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<p>This thesis details a study of saprophytic competence in the Gram-negative bacterium Sinorhizobium meliloti, and comprises three main areas of research. The B-ketoadipate pathway is required for the catabolism of a wide range of aromatic compounds that are released into soil through the degradation of lignin. We demonstrate that S. meliloti encodes enzymes associated with the protocatechuate branch of the B-ketoadipate pathway within two operons (pcaDCHGB and pcaIJF) whose expression is regulated by the LysR-protein PcaQ and the IclR-type regulator PcaR, respectively. We show that purified PcaQ recognizes a motif with partial dyad symmetry (5' ATAACCN4-GGTTAA 3') positioned upstream of the pcaD promoter, and that this site is required for the regulated expression of pcaD in vivo. We report that PcaQ also regulates the expression of a protocatechuate-inducible ABC-type transport system that we infer is involved in the uptake of this aromatic acid, and we extend this analysis to identify PcaQbinding motifs in the genomes of a-,B-, and y-proteobacteria. </p> <p>In addition to protocatechuate, S. meliloti may utilize hydroxyproline as an energy source, as this amino acid is released into soil during the natural decay of plant tissue. We demonstrate that S. meliloti encodes a hydroxyproline-inducible ABC-type transport system that mediates the uptake of trans-4-hydrox-L-proline, as determined via growth and transport assays. </p> <p>As a more comprehensive method of examining saprophytic competence, we assayed the growth of S. meliloti upon inoculation into sterile bulk soil. We screened 40 S. meliloti strains carrying deletions within the pSymA or pSymB megaplasmids for growth in soil, and report that the majority of strains establish a stable population (greater than or equal to 10^8 cells g^(-1) soil) that persists for several weeks. In contrast, two S. meliloti strains exhibited a decreased ability to colonize soil, indicating that loci within the deleted regions play a role in saprophytic competence. </p> / Thesis / Doctor of Philosophy (PhD)

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>

<p>Soybean and related legumes export symbiotically-fixed nitrogen from the nodules to the leaves as ureides. The ureide allantoin is hydrolyzed by allantoinase to allantoate then further degraded by other enzymes, releasing ammonia and carbon dioxide. This study aimed to identify allantoinase genes in soybean and their gene expression as well as enzyme activity patterns. The effects of water limitation and allantoin treatment on the expression and activity of allantoinase in N₂-fixing plants were also evaluated. Enzyme activity and ureide content were evaluated using a spectrophotometric assay. Real time RT-PCR was used to quantify the amount of gene products. Four allantoinase genes were identified and were expressed, with <i>GmALN1</i> and <i>2</i> constantly expressed at higher levels. In seedlings, allantoinase was found to be actively synthesized more in cotyledons than in the embryonic axes, as seen by early enzyme activity and higher <i>GmALN 1</i> and <i>2</i> transcript levels. Allantoate produced in these tissues appeared to be mobilized to the developing axes. <i>GmALN1</i> and <i>2</i> were implicated in post-germination nitrogen assimilation during early seedling growth, while <i>GmALN3</i> and <i>4</i> were consistently expressed at very low levels, with an exception in nodules. Transcript abundance in the nodules of N₂-fixing plants, supported by the high enzyme activity and ureide content observed, suggested an important role in the synthesis and transport of allantoate in these tissues. Allantoinase was also detected in non-fixing tissues but may play a different role in these tissues, most probably functioning in the turnover and salvage of purine nucleotides. The effect of exogenous allantoin during water limitation was investigated. The addition of allantoin prior to water limitation seemed to change the sensitivity of soybean to such stress, prolonging its ureide catabolic activity at least up to 5 days without water. Results of this study will aid in our understanding of how ureide catabolism is regulated during soybean development. This information may help address problems in legume crop improvement specifically in enhancing N₂-fixation and yield capacity and in coping with water limitation stress.</p>

Made available in DSpace on 2014-06-11T19:28:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-08Bitstream added on 2014-06-13T19:36:56Z : No. of bitstreams: 1 vieira_fvr_me_botfmvz.pdf: 300511 bytes, checksum: e122704bc94212fa16d6f5f449d2e702 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / O objetivo deste experimento foi avaliar o efeito da infusão intravenosa de glicose sobre as concentrações séricas de glicose, insulina, IGF-1, P4, expressão de RNAm de GHR1A e IGF-1, e expressão de RNAm das enzimas hepáticas CYP2C e CYP3A, responsáveis pelo catabolismo de P4 no fígado, em vacas leiteiras secas, ovariectomizadas e com dispositivo intravaginal de P4 (CIDR) em diferentes BE. Foram utilizadas 15 vacas mestiças Holandês/Gir ovariectomizadas e secas, aleatoriamente distribuídas em um de dois tratamentos nutricionais: 1) BEN (n=7) e 2) BEP (n=8). O grupo de vacas em BEP recebeu concentrado individualmente uma vez ao dia. Durante a fase de adaptação (d-28 ao d-15,5), cada vaca recebeu um CIDR de terceiro uso, sendo que após esta fase (d-14), cada vaca recebeu um CIDR novo. No d 0, as vacas foram aleatoriamente distribuídas em crossover design contendo dois períodos de 24 horas cada (d 0 e d 1): 1) infusão intravenosa de glicose (0,5g/Kg de PV) ou 2) infusão intravenosa de salina (0,9% NaCl). Imediatamente após jejum de 12 horas, as infusões foram feitas em período de três horas. Amostras de sangue foram coletadas às -12 (início do jeum), 0 (antes da infusão), 3 e 6 horas após início da infusão através da veia coccígea em tubos tipo vacutainer. As biopsias hepáticas foram feitas às 0 e 3 horas nos dias do tratamento (d 0 e d 1). Vacas em BEN perderam mais PV e ECC em relação às vacas em BEP (-23,15 vs. 16,5 kg ± 3,9; -0,200 vs. 0,075 unidades de ECC ± 0,062, respectivamente). Vacas recebendo infusão intravenosa de glicose tiveram maiores concentrações séricas de glicose às 3 horas do início da infusão do que vacas recebendo salina. Vacas em BEN recebendo glicose tiveram maiores concentrações séricas de insulina do que vacas em BEP recebendo glicose às 3 horas pós-infusão... / The objective of this study was to evaluate the effect of intravenous glucose infusion on serum concentrations of glucose, insulin, IGF-1, progesterone (P4), mRNA expression of GHR1A, IGF-1, and mRNA expression of hepatic enzymes CYP 2C and CYP 3A responsible for the catabolism of P4 in the liver in dry cows, ovariectomized with intravaginal device P4 (CIDR) in different energy balances. Fifteen non-lactating, ovariectomized Gir × Holstein cows, and randomly assigned to: 1) negative nutrient balance (NB; n=7)) and 2) positive nutrient balance (PB; n=8). The group of cows in PB was supplemented individually once a day. For the adaptation phase (d-d-28 to 15, 5), each cow received a CIDR of the third use, and after the adaptation phase (d-14), each cow received a new CIDR. On d 0, cows within nutritional treatment were randomly assigned to receive, in a crossover design containing 2 periods of 24 h each (d0 and d1): 1) intravenous glucose infusion (0.5g/Kg of BW), or 2) intravenous saline infusion (0,9% NaCl). Immediately after fasting for 12 hours, infusions were made over a period of three hours. Blood samples were collected at -12 (beginning of fasting), 0 (before infusion), 3 and 6 hours after start of infusion via the coccygeal vein in vacutainer tubes without anticoagulant type. The liver biopsies were performed at 0 and 3 hours in the day of treatment (d 0 and d 1). NB cows lost more BW and BCS than PB cows (-23.15 vs. 16.5 kg ± 3.9, vs. -0.200. 0.075 ± 0.062 BCS units, respectively). Cows receiving intravenous infusion of glucose had higher serum concentrations of glucose to 3 hours for the start of infusion than cows receiving saline. NB cows receiving glucose had higher serum concentrations of insulin than PB cows receiving glucose, however PB cows receiving glucose had higher serum concentrations... (Complete abstract click electronic access below)

Orientador: José Luiz Moraes Vasconcelos / Banca: José Roberto Sartori / Banca: José Buratini Jr. / Resumo: O objetivo deste experimento foi avaliar o efeito da infusão intravenosa de glicose sobre as concentrações séricas de glicose, insulina, IGF-1, P4, expressão de RNAm de GHR1A e IGF-1, e expressão de RNAm das enzimas hepáticas CYP2C e CYP3A, responsáveis pelo catabolismo de P4 no fígado, em vacas leiteiras secas, ovariectomizadas e com dispositivo intravaginal de P4 (CIDR) em diferentes BE. Foram utilizadas 15 vacas mestiças Holandês/Gir ovariectomizadas e secas, aleatoriamente distribuídas em um de dois tratamentos nutricionais: 1) BEN (n=7) e 2) BEP (n=8). O grupo de vacas em BEP recebeu concentrado individualmente uma vez ao dia. Durante a fase de adaptação (d-28 ao d-15,5), cada vaca recebeu um CIDR de terceiro uso, sendo que após esta fase (d-14), cada vaca recebeu um CIDR novo. No d 0, as vacas foram aleatoriamente distribuídas em crossover design contendo dois períodos de 24 horas cada (d 0 e d 1): 1) infusão intravenosa de glicose (0,5g/Kg de PV) ou 2) infusão intravenosa de salina (0,9% NaCl). Imediatamente após jejum de 12 horas, as infusões foram feitas em período de três horas. Amostras de sangue foram coletadas às -12 (início do jeum), 0 (antes da infusão), 3 e 6 horas após início da infusão através da veia coccígea em tubos tipo vacutainer. As biopsias hepáticas foram feitas às 0 e 3 horas nos dias do tratamento (d 0 e d 1). Vacas em BEN perderam mais PV e ECC em relação às vacas em BEP (-23,15 vs. 16,5 kg ± 3,9; -0,200 vs. 0,075 unidades de ECC ± 0,062, respectivamente). Vacas recebendo infusão intravenosa de glicose tiveram maiores concentrações séricas de glicose às 3 horas do início da infusão do que vacas recebendo salina. Vacas em BEN recebendo glicose tiveram maiores concentrações séricas de insulina do que vacas em BEP recebendo glicose às 3 horas pós-infusão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the effect of intravenous glucose infusion on serum concentrations of glucose, insulin, IGF-1, progesterone (P4), mRNA expression of GHR1A, IGF-1, and mRNA expression of hepatic enzymes CYP 2C and CYP 3A responsible for the catabolism of P4 in the liver in dry cows, ovariectomized with intravaginal device P4 (CIDR) in different energy balances. Fifteen non-lactating, ovariectomized Gir × Holstein cows, and randomly assigned to: 1) negative nutrient balance (NB; n=7)) and 2) positive nutrient balance (PB; n=8). The group of cows in PB was supplemented individually once a day. For the adaptation phase (d-d-28 to 15, 5), each cow received a CIDR of the third use, and after the adaptation phase (d-14), each cow received a new CIDR. On d 0, cows within nutritional treatment were randomly assigned to receive, in a crossover design containing 2 periods of 24 h each (d0 and d1): 1) intravenous glucose infusion (0.5g/Kg of BW), or 2) intravenous saline infusion (0,9% NaCl). Immediately after fasting for 12 hours, infusions were made over a period of three hours. Blood samples were collected at -12 (beginning of fasting), 0 (before infusion), 3 and 6 hours after start of infusion via the coccygeal vein in vacutainer tubes without anticoagulant type. The liver biopsies were performed at 0 and 3 hours in the day of treatment (d 0 and d 1). NB cows lost more BW and BCS than PB cows (-23.15 vs. 16.5 kg ± 3.9, vs. -0.200. 0.075 ± 0.062 BCS units, respectively). Cows receiving intravenous infusion of glucose had higher serum concentrations of glucose to 3 hours for the start of infusion than cows receiving saline. NB cows receiving glucose had higher serum concentrations of insulin than PB cows receiving glucose, however PB cows receiving glucose had higher serum concentrations... (Complete abstract click electronic access below) / Mestre

Le maïs est une plante de culture vivrière commerciale très importante, cultivée partout dans le monde. Sa croissance et les rendements en champ sont affectés par de nombreuses contraintes environnementales. Dans ce travail, l'impact du polluant gazeux phytotoxique, l'ozone, a été étudié sur les feuilles de maïs en utilisant une approche moléculaire et biochimique. Les concentrations en ozone dans la troposphère ont considérablement augmenté dans un passé récent, et actuellement l'ozone affecte clairement les cultures. Un système de fumigation récemment développé à l'INRA a été utilisé pour exposer à l'ozone les maïs en champs de façon contrôlée. Soixante et onze jours après le semis, les plantes ont été soumises à une fumigation d'ozone de 98 ppb.h-1 en moyenne. Les feuilles 10 et 12 ont été récoltées après 20, 35 et 50 jours de traitement. L'expression de trois gènes codant les metacaspases (ZmMCII-1, ZmMCII-2 and ZmMCII-3) ainsi que de leur activité enzymatique globale ont été étudiées lors de la sénescence, et en réponse à l'ozone, dans les feuilles. Pour la première fois, nous avons montré une accumulation significative de l'ARNm des trois metacapases en réponse à l'ozone. Par ailleurs, l'activité enzymatique cellulaire globale des metacaspases est également fortement stimulée dans les deux cas (ozone et sénescence). La surexpression des gènes et la stimulation de l'activité des metacaspases pourraient êtres liés à un ajustement des catabolismes protéiques des feuilles lors de la sénescence et en réponse à l'ozone. Au cours d'autres expériences, les mêmes réponses ont été observées dans le cas de la papaïne-like protéase à cystéine (Mor-CP). De plus, un nouvel inhibiteur des protéases à cysteine, une cystatin a été identifiée dans des feuilles de maïs et a été nommée CC11. L'ADNc complet a été isolé et la protéine recombinante CC11 correspondante a été obtenue et purifiée, à partir d'un système bactérien hétérologue (Vecteur pET30 (EK/LIC) et E. coli BL21 (DE3) plyS). Cette nouvelle cystatine présente une activité inhibitrice optimale vis-à-vis de la papaïne (100%) et très importante (57-80%) vis à vis des protéases à cystéine de feuilles de maïs. Par conséquent, son implication dans la régulation de l'activité des protéases à cystéine en réponse à l'ozone, peut être supposée. Enfin, la production de protéines recombinantes à partir d'ADNc isolés a été réalisée, correspondant aux régions actives de la metacaspase ZmMCII-2 et de la protéase à cystéine Mor-CP. Les anticorps polyclonaux ont été obtenus pour une étude future des événements post-transcriptionels. En conclusion, les protéases à cystéine (caspase-like et papaïne-like) pourraient être utilisées comme paramètres nouveaux pour le criblage de géniteurs dans les programmes d'amélioration du maïs pour la tolérance à l'ozone / Maize is a major crop cultivated all over the world. Its yield and growth is affected by numerous environmental stresses. In the present work, the impact of the gaseous phytotoxic pollutant, ozone, on maize leaves was studied, using a molecular and biochemical approach. Concentrations of ozone in the troposphere have increased dramatically in the recent past and it now negatively affects crops growth and yield. In this work, a recently developed fumigation system was used to expose maize plants in the field. Seventy-one days after sowing, the plants were submitted to ozone fumigation at an average concentration of 98 ppb.h-1. The 10th and 12th leaves were harvested after 20, 35 and 50 days of treatment. Expression of three genes coding caspase-like cysteine proteases “metacaspases” (denoted as ZmMCII-1, ZmMCII-2 and ZmMCII-3) and their global activity was studied in senescent and ozone-exposed maize leaves. For the first time, we observed that mRNA accumulation of the three caspase-like cysteine proteases increased significantly in response to ozone exposure. Moreover, the global activity of metacaspases also increased significantly in both senescencing and ozone-exposed plants. The metacaspases activity and mRNA up-regulation could represent control points for leaf tissues to determine the degree and timing of protein catabolism during senescence and ozone treatment. Protein catabolism was indeed stimulated in response to ozone since increases in the expression of a gene coding an ozone-induced papain-like cysteine protease (Mor-CP) and in global papain-like cysteine proteases activities in leaf tissues were observed. In addition, we identified new cysteine protease inhibitor CC11 “cystatin” in maize leaves. After the recombinant CC11 protein was purified from a bacterial heterologous system (E. coli strain BL21 (DE3) plyS with a pET 30 (EK/LIC) vector), it was shown to be active in vitro against commercial papain (100% inhibition) and against total maize leaf cysteine protease extract (57-80% inhibition). Therefore its involvement in the regulation of cysteine protease activity in response to ozone exposure could be supposed. Production of the recombinant proteins corresponding to the active regions of ZmMCII-2 and Mor-CP was achieved. The corresponding polyclonal antibodies were obtained to study post-transcriptional events in maize leaves, in the future. In conclusion, we have identified the new enzymes, metacaspases and cysteine proteases involved in proteolysis which could be used as novel parameters for screening different maize varieties for improved tolerance to ozone pollution

A nutrição é um dos principais fatores que afetam a eficiência reprodutiva por influenciar o crescimento, maturação e capacidade ovulatória do folículo bem como o perfil e estado metabólico do animal, gerando cenários que prejudicam ou corroboram o desenvolvimento e estabelecimento da prenhez. Diferentes fontes energéticas utilizadas na nutrição são capazes de alterar os padrões de fermentação ruminal e causar respostas endócrinas distintas. A partir disso, os objetivos desse estudo foram avaliar de que forma duas fontes energéticas da dieta influenciam a produção embrionária, a expressão gênica de enzimas hepáticas que metabolizam progesterona (P4) e a insulinemia. Em um delineamento em crossover, 22 vacas holandesas não lactantes e não gestantes foram distribuídas em dois grupos: um recebendo milho e outro polpa cítrica como fonte de energia da dieta. A quantidade de alimento fornecida foi de 1,3% do peso corporal em matéria seca por dia. O estudo foi composto por dois períodos de 71 dias de duração e em cada um deles foram realizadas duas superovulações (aos 35 e aos 70 dias) e uma biópsia hepática (aos 71 dias). Amostras sanguíneas foram colhidas imediatamente antes do fornecimento do alimento e 4 horas após, em dias pré-determinados, para dosagem de glicose, insulina e P4. Ao final do estudo as vacas passaram por teste de tolerância à glicose (TTG). Foi quantificada a expressão gênica de enzimas que metabolizam a P4 por RT-qPCR. A análise estatística foi realizada por meio de regressão logística pelo Proc MIXED do SAS 9.3. Os dados de expressão gênica foram avaliados por meio de delta CT. Imediatamente antes do fornecimento do alimento, a insulina circulante foi maior para o grupo milho (P < 0,01) e a P4 circulante foi maior para o grupo polpa cítrica (P < 0,01). Quatro horas após a alimentação, a P4 foi igual entre os tratamentos. A relação da P4 circulante na hora 4 e 0 foi maior para o grupo milho (P < 0,01). Tanto a glicose basal como o Homa-IR (Homeostasis model assessment of insulin resistance) foram maiores para o grupo milho. No TTG, o grupo milho apresentou maior pico de glicose no momento 5 minutos, maior taxa de decaimento da glicose (P = 0,01) e menor tempo de meia vida da glicose (P = 0,05). Não houve efeito de tratamento na resposta superestimulatoria, superovulatória, produção de embriões e na expressão gênica das enzimas que metabolizam a P4, mas as superovulações realizadas aos 70 dias produziram embriões de qualidade inferior em relação às realizadas aos 35 dias, independente de tratamento. Conclui-se que, embora tenha sido possível alterar a insulina circulante através da dieta, a quantidade e qualidade de embriões produzidos não foram alteradas. O aumento pós-prandial da P4 circulante não foi relacionado a menor expressão gênica das enzimas hepáticas que metabolizam a P4. / Nutrition is one of the main factors affecting reproductive efficiency by influencing the growth, maturation and ovulatory capacity of the follicle and metabolic status of the animal, leading to scenarios that impair or corroborate the development and establishment of pregnancy. Different energy sources in the composition of the diet can alter ruminal fermentation patterns and cause different endocrine responses. The objectives of this study were to evaluate how two different energy sources in the diet can influence embryo production, gene expression of liver enzymes that metabolize progesterone (P4) and circulating insulin. In a crossover design, 22 non-lactating and non-pregnant Holstein cows were allocated into two groups: one receiving corn and other receiving citrus pulp as the energy supply of the diet. The amount of feed provided was 1.3% of body weight of dry matter per day. The study consisted of two 71-days periods and cows were superovulated twice on each period (at 35 and 70 days). Liver biopsy was performed after 71 days from the beginning of each replicate. Blood was sampled immediately before feeding and 4 hours later, at predetermined days, for measurement of glucose, insulin and P4. At the end of the study cows underwent a glucose tolerance test (GTT). Gene expression of liver enzymes that metabolize P4 was quantified by RT-qPCR. Statistical analysis was performed using logistic regression of Proc Mixed of SAS 9.3. Gene expression data were evaluated using delta CT. Immediately before feeding, the circulating insulin was greater for the corn group (P < 0.01) and circulating P4 was greater for the citrus pulp group (P < 0.01). Four hours after feeding, circulating P4 was similar. The relationship of circulating P4 between hour 4 and 0 was greater for the corn group (P < 0.01). Both basal circulating glucose and HOMA-IR (Homeostasis model assessment of insulin resistance) were greater for the corn group. The corn group had also greater glucose peak at the time 5 minutes of the GTT, greater glucose rate of decay (P = 0.01) and a shorter half-life of glucose (P = 0.05). There was no treatment effect on superstimulatory and superovulatory response, on embryo production and on gene expression of liver enzymes that metabolize P4. However, superovulations performed at 70 days produced lower embryo quality compared to those performed at 35 days, regardless of treatment. In conclusion, although it was possible to change circulating insulin by feeding different diets, the quantity and quality of embryos produced were not affected by the diets. The postprandial increase in circulating P4 was not associated with altered gene expression of hepatic enzymes that metabolize P4.