bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structure of the Decalone Obtained From the Oxidation of Guaiol
Authors: Carman RM , Garner AC , Robinson WT
Published: December 2006

The structure of the decalone previously reported from the oxidation of guaiol has, after X-ray crystallographic analysis, been revised to structure (3c) which contains a cis -fused ring system. Deuteration studies give insight into the conformation of the transition state during the reaction.
Quinquedentate Coordination of 10-Methyl-1,4,8,12-tetraazacyclopentadecan-10-amine (L) to Rhodium(III). Crystal Structure of cis-[Rh(L)Cl][ClO4]2.H2O And Comparative Activation Volumes for Base Hydrolysis of [M(L)Cl]2+ (M = Rh, Co, Cr)
Authors: Lawrance GA , Martinez M , Skelton BW , Vaneldik R , White AH
Published: December 2006

The pendant-arm macrocycle 10-methyl-1,4,8,12-tetraazacyclopentadecan-10-amine (1) reacts slowly in hot water with rhodium(III) chloride to yield, following cation-exchange chromatography, exclusively cis -[ Rh (l) Cl ]2+.
The cis -complex was crystallized readily as a perchlorate monohydrate
salt, in the orthorhombic space group Pn21a, a 16.854(3), b
13.341(3), c 9.985(1) � , Z = 4, isomorphous with its cobalt(III) counterpart; a single-crystal X-ray structure determination was refined to R 0.027 for 1626 'observed' reflections. The pendant primary arnine and two adjacent secondary amines necessarily occupy an octahedral face, with the chloro
ligand cis to the primary amine, and secondary amines adopt R,R,S,S
stereochemistries. The Rh -N distances [2.056(6) � (pendant primary amine), average 2.08 � (secondary amines)], are at the short end of the range for Rh-N bonds. The Rh-Cl distance is 2.347(2) �. Activation volumes for chloride base hydrolysis were determined for cis -[ Rh (l) Cl ]2+as +19.5( � 1.2), for cis -[Co(l) Cl ]2+ as +27.1(�0.4), and for trans-[Cr(l) Cl ]2+ as +31.1( �0.1) cm3 mol-l, and are consistent with a conjugate base mechanism in each case; variations with metal ion are discussed.
Comparison of Peptide Fragmentation With Different Ionization Techniques: Variation With Sample Preparation Method of the Fragmentation of Luteinizing Hormone-Releasing Hormone Following Plasma Desorption
Authors: Craig AG , Bennich H , Derrick PJ
Published: December 2006

The fragmentation patterns of the peptide luteinizing hormone-releasing hormone (LH-RH) measured in plasma desorption mass spectrometry (PDMS) and keV-atom bombardment have been compared with the field desorption (FD) mass spectrum. Several different methods of preparing the samples for PDMS have been investigated, including application to a nitrocellulose surface which was preadsorbed with a protein. Similar fragment ions were observed in keV-atom bombardment and in PDMS applied to nitrocellulose which had not been preadsorbed with a protein; different fragment ions have been observed in FD mass spectrometry. The PDMS fragmentation when LH-RH was applied to nitrocellulose which had been preadsorbed with thioredoxin was found to differ yet again. The differences in the fragmentation are rationalized on the basis of differences in conformational constraints.
Synthesis of the Fungicides 1-[3-(Para-Tert-Butylphenyl)-2-Methylpropyl]Thiolanium Perchlorate and 1-[3-(Para-Tert-Butylphenyl)-2-Methylpropyl]Thianium Perchlorate
Authors: Wilkie JS , Winzenberg KN
Published: December 2006

Reaction of 4-[3-(p-t-butylpheny1)-2-methylpropylthio]butan-1-ol(3e) and 5-[3-(p-t-butyl-pheny1)-2-methylpropylthio]pentan-1-ol (3f), each prepared from p-t- butylbenzoic acid, with thionyl chloride followed by treatment with silver perchlorate afforded 1-[3-(p-t-butylphenyl)-2-methylpropyl ] thiolanium perchlorate (4b) and 1-[3-(p-t-butylpheny1)-2-methylpropyl] thianium perchlorate (4d), respectively.
Poor incorporation of lime limits grain yield response in wheat
Authors: Scott B. J. , Coombes N. E.
Published: December 2006

Thorough mixing of lime with the soil is a standard recommendation for lime application. However, the implements and passes that may be used to achieve this in Australian cereal farming are unclear. Therefore, 2 experiments were conducted to examine the incorporation of lime applied at 0, 2 and 5 t/ha using a range of different agricultural implements and numbers of cultivation events. Shoot dry matter and grain yield of wheat were measured in the year of lime application in both experiments. The plots were resown to wheat in the following season by direct drilling, and measurements were repeated. In a dry season, high soil disturbance (rotary hoe and disc harrow) improved the response of wheat to lime in the first year of experiment 1. In experiment 2, rainfall was higher, and the advantage from thorough incorporation was less clear. However, the rank order of incorporation methods and lime responsiveness was positively correlated with that in experiment 1 for both dry matter and grain yield; thorough incorporation tended to give better responses to lime than ‘poor’ incorporation (light harrowing). In the second year of experiment 1 there was limited evidence of the influence of incorporation method on lime response. In the second season of both experiments the effects of incorporation method on lime response had dissipated or other effects were more important. We found that to maximise grain yield responses to lime, the most effective incorporation was achieved with a disc harrow or with multiple passes with a tined implement (scarifier). Incorporation limited to a light harrow was inadequate. However, any effects of method of incorporation reduced or disappeared in the following season, even when direct drilling was used and there was limited further soil disturbance.
Early growth of wheat is more sensitive to salinity than boron at levels encountered in alkaline soils of south-eastern Australia
Authors: Nuttall J. G. , Armstrong R. D. , Connor D. J.
Published: December 2006

The early vegetative growth of 3 wheat (Triticum aestivum L.) cultivars, Frame, BT Schomburgk and Schomburgk, was evaluated over a range of soil salinity and soluble boron (B) concentrations in pots. Additions of boric acid and mixed salts to a sandy clay soil produced extractable B levels of 2, 13, 24, 51 and 129 mg/kg and salinities (ECe) of 0.8, 8, 15, 23 and 29 dS/m. In both cases, the levels produced in the first 4 treatments corresponded well with those commonly observed in subsoils of the southern Mallee. Within the ranges tested, wheat cultivars had relatively greater tolerance to B toxicity than to salinity. Significant differences in tolerance also existed among the 3 cultivars for B, but not for mixed salts. For Frame, BT Schomburgk and Schomburgk, critical concentrations of soil soluble B were estimated at 53, 32 and 27 mg/kg, respectively, in the absence of salinity. For salinity tolerance, the 3 wheat cultivars could all tolerate an ECe up to about 9 dS/m equally well. In combination with B, salinity still dictated overall response in growth with the interactive effect of B being to increase sensitivity of plants at low levels of salt. Shoot B concentrations in Frame ranged from 15 to 947 mg/kg for increasing soil B treatments but these responses did not correlate well with growth reduction. Shoot Na contents ranged from 0.02 to 0.58%, but was not a reliable indicator of Na+ toxicity due to interactive effects of B: increasing B reduced Na+ uptake. Generally, differences in B tolerance among the cultivars highlighted the existence of genetic variation in adaptation of wheat to high levels of soil B; however, this does not appear to be the case for salt tolerance in wheat. Because high levels of B and salt usually co-exist in the field, plant tolerance to these limitations need to exist in combination.
Impact of annual legume 'break' crops on the yield and quality of canola in comparison with the impact on yield of wheat
Authors: Evans J. , Murray G. M. , Scott G. , Orchard B. , Brennan J. , Lemerle D. , Kaiser A. , Armstrong E. L.
Published: December 2006

This study investigated the yield, oil and protein content of canola in response to alternative single-season legume crops, and compared the yield response with that of wheat. Two field trials were conducted in consecutive years at Wagga Wagga on the South West Slopes of New South Wales. The soil type was Red Kandasol. The legume treatments were field pea and vetch managed for silage production, vetch managed for green manure, a mixture of aerial seeding clovers (Berseem, Arrowleaf, Persian: 6 : 3 : 3) managed for silage, hay or green manure, and field pea and narrow-leaf lupin managed for grain. There was one wheat treatment managed for grain. In terms of growing season rainfall, the rainfall-use efficiency of canola was low and similar to that achieved on farms (5.1 kg/ha.mm rainfall). Yields of canola were less than potential water-limited yields and the canola equivalent wheat yields were less than comparative wheat yields. The constraint(s) on canola yield meant that nitrogen-limited yield potential was not reached, and in contrast to wheat in adjacent plots, there was no relationship between canola seed yield and soil mineral nitrogen available during seedling establishment of canola. However, relative to other legume treatments, antecedent crops of the clover mix depressed yield and total oil content, particularly in one year. It was concluded that under field conditions that constrained canola productivity to that similar to on-farm productivity, wheat may make more efficient use of legume nitrogen than canola. Were canola to be used as the response crop, antecedent legumes of vetch or pea, rather than aerial seeding clover, would give higher potential yield and total oil content of the canola.
Induction by EDTA of Anthocyanin Synthesis in Spiroldela oligorrhiza
Authors: Elliott D.C
Published: December 2006

EDTA greatly increased anthocyanin synthesis in S. oligorrhiza, the effect being much greater in light than in dark. The effect was not given by ethylene glycol bis(�-aminoethyl ether)- N-N'-tetraacetic acid, possibly because of the different stability constants of the two chelating agents.
Benzyladenine increased anthocyanin levels in the absence of EDTA, being more effective in the dark, but it did not increase the amount accumulated in the presence of EDTA.
Manganese chloride abolished the EDTA effect.
The EDTA effect on anthocyanin synthesis was usually accompanied by a decrease in chlorophyll content but not always. The effect on anthocyanin preceded that on chlorophyll.
Partial Tryptic Digestion of Cucurbitin from Pumpkin Seed
Authors: Blagrove R.J , Lilley G.G , Kortt A.A
Published: December 2006

A stable digestion intermediate was observed when cucurbitin, the crystalline storage globulin of
pumpkin [Cucurbita moschata (Duch.) Poir.] seed, was treated with trypsin. The major digestion
product, termed cucurbitin-T, was isolated as a homogeneous protein of M, 285 000+ 10000 by
chromatography over Sepharose CL-6B. The treatment with trypsin lowered the isoelectric point,
decreased the charge heterogeneity, and increased the solubility of cucurbitin. Electrophoresis of
denatured and reduced cucurbitin-T in 8 M urea at pH 8.5 suggested that the basic polypeptide
chains of cucurbitin were degraded less by the enzyme treatment than were the larger acidic chains.
This difference in susceptibility was confirmed by two-dimensional electrophoresis. The amino acid
analyses of cucurbitin and cucurbitin-T subunit polypeptides are consistent with the changes demonstrated
by electrophoresis. It is suggested that the larger fragments (M, 20000-24000) of the
acidic chains found in cucurbitin-T, together with the more resistant basic chains (M, - 22000) in
both forms of cucurbitin, may constitute the 12 equivalent structural domains observed for cucurbitin
by X-ray diffraction analysis.
Inhibition by Proton Buffers of Photosynthetic Utilization of Bicarbonate in Chara corallina
Authors: Price G.D , Badger M.R
Published: December 2006

A simple model was developed to explain the mechanism by which buffers inhibit HCO*3� utilization in Chara corallina. The chief assumption was that an acidic compartment exists in the peiiplasmic space and that exogenous buffer species must cross the apoplast to dissipate this H+ gradient (�pH). Assuming that HCO*3� utilization depends on H+ extrusion, buffer-induced H+ dissipation should result in a reduction in inorganic carbon (C*i) limited photosynthesis. The model predicts that buffers with a pKa midway between the bulk phase pH (pHo) and an assumed periplasmic pH will be most effective in dissipating the periplasmic �pH. Experiments conducted at pHo = 9.3 and with 5 mM concentrations of various buffers show that buffers in the range pKa 7.5-9.0 are most inhibitory to HCO*3� utilization. The initial slope of the photosynthetic response to C*i (i.e. where C*i availability is rate limiting) is more sensitive to buffers than rates at high C*i levels. Buffer inhibition was reversible. Experimental data correspond well with the model and indicate that HCO*3� utilization sites in the periplast are considerably more acidic than the bulk phase during photosynthetic HCO*3� utilization. Results suggest that during buffer inhibition experiments �pH is around 2 or more units when the bulk phase pH is 9.3.
Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm
Authors: Gardner Allison J. , Evans Janice P.
Published: December 2006

To inhibit fertilisation by more than one sperm (a condition known as polyspermy), eggs have developed preventative mechanisms known as blocks to polyspermy. The block at the level of the egg extracellular coat (the zona pellucida in mammals, the vitelline envelope in non-mammals) has been well characterised in many different animal species and the block at the level of the egg plasma membrane is understood in some non-mammalian species. However, virtually nothing is known about the membrane block to polyspermy in mammalian eggs, despite data dating back 50–90 years that provide evidence for its existence. In the present review, we will discuss the background on blocks to polyspermy used by animal eggs and then focus on the membrane block to polyspermy in mammalian eggs. This will include a summary of classical studies that provide evidence for this block in mammalian eggs, assays used to study the mammalian membrane block and what has been elucidated from recent experimental studies about the cellular signalling events that lead to membrane block establishment and the mechanism of how the membrane block may prevent additional fertilisation.
Equine cloning: applications and outcomes
Authors: Vanderwall Dirk K. , Woods Gordon L. , Roser Janet F. , Schlafer Donald H. , Sellon Debra C. , Tester David F. , White Kenneth L.
Published: December 2006

Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalski’s horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment. Although equine cloning has been successful, like other species, it remains a very inefficient process (<3% success). In most species, the inefficiency of cloning results from a high incidence of embryonic, fetal and/or placental developmental abnormalities that contribute to extremely high rates of embryonic loss, abortion and stillbirths throughout gestation and compromised neonatal health after birth. The present review describes some of the ultrasonographic, endocrinological and histopathological characteristics of successful (produced viable offspring) and unsuccessful (resulted in pregnancy failure) cloned equine (mule and horse) pregnancies we have produced. A total of 21 cloned mule pregnancies were established using fetal fibroblast cells, whereas a total of seven cloned horse pregnancies were established using adult cumulus cells. Three of the cloned mule conceptuses were carried to term, resulting in the birth of three healthy clones. This information adds to an accumulating body of knowledge about the outcome of cloned equine pregnancies, which will help to establish when, and perhaps why, many cloned equine pregnancies fail.
Evaluation of oocyte quality: morphological, cellular and molecular predictors
Authors: Wang Qiang , Sun Qing-Yuan
Published: December 2006

Mounting evidence that oocyte quality profoundly affects fertilisation and subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus–oocyte complex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase l activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-β superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.
104 COMPARING DIFFERENT LEVELS OF OSMOLALITY OF SUCROSE EXTENDER ON THE VIABILITY OF SPERMATOZOA IN BACTRIAN CAMEL (CAMELUS BACTRIANUS)
Authors: Mosaferi S. , Niasari-Naslaji A. , Bahmani N. , Gharahdaghi A. A. , Abarghani A. , Ghanbari A. , Gerami A.
Published: December 2006

Disaccharides have been used as an extender for dromedary camel semen (Bravo et al. 2000 Anim. Reprod. Sci. 62, 173-193). More recently we have investigated the effect of different concentrations of lactose extender on the viability of Bactrian camel spermatozoa (Mosafer et al. 2005 Reprod. Fertil. Dev. 17, 197). Considering the osmolality (316.1 � 1.48 mOsm/kg) and pH (7.4 � 0.03) of Bactrian camel semen (Mosaferi et al. 2005 Theriogenology 63, 92-101), the objective of this study was to investigate the effect of osmolality of sucrose extender on the viability of Bactrian camel spermatozoa. Sucrose at the concentrations of 9, 10, 11, 12, and 13% with osmolalities of 292, 331, 356, 386, and 410 mOsm/kg, respectively, were prepared. All extenders contained 20% egg yolk and antibiotics, with pH adjusted to 6.9. Semen was collected from camels with a sound history of semen quality and fertility (n = 3) using a modified artificial vagina and divided into different treatments after mechanical reduction of semen viscosity (3). Progressive forward motility of spermatozoa was examined at the time of semen collection and at 4, 12, and 24 h after incubation at 4�C. Data were analyzed using the GLM procedure in SAS/STAT after arcsin transformation. At the time of semen dilution, the progressive forward motility of spermatozoa was greater at osmolality of 331 (23%) compared with 292 (1%), 386 (6%), and 410 (3.5%) mOsm/kg (P < 0.05). No progressive forward motility of spermatozoa was noticed after 4 h incubation at 4�C at osmolalities of 292, 386, and 410 mOsm/kg. At this time, a significant decrease (P < 0.05) of progressive forward motility occurred at osmolalities of 331 (4%) and 356 (0.5%) compared with that of the time of dilution. After 12 and 24 h incubation at 4�C, no progressive forward motility of spermatozoa was detected in any of these extenders. In conclusion, 10% sucrose (331 mOsm/kg) at the adjusted pH of 6.9 was the most suitable concentration of this disaccharide for preserving Bactrian camel semen for less than 4 h under chilled conditions.
110 VITRIFICATION DEVICES AFFECT DEVELOPMENTAL COMPETENCE AND BIOCHEMICAL PROPERTIES OF IVM OVINE OOCYTES
Authors: Succu S. , Leoni G. G. , Berlinguer F. , Mossa F. , Galioto M. , Naitana S.
Published: December 2006

This study was designed to evaluate the effects of 3 different vitrification devices on the developmental ability and on the maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of ovine oocytes in vitro-matured. Cumulus-oocytes complex (COCs) were in vitro-matured in TCM-199 supplemented with 10% FCS, 10 �L/mL of FSH/LH, and 1 �g/mL estradiol at 39�C and 5% CO2 atmosphere. For vitrification, oocytes were incubated in HEPES-buffered TCM-199 containing 20% FCS supplemented with DMSO (7.5%) and ethylene glycol (7.5%). After 3 min, oocytes were loaded into the same medium containing 0.5 m sucrose, 16.5% DMSO, and 16.5% EG, and then immersed into N2 using open pull straw (OPS; Vajta et al. 1998 Mol. Reprod. Dev.), cryoloop (CL; Lane et al. 1999 Nat. Biotechnol.), or cryotop (CT; Kuwayama and Kato 2000 J. Assist. Reprod. Genet.). After warming a part of oocytes were fertilized and cultured in vitro up to blastocyst stage in standard conditions (Leoni et al. 2005 Anim. Reprod. Sci., in press). The fertilization (23.8%, 31.6%, and 36.8% vs. 61.5%) and blastocyst rates (0, 12.5, and 0% vs. 50.0%) were lower for oocytes vitrified in OPS, CL, and CT, compared with control group. Control and vitrified IVM oocytes were valued for MPF and MAPK activity at 0 and 2 h after warming. In both post-warming experimental groups, the MAPK activity did not differ from control group. Immediately post-warming, MPF activity was lower in the vitrified groups compared with control oocytes (P < 0.01). If 100% is assigned to MPF activity in the control oocytes, those in the OPS, CL, and CT groups were, respectively, 63.3%, 65.5%, and 26.2%. After warming and culture for 2 h in standard condition, the activity of MPF was restored to values similar to control oocytes (87.0 and 76.8%, respectively) in the OPS and CL groups, whereas it was at the similar value in the CT group. To evaluate if the lowered MPF activity could cause parthenogenetic activation, the vitrified-warmed oocytes were cultured in SOF + 2% oestrus sheep serum in 5% O2, 5% CO2; after 27 h of culture, the oocytes were fixed and stained with propidium iodide to evaluate chromatin configuration. Results showed significantly higher parthenogenetic activation rates in the CT group compared with OPS and CL groups (54.5% vs. 22.6 and 27.4%, respectively). Our results indicate that the success of cryopreservation of the ovine oocyte is still very limited. The use of different vitrification devices not only modifies the ability to survive cryopreservation and developmental competence of oocytes but is also associated with important molecular alterations in the warmed oocyte cytoplasm.
This work was supported by Cofin 2003.
115 SPERM CRYOPRESERVATION IN TRAGELAPHINE ANTELOPES
Authors: Wirtu G. , Pope C. E. , Cole A. , Godke R. A. , Paccamonti D. L. , Dresser B. L.
Published: December 2006

As an integral aspect of our program to develop assisted reproductive technologies in tragelaphine antelopes, we have been evaluating the effects of various extenders on cryosurvival of spermatozoa. Semen was collected by electroejaculation (EEJ) from an eland (Taurotragus oryx, n = 14 EEJs) and a bongo (Tragelaphus euryceros, n = 7 EEJs) male during sedation and standing restraint in a hydraulic chute. Epididymal spermatozoa were collected from the proximal vas deferens, and distal epididymides recovered from four common eland bulls after elective castration at the Hattiesburg Zoo, Mississippi (age = 46 months) and the San Diego Wild Animal Park, California (ages = 18, 20, and 23 months). Testes from a bongo (age = 6.5 yr) and a greater kudu bull (Tragelaphus strepsiceros, age = 7.5 yr) at the Audubon Nature Institute were recovered and processed immediately postmortem. Kudu, bongo, and the Hattiesburg eland testes were transported at ambient temperature whereas the testes from San Diego elands were shipped overnight in a cold-pack container. Sperm processing was done at room temperature (RT = 23°C) in HEPES-buffered Tyrode's solution. Four extenders containing 7% glycerol were evaluated: Biladyl®, TEST Yolk buffer (TYB), Beltsville extender (BF5F), and skim milk. For freezing, sperm samples were initially extended and gradually cooled to 4°C before sequential addition of extender containing glycerol at 4°C; the procedure was then modified by addition of the glycerol fraction at RT before the sample was cooled to 4°C. Spermatozoa were vapor-frozen in 0.5 mL straws placed 5 cm above liquid nitrogen before storage at −196°C. Straws were thawed in a water bath (38°C, 30 s) to evaluate cryosurvival. All eland and bongo electroejaculation procedures produced spermatozoa, although sperm quality varied. Ejaculate volume averaged 5.4 ± 1.2 mL and 3.7 ± 1.1 mL in the bongo and the eland, respectively. Sperm motility at collection, after cooling, and after freezing in the different extenders is presented in Table 1. No immediate decline in sperm motility was observed after adding glycerol to samples, whether at 23°C or 4°C. Bongo spermatozoa lost more motility during freezing than during cooling (15.0 ± 4.4 vs. 5.8 ± 2.0%; P = 0.006); whereas, in eland, motility loss during cooling and freezing was similar (25.3 ± 16.2 vs. 21.1 ± 7.7; P = 0.44). The present results indicate that cooling and freezing tolerance of spermatozoa in tragelaphine antelopes is influenced by species, extender type, and temperature at which a cryoprotectant is added.

Table 1.
Cryosurvival of ejaculated or epididymal spermatozoa of three antelope species in different extenders
124 KNOCKING-IN OF A FOREIGN GENE ON A BOVINE BETA-CASEIN GENE INTO BOVINE PRIMARY FIBROBLASTS USING HOMOLOGOUS RECOMBINATION EVENTS
Authors: Chang M. , Oh K.-B. , Park J.-S. , Koo D.-B. , Shin S.-T. , Lee K.-K. , Han Y.-M.
Published: December 2006

Knocking-in of a foreign gene on a tissue-specific genomic region has not been reported in livestock. In this study, we constructed two knock-in vector cassettes specific for the bovine beta-casein gene which is expressed only in the mammary gland during lactation. The targeting vector cassettes, pBCKI1 and pBCKI2, have homology regions for the bovine beta-casein gene and contain 13.1 kb and 9.1 kb targeting arms with different long arm lengths, respectively. The targeting vector cassettes have unique restriction enzyme sites for insertion of foreign therapeutic genes in front of the neo gene which was inserted into the vector as a selection marker. The human thrombopoietin (hTPO) gene was inserted into the restriction enzyme sites of both targeting vectors, which were named pBCTPOKI1 and pBCTPOKI2. When the two targeting vectors were transfected into bovine ear skin fibroblasts using Lipofectamine (Invitrogen, Seoul, South Korea), 6.3% of neo resistant clones (2/32) were homologously targeted with the pBCTPOKI2 vector. Cells from the targeted colonies were nuclear-transferred into enucleated bovine oocytes and cultured to the blastocyst stage. A total of 66 blastocysts were generated of which 46 were transferred into recipients. No offspring have been produced at this time. Here we first describe knock-in vectors specific for a bovine beta-casein gene, which will be employed to generate animal bioreactors that produce therapeutic proteins secreted into the milk.
128 MITOCHONDRIAL DNA DELETIONS IN RHESUS MACAQUE OOCYTES, EMBRYOS, AND ADULT AND EMBRYONIC STEM CELLS
Authors: Gibson T. , Quebedeaux T. , Rajasekaran S. , Brenner C.
Published: December 2006

Mitochondria are the most abundant organelles in mammalian oocytes and early embryos. Previous data have shown that mitochondrial DNA (mtDNA) deletions are present both in human oocytes and in embryos from in vitro fertilization (IVF) patients and suggest that accumulation of these deletions may contribute to mitochondrial dysfunction and impaired ATP production. In addition, high levels of mitochondrial mutations are present in skeletal muscle fibers from aged rhesus macaques. The specific aims of this study were to determine whether the mitochondrial common deletion is present in non-human primate oocytes and embryos generated by IVF and to determine whether mtDNA mutations are already present in immature oocytes from rhesus ovaries. Using a nested primer polymerase chain reaction (PCR) strategy, we determined the frequency of the rhesus common deletion in immature oocytes compared with stimulated oocytes and embryos. There was a low incidence (21%) of the rhesus common deletion present in immature, unstimulated oocytes derived from necropsied ovaries of 2 to 10-yr-old rhesus macaques. However, there was >3-fold increase (71.4%) in the frequency of deleted mtDNA in stimulated oocytes and IVF embryos from age-matched fertile monkeys. We postulated that, in addition to skeletal muscle, a similar time-dependent accumulation of mtDNA deletions occurs in fertile rhesus macaque oocytes and embryos. We are now investigating the effects of culture and passage number on mtDNA deletions in primate adult and embryonic stem cells. We propose the rhesus monkey to be an excellent model to assess the quality of gametes and embryos, as well as stem cells, and their developmental competence in human and non-human primates.
This study was supported by National Institutes of Health grants RR15395 and HD045966.
143 PRESENCE OF PROSTAGLANDIN F2α RECEPTOR IN IN VITRO-DERIVED MORULA AND BLASTOCYST STAGE BOVINE EMBRYOS
Authors: Scenna F. N. , Edwards J. L. , Pighetti G. M. , Schrick F. N.
Published: December 2006

Culture of in vitro and in vivo-derived embryos in medium containing prostaglandin F2� (PGF) decreased embryonic development to blastocyst stage and reduced hatching rates (Scenna et al. Prostaglandins 73, 215-226). Moreover, administration of an inhibitor of PGF synthesis at the time of embryo transfer in bovine recipients improved pregnancy rates (Schrick et al. 2001 Theriogenology 59, 335 abstr.). These findings indicate a direct negative effect of PGF on embryonic development. However, to our knowledge, no evidence of PGF receptor expression in morula or blastocyst stage bovine embryos is available in the literature. Therefore, the objective of the current study was to determine the presence of PGF receptor mRNA using real-time RT-PCR and protein expression by Western blotting in morula or blastocyst stage in vitro bovine embryos. Briefly, isolated total RNA from compact morula or blastocyst stage embryos and from bovine tongue epithelium (positive control for PGF receptor mRNA) were reverse-transcribed into cDNA. A volume from the RT reaction equivalent to 10 embryos per tube was utilized to determine transcripts for PGF receptor and Histone H2A (standard PCR control). Polymerase chain reaction was performed, and identity of PCR fragments was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis and by DNA sequencing. To determine protein expression, morula and blastocyst stage embryos were lysed in lysis buffer (10% SDS, 1 m Tris pH 7.5, 1 m NaF, 1 m DTT, 0.1 m EGTA with protease inhibitors) and stored at -20�C. Crude proteins isolated from bovine corpora lutea (positive control for PGF receptor protein), embryo samples, and prestained standards were separated by 12% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto a PVDF membrane. Nonspecific binding sites in the PVDF membrane were blocked with 10% nonfat dry milk, and the blot was washed and incubated for 1 h at room temperature with a 1:1000 dilution of the primary antibody (rabbit polyclonal antibody against PGF receptor protein). Subsequently, the blot was washed and incubated for 1 h at room temperature with 1:1000 dilution of mouse anti-rabbit IgG conjugated with horseradish peroxidase. Finally, the blot was washed and revealed by chemiluminescence in a CCD camera. Results indicated that transcripts as well as the protein for PGF receptor were present in early stage bovine embryos. Identification of PGF receptor in morula and blastocyst stage bovine embryos may, in part, explain the increase in pregnancy rates after administration of a PGF synthesis inhibitor at the time of embryo transfer, which opens up the possibility to develop new strategies to prevent detrimental effects of PGF during early embryonic development.
154 EFFECTS OF THE BREED AND SEASON ON THE FERTILITY OF TROPICAL DUAL PURPOSE COWS
Authors: Nava-Trujillo H. , Hernández A. , Hernández-Fonseca H. , Soto-Belloso E. , Perea-Ganchou F.
Published: December 2006

First service fertility is an important factor affecting the calving-conception interval and the number of services per conception. In the present study, first service fertility of 618 multiparous dual purpose cows was analyzed according to breed predominance (Bos indicus and Bos taurus) and service season: dry (December-March); sub-humid (April-August); and humid season (September-November). All cows were located in a sub-humid tropical forest area, characterized by 1950 mm/year of rainfall and a mean daily temperature of 28.3�C. Cows were milked and suckled twice a day. Estrus was detected twice a day with a minimum observation period of 30 min. All cows were serviced according to the international rule AM-PM with semen of one of two Brahman bulls (A and B). All cows received water and mineral salt ad libitum during the entire year. Additionally, during the dry season cows received an energy supplementation. Data were analyzed using the chi-square procedure of SAS (SAS/STAT User's Guide, 8.2 ed. Cary, NC: SAS Institute, Inc., 2001). No bull effects were observed on first service fertility (bull A: 37.70%, 105/305; and bull B: 40.26%, 126/313; P > 0.05). Breed predominance significantly affected the first service fertility, being higher in Bos indicus cows (47.48%) than in Bos taurus cows (32.78%; P < 0.05). The higher first service fertility of Bos indicus cows seen during the year was probably because Bos indicus cattle are more thermotolerant than Bos taurus cattle (Hansen 2004 Anim. Reprod. Sci. 82-83, 349-360). Moreover, season of service did not affect the first service fertility of Bos indicus cows (dry: 48.95%, 70/143; sub-humid: 50%, 33/66; and humid: 41.30%, 19/46; P > 0.05). While in Bos taurus cows, first service fertility was higher in cows serviced during the dry season (43.24%) in comparison with those serviced during the sub-humid (28.26%; P < 0.05) or humid season (23.58%; P < 0.05). No differences were observed between Bos taurus cows serviced in the sub-humid and the humid season (P > 0.05). First service fertility did not differ between Bos indicus and Bos taurus cows serviced during the dry season (P > 0.05), which is likely due to the lower relative humidity and extra energy supplementation. This may improve oocyte quality. In conclusion, fertility of Bos taurus cattle is more sensitive than that of Bos indicus cattle under tropical conditions; therefore, the implementation of a reproductive seasonality scheme is recommended to increase the reproductive efficiency of Bos taurus cattle.
16 OVULATION AND PREGNANCY RATES IN POSTPARTUM BOS INDICUS COWS TREATED WITH PROGESTERONE VAGINAL DEVICES AND ESTRADIOL BENZOATE, WITH OR WITHOUT eCG AND TEMPORARY WEANING
Authors: Maraña D. , Cutaia L. , Peres L. , Pincinato D. , Borges L. F. K. , Bo G. A.
Published: December 2006

Two experiments were designed to compare the effects of eCG treatment and temporary weaning on ovulation and pregnancy rates in postpartum cows. In Experiment 1, 39 lactating multiparous crossbred Bos indicus cows, 60 to 80 d postpartum with a body condition score (BCS) between 2.0 to 2.5 out of 5 were randomly allocated to one of four treatment groups in a 2 � 2 factorial design. On Day 0, all cows received a progesterone (P4) intravaginal device (DIB; Syntex, Argentina) and 2 mg estradiol benzoate (EB) i.m. (Syntex). On Day 8, DIB devices were removed and all cows received 150 �g D(+)cloprostenol i.m. (Ciclase; Syntex) and were randomly divided to receive 400 IU eCG i.m. (Novormon 5000; Syntex) at the same time or no treatment. In addition, half of the cows in each group had their calves weaned temporarily (TW) for 56 h at the time of DIB removal; the other half remained with their calves. All cows received 1 mg EB i.m. on Day 9 and were examined every 8 h by ultrasonography from the time of DIB removal until ovulation. Data were analyzed by ANOVA and chi-square test. The diameter of the dominant follicle on Day 8 (eCG: 7.8 � 0.5 mm vs. no eCG: 8.2 � 0.4 mm, and TW: 7.6 � 0.4 mm vs. no TW 8.4 � 0.5 mm), the proportion of cows that ovulated (eCG: 12/20, 60% vs. no eCG: 9/19, 47%; and TW: 13/20, 65% vs. no TW: 8/19, 42%, and the interval to ovulation (eCG: 72.0 � 1.4 h vs. no eCG: 75.6 � 2.0 h, and TW: 73.8 � 1.6 h vs. no TW: 73.0 � 1.8 h) did not differ among groups (P > 0.05). Although there was no effect of eCG treatment or eCG by TW interaction (P > 0.3) on the size of the preovulatory follicle (eCG: 11.1 � 0.4 mm vs. no eCG: 10.1 � 0.6 mm), it was smaller in cows TW (9.9 � 04 mm), compared to those not TW (11.8 � 0.3 mm; P < 0.05). Nevertheless, the growth rate of the ovulatory follicle was greater (P < 0.02) in cows treated with eCG (1.1 � 0.1 mm/day) than in those not treated with eCG (0.6 � 0.1 mm/day). In Experiment 2 769 lactating multiparous crossbred Bos indicus cows received the same treatments as those in Experiment 1 and were fixed-time inseminated (FTAI) 52 to 56 h after DIB removal. Cows were examined by ultrasonography on Day 0 (22.5% with corpora lutea (CL), 30.0% with follicles >8 mm, and 47.5% with follicles <8 mm) and 42 d after FTAI to determine pregnancy. Data were analyzed by logistic regression. Pregnancy rates were not affected (P > 0.7) by TW (TW: 141/397, 37.2% vs. no TW: 141/390, 36.1%) or the TW by eCG interaction (P > 0.7). However, pregnancy rates were higher (P < 0.05) in cows treated with eCG (154/377, 40.8%) than in those not treated with eCG (128/392, 32.6%). The use of eCG but not TW improved pregnancy rates following FTAI in postpartum Bos indicus cows. Results also suggest that the eCG-related increase in pregnancy rates may be due to the final growth rate of the ovulatory follicle.
161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM
Authors: George F. , Daniaux C. , Genicot G. , Focant F. , Verhaeghe B. , Lambert P. , Donnay I.
Published: December 2006

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed.

Table 1.
Hatching rates post-thawing as a function of the blastocyst size and the culture medium
167 THE SUPPLEMENTATION OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) PROMOTES THE DEVELOPMENT OF NUCLEAR TRANSFERRED BOVINE EMBRYOS
Authors: Kim D. H. , Kim S. W. , Yang B. C. , Im G. S. , Park H. S. , Hwang I. S. , Seo J. S. , Seong H. H. , Yang B. S.
Published: December 2006

Granulocyte-macrophage colony stimulating factor (GM-CSF) is secreted by epithelial cells lining the female reproductive tract in mice and several other species. GM-CSF receptors are present in the fertilized oocyte and in all subsequent stages of development, and in blastocysts it is expressed in both inner cell mass and trophectoderm cells. Recent studies suggest that GM-CSF can act as a survival factor for the developing embryo. The purpose of this study was to examine the effect of GM-CSF, as a medium supplement, on the development of nuclear-transferred bovine embryos. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 mg/mL FSH, and 1 mg/mL estradiol-17� for 20 h. Enucleated oocytes were fused with bovine ear skin fibroblast cells by a DC pulse of 25 V/150 mm for 20 ms in Zimmerman cell fusion medium. For activation, reconstructed embryos were exposed to 10 mM Ca-ionophore for 5 min, followed by 2 mM 6-dimethylaminopurine for 3 h. NT embryos were subsequently cultured in CR2 medium without or with 10 ng/mL recombinant porcine GM-CSF at 39.0�C in 5% O2, 5% CO2 and 90% N2. After 7 days of culture, blastocyst formation was observed. The number of inner cell mass (ICM) and trophectoderm (TE) cells was examined by differential staining. Apoptotic cells in blastocysts were detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick-end labeling (TUNEL) assay. Data were analyzed by chi-square and Student's t-test. Addition of GM-CSF to the medium significantly (P < 0.05) increased the proportion of embryos developing to the blastocyst stage (37.6 � 12.0 and 54.7 � 13.9% for control and GM-CSF groups respectively). No differences in the total cell number and the ratio of ICM to total cells were detected between the control group (125.4 � 35.7 and 38.5 � 9.7%) and the GM-CSF group (123.8 � 35.1 and 34.2 � 13.1%). The mean proportion of apoptotic cells in blastocysts was not different between the control (5.4 � 5.4%) and the GM-CSF (5.3 � 3.9%) group. Our results showed the beneficial effect of GM-CSF on the development of NT bovine embryos. These results suggest that GM-CSF might be a useful molecule for increasing development of NT bovine embryos. Further studies are necessary to verify the mechanism of GM-CSF on the development of bovine NT embryos.
168 IMPROVED MITOCHONDRIAL MEMBRANE POTENTIAL AND PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS BY DIBUTYRYL cAMP TREATMENT
Authors: Kim J.-S. , Koo D.-B. , Song B.-S. , Wee G. B. , Lee K.-K. , Han Y.-M.
Published: December 2006

Mitochondria play a pivotal role in energy metabolism and apoptosis during embryo development. In general, cAMP that exists at high level in GV oocytes inhibits germinal vesicle breakdown (GVBD), and the amount of cAMP in oocyte cytoplasm is gradually decreased for meiotic resumption. We first examined the effects of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryonic development. To determine whether mitocondrial activity is related to embryonic development, mitochondrial membrane potential (ΔΨm) was measured in porcine embryos. Porcine oocytes were cultured in NCSU-23 medium supplemented with 1 mM dbcAMP for 22 h and further cultured without dbcAMP for 22 h. After in vitro fertilization, porcine eggs were cultured in NCSU-23 medium with 4% BSA at 39�C, 5% CO2 in air for 6 d. Porcine embryos were obtained at various developmental stages and stained with the mitochondrial membrane potential-sensitive dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The mitochondrial membrane potential of porcine embryos was quantitatively evaluated by the ratio of green (presumptively alive mitochondria) to red fluorescent signal (presumptively dead mitochondria) using a fluorescence microscope. Acquired images were analyzed using DeltaVision Software (Applied Precision, Inc., Hsin-Chu City, Taiwan, ROC). After completion of IVM, dbcAMP-treated oocytes (91.3 � 0.9%) showed a higher proportion of the metaphase II stage than nontreated ones (72.8 � 2.6%) (P < 0.05). In the dbcAMP-treated group, sperm penetration rate was increased and polyspermic rate was reduced as compared to the nontreated group. Furthermore, the rate (37.3%, 47/126) of blastocyst formation in dbcAMP-treated group was higher than that (19.2%, 28/146) of the nontreated group (P < 0.05). After JC-1 staining, the number of blastomeres having live mitochondria per embryo increased in the dbcAMP-treated group at various developmental stages, whereas the number of blastomeres having dead mitochondria per embryo was enhanced in the nontreated group (43.3% vs. 30.2%). Our results indicate that in vitro maturation of porcine oocytes may affect mitochondrial membrane potential, apoptosis, and embryonic quality during pre-implantation development.