VETERINARY GENETIC

Author: FERNÁNDEZ DEL NACIMIENTO CRISTINA.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The myostatin is a member of the family of growth factors and Transformation (TGFbeta) responsible for the inhibition of development of muscle mass. The cattle with mutations inactivantes in myostatin presents a substantial increase in their muscle mass known as hipertrofía muscle or cularidad. This increase muscle is accompanied by an equally significant decrease in the amount of fat that results in a clear increase in the economic value of their channels. The increased knowledge of the structure and function of myostatin has initiated the development of methodologies to manage your operation or avoid unwanted effects associated with the phenotype culón. In the first line of investigation in this thesis has been raised to create a system that allows explore reversbilidad of hypertrophic phenotype to normal phenotype. This has realiz ate the construction of a "Mstn OFF to ON." In this system the third exon of myostatin is upside down but is likely to return to the position permanently activated by the system Cre / lox, using a variant of lox mutated. This system has been tested in vitro and in a bacterial system, which has demonstrated its ability to move from one configuration inactive to active, keeping also in the latter state. By contrast, when there was a stable integration by homologous recombination in murine ES cells, did not succeed in reversing the current setting, likely due to the use of a promoter too weak for the expression of Cre-recombinasa prove adequate to lox mutated used. Although this line of research has been halted at this point, these results point to the possibility of using vectors Cre better able to achieve expected recombination between lox mutated. The second line of study of this thesis has been to the creation and evaluation of different alleles of myostatin potentially dominant negative. The phenotype culón typical only presents state homocigosis, hence the undoubted interest of obtaining breeding calves heterozygotes culones in industrial crossings, as well as the potential application of these alleles postnatal to eliminate the effects associated with perinatal phenotype culón. We have raised three dominant negative alleles potentially capable of defeating the role of the myostatin wild in his presence. These three variants are designed in a manner that prevents its binding to specific membrane receptor for impedimiento estérico. Since this strategy of dominance negative depend mostly on the ability of dimerización between monomers wild and mutant monomers have conducted two tests iteracción to confirm that the union between monomers has not been disrupted. These tests confirmed that the dominant negative designed three alternatives are perfectly capable of forming dimers to monomers wild, and further, that this union is not significantly different from the union between two monomers wild. Therefore, the alleles designed met the first prerequisite to a strategy of negative dominance.

Author: CALONGE ERREJON M. ESTHER.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The first objective of this thesis is to compare the expression patterns of animals of the bovine race Asturian in the valleys affected pro muscle hypertrophy induced by a deletion of 11pb in the myostatin gene, compared to normal animals. The second objective is the identification of genes that are expressed. Diferencialmente and finally the relationship of these genes and metabolic pathways in which they participate with the emergence of muscle hypertrophy. This uses the technique to study differential expression "Differential Display" and the results are checked by the technique of "QUANTITATIVE PCR." The samples used are muscles of animals Asturian race of the valleys of the three possible genotypes for the deletion 11.Se detect genes that are expressed differently in animals with hypertrophy with respect to normales.Algunos genes arose a increased expression and others show a decrease. Genes found are related to signal transduction, lipid metabolism, cell cycle, and differentiation of muscle metabolism energetico.Se get an overview of the routes activated in skeletal muscle differential of other genes with known functions little or totally unknown, They open new avenues in the study of bovine muscle hypertrophy.

Author: MEDRANO DIAZ ANTONIO ARVELO.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The first objective of this study was to determine the mechanisms of energy use different monosaccharides (glucose, fructose, manosa and sorbitol), by the pig sperm. To do so, the sperm were incubated with no substrate energy and with different hexosas. The concentrations used in the hexosas was 10 mM, provided they wanted to determine the effect of incubation time, and lmM, 5mM, 10mM, 20 mM, 50mM when what was intended was to determine the effect of the concentration of substrate metabolizable . The incubaciones were conducted over 5 min., 15 min., 30 min., And 60 min. In the case of impact time, and for 60 minutes to discuss the effect concentration. The incubaciones were conducted in an environment of Krebs-Ringer-Henseleit devoid of energy substrates. Following these incubaciones "identified both the feasibility and the state of acrosomas as markers of functional state of the sperm. Were also identified intracellular levels of some energy metabolites, such as glucose 6-phosphate, glycogen and adenosine triphosphate ( ATP). addition, we also measured the rate of formation of L-Lactato extracellular from the glucólisis and CO2 as a final product of the Krebs cycle. The most important conclusion drawn from this study was that the monosaccharide glucose is used preferentially by pig sperm. This glucose is metabolized mainly by the way glucolítica (by 99%). Lastly, the control of the means of consumption of glucose is basically on two points, the first modulation of the activity hexoquinasa, and the second modulation of the Pyruvate kinase activity. complementation As the earlier study, also studied the kinetic behavior of the main activities that control the enzymatic route glucolítica, such as hexoquinasa, which regulates the phosphorylation of the initial monosaccharides, and Pyruvate kinase, which regulates the introduction of the final product of the glucólisis in the Krebs cycle. These studies showed that the hexoquinasa has a much higher affinity towards glucose when compared with the other monosaccharides studied, while the Pyruvate kinase shows great its dependence on activity of intracellular balance between levels of bifosfato adenosine diphosphate (ADP) and the ATP, which allows control very sensitive to obtain energy through the flow glucolítico. Lastly, we also assessed the pattern of phosphorylation in waste tyrosine pig sperm. cellular extracts showed a pattern consistent with a band of phosphorylation majority to 45 Kda weight. This pattern suffered no significant changes after incubation with any of the sugars. This result indicates the lack of a action modulating specific sugars on the functionality pig sperm. Another objective was to evaluate the use of substrates not glucolisables, and therefore not usable across the glucólisis, for energy as citrate and ellactato by part of pig sperm. experimental design Merger and incubation times was the same as described above for monosaccharides. incubaciones also were conducted in an environment of Krebs-Ringer-Henseleit devoid of sugars, and outcome measures were the same in the first phase of this work. Moreover, in this case it was determined the kinetic behavior of the activity lactate dehydrogenase (LDH) as a key point of control over the entry of substrates into the Krebs cycle. studied This established the presence of a LDH specific type of semen in the sperm pork, which was evidenced by Western blot analysis and confirmed by the results of immunocytochemistry, which showed a positive reaction in the main piece of the tail, with a given 8 bil mark d8d l in intermediate piece. The main conclusion that could be inferred from the results obtained with the substrates not glucolisables was that the pig sperm can be used efficiently mind as the sole source of energy both citrate as lactate, the use of these substrates takes place. through energy pathways controlled by LDH, ie basically the Krebs cycle, the rate of flow is also closely regulated by intracellular levels of ATP. was also assessed in this case the effect of incubation with citrate or lactate on the pattern of phosphorylation of tyrosine residue. In this case, the hatching with 10 mM citrate or lactate there was a notable increase in the intensity of all the bands, especially that of 45Kda, although not appeared significant changes in the pattern of bands specific phosphorylation, this result also indicates a lack of action on specific substrates functionality sperm. The ultimate goal of our work was the design of four experimental formulas, which combine a monosaccharide at different concentrations with a substrate not glucolisable (citrate at different concentrations or lactate), with the primary aim of studying the impact of these changes on the specific type of energy supply in the long-term cooling. underlying purpose of this study was the to obtain information for obtaining a solvent conservation of semen chilled pork to achieve exceed 7 days of conservation between 15 and 17Â ° C. To evaluate the effectiveness of these four formulas in the preservation of semen chilled pork were measured following parameters: Percentages feasibility and acrosomas altered, as indicators of the state of functional sperm, the rate of lactate formation, the Tests Resistance Osmótica (ORT) and Resistance Hiperosmótica (HRT), as evidence of greater significanción to assess the quality of sperm s, as has been described in previous publications. The final conclusion was that a medium that contains high levels of glucose (always lower than those containing the most widely used commercial thinners) and relatively low levels of lactate as an alternative source of energy allows for preservation of sperm pigs at temperatures between 15-17Â ° C for a period greater than the observed even in commercial thinners used widely, as the BTS. Therefore, a design optimized energy sources glucolíticas rather glucolíticas is essential for the improvement of diluents of porcine semen refrigeration.

Author: TOMÁS SANGENÍS ANNA.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The research of this thesis was developed within the framework of a currently ongoing project, known as the MeIbMAP project, whose main purpose is to gain knowledge about the genetic architecture of economically important traits related to reproduction efficiency in pigs. To address this issue, an F2 experimental population was created from three Iberian boars and 18 Meishan sows. These two parental breeds were selected based on their large phenotypic differences for almost all reproductive traits. This thesis reports the results obtained from the performance of a one-dimensional and a two-dimensional genome scan aimed at identifying quantitative trait loci (QTL) related to litter size traits, and the characterization of three physiological candidate genes and their associations with reproductive performance of sows and physiological and vitality variables related to the survivability of newborn piglets within the first hours of life. The performance of a one-dimensional scan revealed few genome-wide significant QTL with effects on the total number of piglets born (TNB) and the number of piglets born alive (NBA). The joint effect of these QTL explained almost 13% of the total phenotypic variance of both traits. A bidimensional scan allowing for multiple QTL and their interactions permitted the identification of a large number of genomic regions involved in the genetic control of litter size traits. The main effects of these QTL were attributable to dominance and epistasis effects, i.e. to the non-heritable fraction of the genetic variance, providing insight into the complexity of the genetic architecture of reproductive traits that commonly display low heritability values. Complex patterns of interactions, characterized by the involvement of more than one type of epistasis, were shown by almost all interacting QTL pairs. The porcine prolactin receptor (PRLR) gene was selected as a candidate gene because two polymorphisms of this gene had been previously related to litter size by several authors and also because knockout mice deficient for PRLR showed multiple defective reproductive phenotypes. We have sequenced the complete coding region of the porcine PRLR gene and found six non conservative SNPs within exon 10, which entirely encodes the intracytoplasmic domain of the protein, revealing a significant excess of nonsynonymous substitutions. Eight haplotypes were found segregating in the Iberian x Meishan population and in several porcine breeds at different frequencies. Results from the association study between PRLR genotypes and sow reproductive traits revealed significant effects of PRLR haplotypes on the number of corpora lutea. Furthermore, suggestive effects or tendencies of the PRLR polymorphism were also found for heart rate at birth, rectal temperature and oxygen saturation 1 h after birth, and time to first suckle. Our results confirm the effects of the porcine PRLR gene on ovulation rate, and suggest that it may also play a role in the survivability of piglets during the first days of life. The dopamine -hydroxylase (DBH) gene encodes for an enzyme of the catecholamine biosynthetic pathway that catalyzes the hydrolysis of dopamine to norepinephrine. Catecholamines participate in the regulation of ovarian development and in the adult gonadal function. The DBH gene was selected due to the existence of altered reproductive phenotypes in mice with a targeted disruption of this gene. Sequencing of a partial fragment of the porcine DBH cDNA from different porcine breeds has revealed the presence of 6 nucleotide polymorphisms, two of which led to amino acid replacements. These non-conservative mutations have been genotyped in the F2 resource population identifying three different haplotypes. The association study with reproductive and survival traits showed that the DBH gene is significantly correlated with the rectal temperature one hour after birth and the time to the first colostrums intake, suggesting that DBH may be a candidate gene for the early postnatal survivability of newborn pigs. Additional suggestive effects of this gene were also found 8 for numb 6d6 er of teats and birth weight. Finally, the porcine DBH gene was positioned by RH mapping on chromosome 1 near the q-telomere. The bone morphogenetic protein receptor 1 (BMPR1B) gene was selected as a candidate gene because a single amino acid substitution (Q249R) in the ovine BMPR1B was associated to the Booroola phenotype (FecB), which causes hyperprolificity in Merino ewes. The BMP system plays an important role in the regulation of ovarian function by controlling proliferation and cytodifferentiation of granulosa cell as well as oocyte development. We have identified three silent nucleotide polymorphisms within exon 10 of this gene. These polymorphisms leaded to the identification of four haplotypes which were found segregating, at different frequencies, in several porcine breeds. The association study between the BMPR1B polymorphisms and the reproductive performance of Iberian x Meishan sows did not yield significant results. Only suggestive effects were found for prolificacy at first parity, indicating that BMPR1B is not a major gene for reproduction in pigs.

Author: RODRÍGUEZ ALVAREZ ROBERTO.
Year: 2005.
University: LEÓN [www.unileon.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERNARIA.

Summary: The objective of this thesis was to design a method of classifying the characters' body morphology aptitude sheep milk. In an initial experiment explored a total of ten features simple narrative, punctuated according to a linear scale of 1 to 9, and two objective measures, body weight and height at the cross. They also visited 18 herds kernel selection race Churra. We have 4,732 records of 2,809 sheep. The heritabilities estimated for the simple descriptive features were found within the range of 0.05 to 0.17, and repeatabilities between 0.12 and 0.47. Taking into account these parameters, as well as other practical aspects of ease of scoring, we selected four features simple descriptive offering the best features for inclusion in the proposed rating system: height (EST), aplomos of hind paws ( POS), the inclination of the heels (TAL) and the width of the rump (ANC). Furthermore, it added a global nature, called general appearance (APAR). The experiences II and III, took place the implementation of the proposed system, in the first stage and later stage of practical application. The experience II is valued 96 sheep in three consecutive visits. The distribution of scores for the five characters approached normal. The objectivity of method, it was reasonably good, with a repeatability of scores between 0.33 and 0.46. The experience III visited 23 herds kernel selection race Churra. The file consists of 10,189 records, corresponding to 3,977 sheep. For genetic analysis of the data using an animal model of repeatability. The fixed effects considered were, in order of importance, round qualification, the number of birth and month of lactation. The estimated heritabilities were 0.19, 0.18, 0.24, 0.14 and 0.17, for the characters EST, POS, AS, ANC and APAR, respectively. These values allow genetic improvement of these characters through selection. The high correlation coefficient between multiple APAR and the four traits simple descriptive (0.75) and the high genetic correlations estimated (in absolute value between 0.51 and 0.80), show that from the four traits simple descriptive is possible to obtain an index that would reflect an appropriate and sufficient merit overall morphology body. The genetic correlations between the characters of morphology and body morphology mammary are low or moderate in the sense favorable. The estimated genetic correlations between characters dairy and morphological characters were so low that it does not expect significant changes in the morphology of sheep breeds body subjected to a selection process to increase milk production and vice versa. The objective of this thesis was to design a method of classifying the characters' body morphology aptitude sheep milk. In an initial experiment explored a total of ten features simple narrative, punctuated according to a linear scale of 1 to 9, and two objective measures, body weight and height at the cross. They also visited 18 herds kernel selection race Churra. We have 4,732 records of 2,809 sheep. The heritabilities estimated for the simple descriptive features were found within the range of 0.05 to 0.17, and repeatabilities between 0.12 and 0.47. Taking into account these parameters, as well as other practical aspects of ease of scoring, we selected four features simple descriptive offering the best features for inclusion in the proposed rating system: height (EST), aplomos of hind paws ( POS), the inclination of the heels (TAL) and the width of the rump (ANC). Furthermore, it added a global nature, called general appearance (APAR). The experiences II and III, took place the implementation of the proposed system, pr 8 imero in 9a2 experimental stage and later stage of practical application. The experience II is valued 96 sheep in three consecutive visits. The distribution of scores for the five characters approached normal. The objectivity of method, it was reasonably good, with a repeatability of scores between 0.33 and 0.46. The experience III visited 23 herds kernel selection race Churra. The file consists of 10,189 records, corresponding to 3,977 sheep. For genetic analysis of the data using an animal model of repeatability. The fixed effects considered were, in order of importance, round qualification, the number of birth and month of lactation. The estimated heritabilities were 0.19, 0.18, 0.24, 0.14 and 0.17, for the characters EST, POS, AS, ANC and APAR, respectively. These values allow genetic improvement of these characters through selection. The high correlation coefficient between multiple APAR and the four traits simple descriptive (0.75) and the high genetic correlations estimated (in absolute value between 0.51 and 0.80), show that from the four traits simple descriptive is possible to obtain an index that would reflect an appropriate and sufficient merit overall morphology body. The genetic correlations between the characters of morphology and body morphology mammary are low or moderate in the sense favorable. The estimated genetic correlations between characters dairy and morphological characters were so low that it does not expect significant changes in the morphology of sheep breeds body subjected to a selection process to increase milk production and vice versa.

Author: Merchán Acedo Maria Isabel.
Year: 2006.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: Facultat de veterinària.
Place of preparation: Facultat de Vaterinària.

Summary: The research work of this dissertation is framed in a larger project (AGL2001-3068 and AGL2005-07624) whose main objective is to identify what is the largest gene that determines the observed differences in the size of litter into two lines rabbit Selected divergentemente for uterine capacity (CU). This has created a population F2 from crossing experimental 16 rabbits (3 males and 13 females) of the selected lines for the character uterine capacity (CU +) and 8 rabbits (3 males and 5 females) of the lines Selected contradict the uterine capacity (CU-). This thesis presents the results obtained from analysis of 5 candidate genes and the associated physiological study of association 3 of them with the different components of the characteristic size of litter. It also shows the results of the analysis of ESTs produced from gene banks ovary, uterus and oviduct of rabbits in order to characterize the gene expression in these tissues and identify gene sequences not yet described in rabbits. The gene uteroglobina was selected because it encodes the main component of protein secretion by uterine rabbit and its potential physiological role in implantation. We have sequenced a fragment of 3703 bp, which includes part of the promoter region, 3 exons and introns with their respective part of the region 3 'flanqueante. We have identified 6 SNPs in the promoter region of the gene were co-segregando in 2 haplotypes on the lines CU + and CU-, but did not appear to be associated with the lines because they do not have different allelic frequencies. The progesterone receptor gene half the effects of progesterone, speaking on ovulation, facilitates the establishment and maintenance of pregnancy, promotes the development of the mammary gland and regulates sexual behavior. We have sequenced 3702 pb which included part of the promoter, the region 5'UTR, almost the entire coding region and the region 3'UTR. We have identified 5 SNPs: 1 HNS silent in exon 7, 1 SNP in the promoter and 3 in the region 5'UTR that co-segregan two haplotypes in lines CU + and CU- with different frequencies. The study conducted in partnership crossing the F2 of CU + x CU- between the SNP and promoter of the characters reproductive revealed a significant effect on Totals Born and Born Live, bringing the PGR gene could be the largest gene responsible for the differences between lines CU + and CU-. The SNP is in four lines secreting specialized rabbits used to generate commercial stocks. The gene IGF1 was selected based on the results of experiments conducted in vivo and in vitro: for this gene knockout mice showed that the IGF1 was essential for the proper development of the embryo and studies embryo pre-implantacionales of human and rabbit revealed the IGF1 stimulated cell proliferation, promoting embryonic development and prevented apoptosis. It has been sequenced the coding region of the form of the mRNA gene IGF1 rabbit (406 pb), but has not found any nucleotide variation between animals lines CU + and CU-. The gene oviductina encodes a protein that is synthesized in the oviduct of mammals mainly during estrus, fertilization and early embryonic development, and that interacts with eggs, sperm and embryos oviductales and uterine. Despite the fact that the biological function is unknown, is believed to have a positive impact on training, motility and viability of the sperm, fertilization and early embryonic development. It has been determined the structure of the gene OVGP1 rabbit, which stretches about 13 kb and is similar to that described in other species, and has been studied gene expression. The messenger appears mainly in oviduct, but also in the ovary and uterus, and a much lower level even in non - reproductive tissues. His expression seems oviduct not vary significantly during early pregnancy, nor among the animals on both lines, at least in the 62 h post-monta. Of the 11 SNPs present at the messenger, SNPs G/C12903 yC/G12944 of exon 11 identifies two changes aminoacídicos: Gly / Arg and Ala / Gly, respectively. The result of the study of the association between genotype of a microsatellite trialélico present in the promoter and reproductive characters revealed a significant effect on the characters Totals Born and Born Alive. The study of association between the SNP C/G12944 and reproductive characters showed a significant effect on Live Loud and Survival Embrionaria. Although these results suggest that the gene OVGP1 could be a gene for some of the largest components of litter size, it seems to be the gene responsible for the differences between the lines as the favorable alleles are in line CU- . The gene TIMP1 was selected because it encodes a protein that is involved in various reproductive processes (ovulation, embryonic development, the introduction of blastocitos, cervical ripening, the cycle endometrial and uterine involution postpartum) and inhibits apoptosis, promotes steroidogenesis and growth and cell survival. We have determined the sequence of the promoter gene TIMP1 rabbit and we have found a SNP secreting in 2 lines. The study of association between the SNP and reproductive characters indicates a significant effect on the number of embryos Implantados, suggesting that the gene TIMP1 could be higher for a gene implantation. Finally, we generated genebanks of cDNA samples from ovarian, uterus and oviduct of rabbits taken to 5 h, 62 h and 5 days post-monta respectively. We have isolated and sequenced 623 clones, of which 466 were classified as valid, ie sequences were of good quality greater than or equal to 100 bp, and did not correspond to tránscritos of mitochondrial DNA. Of the total valid EST, 386 correspond to different mRNA, of which 36 had not been previously described, 48 showed similarity to sequences previously reported but not scored, and 302 others showed similarity to sequences scored but mostly (262) they had not been described in rabbits. This result is a reflection of the late development of genomics rabbit, compared with other species.