La capacitación espermática puede ser mimetizada in vitro eliminando el plasma seminal por distintos sistemas de lavado. Entre los tratamientos espermáticos empleados habitualmente en los laboratorios para capacitar a los espermatozoides se encuentran los lavados que se realizan con medios enriquecidos con albúmina o a través de gradientes de Percoll. El objetivo de este trabajo ha sido determinar los cambios que acontecen en los espermatozoides (procedentes de epidídimo y eyaculados) sometidos a tres sistemas de capacitación in vitro evaluados mediante una batería de técnicas que determinan distintos estadios de la capacitación espermática.De los resultados obtenidos se desprende que tanto la procedencia de los espermatozoides (epidídimo o eyaculado) como el tratamiento de capacitación al que se les somete afecta en gran medida a los resultados de la penetración in vitro y por lo tanto, la capacitación se produce de manera diferente entre estos grupos. / Sperm capacitation may be defined as a set of molecular modifications that occurs in the spermatozoa, after maturation in the epididymis, which enables them to fertilize the oocyte. In vitro, this process can be imitated in vitro by separation of the seminal plasma by different systems of washing. Between the sperm treatments routinely used in the laboratories, semen samples are washed of albumin or centrifugations through a Percoll. The aim of this work was examine and characterize the changes that happen in the sperms (from epididymis and ejaculated) submitted to three systems of in vitro capacitation, evaluated by means of a battery of tests to determine different levels of the sperm capacitation. Sperm capacitation was dependent on sperm treatment, whetherepididymal or ejaculated and to whichever parameter measured.Nevertheless, all these parameters, in spite of the fact that they have beendescribed as tools to evaluate the sperm capacitation, really are not capable of discriminating or indicating the level of capacitation.

El present treball analitza al microscopi òptic i al microscopi electrònic de transmissió el testicle de Sus domesticus (raça Landrace - varietat anglesa) a partir de mascles reproductors porcins adults i sans. L'objectiu principal de tots els centres d'Inseminació Artificial Porcina i de les Explotacions de Selecció i Multiplicació Porcina és garantir una excel·lent qualitat espermàtica al llarg de la vida reproductiva útil d'un mascle reproductor porcí. Així doncs, un millor coneixement dels patrons estructural i ultraestructural normals del testicle permetrà diagnosticar amb facilitat quina ha estat l'estructura o funció testicular afectada quan s'observa una disminució de la qualitat del semen. Les anàlisis seminals i hormonals són certament crucials en la valoració d'aquests mascles, però, no són totalment informatives de les alteracions testiculars, ja que és necessari conèixer l'organització microscòpica.Diversos estudis sobre testicle han demostrat que els marcadors més sensibles per a l'avaluació de la funció testicular són els següents: (1) la grandària testicular, (2) el gruix i l'organització de la càpsula testicular, (3) el percentatge de túbuls seminífers i de teixit intersticial en el parènquima testicular, (4) el diàmetre dels túbuls seminífers, (5) l'alçada i la composició de cèl·lules germinals de l'epiteli seminífer, (6) el gruix i l'organització de la làmina pròpia i, (7) la morfologia i la grandària de les cèl·lules de Leydig. El primer objectiu concret del present estudi ha estat, per tant, caracteritzar tots aquests paràmetres testiculars en mascles porcins sans i adults. L'organització estructural del testicle i les mesures quantitatives utilitzades com a marcadors no mostren diferències significatives ni entres els mascles porcins (P > 0,01), ni entre el testicle dret i l'esquerre (P > 0,01). Els testicles, de 330,80  16,99 g de pes, estan envoltats per una càpsula, de 2.375,13  246,68 m de gruix, la qual es divideix en tres capes: la túnica vaginalis constitueix l'1,82  0,78 % de la càpsula i està composta per una capa mesotelial externa i una capa interna de teixit conjuntiu dens; la túnica albuginea representa el 37,31  3,27 % i és de teixit conjuntiu dens i, la túnica vasculosa constitueix el 64,24 4,40 % i és de teixit conjuntiu lax. En el parènquima testicular els túbuls seminífers i el teixit intersticial representen el 72,44  2,12 % i el 27,46  2,12 %, respectivament. Els túbuls seminífers, de 226,23  18,08 m de diàmetre, es troben fortament recargolats i empaquetats, i estan compostos per la làmina pròpia i l'epiteli seminífer. La làmina pròpia, de 4-4,5 m de gruix, està formada per la làmina basal i dues capes de cèl·lules peritubulars. L'epiteli seminífer, amb una alçada mitjana de 66,11  10,62 m, és columnar i estratificat amb cèl·lules de Sertoli i diferents generacions d'espermatogònies, espermatòcits i espermàtides. El teixit intersticial és un teixit conjuntiu lax amb abundants cèl·lules de Leydig polièdriques fortament empaquetades (ca. 15 x 12 m).El segon objectiu concret d'aquest estudi ha estat estudiar des del punt de vista morfològic i morfomètric (alçada, longitud, freqüència relativa d'aparició i durada) els estadis del cicle de l'epiteli seminífer en els mascles porcins de la raça Landrace (varietat anglesa), classificats d'acord amb el mètode de la morfologia tubular. Els estadis premeiòtics ( I, II i III) ocupen el 31,9 % del cicle espermatogènic i es caracteritzen, principalment, per la presència de cèl·lules en les fase inicials de la meiosi I. Les primeres etapes de la meiosi I no afecten els paràmetres morfomètrics de l'epiteli seminífer ja que els valors obtinguts per l'alçada de l'epiteli seminífer, la freqüència relativa, la longitud i la durada d'aquests estadis són molt variables. Els estadis meiòtics (IV i V) representen el 16,4 % del cicle espermatogènic i estan constituïts, principlament, per cèl·lules en un estat avançat de la meiosi I i /o cèl·lules en meiosi II. Les últimes fases de la meiosi I i també de la meiosi II tenen lloc ràpidament, la qual cosa resulta en una baixa freqüència relativa d'aparició i, per tant, en una baixa durada dels estadis meiòtics. Els estadis postmeiòtics (VI, VII i VIII) ocupen el 50,6 % del cicle espermatogènic. L'esdeveniment més important que té lloc en aquests estadis és la fase de maduració de l'espermiogènesi. En la fase de maduració, les espermàtides experimenten diverses modificacions morfològiques i estructurals que donen lloc, finalment, als espermatozoides. La complexitat d'aquests processos fa que els estadis postmeiòtics presentin valors més grans de freqüència relativa, longitud i durada.El tercer objectiu concret d'aquest treball ha estat descriure a nivell ultraestructural el procés d'espermiogènesi, i relacionar les transformacions que experimenten les espermàtides en fase d'elongació amb els canvis ultraestructurals que tenen lloc en les diferents cèl·lules que constitueixen el testicle (cèl·lules germinals, de Sertoli i de Leydig, principalment). L'espermiogènesi del mascle porcí de la raça Landrace (varietat anglesa) s'ha dividit en 9 passos que vénen definits per 9 tipus diferents d'espermàtides. Al llarg de l'espermiogènesi no s'observen diferències ultraestructurals significatives (P > 0,01) ni entre els mascles porcins ni entre el testicle esquerre i dret en les cèl·lules que constitueixen el testicle. / The present study describes the structure and ultrastructure of the Sus domesticus testis (Landrace breed -british variety) from healthy adults boars. The main goal of the whole of Porcine Artifitial Insemination Centres and of the Porcine Livestocks is to guarantee an excellent spermatic quality along the boar reproductive life. Therefore, a better knowlegment of the normal structural and ultrastructural patterns of the testis will improve the prognosis of subfertility when a low spermatic quality is observed. Both seminal and hormonal analysis are certainly crucial in the assessment of these males, but it is also necessary to know the microscopic organization.Several studies have demonstrated that the most sensitive markers of impaired function are: (1) the testicular size, (2) the thickness and organization of the testicular capsule, (3) the percentage of seminiferous tubules and interstitial tissue in the testicular parenchyma, (4) the diameter of seminiferous tubules, (5) the height and germ cell composition of the seminiferous epithelium, (6) the thickness and organization of the lamina propria, and (7) the Leydig cell size and morphology. The first aim of this study has been to characterize all these testicular parameters in healthy adults boars. The structural organization of the testis and quantitative measures used as markers did not differ significantly either among boars (P > 0.01), or between left and right testes (P > 0.01). Testes, of 330.80  16.99 g weight, were surrounded by a capsule, of 2,375.13  246.68 m thick, divided into three layers: the tunica vaginalis constituted 1.82  0.78% of the capsule and was composed by an outer mesothelial layer and an inner dense connective tissue layer; the tunica albuginea represented 37.31  3.27% and was of dense connective tissue and the tunica vasculosa constituted 64.26  4.40% and was of loose connective tissue. In the testicular parenchyma, the seminiferous tubules and the interstitial tissue comprised 72.44  2.12% and 27.46  2.12%, respectively. Seminiferous tubules were highly convoluted ducts of 226.23  18.08 m in diameter composed by a lamina propria and the seminiferous epithelium. The lamina propria, of 4-4.5 m thick, was formed by basal lamina and two layers of peritubular cells. The seminiferous epithelium, of 66.11  10.62 m high, was stratified columnar with Sertoli cells and different generations of spermatogonia, spermatocytes and spermatids. The interstitial tissue was loose connective tissue with abundant and closely-packed polyhedral Leydig cells (av. 15 x 12 m).The second aim of this study has been to describe the morphological features of the eight stages of the seminiferous epithelium in Landrace boars (british variety) according to the tubular morphology method, as well as their relative frequency, length, height and duration. Premeiotic stages (I, II and III) occupied the 31.9 % of the spermatogenic cycle and were mainly characterized by the presence of cells in the initial phases of meiosis I. Early meiosis I did not affect the morphometric parameters of the seminiferous epithelium as indicated by the variable values obtained in the seminiferous epithelium height, as well as in the relative frequency, length, and duration of premeiotic stages. Meiotic stages (IV and V) represented the 16.4 % of the spermatogenic cycle and were constituted, mainly, by cells in advanced meiosis I and/or cells in meiosis II. Last phases of meiosis I and also meiosis II occurred rapidly, resulting in low relative frequency and, therefore, in low duration of meiotic stages. Postmeiotic stages (VI, VII and VIII) occupied the 50.6 % of the spermatogenic cycle. The most important event of these stages was the maturation phase of spermiogenesis. The maturation phase included several morphological and ultrastructural modifications in spermatids, resulting in the formation of spermatozoa. The complexity of these processes correlated with the high relative frequency, length, and duration of postmeiotic stages.The third aim of this study has been to describe the spermiogenesis process at ultrastructural level and, to relate the spermatid transformations along the spermiogernesis with the ultraestructural changes undergoing in testicular cells (mainly germinal, Sertoli and Leydig cells).The spermiogenesis of Landrace boars (british variety) was divided into 9 steps, each one characterized by the presence of an specific spermatid type. Significant differences were found neither among the three healthy boars (P > 0.01), nor the left and right testes (P > 0.01) in the ultrastructure of the testiculars cells along spermiogenesis.

A regeneração óssea guiada é usada na reparação de defeitos ósseos com suficiente evidência de sucesso. Dentro desta técnica, os xenoenxertos são uma boa opção devido as suas características e segurança para o paciente. Todavia, estudos para melhorar as propriedades dos substitutos ósseos para a formação adequada de novo osso são constantes. Os bisfosfonatos (BPs) são análogos sintéticos dos pirofosfatos, constituem a primeira linha de tratamento para algumas desordens ósseas e tem sido utilizado com sucesso para reduzir o risco de fratura e melhorar a qualidade de vida dos pacientes. Os efeitos adversos e benefícios dos BPs são amplamente estudados. Está comprovado que os BPs inibem a reabsorção óssea reduzindo a atividade dos osteoclastos, mas também que as células osteoblásticas na presença de BPs que contêm nitrogênio aumentam sua proliferação e sua diferenciação na linhagem osteoblástica, promovendo mineralização além de inibir a apoptose de osteócitos e osteoblastos. Neste trabalho estudou-se o efeito do alendronato administrado local e sistemicamente na regeneração óssea com xenoenxerto porcino em ratos com defeitos críticos no osso parietal. Foram usados sessenta ratos Wistar albinos, distribuídos em três grupos (n=20), sendo que o grupo controle (GC) teve o defeito tratado apenas com xenoenxerto, o grupo experimental XE-ALNL que recebeu o xenoenxerto previamente hidratado com alendronato sódico, e o terceiro grupo, XE-ALNS, recebeu xenoenxerto com administração sistêmica diária de alendronato. As amostras foram fixadas, descalcificadas e processadas para análise histológica em microscopia de luz, histoquímica para fosfatase ácida tartarato-resistente (TRAP), e imuno-histoquímica para osteopontina (OPN). Determinou-se que o uso de alendronato potencializa a formação de novo osso. O que pode ser explicado pelo efeito conservador do alendronato no xenoenxerto prolongando seu efeito osteocondutor. Os resultados do grupo XE-ALNL mostraram que o efeito do alendronato sódico usado na hidratação do xenoenxerto colocado no defeito provocou uma maior formação de novo osso primário, tanto ao redor das bordas que limitavam o defeito como dos grânulos de xenoenxerto, quando comparado ao GC e ao grupo XE-ALNS, para os dois períodos avaliados (30 e 60 dias). Ainda que no grupo XE-ALNS nas amostras avaliadas para o período de 30 dias seu padrão foi similar ao GC, no período de 60 dias se observou maior quantidade de osso novo em relação ao GC no mesmo período. A presença de tecido conjuntivo com suas fibras colágenas entre os grânulos do xenoenxerto foi confirmada com a coloração de tricrômico de Mallory. A imunomarcação de OPN mostrou as áreas de osso primário formadas, bem como a presença de algumas linhas cimentantes. A escassa presença de osteoclastos evidenciou a baixa taxa de reabsorção dos grânulos do xenoenxerto nos períodos avaliados. / Guided bone regeneration is used in treatment for repairing bone defects with proven evidence of success. Within this technique, xenografts are a good alternative because of its characteristics and safety for the patient; however, studies aiming to enhance the properties of bone substitutes for proper formation of new bone is continuous. Bisphosphonates (BPs) are synthetic analogues of pyrophosphates, they represent the first line of treatment for some bone disorders. They have been successfully used to reduce the risk of fracture and improve the quality of life for patients, its adverse effects and benefits are widely studied. It has been established that BPs inhibit bone resorption by reducing osteoclast activity but also that osteoblastic cells in the presence of nitrogen-containing BPs increase their proliferation and differentiation in the osteoblastic lineage, inducing mineralization, and inhibiting apoptosis of osteocytes and osteoblasts. This study investigated the effect of alendronate administered locally and systemically on bone regeneration with porcine xenograft in rats with critical defects (5 mm in diameter) made in the parietal bone. Sixty Wistar albino rats were divided into three groups (n=20): control group (CG) with defect treated only with xenograft, XE-ALNL group received xenograft previously hydrated in 1 mg/ml of alendronate sodium, and the third group, XE-ALNS, received xenograft with daily systemic administration of alendronate (2.5 mg / kg). Each experimental group was randomly divided into two sub-groups (n=10): in the first sub-group of each experimental group the animals were sacrificed after 30 days and in the second after 60 days. The samples were fixed, decalcified and processed for light microscopic analysis, histochemistry for tartrate-resistance acid phosphatase (TRAP), and immunohistochemistry for osteopontin (OPN). The results of the XE-ALNL group showed that the effect of sodium alendronate used in the hydration of the xenograft placed in the defect caused a superior formation of new primary bone, both around the edges that limited the defect and the xenograft granules, when compared to CG and the XE-ALNS groups, for both periods of 30 and 60 days. For the XE-ALNS group even though smaller amount of primary bone was formed when compared to XE-ALNL at 30 and 60 days, its pattern was similar to the CG at 30 days; however, for the 60 days sub-group a greater amount of new bone was observed when compared to the CG in the same period. The presence of connective tissue with its collagen fibers between the granules of the xenograft was confirmed with Mallory\'s trichrome staining. The OPN immunolabeling showed the areas of primary bone formed, as well as the presence of cement lines. The low osteoclast presence indicated a low rate of xenografts reabsorption in the evaluated periods.

Em procedimentos de regeneração óssea guiada (ROG) as membranas de colágeno são os materiais mais utilizados como barreira; porém, sua tendência ao colapso faz indispensável a utilização de materiais de suporte. Para este propósito, os xenoenxertos são substitutos ósseos considerados padrão-ouro, embora apresentem um período longo de reabsorção que impede grande formação do osso. Em contrapartida o ?-fosfato tricálcico (?-TCP) permite boa formação do osso, mas é reabsorvido rapidamente e fracassa quando precisa dar suporte à membrana. O alendronato, um bisfosfonato nitrogenado, é uma droga antirreabsortiva para o tratamento da osteoporose e outras doenças ósseas porque inibe a função dos osteoclastos. O plasma rico em fibrina (PRF) é um concentrado de fibrina sem adição de químicos que consegue estimular processos de cicatrização pelos fatores que fazem parte de sua composição. Neste estudo qualitativo de ROG em defeitos de 5 mm no osso parietal de ratos foi avaliado: 1) o efeito na formação óssea da administração local de 1g/ml de alendronato sódico adicionado a xenoenxerto porcino e a ?-TCP; 2) a adição local de alendronato e PRF a ?-TCP na possibilidade de diminuir a rápida reabsorção do material e impedir o colapso da membrana. Foram usados 100 ratos adultos Wistar distribuídos em 5 grupos (n=20): Xenoenxerto controle (XE-C); xenoenxerto adicionado com alendronato (XE-AL); ?-TCP controle (TCP-C); ?-TCP adicionado com alendronato (TCP-AL); e, ?-TCP adicionado com PRF (TCP-F). Em todos os grupos o enxerto foi coberto com membrana. Dois tempos de estudo de quatro e oito semanas foram considerados para cada grupo (n=10). Ao final de cada tempo, os animais foram sacrificados e as amostras foram fixadas, descalcificadas e processadas para seu estudo em microscopia de luz por meio de análise histológica, histoquímica TRAP e imuno-histoquímica para osteopontina (OPN). Os resultados mostraram maior formação do osso tanto para xenoenxerto como para ?-TCP quando foram adicionados com alendronato local, em ambos tempos de estudo. Nos grupos do ?-TCP a adição de alendronato local permitiu diminuir a reabsorção dos grânulos, melhorando o suporte à membrana ao final dos tempos de estudo; no entanto, no grupo do PRF a reabsorção foi maior e teve pouca formação de osso, provocando colapso da membrana. Adicionalmente, regiões de osso primário subjacentes à membrana de colágeno foram observadas em todos os grupos. / Collagen membranes are the most used materials as a barrier in guided bone regeneration (GBR) procedures; however, its tendency to collapse makes indispensable the use of support materials. For this purpose, xenografts, which are bone substitutes, although they have a long period of resorption that prevents large bone formation, are still considered the gold standard support material. In contrast, tricalcium ?-phosphate (?-TCP) allows good bone formation, but is rapidly reabsorbed and fails when it needs to support the membrane. Alendronate, a nitrogenated bisphosphonate, is an anti-resorptive drug for treatment of osteoporosis and other bone diseases because it inhibits the function of osteoclasts. Fibrin-rich plasma (FRP) is a fibrin concentrate with no added chemicals that can stimulate healing processes by the factors that are part of its composition. In this qualitative study of ROG in 5 mm defects in the rat parietal bone, was evaluated: 1) the effect on bone formation of local administration of 1g / ml sodium alendronate added to porcine xenograft and ?-TCP; 2) the local addition of alendronate and PRF to ?-TCP in the possibility of diminishing the rapid reabsorption of the material and preventing the collapse of the membrane. A 100 adult Wistar rats distributed in 5 groups was used (n = 20): Xenograft control (XE-C); xenograft added with alendronate (XE-AL); ?-TCP control (TCP-C); ?-TCP added with alendronate (TCP-AL); and, ?-TCP added with PRF (TCP-F). In all groups the graft was covered with membrane. Two study times of four and eight weeks were considered for each group (n = 10). At the end of each time, the animals were sacrificed and the samples were fixed, decalcified and processed for light microscopy by histological analysis, TRAP histochemistry and immunohistochemistry for osteopontin (OPN). Results showed higher bone formation for both xenograft and ?-TCP when added with local alendronate at both study times. In the ?-TCP groups the addition of local alendronate allowed to decrease grain resorption, improving membrane support at the end of the study times; however, in the PRF group the resorption was greater and had little bone formation, causing membrane collapse. In addition, primary bone formed in the underlying collagen membrane were observed in all groups.

En aquest treball s'han desenvolupat dos mètodes simples i ràpids pel cultiu de les cèl·lules epitelials de les tres regions de l'epidídim de Sus domesticus. Un es basa en el cultiu de fragments del túbul epididimari intactes durant 8 dies. L'altre mètode es basa en el cultiu de fragments del túbul epididimari digerits amb col·lagenasa que, després de 7 dies, donen lloc a la formació d'una monocapa de cèl·lules epitelials epididimàries que adquireixen el 90-100% de confluència després de 12-16 dies en cultiu. Aquestes cèl·lules es mantenen viables durant més de 60 dies en cultiu i no s'observa proliferació de cèl·lules no epitelials. Per determinar el nivell de conservació de les característiques epididimàries en els cultius s'ha analitzat l'estructura cel·lular, l'activitat de síntesi i secreció proteica, i el manteniment i maduració dels espermatozoides en cocultiu. / In this work, we have developed two simple and quick methods for the culture of boar epididymal epithelial cells. The first one involves the culture of intact epididymal tubule fragments during 8 days. The second one involves the culture of epididymal tubule fragments digested with collagenase that, after 7 days in culture, allows the formation of an epididymal epithelial cell monolayer that reached 90-100% confluence after 12-16 days. These epididymal epithelial cells monolayers were maintained in vitro for more than 60 days and overgrowth of non-epithelial cells was not observed. To estimate the level of preservation of epididymal characteristics in these cultures we have focused on cell morphology, protein secretion activity, and sperm preservation and maturation.

L'objectiu és millorar els gels de plasma porcí induïts per calor a pH àcid utilitzant transglutaminasa microbiana (MTGasa). El tractament millora textura i CRA dels gels a pH 5,5, però les millores no són suficients per recuperar les pèrdues degut a l'acidificació. L'estructura globular de les proteïnes dificulta l'atac enzimàtic. La reactivitat de l'enzim no millora amb l'addició de cisteïna a plasma amb MTGasa. El tractament del plasma amb MTGasa sota alta pressió (HP) millora la duresa dels gels. No obstant, la CRA només millora lleugerament. La duresa es pot incrementar mantenint les solucions de plasma pressuritzat sota refrigeració, encara que no millora la CRA. Es pot concloure que les pèrdues en la textura dels gels de plasma induïts per calor a pH àcid es poden recuperar parcialment tractant amb MTGasa, especialment afegint cisteïna o sota HP. Encara que la CRA només es veu lleugerament millorada en el segon cas. / The objective of the thesis is to improve the heat-induced gel properties of porcine blood plasma at acid pH using microbial transglutaminase (MTGase). The enzymatic treatment enhances texture and WHC of plasma gels at pH 5.5, although increases are not enough to overcome loses on gelling properties. The globular structure of proteins can make the enzymatic attack difficult. The enzyme reactivity is not enhanced when cysteine is added to plasma treated with MTGase. Treating plasma with MTGase under HP increases hardness. However, WHC is only slightly modified. These achievements on hardness can be enhanced by holding pressurised-plasma solutions at refrigeration conditions. In contrast, it has no effects on WHC. Overall, losses in texture of heat-induced plasma gels at acid pH are recovered to an important degree by treating plasma with MTGase, especially with added cysteine or under HP conditions. However, their WHC is only slightly enhanced for the later.

La interacción entre gametos es crucial para la fecundación. La zona pelúcida (ZP) se considera responsable de bloquear la polispermia, pero in vitro estas funciones no son totalmente eficientes. La polispermia es frecuente en fecundación in vitro (FIV) en porcino y bovino, mientras que la interacción heteróloga espermatozoide-ovocito ha sido demostrada. Los objetivos fueron estudiar el bloqueo de la polispermia para mejorar los resultados de FIV e investigar las interacciones heterólogas entre espermatozoide humano y ovocito porcino. Los resultados demuestran que se produce endurecimiento de la ZP de ovocitos bovinos y porcinos de forma previa a la fecundación, utilizando DTSP o fluido oviductal bovino. Cuando se utilizan estos ovocitos en FIV aumenta la monospermia y el rendimiento final. En las interacciones heterólogas los espermatozoides humanos pueden unirse a ZP porcina y sufren la reacción acrosómica, pero no penetran los ovocitos sin ZP. En ICSI activan el ovocito y forman pronúcleos. / The interaction between gametes is crucial to fertilization. The zona pellucida (ZP) is responsible to block of polyspermy, but in vitro these functions are not efficient. The polyspermy is frequently in bovine and porcine in vitro fecundation. Besides the heterologous interaction between spermatozoa-oocyte had been described. The aims were study the block of polyspermy to improve the output of IVF and research the heterologous interactions between human spermatozoa and porcine oocyte.The results show that there is hardening of bovine and porcine ZP previously at fertilization, in vivo and using DTSP or bovine oviductal fluid. When these oocytes are used in IVF improve the monospermy and the output. In heterologus interactions the human spermatozoa could bind to porcine ZP and it triggers the acrosome reaction, but not penetration in ZP-free oocyte was observed. In ICSI the oocyte activation and

La transgénesis es una potente herramienta biotecnológica para la generación de animales modificados genéticamente con aplicaciones en diversas áreas como veterinaria, biomedicina y agricultura. La técnica de transgénesis mediada por espermatozoides (SMGT) se basa en la habilidad intrínseca de las células espermáticas para unir e interiorizar ADN exógeno y permitir su transferencia al interior de los ovocitos tras la fecundación, e integrarse en el genoma del nuevo embrión. El objetivo global de este estudio fue desarrollar un método eficiente de producción in vitro de embriones porcinos y lechones transgénicos aprovechando la capacidad de transferencia de ADN exógeno que presenta el espermatozoide.La combinación de la técnica de ICSI y SMGT es un método eficiente para la producción de lechones y embriones transgénicos, viéndose incrementada su eficiencia mediante el uso de la recombinasa RecA, obteniendo los primeros lechones nacidos en España mediante ICSI, de los cuales 2 fueron transgénicos. / The transgenesis is a powerful biotechnological tool for the generation of genetically modified animal with applications in different areas such as veterinary medicine, biomedicine and agriculture. The technique of sperm mediated gene transfer (SMGT) is based on the intrinsic ability of the spermatozoa to bind exogenous DNA and to allow its transference to the oocytes after fertilization, and to integrate in the genome of the new embryo. The global objective of this study was to develop an efficient method of in vitro production of transgenic embryos and piglets using the spermatozoa capacity to bind to exogenous DNA.The combination of the ICSI technique and SMGT is an efficient method for the production of transgenic embryos and pigs. The efficiency was increased by the use of recombinase RecA, obtaining the first piglets born in Spain by means of ICSI and two of them were transgenic.

Este trabajo se ha centrado en el estudio de diferentes factores que afectan a la capacidad fecundante del semen congelado, como la velocidad de descongelación y el sistema antioxidante del semen porcino. También se ha estudiado la capacidad predictiva de la fertilidad in vivo que tienen las diferentes técnicas de análisis seminal tanto para el semen congelado como en condiciones de campo para el semen refrigerado. Se pudo determinar que la velocidad de descongelación más rápida tiene un efecto positivo sobre la funcionalidad espermática de las muestras evaluada mediante un sistema de FIV. Igualmente se concluyó también que el sistema de FIV parece ser la mejor herramienta disponible para evaluar la calidad del semen congelado-descongelado. Se evidenció que el proceso de crioconservación del semen supuso una pérdida siginificativa en el contenido de GSH intracelular. Finalmente, se demostró que el análisis seminal sólo puede identificar eyaculados con bajo potencial fértil. / Boar frozen-thawed semen is still a valuable tool as a complement to artificial insemination with fresh semen in some conditions. The objectives were firstly, the design of better freezing methods in order to obtain acceptable semen quality (freezing-thawing procedures) and secondly, to address the question of whether differences in farrowing rate and litter size after the use of different ejaculates could be predicted using the standard semen parameters under commercial conditions.We can determine that the IVF fertilization system seems to be a good tool to evaluate the quality of frozen-thawed boar semen previous to its commercial way. In other way, we found that there was a loss in GSH content after cryopreservation of boar semen and the addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Finally, semen analysis, under commercial conditions, allows to identify ejaculates with very low fertility potential. Therefore, it is unlikely to detect fertility differences associated with seminal parameters.

Para reducir la incidencia de las penetraciones polispérmicas en los sistemas de fecundación in vitro porcina (FIV), los objetivos del presente trabajo fueron estudiar: 1) el efecto de tres volúmenes de medio de coincubación (2, 1 y 0'1 ml) y tres números de ovocitos (50, 30 y 15), inseminados con 6 x 105 espermatozoides /ml (primera experiencia) y 2000:1 espermatozoides:ovocito (segunda experiencia); 2) el efecto de la presencia de células del cumulus durante la FIV de ovocitos inseminados con diferentes ratios espermatozoides:ovocito (2000:1; 3000:1; 4000:1, 6000:1 y 8000:1), en 0'1 ml de medio de fecundación, con 30 ovocitos. Ovocitos madurados in vitro y denudados inseminados con 2000 espermatozoides por ovocito fueron el grupo control; y 3) el efecto de tiempos cortos de coincubación ovocitos-espermatozoides durante la FIV (10, 30 y 60 min) sobre la eficiencia de la FIV porcina. / To decrease the high incidence of polyspermy penetration of porcine oocytes fertilized in vitro (IVF), the aim of this study was to evaluate: 1) the effect of three volume of co-incubation medium (2, 1 and 0.1 ml) and three number of oocytes (50, 30 and 15), inseminated with 6 x l05 sperm/ml (first experience) and 2000:1 spermatozoa:oocyte (second experience); 2) the effect of cumulus cells during IVF of oocytes inseminated with different espermatozoa:oocytes rates (2000: 1, 3000: 1, 4000: 1, 6000: 1 and 8000: 1), in 0.1 ml of volume of IVF medium, with 30 oocytes. Denuded matured oocytes inseminated with 2000 spermatozoa:oocyte were the control group; and 3) the effect of short-times that oocytes are exposed to the sperm during IVF (10, 30 and 60 min) on the efficiency of pig IVF.