Use of bayesian inference to correlate in vitro embryo production and in vivo fertility in Zebu Bulls

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP. © 2011 Mateus José Sudano et al.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides. © 2012 Elsevier Ltd. All rights reserved.

Physiology and endocrinology symposium: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature

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

Cyp17a1 and cyp19a1 in the zebrafish testis are differentially affected by oestradiol

Oestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17β-oestradiol (E2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology. © 2013 Society for Endocrinology.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p<0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48h of culture. © 2012 Blackwell Verlag GmbH.

Timing of prostaglandin F2α treatment in an estrogen-based protocol for timed artificial insemination or timed embryo transfer in lactating dairy cows

Objectives were to investigate progesterone concentrations and fertility comparing 2 different intervals from PGF2α treatment and induced ovulation in an estrogen-based ovulation synchronization protocol for timed artificial insemination (TAI) or timed embryo transfer (TET) in lactating dairy cows. A total of 1,058 lactating Holstein cows [primiparous (n=371) and multiparous (n=687)], yielding 34.1±0.33 kg of milk/d at various days in milk were randomly assigned to receive treatment with PGF2α on either d 7 or 8 of the following protocol: d 0: 2mg of estradiol benzoate + controlled internal drug release device; d 8: controlled internal drug release device removal + 1.0mg of estradiol cypionate; d 10: TAI or d 17: TET. Only cows with a corpus luteum at d 17 received an embryo and all cows received GnRH at TET. Pregnancy diagnoses were performed by detection (transrectal ultrasonography) of an embryo on d 28 or a fetus on d 60. Fertility [pregnancy per artificial insemination (P/AI) or pregnancy per embryo transfer (P/ET)] was affected by breeding technique (AI vs. ET) and time of PGF2α treatment (d 7 vs. 8) at the 28-d pregnancy diagnosis for TAI [32.9% (238) vs. 20.6% (168)] and TET cows [47% (243) vs. 40.7% (244)] and at the 60-d pregnancy diagnosis for TAI [30% (238) vs. 19.2% (168)] and TET cows [37.9% (243) vs. 33.5% (244)]. The progesterone (P4) concentration at d 10 altered fertility in TAI cows, with higher P/AI in cows with P4 concentration <0.1 ng/mL compared with cows with P4 concentration ≥0.1 ng/mL, and in ET cows, with higher P/ET in cows with P4 concentration <0.22 ng/mL compared with cows with P4 concentration ≥0.22 ng/mL. Prostaglandin F2α treatment at d 7 increased the percentage of cows with P4 <0.1 ng/mL on d 10 [39.4 (85) vs. 23.2 (54)]. Reducing the period between PGF2α and TAI from 72 to 48h in dairy cows resulted in a clear reduction in fertility in cows bred by TAI and a subtle negative effect in cows that received TET. The earlier PGF2α treatment benefits are most likely mediated through gamete transport, fertilization, or early embryo development and a more subtle effect of earlier PGF2α treatment that may be mediated through changes in the uterine or hormonal environment that manifests itself after ET on d 7. © 2013 American Dairy Science Association.

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. © 2013 Dobbs et al.

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.

Alcohol Impairs Predation Risk Response and Communication in Zebrafish

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism. © 2013 Oliveira et al.

Estudo do efeito de metais em esterases de zebrafish (Danio rerio)

Pós-graduação em Química - IBILCE

Isolate((R)) and Optiprep((R)) minigradients as alternatives for sperm selection in bovine in vitro embryo production

The objective of this study was to evaluate alternatives in small volumes to conventional gradient of Percoll((R)) on semen quality, in vitro embryo production, sex ratio and embryo survival after vitrification. Thawed semen was randomly allocated to one of four density gradient selection methods: (1) conventional Percoll((R)) (P), (2) MiniPercoll (MP), (3) MiniIsolate (MI), and (4) MiniOptiprep (MO). Sperm kinetics and quality were evaluated. Use of P, MP and MI gradients did not affect sperm motility (P > 0.05). However, there was a decrease in total and progressive sperm motility in MO (70.8 and 51.3% vs. 87.3 and 69.5% for P; 87.3 and 73% for MP; 92.3 and 78.8% for MI; P < 0.05). The MO had lower membrane integrity compared with P, MP and MI (39.7 vs. 70.5, 72.3, 63.8%, respectively, P < 0.05). The percentage of blastocysts produced was higher in MI than in MP and MO (21.1 vs. 16.1 and 16.9%, P < 0.05) and similar to P (18.4%; P > 0.05). Sex ratio and embryo survival after vitrification were similar among groups (P > 0.05). Semen selected by Isolate and Optiprep gradient, at the concentrations and small volumes used, demonstrated similar characteristics and in vitro embryo production to conventional Percoll((R)) gradient.

Chemical communication of predation risk in zebrafish does not depend on cortisol increase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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)