bluuebunny/crossref_metadata_2025 huggingface dataset:
52 HYALURONIC ACID IMPROVES CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS
Authors: Boccia L. , Rubessa M. , De Blasi M. , Di Francesco S. , Albero G. , Neglia G. , Gasparrini B.
Published: December 2011

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine fluids, has been shown to successfully support in vitro development of bovine embryos (Stojkovic et al. 2002 Reproduction 124, 141–153). The aim of this study was to evaluate the influence of high concentrations of hyaluronic acid (HA) during late in vitro culture on blastocyst development, as well as on their cryotolerance after cryotop vitrification in buffalos. In vitro matured and fertilized buffalo oocytes (n = 1007) from slaughterhouse ovaries were cultured for 4 days in SOFaa supplemented by 8 mg mL–1 of BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2 and 88% N2, in humidified air, at 38.5°C. On Day 4, cleavage rate was assessed (75.2%) and all of the cleaved elements were divided into 3 different late culture groups: 8 mg mL–1 of BSA (n = 244; group A), 8 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 251; group B) and 1 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 262; group C). On Day 7 after IVF, embryo outcome was assessed and all of the embryos were vitrified by cryotop [De Rosa et al. 2007 Ital. J. Anim. Sci. 6 (Suppl 2), 747–750] and cultured for 24 h. The resistance to cryopreservation was evaluated by assessing the survival rate on the basis of morphological criteria and the percentage of embryos reaching a more advanced developmental stage after 24 h culture. Data were analysed by the chi-square test. No differences in blastocyst rate were recorded among groups (43.9, 44.3 and 40.0%, respectively in A, B and C groups). However, out of the total embryos, a higher percentage of Grade 1 hatched blastocysts (Robertson and Nelson 1998 Manual of the International Embryo Transfer Society 9, 103–16) was observed in group C (P < 0.05) than in groups A and B (14.3, 18.8 and 25.5% in A, B and C groups, respectively). Although the supplementation with HA did not improve the survival rates following vitrification-warming (51.1, 59.4 and 58.4% in A, B and C groups, respectively), the percentage of vitrified-warmed embryos that resumed development and reached a more advanced developmental stage after culture increased (P < 0.01) in group C (20.7, 27.7 and 37.6% in A, B and C groups, respectively). In conclusion, the addition of 6 mg mL–1 of HA, together with a limited protein source (i.e. 1 mg mL–1 of BSA), during late culture improved buffalo embryo quality, indicated by both the greater percentage of advanced-stage embryos and by the resumption of development after post-warming culture.
63 EVALUATION OF HYPOTHALAMIC–PITUITARY RESPONSIVENESS DURING THE POSTPARTUM NELLORE COWS
Authors: Pinheiro V. G. , Cury J. R. L. M. , Satrapa R. A. , Pegorer M. F. , Barros C. M.
Published: December 2011

The presence of calf, body condition score, energy balance, number of births (multiparous vs primiparous) and breed are factors that influence the duration of postpartum anoestrus in beef cows. The objective of the present study was to evaluate, during early postpartum, the time of re-establishment of pituitary LH stocks, measured by the hypothalamic-pituitary-axis responsiveness to exogenous administration of gonadotropin-releasing hormone (GnRH) or oestradiol benzoate (EB). Multiparous lactating Nellore cows (Bos indicus, n = 65) were randomly allocated into 6 groups, according to the hormonal treatment: EB group (1.0 mg of EB, IM, n = 7), GnRH group (50 μg of lecireline, IM, n = 16). The EB-supplemented (SUP; n = 9) and GnRH-SUP (n = 16) groups received the same treatments specified above and were SUP with a balanced diet, based on cotton meal and ground corn (3.5 kg cow–1 per day). Additionally, animals from EB-calf removed (CR; n = 4) and GnRH-CR (n = 13) groups received the same treatments of EB and GnRH group, respectively and had their CR shortly after parturition. The hormones were administered weekly, from 7 days postpartum (±5 days) until the occurrence of the first ovulation, which was determined by the presence of corpus luteum during ovarian ultrasonography performed weekly. Blood samples were collected just before and 2 h (GnRH groups) or 18 h (EB groups) after hormone administration, in order to determine LH concentration by radioimmunoassay. Data were analysed by ANOVA. Mean values in days (± standard error of the means) for the first postpartum LH surge were EB (73.0 ± 5.2); EB-CR (16.7 ± 5.8); EB-SUP (41.7 ± 6.7); GnRH (32.3 ± 3.0); GnRH-CR (11.0 ± 3.5); GnRH-SUP (15.6 ± 2.8). There were significant differences (P < 0.05) between groups EB vs EB-CR; EB vs EB-SUP; BE vs GnRH; GnRH vs GnRH-CR; GnRH vs GnRH-SUP and a tendency between EB-SUP vs EB-CR (P < 0.10). Results indicate that from the second week postpartum, there is sufficient LH in the pituitary to induce ovulation after GnRH or EB administration. However, the cows from the EB group ovulated later than animals from the other groups, possibly due to the sensitivity of the hypothalamus to negative feedback of estrogens, inhibiting the pre-ovulatory LH surge. Additionally, CR and food SUP reduced in 2 to 4 weeks the time of the first postpartum LH surge induced by GnRH or EB in Nellore cows.
A fellowship was received from FAPESP. Support was received from FAPESP.
66 Orai1 IS REQUIRED TO MAINTAIN CALCIUM OSCILLATION AT FERTILIZATION IN PORCINE OOCYTES
Authors: Wang C. , Lee K.
Published: December 2011

During mammalian fertilization, the sperm induces an oscillation in the oocyte's intracellular free calcium concentration that stimulates oocyte activation. The train of calcium spikes is maintained by an influx of calcium across the plasma membrane, probably through a mechanism known as store-operated calcium entry. Despite their importance, little is known about the identity and regulation of the calcium entry channels that mediate calcium influx at fertilization. Previously we have shown that the Orai1 protein is localised in the plasma membrane of pig oocytes and plays an important role in calcium entry after depletion of the intracellular calcium stores. In this study, we investigated the function of Orai1 in signal transduction during fertilization. In Experiment 1, Orai1 levels were down-regulated by injecting immature pig oocytes with small interfering RNA (siRNA) against Orai1; control cells were injected with scrambled siRNA. The injected oocytes were then matured in vitro for 44 h. In Experiment 2, Orai1 was overexpressed by microinjection of mRNA encoding Orai1 conjugated to the enhanced green fluorescent protein (EGFP-Orai1) 36 h after the beginning of maturation; these oocytes were incubated for an additional 8 h. At the end of the incubation period the oocytes were loaded with the calcium indicator dye fura-2 and inseminated. Changes in the intracellular free calcium concentration were monitored by means of a fluorescence imaging system. Some of the fertilized oocytes were cultured for 7 days, at which time embryo development together with the total nuclear number of the embryos were recorded. Data were subjected to 1-way ANOVA; differences between treatments were analysed using the Tukey test. The level of Orai1 protein was significantly lower in Orai1 siRNA-injected oocytes compared to the control group, as determined by Western blot, indicating successful down-regulation of Orai1. During fertilization, control oocytes displayed a series of calcium transients that lasted up to 8 h. However, in Orai1 siRNA-injected oocytes (9 out of 10 measured) only 1 calcium rise could be observed; these oocytes were unable to generate repetitive calcium signals after gamete fusion. Overexpression of Orai1 also disrupted the pattern of the normal fertilization calcium signal; the oscillation ceased after just a few Ca2+ rises in 8 out of 10 oocytes. The percentage of cleaved oocytes was lower in the siRNA-injected group compared to both non-injected or control siRNA-injected oocytes (38.4 ± 4.6% vs 70.5 ± 2.8% and 70.4 ± 2.4%; P < 0.05). The frequency of blastocyst (1.9 ± 0.8% vs11.0 ± 3.1% and 9.9 ± 1.4%) and the total cell number per blastocyst (19.8 ± 0.8 vs 36.2 ± 0.7 and 33.5 ± 5.5) was also significantly lower in oocytes with down-regulated Orai1 levels. These results indicate that an Orai1-mediated calcium influx is required to maintain proper calcium oscillation at fertilization and is also essential to support subsequent embryo development.
69 DIFFERENTIAL AND QUANTITATIVE ANALYSIS OF DOG OVIDUCTAL FLUID
Authors: Reynaud K. , Labas V. , Harichaux G. , Thoumire S. , Tahir M. Z. , Chastant-Maillard S. , Saint-Dizier M.
Published: December 2011

The major reproductive peculiarity of the bitch is that ovulation releases prophase I (germinal vesicle, GV, immature) oocytes. Resumption of meiotic maturation, as well as fertilisation and embryonic development to the morula stage occur in the oviduct. Because the dog is a biomedical model for human diseases and also a model for endangered canid species, the development of assisted reproduction techniques would be of great interest. To date, in vitro-produced canine embryos remain exceptional and no puppy has been born. The main limiting factors of in vitro embryo production are the low oocyte maturation rates, the poor oocyte quality and the high polyspermy. A better knowledge of the composition of oviductal fluid during the periovulatory period may help to mimic the in vivo conditions for in vitro oocyte culture and, thereafter, their fertilisation and embryonic development. The objective of this study was to analyse the oviductal fluid by a label-free quantitative proteomic workflow based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein separation, nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis and quantitative method using spectral counting. Ovarian cycles were followed by vaginal smears, ultrasonography and progesterone blood assays. Oviductal fluids were collected from 3 beagle bitches, after ovariectomies performed 3.5 days after ovulation. After dissection, the ampulla and isthmus were separated and flushed with 50 μL of PBS. Oviductal fluids were submitted to 1D SDS-PAGE and all bands were digested with trypsin. Peptide extracts were analysed on an Ettan multidimensional LC (MDLC) system coupled to a linear ion trap quadrupole (LTQ) mass spectrometer. After protein identification using Mascot server and with Swiss-Prot and National Center for Biotechnology Information (NCBI) databases, bioinformatic processing of data and statistic analysis (t-test with P < 0.05) were performed using the spectral counting quantitative module of the Scaffold software. Using this strategy, 427 proteins were qualitatively identified in canine oviductal fluid. Three proteins were specific of the ampulla, 10 specific of the isthmus and 414 were found in both oviductal parts. Among these common proteins, some were differentially expressed, from 1.25 to 9 times higher (HV303_Human, RLA2_Horse, SPRL1_Human, SODC_CANFA, PROF1_Human, ARF4_Bovin and TRXR1_Bovin). The gene ontology analysis displayed biological pathways specific to the biology of reproduction (6 proteins; RUVB1_Human, OVGP1_Pig, STAT3_Human, PLAK_Human, GPX3_Rat and DYL1_Human). These candidate proteins and especially oviduct-specific glycoprotein and glutathione peroxidase, will now be validated by immunodetection methods.
71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION
Authors: Pontes Godoi C. , Moço P. D. , Cazari B. , Mihara P. T. , Silva P. V. , Emanuelli I. P. , Nogueira M. F. G.
Published: December 2011

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group.
Supported by FAPESP of Brazil.
75 COMPARISON OF THE DG29TM ENZYME-LINKED IMMUNOSORBENT ASSAY KIT COMPARED WITH TRANSRECTAL ULTRASONOGRAPHY FOR EARLY PREGNANCY DIAGNOSIS FOLLOWING TRANSFER OF JAPANESE BLACK CATTLE EMBRYOS
Authors: Nakamura Y. , Urakawa M. , Ideta A. , Shirasawa A. , Oono Y. , Aoyagi Y.
Published: December 2011

In commercial embryo transfer industry, accurate early detection of pregnant and nonpregnant cows plays a key role in achieving an optimum calving-to-conception interval. The DG29TM enzyme-linked immunosorbent assay (ELISA) kit (Conception, Animal Reproduction Technologies, Beaumont, Canada) measures the level of pregnancy-related glycoproteins in blood that are linked to pregnancy. Here, we compared the DG29 kit with transrectal ultrasonography (TU) to evaluate the possibility of the clinical application of the ELISA kit for early pregnancy diagnosis. Embryos recovered from superovulated Japanese Black cows were transferred to 110 recipient Holstein heifers on Day 6 to 8 of the oestrous cycle (oestrus = Day 0). Pregnancy was diagnosed between Day 29 and Day 40 by TU with a 5.0/7.5-MHz linear array transducer (Tringa, Pie Medical Equipment B.V., Maastricht, the Netherlands). Blood samples were collected from the tail vein or artery into vacuum serum tubes after TU and serum was separated and stored at –30°C until the ELISA was performed. The ELISA results were interpreted as positive (pregnant, >1000 pg mL–1) or negative (nonpregnant, <300 pg mL–1). Ninety-nine of the 110 heifers were judged as pregnant or nonpregnant by TU. Seventy-six of the 99 heifers were judged as pregnant, in which fetuses were visualised clearly in the uterine horn. The following measures, sensitivity, specificity, predictive value and accuracy of pregnancy outcomes based on the ELISA results, were assessed by comparing with those based on the definite TU results. The values for sensitivity and specificity were 100% (76/76) and 91.3% (21/23), the positive and negative predictive value were 97.4% (76/78) and 100% (21/21), respectively and accuracy was 98.0% (97/99). On the other hand, chorioallantoic fluids in 11 of the 110 heifers were detected by TU around Day 30 of gestation, but fetuses were not identified or were unclearly visualised, which suggests that the embryos died during the peri-implantation period after transfer. Ten of the 11 heifers were classified as pregnant by the ELISA, but only 3 heifers were identified as pregnant with reexamination by later TU, which indicates that the pregnancy-related glycoproteins residue from embryo mortality was detected by the DG29 kit. However, the negative predictive value of the DG29 kit was 100% in this study. In conclusion, except for early embryonic death, the DG29 kit was highly accurate and suitable for clinical application in early pregnancy determination following transfer of Japanese Black cattle embryos.
80 RECIPIENT SERUM PROGESTERONE CONCENTRATION AND EARLY CONCEPTUS DEVELOPMENT IN BOVINE EMBRYOS PRODUCED IN VIVO OR IN VITRO
Authors: Barnwell C. V. , Whisnant C. S. , Farin C. E. , Alexander J. E. , Farin P. W.
Published: December 2011

The majority of pregnancy loss in cattle occurs during the first 2 to 3 weeks of pregnancy. This loss can be studied by the transfer of in vivo- and in vitro-produced embryos. The objective of this study was to examine the relationship between recipient serum progesterone levels both at the time of embryo transfer and at conceptus recovery on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade-1 blastocysts from each embryo production system were transferred into heifers at Day 7. Conceptuses were recovered at Day 17 of gestation (number recovered/number transferred: IVO, 11/17; IVPS, 16/27; IVPSR, 18/34) and classified as complete, degenerated, or no conceptus. Recipient serum progesterone concentrations were determined by radioimmunoassay and compared with conceptus development outcomes. Sex of conceptus was determined by PCR using a Y-chromosome specific probe. Data were analysed using Fisher's exact test or ANOVA and Duncan's multiple range test. Compared with the IVO group, in vitro-produced embryos had more (P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; IVPSR, 20.6%). There were no differences (P > 0.05) in serum progesterone concentrations in recipients assigned to different treatments at Day 7. There was also no effect (P > 0.05) of treatment on progesterone levels in recipients with either male or female conceptuses at the time of transfer. Interestingly, heifers in the in vitro treatment groups had lower (P < 0.01) progesterone concentrations at Day 7 when no conceptus was recovered at Day 17 (IVPS, 2.1 ± 0.4 ng mL–1; IVPSR, 2.7 ± 0.4 ng mL–1; Least squares means ± standard error of the mean) compared with the IVO group (4.5 ± 0.6 ng mL–1). There was no difference in progesterone concentration between treatment groups for heifers with shorter conceptuses (≤194 mm). However, when longer (>194 mm) conceptuses were recovered, heifers with in vitro produced embryos had lower (P < 0.05) progesterone levels at Day 7 compared with those with in vivo produced embryos (IVPS, 2.2 ± 0.6 ng mL–1; IVPSR, 2.3 ± 0.5 ng mL–1; IVO, 3.9 ± 0.6 ng mL–1). In summary, serum progesterone concentrations in recipients at the time of transfer of in vivo- or in vitro-produced embryos were associated with conceptus development at Day 17 of gestation.
Research supported by NC State University GAANN Biotechnology Fellowship (C. V. Barnwell) and the College of Veterinary Medicine.
85 EFFECT OF FOLLICULAR ASPIRATION JUST PRIOR TO OVULATION ON CORPUS LUTEUM CHARACTERISTICS, CIRCULATING PROGESTERONE CONCENTRATIONS AND UTERINE RECEPTIVITY IN SINGLE-OVULATING BEEF HEIFERS
Authors: O'Hara L. , Scully S. , Maillo-Sevilla V. , Kelly A. K. , Duffy P. , Carter F. , Forde N. , Rizos D. , Lonergan P.
Published: December 2011

Progesterone (P4) has a crucial impact on the transcriptome of the uterine endometrium and the preparation of the uterus to support implantation. The aim of this study was to investigate the effect of follicle aspiration just before ovulation on corpus luteum (CL) development, circulating P4 concentrations and the ability of the uterus to support embryo development and conceptus elongation. We tested the hypothesis that the unavoidable loss of follicular fluid and some granulosa cells during aspiration of the preovulatory follicle would compromise the development and function of the developing CL and that this would be associated with reduced P4 and a poorer uterine environment. Oestrous cycles of crossbred beef heifers were synchronized using an 8-day CIDR treatment with administration of a prostaglandin F2α analogue on the day before CIDR removal to ensure CL regression. Heifers were checked for signs of oestrus 4 times per day commencing 30 h after CIDR withdrawal and only those recorded in standing oestrus (Day 0, n = 20) were used. All heifers received a gonadotropin-releasing hormone agonist (0.01 mg buserelin) 48 h after CIDR removal to induce an LH surge. Half of the animals underwent follicle aspiration 20 h later, while the remainder underwent ovulation. Daily transrectal ultrasonography was carried out from Day 3 to 13 to record CL development. Daily blood samples were collected from Day 0 to 14 for circulating P4 concentrations. To test the ability of the uterus to support embryo development and conceptus elongation, Day 7 in vitro-produced blastocysts were transferred to the uteri of synchronised recipients (7 to 10 blastocysts per recipient). All recipients were slaughtered on Day 14 to assess embryo survival and conceptus size. CL diameter and CL area were significantly reduced in the follicle aspirated group compared with controls from Day 6 onwards (P ≤ 0.05). Similarly, at slaughter on Day 14, CL weight (4.17 ± 0.48 vs 7.05 ± 1.65 mm), diameter (19.89 ± 1.35 vs 24.64 ± 2.07 mm) and area (321.94 ± 45.01 vs 510.18 ± 69.41 mm2) were lower in aspirated heifers (P ≤ 0.05). Circulating P4 concentrations were lower at all time points from Day 3 to Day 14 but were only significantly lower from Day 12 onwards (P ≤ 0.05). Conceptus length (2.08 ± 0.29, n = 56 vs 4.55 ± 0.78 mm, n = 45) and area (2.52 ± 0.39 vs 5.61 ± 1.12 mm2) were lower (P ≤ 0.05) in heifers undergoing follicular aspiration compared with those undergoing ovulation. In conclusion, aspiration of the preovulatory dominant follicle just before expected ovulation was associated with a compromised CL in terms of size and P4 output and this, in turn, was associated with a reduced capacity of the uterus to support the initiation of conceptus elongation.
Supported by Science Foundation Ireland (07/SRC/B1156).
87 BACK TO THE DRAWING BOARD: IN VITRO CULTURE OF BOVINE EMBRYOS FOR FREEZING
Authors: Vigneault C. , Underwood S. L. , Fournier V. , Bousquet D. , Belanger J. , Blondin P.
Published: December 2011

Despite hundreds of scientific papers published, no system has resulted in in vitro embryos comparable to those produced in vivo. We hypothesised that assembling the most pertinent elements of IVF studies into one system would result in a highly efficient in vitro culture system. Here we report the in vitro production of bovine embryos using a culture system with strict environmental conditions that produces very-good-quality embryos at high rates. This system consists of a sequential culture system with media composition based on recent reports that characterise the bovine female reproductive tract (Hugentobler et al. 2007 Mol. Reprod. Dev. 74, 445–454; Hugentobler et al. 2007 Theriogenology 68, 538–548; Hugentobler et al. 2008 Mol. Reprod. Dev. 75, 496–503). This system uses a 3-step culture media to prevent toxicity resulting from ammonium accumulation and nutrient depletion and also to adjust the component concentrations to support embryo needs at different developmental stages. Fatty acid-free BSA is used as the protein source and the culture is in droplets under high-quality paraffin oil at 38.5°C under 6.8% CO2, 5% O2 and 88.2% N2. Numerous other aspects were investigated to limit embryo stresses (Lane et al. 2008 Reprod. Fertil. Dev. 20, 23–32) during manipulations, including the use of mini-incubators and very-high-purity gas combined with stringent laboratory practices. In the first year using this new embryo production system, 2839 oocytes were fertilized, resulting in a transferable blastocyst rate of 51%. Of the 1448 embryos produced, 779 were transferred fresh at our facility with pregnancy rates of 55 and 49% at 28 and 60 days, respectively. Pregnancy rates were directly related to the quality of the embryos transferred as 61% of grade 1 embryos transferred induced a pregnancy at Day 28, compared with 41% of grade 2 embryos. Pregnancy induction is not the only indication of good embryo quality. As is well-documented, in vitro-produced bovine embryos do not tolerate slow freezing, so vitrification was applied to surmount this intolerance. However, this is difficult to apply to industry because direct transfer of vitrified embryos is challenging. We hypothesised that the improvement of embryo culture would result in embryos that could tolerate slow freezing. Grade 1 blastocysts (n = 229) were frozen in 1.6M ethylene glycol and 0.1 M sucrose using standard slow freezing procedures. A very high proportion (91%) of frozen–thawed in vitro-produced embryos re-expanded after 24 h of culture with a good quality inner cell mass. Subsequently, 45 grade 1 blastocysts were frozen and transferred, giving pregnancy rates of 58% at Day 60. In conclusion, combining good-quality culture media and conditions resulted in the production of in vitro embryos that were very efficient at inducing pregnancies and tolerating slow freezing, which makes it now possible to consider direct transfer of frozen in vitro-produced bovine embryos.
94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS
Authors: Pradiee J. , Viana L. L. , Santos E. C. S. , Gonçalves A. , Mondadori R. G. , Vieira A. D. , Lucia T. , Pegoraro L. M. C.
Published: December 2011

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos.
CAPES, MARFRIG Group.
Antigen-specific induced Foxp3+regulatory T cells are generated following CD40/CD154 blockade
Authors: Ferrer Ivana R. , Wagener Maylene E. , Song Minqing , Kirk Allan D. , Larsen Christian P. , Ford Mandy L.
Published: December 2011

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4+and CD8+T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4+and CD8+TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8+T-cell expansion, delaying differentiation into IFN-γ+TNF+multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4+CD25hiFoxp3+T-cell population, which did not arise from endogenous natural Tregbut rather were peripherally generated from naïve Foxp3−precursors.
Global population trajectories of tunas and their relatives
Authors: Juan-Jordá Maria José , Mosqueira Iago , Cooper Andrew B. , Freire Juan , Dulvy Nicholas K.
Published: December 2011

Tunas and their relatives dominate the world's largest ecosystems and sustain some of the most valuable fisheries. The impacts of fishing on these species have been debated intensively over the past decade, giving rise to divergent views on the scale and extent of the impacts of fisheries on pelagic ecosystems. We use all available age-structured stock assessments to evaluate the adult biomass trajectories and exploitation status of 26 populations of tunas and their relatives (17 tunas, 5 mackerels, and 4 Spanish mackerels) from 1954 to 2006. Overall, populations have declined, on average, by 60% over the past half century, but the decline in the total adult biomass is lower (52%), driven by a few abundant populations. The trajectories of individual populations depend on the interaction between life histories, ecology, and fishing pressure. The steepest declines are exhibited by two distinct groups: the largest, longest lived, highest value temperate tunas and the smaller, short-lived mackerels, both with most of their populations being overexploited. The remaining populations, mostly tropical tunas, have been fished down to approximately maximum sustainable yield levels, preventing further expansion of catches in these fisheries. Fishing mortality has increased steadily to the point where around 12.5% of the tunas and their relatives are caught each year globally. Overcapacity of these fisheries is jeopardizing their long-term sustainability. To guarantee higher catches, stabilize profits, and reduce collateral impacts on marine ecosystems requires the rebuilding of overexploited populations and stricter management measures to reduce overcapacity and regulate threatening trade.
Characteristics ofBorrelia hermsiiinfection in human hematopoietic stem cell-engrafted mice mirror those of human relapsing fever
Authors: Vuyyuru Raja , Liu Hongqi , Manser Tim , Alugupalli Kishore R.
Published: December 2011

Rodents are natural reservoirs for a variety of species ofBorreliathat cause relapsing fever (RF) in humans. The murine model of this disease recapitulates many of the clinical manifestations of the human disease and has revealed that T cell-independent antibody responses are required to resolve the bacteremic episodes. However, it is not clear whether such protective humoral responses are mounted in humans. We examinedBorrelia hermsiiinfection in human hematopoietic stem cell-engrafted nonobese diabetic/SCID/IL-2Rγnullmice: “human immune system mice” (HISmice). Infection of these mice, which are severely deficient in lymphoid and myeloid compartments, withB. hermsiiresulted in persistent bacteremia. In contrast, this infection in HISmice resulted in recurrent episodes of bacteremia, the hallmark of RF. The resolution of the primary episode of bacteremia was concurrent with the generation ofB. hermsii-specific human IgM. Remarkably, HISmice generated antibody responses to theB. hermsiiouter-membrane protein Factor H binding protein A. Sera from humans infected byB. hermsiihave a similar reactivity, and studies in mice have shown that this response is generated by the B1b cell subset. HISmice contain several B-cell subsets, including those with the phenotype CD20+CD27+CD43+CD70−, a proposed human equivalent of mouse B1 cells. Reduction of B cells by administration of anti-human CD20 antibody resulted in diminished anti-B. hermsiiresponses and persistent bacteremia in HISmice. These data indicate that analysis ofB. hermsiiinfection in HISmice will serve as a model in which to study the cellular and molecular mechanisms involved in controlling human RF.
Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases
Authors: Giardina Giorgio , Montioli Riccardo , Gianni Stefano , Cellini Barbara , Paiardini Alessandro , Voltattorni Carla Borri , Cutruzzolà Francesca
Published: December 2011

DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5′-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.
On the recombination of hydronium and hydroxide ions in water
Authors: Hassanali Ali , Prakash Meher K. , Eshet Hagai , Parrinello Michele
Published: December 2011

The recombination of hydronium and hydroxide ions following water ionization is one of the most fundamental processes determining the pH of water. The neutralization step once the solvated ions are in close proximity is phenomenologically understood to be fast, but the molecular mechanism has not been directly probed by experiments. We elucidate the mechanism of recombination in liquid water with ab initio molecular dynamics simulations, and it emerges as quite different from the conventional view of the Grotthuss mechanism. The neutralization event involves a collective compression of the water-wire bridging the ions, which occurs in approximately 0.5 ps, triggering a concerted triple jump of the protons. This process leaves the neutralized hydroxide in a hypercoordinated state, with the implications that enhanced collective compressions of several water molecules around similarly hypercoordinated states are likely to serve as nucleation events for the autoionization of liquid water.
Visuoauditory mappings between high luminance and high pitch are shared by chimpanzees (Pan troglodytes) and humans
Authors: Ludwig Vera U. , Adachi Ikuma , Matsuzawa Tetsuro
Published: December 2011

Humans share implicit preferences for certain cross-sensory combinations; for example, they consistently associate higher-pitched sounds with lighter colors, smaller size, and spikier shapes. In the condition of synesthesia, people may experience such cross-modal correspondences to a perceptual degree (e.g., literallyseeingsounds). So far, no study has addressed the question whether nonhuman animals share cross-modal correspondences as well. To establish the evolutionary origins of cross-modal mappings, we tested whether chimpanzees (Pan troglodytes) also associate higher pitch with higher luminance. Thirty-three humans and six chimpanzees were required to classify black and white squares according to their color while hearing irrelevant background sounds that were either high-pitched or low-pitched. Both species performed better when the background sound was congruent (high-pitched for white, low-pitched for black) than when it was incongruent (low-pitched for white, high-pitched for black). An inherent tendency to pair high pitch with high luminance hence evolved before the human lineage split from that of chimpanzees. Rather than being a culturally learned or a linguistic phenomenon, this mapping constitutes a basic feature of the primate sensory system.
Kinetic analysis reveals the ordered coupling of translation termination and ribosome recycling in yeast
Authors: Shoemaker Christopher J. , Green Rachel
Published: December 2011

Although well defined in bacterial systems, the molecular mechanisms underlying ribosome recycling in eukaryotic cells have only begun to be explored. Recent studies have proposed a direct role for eukaryotic termination factors eRF1 and eRF3 (and the related factors Dom34 and Hbs1) in downstream recycling processes; however, our understanding of the connection between termination and recycling in eukaryotes is limited. Here, using an in vitro reconstituted yeast translation system, we identify a key role for the multifunctional ABC-family protein Rli1 in stimulating both eRF1-mediated termination and ribosome recycling in yeast. Through subsequent kinetic analysis, we uncover a network of regulatory features that provides mechanistic insight into how the events of termination and recycling are obligately ordered. These results establish a model in which eukaryotic termination and recycling are not clearly demarcated events, as they are in bacteria, but coupled stages of the same release-factor mediated process.
Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development
Authors: Haas Ulrike , Raschperger Elisabeth , Hamberg Mats , Samuelsson Bengt , Tryggvason Karl , Haeggström Jesper Z.
Published: December 2011

Lipoxygenases (LO) are a class of dioxygenases, which form hydroperoxy, hydroxy, and epoxy derivatives of arachidonic acid with distinct positional and stereochemical configurations. In man, there are two known types of 12-LO that are distinguished by their expression patterns and catalytic properties. The platelet 12S-LO plays a role in platelet aggregation and 12R-LO seems to be important for normal skin function. Using BLAST searches of the zebrafish (zf) genome we identified one candidate zf12-LO gene with 43% identity with human 12R-LO at the mRNA level and the deduced primary sequence carried the so called “Coffa” structural determinant (Gly residue) for R stereoselectivity of LOs. However, incubations of recombinant, purified, zf12-LO with arachidonic acid revealed exclusive formation of 12(S)-hydroperoxy-eicosatetraenoic acid. Further studies with immunohistochemistry showed prominent expression of zf12-LO in the cell nuclei of skin epithelium, the epithelial lining of the stomodeum, and the pharyngeal pouches in zf embryos. To probe its function, zf12-LO was subjected to targeted knock-down in zf embryos, resulting in the development of a severe phenotype, characterized by abnormal development of the brain, the eyes, and the tail as well as pericardial and yolk sac edema. Hence, we have identified a unique vertebrate 12S-LO that breaks the current structure-function paradigms for S and R stereo-specificity and with critical roles in normal embryonic development.
Shs1 rounds out the septin repertoire
Authors: Short Ben
Published: December 2011

Study reveals how changes in subunit composition and phosphorylation alter the organization of septin filaments.
Learning from the 2009 Pandemic
Authors: Young Peter
Published: December 2011

When swine flu (H1N1) arrived into Glasgow airport from Mexico on 27 April 2009, the UK was catapulted into its most extensive viral management programme to date. Following the emergence of H5N1 avian flu in the Far East over a decade earlier, health services had been alert to the possibility of a new worldwide pandemic. Although not the anticipated virus, it was thought that this mongrelized swine and human strain could emulate the Spanish flu of 1918. Working as a general practice registrar during this period was challenging and offered a unique learning experience. Here are some important points to help trainees prepare for similar events in future.
A phylogeny of the temperate seabasses (Moronidae) characterized by a translocation of the mt‐nd6 gene
Authors: Williams E. P. , Peer A. C. , Miller T. J. , Secor D. H. , Place A. R.
Published: December 2011

The entire mitochondrial genome of the striped bass Morone saxatilis was sequenced together with the mitochondrial (mt) control regions of the white bass Morone chrysops, white perch Morone americana, yellow bass Morone mississippiensis, spotted seabass Dicentrarchus punctatus, European seabass Dicentrarchus labrax and the Japanese seabass Lateolabrax japonicus. The resultant 17 580 base pair circular genome of M. saxatilis contains 38 genes (13 proteins, 23 transfer RNAs and two ribosomal RNAs) and a control region bordered by the proline and phenylalanine mitochondrial tRNAs. Gene arrangement was similar to other vertebrates, except that the mt‐nd6 gene was found within the control region rather than the canonical position between the mt‐nd5 and mt‐cyb genes. This translocation was found in all the Morone and Dicentrarchus species studied without functional copies or pseudogenes in the ancestral position. In L. japonicus, the mt‐nd6 gene was found in the canonical position without evidence of an mt‐nd6 gene in the control region. A Bayesian analysis of these and published mt‐nd6 sequences from 45 other Perciformes grouped the Morone and Dicentrarchus species monophyletically with a probability of 1·00 with respect to L. japonicus and all other perciforms, and placed the Dicentrarchus species in the basal position. These data reinforce current placement of L. japonicus outside the Moronidae and provide a clear evolutionary character to define this family. The phylogeny of the Moronidae presented here also supports the hypothesis of an anadromous common ancestor to this family that gave rise to the North American estuarine and freshwater species. A series of tandem repeats previously reported in M. saxatilis was found in the control region of all Morone species between the mt‐nd6 and mt‐rnr1 genes, but not in either Dicentrarchus species, which reinforces the continued use of these two separate genera.
Genomic profiling of renal cell carcinoma in patients with end‐stage renal disease
Authors: Inoue Toru , Matsuura Keiko , Yoshimoto Taichiro , Nguyen Lam Tung , Tsukamoto Yoshiyuki , Nakada Chisato , Hijiya Naoki , Narimatsu Takahiro , Nomura Takeo , Sato Fuminori , Nagashima Yoji , Kashima Kenji , Hatakeyama Shingo , Ohyama Chikara , Numakura Kazuyuki , Habuchi Tomonori , Nakagawa Masayuki , Seto Masao , Mimata Hiromitsu , Moriyama Masatsugu
Published: December 2011

The purpose of the present study was to determine the genomic profile of renal cell carcinoma (RCC) in end‐stage renal disease (ESRD) by analyzing genomic copy number aberrations. Seventy‐nine tumor samples from 63 patients with RCC‐ESRD were analyzed by array comparative genomic hybridization using the Agilent Whole Human Genome 4 × 44K Oligo Micro Array (Agilent Technologies Inc., Palo Alto, CA, USA). Unsupervised hierarchical clustering analysis revealed that the 63 cases could be divided into two groups, Clusters A and B. Cluster A was comprised mainly of clear cell RCC (CCRCC), whereas Cluster B was comprised mainly of papillary RCC (PRCC), acquired cystic disease (ACD)‐associated RCC, and clear cell papillary RCC. Analysis of the averaged frequencies revealed that the genomic profiles of Clusters A and B resembled those of sporadic CCRCC and sporadic PRCC, respectively. Although it has been proposed on the basis of histopathology that ACD‐associated RCC, clear cell papillary RCC and PRCC‐ESRD are distinct subtypes, the present data reveal that the genomic profiles of these types, categorized as Cluster B, resemble one another. Furthermore, the genomic profiles of PRCC, ACD‐associated RCC and clear cell papillary RCC admixed in one tissue tended to resemble one another. On the basis of genomic profiling of RCC‐ESRD, we conclude that the molecular pathogenesis of CCRCC‐ESRD resembles that of sporadic CCRCC. Although various histologic subtypes of non‐clear cell RCC‐ESRD have been proposed, their genomic profiles resemble those of sporadic PRCC, suggesting that the molecular pathogenesis of non‐CCRCC‐ESRD may be related to that of sporadic PRCC. (Cancer Sci 2012; 103: 569–576)