<p>Animal mitochondrial DNA (mtDNA) has several features that give it great utility in the study of geographic structure of natural populations. Its small size and covalently closed circular conformation make it easy to purify. Strict maternal inheritance and homoplasmy makes the effective copy number of mtDNA as little as 1/4 that of nuclear loci; this renders populational complements of mtDNA less susceptible to the homogenizing effects of gene flow due to migration, and more susceptible to founder effects due to fluctuations in effective population size. The absence of recombination allows for comparatively simple reconstruction of genealogies of mtDNA variants. A comparatively high rate of base substitution assures that most species will have enough mtDNA variants to make population genetic inferences meaningful.</p> <p>Finally, sequence variants of mtDNA harboured in natural populations are presumed to be selectively neutral (i.e. has no effect on organismal fitness). This means that the distribution of mtDNA variants in populations will be due entirely to the stochastic and deterministic effects of species history, and not to natural selection. As a result, mtDNA can be used to infer species history directly and, after allowing for differences in modes of transmission, to infer the action of natural selection on other genetically determined factors like allozymes. The research of this dissertation was to survey world-wide natural populations of Drosophila melanogaster and its sibling species D. simulans for restriction fragment length polymorphisms of mtDNA. These cosmopolitan species have been widely studied for variation of allozymes and many other genetically determined factors (e.g. chromosomal inversions, morphometric characters).</p> <p>A total of 144 isofemale lines of D. melanogaster were analyzed from 18 geographic populations. Considerable size variation was observed in this sample. Most size variation occurs in the major non-coding region (A+T-rich region), yielding a total size range of 18.2 kbp to 19.9 kbp. The occurrence of several size variants among all haplotypes indicates that the rate of size mutation is quite high. Further, the frequency distribution of size variants suggests that there is selection against larger sized mtDNA molecules; a replication advantage to smaller sized molecules is a likely explanation. Finally, there is evidence for 'small-scale' size variation (i.e. a total range of 20 bp) in the coding region of mtDNA. However, it could not be determined if the observed mobility variation of the fragments in question was actually due to addition/deletion of DNA or due to conformational effects of particular base substitutions.</p> <p>Using 10 restriction enzymes, 23 restriction haplotypes were observed in D. melanogaster mtDNA. The phylogeographic distribution of these haplotypes allowed populations to be roughly divided into three longitudinal regions. Euro-African populations showed high intra- and inter-populational diversity and were inferred to be the oldest. Far East populations showed low intra-populational diversity but high inter-populational diversity; these populations similarly have a lengthy, but more complex, history. Western Hemisphere populations have low intra- and inter-populational diversity, and were inferred to be the youngest. A colonization history of this species is proposed. The species history suggested by mtDNA alone is quite similar to one proposed from the collective results of studies on several genetically-influenced traits in D. melanogaster (David and Capy 1988). mtDNA analysis is therefore shown to be a very efficient means by which to study species history. The very different histories of these regions reinforces the notion that the parallel latitudinal clines of allozyme alleles observed in this species (Singh et al 1982) are due to natural selection and are not historical.</p> <p>A total of 79 isofemale lines of D. simulans from 14 geographic populations (13 continental populations, and the Seychelles Islands) were surveyed. The 10 restriction enzymes revealed a very discontinuous distribution of variation. The continental populations harboured only four haplotypes, while the Seychelles population harboured two haplotypes that were very different from the continental ones. The discontinuous distribution suggests that D. simulans evolved as a series of allopatric ancestral populations, as has been previously suggested. The relative lack of diversity among the continental mtDNAs, coupled with the observation that none of the four continental haplotypes are unique to a single population, strongly suggests that continental populations of D. simulans result from a recent world-wide expansion from one of the ancestral populations, possibly concurrent with the expansion into the western Hemisphere by D. melanogaster. A recent expansion would explain the lower degree of allozyme population structure seen in D. simulans than in D. melanogaster, and is more parsimonious than the alternative explanations of a narrower niche-width or the adoption of a general purpose genotype.</p> / Doctor of Philosophy (PhD)

Biocompatible polymers are used exhaustively within the biomedical arena, demonstrating a mechanical and chemical diversity that few other materials possess. As polymer technologies evolves to cater for new medical demands, even the most niche biomedical application becomes an achievable reality. However, the discovery of new polymers is hindered by the complexity and intricacy in which the biological milieu interacts with a new substrate, reducing the ability to predict the appropriateness of a certain polymer for a specific application. This drawback can be countered by the high-throughput evaluation of large numbers of chemically diverse polymer candidates. In this thesis, the use of polymer microarrays is invoked to address two separate medically-relevant issues: the control of inflammation, and the improvement of cancer screening. In addition, I provide details of how polymer microarray techniques and technology can be employed to expand the repertoire of biomaterials research. Mitochondrial DNA (mtDNA) is an alarm molecule that contributes to the cytokine storm observed during severe tissue injury. An application where control of this systemic inflammation is achieved through scavenging of mtDNA by a polymer was proposed. Primary screening highlighted that 166 out of the 380 polymers evaluated bound to blood cells, making them unsuitable for a blood-based application. The remaining 214 blood-compatible polymers were cross-examined for mtDNA binding. Through polymer microarray and subsequent scale-up of promising candidates, a poly(methoxyethyl methacrylate-co-di(ethylamino)ethyl acrylate-co-methoxyethyl acrylate) was found to have a remarkable ability to scavenge mtDNA. Removal of cell-free mtDNA using this polymer is proposed to remove a key trigger of systemic inflammation. Cervical cancer screening includes the cytological evaluation of patient material for developed or developing abnormalities. An application was sought that would enrich for cancerous/pre-cancerous cells and improve upon current standards for detection. Four cancerous cervical cell lines (HeLa, CaSki, SiHa, and C33a) and four precancerous cell lines (W12E, W12G, W12GPX, and W12GPXY) were interrogated to identify polymers with consistent binding that may improve routine cytological evaluation. A short-list of 24 polymers was assembled, and cells from liquid based cytology samples from healthy patient were spiked with DiI-labelled cancerous/precancerous cells and the short-listed polymers were re-evaluated for preferential binding. An enrichment of abnormal cervical cells was observed with three polymers, which could form the foundation for improved screening resources. Inkjet printing can be a useful tool in developing patterned substrates, such as polymer microarrays. A piezoelectric drop-on-demand printer was used to explore the methods in which these can be fabricated. A wettability assay using picolitre volumes was developed and used to characterise O2 plasma treatment of glass slides. Additionally, the printing of a cell-binding polymer using this approach enabled the decoration of cells with precise spatial resolution.

This thesis uses mitochondrial DNA (mtDNA) phylogenies to explore patterns of past human mobility in Oceania. To extend the current knowledge of mtDNA variation in Oceania, 20 entire mt genomes were sequenced and analysed in a data set of more than 144 sequences from Australia, Oceania, Island Southeast Asia and Taiwan. The MinMax Squeeze method enabled this large data set to be analysed with an optimality criterion (Pierson et al. 2006). The analysis revealed two major groups of haplogroups in Oceania, distinguished by the relationships to others outside of the region: an 'ancient' set of types whose phylogenies and distributions suggest they are descended from the Pleistocene-era settlers of Near Oceania, and a second 'young' group whose presence in Oceanic populations may reflect more recent movements into Near Oceania. The detailed phylogenies of these haplogroups presented here will aid in future investigations of human mtDNA in Oceania, allowing samples to be screened by defining mutations to target haplogroups of interest. A large data set of global entire human mt DNA sequences was assembled from public data bases and tested for evidence of selection and recombination. These tests, and phylogenetic analyses of random subsets of the data set, found high levels of homoplasy in the sequences. Homoplasy in the control region of the mtDNA molecule was examined in particular, resulting in a relative scale of mutability at each position of the ~1kb sequence. Subsequent phylogenetic tests of weighting schemes derived from this analysis for the control hypervariable region I (HVR-I) did not show demonstrable improvements over the unweighted examples, but did highlight instances in which the HVR-I sequence failed to predict the more robust trees generated by the coding region. Finally, the HVR-I and diagnostic SNPs were sequenced in a set of 46 Polynesian samples from Auckland, and this data was analysed within a large set of HVR-I sequences (more than 4000) from Oceanic, Asian and the American populations available from public data bases. These analyses were informed by the whole mtDNA phylogenies generated earlier in the project, and add population level data to the emerging picture of prehistoric female mobility gained from entire mtDNA analyses.

In fishes, elopomorphs have a leptocephalous stage in the life cycle. Elopomorpha includes tenpounders (Elops), tarpons (Megalops), bonefishes (Albula), spine eels (Notacanthus), apodes and gulper eels. They are highly diversified in morphology and habitat utilization, Their monophyly is based on sharing of this larval stage. However, not all researches on these group accept the idea that this stage is of relationship. If this is true, the concept of elopomorpha must be re-evaluated. In attempt to elucidate their phylogenetic relationships, mtDNA 12S rRNA sequences were analyzed and data suggest that: (1)Elopomorpha is a monophyletic group. In other word, leptochphalous stage is a valid phylogenetic indictor, and it is not the result of convergency to environment; (2)Elops and Megalops share a common ancestor, and is a primitive group for Elopomorpha; (3)Megalops is the primitive lineage of Elopomorpha; (4)Albula and Notacanthus share a common ancestor, and they are the sister group of Anguilliformes; (5)Anguilliformes is a monophyletic group; (6)Muraenidae is a primitive group of Anguilliformes; (7)A high speciation rate might have taken place in Apodes within a short period of time; (8)Synaphobranchidae is a monophyletic group and it is the primitive group of Congroidei. (10) Synaphobranchid species have short interspecific genetic distance among species.

Mip1p is the highly processive monomeric mitochondrial DNA polymerase in Saccharomyces cerevisiae. Despite differences in enzyme structure, substrate topology, and possible nucleoid interactions, Mip1p continues to be used as a model for human mitochondrial DNA polymerase (POLG) variants associated with various human mitochondrial diseases. Structurally, Mip1p functions as a monomer, whereas, the POLG holoenzyme contains a catalytic subunit (POLGA) complexed with a dimeric form of an accessory subunit (POLGB) which functions by loading the enzyme onto mitochondrial DNA and enhancing processivity. However, Mip1p does contain a 279-residue carboxyl-terminal extension (CTE) absent in the structure of POLG. The function of the CTE has not yet been determined although studies of truncation variants identify 74 N-terminal residues are essential for Mip1p wild-type activity. Furthermore, regions encompassing Mip1p residues N1033 – E1038 and Y1039 – A1049 are suggested to function in mitochondrial DNA maintenance and fidelity, respectively. This study has developed a mutagenic strategy to systematically replace the residues in the mitochondrial DNA maintenance region with glycine in order to identify residues critical for Mip1p function. Using in vivo respiratory competence and erythromycin resistance assays accompanied by an in vitro non-radioactive DNA polymerase assay, this study has identified two key residues, E1036 and D1037 that may function in the exonuclease-polymerase coupling mechanism of Mip1p.

Fungal mitochondria are variable in size due to the presence of potential mobile elements such as group I and group II introns and homing endonuclease genes (HEGs). In this work the mitochondrial large ribosomal subunit gene (mt-rnl) of Ophiostoma ulmi and related species have been screened for the presence of introns and intron encoded proteins. Five introns have been noted in different regions of the rnl gene of O. ulmi and related species. Based on this rnl survey and rnl data from Genbank, an rnl intron landscape for ascomycetous and basidiomyctous fungi was generated by using bioinformatic based analysis. A total number of 23 possible intron insertion sites were found in the rnl gene of ascomycetous and basidiomycetous fungi. The results also indicate that regions of the rnl gene are more prone to intron invasion then others. The second project dealt with the evolution of mitochondrial ribosomal protein S5 (rps3) gene within the filamentous ascomycetes fungi. Within members of this group of fungi the rps3 gene typically is a component of the mL2449 group I intron but there are free-standing forms of rps3. The study examined if these free standing forms evolved only once due to an as of yet unknown recombination event or if the rps3 gene was transferred from the mL2449 intron to a new mtDNA locus several times during the evolution of the filamentous ascomycetes fungi. The third project was to sequence and characterize the intron and HEG found in the mitochondrial NADH dehydrogenase 4 (nad4) gene of an undescribed species of Pesotum. A 1.4 kb group IC2 intron has been identified in the nad4 gene of Pesotum strain WIN (M)1630. Overall the three studies demonstrate the invasive nature of introns and their associated ORFs and the potential of these introns to influence gene structure and size variation among the fungal mtDNAs.

Mip1p is the highly processive monomeric mitochondrial DNA polymerase in Saccharomyces cerevisiae. Despite differences in enzyme structure, substrate topology, and possible nucleoid interactions, Mip1p continues to be used as a model for human mitochondrial DNA polymerase (POLG) variants associated with various human mitochondrial diseases. Structurally, Mip1p functions as a monomer, whereas, the POLG holoenzyme contains a catalytic subunit (POLGA) complexed with a dimeric form of an accessory subunit (POLGB) which functions by loading the enzyme onto mitochondrial DNA and enhancing processivity. However, Mip1p does contain a 279-residue carboxyl-terminal extension (CTE) absent in the structure of POLG. The function of the CTE has not yet been determined although studies of truncation variants identify 74 N-terminal residues are essential for Mip1p wild-type activity. Furthermore, regions encompassing Mip1p residues N1033 – E1038 and Y1039 – A1049 are suggested to function in mitochondrial DNA maintenance and fidelity, respectively. This study has developed a mutagenic strategy to systematically replace the residues in the mitochondrial DNA maintenance region with glycine in order to identify residues critical for Mip1p function. Using in vivo respiratory competence and erythromycin resistance assays accompanied by an in vitro non-radioactive DNA polymerase assay, this study has identified two key residues, E1036 and D1037 that may function in the exonuclease-polymerase coupling mechanism of Mip1p.

This thesis uses mitochondrial DNA (mtDNA) phylogenies to explore patterns of past human mobility in Oceania. To extend the current knowledge of mtDNA variation in Oceania, 20 entire mt genomes were sequenced and analysed in a data set of more than 144 sequences from Australia, Oceania, Island Southeast Asia and Taiwan. The MinMax Squeeze method enabled this large data set to be analysed with an optimality criterion (Pierson et al. 2006). The analysis revealed two major groups of haplogroups in Oceania, distinguished by the relationships to others outside of the region: an 'ancient' set of types whose phylogenies and distributions suggest they are descended from the Pleistocene-era settlers of Near Oceania, and a second 'young' group whose presence in Oceanic populations may reflect more recent movements into Near Oceania. The detailed phylogenies of these haplogroups presented here will aid in future investigations of human mtDNA in Oceania, allowing samples to be screened by defining mutations to target haplogroups of interest. A large data set of global entire human mt DNA sequences was assembled from public data bases and tested for evidence of selection and recombination. These tests, and phylogenetic analyses of random subsets of the data set, found high levels of homoplasy in the sequences. Homoplasy in the control region of the mtDNA molecule was examined in particular, resulting in a relative scale of mutability at each position of the ~1kb sequence. Subsequent phylogenetic tests of weighting schemes derived from this analysis for the control hypervariable region I (HVR-I) did not show demonstrable improvements over the unweighted examples, but did highlight instances in which the HVR-I sequence failed to predict the more robust trees generated by the coding region. Finally, the HVR-I and diagnostic SNPs were sequenced in a set of 46 Polynesian samples from Auckland, and this data was analysed within a large set of HVR-I sequences (more than 4000) from Oceanic, Asian and the American populations available from public data bases. These analyses were informed by the whole mtDNA phylogenies generated earlier in the project, and add population level data to the emerging picture of prehistoric female mobility gained from entire mtDNA analyses.

O objetivo do presente estudo foi comparar o efeito da idade de doadores de oócitos sobre os parâmetros reprodutivos, a taxa de recuperação e qualidade de oócitos após ICSI utilizando sêmen sexado. Éguas Pôneis de Polo (n = 79) e Puro-Sangue (n = 23) doadoras de oócitos (400-600kg e 3-29 anos) foram utilizados durante as estações reprodutivas de 2009/2010 e 2011 no hemisfério Sul (San Luis, Argentina) e norte (Kentucky, EUA), respectivamente. As éguas foram divididas em três categorias experimentais: Jovens (YM: 3-10 anos), Meia Idade (MA: 11-17a) e Idosas (OM&ge;18a). Um total de 326 oócitos recuperados in vivo a partir de folículos pré-ovulatórios (n=279) e folículos imaturos em crescimento (n=47). Desses, 224 oócitos foram recuperados, classificado e dirigido a um programa comercial ICSI com Pôneis de Polo (primeira estação). Durante a segunda estação, 57 oócitos foram recuperados a partir de folículos pré-ovulatórios e 47 oócitos de folículos em crescimento e congelados em nitrogênio líquido para ser usado posteriormente em outro estudo. Os pontos experimentais avaliados foram: intervalos (dias) entre a aspiração ou a ovulação (AS/OV) ao PGF, PGF ao GnRH (Dia 0=GnRH), AS/OV ao Dia 0, e AS/OV ao Dia&#43;1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D&#43;1, e a taxa de crescimento folicular. O número total de aspirações, o número de folículos aspirados e oócitos por aspiração e/ou folículo, o grau de expansão das células do cúmulos, a presença e qualidade do corpo polar (PB), o volume ooplasma, o intervalo de GnRH a aspiração, a GnRH a ICSI, e a taxa de clivagem (CR) depois da ICSI também foram avaliados. Foram observadas diferenças significativas entre as três categorias experimentais (YM, MA e OM) para: intervalos (dias) entre PGF-GnRH, AS/OV-GnRH, AS/OV-Day 1, edema uterino no Dia 0, CT em Dia 1, MdF1 no D0 e D&#43;1 e volume de ooplasma. A taxa de recuperação in vivo (RR) não foi afetada pela idade (média de 102%, 85% e 73,4% de oócitos recuperados por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as categorias experimentais. Em conclusão, um efeito de envelhecimento pode ser observado em vários parâmetros reprodutivos em éguas. Os ovócitos no presente estudo tiveram um menor volume de ooplasma para OM comparados com YM, mas isso não foi eficaz em predizer a viabilidade dos oócitos ou o potencial para o desenvolvimento para a avaliação da taxa de clivagem após ICSI utilizando sêmen sexado / The aim of the present study was to compare the effect of oocyte donors` age on reproductive parameters, recovery rate and quality of oocytes after ICSI using sexed semen. Polo Ponies (n=79) and Thoroughbred (n=23) oocyte donor mares (400-600kg and 3-29 years) were used during the 2009/2010 and 2011 reproductive seasons in the Southern (San Luis, Argentina) and in the Northern hemispheres (Kentucky, USA), respectively. Mares were divided into three experimental categories: Young Mares (YM: 3-10&#1059;), Middle Age Mares (MA: 11-17&#1059;) and Old Mares (OM18&#1059;). A total of 326 oocytes were recovered in vivo from pre-ovulatory follicles (n=279) and immature-growing follicles (n=47). From those, 224 oocytes were recovered, sorted and directed to a commercial ICSI program with Polo Ponies (first season). During the study`s second season, 57 oocytes were recovered from pre-ovulatory follicles and 47 oocytes from growing follicles and frozen in liquid nitrogen to be used in another study. The evaluated experimental end-points were: Intervals (days) between aspiration or ovulation (AS/OV) to PGF, PGF to GnRH (Day 0=GnRH), AS/OV to Day 0, and AS/OV to Day &#43; 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D&#43;1, and follicular growth rate. Total number of aspirations, number of follicles aspirated and oocytes recovered per aspiration and/or follicle, the degree of cumulus cell expansion, the presence and quality of polar body (PB), the ooplasm volume, the interval from GnRH to aspiration, GnRH to ICSI, and the cleavage rate (CR) after ICSI were also evaluated. Significant differences between the three experimental categories (YM, MA and OM) were observed for: intervals (days) between PGF-GnRH, AS/OV-GnRH, AS/OV-Day &#43;1, uterine edema on Day 0, CT on Day &#43;1, MdF1 on D0 and D&#43;1 and ooplasm volume. The in vivo recovery rate (RR) was not affected by age (average of 102%, 85% and 73.4% of oocyte recovered per cycle, per F1 and per follicle, respectively; P>0.05). The CR did not differ among experimental categories. In conclusion, an effect of aging could be observed in several reproductive parameters in mares. The oocytes in the present study had a smaller ooplasm volume for OM compared to YM, but this was not effective in predicting oocyte viability or the potential to development through evaluation of the cleavage rate after ICSI using sexed semen

O objetivo do presente estudo foi comparar o efeito da idade de doadores de oócitos sobre os parâmetros reprodutivos, a taxa de recuperação e qualidade de oócitos após ICSI utilizando sêmen sexado. Éguas Pôneis de Polo (n = 79) e Puro-Sangue (n = 23) doadoras de oócitos (400-600kg e 3-29 anos) foram utilizados durante as estações reprodutivas de 2009/2010 e 2011 no hemisfério Sul (San Luis, Argentina) e norte (Kentucky, EUA), respectivamente. As éguas foram divididas em três categorias experimentais: Jovens (YM: 3-10 anos), Meia Idade (MA: 11-17a) e Idosas (OM&ge;18a). Um total de 326 oócitos recuperados in vivo a partir de folículos pré-ovulatórios (n=279) e folículos imaturos em crescimento (n=47). Desses, 224 oócitos foram recuperados, classificado e dirigido a um programa comercial ICSI com Pôneis de Polo (primeira estação). Durante a segunda estação, 57 oócitos foram recuperados a partir de folículos pré-ovulatórios e 47 oócitos de folículos em crescimento e congelados em nitrogênio líquido para ser usado posteriormente em outro estudo. Os pontos experimentais avaliados foram: intervalos (dias) entre a aspiração ou a ovulação (AS/OV) ao PGF, PGF ao GnRH (Dia 0=GnRH), AS/OV ao Dia 0, e AS/OV ao Dia&#43;1; edema uterino (UE); tonus cervical (CT); diâmetro máximo do folículo dominante (MdF1) em D0 e D&#43;1, e a taxa de crescimento folicular. O número total de aspirações, o número de folículos aspirados e oócitos por aspiração e/ou folículo, o grau de expansão das células do cúmulos, a presença e qualidade do corpo polar (PB), o volume ooplasma, o intervalo de GnRH a aspiração, a GnRH a ICSI, e a taxa de clivagem (CR) depois da ICSI também foram avaliados. Foram observadas diferenças significativas entre as três categorias experimentais (YM, MA e OM) para: intervalos (dias) entre PGF-GnRH, AS/OV-GnRH, AS/OV-Day 1, edema uterino no Dia 0, CT em Dia 1, MdF1 no D0 e D&#43;1 e volume de ooplasma. A taxa de recuperação in vivo (RR) não foi afetada pela idade (média de 102%, 85% e 73,4% de oócitos recuperados por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as por ciclo, por F1 e por folículo, respectivamente, P> 0,05). O CR não diferiu entre as categorias experimentais. Em conclusão, um efeito de envelhecimento pode ser observado em vários parâmetros reprodutivos em éguas. Os ovócitos no presente estudo tiveram um menor volume de ooplasma para OM comparados com YM, mas isso não foi eficaz em predizer a viabilidade dos oócitos ou o potencial para o desenvolvimento para a avaliação da taxa de clivagem após ICSI utilizando sêmen sexado / The aim of the present study was to compare the effect of oocyte donors` age on reproductive parameters, recovery rate and quality of oocytes after ICSI using sexed semen. Polo Ponies (n=79) and Thoroughbred (n=23) oocyte donor mares (400-600kg and 3-29 years) were used during the 2009/2010 and 2011 reproductive seasons in the Southern (San Luis, Argentina) and in the Northern hemispheres (Kentucky, USA), respectively. Mares were divided into three experimental categories: Young Mares (YM: 3-10&#1059;), Middle Age Mares (MA: 11-17&#1059;) and Old Mares (OM18&#1059;). A total of 326 oocytes were recovered in vivo from pre-ovulatory follicles (n=279) and immature-growing follicles (n=47). From those, 224 oocytes were recovered, sorted and directed to a commercial ICSI program with Polo Ponies (first season). During the study`s second season, 57 oocytes were recovered from pre-ovulatory follicles and 47 oocytes from growing follicles and frozen in liquid nitrogen to be used in another study. The evaluated experimental end-points were: Intervals (days) between aspiration or ovulation (AS/OV) to PGF, PGF to GnRH (Day 0=GnRH), AS/OV to Day 0, and AS/OV to Day &#43; 1; uterine edema (UE); cervical tone (CT); maximum diameter of dominant follicle (MdF1) on D0 and D&#43;1, and follicular growth rate. Total number of aspirations, number of follicles aspirated and oocytes recovered per aspiration and/or follicle, the degree of cumulus cell expansion, the presence and quality of polar body (PB), the ooplasm volume, the interval from GnRH to aspiration, GnRH to ICSI, and the cleavage rate (CR) after ICSI were also evaluated. Significant differences between the three experimental categories (YM, MA and OM) were observed for: intervals (days) between PGF-GnRH, AS/OV-GnRH, AS/OV-Day &#43;1, uterine edema on Day 0, CT on Day &#43;1, MdF1 on D0 and D&#43;1 and ooplasm volume. The in vivo recovery rate (RR) was not affected by age (average of 102%, 85% and 73.4% of oocyte recovered per cycle, per F1 and per follicle, respectively; P>0.05). The CR did not differ among experimental categories. In conclusion, an effect of aging could be observed in several reproductive parameters in mares. The oocytes in the present study had a smaller ooplasm volume for OM compared to YM, but this was not effective in predicting oocyte viability or the potential to development through evaluation of the cleavage rate after ICSI using sexed semen