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.

Control of Buffalo Follicular Dynamics for Artificial Insemination, Superovulation and In Vitro Embryo Production

Currently, timed ovulation induction and timed artificial insemination (TAI) can be performed in buffalo using GnRH or estradiol plus progesterone/progestin (P4)-releasing devices and prostaglandin F-2 alpha (PGF(2 alpha)). The control of the emergence of follicular waves and of ovulation at predetermined times, without the need for estrus detection, has facilitated the management and improved the efficiency of AI programs in buffalo during the breeding and nonbreeding season. Multiple ovulations, embryo transfer, ovum collection and in vitro embryo production have been shown to be feasible in buffalo, although low efficiency and limited commercial application of these techniques have been documented as well. These results could be associated with low ovarian follicular pools, high levels of follicular atresia and failures of the oocyte to enter the oviduct after superstimulation of follicular growth. This review discusses a number of key points related to the manipulation of ovarian follicular growth to improve pregnancy rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in buffalo.

Plasma anti-mullerian hormone: an endocrine marker for in vitro embryo production from Bos taurus and Bos indicus donors

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

Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro

Contents Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22h in TCM-199 supplemented with 0, 2.5, 10 or 50ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5ng/ml FGF10 increased (p<0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis

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

Paradoxical effects of bovine somatotropin treatment on the ovarian follicular population and in vitro embryo production of lactating buffalo donors submitted to ovum pick-up

The aim of the present study was to evaluate the effect of bovine somatotropin (bST; 500 mg) administration on lactating buffalo donors submitted to two different ovum pick-up (OPU) and in vitro embryo production schemes with a 7 or 14 d intersession OPU interval. A total of 16 lactating buffalo cows were randomly assigned into one of four experimental groups according to the bST treatment (bST or No-bST) and the OPU intersession interval (7 or 14 d) in a 2 x 2 factorial design (16 weeks of OPU sessions). The females submitted to OPU every 14d had a larger (P < 0.001) number of ovarian follicles suitable for puncture (15.6 +/- 0.7 vs. 12.8 +/- 0.4) and an increased (P = 0.004) number of cumulus-oocyte complexes (COCs) recovered (10.0 +/- 0.5 vs. 8.5 +/- 0.3) compared to the 7 d interval group. However, a 7 or 14 d interval between OPU sessions had no effect (P = 0.34) on the number of blastocysts produced per OPU (1.0 +/- 0.1 vs. 13 +/- 0.2, respectively). In addition, bST treatment increased (P < 0.001) the number of ovarian follicles suitable for puncture (15.3 +/- 0.5 vs. 12.1 +/- 0.4) but reduced the percentage (18.9% vs. 10.9%; P = 0.009) and the number (1.4 +/- 0.2 vs. 0.8 +/- 0.1; P = 0.003) of blastocysts produced per OPU session compared with the non-bST-treated buffaloes. In conclusion, the 14d interval between OPU sessions and bST treatment efficiently increased the number of ovarian follicles suitable for puncture. However, the OPU session interval had no effect on embryo production, and bST treatment reduced the in vitro blastocyst outcomes in lactating buffalo donors.

Manifestation of estrous behavior and subsequent progesterone concentration at timed-embryo transfer in cattle are positively associated with pregnancy success of recipients

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of cell cycle synchronization on the accuracy of murine and bovine embryo sex determination

Different cell cycle synchronization methods were used to increase the mitotic index and accuracy of sex determination in murine and bovine embryos. For sexing purposes, colchicine treatment for 2, 4, 6 and 8 h and the FdU-thymidine-colchicine combination were tested in murine embryos. The best results were obtained with colchicine treatment for 8 h (96.88% accuracy) and with FdU-thymidine-colchicine (97.22% accuracy). Mitotic indexes differed significantly between the 2 treatments (21.71% for colchicine and 32.95% for FdU-thymidine-colchicine). For sex identification of murine and bovine demi-embryos, both treatments were demonstrated to be equally effective (nearly 90%). The mitotic index for the FdU-treated murine demi-embryos (19.04%) was higher than the one obtained for the 8-h colchicine treatment (15.62%).

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryo

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryoSpecification of a unique cell fate during development of a multicellular organism often is a function of its position. The Drosophila central nervous system (CNS) provides an ideal system to dissect signalling events during development that lead to cell specific patterns. Different cell types in the CNS are formed from a relatively few precursor cells, the neuroblasts (NBs), which delaminate from the neurogenic region of the ectoderm. The delamination occurs in five waves, S1-S5, finally leading to a subepidermal layer consisting of about 30 NBs, each with a unique identity, arranged in a stereotyped spatial pattern in each hemisegment. This information depends on several factors such as the concentrations of various morphogens, cell-cell interactions and long range signals present at the position and time of its birth. The early NBs, delaminating during S1 and S2, form an orthogonal array of four rows (2/3,4,5,6/7) and three columns (medial, intermediate, and lateral) . However, the three column and four row-arrangement pattern is only transitory during early stages of neurogenesis which is obscured by late emerging (S3-S5) neuroblasts (Doe and Goodman, 1985; Goodman and Doe, 1993). Therefore the aim of my study has been to identify novel genes which play a role in the formation or specification of late delaminating NBs.In this study the gene anterior open or yan was picked up in a genetic screen to identity novel and yet unidentified genes in the process of late neuroblast formation and specification. I have shown that the gene yan is responsible for maintaining the cells of the neuroectoderm in an undifferentiated state by interfering with the Notch signalling mechanism. Secondly, I have studied the function and interactions of segment polarity genes within a certain neuroectodermal region, namely the engrailed (en) expressing domain, with regard to the fate specification of a set of late neuroblasts, namely NB 6-4 and NB 7-3. I have dissected the regulatory interaction of the segment polarity genes wingless (wg), hedgehog (hh) and engrailed (en) as they maintain each other’s expression to show that En is a prerequisite for neurogenesis and show that the interplay of the segmentation genes naked (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment of NB 7-3 and NB 6-4 cell fate. I have shown that in the absence of either nkd or gsb one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.

Religion and assisted and non-assisted suicide in Switzerland: National Cohort Study

Background In the 19th century, eminent French sociologist Emile Durkheim found suicide rates to be higher in the Protestant compared with the Catholic cantons of Switzerland. We examined religious affiliation and suicide in modern Switzerland, where assisted suicide is legal. Methods The 2000 census records of 1 722 456 (46.0%) Catholics, 1 565 452 (41.8%) Protestants and 454 397 (12.2%) individuals with no affiliation were linked to mortality records up to December 2005. The association between religious affiliation and suicide, with the Protestant faith serving as the reference category, was examined in Cox regression models. Hazard ratios (HRs) with 95% confidence intervals (CIs) were adjusted for age, marital status, education, type of household, language and degree of urbanization. Results Suicide rates per 100 000 inhabitants were 19.7 in Catholics (1664 suicides), 28.5 in Protestants (2158 suicides) and 39.0 in those with no affiliation (882 suicides). Associations with religion were modified by age and gender (P < 0.0001). Compared with Protestant men aged 35–64 years, HRs (95% CI) for all suicides were 0.80 (0.73–0.88) in Catholic men and 1.09 (0.98–1.22) in men with no affiliation; and 0.60 (0.53–0.67) and 1.96 (1.69–2.27), respectively, in men aged 65–94 years. Corresponding HRs in women aged 35–64 years were 0.90 (0.80–1.03) and 1.46 (1.25–1.72); and 0.67 (0.59–0.77) and 2.63 (2.22–3.12) in women aged 65–94 years. The association was strongest for suicides by poisoning in the 65–94-year-old age group, the majority of which was assisted: HRs were 0.45 (0.35–0.59) for Catholic men and 3.01 (2.37–3.82) for men with no affiliation; 0.44 (0.36–0.55) for Catholic women and 3.14 (2.51–3.94) for women with no affiliation. Conclusions In Switzerland, the protective effect of a religious affiliation appears to be stronger in Catholics than in Protestants, stronger in older than in younger people, stronger in women than in men, and particularly strong for assisted suicides.