Optimal single-embryo mass spectrometry fingerprinting

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P < 0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P < 0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P > 0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P > 0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P > 0.05) in the presence of heparin and PHE (P < 0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development. © 2013 The Society for In Vitro Biology.

Reproductive characteristics and the weight-length relationship in Anableps anableps (Linnaeus, 1758) (Cyprinodontiformes: Anablepidae) from the Amazon Estuary

O presente estudo analisou a relação peso-comprimento e uma série de aspectos da biologia reprodutiva de Anableps anableps da foz do rio Maracanã, no estado brasileiro do Pará. Os espécimes foram coletados por meio de duas redes de emalhar de 30 m de comprimento, com malhas de 15 e 20 mm, e com um puçá com um metro de diâmetro, com malha de 10 mm. Foram capturados 865 espécimes, os quais foram analisados para estabelecer a razão sexual, que foi de 2,12 fêmeas por macho (χ² = 13,07; p<0,05). As fêmeas apresentaram crescimento alométrico positivo, enquanto que os machos foram alométricos negativos. Nas análises adicionais, as gônadas de 371 fêmeas foram observadas ao microscópio. O desenvolvimento gonadal foi classificado em três estádios: imaturo (5,0%), em maturação (12,0%) e maturo (83,0%). O desenvolvimento embrionário foi classificado em cinco fases, de acordo com o tamanho do embrião e do saco vitelino. A fecundidade média foi de 12 ovos / embriões por fêmea (variando de 1-37 ovos / embriões). Uma relação significativa foi verificada entre o comprimento padrão das fêmeas, o peso de seus ovários (R² = 0,257, p < 0,001), e o número de embriões (R² = 0,573, p < 0,001). O tamanho médio da primeira maturação sexual (L₅₀) para as fêmeas foi estimado em 11,7 cm. A espécie se reproduz durante todo o ano.

In vivo embryo production in cows superovulated 1 or 2 days after ovum pick-up

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seed development in Malpighiaceae species with an emphasis on the relationships between nutritive tissues

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ovule, fruit and seed development in Abolboda (Xyridaceae, Poales): implications for taxonomy and phylogeny

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Maternal Thyroid and Glucocorticoid Hormone Interactions in Larval Fish Development, and Their Applications in Aquaculture

Thyroid hormones (THs) have long been known to have regulatory roles in the differentiation and maturation of vertebrate embryos, beginning with the knowledge that hormones of maternal origin are essential for human fetal central nervous and respiratory system development. Precise measurements of circulating THs led to insights into their critically important actions throughout vertebrate growth and development, initially with amphibian metamorphosis and including embryogenesis in fishes. Thyroid cues for larval fish differentiation are enhanced by glucocorticoid hormones, which promote deiodinase activity and thereby increase the generation of triiodothyronine (T-3) from the less bioactive thyroxin (T-4). Glucocorticoids also induce the expression of thyroid hormone receptors in some vertebrates. Maternally derived thyroid hormones and cortisol are deposited in fish egg yolk and accelerate larval organ system differentiation until larvae become capable of endogenous endocrine function. Increases in the T-3/T-4 ratio during larval development may reflect the regulatory importance of maternal thyroid hormones. Experimental applications of individual hormones have produced mixed results, but treatments with combinations of thyroid and corticoid hormones consistently promote larval fish development and improve survival rates. The developmental and survival benefits of maternal endocrine provisioning are increased in viviparous fishes, in which maternal/larval chemical contact is prolonged. Treatments with exogenous thyroid and corticoid hormones consistently promote development and reduce mortality rates in larval fishes, with potential hatchery-scale applications in aquaculture.

Developmental aspects of early pregnancy in mares

In mares, the embryo migrates to the uterus between days 5 and 6 postovulation beginning its mobility through all uterine segments, which is essential for the maternal recognition of pregnancy. During the mobility phase, the embryonic vesicle shows a linear growth rate until its fixation between days 15 and 17, when the orientation phenomenon occurs. From fixation to day 28 of pregnancy, the embryonic growth is less evident (plateau) by cross-section ultrasound examination. After this period the linear growth rate is reestablished until day 46. This plateau is attributed to the increased uterine tone that compresses the vesicle and to volume expansion, making it difficult to detect the conceptus growth only by the cross-section diameter. Around day 20, the embryo proper is visualized as an echogenic spot in the ventral aspect of the vesicle. Additionally, development of allantoic sac, embryonic heartbeat, yolk sac regression and posterior umbilical cord formation also can be visualized from days 20 to 40. An intimate interaction between uterus and conceptus is essential for the normal pregnancy development. Color-and spectral-Doppler ultrasonography can be useful for the evaluation of this interface. A gradual increase on uterine vascularity during the early pregnancy and transient changes in endometrial vascularity accompanying the vesicle location during the mobility phase have been described. Around day 38 of gestation, the formation of the endometrial cups begins and, consequently, the synthesis of the equine chorionic gonadotropin (eCG) induces the formation and development of supplementary corpora lutea, which are important to secrete progesterone and to maintain pregnancy until around day 120.

State of the art on animal embryonic chimeras

Embryonic chimerism is generally used in basic research and in vivo diagnosis of undifferentiated embryonic stem cells (ESC), mostly using mice embryos, although there have been reports in the literature on using rat, rabbit, sheep, chicken, primate, bovine, goat and pig embryos. Several techniques can currently be used to produce chimeric embryos, including microinjection, co-culture with ESC, fusion and aggregation. Although microinjection is the most commonly used method in mice, the mere aggregation of embryos with ESC may result in viable chimeras and be as efficient as microinjection. In mice, this chimerism technique has been shown to have the advantage of aggregating embryos in different stages of development with different ploidy, in addition to using ESC in the tetraploid complementation assay. Compared to other techniques for producing chimeras, the aggregation technique is a cheaper, faster and easier methodology to be performed. Moreover, aggregation can be simplified by chemically removing the zona pellucida with pronase or acidic Tyrode’s solution and be enhanced by using the Well of the Well culture system in combination with adhesion molecules, such as phytohemagglutinin. The most commonly used stages for chimerism by aggregation are those that precede the full compaction of the morula. In these stages, embryos have low-tension adherent junctions at the tangential point between two blastomeres. During the embryonic development of mice, the inner cell mass differentiates into epiblast and hypoblast. These layers will originate the fetal tissues and a portion of the extraembryonic tissues (yolk sac, allantois and amnion), whereas the trophectoderm (TE) gives rise to the chorion. A functional TE is essential for the complex molecular communications that occur between the embryo and the uterus. Embryos produced by somatic cell nuclear transfer, such as commercial cattle clones or endangered species, are subject to large fetal and neonatal losses. Hence embryo complementation with heterologous TE could be of assistance to decrease these losses and might as well assist development of high-value embryos in other approaches.

Ultrasonography of the conceptus development from days 15 to 60 of pregnancy in non-cyclic recipient mares

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Early development of Betta splendens under stereomicroscopy and scanning electron microscopy

Betta splendens is a very important ornamental species. The current paper describes the embryonic and larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0-2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2-3 hpIC and the early gastrula phase was observed at 3-4 hpIC with 20% epiboly, which was finalized after 13-18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23-25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 +/- 0.2 degrees C. The newly hatched larvae measured 2.47 +/- 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of B. splendens and provides relevant information for its reproduction, rearing and biology.