In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Spilled heavy fuel oil (HFO) sinks within the water column and accumulates in sediments, affecting aquatic organisms that are not typically exposed to oils that float. Previously, the 3-4 ring alkyl substituted polycyclic aromatic hydrocarbons (PAHs) have been identified as the major toxic components in crude oil. Since HFO is comprised of higher concentrations of 3-4 ringed alkyl PAH and an abundance of 5-6 ringed PAH relative to crude oil, it is predicted to be more toxic to the early life stages of fish. An effects-driven chemical fractionation (EDCF) of HFO 7102 was undertaken to establish the toxicity relative to crude oil, and to identify the compounds that are bioavailable and chronically toxic to the early life stages of fish. In this EDCF, the complex HFO 7102 mixture was separated by low temperature vacuum distillation into three distinct fractions, 2, 3 and 4. Each fraction was assessed using a chronic bioassay to determine whether it contained components that caused toxicity to rainbow trout embryos similar to that of the whole oil. Acute bioassays with juvenile trout demonstrated the presence of compounds that induce cytochrome P450 enzymes, an indicator of exposure to PAH. Fraction 3, the fraction more toxic than the parent mixture, was further separated by cold acetone extraction into fraction 3-1 (PAH-rich extract) and fraction 3-2 (wax residue), and assessed with the same bioassays. Simultaneous chemical analysis with bioassays guided the fractionation, and identified compounds abundant and consistently present in toxic fractions. Due to resistance to dispersion of HFO, a chemical dispersant was used with vigorous mixing to drive the maximum amount of oil into solution to minimize the potential for false negatives and the volume of test material used. The potency of HFO 7102 and its fractions were also measured using water accommodated fractions (WAFs) produced by a continuous flow system of water flowing through oil coated gravel. Both exposure methods traced the toxicity from whole oil into fractions containing higher concentrations of 3-4 ring alkyl PAH, similar to crude oil. This research is the first toxicological assessment of HFO 7102, which is essential for determining the risk of spills of HFO to fish, and whether the risk of oils can be predicted from their alkyl PAH composition. / Thesis (Master, Biology) -- Queen's University, 2013-01-24 14:14:16.278

Durante o cultivo in vitro, as células são submetidas a uma série de estímulos estressores e em geral, a heterogeneidade da resposta a estes estímulos não é levada em consideração. O presente estudo foi baseado em um modelo fatorial (2x2x3) criado para avaliar como embriões com cinética de desenvolvimento distinta respondem à combinação de estresse ambiental e metabólico durante o cultivo in vitro. Para isso, embriões rápidos (4 ou mais células às 22 horas de cultura) e Lentos (2 ou 3 células) foram produzidos in vitro, cultivados em 20% ou 5% O2 e também em suplementações de glicose distintas (0.6, 2 e 5mM), resultando em 12 grupos de estudo. Os embriões em estágio de blastocisto foram avaliados em 95 caracteres, incluindo 82 genes, 7 evidências bioquímicas (consumo de glicose, glutamato e piruvato; produção de lactato e ATP), geração de espécies reativas de oxigênio (EROs), atividade mitocondrial e, finalmente, o conteúdo lipídico. Os dados foram normalizados e reunidos em uma matriz que foi analisada em parcimônia, com 500 repetições e Tree-Bissection Reconection como algoritmo (software TNT) em busca de comportamentos semelhantes entre os grupos. Todos os caracteres foram aditivos e igualmente ponderados. Esta análise resultou em uma única árvore ideal, totalmente resolvida. Os resultados mostram os grupos dispostos em dois arranjos principais de acordo com a tensão de oxigênio, exceto os grupos de embriões lentos cultivados em um ambiente de alta glicose (5mM). Num segundo momento, cada um dos grupos pertencentes aos primeiros arranjos foi novamente analisado. A 5% O2 (mais semelhante ao encontrado no útero), os embriões se agruparam de acordo com a cinética de desenvolvimento, independentemente da concentração de glicose no meio de cultura. Nesta condição, embriões rápidos foram mais capazes de atingir o estágio de blastocisto sem sobrecarregar o Metabolismo. Já a 20% O2, a suplementação de glicose foi mais importante para o agrupamento. Em um ambiente de alta glicose, em especial os embriões lentos, apresentaram metabolismo alterado e bloqueio de desenvolvimento. No entanto, embriões cultivados em baixas concentrações de glicose foram mais capazes de ativar mecanismos adaptativos e superar as injúrias causadas pelo estresse. A plasticidade embrionária é uma característica única e com esse trabalho conseguimos demonstrar como o 13 metabolismo se modula para ajudar os embriões a enfrentar condições estressantes enquanto tentam sobreviver a qualquer custo. / During in vitro culture cells are submitted to many stressful stimuli, however, the heterogeneity of the response to those stimuli is usually not considered. This study was based on a factorial experimental design (2x2x3) created to evaluate how embryos with distinct developmental kinetics respond to the combination of environmental and metabolic stress during in vitro culture. For this purpose, Fast (4 or more cells at 22 hours of culture) and Slow embryos (2-3 cells) were produced in vitro using standard protocols, cultured in 20% or 5% O2 and also in distinct glucose concentrations (0, 2 and 5mM), resulting in 12 groups. Blastocysts were evaluated for 95 characters including 82 genes, 7 biochemical evidences: consumption of glucose, glutamate and pyruvate; production of lactate and ATP; generation of reactive oxygen species (ROS), mitochondrial activity and finally the lipid content. Data were normalized and gathered in a matrix that was analyzed under parsimony, with 500 replicates and Tree-Bissection Reconection as the swapping algorithm (TNT software), searching for similar behaviors. All characters were additive and equally weighted. This analysis resulted in a single optimal tree, fully resolved. Results show the groups arranging in two main clusters according to oxygen tension regardless of developmental kinetics and glucose, except the groups of slow embryos cultured in a high glucose environment (5mM). Each cluster was separately analyzed and at 5% of oxygen (more similar to what is found in the uterus), embryos clustered according to developmental kinetics and independently of glucose concentration in culture media. On that condition, embryos with a faster kinetics were more capable of reaching blastocyst stage without overloading the metabolism. At 20% of oxygen, glucose supplementation seems to be more important for the clustering. In a high glucose environment embryos, in special de slow ones, show altered metabolism and block. However embryos cultured in lower glucose concentrations are more capable of activating adaptive mechanisms and overcome stress injuries. The embryonic plasticity is a unique feature and with this work we were able to demonstrate how metabolism modulates to help the embryos face stressful conditions while trying to survive at any cost.

O momento das primeiras divisões celulares pode predizer o potencial de desenvolvimento de um embrião, incluindo sua capacidade de estabelecer prenhez. Além de diferenças relacionadas ao metabolismo, estresse e sobrevivência, embriões com diferentes velocidades de desenvolvimento apresentam padrões transcricionais distintos, principalmente relacionados ao metabolismo energético e lipídico. Como o padrão transcricional é regulado por fatores epigenéticos este estudo visou caracterizar os mecanismos epigenéticos envolvidos neste fenótipo. Para isto, embriões bovinos foram produzidos in vitro utilizando sêmen sexado (fêmeas) e as 40 horas pós inseminação (hpi) foram classificados como Rápidos (4 ou mais células) ou Lentos (2 ou 3 células), permanecendo em cultivo até o estágio de blastocisto (168 hpi) (Capítulo 1) ou sendo avaliados com 40 hpi, 96 hpi ou 168 hpi (Capítulo 2). No capítulo 1, a análise da metilação global do genoma pelo sistema EmbryoGENE Methylation DNA Array (EDMA) identificou 11.584 regiões diferencialmente metiladas (DMRs) (7.976 regiões hipermetiladas em blastocistos derivados do grupo de clivagem rápida FBL - e 3.608 regiões hipermetiladas em blastocistos derivados de embriões de clivagem lenta - SBL). Os FBL apresentaram mais regiões classificadas como hipermetiladas, distribuídas ao longo do genoma, como nos íntrons, exons, promotores e elementos de repetição, enquanto em SBL, as regiões hipermetiladas estavam mais presentes nas ilhas CpG. As DMRs foram agrupadas em relação aos processos biológicos a que estavam envolvidas, e algumas das vias mais afetadas foram relacionadas à sobrevivência/diferenciação celular e metabolismo energético/lipídico. Os perfis dos transcritos dos genes diferencialmente metilados (DMs) relacionados com estas vias também foram avaliados, e a maior parte revelou mudanças na quantificação relativa. No capítulo 2, foi avaliada ao longo do desenvolvimento a presença de metilações e hidroximetilações do DNA e modificações pós-traducionais de histonas (acetilação e trimetilação da lisina 9 da histona 3 e trimetilação da lisina 27 da histona 3 H3K9ac, H3K9me3 e H3K27me3, respectivamente). A cinética das primeiras clivagens influenciou a maioria das modificações epigenéticas estudadas desde os estágios iniciais até o blastocisto (exceto pela hidroximetilação do DNA e H3K27me3). A análise dos transcritos relacionados a inclusão ou remoção dessas modificações corroborou o padrão de modificações encontrado. É possível concluir que a cinética das primeiras clivagens apresenta relação com modificações epigenéticas nos embriões e que se manterão até o final do desenvolvimento pré-implantacional, resultando em blastocistos de padrão transcricional e metabólico distintos, podendo influenciar na viabilidade embrionária. / The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this, bovine embryos were produced in vitro using sexed semen (females) and the 40 hours post insemination (hpi) were classified as Fast (4 or more cells) or Slow (2 or 3 cells), remaining in culture until the blastocyst stage (168 hpi) (Chapter 1) or evaluated at 40 hpi, 96 hpi or 168 hpi (Chapter 2). In Chapter 1, the analysis of global DNA methylation by EmbryoGENE DNA Methylation Array (EDMA) identified 11,584 differentially methylated regions (DMRs) (7,976 regions hypermethylated in blastocysts derived from the fast cleavage group -FBL- and 3,608 hypermethylated regions in blastocyst derived from the slow cleavage group -SBL). FBL presented more regions classified as hypermethylated, distributed throughout the genome, as in introns, exons, promoters and repeating elements, whereas in SBL, hypermethylated regions were more present in the CpG islands. DMRs were grouped in relation to the biological processes to which they were involved, and some of the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Profiles of transcripts of differentially methylated (DMs) genes related to these pathways were also evaluated, and most presented changes in relative quantification. In Chapter 2, the presence of DNA methylations and hydroxymethylations and post-translational histone modifications (histone 3 lysine 9 acetylation and trimethylation and histone 3 lysine 27 trimethylation - H3K9ac, H3K9me3 and H3K27me3, respectively). The kinetics of the first cleavages influenced most of the epigenetic modifications studied from the initial stages to the blastocyst (except DNA hydroxymethylations and H3K27me3). The analysis of the transcripts related to insertion or removal of these modifications corroborated the pattern of modifications. It is possible to conclude that the kinetics of the first cleavages is related with epigenetic modifications in embryos that will be maintained through the pre-implantation development, resulting in blastocysts with different transcriptional and metabolic patterns, which might influence the embryonic viability.

A progesterona desempenha uma função de extrema importância para o desenvolvimento embrionário inicial, por meio da regulação do ambiente uterino no período prévio à adesão dos embriões ao endométrio. Nesse contexto o objetivo do presente estudo foi avaliar os efeitos da suplementação com progesterona ou progestágeno durante a fase inicial da gestação sobre o ambiente uterino e desenvolvimento embrionário de suínos, bem como seus efeitos no desempenho da leitegada nascida. Foram realizados dois experimentos. No experimento 1 utilizou-se 40 porcas e 28 marrãs que no 6º dia de gestação foram distribuídas em um dos três grupos experimentais: fêmeas suplementadas com 20 mg de altrenogest (Regumate®) do 6º ao 12º dia de gestação (RU; n = 23); fêmeas suplementadas com 2,15 mg/kg de progesterona de longa ação (Sincrogest®), injeção única no 6º dia de gestação (PG; n = 24); fêmeas não suplementadas (CON; n = 21). Esse experimento foi delineado de maneira inteiramente casualizada em um arranjo fatorial, sendo que a categoria (marrã ou porca) foi considerada fator 1 e os grupos (CON, RU e PG) fator 2. 18 fêmeas foram eutanasiadas no 13º dia de gestação, e 50 fêmeas no 28º dia de gestação. Foram analisados: taxa de prenhez, taxa de ovulação, sobrevivência embrionária, tamanho e peso de embriões e útero, volume e peso de corpos lúteos, volume das vesículas embrionárias, dosagem sérica de progesterona e 17β- estradiol, morfometrias glandular e de epitélio luminal do uterino. No experimento 2 foram utilizadas 75 matrizes, que no 6º dia de gestação foram alocadas de maneira interiramente casualisada em um dois grupos: fêmeas suplementadas com 20 mg de altrenogest (Matrix®) do 6º ao 12º dia de gestação (ALT; n = 36); fêmeas não suplementadas (CTR; n = 36). Analisadas: taxa de prenhez, período gestacional, peso médio e homogeneidade da leitegada, número de leitões mumificados, natimortos e nascidos vivos, quantidade de leitões nascidos com menos de 800 gramas. Não houve influência dos tratamentos sobre a taxa de prenhez e a sobrevivência embrionária foi prejudicada apenas para marrãs do grupo RU. Para o desenvolvimento embrionário os resultados divergiram entre as categorias, as marrãs do grupo CON apresentaram embriões maiores e mais pesados quando comparados aos grupos suplementados, bem como vesículas embrionárias maiores. Para as porcas o grupo RU apresentou embriões maiores e mais pesados. De maneira geral as suplementações com progesterona ou progestágeno estimularam o crescimento do epitélio glandular aos 13 dias de gestação, mas não tiveram efeito sobre epitélio luminal. Já aos 28 dias de gestação o efeito da estimulação foi apenas observado para marrãs do grupo PG. Os tratamentos estimularam também o crescimento dos corpos lúteos que foram maiores e mais pesados para os grupos suplementados. Em relação ao desempenho da leitegada, analisado no experimento 2, não houve efeito de tratamento para nenhuma das variáveis analisadas. A suplementação de progesterona/progestágeno a partir do 6º dia de gestação estimulou o crescimento do epitélio glandular uterino e afetou o desenvolvimento embrionário inicial, mas não exerceu efeito significativo sobre o desempenho da leitegada. / Progesterone plays a role of extreme importance for early embryonic development by regulating the uterine environment in the period prior to the adhesion of the embryos to the endometrium. In this context, the objective of the present study was to evaluate the effects of progesterone or progestogen supplementation during early gestation on the uterine environment and embryo development of pigs, as well as their effects on litter performance. Two experiments were carried out. In the experiment 1, 40 sows and 28 gilts were used, which were distributed in one of the three experimental groups: females supplemented with 20 mg altrenogest (Regumate®) from the 6th to the 12thh day of gestation (RU; n = 23); females supplemented with 2.15 mg / kg long acting progesterone (Sincrogest®), single injection at 6th day of gestation (PG; n = 24); females not supplemented (CON; n = 21). This experiment was completely randomized in a factorial arrangement, with the category (gilt or sow) being considered as factor 1 and the groups (CON, RU and PG) factor 2. 18 females were euthanized on the 13th day of gestation, and 50 females on the 28th day of gestation. Pregnancy rate, ovulation rate, embryo survival, embryo and uterus size and weight, volume and weight of corpora lutea, volume of embryonic vesicles, serum progesterone and 17β-estradiol concentrations, morphometric of uterine glandular epithelium and uterine luminal epithelium. In the experiment 2, 75 sows were used, which at the 6th day of gestation were allocated in a randomized manner in one of two groups: females supplemented with 20 mg of altrenogest (Matrix®) from 6 to 12 days of gestation (ALT; = 36); females not supplemented (CTR; n = 36). The variables analyzed were: pregnancy rate, gestation length, average of litter weight, within-litter variation, number of mummified, stillborn and live born piglets, number of piglets born with less than 800 grams. There was no influence of treatments on the pregnancy rate and embryo survival was impaired only for gilts in the RU group. For embryonic development the results differed among the categories, the gilts of the CON group had larger and heavier embryos when compared to the supplemented groups, as well as larger embryonic vesicles. For the sows the RU group presented larger and heavier embryos. In general, progesterone or progestogen supplementation stimulated the growth of the glandular epithelium at 13 days of gestation, but had no effect on luminal epithelium. However, on day 28 of gestation the stimulatory effect was only observed for gilts of the PG group. Treatments also stimulated the growth of corpora lutea that were larger and heavier for the supplemented groups (RU and PG). Regarding the performance of the litter, analyzed in experiment 2, there was no treatment effect for any of the variables analyzed. In conclusion, Progesterone / progestogen supplementation from day 6 of gestation affected the uterine glandular epithelium area, and early embryonic development, but did not have a significant effect on the litter performance.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Insects are confronted by a large variety of potentially harmful microorganisms to which they are resistant as they are able to build up an efficient innate defense system that relies on three tightly interconnected reactions. One of the reactions is the transient and rapid synthesis of a battery of antimicrobial peptides (AMPs). Development of antimicrobial therapeutic drugs and vaccines is very crucial due to factors such as the emergence of multiple-drug resistance. AMPs have been termed natural antibiotics because of their large spectrum of activity. The current study focused on the isolation and identification of cationic antimicrobial peptides and the analysis of immune response in the South African Euoniticellus intermedius embryonic (SAEIE08) cell line upon exposure to pathogens. E. intermedius is of the Coleopteran order in the Scarabaeoidea superfamily. Liquid growth inhibition assay showed higher antimicrobial activity in SAEIE08 that was treated with heat-killed E. coli compared to untreated. Further evidence for antimicrobial activity was seen as a clear zone of inhibition in solid growth inhibition assay when a gel run with protein extracts was plated and overlayed with live E. coli. Changes in protein expression patterns that were analysed in SDS-PAGE and 2-D PAGE indicated the most intense bands and spots at low molecular weight sizes around 10 kDa and/or 16 kDa which implicated increased induction of AMP expression upon exposure to pathogen. Homologues of Saccharomyces cerevisiae proteins were found in some of the 5′/3′ RACE sequences. Possible explanation for matches to these homologues could be that short sequences were used for database searches. The proteins were identified as flavin-containing monooxygenase, long-chain fatty acyl-CoA synthetase, severe depolymerization of actin protein and serine/threonine protein kinase. Interestingly, these proteins play roles in metabolism, cell proliferation and/or molecular pathways which do occur when cells are exposed to stress. There was also an insect peptide allatotropin from Spodoptera frugiperda. The results show that there is inducible antimicrobial activity in embryonic E. intermedius cell line.

Orientador: Roberto Sartori Filho / Banca: João Carlos Pinheiro Ferreira / Banca: Margot Alves Nunes Dode / Resumo: Objetivou-se com o presente estudo avaliar a criotorerância de mórulas e blastocistos produzidos in vivo de doadoras da raça Sindi e Nelore (Bos indicus) e Holandês Preto e Branco (HPB - Bos taurus). No Experimento 1, 24 fêmeas lactantes e não lactantes da raça Sindi foram superovuladas com 100 mg de FSH suíno com protocolos em que as duas últimas aplicações de FSH foram substituídas ou não por 300 UI de eCG. Sete dias após a indução da ovulação, os embriões foram colhidos e avaliados quanto ao estágio de desenvolvimento embrionário e grau de qualidade. Dois terços dos embriões foram destinados à criopreservação, dos quais metade foi congelada pelo método convencional, com curva de resfriamento de -0,5ºC/min e a outra metade foi vitrificada pela técnica de Cryotop. Em seguida, foram descongelados e transferidos para receptoras sincronizadas, de maneira contemporânea aos embriões frescos. No Experimento 2, 31 fêmeas da raça Nelore foram superovuladas com 133 mg de FSH suíno e os dois terços dos embriões colhidos foram criopreservados e transferidos semelhantemente ao Experimento 1. No Experimento 3, 67 vacas lactantes e novilhas da raça HPB foram superovuladas com 500 e 300 UI de FSH suíno, respectivamente, e após a colheita dos embriões foram adotados os mesmos procedimentos realizados nos Experimentos 1 e 2. Os resultados foram analisados por modelos lineares generalizados e estão apresentados na forma de média dos quadrados mínimos ± erro padrão. Nos Experimentos 1 e 2, os embriões transferidos a fresco apresentaram maior taxa de concepção aos 30 dias do que os vitrificados e congelados (54,8±7,4a, 17,7±7,3b e 19,5±6,6%b; respectivamente; P 0,0013) na raça Sindi (n=231 embriões) e (46,0±6,1a; 31,2±5,4b e 28,1±5,3%b; respectivamente, P 0,04) na raça Nelore (n=297 embriões). O estágio de desenvolvimento embrionário parece não... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the cryotolerance of morulae and blastocysts produced in vivo in Sindhi and Nelore (Bos indicus) and Holstein (Bos taurus) donnors. In Experiment 1, 24 lactating and non-lactating Sindhi donnors were superovulated with 100 mg porcine FSH with protocols in which the last two FSH treatments were replaced or not by 300 IU eCG. Embryos were collected 7 days after ovulation induction and embryo development and quality degree were accessed. Two thirds of the embryos were cryopreserved, by the conventional freezing or Cryotop method. After that, embryos were thawed and transferred to synchronized recipients, simultaneously to fresh embryos. In Experiment 2, 31 Nelore cows were superovulated with 133 mg porcine FSH and two thirds of the embryos were cryopreserved and transferred similarly to Experiment 1. In Experiment 3, 67 Holstein lactating dairy cows and heifers were superovulated with 500 and 300 IU pFSH, respectively, and the same procedures were performed as in Experiments 1 and 2. Results were analyzed using generalized linear models and are presented as least squares means ± standard error. In Experiments 1 and 2, fresh embryos had a higher conception rate at Day 30 than those vitrified and frozen (54.8±7.4a, 17.7±7.3b and 19.5±6.6%b; respectively; P 0.0013) in Sindhi donnors (n=231 embryos) and (46.0±6.1a; 31.2±5.4b and 28.1±5.3%b; respectively; P 0.04) in Nelore donors (n = 297 embryos). Embryo developmental stage seems to have not influenced conception rates of Zebu embryos, especially cryopreserved ones. Finnaly, in Experiment 3, conception rates of fresh and cryopreserved embryos were similar (37.4±12.3, 24.5±11.3 and 21.0±9.5%; respectively, P>0.15) and also no difference was detected for the cryotolerance of morulae versus blastocysts (P>0.10) / Mestre