bluuebunny/crossref_metadata_2025 huggingface dataset:
7 ARTIFICIAL INSEMINATION IN BOS INDICUS WITH SEMEN IN SIMULTANEOUS POOL OF MULTIPLE BULLS
Authors: Torres-Júnior J. R. S. , Souza J. A. T. , Silva M. I. S. , Costa J. S. , Lima G. S. , Pereira Júnior A. C. , Melo W. O. , Aguiar H. M. U. S. B.
Published: August 2010

The duration of spermatic viability in the female reproductive system in cattle is 18 to 24 hours. Therefore, the time required for sperm capacitation in different bulls demonstrated to be highly variable and may affect the adequate amount of capacitated sperm within an optimal window of time for fertilization. The aim of this study was to evaluate the conception rate in Nelore cows inseminated with semen from a single bull compared with semen from a pool of 3 bulls (Fertility Plus®, ABS Pecplan, Minas Gerais, Brazil). On a random day of the estrous cycle (Day 0), 400 Nelore multiparous cows received a progesterone intravaginal device (1 g of P4, DIB®, Intervet/Schering-Plough, Cotia, Brazil) plus 2mg of estradiol benzoate (i.m. Estrogin®, Farmavet, São Paulo, Brazil). At the time of P4 device withdrawal (Day 8), 150 (ig of d-cloprostenol (i.m. Preloban®, Intervet/Schering-Plough), 1 mg of estradiol cypionate (i.m. ECP®, Pfizer, Brazil), and 300IU of eCG (i.m. Folligon®, Intervet/Schering-Plough) were administered. At 48 to 52 h after implant removal, all cows were fixed-time inseminated (FTAI). Then, cows were homogeneously distributed based on the semen used for FTAI in 4 groups: CONV1 (conventional dose of semen from bull 1), CONV2 (conventional dose of semen from bull 2), CONV3 (conventional dose of semen from bull 3), FERT (pool of semen from bulls 1, 2 and 3 homogeneously mixed in a single dose). Both conventional and Fertility Plus batches of semen contained a total dose of 30 × 106 spermatozoa. For pregnancy diagnosis, an ultrasonographic (CHISON 500VET, USP Brasil Eletromedicina, São Paulo, Brazil) examination was performed 30 days after the FTAI. Data were analyzed using the GENMOD procedure of SAS (SAS Institute, Cary, NC, USA). There was no interaction between treatment and body condition score [using a 1 (very thin) to 5 (very fat) point scale] or AI technician. The conception rates for CONV1, CONV2, CONV3, and FERT were, respectively, 40/101 (39.6%)b, 50/99 (50.5%)ab, 46/99 (46.5%)ab, 54/101 (53.5%)a; P < 0.05. In addition, the mean of conventional v. Fertility Plus treatments presented conception rates of 136/299 (45.5%) v. 54/101 (53.5%), respectively (P = 0.14). No significant statistical differences were observed between treatments. However, this trial showed that the utilization of the new Fertility Plus technology increased the overall conception rate by 8.0%. This probably implicates a complementary effect among semen samples and provides a greater time of sperm viability in oviduct. Also it provides evidence of the individual variation in capacitation time of bulls used for composition of the pool. Pooled semen of multiple sires may improve the reproductive performance at AI in cattle and increase the potential of the beef industry to produce pregnancies and born calves.
76 DEVELOPMENTAL CAPABILITY OF TRANSGENIC NUCLEAR-TRANSFERRED PIG EMBRYOS PRODUCED USING THE NOVEL METHOD OF PSEUDOPHYSIOLOGICAL ACTIVATION
Authors: Samiec M. , Skrzyszowska M. , Slomski R.
Published: August 2010

The physicochemical stimuli, which are commonly used for artificial activation of porcine nuclear-transferred (NT) oocytes, can affect detrimentally or cytotoxically the clonal cybrids and thereby inhibit the development or decrease the quality of cloned embryos. Therefore, we have recently developed a novel method of pseudophysiological transcomplementary (transcytoplasmic) activation to stimulate the developmental program of porcine oocytes reconstructed by somatic cell nuclear transfer. The mechanism underlying this original technique of activation is transcytoplasmic influx of sperm-derived proteins triggering intracellular calcium oscillations, which is mediated via heterologous (rabbit) zygote-descended cytoplasts. The purpose of our study was to estimate the in vitro developmental competences of porcine transgenic cloned embryos following pseudophysiological activation of oocytes receiving pWAPhGH-GFPBsd gene construct-nucleofected fetal fibroblast cell nuclei. In the cloning procedure, IVM pig oocytes were used as recipient cells for cell nuclei of positively selected transgenic fibroblast cells. The reconstruction of enucleated oocytes was performed by intracytoplasmic injection of either the somatic cell-derived karyoplast or whole tiny nuclear donor cell. The activation of porcine NT oocytes was achieved by electrofusion of them with the xenogeneic cytoplasts isolated from in vivo-derived rabbit zygotes (i.e. with the so-called zygoplasts), which led to the formation of triple xenocytoplasmic hybrids (xenocybrids). The rabbit zygotes had been flushed postmortem from the separated oviducts of superovulated postpubertal female donors 18 to 20 hafter administration of hCG and copulation. Single rabbit zygote-descended cytoplasts were inserted into the perivitelline space of previously reconstructed pig oocytes. The resulting zygoplast-NT oocyte couplets underwent fusion, which was induced by generation of 2 successive DC pulses of 1.2 kV cm-1 for 60 μs. The electrofusion medium consisted of 0.3 M Ca2+-deprived mannitol supplemented with 0.1 mM MgSO4 and 0.2 mg mL-1 fatty-acid-free BSA. The transcytoplasmically activated xenocybrids were cultured in vitro for 6 to 7 days up to morula/blastocyst stages. A total of 183/207 (88.4%) oocytes reconstructed with nucleofected fibroblast cell nuclei were successfully fused with zygoplasts. Out of 183 cultured NT embryos, 138 (75.4%) were cleaved. The rates of transgenic NT embryos that reached the morula and blastocyst stages yielded 106/183 (57.9%) and 65/183 (35.5%), respectively. In conclusion, the original method of pseudophysiological activation of porcine NT oocytes turned out to be relatively efficient, which has been confirmed by the high percentages of pWAPhGH-GFPBsd transgenic embryos developing in vitro to morula and blastocyst stages.
88 SURVIVAL AFTER FREEZING OF IN VITRO AND IN VIVO BOVINE EMBRYOS
Authors: Cho S. R. , Choi S. H. , Choe C. Y. , Son J. J. , Kim H. J. , Jin H. J. , Son D. S.
Published: August 2010

The present study was conducted to investigate the survivability of post-thawed bovine embryos for direct transfer. Bovine ovaries were collected at a local slaughterhouse. The cumulus-oocyte complexes (COC) were aspirated from 2 to 8 mm antral follicles using a syringe with an 18-gauge needle. Selected COC were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 15 COC were matured for 22 h in 50-μL droplets of TCM-199 supplemented with 5% FBS, 10 μg mL-1 of LH, 10 μg mL-1 of FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Mature COC were fertilized with frozen-thawed semen treated with BO medium (Brackett and Oliphants Biol. Reprod. 12, 260-274). All oocytes and embryos were placed in CR1aa medium culture system for in vivo embryo production. The Korean native cows that were between days 8 and 12 of their estrous cycles were superovulated with 28 mg of porcine follicle stimulating hormone (FSH, Antorine-R10; Kawasaki Mitaka Pharmaceutical, Tokyo, Japan) in twice daily i.m. injections, with a gradual decrease over 4 days. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.5 M, and 1.8 M ethylene glycol(EG) was used as a cryoprotectant. Embryo was loaded into 0.25 mL straw and directly into a cooling chamber (CL-863, USA) and kept at -7°C for 10 min, including time for seeding, and further cooled to -35°C at -0.3°C. After 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in water bath at 35°C and 37°C. Embryos were evaluated at 24, 48, and 72 h post thawing. Embryos that survived were recorded as either blastocysts that had expanded or hatched at 24 h or had hatched at 72 h. All of the results were analyzed by ANOVA using the STATVIEW program. After frozen the blastocysts cultured without serum, better survivability for frozen embryos was seen in the 1.8 M EG with 0.5% BSA (bovine serum albumin) group than 1.5 M EG with 0.5% BSA (75.7 v. 72.7). The survivability of frozen-thawed embryos was significantly higher in the 37°C water bath than 35°C (85.7% v. 70.8%). However, there was no difference in the total cell number of thawed embryos (142 ± 13 v. 137 ± 12), and chromosome abnormality higher than in vivo frozen-thawed embryos. In conclusion, the results suggest that the thawing temperature at 37°C may be optimal for better in vitro survival of frozen-thawed embryos produced in vitro and in vivo. Furthermore, the data suggest that embryo freezing system may provide reasonable conditions for embryo transfer.
89 HIGH PREGNANCY RATES AFTER TRANSFER OF LARGE EQUINE BLASTOCYSTS COLLAPSED VIA MICROMANIPULATION BEFORE VITRIFICATION
Authors: Choi Y. H. , Hartman D. L. , Bliss S. B. , Hayden S. S. , Blanchard T. L. , Hinrichs K.
Published: August 2010

Whereas small equine embryos may be successfully cryopreserved, pregnancy rates after cryopreservation of large equine blastocysts (>300 μm) are low. This has been attributed to their large diameter, failure of cyoprotectants to diffuse into the blastocoele, and/or impedance from the equine embryonic capsule. Previously, we successfully biopsied the trophoblast of equine in vivo-recovered expanded blastocysts (>300 μm; Choi et al. 2009 Reprod. Fertil. Dev. 21, 166-167). In that study, we observed embryonic growth in vitro after warming of a blastocyst vitrified immediately after biopsy. An efficient vitrification method was recently described for ferret embryos, which, like horse embryos, contain a large amount of lipid. This method utilizes very small (250 μm inner diameter) pipettes (Femtotips, Eppendorf; Sun et al. 2008 Biol. Reprod. 79, 832-840). We utilized this technique, with minor modifications, to vitrify equine embryos. In Study 1, the vitrification procedure was performed on equine in vitro-produced blastocysts (<200 μm diameter). All 11 embryos vitrified with the procedure and subsequently warmed grew in culture, showing an increase in diameter over the first 24 h, followed by protrusion of embryonic tissue through holes in the zona pellucida. In Study 2, 10 in vivo-recovered expanded blastocysts, >300 μm diameter, were biopsied using a Piezo drill, causing blastocoele collapse, and were then immediately vitrified. Three of these vitrified blastocysts (346, 399, and 514 μm in diameter) were warmed and transferred to culture; all 3 expanded within 8 h and subsequently proliferated through holes in the embryonic capsule. Seven vitrified in vivo-recovered blastocysts were warmed at the laboratory, shipped to a transfer facility, and transferred transcervically to recipient mares. Five out of 7 transferred blastocysts (71%) resulted in pregnancy at Day 14. Diameters of these embryos before biopsy and vitrification were 360, 398, 415, 434, and 599 μm. The 2 biopsied and vitrified embryos that did not result in pregnancy after transfer were originally 376 and 515 μm in diameter. To our knowledge, this represents the first report of normal (>50%) pregnancy rates achieved after transfer of cryopreserved expanded equine blastocysts. Our findings suggest that biopsy before cryopreservation, which punctures the embryonic capsule and causes blastocoele collapse, combined with vitrification in a small-diameter pipette, may provide an efficient procedure for cryopreservation of large equine blastocysts. Further studies are needed to explore the different factors associated with success of this procedure.

This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.
9 EFFECT OF FLUSHING WITH PROTEIN SOURCES OF DIFFERENT DEGRADABILITY ON OVULATION NUMBER IN Santa Inês EWES
Authors: Alves N. G. , Saunders G. A. , Pérez J. R. O. , Souza J. C. , Muniz J. A. , Lazarin G. B. , Bitencourt L. L. , José Neto A. , Moraes E. A.
Published: August 2010

Flushing provides sheep a diet dense in nutrients before the breeding season to increase the ovulation rate and is usually accompanied by increase in body weight (BW) and body condition score (BCS). Although the effect of flushing with high-energy diets is documented, the influence of flushing with high-protein diets on ovulation rate and on plasma urea nitrogen (PUN) is not well established. The aim of this study was to evaluate the ovulation rate of Santa Ines ewes subjected to flushing with protein sources with different ruminal degradability. Forty-eight ewes were assigned to 2 treatments in a randomized block design, based on initial BCS (2.25 to 2.5; 2.75; and 3.0 to 3.25; scale 1 = thin and 5 = fat). The treatments consisted of 2 isonitrogenous (12% crude protein) and isocaloric (3.9 Mcal of metabolizable energy per day) diets, but with different proportions of rumen-degradable protein; one with soybean meal (SM, n = 24) and the other with corn gluten meal and cottonseed meal (CG + CM, n = 24), provided for 28 d before ovulation. The BW and BCS were evaluated weekly. Estrus synchronization was initiated on Day 17 after beginning the treatment diets. An intravaginal sponge with 60 mg of medroxyprogesterone acetate (MAP-60®, Tecnopec, São Paulo, Brazil) was used for 11 d, and an injection of 300 IU of eCG (Novormon®, Syntex SA, Buenos Aires, Argentina) and 50 μg of cloprostenol (Prolise®, ARSA S.R.L., Buenos Aires, Argentina) was given 9 d after beginning the protocol. Estrus was monitored twice daily. The ovulation rate was evaluated by transrectal ultrasonography and was characterized by the disappearance of dominant follicles in the ovaries. Blood samples for determination of PUN were collected on Days 7, 14, 21, and 28 after the start of flushing. Quantitative data were subjected to ANOVA and the rate of estrus detection was analyzed by chi-square test. Treatment differences were considered significant if P < 0.05. The sheep consumed the equivalent to 1.60 times the metabolizable energy requirement for maintenance and 1.86 times the requirement of protein for maintenance. Estrus detection rate (95.83 v. 83.33%), BW (3.14 ± 0.55 v. 2.94 ± 0.61 kg) and BCS (0.04 ± 0.05 v. 0.10 ± 0.06) gain before ovulation, BW (54.46 ± 1.31 v. 53.36 ± 1.44 kg) and BCS (2.78 ± 0.05 v. 2.85 ± 0.05) on the 28th experimental day, PUN (17.25 ± 0.60 v. 16.23 ± 0.66 mg dL-1), ovulation rate (2.25 ± 0.16 v. 2.24 ± 0.18) and ovulatory follicle diameter (6.61 ± 0.17 v. 6.72 ± 0.19 mm) did not differ (P > 0.05) between ewes feed SM or CG + CM, respectively. Flushing with protein sources with different ruminal degradability did not alter the ovulation rate or the ovulatory follicle diameter in Santa Ines ewes.

Funded by FAPEMIG, CNPq, TECNOPEC.
90 EFFECT OF EGG YOLK ON THE KINEMATICS AND ACROSOME MEMBRANE INTEGRITY OF COOLED-REWARMED CANINE SPERMATOZOA
Authors: Dorado J. , Galvez M. J. , Murabito M. R. , Demyda S. , De Luca L. J. , Moreno M. , Hidalgo M.
Published: August 2010

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in either Tris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5°C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Data were statistically analysed by ANOVA. Dependent variables expressed as percentages were arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean ± SEM. Differences were considered significant when P < 0.05. Analyses were performed using the statistical package SPSS 12.0. A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10. After 24, 48, and 72 h of preservation, MS and PMS were statistically higher (P < 0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P < 0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P < 0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P < 0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P < 0.001) in EY20. At hour 72, higher acrosome integrity (P < 0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.
93 POSTCRYOPRESERVATION VIABILITY OF EMBRYOS FROM NELLORE HEIFERS SUPPLEMENTED WITH RUMEN-PROTECTED FAT
Authors: Guardieiro M. M. , Machado G. M. , Bastos M. R. , Mourão G. B. , Carrijo L. H. D. , Dode M. A. N. , Sartori R.
Published: August 2010

Polyunsaturated fatty acids seem to exert an extra-caloric positive effect on ruminant reproduction, although the reasons for that are still unclear. Although some studies have detected a positive effect of feeding unsaturated fatty acids on embryo development of superovulated Bos taurus cattle (Thangavelu et al. 2007 Theriogenology 68, 949-957), others have not (Petit et al. 2008 J. Dairy Sci. 91, 1786-1790). Our hypothesis was that, although number and quality of embryos from superovulated heifers would not be affected by diet, supplemental fat would improve embryo cryotolerance. Therefore, this study evaluated superovulatory response and embryo production, as well as cryotolerance of embryos cryopreserved through freezing or vitrification in Nellore heifers supplemented with rumen-protected fat. Forty heifers (24 to 36 mo old) were kept in pasture and randomly divided into 2 experimental groups according to supplemental source [F = concentrate with rumen-protected fat (100 g/d of Megalac-E®) and C = control, without fat supplementation]. Supplements were formulated to be isocaloric and isoproteic. Each female underwent both treatments in a cross-over design with approximately 68 d between replicates. After 50 d of feeding, emergence of the wave was synchronized with the aid of hormones to initiate the superovulation protocol. Recovered embryos were frozen or vitrified, and subsequently in vitro embryo development evaluation was accomplished. Data were analyzed using generalized linear models. There was no difference between F and C groups (P > 0.10) regarding superstimulatory response, number of total embryos/ova, viable embryos, degenerate embryos, or unfertilized oocytes recovered. However, group C had a greater superovulatory response than F (18.0 ± 1.3 v. 15.7 ± 1.2 CL; P = 0.06). Group C embryos presented greater hatching rate, independently of the cryopreservation method, at 48 h (33.1 ± 4.0%; n = 148 v. 17.3 ± 3.3%; n = 137; P = 0.009) and at 72 h (44.3 ± 4.2%; n = 148 v. 30.9 ± 4.0%; n = 137; P = 0.04) of in vitro culture. Under the conditions of the present study, supplementation with protected fat did not affect superstimulatory response and quantity or quality of embryos. However embryos from the F group were less tolerant to cryopreservation.

Financial support from FAPESP, EMBRAPA, Arm & Hammer, Integral Produbon, and Pfizer of Brazil.
94 VITRIFICATION OF WHOLE OVARIES IN YOUNG RATS
Authors: Hoshina M. , Furugaichi A. , Kuji N. , Ito J. , Kashiwazaki N.
Published: August 2010

Cryopreservation of reproductive organs is an important technology for preservation of genetic resources of experimental, domestic, and wild animals. In addition, cryopreservation of the ovary could be applied to restore the fertility of young women diagnosed with cancer because it could not only provide future fertility, but could also decrease the emotional consequences of cancer therapy for women afflicted with such devastating diseases. Cryopreservation of whole ovary particularly would enable such females to be pregnant by natural mating after transplantation. The aim of the present study was to evaluate the possible of vitrification for young rat ovaries as a possible model for human ovaries using a Cryotop method. Whole ovaries were collected from 10-day-old female rats (Brown-Norway × Wistar) and then washed several times in PB1 medium containing 20% (v/v) fetal calf serum (FCS). The ovaries were submerged in equilibration solution [10% (v/v) dimethyl sulfoxide (DMSO), 10% (v/v) ethylene glycol (EG), and 20% (v/v) FCS in PB1] for 1, 3, 5 or 10 min and then vitrified in vitrification solution (20% DMSO, 20% EG, and 20% FCS in PB1) for 5 min, being plunged into liquid nitrogen on Cryotops. Vitrified ovaries were later warmed in 37°C in PB1 containing 1 M sucrose and 20% FCS for 5 min and then PB1 containing 0.5 M sucrose and 20% FCS for 5 min. Eight ovaries were embedded in paraffin and cut into 4 μm slices. The slices were stained by hematoxylin-eosin (HE) and integrity of nuclei and cytoplasm in the follicles was histologically evaluated. In each group, 3 ovaries were used for the staining of live/dead (Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, #L3224). Follicles were separated from the ovary with enzyme and then classified into 2 categories by staining. Follicles possessing more than 50% of green-stained and red-stained granulosa cells were divided as live and dead, respectively. Data were analyzed by Tukey’s test. Results of the HE staining showed that integrity of nuclei and cytoplasm in ovaries equilibrated for 10 min (11%) was lower than those for 1 min (25%), 3min (42%), and 5 min (30%). As for evaluation by live/dead staining, most of follicles in ovaries equilibrated for 10 min (25%) were stained with red (dead). In vitrified ovaries equilibrated for 1 min, 3 min, and 5 min (18%, 16%, and 18%, respectively), there were no significant differences in red-stained follicles. These results suggest that optimal equilibration time is 3 to 5 min for vitrification of whole ovaries of 10-day-old rats. Using this equilibration time, we are now trying to evaluate the capability of vitrified/warmed ovaries after transplantation to recipient rats.

The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J. I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.
97 BOER GOAT OFFSPRING BORN FOLLOWING TRANSFER OF CRYOPRESERVED EMBRYOS
Authors: Lehloenya K. C. , Greyling J. P. C.
Published: August 2010

Cryopreservation of embryos is an important technique in the whole MOET program, which could help improve the transportation of genetic material across South Africa and globally. This trial evaluated the survival rate of goat embryos following transfer with cryopreserved Boer goat embryos. Twenty-seven multiparous Boer goat recipients were synchronized with CIDR for 16 days and injected with 300 IU of eCG at CIDR withdrawal. The recipients were allocated into 3 groups (n = 9). Group 1 received fresh embryos; Group 2 received slow frozen embryos; and Group 3 received vitrified embryos. Expanded blastocysts used were surgically collected from donors superovulated with pFSH on 7 following AI. Two blastocysts were transferred laparoscopically to the uterine horn ipsilateral to functional CL. A pregnancy rate of 85.7% (6) was obtained following the transfer of fresh embryos and tended to be better than in the does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively) with no significant differences. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and slow frozen groups, respectively. The embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was obtained and was not affected by the number of CL present on the respective ovaries at the time of embryo transfer. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of fresh or cryopreserved embryos tended to be lower.

The authors acknowledge the University of the Free State for financial and facility support and National Research Foundation (Thuthuka) for financial support for conducting this trial.
The evolution of electronic structure in few-layer graphene revealed by optical spectroscopy
Authors: Mak Kin Fai , Sfeir Matthew Y. , Misewich James A. , Heinz Tony F.
Published: August 2010

The massless Dirac spectrum of electrons in single-layer graphene has been thoroughly studied both theoretically and experimentally. Although a subject of considerable theoretical interest, experimental investigations of the richer electronic structure of few-layer graphene (FLG) have been limited. Here we examine FLG graphene crystals with Bernal stacking of layer thicknesses
N
 = 1,2,3,…8 prepared using the mechanical exfoliation technique. For each layer thickness
N
, infrared conductivity measurements over the spectral range of 0.2–1.0 eV have been performed and reveal a distinctive band structure, with different conductivity peaks present below 0.5 eV and a relatively flat spectrum at higher photon energies. The principal transitions exhibit a systematic energy-scaling behavior with
N
. These observations are explained within a unified zone-folding scheme that generates the electronic states for all FLG materials from that of the bulk 3D graphite crystal through imposition of appropriate boundary conditions. Using the Kubo formula, we find that the complete infrared conductivity spectra for the different FLG crystals can be reproduced reasonably well within the framework a tight-binding model.
T-cell receptor complex is essential for Fas signal transduction
Authors: Akimzhanov Askar M. , Wang Xinmin , Sun Jiaren , Boehning Darren
Published: August 2010

The Fas receptor (also known as CD95 and APO-1) is a member of the tumor necrosis factor α-family of death receptors that mediate T-cell responses. Here, we show that Fas receptor signaling requires a functional T-cell receptor (TCR) complex. Fas receptor directly binds to and activates TCR components in a stimulus-dependent manner. Fas receptor stimulation does not activate canonical downstream TCR pathways, but instead the TCR complex is required specifically for Fas-mediated calcium release. Importantly, null mutations in Lck, ZAP70, and the TCR α- and β-chains abrogate Fas signaling. Our results reveal a direct role for the TCR complex in mediating Fas-specific signaling events critical for T-cell homeostasis.
Proangiogenic scaffolds as functional templates for cardiac tissue engineering
Authors: Madden Lauran R. , Mortisen Derek J. , Sussman Eric M. , Dupras Sarah K. , Fugate James A. , Cuy Janet L. , Hauch Kip D. , Laflamme Michael A. , Murry Charles E. , Ratner Buddy D.
Published: August 2010

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30–40 μm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.
PARK2
deletions occur frequently in sporadic colorectal cancer and accelerate adenoma development in
Apc
mutant mice
Authors: Poulogiannis George , McIntyre Rebecca E. , Dimitriadi Maria , Apps John R. , Wilson Catherine H. , Ichimura Koichi , Luo Feijun , Cantley Lewis C. , Wyllie Andrew H. , Adams David J. , Arends Mark J.
Published: August 2010

In 100 primary colorectal carcinomas, we demonstrate by array comparative genomic hybridization (aCGH) that 33% show DNA copy number (DCN) loss involving
PARK2
, the gene encoding PARKIN, the E3 ubiquitin ligase whose deficiency is responsible for a form of autosomal recessive juvenile parkinsonism.
PARK2
is located on chromosome 6 (at 6q25–27), a chromosome with one of the lowest overall frequencies of DNA copy number alterations recorded in colorectal cancers. The
PARK2
deletions are mostly focal (31% ∼0.5 Mb on average), heterozygous, and show maximum incidence in exons 3 and 4. As
PARK2
lies within FRA6E, a large common fragile site, it has been argued that the observed DCN losses in
PARK2
in cancer may represent merely the result of enforced replication of locally vulnerable DNA. However, we show that deficiency in expression of
PARK2
is significantly associated with adenomatous polyposis coli (
APC
) deficiency in human colorectal cancer. Evidence of some
PARK2
mutations and promoter hypermethylation is described.
PARK2
overexpression inhibits cell proliferation in vitro. Moreover, interbreeding of
Park2
heterozygous knockout mice with
Apc
Min
mice resulted in a dramatic acceleration of intestinal adenoma development and increased polyp multiplicity. We conclude that
PARK2
is a tumor suppressor gene whose haploinsufficiency cooperates with mutant
APC
in colorectal carcinogenesis.
Helicobacter pylori-induced acquired immunity and immunoregulation.
Authors: Robinson K. , Atherton J. C.
Published: August 2010

This chapter reviews acquired immune response to Helicobacter pylori and its role in modulating gastroduodenal pathology and disease. The H. pylori-mediated immunomodulation of allergies and autoimmunity is also discussed.
Helicobacter pylori vaccines.
Authors: Blanchard T. G. , Nedrud J. G.
Published: August 2010

This chapter discusses several candidate antigens for immunization against Helicobacter pylori, as well as various vaccine adjuvants/delivery systems and immunization routes. The mechanisms of vaccine-induced H. pylori immunity and the induction of protective immunity through bypassing host regulatory immune response are described. Animal models and human clinical trials evaluating the efficacy of these vaccines are presented.
Helicobacter pylori adhesion to the gastric surface.
Authors: Lindén S. K. , Arnqvist A. , Teneberg S. , Moran A. P.
Published: August 2010

This chapter discusses the adhesins and other structures that Helicobacter pylori uses to adhere to the gastric surface, as well as how these adhesins are regulated. The host structures (mucus layer and glycosphingolipids) to which H. pylori binds are described.
An endogenous bioeconomic optimization algorithm to evaluate recovery plans: an application to southern hake
Authors: Da Rocha José-María , Cerviño Santiago , Gutiérrez María-José
Published: August 2010

Da Rocha, J-M., Cerviño, S., and Gutiérrez, M-J. 2010. An endogenous bioeconomic optimization algorithm to evaluate recovery plans: an application to southern hake. – ICES Journal of Marine Science, 67: 1957–1962. Recovery plans were analysed by introducing social and economic behaviour and endogenous disinvestment decisions into bioeconomic models. Considering these endogenous constraints, a dynamic optimization problem was solved to find fishing mortality (F) trajectories that maximize discounted profits per vessel, subject to recovery of the stock to a spawning-stock biomass (SSB) target in 2015. The algorithm developed was used to assess the southern hake recovery plan. Three scenarios were analysed: (1) represents the current plan with an annual 10% reduction in F; (2) represents the optimum trajectory where profits must be positive all along and the SSB target is reached no later than 2015, and (3) represents the optimum trajectory allowing profits to be negative. The results from (3) indicate that if economic and social restrictions are not considered a prior condition, the optimum solution implies a fleet reduction in 2010 and 2011. Comparing (1) and (2), our results suggest that reducing F to 0.30 by 2010 achieves the recovery target in 2012, increases the net present profits by 7.7% relative to the current plan, and is compatible with maintaining the current fleet size.
Specific Light-Emitting Diodes Can Suppress Sporulation of Podosphaera pannosa on Greenhouse Roses
Authors: Suthaparan A. , Torre S. , Stensvand A. , Herrero M. L. , Pettersen R. I. , Gadoury D. M. , Gislerød H. R.
Published: August 2010

When rose plants bearing colonies of Podosphaera pannosa were placed in a wind tunnel, the number of conidia trapped was directly proportional to intensity of daylight-balanced (white) light from 5 to 150 μmol m–2 s–1. Illumination of samples using blue (420 to 520 nm) light-emitting diodes (LEDs) increased the number of conidia trapped by a factor of approximately 2.7 over white light but germination of conidia under blue light was reduced by approximately 16.5% compared with conidia germination under white light. The number of conidia trapped under far-red (>685 nm) LEDs was approximately 4.7 times higher than in white light, and 13.3 times higher than under red (575 to 675 nm) LEDs, and germination was not induced compared with white light. When mildewed plants were exposed to cycles of 18 h of white light followed by 6 h of blue, red, far-red light, or darkness, light from the red LEDs reduced the number of conidia trapped by approximately 88% compared with darkness or far-red light. Interrupting the above dark period with 1 h of light from red LEDs also reduced the number of conidia trapped, while a 1-h period of light from far-red following the 1 h of light from red LEDs nullified the suppressive effect of red light. Our results indicate that brief exposure to red light during the dark interval may be as effective as continuous illumination in suppressing powdery mildew in greenhouse rose plant (Rosa × hybrida).
First Report of Southern Blight on Canadian Goldenrod (Solidago canadensis) Caused by Sclerotium rolfsii in China
Authors: Tang W. , Zhu Y. Z. , He H. Q. , Qiang S.
Published: August 2010

Canadian goldenrod (Solidago canadensis L., Asteraceae) is a rhizomatous perennial plant native to North America that has invaded eastern China and continues to spread northward and westward. It is quite common on field borders, roadsides, and in undeveloped areas, posing a serious threat to native ecosystems and their biodiversity. During the late summers of 2007 and 2008, wilted Canadian goldenrod plants were occasionally found in invasive populations in the suburb of Nanjing city. Wilted plants were transplanted and maintained in a greenhouse at Nanjing Agricultural University. A white mass of fungal hyphae, which grew on the soil surface around the stem of the symptomatic S. canadesis plants and eventually covered the stem, was observed. Initially, the base of the stem became yellow, turned brown, and the light brown discoloration extended up the stem to a height of 3 to 7 cm. The leaves then collapsed, starting from the top until the entire plant wilted. The fungus produced numerous, small, roundish sclerotia of uniform size (0.7 to 2.0 mm in diameter), which were white at first and then became brown to dark brown. The fungus grew into the stems and downward into the rhizome area, but no sclerotia were detected inside the stem or root. Diseased tissue with sclerotia was disinfested for 1 min in 1% NaOCl and plated on potato dextrose agar amended with 100 mg/liter of streptomycin sulfate. On the basis of sclerotia morphology and the presence of clamp connections at hyphal septa, the fungus was identified as Sclerotium rolfsii. Pathogenicity of the isolate was confirmed by inoculating 1-year-old S. canadensis plants (average 1.5 m high) grown in pots. The inoculum consisted of cottonseed hulls infested with mycelium and sclerotia of the pathogen and was placed on the soil surface around the base of each unwounded plant. Noninoculated plants served as controls. The pathogenicity test was conducted twice. After inoculation, the plants were maintained at high humidity and 30°C for 3 days and then transferred to a greenhouse. All inoculated plants developed symptoms of southern blight. Inoculated plants developed symptoms of wilting 5 to 7 days after inoculation and were completely wilted within 15 to 20 days. Symptoms of wilting were soon followed by the appearance of white-to-light brown sclerotia on the collar region. Control plants remained symptomless and Sclerotium rolfsii was reisolated from inoculated plants. To our knowledge, this is the first report of southern blight of Canadian goldenrod caused by Sclerotium rolfsii in China.
Absorption of polar drugs following caecal instillation in healthy volunteers
Authors: RILEY S. A. , KIM M. , SUTCLIFFE F. , ROWLAND M. , TURNBERG L. A.
Published: August 2010

We have investigated colonic drug absorption in man by the caecal instillation of a multi‐component solution of atenolol, cimetidine, frusemide, hydrochlorothiazide and salicylic acid. We found that salicylic acid absorption from this solution was delayed but complete whereas the absorption of atenolol, cimetidine, frusemide and hydrochlorothiazide was four‐ to five‐fold lower than expected from oral bioavailability studies.
Safety issues relating to long‐term treatment with histamine H2‐receptor antagonists
Authors: SABESIN S. M.
Published: August 2010

H2‐receptor antagonist therapy is associated with a low incidence of adverse reactions. Adverse events reported in clinical trials of ranitidine in daily doses of up to 1200 mg include headache, tiredness and mild gastrointestinal disturbances, but the incidence is similar to or less than that for placebo. High doses of cimetidine (> 5 g/day) can cause reversible impotence or gynaecomastia. While ranitidine exhibits no clinically significant drug‐drug interactions, cimetidine interacts with many drugs metabolized by cytochrome P450. In contrast to ranitidine and cimetidine, where safety data are available for up to 10 years of continuous therapy, experience with famotidine and nizatidine is limited. The safety of long‐term H2‐receptor antagonist therapy needs to be considered in relation to the potential consequences of prolonged acid suppression, including the risk of proliferation of gastric flora and the risk of developing enterochromaffin‐like cell hyperplasia, which could in turn, theoretically, lead to gastric malignancy. Such problems have not been observed in patients during long‐term therapy at low or full doses of H2‐receptor antagonists. Standard doses of currently available H2‐receptor antagonists permit acid secretion in response to food and other stimuli, and this daily acid tide prevents persistent bacterial colonization.
Molecular Properties of Post‐Mortem Muscle. I. Myofibrillar Nucleosidetriphosphatase Activity of Bovine Muscle
Authors: GOLL DARREL E. , ROBSON R. M.
Published: August 2010

SUMMARY—

A study was made of the nucleosidetriphosphatase (NTPase) activities of myofibrils isolated from pre‐rigor, rigor, and post‐rigor bovine semitendinosus and psoas muscles with three different extracting solutions: 1) 0.25M sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.05M trischydroxy‐methyl)‐aminomethane (Tris), pH 7.6; 2) 0.15M KCI, 1 mM EDTA, O.OSM Tris, pH 7.6; and 3) 50% glycerol, 1mM EDTA, 0.05M Tris, pH 7.6. Post‐mortem storage was at 2 and 16°C. By 6 hr post‐mortem, the Ca++‐ and Mg++‐modified adenosine‐triphosphatase (ATPase) activities at an ionic strength (I‐/2) of 0.18 had increased 20–50% over the O‐time level. An increase in Ca++‐modified ATPase of Γ/2 = 0.52 was also found. The Mg++‐modified inosinetriphosphatase (ITPase) at Γ/2 = 0.18 increased with post‐mortem time, but the Cat+‐modified ITPase at Γ/2 = 0.18 did not change.

The Ca++‐modified ITPase activity at Γ/2 = 0.52 increased for the first 24 hr post‐mortem, but after 312 hr post‐mortem, it had decreased back to the O‐time level. No differences in NTPase activities were found between the semitendinosus and the psoas muscles or between post‐mortem storage at 2 and 16°. Use of sucrose or KCI extracting solutions gave the most consistent NTPase results. The low Mg++‐modified NTPase activity at Γ/2 = 0.52 suggests that it is possible to dissociate thick and thin filaments from rigor muscle through the use of 5mM ATP or ITP. The fact that NTPase activities in the presence of certain modifiers did not change with post‐mortem time suggests that the increased NTPase activities did not result from a proteolytic loss of part of the enzyme molecule, causing an increased activity per unit of protein. Mg++‐modified ATPase activities in the presence of a Ca++ chelator indicated that tropomyosin, the most sensitive of the three myofibrillar proteins to proteolytic degradation, had not undergone significant proteolysis, even after 312 hr post‐mortem.
QUANTITATIVE METHODS FOR ANTHOCYANINS
Authors: LEES D. H. , FRANCIS F. J.
Published: August 2010

SUMMARY –A qualitative technique for the determination of the individual flavonol pigments of cranberries was developed, based on cellulose thin‐layer chromatography. The method was such that it was suitable for quantitative use by removing the pigment spots and measurement of the selected pigments by spectrophotometry. A quantitative technique for total flavonols, expressed as quercetin, based on spectrophotometry at 374 nm was developed. Total flavonols and total anthocyanins can be determined on the same extract.
THE LIPIDS OF EGG YOLK. 5. Presence of Ceramide
Authors: FUJINO Y. , MOMMA H.
Published: August 2010

SUMMARY —Ceramide (an N‐acyl sphingosine) was isolated from egg yolk lipid and examined for composition of fatty acids and long‐chain base constituents. Yolk ceramide was observed to contain sixteen fatty acids; C24:0 (35.7%), C22:0 (22.7%), C23:0 (15.1%) and C24:1 (13.5%) were predominant. The component long‐chain bases were identified as sphingosine and dihydrosphingosine, the former being the major component (88.6%).
RHEOLOGICAL PROPERTIES OF SINGLE‐CELL PROTEIN CONCENTRATE: DOPE FORMATION AND ITS FLOW BEHAVIOR
Authors: HUANG FRANK , RHA C. K.
Published: August 2010

SUMMARY –Protein solution was made with single‐cell protein (SCP) concentrate obtained by alkali extraction of Torula dried yeast. The minimum coagulation temperature for the dope was determined. The dope containing 10–25% of the extracted protein having a pH range of 9–12 was subjected to viscosity measurement at a temperature lower than the coagulation temperature. The optimum protein content of the SCP concentrate dope for spinning seems to be in the range of 15–20% At pH 9, the dope attained maximum viscosities without gelation.
EFFECT OF NEODIOSMIN ON THRESHOLD AND BITTERNESS OF LIMONIN IN WATER AND ORANGE JUICE
Authors: GUADAGNI D. G. , MAIER V. P. , TURNBAUGH J. G.
Published: August 2010

The thresholds of limonin and naringin in water and limonin in orange juice were determined in the presence of varying amounts of neodiosmin (NEO), the flavone analog of the bitter flavanone neohesperidin. Up to 40 ppm of NEO in water solution was essentially tasteless, but as little as 10 ppm in water increased the threshold of naringin and limonin 3.5‐ and 4.0‐fold, respectively. Regression analysis showed the relation between limonin and naringin threshold and NEO concentration in water was best described by a power function with similar slope exponents for naringin and limonin. This indicated that increasing concentrations of NEO had a similar bitter suppressive effect for both compounds. The data for limonin threshold and NEO concentration in orange juice was also described best by a power function but with a much smaller slope exponent. Nevertheless, NEO was effective in significantly reducing bitterness in orange juice at threshold and suprathreshold levels of limonin. The addition of 60‐100 ppm of NEO to orange juice containing 10 ppm of added limonin reduced limonin bitterness to the equivalent of 4–5 ppm of limonin in orange juice.
MEAT WITH HIGH LINOLEIC ACID CONTENT: OXIDATIVE CHANGES DURING FROZEN STORAGE
Authors: BREMNER H. A. , FORD A. L. , MACFARLANE J. J. , RATCLIFF D. , RUSSELL N. T.
Published: August 2010

“High linoleic” meat with fat containing up to 20% linoleic acid was produced by feeding a protected lipid supplement to sheep and steers. A comparison was made of the frozen storage life of this meat and of conventional meat, packaged in sealed polyethylene film pouches and stored at ‐10°C or at ‐20°C. Peroxide development was much more rapid in adipose tissue from high linoleic meat stored at ‐10°C. Rate of peroxide development in high hnoleic meat was greatly decreased when stored at ‐20°C. Taste panel assessments indicated that high linoleic meat stored at ‐10°C developed rancid odors and flavors 2‐3 times more rapidly than did conventional meat.
Aciduric and Heat Resistant Microorganisms in Apple Juice and Cider Processing Operations
Authors: SWANSON K. M. J. , LEASOR S. B. , DOWNING D. L.
Published: August 2010

Site visits to 15 apple juice or cider facilities were conducted in New York State. Washed apples, pulp, press juice, final product, and other samples, when appropriate, were sampled aseptically. Samples were plated on acidified potato dextrose agar, before and after heating at 70°C for 1‐2 hr. For unheated samples, molds predominated in fruit, yeasts and molds in pulp, and yeasts in press and final juice samples. Average counts were 2.8 × 104, 7.3 × 104, 1.7 × 105, and 1.4 × 105/g, respectively. Heat resistant yeasts, molds, and bacteria were isolated frequently, generally at a level of less than 1 per log.
Differential Scanning Calorimetry of Fish Muscle: The Effect of Processing and Species Variation
Authors: HASTINGS ROSEMARY J. , RODGER GRAHAM W. , PARK ROBIN , MATTHEWS ANTHONY D. , ANDERSON EVELYN M.
Published: August 2010

Differential Scanning Calorimetry (DSC) has been used to study the thermal properties of fish muscle proteins and to measure the extent of their denaturation under various processing conditions. Fish myosin was susceptible to denaturation by frozen storage and dehydration. Denaturation of certain fish proteins was partially reversible. Although fish myosin was very unstable, its thermal stability was found to increase in species adapted, to higher environmental temperatures.