MSCAGR (Animal Science) / Department of Animal Science / The Nguni is a transboundary indigenous Southern African cattle breed. The breed has distinct populations that are adapted to the different ecological zones of Southern Africa. Previous work on characterising the Nguni has been limited to within-country studies. Thus, the aim of the current study was to genetically characterise South African (SA) Nguni, Mozambican Nguni (Landim) and Swazi Nguni populations across Southern African region using a panel of 25 microsatellite markers, recommended by FAO and ISAG for genetic diversity studies. Genotypic data were generated from 90 unrelated autosomal DNA samples of the three cattle populations (SA Nguni n=30, Mozambican Nguni (Landim) n=30 and Swazi Nguni n=30) collected from government research stations and stud herds. Five South African beef cattle breeds’ DNA profiles were obtained from the ARC-DNA database and used as reference populations. A majority of the microsatellite markers were highly polymorphic across the studied populations. High genetic diversity was detected and expected heterozygosity varied from 71% (Landim) to 75% (SA Nguni) with a higher mean number of alleles (MNA) in the SA Nguni (7.52±0.42) compared to the Swazi Nguni (6.92±0.40) and Landim (7.16±0.43) populations. Observed heterozygosity (Ho) (0.597±0.046) compared to expected heterozygosity (He) (0.719±0.022) was lowest for the Swazi Nguni, confirming a relatively high level of inbreeding (FIS=0.158) in that population. An analysis of molecular variance (AMOVA) revealed that 9.61% of the total variation occurred among populations, while 90.39% occurred within populations. Short genetic distance (29.9%) was observed between Landim and Swazi Nguni, with the SA Nguni (>50%) being the most genetically distant population. The distant relationship between SA Nguni and the other two Nguni cattle populations was further confirmed by neighbor-joining (NJ) tree, Principal Coordinates Analyses (PCoA) and Factorial Corresponding Analysis (FCA). The structure of the three Nguni cattle populations clustered independently, despite some evidence of admixture. Additionally, genetic differentiation and population structure within four Mozambican indigenous cattle populations were investigated using the same panel of microsatellite markers. The analysis of unrelated autosomal DNA was performed on 120 animals (Angone n=30, Bovine de Tete n=30, Landim n=30 and Namaacha Nguni n=30), which presented sufficient genetic diversity across all populations. Estimates of mean number of alleles, observed and expected heterozygosities were 6.920±0.20, 0.68±0.02 and 0.71±0.01, respectively. Genetic differentiation among the populations accounted for 8.02% of total genetic variability. Negative (-0.025±0.029) to low positive (0.073±0.050) levels of inbreeding were observed within the four populations. The genetic distance, NJ tree, PCoA and FCA revealed a close relationship between Bovine de Tete and Landim as opposed to Angone and Namaacha Nguni. STRUCTURE analysis assigned the four Mozambican populations independently; however Bovine de Tete and Landim showed relatively higher levels of admixture with each other than Angone and Namaacha Nguni. It can be concluded that SA Nguni, Landim and Swazi Nguni populations accomplish high genetic diversity and they are genetically distant; however, the two latter populations are closely related. These results present useful information / NRF

No description available.

Cette thèse s’inscrit dans le cadre du projet CRB-Anim (Centre de Ressources Biologiques d’Animaux Domestiques) dont le but est de constituer une cryobanque nationale et d’améliorer les techniques de cryoconservation. Aujourd’hui, les ressources biologiques de type reproductif (embryons, sperme, ovocytes) et de type somatique (fibroblastes et cellules souches pluripotentes) sont conservées dans des milieux contenant des produits d’origine animale (POA). L’utilisation actuelle des POA (sérums, lait, jaune d’œuf) pose des problématiques sanitaires (risque de contamination) et scientifiques (manque de reproductibilité liée à la variabilité de composition des POA). Cette étude a pour objectif d’évaluer l’effet d’un milieu de préservation synthétique, chimiquement défini, breveté pour la congélation de cellules de sang de cordon (STEMALPHA.CRYO3) sur la cryoconservation de sperme ovin et bovin, et de cellules souches pluripotentes de lapin. Pour cela, une approche biologique (étude in vitro et in vivo sur le terrain), alliée à une approche physique (étude des cinétiques de refroidissement et caractérisation des propriétés thermodynamiques des milieux de congélation) ont été mis en place.Nos résultats montrent l’intérêt de l’utilisation du STEMALPHA.CRYO3, en tant que substituant aux sérums, dans les solutions de cryoconservation des cellules souches pluripotentes. Néanmoins, notre étude indique que ce produit n’est pas efficace pour protéger le sperme lors de la congélation. Cette thèse confirme l’intérêt de standardiser les procédures de cryoconservation afin d’assurer la qualité des ressources biologiques pour les cryobanques et les échanges internationaux / Nowadays, reproductive (embryos, sperm, oocytes) and somatic (fibroblasts and pluripotent stem cells) resources are cryopreserved in media containing animal-derived products (serum, egg yolk, milk). Using these products raises sanitary (risk of contamination) as well as scientific concerns (reproducibility limits due to the variability of their composition). This study aims to replace animal derived-product in assessing the effect of a synthetic and chemically defined medium, STEMALPHA.CRY03® (Stem Alpha, France), on the cryopreservation of ovine and bovine sperm, and on rabbit pluripotent stem cells. First, a physical approach permitted to study the cooling rates and the characterization of thermodynamic properties of the freezing media. The differential scanning calorimetry allowed us to define their phase transition temperatures (crystallization temperature, melting temperature and enthalpy variation of crystallization, proportional to the amount of crystallized ice). Second, a biological approach was used for the cryopreservation of bovine and ovine sperm, as well as rabbit pluripotent stem cells. Flow cytometry and computer- assisted sperm analyses showed that STEMALPHA.CRY03® impaired bovine sperm, compared to a medium containing animal derived-product. These last results were confirmed in ovine species. Nevertheless, artificial insemination by laparoscopy (n = 270 ewes) counteracts this impairment and allowed an average pregnancy rate of 70 %. Moreover, without any additive in the freezing medium, a similar pregnancy rate was obtained. The study of pluripotent gene expression profile, and analyses of viability and growth rates for the cryopreservation of rabbit pluripotent stem cells confirmed that synthetic media, STEMALPHA.CRY03® (with 4, 5 or 10 % of cryoprotectant) and CryoStor® CS10 (containing 10 % of cryoprotectant) were more efficient than serum-based media. We demonstrate that it is possible to cryopreserve sperm cells and pluripotent stem cells in synthetic and chemically defined media. 0ur results confirmed the interest of a standardized approach for cryopreservation procedures of genetic resources in mammals. This work meets the needs of cryobanking activities (quality policy) and of the regulation development within the framework of international trade

Les milieux de cryopréservation d'embryons contiennent généralement des produits d'origine animale, qui présentent des inconvénients majeurs : une composition variable et insuffisamment définie, et un risque de transmission d'agents pathogènes. La substitution de ces produits par des composés synthétiques chimiquement définis pourrait contribuer à l’amélioration des procédures de cryopréservation d’embryons, en réduisant la variabilité de composition des milieux, et le risque de contamination des ressources conservées.L’objectif de ce travail était d’évaluer l’effet du remplacement de l’albumine sérique bovine utilisée dans les milieux de congélation lente ou de vitrification d’embryons cunicoles et ovins par un milieu synthétique formulé à base d’acide hyaluronique (STEM ALPHA.Cryo3 – « CRYO3 »). Dans un premier temps, les propriétés thermodynamiques des substituts potentiels ont été évaluées à l’aide de la calorimétrie différentielle à balayage. Parallèlement, nous avons optimisé les différents outils expérimentaux dont nous avions besoin pour cette étude. Nous avons adapté un protocole d’évaluation de l'activité mitochondriale (JC-1) pour complémenter l'évaluation morphologique in vitro des embryons de lapin, et nous avons évalué l’efficacité de différents protocoles de superovulation sur la production d’embryons chez la brebis. Dans un second temps, nous avons procédé à la substitution de l’albumine sérique bovine (BSA) utilisée dans des milieux de congélation lente et de vitrification d’embryons cunicoles et ovins par du CRYO3. Une approche in vitro a été utilisée pour les protocoles de congélation et de vitrification, puis complétée par une approche in vivo pour les protocoles de vitrification.Nos résultats confirment que la BSA peut être efficacement remplacée par le CRYO3 dans des protocoles de cryoconservation d‘embryons de lapin et d’embryons ovins, qu’il s’agisse de congélation lente ou de vitrification / Embryo cryopreservation media usually contain animal-derived products, such as bovine serum albumin (BSA). These products present two major disadvantages: an undefined variable composition and a risk of pathogen transmission. The substitution of animal products of embryo cryopreservation media by synthetical products may improve procedure standardization (by avoiding variability in media composition) and avoid sanitary concerns inherent to animal-derived products.We aimed to evaluate the effect of replacing BSA in rabbit and ovine embryo slow freezing and vitrification media with a synthetic animal products free medium composed of synthetic hyaluronic acid: STEM ALPHA.Cryo3 (« CRYO3 »).During the first part, we evaluated the substitution candidates through a thermodynamic approach, using differential scanning calorimetry. In paralel, we adapted a mitochondrial activity evaluation protocol (JC-1) to rabbit embryo, which allowed us to complement morphological in vitro evaluation, and evaluated ewe superovulation protocols.During the second part, we used a biological approach to evaluate the replacement of BSA with synthetical products (containing CRYO3) in rabbit and ovine embryo slow freezing and vitrification media, using in vitro (slow freezing and vitrification) and in vivo (vitrification) evaluation methods.Our results seem to demonstrate that the chemically defined substitute CRYO3 can successfully replace BSA during rabbit embryo and ovine embryo cryopreservation (slow-freezing and vitrification)

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-12-20T10:33:59Z No. of bitstreams: 2 Tese - Gisele Aparecida Felix - 2016.pdf: 1558360 bytes, checksum: 761cc39345caea8787f3f1e6a9816a8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-12-27T12:47:48Z (GMT) No. of bitstreams: 2 Tese - Gisele Aparecida Felix - 2016.pdf: 1558360 bytes, checksum: 761cc39345caea8787f3f1e6a9816a8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-27T12:47:48Z (GMT). No. of bitstreams: 2 Tese - Gisele Aparecida Felix - 2016.pdf: 1558360 bytes, checksum: 761cc39345caea8787f3f1e6a9816a8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-24 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Outro / Trichology is a method to analyze micro-structural patterns of scarf-skin scales arrangements and medullary cells found in mammals guard hair. The present study was conducted to identify Caracu, Curraleiro Pé-Duro, Pantaneiro and Nellore bovine breeds by trichological morphology and morphometry, as well as validate the technique compared with genetic characterization. This study was presented in chapters. First, a review on trichology application on mammal species and breeds identification with basic concepts necessary for understanding trichology analysis and its applications. The second chapter was composed by characterization of guard hair of Caracu, Curraleiro Pé-Duro and Pantaneiro (Bos taurus taurus) and of Nellore (Bos taurus indicus), Brazilian bovine breeds, through trichology analysis, to assess the possibility of using this technique as a racial marker. Morphological and morphometric descriptions were held by 160 cattle (40 animals/breeds). Sub-samples of each bovine were made until quality slides were obtained. Morphology was determined through dichotomous keys and measures were carried out in the cuticle scale and in the hair shield matrix. Results indicated that trichology and genetic evaluation were similar in the point of view of classification of individuals within racial groups. Therefore, the technique can be considered useful as a marker for bovine breeds. / A tricologia é um método que permite analisar os padrões microestruturais de arranjos de escamas cuticulares e células medulares encontradas em pelos guarda de mamíferos. O presente estudo foi conduzido com o objetivo de identificar as raças bovinas Caracu, Curraleiro Pé-Duro, Pantaneiro e Nelore por meio da morfologia e morfometria tricológica, bem como validar a técnica comparando com resultados da caracterização genética. Esse estudo foi apresentado em forma de capítulos, com uma revisão de literatura sobre a aplicação da tricologia na identificação de espécies de mamíferos e de raças de interesse zootécnico, onde foram disponibilizadas informações sobre os conceitos básicos necessários ao entendimento da análise tricológica e suas aplicações. O capítulo seguinte foi composto pela caracterização de pelos guarda das raças bovinas Caracu, Curraleiro Pé-Duro e Pantaneiro (Bos taurus taurus) e da raça Nelore (Bos taurus indicus), por meio de análise tricológica, a fim de avaliar a possibilidade de uso desta técnica como marcador racial. Realizaram-se descrições morfológicas e morfométricas dos pelos de 160 bovinos (40 animais/raça). De cada bovino foram feitas subamostras de pelos até serem obtidas lâminas de qualidade. A morfologia foi determinada por meio de chave dicotômica e as medidas foram realizadas na escama cuticular e na medula e os resultados dessa metodologia foram comparados à caracterização genética dos animais amostrados. Os resultados indicaram que a tricologia e a avaliação genética foram similares no ponto de vista de classificação dos indivíduos dentro dos grupos raciais. Portanto, a técnica pode ser considerada útil como marcador racial para raças bovinas.