Impact of different irrigation strategies on grapes and wine quality of four grapevine cultivars (Vitis sp.) in cool climate conditions. An investigation into the relationships among ABA, water status, grape cultivar and wine quality

Niagara Peninsula of Ontario is the largest viticultural area in Canada. Although it is considered to be a cool and wet region, in the last decade many water stress events occurred during the growing seasons with negative effects on grape and wine quality. This study was initiated to understand and develop the best strategies for water management in vineyards and those that might contribute to grape maturity advancement. The irrigation trials investigated the impact of time of initiation (fruit set, lag phase and veraison), water replacement level based on theoretical loss through crop evapotranspiration (ETc; 100,50 and 25%) and different irrigation strategies [partial root zone drying (PRD) versus regulated deficit irrigation (RD!)] on grape composition and wine sensory profiles. The irrigation experiments were conducted in a commercial vineyard (Lambert Vineyards Inc.) located in Niagara-on-the-Lake, Ontario, from 2005 through 2009. The two experiments that tested the combination of different water regimes and irrigation time initiation were set up in a randomized block design as follows: Baco noir - three replicates x 10 treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set, lag phase and veraison) + control]; Chardonnay - three replicates x seven treatments [(25%, 50% and 100% of ETc) x (initiation at fruit set and veraison) + control]. The experiments that tested different irrigation strategies were set up on two cultivars as follows: Sauvignon blanc - four replicates x four treatments [control, fully irrigated (100% ETc), PRD (100% ETc) and RDI (25% ETc)]; Cabemet Sauvignon - four replicates x five treatments [control, fully irrigated (100% ETc), PRD (100% ETc), RDI (50% ETc) and RDI (25% ETc)]. The controls in each experiment were nonirrigated. The irrigation treatments were compared for many variables related to soil water status, vine physiology, berry composition, wine sensory profile, and hormone composition [(abscisic acid (ABA) and its catabolites]. Soil moisture profile was mostly affected by irrigation treatments between 20 and 60 em depth depending on the grapevine cultivar and the regime of water applied. Overall soil moisture was consistently higher throughout the season in 100 and 50% ETc compare to the control. Transpiration rates and leaf temperature as well as shoot growth rate were the most sensitive variables to soil water status. Drip irrigation associated with RDI treatments (50% ETc and 25% ETc) had the most beneficial effects on vine physiology, fruit composition and wine varietal typicity, mainly by maintaining a balance between vegetative and reproductive parts of the vine. Neither the control nor the 100 ETc had overall a positive effect on grape composition and wine sensory typicity. The time of irrigation initiation affected the vine physiology and grape quality, the most positive effect was found in treatments initiated at lag phase and veraison. RDI treatments were overall more consistent in their positive effect on grape composition and wine varietal typicity comparing to PRD treatment. The greatest difference between non-irrigated and irrigated vines in most of the variables studied was found in 2007, the driest and hottest season of the experimental period. Soil water status had a greater and more consistent effect on red grapevine cultivars rather than on white winegrape cultivars. To understand the relationships among soil and plant water status, plant physiology and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA (TOH-ABA), 8' -hydroxy-ABA, neophaseic acid and abscisic acid glucose ester (ABA-GE)] were analyzed in leaves and berries from the Baco noir and Chardonnay irrigation trials over two growing seasons. ABA and some of its catabolites accurately described the water status in the vines. Endogenous ABA and some of its catabolites were strongly affected in Baco noir and Chardonnay by both the water regime (i.e. ET level) and timing of irrigation initiation. Chardonnay grapevines produced less ABA in both leaves and berries compared to Baco noir, which indicated that ABA synthesis is also cultivar dependant. ABA-GE was the main catabolite in treatments with high water deficits, while PA and DPA were higher in treatments with high water status, suggesting that the vine produced more ABA-GE under water deficits to maintain rapid control of the stomata. These differences between irrigation treatments with respect to ABA and catabolites were particularly noticeable in the dry 2007 season. Two trials using exogenous ABA investigated the effect of different concentrations of ABA and organs targeted for spraying, on grape maturation and berry composition of Cabemet Sauvignon grapevines, in two cool and wet seasons (2008-2009). The fIrst experiment consisted of three replicates x three treatments [(150 and 300 mg/L, both applications only on clusters) + untreated control] while the second experiment consisted in three replicates x four treatments [(full canopy, only clusters, and only leaves sprayed with 300 ppm ABA) + untreated control]. Exogenous ABA was effective in hastening veraison, and improving the composition of Cabemet Sauvignon. Ability of ABA to control the timing of grape berry maturation was dependant on both solution concentration and the target organ. ABA affected not only fruit composition but also yield components. Berries treated with ABA had lower weight and higher skin dry mass, which constitutes qualitative aspects desired in the wine grapes. Temporal advancement of ripening through hormonal control can lead to earlier fruit maturation, which is a distinct advantage in cooler areas or areas with a high risk of early frost occurrence. Exogenous ABA could provide considerable benefits to wine industry in terms of grape composition, wine style and schedule activities in the winery, particularly in wet and cool years. These trials provide the ftrst comprehensive data in eastern North America on the response of important hybrid and Vitis vinifera winegrape cultivars to irrigation management. Results from this study additionally might be a forward step in understanding the ABA metabolism, and its relationship with water status. Future research should be focused on ftnding the ABA threshold required to trigger the ripening process, and how this process could be controlled in cool climates.

HEAVY METALS AND MICRONUTRIENTS IN THE SOIL AND GRAPEVINE UNDER DIFFERENT IRRIGATION STRATEGIES

Soils under natural conditions have heavy metals in variable concentrations and there may be an increase in these elements as a result of the agricultural practices adopted. Transport of heavy metals in soil mainly occurs in forms dissolved in the soil solution or associated with solid particles, water being their main means of transport. In this context, the aim of this study was to evaluate the heavy metal and micronutrient content in the soil and in the grapevine plant and fruit under different irrigation strategies. The experiment was carried out in Petrolina, PE, Brazil. The treatments consisted of three irrigation strategies: full irrigation (FI), regulated deficit irrigation (RDI), and deficit irrigation (DI). During the period of grape maturation, soil samples were collected at the depths of 0-10, 10-20, 20-40, 40-60, and 60-80 cm. In addition, leaves were collected at the time of ripening of the bunches, and berries were collected at harvest. Thus, the heavy metal and micronutrient contents were determined in the soil, leaves, and berries. The heavy metal and micronutrient contents in the soil showed a stochastic pattern in relation to the different irrigation strategies. The different irrigation strategies did not affect the heavy metal and micronutrient contents in the vine leaves, and they were below the contents considered toxic to the plant. In contrast, the greater availability of water in the FI treatment favored a greater Cu content in the grape, which may be a risk to vines, causing instability and turbidity. Thus, adoption of deficit irrigation is recommended so as to avoid compromising the stability of tropical wines of the Brazilian Northeast.

Influences of the harvest season on analytical characteristics of syrah grapes and wines produced in the northeast region of Brazil

The present study evaluated the main physical and chemical characteristics of Syrah grapes, coming from the tropical region of São Francisco river valley, harvested at different times and their relationship with analytical characteristics of resulting wines. Grapes came from the first half of 2009 harvest, collected at Casa Nova - Bahia, a semi-arid and hot region, comprising an interval from 84 days after pruning (84 dap) to the beginning of grape over-ripening, 133 days after pruning (133 dap). Harvests at 84, 91, 98, 105, 112, 119, 126 and 133 dap, were analyzed for pH, soluble solids and acidity in grapes, which were then processed for wine production. Maximum sugar/acidity ratio (s/a = 56) were observed in grapes harvested between 126 and 133 dap, coincided with the highest concentration of anthocyanins (851 mg L-1 ) and total tannins (2.6 g L-1 ) in the resulting wines, indicating that grapes collected between 126 and 133 dap showed the best potential for winemaking in the tropical climate of São Francisco river valley. This result was confirmed by the analytical characterization of wines, that, between 126 and 133 dap, showed 12.8% alcohol, 31 g L-1 dry extract, 5.6 g L-1 ashes and a TPI of 58, using only one pumping per day, for 5 days maceration. Syrah grapes considered in the present work presented a good evolution of maturation, and grapes harvested between 126 and 133 days after pruning, showed the best oenological potential for the development of red wine. The use of pH and soluble solids appeared useful parameters in the estimation of maturation degree and quality potential of grapes for winemaking.

Effects de l'application d'acide gibbérellique (GA3) sur la qualité de raisins et de vins produits en climat tropical au Nord-Est du Brésil.

The honeydew moth Cryptoblabes gnidiella is the main problem for the wineries in the Northeast of the Brazil, because it attacks the bunch and reduces the quality of the grapes and the wines. In order to stretch out the bunch to facilitate the penetration of the insecticides, it was used gibberellic acid. Six treatments with different concentrations and different dates of application, and the control were compared. The bunches are compact, characteristic of the "Syrah" grapes in the region. The grape berries were analysed at harvest and wines were made by microvinifications. The grape berries showed different qualitative characteristics, as berry weight, number of berries. °Brix, total acidity and heterogeneity of the maturation. The microvinifications were carried out with 50 kg of grapeberries into glass bottles of 20 L at 22°C, for the alcoholic and malolactic fermentations, then stabilized and bottled. The wines were tasted by a panel of ten people and compared on smell and taste plans. The tasting results showed that the control treatment was the best graded wine. The application of gibberellic acid allowed to control the honeydew moth attack, but it caused a heterogeneity on grape maturation, with a lower quality of the grapes and wines compared to the control.

WINE GRAPE QUALITY OF GRAPEVINES GROWN IN THE CERRADO ECOREGION OF BRAZIL

Aims: Geographical indication plays an important role in the improvement of wine quality. In this context, the search for new grape growing areas has been constant. The Sao Francisco River Valley in the cerrado of Minas Gerais State (Brazil) has been pointed out in the Geoviticulture Multicriteria Climatic Classification System (MCC System) as a potentially winegrowing region, especially considering the autumn-winter period when night temperatures are favorable to grape ripening. In this work, we studied the maturation curves and fruit composition of four wine grape varieties (Syrah, Merlot, Cabernet-Sauvignon and Cabernet Franc) in two growing seasons in order to validate the state of Minas Gerais as a new winegrowing region in Brazil. Methods and results: Quality parameters (berry weight, pH, titratable acidity and total soluble solids) were measured weekly from veraison to harvest, and sugar, organic acid, anthocyanin and phenolic concentrations were determined in must and berry skins and seeds at harvest. Syrah berries showed the highest weight throughout maturation which contributed to higher yield (8.92 ton ha(-1)), followed closely by Merlot (8.07 ton ha(-1)). Bern, sugar concentrations were higher and malic acid levels were lower than the values usually observed in wine grapes harvested during summer in traditional winegrowing regions in Brazil. Cabernet Franc showed lower levels of anthocyanins and skin phenolics per kg berries and the highest values of seed phenolics, which were not affected by growing season. Conclusion: Weather conditions of the cerrado of Minas Gerais State in Brazil during winter allowed complete maturation of Cabernet-Sauvignon, Cabernet Franc, Merlot and Syrah cultivars as revealed by the satisfactory sugar, anthocyanin and skin phenolic accumulation. Significance and impact of the study: This study revealed the potential of the cerrado ecoregion in the northeast of Minas Gerais to become a new winemaking region in Brazil.

Wine and grape polyphenols — a chemical perspective

Phenolic compounds constitute a diverse group of secondary metabolites which are present in both grapes and wine. The phenolic content and composition of grape processed products (wine) are greatly influenced by the technological practice to which grapes are exposed. During the handling and maturation of the grapes several chemical changes may occur with the appearance of new compounds and/or disappearance of others, and consequent modification of the characteristic ratios of the total phenolic content as well as of their qualitative and quantitative profile. The non-volatile phenolic qualitative composition of grapes and wines, the biosynthetic relationships between these compounds, and the most relevant chemical changes occurring during processing and storage will be highlighted in this review.

Importance of canopy porosity into vineyard and the relationship with the grape maturity. Digital estimation method

In warm and dry climates, the use of porous systems should be required in order to allow a better leaf distribution inside the plant, causing more space in the clusters area and enhancing determined physiological processes so in the leaf (photosynthesis, v entilation, transpiration) as in berry (growth and maturation). Plant geometry indexes, yield and must composition have been studied in three different systems: sprawl with 12 shoots/m (S1); sprawl system with 18 shoots/m (S2) and vertical positioned syste m or VSP with 12 shoots/m (VSP1). Total leaf area increases as the crop load does, whoever surface area depends on to two factors: crop load and the training system (VSP vs. sprawl), which can provide differences in leaf exposure efficiencies. The main objective of this study was to validate digital photography measurements used to compare porosity differences among treatments and, as they affect plant microclimate and, therefore, yield and berry quality. Also, all previous studied indexes (LAI, SA, SFEr) tended to overestimate the relationship between exposed leaf surface and porosity of each treatment, but the use of digital method proved to be an effective tool in order to assess canopy porosity. Results showed that not positioned and free systems (sprawl) scored between 25- 50% more porosity in the clusters area than the fixed vertical system (VSP), which resulted in a better plant microclimate for test conditions, mainly by improving the exposure of internal clusters and internal canopy ventilation. On the other hand, higher crop load treatment (S2) showed a real increase in yield (16%) without any relevant change into must composition, even improving total anthocyanin content into berry during ripening

Importance of canopy porosity into vineyard and the relationship with the grape maturity. Digital estimation method

In warm and dry climates, the use of porous systems should be required in order to allow a better leaf distribution inside the plant, causing more space in the clusters area and enhancing determined physiological processes so in the leaf (photosynthesis, ventilation, transpiration) as in berry (growth and maturation). Plant geometry indexes, yield and must composition have been studied in three different systems: sprawl with 12 shoots/m (S1); sprawl system with 18 shoots/m (S2) and vertical positioned system or VSP with 12 shoots/m (VSP1). Total leaf area increases as the crop load does, whoever surface area depends on to two factors: crop load and the training system (VSP vs . sprawl), which can provide differences in leaf exposure efficiencies. The main objective of this study was to validate digital photography measurements used to compare porosity differences among treatments and, as they affect plant microclimate and, therefore, yield and berry quality. Also, all previous studied indexes (LAI, SA, SFEr) tended to overestimate the relationship between exposed leaf surface and porosity of each treatment, but the use of digital method proved to be an effective tool in order to assess canopy porosity. Results showed that not positioned and free systems (sprawl) scored between 25 - 50% more porosity in the clusters area than the fixed vertical system (VSP), which resulted in a better plant microclimate for test conditions, mainly by improving the exposure of internal clusters and internal canopy ventilation. On the other hand, higher crop load treatment (S2) showed a real increase in yield (16%) without any relevant change into must composition, even improving total anthocyanin content into berry during ripening

Régime thermique en période de maturation du raisin dans le géoclimat viticole.

Mots clés :indice de fraîcheur des nuits, amplitude thermique, Système CCM Géoviticole, zonage, qualité. Key words: cool night index, thermal amplitude, MCC System for World Viticulture, zoning, quality. RESUME Le régime thermique en période de maturation du raisin est l?une des variables déterminantes de la coloration du raisin et de la richesse en arômes, anthocyanes et polyphénols des vins. L?objectif du travail est de caractériser le régime thermique, notamment la fraîcheur des nuits et l?amplitude thermique au cours de la maturation, au niveau du climat viticole mondial, sur une base des données de 100 régions viticoles dans 30 pays, obtenue par l?intermédiaire de l?Organisation Mondiale de la Météorologie - OMM. Plusieurs indices climatiques viticoles ont été calculés: l?Indice de Fraîcheur des Nuits ?IF (ºC), l?Indice Héliothermique de Huglin ?IH (ºC) et l?Indice de Sécheresse ?IS (mm) du Système de Classification Climatique Multicritères Géoviticole, et l?amplitude thermique moyenne en août et septembre Aa-s (ºC). Egalement, sur la période véraison-récolte ?v-r(moyenne des 30 jours précédant la date de récolte, estimée sur la base d?un Indice Héliothermique de HUGLIN égal à 1.900 - approximatif pour la maturation du Cabernet-Sauvignon): la fraîcheur des nuits (FNv -r), la température moyenne de l?air (Tv-r), la température maximale de l?air (Txv-r) et l?amplitude thermique (Av-r). Les résultats montrent que IH est corrélé avec Tv-r (r=0,79**) et avec Txv-r (r=0,80**). IH représente donc bien les conditions thermiques générales de la période de maturation en ce qui concerne la température moyenne et maximale de l?air. Mais IH n?est pas corrélé ni avec Aa-s ni avec Av-r. Par contre, IF est corrélé avec Aa-s (r=-0,70**) et FNv-r est corrélé avec Av-r (r=-0,69**). Cette corrélation doit justifier, en partie, l?usage assez courant de l?amplitude thermique comme indicateur de bonnes conditions thermiques de maturation pour les régions qui présentent des valeurs élevées. Mais ce raisonnement peut amener à des caractérisations erronées. Le travail met en évidence, également, ?importance de considérer le bilan hydrique des régions (IS) dans l?analyse du régime thermique sur la qualité du raisin. On peut onclure que pour avoir une bonne caractérisation du régime thermique en période de maturation il faut considérer la fraîcheur des nuits (IF étant un bon indicateur de FNv-r moyen des régions, avec un r=0,80**), caractérisation qui peut être améliorée avec l?information des températures maximales et de sur 11l?amplitude thermique en période de maturation du raisin. Les éléments présentés peuvent servir à améliorer les indices climatiques pour estimer le potentiel qualitatif du raisin des différentes régions viticoles, notamment en complément de IF. ABSTRACT The thermal conditions during the grape ripening period are important variables related to colour of the grapes, anthocyanins, polyphenols and flavour of the wine. The main purpose of this work was to characterise the thermal conditions, especially the night coolness and the thermal amplitude during maturation, in the geoclimate of the world vine culture. A database of 100 grape-growing regions of 30 countries obtained from the World Meteorology Organisation (WMO) was used. Some climatic indexes were calculated: Cool Night Index ?IF (°C), Huglin?s Heliothermal Index ?IH (°C) and Dryness Index ?IS (mm), from the Multicriteria Climatic Classification System for World Viticulture, and the thermal amplitude in August and September Aa-s (°C). Over véraison-harvest period ?v-r(mean of the 30 days before harvesting date, estimated on the basis of HUGLIN Heliothermal Index equal to 1,900 ? approximately value to ripen Cabernet-Sauvignon) similar indexes were obtained: the cool night (FNv-r), the mean air temperature (Tv-r), the maximal air temperature (Txv-r) and the thermal amplitude (Av-r). The results showed that IH is positively correlated with Tv-r (r=0.79**), Txv-r (r=0.80)and IF (r=0.67**). Therefore, IH represents well the general thermal conditions during maturation period, specially concerning the mean and the maximal air temperature. owever, IH was correlated neither with Aa-s nor to Av-r. IF was negatively correlated with Aa-s (r= -0.70**) and FNv-r was negatively correlated with Av-r (r=-0.69**). The correlation to some extent explains the current use of the thermal amplitude to predict good ripening thermal conditions for those regions that show high values. As here we have described, this thinking may give incorrect results. This work has also showed the importance to consider the water balance of the regions (IS) in the effect of the thermal conditions in grape quality. We conclude that the characterisation of the thermal conditions during the ripening period do need the cool night index (in this case, IFis a good index to provide the mean FNv-r of the regions, r=0,80**). Factors other than cool night which influence this characterisation are both maximal air temperature and thermal amplitude data. The elements presented in this work, in addition to IF , may improve the climatic indexes to be used to predict the qualitative potential of grapes from different regions.

Maturação somática e desempenho físico em jovens futebolistas = : Somatic maturation and physical performance in young soccer players

Universidade Estadual de Campinas . Faculdade de Educação Física

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

Melatonin (MEL) acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM) on bovine embryos. This study tested the addition of MEL to maturation medium (MM) with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS) at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL) or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.

Differential gene expression profiling in mucus glands of honey bee (Apis mellifera) drones during sexual maturation

The mating sign that each drone leaves when mating with a queen essentially consists of mucus gland proteins. We employed a Representational Difference Analysis (RDA) methodology to identify genes that are differentially expressed in mucus glands during sexual maturation of drones. The RDA library for mucus glands of newly emerged drones was more complex than that of 8 day-old drones, with matches to 20 predicted genes. Another 26 reads matched to the Apis genome but not to any predicted gene. Since these ESTs were located within ORFs they may represent novel honey bee genes, possibly fast evolving mucus gland proteins. In the RDA library for mucus glands of 8 day-old drones, most reads corresponded to a capsid protein of deformed wing virus, indicating high viral loads in these glands. The expression of two genes encoding venom allergens, acid phosphatase-1 and hyaluronidase, in drone mucus glands argues for their homology with the female venom glands, both associated with the reproductive system.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1 beta maturation

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1 beta and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE(2) and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1 beta and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)alpha and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1 beta and PGE(2) did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-alpha and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1 beta was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE(2) production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1 beta maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE(2) production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.