Improvement of polyploidy induction in Eriocheir sinensis

An incubating temperature of 15 degreesC is used to induce triploidy in Etiocheir sinensis through inhibition of the release of polar body H, and that of 18 degreesC to induce tetraploidy through inhibition of the first cleavage. Flow cytometry is used to identify the ploidy in different developmental stages. For induction of triploidy in fertilized eggs in vitro, the highest induction rate observed in blastula by cytochalasin B, 6-DMAP and KCI is 49.1%, 51.7% and 77.5%, respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest triploid induction rate observed in the zoea is 85.3%. For induction of tetraploidy, the highest induction rate observed in the blastula by cytochaslasin 13, 6-DMAP and KCI is 50.3%, 54.9% and 79.8% respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest induction rate in zoea is 27.3%. Through this study such difficulty as in vitro culture is overcome. Triploid zoea Etiocheir sinensis has been developed for the first time. The induction rate of tetraploid zoea has also been greatly improved.

几种对虾染色体及多倍体诱导研究

本文利用染色体制片、流式细胞术、显微荧光、组织学切片、透射电镜、及荧光原位杂交等技术，从细胞遗传学角度，研究了对虾的染色体和多倍体诱导等问题。以胚胎、无节幼体、卵巢、精巢等为材料，采用空气干燥法制片，获得了刀额新对虾、凡纳对虾和细角对虾的体细胞和减数分裂染色体中期分裂相。结果表明，刀额新对虾的染色体数目为2n = 78, n = 39，核型为2n = 78 = 40M + 10SM + 14ST + 14T。凡纳对虾和细角对虾染色体数目相同，为2n = 88, n = 44。以鸡血细胞为对照，用流式细胞仪（Partec CCAII)对刀额新对虾的细胞核DNA含量进行了测定，其值为鸡血细胞的1.75倍，绝对含量为4.025pg/2c。对细角对虾、日本对虾、刀额新对虾和中国对虾的基因组大小进行了比较，发现四种对虾基因组由大到小的顺序为细角对虾、日本对虾、刀额新对虾、中国对虾，由此推导出对虾科在进化过程中DNA发生丢失的假设。应用荧光原位杂交技术研究了中国对虾18SrDNA序列和（AG）n微卫星序列在染色体上的定位。初步研究的结果发现18SrDNA序列和（AG）n序列分布在中国对虾一对染色体的端粒区域；（AG）n微卫生序列分布在5对染色体上，着丝粒和端粒区域都有分布。本文对分布在热带和亚热带的日本对虾，斑节对虾及刀额新对虾的多倍体诱导进行了研究，成功地诱导出了三种对虾的三倍体幼体，诱导率最高分别达到68.52%、59.99%、49.70%。并进一步探讨了热带对虾多倍体诱导的特点，认为对产卵温度较市制热带和亚热带对虾，冷休克的诱导效果较好。采用热休克、冷休克、CB、6-DMAP、秋水仙素及咖啡因等方法，进行了中国对虾四倍体诱导研究。通过抑制第一极体排放或抑制第一次卵裂等途径，上述方法都获得了四倍体胚胎，最高诱导率达100%。实验中优化了诱导条件，筛选了诱导方法，认为热休克方法最佳。利用显微荧光技术研究精子入卵，原核形成、移动、结合以及再分离的过程，微管、微丝的活动及纺锤体等的动态变化，有助于深入了解多倍体的诱导机理。本文在中国对虾三倍体、四倍体诱导过程中，观察了诱导处理后染色体行为的变化，进一步分析了多倍体的形成过程。实验中发现四倍体诱导中存在细胞形态的特殊变化，认为是由于诱导处理导致的中心体复制异常所致，这也是诱导率降低的主要原因。文中讨论了四倍体诱导的最佳时机的选择即选择染色体已经分开，中心体尚未复制的时刻，并由此讨论了中心体的复制事件。利用组织学切片和透射电镜观察了三倍体成虾不同时期的性腺。结果表明，中国对虾三倍体的卵巢外观上退化明显，在组织学上可分为增殖期、阻滞期和退化期，卵巢细胞的发育大多停滞在卵原细胞阶段，尚未观察到有形态成熟的卵子形成，除少数生殖细胞进入卵黄形成前期，大多数为不定性细胞。三倍体精巢形态发育和组织学结构与二倍体相近，但产生的精子数明显少于二倍体。且输精管、贮精囊也呈退化趋势。精巢中所发现的染色体数在60-80、100-120和230-250区间的非整倍体细胞所占比例很大，其原因可能是减数分裂过程中，染色体发生了丢失。在超微结构上，观察到三倍体卵巢内的卵原细胞线粒体退休。核糖体较少，内质网和高尔期体比较发达，在晚期少数卵母细胞有卵黄颗粒存在。三倍体精巢中精原细胞和精母细胞的细胞器不发达，线粒体少，高尔基体较多；滤泡腔和输精管内精子很少，大小不一。

虾夷扇贝Patinopecten yessoensis (Jay)和仿刺参Apostichopus japonicus (Seienka)多倍体诱导研究

本文采用化学方法诱导了虾夷扇贝Patinopecten yessoensis (Jay)和仿刺参Apostichopus japonicus (Selenka)的多倍体。采用6－二甲基氨基嘌呤（6－dimethylaminopurine）抑制虾夷扇贝极体释放，研究了诱导三倍体的实验条件、技术工艺以及三倍体虾夷扇贝幼虫的生长和培育方法。结果表明，使用6－二甲基氨基嘌呤（6－DMAP）可诱导17.3%-100.0%的三倍体虾夷扇贝面盘幼虫。筛选并优化了生产中操作简便、高效、成活率高的诱导条件，并研究出在扇贝三倍体诱导中批量获得受精卵、处理及培育的技术工艺，对扇贝三倍体的规模化诱导及培育等生产性应用具有较高的参考价值。报道了采用细胞松弛素B（cytochalasin B）、秋水仙素（colchicine）、6－DMAP以及咖啡因（coffeine）等药物抑制虾夷扇贝第一极体（PB_1）释放、PB_1和第二极体（PB_2）释放以及抑制第一次卵裂等方法诱导四倍体的结果。结果表明，CB、6－DMAP和秋水仙素抑制扇贝第一次有丝分裂诱发四倍体的比例低于25%，而采用CB抑制PB_1可有效地诱导产生四倍体，从授精后42min提前到15-22min开始处理，抑制PB_1的放出有助于提高四倍体的比例，在12 ℃，处理开始和终上时间分别在授精后20－22min和60－62min时，面盘幼虫四倍体率最高，为56.5%。对CB处理抑制受精卵PB_1释放的处理组胚胎的染色体分离状况进行了观察研究。对照组受精卵具有19条四分体染色体，经过减数分裂I期（meiosis I）和减数分裂II期（meiosis II），放出PB_1和PB_2，受精卵的发育具有不同步性。处理组受精卵在第二次减数分裂中出现了“三级分离“、“联合二级分离“和“独立二级分离“等特殊的分离类型，初步分析了CB抑制第一极体放出的机理。对三倍体虾夷扇贝的繁殖潜力和卵子的大小进行了观察研究，三倍体扇贝具一定的繁殖能力，三倍体雌贝平均产卵3.26 * 10~6个，而相同壳长二倍体贝为1.45 * 10~7，三倍体产卵量仅为二倍体产卵量的22.5%。三倍体卵子产出后，形状不规则，卵子平均直径为87.25μm，比二倍体大11.7%，卵子体积比二倍体大39.4%。利用CB抑制第一极体释放诱导了虾夷扇贝的四倍体，诱导率为41.5%。首次报道了仿刺参Apostichopus japanicus (Selenka)多倍体诱导的结果。采用紫外线照射的海水成功地使海参分别单独产卵、排精，从而准确地控制了海参的人工授精后处理的时间。采用0.2、0.4mg/L CB 抑制受精卵第一极体释放以及10、20、30和40mg/L 的6－DMAP 分别抑制PB_1、PB_2放出的方法诱导了仿刺参的多倍体。研究了诱导的药物浓度、处理时间以及处理开始时间等，同时对幼体的成活率等进行了探讨。结果表明，2种药物均可诱导仿刺参产生三倍体和四倍体，从效果上看，采用CB抑制PB_1诱导，到达小耳幼体时，可产生9.7%-21.3%的四倍体。6－DMAP抑制PB_2放出诱导三倍体，三倍体诱导率介于7.5%-25.8%之间。

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for

Precursor adsorption on copper surfaces as the first step during the deposition of copper: a density functional study with van der Waals correction

Copper dimethylamino-2-propoxide [Cu(dmap)2] is used as a precursor for low-temperature atomic layer deposition (ALD) of copper thin films. Chemisorption of the precursor is the necessary first step of ALD, but it is not known in this case whether there is selectivity for adsorption sites, defects, or islands on the substrate. Therefore, we study the adsorption of the Cu(dmap)2 molecule on the different sites on flat and rough Cu surfaces using PBE, PBE-D3, optB88-vdW, and vdW-DF2 methods. We found the relative order of adsorption energies for Cu(dmap)2 on Cu surfaces is Eads (PBE-D3) > Eads (optB88-vdW) > Eads (vdW-DF2) > Eads (PBE). The PBE and vdW-DF2 methods predict one chemisorption structure, while optB88-vdW predicts three chemisorption structures for Cu(dmap)2 adsorption among four possible adsorption configurations, whereas PBE-D3 predicts a chemisorbed structure for all the adsorption sites on Cu(111). All the methods with and without van der Waals corrections yield a chemisorbed molecule on the Cu(332) step and Cu(643) kink because of less steric hindrance on the vicinal surfaces. Strong distortion of the molecule and significant elongation of Cu–N bonds are predicted in the chemisorbed structures, indicating that the ligand–Cu bonds break during the ALD of Cu from Cu(dmap)2. The molecule loses its initial square-planar structure and gains linear O–Cu–O bonding as these atoms attach to the surface. As a result, the ligands become unstable and the precursor becomes more reactive to the coreagent. Charge redistribution mainly occurs between the adsorbate O–Cu–O bond and the surface. Bader charge analysis shows that electrons are donated from the surface to the molecule in the chemisorbed structures, so that the Cu center in the molecule is partially reduced.

Computational study of the growth of copper thin films by atomic layer deposition

Copper is the main interconnect material in microelectronic devices, and a 2 nm-thick continuous Cu film seed layer needs to be deposited to produce microelectronic devices with the smallest features and more functionality. Atomic layer deposition (ALD) is the most suitable method to deposit such thin films. However, the reaction mechanism and the surface chemistry of copper ALD remain unclear, which is deterring the development of better precursors and design of new ALD processes. In this thesis, we study the surface chemistries during ALD of copper by means of density functional theory (DFT). To understand the effect of temperature and pressure on the composition of copper with substrates, we used ab initio atomistic thermodynamics to obtain phase diagram of the Cu(111)/SiO2(0001) interface. We found that the interfacial oxide Cu2O phases prefer high oxygen pressure and low temperature while the silicide phases are stable at low oxygen pressure and high temperature for Cu/SiO2 interface, which is in good agreement with experimental observations. Understanding the precursor adsorption on surfaces is important for understanding the surface chemistry and reaction mechanism of the Cu ALD process. Focusing on two common Cu ALD precursors, Cu(dmap)2 and Cu(acac)2, we studied the precursor adsorption on Cu surfaces by means of van der Waals (vdW) inclusive DFT methods. We found that the adsorption energies and adsorption geometries are dependent on the adsorption sites and on the method used to include vdW in the DFT calculation. Both precursor molecules are partially decomposed and the Cu cations are partially reduced in their chemisorbed structure. It is found that clean cleavage of the ligand−metal bond is one of the requirements for selecting precursors for ALD of metals. 2 Bonding between surface and an atom in the ligand which is not coordinated with the Cu may result in impurities in the thin film. To have insight into the reaction mechanism of a full ALD cycle of Cu ALD, we proposed reaction pathways based on activation energies and reaction energies for a range of surface reactions between Cu(dmap)2 and Et2Zn. The butane formation and desorption steps are found to be extremely exothermic, explaining the ALD reaction scheme of original experimental work. Endothermic ligand diffusion and re-ordering steps may result in residual dmap ligands blocking surface sites at the end of the Et2Zn pulse, and in residual Zn being reduced and incorporated as an impurity. This may lead to very slow growth rate, as was the case in the experimental work. By investigating the reduction of CuO to metallic Cu, we elucidated the role of the reducing agent in indirect ALD of Cu. We found that CuO bulk is protected from reduction during vacuum annealing by the CuO surface and that H2 is required in order to reduce that surface, which shows that the strength of reducing agent is important to obtain fully reduced metal thin films during indirect ALD processes. Overall, in this thesis, we studied the surface chemistries and reaction mechanisms of Cu ALD processes and the nucleation of Cu to form a thin film.

A Mild New Method for the C-Acylation of Ketone Enolates. A Convenient Synthesis of β-Keto Esters, -Thionoesters and Thioesters

Abstract image.

A new method for ketone enolate C-acylation is described which utilizes alkyl pentafluorophenylcarbonates, thiocarbonates and thionocarbonates as the reactive acylating agents, and MgBr2.Et2O, DMAP and i-Pr2NEt as the reagents for enolization. A wide range of ketones have been observed to undergo clean C-acylation via this protocol.

Synthesis, properties and characterization of N-Alkyl substituted b-Diketiminato copper(I) Complexes

Le ligand nacnacxylH (xyl = C6Me2H3) et les ligands dikétimines N-alkyle substitués (nacnacCH(Me)PhH, nacnacBnH and nacnaciPrH) ont été préparés avec de bons rendements à l’exception du nacnaciPrH (23%) en utilisant un protocole en une étape et à l’aide d’un montage Dean-Stark. La réaction du S,S-nacnacCH(Me)PhH et du nacnacBnH avec le nBuLi dans le THF conduit au S,S-nacnacCH(Me)PhLi(THF) et au nacnacBnLi(THF). Les tentatives de bromation de ces composés par le N-bromosuccinimide conduisent plutôt aux ligands S,S-succnacnacCH(Me)PhH et succnacnacBnH (succ = succinimido) substitués par un groupement succinimido sur le carbone  La chloration par le N-chlorosuccinimide conduit au produit désiré, mais avec des impuretés. La réaction de ces ligands avec le CuOtBu (ou bien MesCu, où Mes = C6Me3H2, et une quantité catalytique de CuOtBu) en présence de bases de Lewis donne les (nacnacxylCu)2(-toluène), nacnacxylCuCNC6H3(Me)2, nacnacCH(Me)PhCuL (L = PPh3, PMe3, CNC6H3(Me)2, DMAP, lutidine, Py, MeCN), nacnacBnCuL (L = PPh3, CNC6H3(Me)2, styrène, trans-stilbene, phenylvinylether, acrylonitrile, diphenylacetylène), nacnaciPrCuL (L = PPh3, CNC6H3(Me)2, MeCN) et le succnacnacCH(Me)PhCuL (PPh3, CNC6H3(Me)2, pyridine). Tous ces complexes sont jaunes et sensibles à l’air et à l’humidité. En l’absence de fortes bases de Lewis, on n’observe pas de réaction entre les précurseurs de cuivre et les ligands N-alkyle substitués. Les études RMN des complexes dans le C6D6 ne présentent pas de complexe de toluène mais un mélange à l’équilibre du (nacnacxylCu)2(-C6D6) et nacnacxylCu(C6D6) dans une proportion de 2 pour 1. Alors que l’addition de plus de cinquante équivalents soit de THF, soit de toluène n’induit aucun changement des spectres RMN, l’addition de 2 équivalents de MeCN conduit instantanément au complexe nacnacxylCu(MeCN). De plus, le (nacnacxylylCu)2(-C6D6) ne se coordone ni ne réagit avec le N2O, même après avoir été chauffé à 60°C pendant treize jours. En présence de DPA (diphenylacétylène), la réaction du nacnacBnH avec le CuOtBu conduit au dimère ponté (nacnacBnCu)2(µ-DPA). L’addition d’un excès de DPA (10-12 équivalents) transforme le dimère ponté en complexe lié en position terminale nacnacBnCuDPA. Les nacnacRH (R = CH(Me)Ph et i-Pr) ne forment pas de complexe ni avec les oléfines ni avec le DPA. Une réactivité similaire a été observée avec les complexes de nacnacCH(Me)PhCu(NCMe) et nacnaci-PrCu(NCMe). Tandis que le complexe lié en position terminale par MeCN a été isolé et caractérisé, l’équilibre en solution nous laisse suspecter la formation d’un complexe d’acétonitrile ponté. Des études de réactivité comparatives ont été menées sur quelques complexes de cuivre. La Morpholine ne réagit pas avec le nacnacBnCu(acrylonitrile) contrairement à l’acrylonitrile libre. L’expérience de l’échange d’oléfine montre que l’acrylonitrile (une oléfine électro-attractrice) se lie plus fortement que les autres oléfines, mettant ainsi en évidence l’importance de la rétrodonation  face à la donation La rétrodonation est cependant faible comparée aux autres complexes de styrène structurellement caractérisés. Les complexes nacnacCH(Me)PhCuL (L = PPh3 et MeCN) ont été employés dans la cyclopropanation catalytique du styrène et dans l’addition conjuguée du ZnEt2 sur la 2-cyclohexénone, mais les résultats indiquent que le ligand dikétimine est éliminé avant son entrée dans le cycle catalytique. Par conséquent, il n’y a pas d’induction chirale. Les complexes tétra coordinées de cuivre avec les nacnacRCu(phen) (R = Bn, CH(Me)Ph et Phen = 1,10-phenanthroline, 2-Mes-1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline (dmp) et 2,9-diphenyl-1,10-phenanthroline (dpp)) ont été synthétisés. Ces complexes sont d’une intense couleur bleue et des interactions d’empilement entre l’un des cycles phényle des ligands nacnac et la phénanthroline ont été observées dans les structures à l’état solide. Les mesures en absorption UV-visible ont été effectuées dans le toluène et les bandes MLCT sont déplacées vers le rouge par rapport à celles des complexes de cuivre et bisphénanthroline. Tous ces composés émettent à l’état solide mais les complexes 1,10-phenanthroline et 2-Mes-1,10-phenanthroline n’émettent pas en solution. Pour renforcer les interactions d’empilement , les nouveaux ligands nacnacRH (R = CH2C6H2(OMe)3, CH2C6F5) et leurs complexes de cuivre respectifs ont été préparés avec du dmp et dpp. Afin de permettre la comparaison, le nacnaciBuCu(dmp) a été synthétisé. Alors que les complexes dmp montrent une augmentation des interactions intramoléculaires - avec les substituants phényle du ligand dikétimine et de la phénanthroline, les complexes dpp ne révèlent pas de telles interactions. Les complexes perfluorés montrent, en absorption et en émission, un déplacement significatif vers le bleu, alors que les complexes substitués par un groupements isobutyle présentent des transitions déplacées vers le rouge. Alors que les intensités de luminescence et les durées de vie sont faibles, les déplacements réduits de Stokes et les pics étroits de luminescence comparables indiquent une réduction des distorsions de l’état excité.

Dinitrogen release from arylpentazole: A picosecond time-resolved infrared, spectroelectrochemical, and DFT computational study

p-(Dimethylamino)phenyl pentazole, DMAP-N5 (DMAP = Me2N−C6H4), was characterized by picosecond transient infrared spectroscopy and infrared spectroelectrochemistry. Femtosecond laser excitation at 310 or 330 nm produces the DMAP-N5 (S1) excited state, part of which returns to the ground state (τ = 82 ± 4 ps), while DMAP-N and DMAP-N3 (S0) are generated as double and single N2-loss photoproducts with η ≈ 0.14. The lifetime of DMAP-N5 (S1) is temperature and solvent dependent. [DMAP-N3]+ is produced from DMAP-N5 in a quasireversible, one-electron oxidation process (E1/2 = +0.67 V). Control experiments with DMAP-N3 support the findings. DFT B3LYP/6-311G** calculations were used to identify DMAP-N5 (S1), DMAP-N3 +, and DMAP-N in the infrared spectra. Both DMAP-N5 (S1) and [DMAP-N5]+ have a weakened N5 ring structure.

Parthenogenetic activation of bovine oocytes using single and combined strontium, ionomycin and 6-dimethylaminopurine treatments

In vitro-matured (IVM) bovine oocytes were activated with single and combined treatments of strontium (S), ionomycin (1) and 6-DMAP (D). Using oocytes IVM for 26 h, we observed that activation altered cell cycle kinetics (faster progression, MIII arrest, or direct transition from MII to pronuclear stage) when compared to in vitro fertilization. The effect of oocyte age on early parthenogenesis was assessed in oocytes IVM for 22, 26 and 30 h. Better results in pronuclear development were obtained in treatments ISD (81.7%) at 22 h; D (66.7%), IS (63.3%), ID (73.3%) and ISD (76.7%) at 26 h; and D (86.7%), IS (85.0%) and ID (78.3%) at 30 h. Higher cleavage occurred on ISD (80.0%) at 22 h; ID (83.3%) and ISD (91.7%) at 26 h; and 1 (86.7%), IS (90.0%), ID (85.0%) and ISD (95.0%) at 30 h. More blastocysts were achieved in ID (25.0%) and ISD (18.3%) at 22h; and in ID at 26h (45.0%) and 30h (50.0%). We also observed that IS allowed higher haploid (77.4%) embryonic development, whilst ID was better for diploid (89.1%) development. It was concluded that association of S and D without I was not effective for blastocyst development; treatments using S were less influenced by oocyte age, but when S was associated with D there was a detrimental effect on aged oocytes; treatment ISD promoted higher activation and cleavage rates in young oocytes and ID protocol was the best for producing blastocysts.

Produtividade das culturas de alface e rabanete em função da época de estabelecimento do consórcio

Avaliou-se a produtividade das culturas de rabanete e alface, e a qualidade de seus produtos, em função da época de estabelecimento do consórcio, na UNESP em Jaboticabal, de março a maio de 1999. Os tratamentos foram constituídos por consórcios estabelecidos aos 0; 7 e 14 dias após o transplantio (DAT) da alface e monocultivos implantados nestas mesmas épocas. As cultivares de rabanete (Raphanus sativus L.) e alface (Lactuca sativa L.) utilizadas foram, respectivamente, Crimson Gigante e Carolina. Maior altura das plantas de rabanete foi observada quando consorciada com alface. As plantas de rabanete em monocultivo apresentaram acúmulo de massa seca da parte aérea (MSPA) 29,4% menor do que em consorciação, independente da época de semeadura. Para massa seca de raízes tuberosas, maior acúmulo foi obtido na presença de alface, independentemente da época de instalação do consórcio. Quanto a MSPA de alface, maior acúmulo ocorreu em consórcio, quando a semeadura do rabanete foi realizada até 7 DAT. Além de menor acúmulo de MSPA, quando a semeadura do rabanete foi realizada aos 14 DAT, a alface apresentou perda na qualidade comercial. O maior valor da razão de área equivalente (1,6) no sistema de consórcio, foi obtido quando o rabanete foi semeado aos 7 dias após o transplantio da alface.

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylammopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

Micorrização e crescimento de progênies de hymenaea stignocarpa mart. ex. Hayne em subsolo de área degradada degraded area

In tropical ecosystems, little is known about the relationship between arbuscular mycorhizal fungi (AMF) and the host genetic variability, especially among tree species. This study aimed to examine the response of 19 progenies of jatobá-do-cerrado (Hymenaea stignocarpa Mart. Ex. Hayne) inoculated with AMF, the root colonization and seedlings early growth were evaluated, growing in cultivated in subsoil from the loading area, under greenhouse conditions. The seedlings, germinated in the laboratory, were transferred to plastic bags containing subsoil and sand mixture (4:1). For the inoculation, each replicate received 100 g of soil (with about 48 spores of AMF) from a preserved Cerrado area, to reintroduce microorganisms. After 120 days, the mycorrhiza colonization (COL), the number of spores of AMF, the plant height (PH), the weight of shoot dry matter (SDM) and the root fresh matter (RFM) were assessed. The highest values of COL, SDM and RFM were observed in the progenies JC7, JC18, JC29, JC27 and JC14; the JC7 andJC18 also increased number of spores. There were significant and positive correlations between COL and the others variables (AP, NE, RFM and SDM, and between RFM and other variables (NE, AP and SDM). The conclusion is that, there is variability among the genotypes of Hymenaea stignocarpa growth (AP MFSR and DMAP) and mycorrhization (COL and NE), with emphasis on two progenies (JC7 and JC18), that showed the highest values, which can be resulted of a greater affinity to strains of AMF- soil inoculum.

Ativação partenogenética de oócitos maturados in vitro de Cebus apella

O objetivo deste trabalho foi avaliar a maturação oocitária in vitro (MIV) na espécie C. apella, relacionando com a expansão das células do cumulus e promovendo a produção in vitro de embriões (PIVE), por meio da ativação partenogenética e fecundação in vitro (FIV). Os oócitos puncionados dos folículos antrais medindo 2-9 mm de diâmetro foram classificados em desnudos (OD), com poucas células do cumulus (PCC) e complexo cumulus-oophorus intacto (CCO intacto). A expansão das células do cumulus foi analisada nos tempos de 0, 36 e 40 h de MIV e classificada em cinco categorias de expansão. A competência meiótica nos oócitos foi verificada pela extrusão do 1º corpúsculo polar (CP) após 40 h de MIV. Os oócitos foram divididos em 3 grupos para PIVE: grupo controle (FIV), ativação partenogenética utilizando 5 μM de ionomicina em associação com 2 mM de 6-DMAP ou em associação com 50 μM de roscovitina. Para a FIV, os espermatozóides obtidos do coágulo seminal, foram diluídos em água de coco em pó (ACP-118®) e submetidos ao resfriamento para serem levados posteriormente ao cultivo in vitro com os oócitos. O aumento no tempo da MIV e a presença de várias células do cumulus associadas aos oócitos proporcionaram maior expansão das células do cumulus, pois somente os CCO intactos alcançaram a expansão total em 40 h de MIV (p < 0,005). A presença das células do cumulus nos oócitos (PCC e CCO intactos) maturados in vitro por 40 h promoveu a competência meiótica (metáfase II) significamente mais elevada que os OD (p < 0,005). Após 6 h de resfriamento em ACP-118® e separação pelo método do swin-up, os espermatozóides utilizados na FIV possuíam 80% de motilidade e vigor 4. O tratamento com ionomicina/roscovitina promoveu a extrusão do 2º CP e formação pronuclear e com ionomicina/6-DMAP formou pronúcleos, sem extrusão do 2º CP. O protocolo utilizando a ionomicina/6-DMAP e da FIV originou as primeiras divisões embrionárias, observada pela taxa de clivagem. Os resultados encontrados sugerem que em C. apella a presença e a expansão total das células do cumulus estão relacionadas com a competência oocitária. A utilização dos oócitos maturados in vitro e dos espermatozóides diluídos e resfriados em ACP-118® promoveu a PIVE por diferentes métodos nesta espécie.

Interação núcleo-citoplasmática em embriões e expressão de genes imprinted em fetos bovinos produzidos in vivo, in vitro e partenogenéticos

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