bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structure of [PdCl2{(Py)2(PyH)CH-N′,N″}][PdCl4]0.5.2H2O (Py = Pyridin-2-yl), a Complex With Protonated 2,2′,2″-Methylidynetrispyridine as a Bidentate Ligand
Authors: Canty AJ , Minchin NJ , Skelton BW , White AH
Published: December 2006

Crystals of dichloro[2,2′,2″-methylidynetris(pyridin)-1-ium-N′,N″]palladium(II) hemi[tetra-chloropalladate(II)] dihydrate are monoclinic, P21/c, a 8.558(5), b 15.516(7), c 16.153(5) a, P 112.07(4)°, Z 4. The cations [PdCl2{(Py)2(PyH)CH-N′,N″}]+ have square-planar geometry for palladium, with the ligand in a boat conformation and having an uncoordinated protonated pyridine group.
The Absolute Structure of Latifoline
Authors: Culvenor CCJ , Mackay MF
Published: December 2006

The absolute structure of latifoline, a pyrrolizidine diester alkaloid of retronecine and latifolic and angelic acids, has been defined by single-crystal X-ray crystallographic analysis of the hydrobromide salt hydrate. Orthorhombic crystals of C20H27NO7.HBr.H2O belong to the space group P 2′2′2′ with a 7.279(1), b 17.898(3), c 17.922(2) � and V 2334-9(8) � 3 . The structure has been refined to an R index of 0.054 for 1725 observed terms. The absolute configuration has been assigned by comparison with that of retronecine and also determined independently by X-ray examination of selected Bijvoet pairs. Our results have shown that the absolute structure of latifolic acid is 13S,14R,19S (2R,3S,4S in latifolic acid numbering), and not the stereoisomer reported recently by Roitman and Wong.
Aromatization Reactions of Castanospermine Tetraacetate
Authors: Brown RFC , Collins DJ , Lagniton LM , Smith RJ , Wong NR
Published: December 2006

The Polonovski reaction of castanospermine tetraacetate N-oxide with acetic anhydride gave (�)-1-acetoxy-8-hydroxy-2,3-dihydro-lH-indolizinium acetate (10) by aromatization of the piperidine ring. N- Bromosuccinimide oxidation of castanospermine tetraacetate gave a low yield (4%) of (+)-2-bromo-5,6,7,8-tetrahydroindolizine-6,7,8-triyl triacetate (13), by aromatization of the pyrrolidine ring.
Variation in molasses composition from eastern Australian sugar mills
Authors: Bortolussi G. , O'Neill C. J.
Published: December 2006

Variation in the composition of eastern Australian molasses was examined. Data on molasses composition for 1997–2001 were collected from all 28 eastern Australian sugar mills. Since the last detailed study in 1975, there have been decreases in the concentration of reducing sugars (–6.2%), sucrose (–0.9%), total sugars (–2.5%), other organic matter (–11%) and calculated metabolisable energy (ME) (–2.8%). Dry matter (DM, +0.1%) and ash (+29%) concentrations have increased. Differences between milling regions were observed for DM (P<0.001), reducing sugars (P<0.05) and sucrose (P<0.01) concentrations. Molasses DM was highest for the Northern milling region and lowest for the Southern milling region. Reducing sugar concentrations were lower whereas sucrose concentrations were higher in molasses from the Southern milling region than the Northern region. The concentration of reducing sugars, total sugars and calculated ME was significantly (P<0.05) lower in mid-season molasses than early and late season molasses. Mid-season molasses ash concentration was highest (P<0.05). Significant (P<0.05) region × year interactions were found for all molasses components. Significant (P<0.05) region × season interactions were found for reducing sugars, sucrose, total sugars, ash and the calculated ME. Significant (P<0.001) nested effects for mills within a region were found for all molasses components. Mill within-region effects (P<0.001) accounted for much (31–62%) of the total variation for all molasses components. Significant (P<0.01) nested effects for season within year were found for all molasses components. Depending on the molasses component, season within year accounted for 3–19% of variation. Calculated ME in molasses was most correlated with total sugars (r = –0.97; P<0.001) and ash concentration (r = –0.73; P<0.001). Commercial cane sugar (percentage sugar in juice) was negatively (r = –0.43; P<0.001) correlated with calculated molasses ME. Generally, the observed variation in the calculated ME of molasses would only be sufficient to reduce average daily gains by up to 0.05 kg/day in cattle fed diets containing 60% molasses. Compared with the calculated molasses ME of molasses in 1975, a similar reduction in animal performance could be expected with current molasses composition. Such variation in ME also has implications for the use of molasses in the dairy industry and for ethanol production.
Forecasting the risk of crown rot between successive wheat crops
Authors: Backhouse D.
Published: December 2006

Published data from long-term trials at Moree, New South Wales (1986–1996), and Billa Billa, Queensland (1986–1993), were analysed to determine the factors that influence the incidence of crown rot, caused by Fusarium pseudograminearum, in successive stubble-retained, no-till wheat crops and to examine the feasibility of developing a forecasting system for the disease. Polyetic progress of the epidemics could be described by a form of the logistic growth model with a carrying capacity (K) about 5% higher than the maximum recorded incidence at each site. Infection rate between seasons was positively correlated with yield and in-crop rainfall in the previous season, both of which were indicators of biomass. Infection rate was negatively correlated with rainfall parameters during the summer fallows, which were indicators of conditions favouring residue decomposition. In-crop rainfall, stored soil moisture and temperature parameters were not significantly correlated with infection rates. Multiple regressions based on incidence in the previous season, summer rainfall and either yield or in-crop rainfall in the previous season accounted for 65–81% of the variation in disease incidence at Moree and 86% of the variation in incidence at Billa Billa. Simplified parameters for use in on-farm forecasting systems were explored. The most useful of these was the square root of the product of incidence and either yield or in-crop rainfall, which gave sufficiently accurate predictions at each site to estimate the qualitative risk of crown rot in the following crop. This could be used to decide whether management options such as resistant varieties, rotations or burning were required.
Landowners' perception of the benefits and barriers to adopting centre pivot and lateral move irrigators on dairy farms
Authors: Maskey R. , Bethune M. , Lawler D.
Published: December 2006

Maintaining profitability under conditions of limited water availability is a key issue facing dairy farmers in the Shepparton Irrigation Region. These farmers predominantly use border-check irrigation. Pressurised irrigation methods are seen as one option for increasing productivity from the limited water resources. This study reports on landowners’ perceptions towards the adoption of centre pivot and lateral move irrigators, based on survey and factor analysis. The survey was of 20 landowners using face-to-face interviews. Factor analysis was used to identify broad categories of benefits and barriers that best summarised farmers’ responses to survey questions. Improved farm management, productivity benefits, lifestyle benefits and improved marketability of farms were identified as the key benefits of adopting centre pivot and lateral move irrigators perceived by farmers. Capital cost, operation and maintenance costs, layout of the farm and unreliability of systems were seen as the key barriers. An understanding of these perceived benefits and barriers is the first step towards the development of extension information relating to the adoption of centre pivot and lateral move irrigation systems.
Carbon Dioxide and the Regulation of Photosynthesis: Activities of Photosynthetic Enzymes and Carbonate Dehydratase (Carbonic Anhydrase) in Chlorella after Growth or Adaption in Different Carbon Dioxide Concentrations
Authors: Reed M.L , Graham D
Published: December 2006

The unicellular green alga Chlorella pyrenoidosa, after growth in air or 5 % (v/v) CO*2, has markedly differing rates of photosynthesis in the presence of very low concentrations of CO*2. It was proposed that the differences could be accounted for by differences in the activities of ribulosebisphosphate carboxylase or other enzymes of the reductive pentose phosphate pathway. This hypothesis was tested and it was shown that between the two types of cells there were no significant differences in the activities of ribulosebisphosphate carboxylase (EC 4.1.1.39), phosphoenolpyruvate carboxylase (EC 4.1.1.31), phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49), glyceraldehyde-phosphate dehydrogenase (NADP) (EC 1.2.1.9), hexosebisphosphatase (EC 3.1.3.11), fructose-bisphosphate aldolase (EC 4.1.2.13), ribosephosphate isomerase (EC 5.3.1.6) and phosphoribulokinase (EC 2.7.1.19). There was also no difference between air-grown and 5% CO*2-grown cells in the content of fraction I protein isolated by disc gel electrophoresis and measured by a colorimetric procedure.
A fivefold or sixfold increase in rate of photosynthesis occurs during about 90 min when 5% CO*2-grown Chlorella is illuminated in the presence of a very low CO*2 concentration (approximately 1 �M free CO*2, which is about one-tenth the concentration of CO*2 in distilled water in equilibrium with air). During this increase, the only photosynthetic enzyme to change in activity was carbonate dehydratase (carbonic anhydrase, EC 4.2.1.1). This enzyme was absent from cells grown with 5% CO*2 but was present in air-grown cells. The enzyme appears to be induced by low CO*2 concentrations and repressed by high concentrations.
It was concluded that carbonate dehydratase plays a major regulatory role in photosynthesis in Chlorella at low CO*2 concentrations.
Velocity Distributions of Characean Cytoplasmic Streaming are Consistent with Random Cross-bridge Contractions
Authors: Piddington R.W , Ross D.A
Published: December 2006

We have obtained velocity distributions for cytoplasmic streaming in Chara and Nitella by three separate
techniques: laser light scattering, where a Doppler shift is measured; clocking of organelles over a
standard distance using low-power microscopy; and frame-by-frame analysis of cine film taken at highpower.
The laser-derived distribution is a symmetrical Lorentzian centred at the average velocity, with
half-bandwidth 12% of the average. The distribution as seen by low-power microscopy is different: it
is skewed, showing a sharp drop off at the high-velocity end. The cine distribution is more symmetrical
and indicates by its spread that streaming is not a smooth process when viewed on a small time and
distance scale. We propose that organelles advance in a jerky fashion caused by brief cross-bridge
contractions which are randomly timed. Streaming would nevertheless appear smooth under low-power
as each organelle would undergo a larger number of contractions to cover the longer distances involved.
Velocity spread in the laser distribution, where measurements are made over distances the order of a
wavelength, cannot reasonably be attributed to Brownian motion.
Moving to the beat: a review of mammalian sperm motility regulation
Authors: Turner Regina M.
Published: December 2006

Because it is generally accepted that a high percentage of poorly motile or immotile sperm will adversely affect male fertility, analysis of sperm motility is a central part of the evaluation of male fertility. In spite of its importance to fertility, poor sperm motility remains only a description of a pathology whose underlying cause is typically poorly understood. The present review is designed to bring the clinician up to date with the most current understanding of the mechanisms that regulate sperm motility and to raise questions about how aberrations in these mechanisms could be the underlying causes of this pathology.
Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation
Authors: Malcuit Christopher , Maserati Marc , Takahashi Yoshiyuki , Page Raymond , Fissore Rafael A.
Published: December 2006

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.
Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction
Authors: Suarez S. S.
Published: December 2006

Artificial insemination with sexed semen, in vitro fertilisation and intracytoplasmic sperm injection have been used to reproduce animals, but often not as successfully as natural mating. Learning more about how spermatozoa normally interact with the female tract can provide inspiration for developing improvements in assisted reproduction. The present review focuses on Bos taurus, because more is known about this species than others. At coitus, bull spermatozoa are deposited into the anterior vagina, where they rapidly enter the cervix. Cervical mucus quickly filters out seminal plasma from spermatozoa, unlike most assisted reproduction protocols. Spermatozoa that reach the uterus may require certain cell surface proteins to swim through the uterotubal junction. Shortly after passing through the junction, most spermatozoa are trapped in a storage reservoir by binding to oviducal epithelium, in the case of cattle via bovine seminal plasma (BSP) proteins coating the sperm head. As ovulation approaches, spermatozoa capacitate and shed BSP proteins. This reduces sperm binding to the epithelium and releases them from storage. Motility hyperactivation assists spermatozoa in leaving the storage reservoir, swimming through oviducal mucus and the cumulus oophorus, and penetrating the oocyte zona pellucida. Chemotactically regulated switching between asymmetrical (i.e. hyperactivated) and symmetrical flagellar beating may also guide spermatozoa to the oocyte.
State of the art in farm animal sperm evaluation
Authors: Rodríguez-Martínez H.
Published: December 2006

Our ability to screen the structural and functional integrity of the spermatozoon in vitro has increased markedly over the past decades, but our capacity to estimate the fertility of a semen sample or of the sire from which it has been collected, especially in selected farm animal breeders, has not. The estimation of fertility is constrained by several factors (e.g. type of cell, analysis strength, sperm deposition strategies, recordings of fertility), including the fact that the ejaculate is composed of a diverse sperm population. Such cell heterogeneity is reflected not only in differences in the intactness of attributes needed for fertilisation, such as motility or morphology, but also in the relative ability of the spermatozoa to remain fertile over time, to sustain selection steps and responses to exogenous stimuli similar to those during sperm transport in the female genital tract, all of which account for innate variations in the fertilising ability among doses, ejaculates and sires. Determination of how large such a sperm population with competence for fertilisation and in-built ability to display these attributes under physiological signalling is would allow for a better estimation of fertility, provided that the particular sire produces this sub-population in a repeatable manner. The value of these analyses is discussed in the present paper.
Gene expression and maintenance of pregnancy in bovine: roles of trophoblastic binucleate cell-specific molecules
Authors: Hashizume Kazuyoshi , Ushizawa Koichi , Patel Osman V. , Kizaki Keiichiro , Imai Kei , Yamada Osamu , Nakano Haruo , Takahashi Toru
Published: December 2006

Cell to cell interaction plays a pivotal role in the regulation of placentogenesis and exchange of stage-specific developmental signals between the fetal and maternal units. Specifically, these interactions are paramount for programmed fetal growth, maternal adaptation to pregnancy and coordination of parturition. However, little is known about the precise regulation of placentation and maintenance of gestation in cattle. Therefore, the aim of the present study was to decipher the complex networks of cell communication to gain an insight into the multifaceted developmental process and understand the profound consequences of flawed communication. In the ruminant, the binucleate cell plays a central role in forming the structures and secretions at the fetomaternal interface that are crucial in establishing and maintaining pregnancy. Herein, we summarise differences in the abundance of specific RNA transcripts in the bovine cotyledon and caruncle using global gene expression profiling and further investigate the relationship of mRNA abundance for selected pregnancy-specific genes of interest (identified from microarray studies) that are localised exclusively to the binucleate cell, such as placental lactogen, prolactin-related proteins and pregnancy-associated glycoproteins. The results suggest that a well-orchestrated transcriptional command from binucleate cells is pivotal to the establishment and progression of pregnancy in cattle.
Temporal and spatial control of gene expression in early embryos of farm animals
Authors: Brevini Tiziana A. L. , Cillo Fabiana , Antonini Stefania , Tosetti Valentina , Gandolfi Fulvio
Published: December 2006

A gradual transition from oocyte-derived mRNA and proteins to full embryonic transcription characterises early embryonic development. Messenger RNAs and proteins of maternal origin are accumulated into the oocyte throughout its growth in the ovary. Upon fertilisation, several mechanisms are activated that control the appropriate use of such material and prepare for the synthesis of new products. The present review will describe some of the mechanisms active in early embryos of domestic species. Data will be presented on the control of gene expression by the 3′ untranslated regions and their interaction with specialised sequences at the 5′ cap end. The process of RNA sorting and localisation, initially described in different cell types and in oocytes of lower species, will also be discussed, particularly in relation to its possible role in regulating early pig development. Finally, specific genes involved in the activation of cattle embryonic transcription will be described. This brief overview will provide some suggestions on how these different mechanisms may be integrated and cooperate to ensure the correct initiation of embryonic development.
111 ESTIMATION OF SPERM QUALITY IN FRESH AND FROZEN - THAWED SEMEN FROM ASTURIANA DE LOS VALLES BULLS
Authors: Tamargo C. , Carbajo M. , Diez C. , Martin D. , Hidalgo C. O.
Published: December 2006

Artificial insemination and semen cryopreservation have significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality (Januskauskas et al. 1999 Theriogenology 52, 641-658), and surviving cells are affected post-thaw either structurally or functionally (Nagy et al. 2004 Anim. Reprod. Sci. 80, 225-235). In this work we analyze the impact of cryopreservation on Asturiana de los Valles bull sperm. Ejaculates (n = 373) from seven adult bulls were weekly collected by means of artificial vagina. Immediately after collection, routine parameters including volume (V), mass motility (MM), and concentration (C) of sperm cells were evaluated. Then the semen was extended with a commercial extender, loaded into 0.25-mL plastic straws at a concentration of 23 � 106 per straw, frozen and stored for further analysis. Four straws per ejaculate were thawed, pooled and analyzed for motion characteristics by means of a CASA system (Sperm Class Analyzer, SCA 2002� Microptic S. L., Barcelona, Spain) added to an optical phase-contrast microscope with heatable (37�C) stage. Immediately after thawing, we analyzed the % of motile spermatozoa (MS) and the % of progressively motile spermatozoa (PMS); then samples were incubated for 3 h at 37�C and MS and PMS were measured again (MS3 and PMS3, respectively). Functional integrity of the plasmallema was evaluated by the hypoosmotic swelling test (HOST) together with the % of typical tail coiling/swelling (percentage of HOST-positive spermatozoa, HOST-PS). The % of viable spermatozoa (VS) [membrane integrity was evaluated by fluorescence microscopy with a dual staining system (propidium iodide (PI) and 6-carboxyfluorescein diacetate (CFDA)]. Sperm showing partial or complete red fluorescence (PI staining) were considered nonviable, whereas sperm showing complete green fluorescence were considered viable. Altered acrosomes (AA) and morphological abnormalities were also determined. The % of morphological abnormalities was classified according to their location in head (HA), midpiece (MA), and tail (TA). Proximal and distal cytoplasmic droplets were counted as separate abnormalities (CD). Data were analyzed by the MEANS procedure of SAS (SAS Institute, Inc., Cary, NC, USA). A significant (P < 0.05) decrease in the sperm motility was observed after freezing/thawing (MS: 80.20 � 0.75 vs. 47.36 � 1.04, and PMS: 68.73 � 0.73 vs. 42.14 � 0.96 for fresh and frozen-thawed semen, respectively). Also, the frozen-thawed sperm showed increased % of HA, MA, AA, HOST-PS, and VS (P < 0.05). These morphological abnormalities could contribute to decreasing sperm motility. The new computer and video technologies provide useful information about sperm quality and can be used in the daily routine of processing semen.
This work was performed in collaboration with ASEAVA.
116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES
Authors: Yamada C. , Goissis M. D. , Caetano H. V. A. , Coutinho A. R. S. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2006

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes.

Table 1.
Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups

This work was supported by FAPESP 03/08543-1.
119 EARLY BOVINE GESTATIONAL DEVELOPMENT: MANIPULATED (IVF) AND NOT MANIPULATED IN LABORATORY
Authors: Assis Neto A. C. , Verechia F. T. , Ambrosio C. E. , Alberto M. L. V. , Meirelles F. V. , Garcia J. M. , Santos J. M. , Miglino M. A.
Published: December 2006

High embryonic and fetal death rates in manipulated embryos in laboratory suggest that the process of early placentation can be inefficient. This investigation aimed to evaluate the development of placenta, and organogenesis of Nelore bovine embryos, and fetuses by natural stud and in vitro fertilization (IVF) over the period from 15 to 70 days of pregnancy. Fifty-nine embryos (15 to 50 days of gestation), 9 fetuses in initial period (60 to 70 days of gestation), and 10 embryos originated by IVF technique (35 to 46 days of gestation) were used. The same semen was used for all IVF, except the embryos originated by natural stud. The embryos were prepared by serial sections and the fetal membranes were fixed in 4% paraformaldehyde for light and scanning electron microscopy (SEM) and 2.5% glutaraldehyde for transmission electron microscopy (TEM). All material was routinely processed and stained. The embryos and fetuses originating from natural stud showed an increase in growth based on the weight of the gestational sac, and related with cranio-caudal and dorso-ventral length of the chorioallantois and amniotic membrane in periods from 20 to 30 and from 50 to 60 days of the pregnancy. The gross appearance of the first cotyledons in development (9.39 � 0.73 cm) was quantified at 30 to 40 days of pregnancy. The IVF embryos on days 35 to 46 of gestation showed discreet cotyledons presenting a functional decrease of development related to embryo derived naturally. Ultrastructurally, the trophoblast showed binucleate trophoblast giant cells with a cytoplasm rich in electrodense vesicles and few mitochondria located in the apical poles suggesting lower cell activity. The yolk sacs of IVF embryos were shorter (1.07 � 0.55 cm) when compared with the natural group (5.53 � 3.14 cm) over 30 to 40 days of pregnancy. In both groups, the epithelium of the yolk sac presented cells with round nuclei, hemangioblast cells, and blood islands with a great number of primitive mononuclear cells. Embryos by natural stud (10% of analyzed ones) showed malformations in cephalic and frontal curvature, encephalocele, gastroschisis, and hepatomegaly. In conclusion, these results indicate occurrence of alteration in the organogenesis in bovine embryos originated by natural stud and retardation of fetal membrane development in bovine embryos by IVF.
This work was supported by FAPESP and CAPES.
137 IRON TRANSFER ACROSS THE LLAMA PLACENTA (LAMA GUANICOE GLAMA)
Authors: Miglino M. A. , Iturrizaga D. , Morini A. C. , Verechia F. T. , Kfoury Jr J. R.o , Monteiro J. M. , Bazer F. W.
Published: December 2006

The placenta of the llama has been described as epitheliochorial in type, but recent studies have not shown extensively the fetal nutrition aspects in this animal. In epitheliochorial placentation there is development of structures called areolae, as well as inter-microvillous attachment of the trophoblast, with irregular contact, to the uterine epithelium. This attachment is interrupted and the transfer of substances between the mother and the fetus takes place across the areolar cavity. These areolae appeared as small rounded or dome-shaped elevated areas of the chorioallantoic membrane over the narrow uterine gland openings. In order to detail their mechanisms of iron transfer in the llama placenta, we collected the samples of nine uteri between 28 to 36 weeks of pregnancy in association with fetal membranes. These samples were fixed in 4% paraformaldehyde in PBS, processed, and stained for light microscopy (HE, picrosirius, and Masson's trichrome), histochemistry (Perls, acid phosphatase, and PAS reactions) and immunohistochemistry with rabbit anti pig uteroferrin antibody to confirm the iron transfer, because the uteroferrin is an iron transporter and a progesterone-induced hematopoietic growth factor. The trophoblast formed a columnar-type single layer that was comprised of cells of various sizes and shapes with basal nuclei, including the giant binucleate cells. The trophoblast formed chorionic projections which presented ramifications in number from 4 to 5. A great quantity of blood vessels were found in the materno-fetal interface, between the cells of uterine epithelium and around of the chorionic projections. A PAS-positive reaction was observed with diffuse cytoplasmic PAS staining at the apical region of the trophoblast at the materno-fetal interface as well as in the endometrial glands. Collagen fibers were observed in the mesenchyme and inside the chorionic projections. In the areolae we confirmed the positive reaction of the acid phosphatase enzyme that detects phagocytic activity. In the basal region of the uterine gland epithelium, which is columnar type, and in the gland lumina, this reaction demonstrated a strong positive stain. The Perls histochemical reaction that reveals ferric iron was positive in the areola, as well as in the uterine glands. The uteroferrin immunohistochemistry showed a strong stained in the areolae and in the epithelium and lumina of the uterine glands. Our findings suggest that the areola region and the endometrial glands play an important role in histiotrophic nutrition in llamas, and in fetal red blood cell formation by iron transfer from mother to the fetus.
This work was supported by FAPESP, CNPq, CAPES, PRONEX, Brazil.
148 INDUCTION OF HYPOMETHYLATION BY EPIGENETIC ALTERATION OF SOMATIC NUCLEI IN CLONED BOVINE BLASTOCYSTS
Authors: Wee G. , Shim J.-J. , Song B.-S. , Kim J.-S. , Koo D.-B. , Lee K.-K. , Han Y.-M.
Published: December 2006

Epigenetic reprogramming such as DNA methylation is incomplete in cloned embryos during early development as compared with normal embryos. The increased methylation levels of cloned bovine blastocysts are showed in centromeric heterochromatin. The aim of the present study was to investigate the change of methylation state by treatment of trichostatin A (TSA), a specific inhibitor of histone deacetylase in somatic donor nuclei and cloned blastocyst reconstructed with TSA-treated cells or nontreated cells. Bovine ear skin fibroblast cells (bESF) were used as donor cell and treated with TSA for 60 h at a final concentration of 1 �M. To methylation analysis of satellite I as specific DNA sequence, genomic DNA from 7 � 104 cells and a blastocyst were isolated, and then the genomic DNA was analyzed by bisulfite sequencing. The reduction of HDAC1, 2 and Dnmt family such as Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L after TSA treatment were shown by Western blot in bESF, but histone acetyltransferases such as Tip60 and HAT1 were not changed. Satellite I DNA in nontreated cells was highly methylated in CpG sequences, whereas methylation level of TSA-treated cells was significantly decreased (64 vs. 48%, P < 0.05). After nuclear transfer using normal or altered donor cells, methylation levels of satellite were measured at the blastocyst stage of NT and TSA-NT embryos as compared with IVF embryos. In nontreated NT blastocysts, methylation levels were significantly higher than IVF blastocysts (66 vs. 29%, P < 0.05) and were similar to that of nontreated bESF cells. The reduction of methylation levels in TSA-NT blastocysts were showed and were significantly lower than NT blastocyst derived with nontreated cells (37 vs. 66%, P < 0.05), but no significant differences were found between TSA-NT and IVF blastocysts. Also, the levels of methylation were similar to that of TSA-treated donor cells. In blatocyst formation, TSA-NT embryos were improved significantly compared with NT or IVF embryos (45.9 vs. 31.7 or 28%, P < 0.05). These results demonstrated that somatic methylation status after epigenetic alteration affect in early cloned embryo development, suggesting epigenetic control may help to solve of inherent problems in cloning.
151 PROTEOMICS ANALYSIS OF PREGNANCY-SPECIFIC SERUM PROTEINS IN BOVINE
Authors: Jin D. I. , Lee H. R. , Kim H. R. , Lee H. J. , Yoon J. T. , Park C. S.
Published: December 2006

To identify early pregnancy-specific serum proteins in bovine, we performed proteomics analysis using blood serum samples of pregnant and non-pregnant Holstein dairy cattle Days 21 and 35 after AI. A total of eight pregnant and eight non-pregnant cattle were used for collection of the blood samples. The global proteomics approach was exploited by the use of 2-D gel electrophoresis and mass spectrometry to sort out pregnancy-specific proteins. Serum proteins within isoelectric point ranges of 4.0 to 7.0, 6.0 to 9.0, and 5.5 to 6.7 were analyzed separately by 2-D electrophoresis with three replications of each sample. The stained gels were scanned and calibrated at an optical resolution of 63.5 �m/pixel using a GS-710 imaging densitometer (Bio-Rad Laboratories, Philadelphia, PA, USA). A total of approximately 1200 spots were detected in 2-D gels stained with Coomassie-blue. In the comparison of serum samples from pregnant and non-pregnant cattle, nine pregnancy-specific spots were detected unanimously in Day 21 and Day 35 serum samples. Pregnancy-specific proteins were identified as transferrin, albumin, IgG2a heavy chain constant region, and immunoglobulin gamma heavy chain variable region by means of MALDI-TOF-MS (PerSeptive Biosystems, Framingham, MA, USA). Even though the identified spots were abundant serum proteins, their molecular weights and pI values were different from those of the main serum proteins. Most proteins identified in this analysis appeared to be related with pregnancy-specific subunits or fragments of transferrin, albumin, and IgG. One of the pregnancy-specific proteins, transferrin, is known to be related to iron transport during pregnancy. Western blot analysis using polyclonal anti-transferrin antibody revealed specific transferrin expression in the serum samples from the pregnant cattle but no detectable expression in the serum samples from the non-pregnant cattle. Our results revealed composite profiles of key proteins involved in early pregnancy and suggest the potential use of identified proteins to detect early pregnancy in bovine.
157 EFFECTS OF FETAL CALF SERUM OR PHENAZINE ETHOSULFATE AND FRUCTOSE OR GLUCOSE ON EMBRYONIC DEVELOPMENT AND LIPID ACCUMULATION OF BOVINE EMBRYOS
Authors: Barcelo-Fimbres M. , Seidel Jr G.
Published: December 2006

Slaughterhouse oocytes (n = 6222) were maturated in a chemically defined medium (CDM) similar to SOF plus 0.5% fatty acid-free BSA (FAF-BSA) and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Oocytes and frozen-thawed sperm, centrifuged through a Percoll gradient, were co-cultured for 18 h in F-CDM (CDM + heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 (CDM + nonessential amino acids, 10 μM EDTA, 0.5% FAF-BSA, and 0.5 mM fructose or glucose in Expt 1 and glucose in Expt 2). In both experiments, after 48 h, 8-cell embryos were cultured 135 h in CDM-1 (CDM-1 + essential amino acids, no EDTA, and 2 mM fructose or glucose). A factorial design with two hexoses and three additives in CDM-2 (control; 10% fetal calf serum (FCS); and 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH) was used for both experiments, each replicated eight times. For Expt 1, Day 7.5 blastocysts were fixed and stained with Sudan Black B to quantify cytoplasmic lipid droplets. A digital photo at 600× of the equatorial part of the embryo was evaluated by classifying lipophilic droplet diameters as small (S, <2 μm), medium (M, 2 to 6 μm), or large (L, >6 μm), reported as number of lipid droplets (LD) per 1000 μm2. Data were analyzed by ANOVA. For Expt 1, 8-cell embryo production per oocyte matured was not affected by fructose or glucose (P > 0.1) (70 vs. 68%, respectively); however, blastocyst rates per oocyte matured (B/O) and per 8-cell embryo (B/E) were higher (P < 0.01) for fructose than glucose (Table 1). There were no differences between control, PES, and FCS (P > 0.1) for B/O, or B/E. For Expt 2, B/O and B/E were higher (P < 0.01) for fructose than for glucose. No differences were found for additives (P > 0.1) control, FCS, or PES for B/O or B/E. There was an interaction (P < 0.05) between additives and hexoses for blastocyst production, because the benefit of fructose compared to glucose was greater for controls than for FCS or PES (means not presented). Accumulations of each size of LD were less for PES (P < 0.05) than for control and FCS. Control and PES were lower than FCS (P < 0.05) for S, M, and L droplets. There was no effect of fructose or glucose (P > 0.1) on numbers of S, M, or L droplets (Table 1). In conclusion, fructose produced more blastocysts than glucose after 8-cell development, but there was no hexose effect either before this stage or in lipid accumulation. PES reduced and FCS increased lipid accumulation relative to controls.

Table 1.
Main effects of additives and hexoses on development of bovine embryos (±SE)
158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS - OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS
Authors: Choi S.-H. , Cho S.-R. , Han M.-H. , Kim H.-J. , Choe C.-Y. , Son D.-S. , Kim Y.-K. , Chung Y.-G. , Hoshi H.
Published: December 2006

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 �g/mL apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 �g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 �g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 �g/mL and 10 �g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 �g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.
172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES
Authors: Ozawa M. , Nagai T. , Fahrudin M. , Karja N. W. K. , Kaneko H. , Noguchi J. , Ohnuma K. , Kikuchi K.
Published: December 2006

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H2O2 level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H2O2. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 �M GSH (29.6 � 2.7% or 30.4 � 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 � 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 � 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 � 2.1 to 49.2 � 2.1) and lowered ratios of apoptotic cells (6.2 � 0.6% to 7.0 � 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 � 2.0, apoptosis rate = 11.1 � 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.
174 DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER EXPOSURE TO TRYPLE™ EXPRESS (RECOMBINANT TRYPSIN)
Authors: Pryor J. H. , Bondioli K. R. , Wood S. K. , Givens M. D. , Looney C. R.
Published: December 2006

TrypLE™Express (1X; GIBCO, Grand Island, NY, USA) is a recombinant fungal trypsin-like protease that may provide an alternative to animal-origin trypsin for inactivation of embryo-associated virus. This experiment was designed as an embryo safety study to determine if different exposure times of TrypLE™Express on 7-day bovine in vitro-fertilized (IVF) embryos would inhibit normal embryonic development in vitro. Good quality oocytes were harvested from ovaries of slaughtered animals and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). Oocytes were fertilized (Day 0) with frozen-thawed, Percoll-separated Brangus spermatozoa in TALP-fertilization medium for 18 h. Presumptive zygotes were cultured in 0.5 mL of G1.3/G2.3 medium (Lane et al. 2003 Theriogenology 60, 407) supplemented with 8 mg/mL of Pentex BSA (Serologicals Corporation, Norcorss, GA, USA) under oil for 3 days in a 5% CO2, 5% O2, 90% N2 humidified modular incubator (Billups-Rothenberg, Inc., Del Mar, CA, USA) at 38.5°C. On Day 4, embryos were washed and moved to new culture wells containing G2.3 medium. Cleavage rates of 74% (750/1014) from three replicates produced 322 viable embryos on Day 7 which were then evenly distributed by grade and stage into three treatment groups (1-, 5-, and 10-minute exposure times to TrypLE™Express). Embryos were washed in accordance with International Embryo Transfer Society standards (Stringfellow, IETS Manual, 1998, 79–84) substituting TrypLE™Express with phenol red for the porcine-origin trypsin. Washed embryos were cultured in G2.3 medium with hatched rates assessed at 24, 48, and 72 h. Hatched embryos were fixed and stained using a modified Hoechst method (Damiani et al. 1996 Mol. Reprod. Dev. 45, 521–534) to count cells. The results are presented in Table 1. There were no significant differences (P < 0.05, chi-square analysis) between treatment groups for hatched rates or average cell counts. The results indicate that exposure to TrypLE™Express did not inhibit normal embryonic development. The efficacy of TrypLE™Express to remove or inactivate certain embryo-associated viruses is being investigated. Establishing an appropriate protocol for treatment of bovine embryos with TrypLE™Express will depend on the safety and efficacy of exposing embryos to this recombinant protease.

Table 1.
Comparison of hatched IVF bovine embryo rates and average cell counts after exposure to TrypLE™Express

This work was funded by Invitrogen Corporation.