negatiu (8,2 ± 0,22 vs. 9,3 ± 0,23 i 9,2 ± 0,26 catxaps per part, respectivament). D'aquest capítol, es pot concloure que l'addició d'una àmplia varietat d'inhibidors de proteases en el diluent de semen de conill afecta negativament la taxa de prolificitat i que en el futur seria aconsellable provar inhibidors específics de aminopeptidasas (AMIS). En el capítol II, suplementàrem el DI amb AMIs (bestatina i EDTA), i vam estudiar el seu efecte sobre la qualitat seminal i el rendiment reproductiu. La inclusió de AMIs no va afectar la qualitat seminal, ni la fertilitat (85,3 vs. 88,6%), ni la prolificitat (10,12 vs. 10,51 catxaps per part) en comparació amb el grup control. Per tant, concloem que els AMIs es poden utilitzar en els DIs de conill per inhibir part de l'activitat aminopeptidasa del plasma seminal (PS). En el capítol III, vam provar nous DIs de conill que contenien AMIs amb o sense nanopartícules de quitosà (CS)-sulfat de dextrà (DS) que atrapen l'anàleg de GnRH. Es van estudiar els següents diluents: C4 (4 µg de buserelina/conilla en medi control (MC): tris-àcid cítric-glucosa suplementat amb AMIs), C5 (5 µg de buserelina/conilla en MC), Q4 (4 µg de buserelina/conilla en nanopartícules CS-DS en MC) i grup Q5 (5 µg de buserelina/conilla en nanopartícules CS-DS en MC). La fertilitat va ser significativament menor en el grup C4 en comparació amb els grups C5, Q5 i Q4 (0,7 enfront de 0,85, 0,85 i 0,82, respectivament). Per contra, la prolificitat va ser similar en els quatre grups experimentals. Per tant, les nanopartícules de CS-DS com a transportador d'acetat de buserelina permeten reduir la concentració de l'hormona en diluents amb AMIs sense afectar la fertilitat ni la prolificitat. Per això, la nanoencapsulació sembla ser un sistema prometedor per protegir l'anàleg de GnRH en els diluents d'IA de conills. D'altra banda, l'objectiu dels últims tres capítols va ser caracteritzar les proteïnes del semen de conill. En els capítols IV i V, es van estudiar les proteïnes del PS de conill. Les mostres de semen es van recuperar utilitzant 6 mascles de cada línia genètica (A i R) i seleccionant una mostra heteroespérmica del començament, del mitjan i del final de cada estació i de cada línia (24 mostres en total). En el capítol IV, utilitzàrem la tècnica d'electroforesi en gel de poliacrilamida 1D. Set bandes proteiques van ser significativament diferents entre les línies genètiques i tres bandes entre les estacions. En el capítol V, es va sotmetre el PS a nano LC-MS / MS i es van identificar i quantificar 402 proteïnes. 23 proteïnes es van expressar diferencialment entre genotips. Pel que fa a l'efecte de l'estació en el proteoma del PS de conill, els resultats van mostrar que no hi havia un patró clar de variació proteica al llarg de l'any. En el capítol VI, es van caracteritzar les proteïnes de l'espermatozoide de conill. Es van recuperar 6 mostres espermàtiques utilitzant 5 mascles de cada genotip i es van sotmetre a nano LC-MS / MS, identificant i quantificant 487 proteïnes. 40 proteïnes es van expressar diferencialment entre genotips. En conclusió, els resultats dels tres últims capítols evidencien que el genotip està relacionat amb una abundància específica de proteïnes del PS i de l'espermatozoide. Finalment, es va crear la primera base de dades d'accés públic del proteoma / Casares Crespo, L. (2018). Development of new artificial insemination extenders supplemented with GnRH analogues to induce ovulation and proteomic characterization of rabbit semen [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/100854 / Compendio

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.

A espécie Callithrix jacchus pode ser utilizada como modelo experimental para outras espécies de primatas neotropicais, principalmente Callithriquídeos. Colheu-se sêmen desta espécie com os objetivos de otimizar a colheita de sêmen por vibro estimulação peniana, validar técnicas de avaliação espermática que possam ser utilizadas a campo, comparar as características seminais de indivíduos de duas colônias (Brasil e Alemanha), testar diferentes protocolos de congelação espermática e avaliar a atividade citoquímica mitocondrial (DAB) e a fragmentação de DNA de espermatozóides congelados. As técnicas de coloração para avaliação espermática utilizadas foram: eosina-nigrosina, coloração dupla trypan-blue giemsa, coloração simples para acrossoma, FITC-PSA, SpermOscan®, Spermac®, coloração com 3,3&#39;-diaminobenzidina (DAB) e ensaio Cometa alcalino. A congelação foi realizada com 100 µL de sêmen com meio TEST-Gema de ovo clarificado com glicerol 4% em palhetas 0,25 mL, e os protocolos de congelação testados foram: 1) fase de equilíbrio com curva de resfriamento (25°C-4°C) em 2 horas, 10 minutos no vapor de nitrogênio e finalmente imersão em nitrogênio líquido; 2) como Protocolo 1, mas sem fase de equilíbrio/resfriamento; e 3) diretamente no nitrogênio líquido. As palhetas foram descongeladas em água a 37°C por 5-10 segundos. A técnica de colheita resultou em 83,33% dos procedimentos com ejaculação. A coloração simples para acrossomo foi eficiente e pôde ser validada para utilização a campo. Não houve diferença significante (p<0,05) nas características seminais avaliadas entre as colônias do Brasil e da Alemanha. As análises pós-descongelação demonstraram queda nas variáveis motilidade e integridade da membrana plasmática com discreta superioridade no Protocolo 1, não houve diferença significante (p<0,05) entre os protocolos para a atividade citoquímica mitocondrial e o Protocolo 2 apresentou a menor taxa de fragmentação de DNA. / The Callithrix jacchus can be used as a model species for other Neotropical primates species, mainly Callithriquids, for reproductive studies. Semen samples were collected with the following objectives: to improve the penile vibrostimulation technique, validate sperm evaluation techniques for field work, analyse the semen characteristics from animals at 2 colonies (Brazil and Germany), use 3 different freezing protocols in common marmosets sperms, evaluate a cytochemical technique for mitochondrial activity demonstration and evaluate DNA damage in frozen-thawed sperms. The staining techniques used in this work were: Eosin-nigrosin, Trypan-blue Giemsa, simple staining for acrosome, FITC-PSA, SpermOscan®, Spermac®, demonstration of cytochrome c oxidase activity in situ by the oxidation of 3,3&#39;-diaminobenzidina (DAB) and the Comet assay. Freezing was done in 100 µL semen with TESt-Yolk clarified medium plus 4% glycerol using 0.25 mL straws. The freezing protocols tested were: 1) straws at equilibrium phase with a 25°C to 4°C curve in 2 hours, 10 minutes at nitrogen vapour and finally into liquid nitrogen; 2) same as Protocol 1 without the equilibrium phase; and 3) straws directly into liquid nitrogen. All attempts resulted in 83,33% ejaculates. The simple staining technique for acrosome was validated and resulted in an obvious differentiation of the intact acrosome. Semen characteristics evaluation showed no difference between the 2 colonies. Post-thaw evaluation showed all protocols had negative effects on motility and plasmatic membrane integrity with a slightly superiority on Protocol 1. There was no difference between protocols in the mitochondrial activity demonstration and Protocol 2 presented the lowest DNA damage.

No Brasil, assim como em outros países tropicais, a criação de bovinos é feita de maneira extensiva e, portanto é suscetível às intempéries climáticas. Esse tipo de criação pode levar ao estresse térmico, mesmo em animais zebuínos. Sob condições de estresse térmico é possível a ocorrência de degeneração testicular, e por isso, há a procura por animais tolerantes ao calor. Desta maneira, este trabalho objetivou a avaliação da tolerância ao calor de touros da raça Brahman utilizando o teste de tolerância ao calor, e também a análise da qualidade seminal relacionada à disponibilidade de sombra nos pastos. Para tal, foram utilizados 10 touros da raça Brahman com idades entre 24 e 30 meses. Duas semanas antes do início das análises, três amostras de sêmen foram colhidas de cada animal para o nivelamento biológico do sêmen, e então os touros foram separados em dois grupos: 5 touros foram alocados em pasto com disponibilidade de sombra enquanto 5 touros foram alocados em pasto sem qualquer tipo de sombra. O teste de tolerância ao calor foi realizado em três dias consecutivos típicos de verão, e as colheitas de sêmen foram realizadas em 14 dias durante 2 meses, totalizando 4 colheitas. Anteriormente a todas as colheitas foram avaliadas a consistência testicular e o perímetro escrotal. As características seminais avaliadas foram volume, aspecto, turbilhonamento, motilidade, vigor e concentração e morfologia espermáticas. Os dados obtidos foram analisados com o pacote estatístico Statistical Analysis System (SAS Institute Inc., 2004). Não houve diferenças estatísticas entre os dois grupos de animais. O resultado do teste de tolerância ao calor mostrou que os animais testados são tolerantes ao calor e apresentam boa capacidade termolítica. / In Brazil, similarly to others tropical countries, bovines are bred under environmental conditions of extensive system. This breed system type may lead to heat stress even on zebu animals. Under heat stress conditions, testicular degeneration can occur and, therefore, there is a search for animals that are heat tolerant. Therefore, the aim of this work was to evaluate the heat tolerance of Brahman bulls using the heat tolerance test and to verify the occurrence of any possible effect of shadow availability on pasture on semen quality. Ten Brahman bulls aging between 24 and 30 months were used in this work. For biological semen evaluation, three semen samples were collected from each animal two weeks before beginning. The bulls were then separated into two groups: 5 animals were allocated on pasture with shadow availability and 5 bulls were allocated on pasture without any kind of shadow. The heat tolerance test were performed in three consecutive typical summer days and the semen samples were collected each 14 days during 2 months, in a total of 4 semen samples per animal. Testicular consistence and scrotal circumference were measured just before every semen collection. The semen\'s characteristics evaluated were volume, aspect, mass movement, motility, straight movement, sperm concentration and morphological exam. Data obtained from experimental proceedings were analyzed by Statistical Analysis System program (SAS Institute Inc., 2004). No difference was observed (P>0.05) in any of the characteristics analyzed when Brahman bulls were maintained on pastures with or without shadow availability. The performance of tested animals on the heat tolerance test shows that these animals are heat tolerant and present good thermolitic ability.

Em sete adolescentes com dermatomiosite (DM) juvenil (DMJ) foi avaliada a função gonadal através do estadiamento puberal, aspectos da sexualidade, exame físico da genitália e exames complementares: análise seminal (duas amostras com intervalo de um mês), anticorpos anti-espermatozóides, ultra-sonografia escrotal e dosagens hormonais (testosterona, hormônio estimulante do folículo, hormônio luteinizante, prolactina, T3, T4, T4 livre e TSH). Todos os pacientes apresentaram terazospermia, dois tiveram varicocele e um anticorpo anti-espermatozóide localizado em peça intermediária. A futura fertilidade destes pacientes é incerta e estudos de prevalência de função gonadal em populações de jovens e adultos do sexo masculino com DM são necessários / In seven adolescents with dermatomyositis (MD) juvenile (JDM), gonadal function was evaluated through the puberal estadiamento, aspects of the sexuality, examination of the genitalia, semen analysis (two semen samples over a period of one month), anti-sperm, testicular ultrasound and hormones (testosterone, follicle stimulating hormone, luteinizing hormone, prolactin, T3, T4, free T4 and TSH).