Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Response of weed populations to tillage, reduced herbicide and fertilizer rates in wheat (Triticum aestivum) production

Field experiments were carried out in 1999 and 2000 to investigate the effects of conventional (CT) and no-tillage (NT) systems, interacting with three herbicide dose levels and three nitrogen (N) levels on weed growth and wheat production of two varieties. There was a higher grain yield for NT system compared with CT in one year. CT weed biomass was lower than from NT weed biomass, in both varieties. No differences on wheat biomass and grain yield were observed between full and reduced herbicide rates. N fertilizer increased wheat biomass and grain yield significantly. Only N medium level had an effect upon weed biomass with respect to non-fertilized plots, while the highest fertilization rate lowered weed biomass. Conventional tillage, reduced herbicide rates and nitrogen fertilization were effective ways of limiting weed production in wheat.

Voimalaitostuhkan Hyödyntämismahdollisuudet - Case: Kouvolan Seutu

Maailman laajuisen kasvihuoneilmiön kiihtyminen ja EU:n tiukentuneet jäsenmailleen asettamat tavoitteet uusiutuvien polttoaineiden käytön lisäämiseksi ovat lisänneet puupolttoaineiden käyttöä Suomessa. Lisääntynyt puupolttoaineiden käyttö ja entistä tehokkaampi hakkuutähteiden hyödyntäminen on lisännyt metsistä poistuvien ravinteiden määrää. Huoli metsien maaperän ravinteiden niukkenemisesta sekä tiukentunut lainsäädäntö tuhkien kaatopaikkasijoittamisesta ovat lisänneet energiayhtiöiden kiinnostusta lisätä tuhkien hyötykäyttöä. Tuhka täytyy esikäsitellä, eli stabiloida ennen hyötykäyttöä. Stabiloinnissa tuhkaa kostutetaan, jolloin siitä muodostuu rakeita. Hyödynnettäessä tuhkaa metsälannoitukseen tuhka tulee usein myös terästää typellä, sillä tuhka ei sisällä lainkaan typpeä. Muita tuhkan hyötykäyttömahdollisuuksia ovat peltolannoitus, betonin valmistus, maarakentaminen sekä jätevedenpuhdistus. Näistä vaihtoehdoista betonin valmistus ja maarakentaminen ovat melko yleisiä. Kouvolassa tuhkaa muodostuu noin 14 000 tonnia vuodessa. Kaupungin omistama metsäalue Saaramaalla voitaisiin lannoittaa lähes samoilla kustannuksilla kuin mitä kaatopaikkasijoittaminen maksaisi. Sen sijaan tuhkan käytöllä tierakentamiseen Tähteen alueella saavutetaan tuhansien eurojen säästöt kaatopaikkasijoitukseen verrattuna ja samalla tuhka saadaan hyötykäyttöön kiviaineksia säästäen.  

Reflectance indices as a diagnostic tool for weed control performed by multipurpose equipment in precision agriculture

Several tools of precision agriculture have been developed for specific uses. However, this specificity may hinder the implementation of precision agriculture due to an increasing in costs and operational complexity. The use of vegetation index sensors which are traditionally developed for crop fertilization, for site-specific weed management can provide multiple utilizations of these sensors and result in the optimization of precision agriculture. The aim of this study was to evaluate the relationship between reflectance indices of weeds obtained by the GreenSeekerTM sensor and conventional parameters used for weed interference quantification. Two experiments were conducted with soybean and corn by establishing a gradient of weed interference through the use of pre- and post-emergence herbicides. The weed quantification was evaluated by the normalized difference vegetation index (NDVI) and the ratio of red to near infrared (Red/NIR) obtained using the GreenSeekerTM sensor, the visual weed control, the weed dry matter, and digital photographs, which supplied information about the leaf area coverage proportions of weed and straw. The weed leaf coverage obtained using digital photography was highly associated with the NDVI (r = 0.78) and the Red/NIR (r = -0.74). The weed dry matter also positively correlated with the NDVI obtained in 1 m linear (r = 0.66). The results indicated that the GreenSeekerTM sensor originally used for crop fertilization could also be used to obtain reflectance indices in the area between rows of crops to support decision-making programs for weed control.

How glyphosate may affect transgenic soybean in different soil and phosphorus levels

The technology that employs genetic modifications brought a significant increase in the utilization of glyphosate. Transgenic soybean has been suffering injury, even though it possesses a resistance mechanism to glyphosate. Currently, there are only a few studies on the dynamics of glyphosate in transgenic soybean planted in soils with different textures interacting with phosphorus concentrations. This study focused on assessing the effects of glyphosate in transgenic soybean plants on different types of soil and at different phosphorus levels. The experimental design was completely randomized, in factorial design: 2 x 6 x 3, that being 2 soil types, 6 doses of glyphosate and 3 levels of phosphorus, and four replications. Plants were cultivated for thirty days in pots with two types of soil, one being clayey (Red-Yellow Latosol) and the other sandy (Quartzarenic Neosol). They received one, two, and three times the maintenance dose of fertilization of phosphorus, corresponding to: 170, 250 and 330 kg of P2O5 ha-1 to QN, and 380, 460 and 540 kg P2O5 ha-1 to RYL, respectively. Glyphosate was applied at six different doses: 0, 1,200, 2,400, 12,000, 60,000 and 120,000 g ha-1 of active ingredient. Plant height, a and b chlorophyll, and shoot were lower for the plants that received lower doses of glyphosate, regardless of the type of soil. Greater availability of phosphorus and lower amount of glyphosate used in Quartzarenic Neosol soil provided for less phytointoxication symptoms in transgenic soybean.

Phenology, fruit set and dispersal of Cordia multispicata Cham., an important weed shrub of abandoned pastures in eastern Amazonia

ABSTRACT - (Phenology, fruit set and dispersal of Cordia multispicata Cham., an important weed shrub of abandoned pastures in eastern Amazonia). The reproductive ecology of the distylous tropical shrub Cordia multispicata was studied in an abandoned pasture in Paragominas County , Pará state, Brazil. It is a common species in the Amazon basin where it occurs as a weed in open and disturbed habitats. C. multispicata has many flowers per inflorescence (85 ± 12) but 84% abort before fertilization. Flowering occurs throughout the year. Fruits are small, with a red fleshy pericarp (skin-pulp) attractive to birds. Fruit set is lower during the dry season (less than 30%) and higher during the rainy season when there are many visits of insects to the flowers. Fruiting has a peak between the end of the dry season and the middle of the rainy season. Nineteen bird species were observed foraging for the fruits of C. multispicata, and 79% of those species can be considered as potential dispersal agents. The efficient seed dispersal and aggregated spatial distribution associated with some characteristics of the dispersors greatly contributed to the success of this species in abandoned pastures of eastern Amazonia.

Late-acting self-incompatibility in Capparis retusa (Capparaceae), a species of Chaco woodland in NE Argentina

The reproductive biology of Capparis retusa was studied by means of controlled pollination experiments and fluorescence microscopy observations of post-pollination events. Self-pollinated flowers mostly failed to form fruits despite the fact that self-pollen tubes grew to the ovary and penetrated ovules within 24 h. Since embryo development to globular stage was observed in some self-fertilized ovules it was concluded that control of self-fertilization in this species occurs by some kind of post-zygotic mechanism.

Ovule ontogeny of Tabebuia pulcherrima Sandwith (Bignoniaceae): embryo sac development

The chalazal megaspore develops in a Polygonum-type embryo sac. The amyloplast-rich endothelium is partially degraded during the expansion of the micropylar portion of the megagametophyte. Organization of the mature embryo sac is determined by the patterns of vacuolation, nuclear migration, spindle orientation and cellularization. The egg cell is slightly chalazal in relation to the synergids, and its micropylar end does not touch the micropylar channel. At the chalazal pole of the egg apparatus, the common walls between the synergids, the egg and central cells, despite their tenuity, are present in the mature megagametophyte. The polar nuclei do not fuse before fertilization and the antipodals are persistent until the first stages of endosperm formation. The taxonomic significance of some embryological characters for the Bignoniaceae is discussed.

The site of self-incompatibility action in cupuassu (Theobroma grandiflorum)

Cupuassu (Theobroma grandiflorum (Willd. ex Spreng.) Schumann) is a fruit tree that is attracting attention in Brazil and also in other tropical countries. Its pulp is used to prepare ice-cream, juices, jellies and many other home-made sweets. This species has a very low fecundity, attributed to problems with pollination and self-incompatibility, noted as being restrictive to the agronomic productivity of the species. Controlled pollinations between compatible and incompatible parents were made and flowers were collected at three times: 24, 48 and 72 h after pollinations, during the floral seasons of 1995 and 1998. In flowers collected 24 h after pollination, in both compatible and incompatible crosses, around 70% of ovules showed egg apparatus without evidences of sperm cell delivery; nevertheless some pollen tubes had been observed. Ovaries collected at 48 h showed different behaviors according to the type of cross. In the compatibles, 35% of ovules did not show evidences of gamete fusion. In incompatible crosses this number rose to 50%. Collected ovaries with 72 h, from compatible and incompatible crosses, showed respectively 1.2% and 14.2% of the ovules with the egg apparatus and polar nuclei only. In these ovaries, for the two types of crosses, the presence of sperm nuclei was common and the zygote was still undivided. The incompatibility action is occurring after fertilization, in a late self-incompatibility action.

Sexual behavior and fertility of male rats submitted to prolonged immobilization-induced stress

In order to investigate whether prolonged stress interferes with the onset of sexual behavior at puberty and with fertility at adulthood, prepubertal male Wistar rats (40 days of age) were immobilized 6 h a day for 15 days (up to early puberty) or for 60 days (until sexual maturity). Pubertal stressed rats showed a two-fold increase in the latency for the first mount (probably due to repeated aversive experience in which a change of environment was always followed by immobilization) and a 2.5-fold increase in the frequency of thrusting (indicative of enhanced sexual performance). The apparently stimulatory effect of prolonged stress on the onset of sexual behavior is discussed in terms of increased testosterone level and interference with the complex interchanges between the neurotransmitters/neuropeptides involved in the central control of male sexual activity. Adult stressed animals were mated with normal females, which became pregnant but exhibited a more than two-fold increase in both pre-implantation and post-implantation loss, probably due to a smaller rate of fertilization and/or fertilization with damaged spermatozoa.

Interactions between intracellular Ca2+ stores: Ca2+ released from the NAADP pool potentiates cADPR-induced Ca2+ release

Cells possess multiple intracellular Ca2+-releasing systems. Sea urchin egg homogenates are a well-established model to study intracellular Ca2+ release. In the present study the mechanism of interaction between three intracellular Ca2+ pools, namely the nicotinic acid adenine dinucleotide phosphate (NAADP), the cyclic ADP-ribose (cADPR) and the inositol 1',4',5'-trisphosphate (IP3)-regulated Ca2+ stores, is explored. The data indicate that the NAADP Ca2+ pool could be used to sensitize the cADPR system. In contrast, the IP3 pool was not affected by the Ca2+ released by NAADP. The mechanism of potentiation of the cADPR-induced Ca2+ release, promoted by Ca2+ released from the NAADP pool, is mediated by the mechanism of Ca2+-induced Ca2+ release. These data raise the possibility that the NAADP Ca2+ store may have a role as a regulator of the cellular sensitivity to cADPR.

Estradiol and testosterone concentrations in follicular fluid as criteria to discriminate between mature and immature oocytes

The objective of the present study was to examine the association between follicular fluid (FF) steroid concentration and oocyte maturity and fertilization rates. Seventeen infertile patients were submitted to ovulation induction with urinary human follicle-stimulating hormone, human menopausal gonadotropin and human chorionic gonadotropin (hCG). A total of 107 follicles were aspirated after hCG administration, the oocytes were analyzed for maturity and 81 of them were incubated and inseminated in vitro. Progesterone, estradiol (E2), estrone, androstenedione, and testosterone were measured in the FF. E2 and testosterone levels were significantly higher in FF containing immature oocytes (median = 618.2 and 16 ng/ml, respectively) than in FF containing mature oocytes (median = 368 and 5.7 ng/ml, respectively; P < 0.05). Progesterone, androstenedione and estrone levels were not significantly different between mature and immature oocytes. The application of the receiver-operating characteristic curve statistical approach to determine the best cut-off point for the discrimination between mature and immature oocytes indicated levels of 505.8 ng/ml for E2 (81.0% sensitivity and 81.8% specificity) and of 10.4 ng/ml for testosterone (90.9% sensitivity and 82.4% specificity). Follicular diameter was associated negatively with E2 and testosterone levels in FF. There was a significant increase in progesterone/testosterone, progesterone/E2 and E2/testosterone ratios in FF containing mature oocytes, suggesting a reduction in conversion of C21 to C19, but not in aromatase activity. The overall fertility rate was 61% but there was no correlation between the steroid levels or their ratios and the fertilization rates. E2 and testosterone levels in FF may be used as a predictive parameter of oocyte maturity, but not for the in vitro fertilization rate.

Prolactin and cortisol levels in women with endometriosis

Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997). Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis). Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 ± 2.1 ng/mL) than in healthy controls (13.2 ± 2.1 ng/mL). Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 ± 1.3 ng/mL) than in controls (10.5 ± 1.4 ng/mL). Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

Feasibility of the use of recycled nutrients in fast growing pulpwood production

The application of pulp and paper mill (PPM) sludge in agriculture and forestry has been acknowledged as soil amendments and a plant nutrient source. The main objectives of this study were to evaluate the total cost of the use of recycled nutrients from PPM sludge in fast growing pulpwood production, and the financial profitability of fast growing pulpwood production with the use of these recycled nutrients. The investment and production costs of fast growing pulpwood plantation were directly acquired from a previous research, while the other data was compiled through different studies. The total cost of the use of PPM sludge was evaluated based on assumed factors. Discounted cash flow method was used to evaluate the financial profitability, using NPV and IRR as indicators. The results of estimated sludge nutrient contents were 16.2 g N, 2.9 g P, and 2.4 g K kg-1 of dry sludge. The sludge application rate was estimated at 1.36 Mg/ha in the first year. The total cost of the use of PPM sludge involved transport and spreading cost of US$49.15/dry ton. The fertilization cost applied in the financial model was designed in 3 different options and their results were as follows: option (1) was taken directly from the reference research (US$97/ha); option (2) was the use of sludge alone (US$66.75/ha); and option (3) was the use of sludge and TSP fertilizer (US$83.80/ha). The average NPV without discounting was US$248,180 while the IRRs ranged between approximately 3-4% with an average of 3.63%. Although option (2) and (3) contributed to higher IRRs compared to option (1), this increase was still not significant as the IRR was not sensitive to the total fertilization cost. The advantages are that this practice can be performed at a lower cost and the application rate can be still increased if necessary. It is better for forest plantations compared to agriculture and consequently supports reforestation program. In addition, it can be similarly applied in wood biomass production. A disadvantage is that the IRRs were not very favorable compared to the criterion of 11%. The sludge high in C:N ratio can cause nitrogen immobilization, and regulatory concerns may restrict and complicate the use of sludge landspreading and contribute to additional costs and processes.