ABSTRACT: Bovine spongiform encephalopathy (BSE) is a transmissible progressive neurodegenerative disease characterized by the accumulation of a pathological isoform (PrpSC) of the cellular prion protein (PrpC) in the brain of cattle. Two insertion/deletion polymorphisms in the PRNP gene (23bp in the promoter and 12bp in intron 1) have been associated with resistance or susceptibility to the disease. The aim of this study was to analyze the distribution of these polymorphisms in 214 healthy bovines belonging to four different breed groups (Aberdeen Angus, Aberdeen Angus x Hereford, Holstein Friesian and Uruguayan Creole cattle). DNA samples were amplified by end-point PCR. A high frequency of the alleles and haplotype associated with susceptibility to BSE (del12 and del23, and del12-del23, respectively) were found in the Aberdeen Angus, Aberdeen Angus x Hereford and Holstein Friesian animals. At the same time, the Uruguayan Creole cattle presented a higher frequency of the alleles and haplotype associated with resistance to BSE (ins12 and ins23, and ins12-ins23, respectively). These data could indicate a greater genetic resistance of the Uruguayan Creole cattle to BSE compared to other analyzed breeds, reinforcing its value as a zoogenetic resource.

RESUMO: A encefalopatia espongiforme bovina (EEB) é uma doença neurodegenerativa progressiva transmissível dos bovinos, caracterizada pelo acúmulo no cérebro de uma isoforma patológica (PrpSC) da proteína priônica celular (PrpC). Dois polimorfismos de inserção/deleção no gene PRNP (23bp no promotor e 12bp no íntron 1) foram associados à resistência ou suscetibilidade à doença. O objetivo deste trabalho foi analisar a distribuição desses polimorfismos em 214 bovinos sadios, pertencentes a quatro diferentes grupos raciais (Aberdeen Angus, Aberdeen Angus x Hereford, Holstein Friesian e Crioulo Uruguaio). As amostras de DNA foram amplificadas por PCR de tempo final. Uma alta frequência dos alelos e haplótipos associados à suscetibilidade à BSE (del12 e del23 e del12-del23, respectivamente) foram encontrados nos animais Aberdeen Angus, Aberdeen Angus x Hereford e Holstein Friesian, enquanto o gado Crioulo Uruguaio apresentou maior frequência dos alelos e haplótipos associados à resistência à BSE (ins12 e ins23 e ins12-ins23, respectivamente). Esses dados podem indicar uma maior resistência genética do gado Crioulo Uruguaio à BSE em comparação com as outras raças analisadas, reforçando seu valor como recurso zoogenético.

RESUMO: Os distúrbios genéticos nos bovinos da raça Holandesa são um problema de saúde que cresceu nos últimos anos a nível mundial, comprometendo a sustentabilidade da produção leiteira moderna. No Uruguai, a produção leiteira com base na raça Holstein é um dos setores mais importantes da economia do país, mas altos níveis de endogamia diminuíram a fertilidade da raça nas últimas décadas. O objetivo deste estudo foi investigar a presença e difusão de alelos letais e semi-letais causando morte de embriões, abortos, malformações fetais e doenças neonatais em bezerros da raça Holandesa. Usando o BeadChip Bovino 50K GeneSeek® Genomic Profiler™, genotipamos 383 bezerros (menos de um mês) de 27 fazendas localizadas na principal região leiteira do Uruguai. Os resultados mostraram uma alta prevalência de fazendas (85%) e bezerros portadores (21%), incluindo um ou mais dos seguintes alelos letais ou semi-letais: sindactilismo (4,18%), braquipespina (3,39%), haplótipo de deficiência de colesterol (2,61%), malformação vertebral complexa (2,09%), deficiência de adesão de leucócitos bovinos (1,04% s) e haplótipos de Holstein HH1 (4,44%), HH3 (3,13%), HH4 (1,04%) e HH5 (0,26%). A maioria desses alelos não havia sido reconhecida anteriormente no país. Concluímos que as mutações letais e semi-letais são comuns na raça Holstein no Uruguai. Mais estudos são necessários para determinar seu impacto na fertilidade do gado leiteiro.

ABSTRACT: Genetic disorders in Holstein cattle are a health problem that has grown worldwide in recent years, compromising the sustainability of modern dairy production. In Uruguay, Holstein-based milk production is one of the most important sectors of the country’s economy, but high levels of inbreeding have decreased the breed’s fertility in recent decades. This study investigated the presence and diffusion of lethal and semi-lethal alleles causing embryo death, abortions, fetal malformations, and neonatal diseases in Holstein calves. Using the GeneSeek® Genomic Profiler™ Bovine 50K BeadChip, we genotyped 383 calves (1-30 days-old) from 27 farms located in the main dairy region of Uruguay. Results showed a high prevalence of farms (85%) and carrier calves (21%), including one or more of the following semi-lethal or lethal alleles: Syndactylism (4.18%), brachyspina (3.39%), cholesterol deficiency haplotype (2.61%), complex vertebral malformation (2.09%), bovine leukocyte adhesion deficiency (1.04%s), and Holstein haplotypes HH1 (4.44%), HH3 (3.13%), HH4 (1.04%), and HH5 (0.26%). Most of these alleles had not been recognized previously in Uruguay. We concluded that lethal and semi-lethal mutations are widespread in the Holstein breed in Uruguay. More studies are required to determine their impact on dairy cattle fertility.

RESUMO: A síndrome de Brachyspina (BS) é um defeito hereditário monogênico autossômico recessivo raro da raça Holstein Friesian causado por uma exclusão de 3,3 KB no gene FANCI localizado no cromossomo bovino 21, o que leva a um deslocamento de quadro e um códon de parada prematuro. Uma consequência da BS é a eficiência de reprodução reduzida e a produção de leite. O objetivo deste estudo foi o desenvolvimento de um método simples, rápido e sensível, baseado em PCR em tempo real e análise da curva de fusão para identificar animais portadores de BS. Sessenta e oito genótipos homozigotos normais e quatro heterozigotos foram detectados e confirmados através da análise tradicional de PCR e electophorese. Concluímos que o novo método é uma ferramenta confiável, altamente precisa e de baixo custo, que poderia ser usado para monitorar a presença da mutação BS na raça Holandês uruguaia.

ABSTRACT: Brachyspina syndrome (BS) is a rare monogenic autosomal recessive hereditary disorder of the Holstein Fresian breed caused by a deletion of 3.3Kb in the Fanconi anemia complementation group I (FANCI) gene on BTA-21, which leads to a frame-shift and premature stop codon. Some of the consequences of BS are the reduction of the fertility rate and milk production. This study developed a simple, sensitive, rapid cost- effective assay method based on real time PCR and melting curve analysis for the detection of BS carrier animals. Sixty-eight normal homozygous and four heterozygous carrier genotypes were detected and confirmed through traditional PCR- electrophoresis analysis. We concluded that the assay we have developed proved to be a reliable, highly precise and low-cost tool, which could be used to monitor the presence of the BS mutation in uruguayan Holstein breed.

Abstract To know the genetic situation of the Pampa Rocha, Celta, Bizaro Portuguese, Duroc, Iberian Extremeño and Iberian Andalusian porcine populations, their genetic structure and population dynamics were studied on the IGF2 and MC4R genes, which determine meat characteristics and quality. The degree of genetic variability (He = 0.2511 in Pampa Rocha; 0.0278 in Celta; 0,1453 in Bizaro Portuguese; 0.3719 in Duroc; 0.0764 in Iberian Extremeño and 0.0384 in Iberian Andalusian), genetic distance, and the presence or absence of consanguinity were studied. The Fis values were positive for the Duroc population (0.00426) indicating a very low inbreeding, the rest of the populations did not present consanguinity. Significant deviations (P ≤ 0.05) in the Hardy-Weinberg (HW) equilibrium were obtained for the IGF2 locus in Celta, Iberian Extremeño, and Iberian Andalusian populations with the G allele fixed, while the Bizaro Portuguese, Pampa Rocha, and Duroc populations presented polymorphism, the G allelic frequency was much higher than A allele, except in the Duroc breed (0.15). These findings could help breeders to increase the presence of the A allele for the improvement of muscle mass and reduction in the back-fat thickness in this breed. All the studied populations presented polymorphism for the MC4R locus with different frequencies for each allele. Furthermore, these results could allow developing strategies against anthropogenic activities that hinder the conservation of the biodiversity of these porcine breeds.

Resumen El objetivo de este estudio consistió en un relevamiento de las principales enfermedades hereditarias BLAD, DUMPs y Citrulinemia, en una población representativa de terneras Holando de la región este del Uruguay utilizando el panel GGP-BovineLDv3, así como en el análisis de la frecuencia del alelo mutante de BLAD desde que fue reportado por primera vez en Uruguay. El ADN fue extraído a partir de sangre fresca de 190 terneras Holando de la cuenca lechera de Cerro Largo (Uruguay) y genotipado con el mencionado panel, validando así técnicas previamente utilizadas tales como PCR-RFLP y PCR-secuenciación. Se detectó la presencia del alelo mutante para BLAD en una ternera y se estimó una frecuencia alélica de 0.28 %. Las frecuencias reportadas del alelo BLAD durante los últimos 15 años fueron comparadas, encontrándose una disminución de la misma en los trabajos reportados hasta llegar a cero en 2018, lo cual podría deberse al uso de semen genotipado libre de estas enfermedades durante los últimos años. No se encontraron alelos mutantes para DUMPs ni para Citrulinemia. El uso de paneles de baja densidad sería una herramienta muy útil para establecer estrategias de cría en tambos y monitorear las enfermedades hereditarias en la raza Holando.

Abstract The objective of this study consisted in a screening of the main hereditary diseases BLAD, DUMPs and Citrullinaemia using the GGP-BovineLDv3 panel in a representative population of Holstein heifers of the eastern area of Uruguay, as well as the analysis of BLAD mutant allele frequency since it was first reported in Uruguay in 2003. DNA was extracted from fresh blood of 190 Holstein heifers in the dairy area of Cerro Largo (Uruguay) and genotyped using the above mentioned panel, thus validating previously used techniques, suhc as PCR- RFLP and PCR- sequencing. We detected the presence of the BLAD mutant allele in one heifer, and allele frequency was estimated as 0.28%. Reported frequencies of BLAD allele during the last 15 years were compared and the reported studies revealed that the frequency progressively decreased and fell to zero in 2018, which can be due to the use of genotyped semen free of these diseases during the last years. No mutant alleles were found for DUMPs or Citrulinaemia. The use of low-density panels would be a useful tool to develop breeding strategies in dairly farms and to monitor hereditary diseases in Holstein breed.

Resumen La abiotrofia cerebelar (AC) es una enfermedad neurodegenerativa causada por una mutación en el ADN que afecta a las células de Purkinje del cerebelo, produciendo principalmente problemas en el balance y la coordinación de los animales. Entre las especies que se ven afectadas se encuentra la equina, particularmente la raza Árabe. En este trabajo se presenta el caso de un potrillo árabe que presentó sintomatología compatible con AC. Los hallazgos clínicos se confirmaron mediante estudios moleculares y confirmación por histopatología de las lesiones. Los estudios moleculares permitieron confirmar la presencia de la mutación causal. Dados estos hallazgos y que la sintomatología neurológica compatible con la abiotrofia cerebelar en equinos no es nueva en nuestro país, se considera que sería importante estudiar a nivel poblacional la raza Árabe, con el fin de conocer la prevalencia y casuística de AC en Uruguay.

Summary Cerebellar abiotrophy (CA) is a neurodegenerative disease caused by a mutation in the DNA that affects the Purkinje cells of the cerebellum, mainly causing problems in the balance and coordination of animals. Among the species that are affected is equine, mainly the Arabian breed. In this study we presented the case of an Arabian foal with clinic signs compatible with the pathology mentioned before. Histopathological and molecular studies were carried out to confirm the clinical findings, giving results that were positive for CA in both cases. Considering these findings and that the neurological symptomatology compatible with CA in horses is not new in our country, it is important to study the Arabian breed at a population level, in order to know the prevalence of CA in Uruguay.

Resumen Las células madre mesenquimales (CMM) presentan gran interés en veterinaria por su potencial uso en medicina regenerativa. Este interés se debe a que presentan dos atractivas características, una es el poder promover la regeneración tisular y la segunda es poseer la capacidad de inmunomodulación, además, se pueden extraer fácilmente de animales adultos. El objetivo del presente trabajo fue el aislamiento y caracterización in vitro de las CMM derivadas de tejido adiposo (TA) provenientes de las especies canina, equina y felina. Se consiguió aislar CMM-TA provenientes de 5 caninos, 4 equinos y 6 felinos. Las células de las tres especies mostraron capacidad de formar colonias fibroblastoides y tridiferenciación in vitro y fue posible criopreservarlas. En conclusión, se logró el aislamiento y caracterización de las CMM de las tres especies. Además, se generó un banco de CMM de origen animal criopreservadas con el propósito de futuras aplicaciones terapéuticas en medicina veterinaria y/o para otros estudios in vitro.

Summary Mesenchymal stem cells (MSCs) are of great interest in veterinary medicine due to their potential use in regenerative medicine. The attractive characteristics are the ability to promote tissue regeneration, to immunomodulate and easiness of extraction from adult animals. The objective of the present work was the isolation and in vitro characterization of adipose tissue-derived MSCs (AD-MSCs) of canine, equine and feline species. The AD-MSCs from 5 canines, 4 equines and 6 felines, were isolated. The cells of each species showed the capacity of forming fibroblast colonies, tridifferentiation in vitro and cells were cryopreserved. In conclusion, it was possible to isolate and in vitro characterize AD-MSCs. A bank of cryopreserved animal MSCs was created, for future therapeutic applications in veterinary medicine and other in vitro studies.

Bovine presents a complex genome due to high number of repetitive sequences involved in macro and micro-rearrangements that are essential in the bovids evolution. However, it has a morphologically simple karyotype, with acrocentric autosomes (2n=60,XX and 60,XY). The Robertsonian translocation rob(1;29), which produces embryo mortality, has been widely described in different breeds of Bos taurus but the complexity of its mechanism and its consequences in chromatin changes, are still not clear. Clastogenic agents are used as an approach to study this rearrangement in chromatin structure. Particularly, the aphidicolin (APC) inhibits the eukaryotic DNA polymerase alpha during replication. This fact permit to identify regions rich in dCTP due to a competition and spreading of the enzyme. Lymphocyte macro culture of 2 female Uruguayan Creole cattle carriers of rob(1;29) were APC-inducted (0,3uM) in one cell cycle. A new break point, rob(1;29)p13/21 (relative distance: p2c/p1=0.45), is located in a late replication region (RBG- band). Two fragile sites (c-fra) in the long arm of rob(1;29) (q13/ 21 and q43) and two other c-fra in BTAX (q12 and q31) were scored. C-fra: rob(1:29)q13/21 y Xq12 were located proximal to centromere in later replication region while rob(1;29)q43 and Xq31 were located proximal to telomere in early replication region. A high incidence in c-fra proximal to centromere in both biarmed chromosomes (BTAXc2=11.3 P<0.001; rob(1;29)c2=4.66P<0.05) was found. Comparing APC-effect: BTA1q13/21 and rob(1;29)q13/21, the high damage found in the second was equivalent to 75% of heterozygosis. This region (RBG- band) has a microsatellite associated to this rearrangement and tissue specific genes.

Los bovinos presentan un genoma complejo debido al número alto de secuencias repetidas involucradas en la formación de macro y micro-rearreglos, que fueron esenciales en la evolución de los bóvidos. Sin embargo presentan un cariotipo de morfología sencilla, con autosomas acrocéntricos (2n=60,XX y 60,XY). La translocación Robertsoniana rob(1;29) está ampliamente descripta en diferentes razas de Bos taurus, determinando mortalidad embrionaria temprana. La complejidad del reordenamiento y sus consecuencias en cambios de la cromatina, no son bien conocidas. Para estudiar este reordenamiento se utilizó afidicolina (APC), un agente clastogénico que inhibe a la ADN polimerasa alfa durante la replicación y permite identificar regiones ricas en dCTP debido a competencia y desplazamiento de la enzima. Se realizaron macro cultivos linfocitarios de 2 hembras de bovinos Criollos Uruguayos portadores de la rob(1;29). Se indujeron con APC (0,3uM) durante un ciclo celular. Se localizó un nuevo punto de ruptura, rob(1;29)p13/21 (distancia relativa: p2c/p1=0.45) en una región de replicación tardía (RBG-). Se analizaron dos sitios frágiles (c-fra) en el brazo largo de la rob(1;29) (q13/21 y q43) y otros dos c-fra en BTAX (q12 y q31). Los c-fra: rob(1:29)q13/21 y Xq12 se ubicaron próximos al centrómero en regiones de replicación tardía, mientras que rob(1;29)q43 y Xq31 se ubicaron próximos al telómero en regiones de replicación temprana. Se encontró una alta incidencia de c-fra proximal al centrómero en ambos cromosomas (BTAXc²=11.3 P<0.001; rob(1;29)c²=4.66P<0.05). Comparando los efectos de APC: BTA1q13/2 y rob1;29q13/21, se encontró mayor daño en el segundo, equivalente a un 75% de heterocigosis. Esta región (RBG-) contiene un microsatélite asociados al reordenamiento y genes tejido-específicos.

La azacitidina (5-azaC) es una sustancia que actúa inhibiendo la metilación del ADN, es utilizada en experiencias de clonación, transgénesis por transferencia nuclear y estudios de fragilidad cromosómica. Se ha observado la manifestación de diversas patologías en animales domésticos nacidos de experiencias de clonación. Ellas estarían asociadas a una inexacta reprogramación nuclear relacionada con fenómenos epigenéticos de metilación del ADN. En el presente trabajo se realizó una curva de inducción con diferentes concentraciones finales de 5-azaC (0 uM, 5 uM, 10 uM, 20 uM, 50 uM y 70 uM) con el objetivo de estudiar los efectos sobre la estructura cromosómica en cultivos linfocitarios bovinos. Se utilizó la técnica de macrocultivo para análisis cromosómico a partir de sangre de un bovino macho (Bos taurus, Holstein Friesian). Se analizó un total de 50 placas metafásicas por tubo de cultivo y se encontraron diferencias significativas (p<0.01) en cuanto a las fracturas del cromosoma X en el cultivo control frente al cultivo con mayor concentración de 5-azaC. El cromosoma Y no presentó fracturas. En los cultivos con concentraciones de 50 uM y 70 uM se observó descondensación cromosómica (12% y 16%) y fracturas centroméricas en autosomas (2% y 4%).

Azacytidine (5-azaC) is a substance that inhibits the metilation of DNA, being used in cloning experiments, nuclear transference transgenesis and in chromosomal fragility studies. The manifestation of diverse pathologies has been observed in domestic animals born from cloning experiments. These would be associated to an incorrect nuclear reprogramation related to epigenetic metilation phenomena of the DNA. In the present work an induction curve with different final concentrations of 5-azaC (0 uM, 5 uM, 10 uM, 20 uM, 50 uM and 70 uM) were carried out, with the objective of studying the effects on chromosome structure in bovine lymphocyte cultures. The macroculture technique was used for chromosomic analysis, from a blood sample of a male bovine (Bos taurus, Holstein Friesian). A total of 50 metaphases were analyzed from each culture. Significative differences (p<0.01) were found regarding the break points of chromosome X in the control culture with respect to the culture with higher concentration of 5-azaC. Chromosome Y did not show break points. The cultures with higher concentration of 5-azaC (50 uM and 70 uM) presented chromosomic decondensation (12% and 16%) and autosomic centromeric break points (2% and 4%).

La translocación Robertsoniana (rob1; 29) está ampliamente distribuida en razas comerciales y en reservas genéticas de bovinos Criollos americanos. Se ha descrito un enlentecimiento en el desarrollo de embriones portadores de esta aneuploidía, frente a embriones normales. La acción de la 5-aza-C, como agente desmetilante, permitiría descondensar la heterocromatina constitutiva o facultativa. En este trabajo se realiza inducción con el análogo de base 5-aza-C(10mM, 2 hrs) en cultivos linfocitarios de una hembra y un macho portadores de la rob1; 29, frente a bovinos Criollos normales. Se controla la acción desmetilante del inductor al identificar la despiralización del cromosoma X de replicación tardía en hembras y permitir el análisis de la despiralización de la cromatina en múltiples regiones de los autosomas (grandes, medianos, pequeños); de la rob1; 29 y del BTA1. Se discute la correlación existente entre regiones desmetiladas con la descondensación de la heterocromatina facultativa (condicional), relacionándola con la inestabilidad genómica, y la reprogramación epigenética.

The Robertsonian translocation (rob1; 29) is widely spread in commercial breeds and specially in genetic reserve of American Creole cattle. It is also described a delay on embryo development in front of normal ones. The action of 5-aza-C, as an hypomethylated agent, could permitted to decondensate the constitutive or facultative heterochromatin. In this work we made induction with the 5-aza-C(10mM, 2 hrs) analogs, in lymphocyte cultures of female and male carriers and normal Creole cattle. The DNA hypometilation is found in the inactive X chromosome of late replication as it is incorporated during the last hours of cell culture. The decondensing effects of 5-aza-C analogs is observed in multiple regions of the autosomes chromatin, the rob1; 29 and the BTA1. A correlation between hypometilated regions and decondensed of facultative (conditional) heterochromatin is related with genomic instability, and epigenetic reprogramming.