ANIMAL BREEDING

Author: URDANETA VARGAS AIXA EFRAILDA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE VETERIANARIA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: In order to improve the production in vitro embryos (PIVE) oocytes from goat perpúber, three studies were designed in this investigation. The aim of the first study was to determine in oocytes selected by the test cresol bright blue (BCB), the effect of the addition of glutathione (GSH) alone or in combination with glusoca the culture medium in vitro (VIC), on development embryonic of oocytes goat perpúber. The oocytes were exposed to test BCB and were classified as: oocyte cytoplasm with blue (BCB +) and oocyte cytoplasm without the blue (BCB-). The oocytes BCB + showed higher percentage of nuclear maturing oocytes BCB- and the control group (82.6%, 55.7% and 74.7% respectively). The percentage of oocytes poliespérmicos was higher in oocytes BCB- that the BCB +. Supplementation of culture medium (VIC) with 1 mM GSH did not affect embryonic development, but the total percentage of embryos developed after the crop was higher in oocytes BCB + in BCB-, regardless of supplementation with GSH. The addition of glucose, either alone or with GSH did not affect embryonic development. The aim of the second study was to assess the effect of adding different concentrations of cysteamine (100uM, 200uM or 400uM) Middle d eMIV and means of VIC (50uM or 100uM) on the embryonic development of oocytes goat perpúber selected by the BCB test . The addition of 400 uM cysteamine Middle MIV improved fertilization and normal embryonic development of oocytes BCB- to the same levels of oocytes BCB +. The proportions of morulae more developed blastocyst were not affected by the treatments. Finally, it was studied the effect of the addition of cysteamine (400uM) to the middle of MIV, glutathione (1mM) Middle IVF and ionomicina the average sperm capacitation. This treatment improved fertilization normal zygotes with pronúcleos male and embryonic development of oocytes goat prepuber, however did not improve the developing blastocyst.

Author: ANDRADE MARTINS DE CARVALHO BESSA ANA CELESTE.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The objective of this study was to assess the effects on motility, integrity of the plasma membrane and acrosomal, level of training, and mitochondrial activity acrosomal reaction, introduced incorporating different crioprotectores to diluents for refrigeración'y frozen canine semen. For this purpose were conducted 4 experiments, in which the sperm preserved by refrigeration and freezing were analyzed by conventional methods, and depending on the experiment, additional tests for assessing the functionality of the spermatozoon, including: -motilidad determined by the computer system SCA @ 2002, -integridad the plasma membrane determined by test endósmosis or staining with fluoroeromo iodide propididum (IP) and eosin staining of, -integridad of acrosoma determined by staining specific Spennac @ lectin or fluorescent Pisum sativum (PSA ) and Arachis hypogea (NPA), -cstado training elortetraciclina (CTC), -actividad mitochondrial by tineión with Rodaminal23 (R123) - ability and experience acrosomal reaction by incubation with calcium ionóforo A23187 and leetinas fluorescent. The results for each variable is eon'elacionaron one another in each experiment. To run these experiments were used pools of semen obtained from the fraction espennática different eyaeulados from 19 penos between 2 and 7 years old, of different races and fertility tested earlier. The selection of eyaeulados was performed taking into account a motility total more than 80%, progressive motility of over 70% and the percentage of morphological abnormalities less than 20%. In the experiment I appreciated the incorporation of the amino acid solvent cooling Tris-fructosa-ácido citric and 20% yolk (Half control) and was proposed rcduceión the concentration of yolk. The results suggest that supplementation with taurine to 30 and 5OmM provides only slight improvements motility espem1átiea and that the addition of 30mM of Taurine has helped reduce the S'10el proportion of yolk, no significant differences with the means to control the day 7 . Only significant differences were observed between the thinners containing a 20 and a 10% peak for the visually estimated total motility in the days 2 and 3 (p less O OO1). There have been high and significant freezes catre results motility estimated visually and through computer analysis, as with the test hipo-ósmosis (r between 0703 and 0976, higher p O Ol). The marking with CTC allowed to differentiate statistics for the pattern SR sperm chilled in diluents with 20% yolk O, 30 YSOmM of taurine, being thinner with 30mM which induces higher percentage in the shortest time (14.67%, the cn day 3, p = 0007). The second experiment was conducted to assess the ability of etilénglieol as eríoprotector in the solvent freezing of canine sperm, or alternately associated with glycerol or Pasta Equex @. Using etilénglieol a14% motility provides similar results to those obtained with 8% glycerol, but is harmful when used at 8% (p greater O O5). Adding Equex STM Paste @ S with a solvent% etilénglicol improves outcomes compared a14% etilénglicol or 8% glycerol (p greater O O5). Using etilénglicol not got better results than the solvent Uppsala-Equex (dc Glycerol% S and O, 5% of Equex-STM Paste), which pennite increased cell survival post-descongelac 8 ion. In 839 the third experiment were compared with results of evaluation of the activity mitoeondrial espelmátiea, using eitometda flow after staining with RI23 associated with IP, with the valuation of vitality by tineión with eosina-nigrosina and motility estimated subjectively; being able to establish high eonelaeioncs including (1'de 0992 to 0999, p higher O O1). In the fourth experiment studied the impact of supplemental diluent freezing Uppsala-Equex with the amino acid taurine or hipotaurina aO, 25, 5Oy 75mM in various aspects of the functions espelll1áticas. There was obtained significant influence on the motility espennátiea however, the addition of 25mM of hipotaurina introduced a value significantly lower factor ALH after 2h in incubation at 38Â ° C (p = 0021), while adding 5OmM of hipDtaurina gave place a larger% of cells espontáneamcnte reaccionadas (PI-PSA +) immediately upon thawing after 1:30 pm in and incubation at 38Â ° C (p = O OOIy 0004, respectively). Moreover, the addition of 2.5 and 10 1Mde calcium ionóforo to canine sperm thawed and incubated for 20min at CCM to 38Â ° C allowed induce an increase in reaction aerosómica vcrdadera (PI-PNA +) after S, IS and 30 minutes but in ineubaeión, compared with Ou 1M (p greater O OO1). It was possible ohservar differences (p = O, FS) in the percentage of reactions acrosómicas to ISmin incubation with 2.5 Â ¡1Mde ionóforo; reaching solvent with 5OmM-Taurina highest percentage compared with diluycntes with 7SmM-Taurina, 25 and 50mM-Hipotaurina and no differences compared with solvent control. These diluents were between 19 and 22% less than control RA.

Author: FERNANDEZ SANTOS MARIA DEL ROCIO.
Year: 2005.
University: CASTILLA-LA MANCHA [www.uclm.es].
Place of defense: E.T.S. DE ING. AGRO. DE ALBACETE.
Place of preparation: INST. DE INVES. EN REC. CINEG (IREC).

Summary: The protocol for freezing samples espermáticas epididimarias used so far has been virtually the same as for the seminal samples from eyaculados pets. The overall objective of this work was oriented towards adapting these protocols for use with samples espermáticas epididimarias of Iberian red deer in order to improve the effectiveness of the same for this type of sample. First, we studied the combined effect of two variable-speed cooling and concentration of egg yolk in the solvent on the survival of sperm epididimarios chilled. Once known the effects of egg yolk on sperm epididimarios in refrigeration, we decided to study them at the stage of cryopreservation. To this end, on the one hand, an experiment was designed to determine what concentration and method of preparation of the egg yolk (vs. centrifuged complete) were the most suitable for freezing sperm epididimarios of deer. On the other hand, we studied the combined effects of three variables on congelabilidad of samples espermáticas epididimarias Deer Iberian: concentration of yolk clarified final concentration of glycerol and cooling rate from 22 Â ° C to 5 Â ° C. Next, we study the influence of various crioprotectores well as temperature adding crioprotector (22 Â ° C -temperatura ambiente- or 5 Â ° C) on the congelabilidad such sperm. Continuing with our goal of optimizing the protocol for freezing samples espermáticas epididimarias of deer, we carried out two other experiments. The first, designed to determine the most suitable solvent osmolality (Tris-citrato-fructosa) and the second, to study the effects of adding sugar to solvent freezing. Finally, we studied the different possible protective effect of antioxidants during cryopreservation of sperm epididimarios Deer Iberian. After the results of this thesis, we propose that the freezing of sperm epididimarios Deer Iberian is performed using a solvent Tris-citrato-fructosa supplemented with a 20% yolk clarified and a final concentration of a 6% glycerol. The glycerol can be added at room temperature (22 Â ° C), and cooling should be quick (? 4.2 Â ° C / min). We suggest the addition of the antioxidant enzyme thinner freezing, thereby providing greater protection to the doses frozen.

Author: CIUDAD LADRERO MARIA ANGELES.
Year: 2005.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The main objective of the work was to determine the factors surrounding AI sheep race Rasa Aragonesa within the Choice Program for prolificacy of the UPRA-Oviaragón, which influence their reproductive outcomes. These factors have been grouped into 3 types: those that affect one's own AI female (age, body condition, the interval between birth and previous AI), which they refer to the very technical (treatment duration progestativo, when the insemination regard to the preparation of the doses seminal and respect at the end of treatment progestativo, solvent used, inseminador, etc..), and those that have to do with factors related to the exploitation and management in farming (average fertility of the farm, margin gross farmer's experience in the use of the technique, and so on.). Of these factors, the most influential were the age of the sheep (better results from 2 to 3 years), the solvent (better results with skim milk that Tris-tes over 6% of egg yolk and 3% glycerol) ; the age of stallions (better results with ages from 27 to 72 months), the CC of the sheep (better results with CHD between 3 and 3.5); time of insemination (AI better results if carried out in the Top 5 hours after the preparation of semen and by 54.75 hours after the end of treatment progestativo). Finally, we studied the effect of season on the reproductive outcomes of AI, such as better results between July and October.

Author: CARDOZO CERQUERA JAIME ANTONIO.
Year: 2005.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: UNIVERSIDAD DE ZARAGOZA.

Summary: The overall objective of this thesis was to deepen the knowledge of seminal plasma proteins and plasma membrane of sperm sheep, components essential for the proper functioning of sperm. The technique used, allows two-dimensional electrophoresis characterize complex mixtures of proteins, identifying qualitative and quantitative changes in the cell response to variations ambiéntales. The characterization of the protein profile of both the seminal plasma, as the plasma membrane of sperm sheep, and their variation in response to seasonal factors and agents stressful, constituted the basics of this study. Initial work possible, standardization of the various protocols to establish the proper amount of protein, both of seminal plasma and plasma membrane, which together with the use of tampons of solubilization specific for the different samples studied, allowing obtaining gels, high-resolution points protein. The seminal plasma of mammals is a complex fluid that serves as a means to transport sperm from the testis to meet with the oocyte (Thomas 2003), which contains a variety of biochemical components, some of them relatively specific regulation of the operation the spermatozoon. It has been described significant shares of seminal plasma proteins, in areas such as motility and viability of the sperm ejaculated, and also in key areas such as training and interaction among gametes. Therefore, in this paper we established a map reference] 2D of seminal plasma proteins and possible relations with the percentages of viable sperm and mótiles, and the concentration of sperm ejaculated. It identified 252 "points of protein," established that the vast majority (86%) have an acid pH and are glicosiladas. Similarly, there was a significant influence of the station (or reproductive no-reproductiva) in the concentration of some of them, and the appearance and disappearance of some other in one or another station. The correlations positive and / or negative points were established between some protein and variable quality seminal mentioned suggest that could interact together to ensure a proper functioning of sperm. The results of the analysis by 2D-PAGE and inmunodetección established the composition of the bands of protein RSVP14 and RSVP20 of seminal plasma, which was proposed in previous studies as able to protect and / or repair the damage to the membrane of the sperm by heat shock by cold, highlighting points that are made up of protein and low molecular weight acid pH. The band RSVP14 is formed by six points protein, "while the band RSVP20 this constituted by two points, possibly with a different degree of glycosylation and / or phosphorylation. The analysis of sequences through internal sequencing "de novo" full information previously obtained by automated Edman degradation, and concluded that RSVP14 is related to the family of proteins majority in the seminal plasma veal (BSP), while one of the items that relate to the RSVP20 no homology to any protein contained in the databases of mammals. The electrophoretic analysis of the protein in the plasma membrane of the sperm revealed the presence of 150 points protein "on the map 2D referential, with seasonal variations in the concentration of some items, as well as the exclusive presence of two points in the mating season, which might suggest its importance in the reproductive process. Studies by western blot evidenci 8 aron the 790 response of six points with the antibody generated against Banda RSVP14 of seminal plasma, a result that confirms that these proteins derived from seminal plasma and adsorb to the membrane of sperm as has been demonstrated in previous work. Finally, it was found that cryopreservation causes variation in the protein profiles of the plasma membrane of sperm, loss of points and some other emerging with respect to maps 2D protein fresh semen. The addition of seminal plasma proteins in the middle of cryopreservation led to a significant increase in the concentration of one of the points that form part of the band RSVP 14 confirming again its adsorption to the membrane of the spermatozoa. In summary, these studies have tried to help through technical 2D-PAGE to generate basic knowledge about the major proteins present in the seminal plasma and the plasma membrane of the spermatozoa. In addition, it has provided new data about the effect of seasonal and stressful agents in electrophoretic profiles of proteins, in order to be able to establish management strategies aimed at increasing the sperm reproductive efficiency of the production system sheep.

Author: GARCÍA TOMÀS MÓNICA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Author: APARICIO DONOSO INÉS MARÍA.
Year: 2006.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: When the sperm is deposited in the female genital tract, will undergo a series of physiological and biochemical changes that will enable it to reach the egg and fecundarlo. These changes represent an increase in motility, training, and finally the acrosomal reaction. While these changes are taking place in vivo, in vitro can also be achieved using an average incubation chemically defined, which allows the foundation to study physiological and biochemical governing these processes. Thus, it has been shown that both motility, training as the acrosomal reaction are regulated or associated with changes in the state of phosphorylation of various proteins. In this thesis we have set ourselves the following objectives:-To study the possible relationship between intracellular signaling pathways mediated by protein tyrosine phosphorylation and training process of the sperm of boar. B-To determine the role of intracellular route of the Fosfatidilinositol 3-Quinasa (Pl3K) in the regulation of motility, training, acrosomal reaction and viability of the sperm of boar. C-Consider the possible role of Glucógeno Sintasa Quinasa 3 (GSK3) in the regulation of motility, training and acrosomal reaction of the sperm of varraco. D-To determine the relationship between the tracks of intracellular cAMP / protein Quinasa Woe to the Fosfatidilinositol 3-Quinasa/Proteína Quinasa B in the regulation of the activity of the Glucógeno Sintasa Quinasa 3alfa and motility of the sperm of boar. Our results allow us to conclude that the training process of the sperm of boar this partially regulated by intracellular signaling pathways involving the performance of tirosinas kinases and serine / reonina kinase GSK3. Similarly, in tyrosine phosphorylation of a protein of 32 kDa (called p32) can be used to more accurately processes and training of the sperm acrosomal reaction of boar. In these cells, the processes of training and pseudocapacitación induced by heat shock are regulated by different intracellular signaling pathways. Moreover, the survival of the sperm cell of boar is regulated, at least in part, by PI3K. The motility of the sperm of boar is negatively regulated by PI3K and positively by PKA. Finally, the GSK3alfa represents a common effector converging different signaling pathways that regulate intracellular motility of spermatozoa in boar.