bluuebunny/crossref_metadata_2025 huggingface dataset:
239 MAC-T CELLS AS A TOOL TO EXAMINE GENOME EDITING USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)
Authors: Lotti S. N. , Tasan I. , Zhao H. , Wheeler M. B.
Published: December 2015

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.
243 EFFICIENT EDITION OF THE BOVINE PRNP PRION GENE IN SOMATIC CELLS AND IVF EMBRYOS USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 SYSTEM
Authors: Bevacqua R. J. , Fernandez-Martín R. , Savy V. , Canel N. G. , Gismondi M. I. , Kues W. , Carlson D. F. , Fahrenkrug S. C. , Niemann H. , Taboga O. A. , Ferraris S. , Salamone D. F.
Published: December 2015

The rapid introduction of engineered nucleases technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR), provides new opportunities for editing genes in a targeted and rather simple fashion. Few reports are available regarding CRISPR efficiency in domestic species. Here, the CRISPR/Cas9 system was employed to develop knockout and knock-in alleles of the bovine PRNP gene, responsible for bovine spongiform encephalopathy (mad cow disease), both in bovine fetal fibroblasts and in IVF embryos. Five sgRNAs were designed to target a 875-bp region within prnp exon 3; all 5 were co-delivered with hCas9 and a homologous recombination vector carrying gfp (pHRegfp). For cells, 3 transfection conditions were compared: 2 μg of hCas9 + 1 μg of sgRNAs mix ± 2 μg pHREGFP (1X) versus 4 μg of hCas9 + 2 μg of sgRNAs mix ± 4 μg of pHREGFP (2X). For IVF zygotes, cytoplasmic injection was conducted with 2 RNA concentrations: (a) 50 ng μL–1 hCas9 RNA + 25 ng μL–1 sgRNAs mix (RNA1X), ±50 ng μL–1 pHREGFP, and (b) 100 ng μL–1 hCas9 + 50 ng μL–1 sgRNAs mix (RNA2X), ±100 ng μL–1 pHREGFP, which were compared with plasmid injections with 100 ng μL–1 pCMVCas9 + 50 ng μL–1 pU6sgRNAs mix (DNA2X), ±100 ng μL–1 pHREGFP. The pHREGFP was always injected as plasmid, under the same conditions as hCas9. DNA from cells was subjected to PCR, Surveyor assay, and sequence analysis. Embryo analysis was conducted on whole-genome-amplified DNA from blastocysts, followed by PCR assays and sequencing. In cells, 2X transfection resulted in indels and amplification of PCR products of lower MW than the wild-type, indicative of the deletion of a part of the targeted PRNP region. However, it was not possible to detect an effect for 1X transfection. For the group transfected with pHREGFP, insertion of a partial EGFP sequence was detected (383 bp). Regarding embryo injection, higher blastocyst rates were obtained in all groups injected with RNA (Table 1). In 48% (21/43) of the sequenced blastocysts specific gene editing was detected (Table 1). Modifications varied among single base pair shift (3/43; 7%), high level of mismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletion of the 875-bp region (1/43; 2.3%), and partial insertion of 100–498 bp pHREGFP fragments between the HR arms (5/24; 20.8%). Most of these modifications occurred in a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficiently applied for site-specific edition of domestic species genomes.

Table 1.In vitro development and gene editing efficiency of embryos injected with plasmids or RNA coding for CRISPR/Cas9 system targeting PRNP
246 INFLUENCE OF BREED AND SEASON ON IN VITRO EMBRYO PRODUCTION
Authors: Bernal B. , Revol J. , Oviedo J. M. , Tribulo A. , Tribulo H. , Moreria G. , Bó G. A.
Published: December 2015

A retrospective analysis of in vitro production (IVP) data was done to determine the influence of breed and season on the production of viable oocytes and embryos. Cumulus‐oocyte complexes (COC) were obtained from 1946 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in Bos taurus and Bos indicus donors in commercial IVP in Argentina. Frozen-thawed conventional semen was used in beef cattle and conventional (n = 139) and sexed-selected (n = 481) semen in dairy cattle. The COC were classified, matured in B-199 medium, fertilized in IVF-SOF medium (Day 0), and cultured in SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, and saturated humidity for 7 days. The number of viable COC and transferable embryos in each breed and season were compared by ANOVA and means were compared by Fisher’s Least Significant Difference test. Proportions were first transformed by arcsin and then analysed by ANOVA. To simplify the interpretation of the results, breeds were grouped as follows: dairy Bos taurus (Holstein, n = 620), beef Bos taurus (Angus and Bonsmara, n = 229), Bos taurus × Bos indicus (Brangus and Braford, n = 1045), and Bos indicus (Brahman, n = 52). There was no interaction between breed and season for any of the end points analysed (P > 0.1). Mean (± standard error of the mean) numbers of viable COC and transferable embryos were higher (P < 0.01) in Bos indicus × Bos taurus (19.3 ± 0.4 and 5.3 ± 0.2, respectively) and Bos indicus (15.8 ± 1.4 and 6.8 ± 0.9, respectively) than in beef (11.6 ± 0.5 and 3.0 ± 0.2, respectively) and dairy (8.0 ± 0.2 and 1.6 ± 0.1, respectively) Bos taurus donors. Cleavage rates were higher (P < 0.01) in Bos indicus (72%) than in the other breeds (57% for Bos indicus × Bos taurus and dairy Bos taurus and 54% for beef). Transferable embryo rates were higher (P < 0.01) in Bos indicus (41%) and Bos indicus × Bos taurus (30%) than in beef Bos taurus (26%). Dairy Bos taurus had the lowest (P < 0.01) embryo rates of all breeds (21%). In dairy Bos taurus, cleavage rates, the number of embryos produced, and transferable embryo production rates were higher (P < 0.01) when conventional semen was used (62%, 2.8 ± 0.15, and 27%, respectively) compared to sexed-selected semen (55%, 1.3 ± 0.1, and 19%, respectively). With regards to season, the number of viable COC was highest (P < 0.01) in the spring (14.3 ± 0.5), lowest in the summer (11.3 ± 1.0), and intermediate in the fall (12.2 ± 1.2) and winter (13.7 ± 1.2), which did not differ. Although not affected significantly by season, the number of embryos produced was numerically lower in the summer (2.8 ± 0.4) than in the spring (4.2 ± 0.2), winter (4.5 ± 0.5), or fall (4.6 ± 0.5). In conclusion, in vitro embryo production was directly influenced by breed and season. Bos indicus influenced cattle and the spring season were preferable for commercial IVP programs that did not include superstimulation.
247 THE IMPORTANCE OF FOLLICULAR DIFFERENTIATION TO OBTAIN FULLY COMPETENT BOVINE OOCYTES
Authors: Sirard M. A.
Published: December 2015

Making an oocyte with the capacity to form a Day-7 embryo that will implant and produce a live healthy calf is a long and complex process. Since the beginning of bovine IVF, it has been clear that the follicle from which an oocyte is obtained is the most important factor to predict outcome. The blastocyst rate is above 75% with in vivo-matured oocytes, but success varies greatly following in vitro maturation, depending more on follicular status than size. Indeed, aspirating oocytes from non-preovulatory follicles can result in 30% blastocyst development following fertilization but rarely more, unless the follicle is selected. Follicles leading to improved development are either early atretic or exposed to FSH withdrawal. If no exogenous FSH is given, natural early atresia will occur the day after the dominant follicle is selected, but will be limited to a few follicles per wave. If FSH is given to generate a wave of dominant follicles, and then withdrawn, most of the follicles will continue growth and differentiation under basal LH and a maximal oocyte quality will be reached between Day 2 and 3 after the last FSH injection. Under these conditions, the oocyte has the right cascade of signaling to condense its chromatin and to prepare for fertilization. Oocytes obtained from less-prepared follicles are of lower quality, as demonstrated by lower blastocyst rates, lower blastocyst quality, lower implantation rates, and higher abortion rates. Genomic analysis of embryos generated from slaughterhouse-derived oocytes indicates a progressive dysfunction in direct link with the in vitro exposure time. The best way to minimize in vitro-related stress is to begin with a competent oocyte and the best demonstration of that is when some animals (treated with FSH and then coasted) generate 100% blastocyst rate for a recovery of more than 5 oocytes, indicating that the initial quality of oocytes is not much affected by the in vitro culture. It is surprising that blastocyst rates following coasting and in vitro maturation can surpass that observed with Day-7 embryo collections based on the number of ovulations. Finally, new epigenetic analysis will progressively unravel differences of oocytes obtained from different sources and will become an important research tool to assess follicular conditions that will lead to minimal culture stress and optimal embryo quality. If epigenetic programming of the embryo depends on its capacity to deal with the stress created by culture, as has been shown in other species, the initial quality of the oocyte will potentially impact the health status of the newborn calf and resulting adult.
255 SEMINAL PLASMA PROTEINS: FUNCTIONAL ATTRIBUTES AND POTENTIAL MARKERS OF FERTILITY
Authors: Moura A. A.
Published: December 2015

Mammalian seminal plasma is a complex milieu containing, mostly, secretions of the epididymides and accessory sex glands. After ejaculation, proteins of the seminal fluid interact with sperm and participate in several events, including sperm motility, protection against immune reactions, oxidative processes and microorganisms, interaction with the oviduct epithelium, capacitation, acrosome reaction, and fertilization. Given these aspects, the present talk is focused on seminal plasma proteins and cross-species comparisons of their functional attributes. Experiments that will be presented used gel-based and shotgun proteomics. Binder of sperm proteins are major components of ruminant seminal plasma, while spermadhesins are predominant in the boar and deer (Mazama gouazoubira; Mazama nemorivag) seminal fluid. Annexins (rabbits), clusterin (peccaries: Pecari tajacu), and arginine esterase (coatis: Nasua nasua; dogs) are the most abundant proteins in seminal fluid of other species. Such diversity may reflect differences in how paternal factors modulate fertility. Studies have also reported empirical associations between seminal proteins, sperm freezability, and fertility indexes in ruminants and other domestic species. As evidenced by experiments using IVF, mainly in the bovine, specific seminal proteins influence the fertilizing capacity of both epididymal and ejaculated sperm, as well as early embryo development. In summary, fundamental knowledge gathered by studies of seminal plasma proteome can be used to identify molecular markers of fertility. Availability of such markers will potentially improve the outcome of artificial reproductive technologies. Diversity of seminal plasma composition, however, represents challenges for discovery of fertility markers in unique species. Seminal plasma proteins may also be related to new phenotypes, such as bull differences in sperm survival after sexation.
This research was funded in part by the Brazilian Research Councils (CAPES and CNPq) and Ceara State Foundation for Scientific and Technological Development (FUNCAP).
31 INTRAFOLLICULAR TRANSFER OF FRESH AND VITRIFIED IMMATURE BOVINE OOCYTES: AN OPTION FOR EMBRYO PRODUCTION
Authors: Spricigo J. F. W. , Netto S. B. S. , Muterlle C. V. , Rodrigues S. A. D. , Leme L. O. , Guimaraes A. L. , Caixeta F. , Pivato I. , Dode M. A. N.
Published: December 2015

The association of a technique that guarantees the embryo quality of in vivo blastocyst and otherwise allows the increment of embryo production, such as the in vitro model, would result in a healthier and cheaper embryo. Immature oocytes intrafollicular transfer (IOIFT) is a technique in which immature oocytes obtained by ovum pickup are injected into a dominant follicle of a synchronized recipient. We hypothesised that IOIFT could support embryo development even after oocyte vitrification. We aimed to compare IOIFT or traditional in vitro embryo system using fresh and vitrified immature oocytes. Cumulus-oocyte complexes (COC) were obtained from slaughterhouse ovaries; after selection, half of COC were vitrified by cryotop method. Vitrified and fresh oocytes were either cultured in vitro or transferred to a follicle on the recipient ovary. Four groups were used: (1) fresh immature oocytes (VitroF); (2) vitrified/warmed immature oocyte (VitroV), both (1) and (2) were in vitro matured, fertilized, and cultured; (3) fresh immature oocytes submitted to IOIFT (VivoF), and (4) vitrified/warmed immature oocytes submitted to IOIFT (VivoV). Recipients heifers (n = 12) were synchronized with the following protocol: on Day –10 a progesterone device (P4, Primer) was inserted together with the administration of 2 mL of oestradiol benzoate (RIC-BE); at Day –8 the devices were removed simultaneously to the administration of 2 mL of prostaglandin (Veteglan); Day –1, 1 mL of oestradiol benzoate was administered. The COC from VivoF or VivoV groups were injected into dominant follicle (>10 mm), 58 h after P4 removal. The intrafollicular injections were guided by a 7.5-MHz ultrasound vaginal probe (Aloka) using a modified aspiration system. For the injection, 25 COC were placed into a needle (27 G), with 80 μL of follicular fluid. An insulin syringe served to perform the injections. A single dose of semen was used for AI, soon after IOIFT, and embryos were recovered by uterine flushing 7 days later. The results of embryo development and total cell number and apoptotic cells (TUNEL) are present in Table 1. The results obtained for fresh oocytes suggest that IFIOT technique may be an option for bovine embryo production. Despite, it does not improve embryo development or increase total cell number when vitrified and warmed immature oocytes are used.
Table 1.Cleaved and blastocyst rates, total cell number, and apoptotic cell counting of expanded blastocyst of fresh (F) and vitrified (V) cumulus-oocyte complexes that were in vitro (Vitro) or immature oocytes intrafollicular transfer (Vivo) produced
32 EFFECTS OF HIGH HYDROSTATIC PRESSURE ON EXPRESSION PROFILES OF IN VITRO-PRODUCED, VITRIFIED BOVINE BLASTOCYSTS
Authors: Jiang Z. , Harrington P. , Zhang M. , Marjani S. , Kuo L. , Pribenszky C. , Tian X.
Published: December 2015

High hydrostatic pressure (HHP) has been used to enhance stress tolerance and to promote embryo survival before they are subjected to insulting procedures such as cryopreservation. However, the molecular mechanisms of the beneficial effects of HHP are poorly understood. Here in vitro-produced bovine blastocysts were treated with 40, 60, and 80 MPa of HHP for 1 h at either 25 or 37°C, followed by 3 different recovery periods (0, 1, and 2 h) after HHP before vitrification by the solid surface vitrification method (Dinnyes et al. 2000). The re-expansion rates after vitrification-warming were significantly (P < 0.05) higher in embryos treated with 40 or 60 MPa than controls, demonstrating that HHP promotes the in vitro developmental competence of vitrified bovine embryos. However, 80 MPa resulted in significantly reduced re-expansion rates, suggesting that this pressure started to be lethal to bovine blastocysts. In addition, no significant difference was found on re-expansion rates between 25 and 37°C; data were therefore combined for the 2 temperatures. Microarray analysis revealed a total of 399 differentially expressed transcripts, representing 254 unique genes, among different treatment groups. Gene ontology analysis revealed that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes involved in cell death and apoptosis, and up-regulation of RNA processing, cellular growth, and proliferation. Moreover, gene expression was also changed by the length of the recovery time after HHP. The significantly over-represented groups are apoptosis and cell death in the 1-h group, and protein folding, response to unfolded protein, and cell cycle in the 2-h group. Although 80 MPa also up-regulated expression of genes for apoptosis, but it also significantly down-regulated genes for protein folding and cell cycle, which may explain why these embryos stopped developing. Taken together, these data suggest that HHP induces specific responses in vitrified bovine blastocysts and promotes their developmental competence through modest transcriptional reprogramming.
33 POST-THAW VIABILITY OF BOVINE EMBRYOS PRODUCED IN VITRO FOLLOWING TREATMENT WITH ASCORBIC ACID, DITHIOTHREITOL, AND CASPASE-3 INHIBITOR DURING CRYOPRESERVATION
Authors: Carrascal-Triana E. L. , Zolini A. M. , Ruiz A. , Penitente-Filho J. M. , Torres C. A. A. , Block J.
Published: December 2015

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P < 0.05) re-expansion and hatching rates at 24, 48, and 72 h compared with embryos not treated with ascorbic acid (Table 1). The optimal concentration of ascorbic acid for post-thaw cryosurvival was 0.1 mM. In particular, embryos treated with 0.1 mM ascorbic acid during cryopreservation had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h, as well as hatching rates at 48 and 72 h, compared with control-treated embryos (Table 1). There was no effect of treatment with dithiothreitol or Z-DEVD-FMK on re-expansion or hatching rates at 24, 48, or 72 h after thaw. In conclusion, addition of ascorbic acid to freezing medium improves the cryosurvival of bovine embryos produced in vitro. Further research is necessary to determine whether treatment with ascorbic acid can increase pregnancy rates.
Table 1.Effect of ascorbic acid during cryopreservation of bovine embryos (%, means ± standard error of the mean)
35 EFFECTS OF THAWING TEMPERATURE OF FROZEN SEMEN ON VIABILITY OF REFROZEN AND THAWED CHICKSO (KOREAN BRINDLE CATTLE) AND KOREAN ALBINO CATTLE SPERMATOZOA
Authors: Kim S. W. , Choe C. Y. , Kim D. K. , Choi A. R. , Seong H. H.
Published: December 2015

Germplasm cryopreservation from a desired species with agricultural and genetic importance would protect them from the risk for extinction. Semen freezing from Korean native cattle would be a good approach for protecting genetic resources due to their limited numbers. It has been known that sperm could resist cryo-damages by freeze-thaw cycles. Thus, we performed 2 refreezing experiments with different initial thawing temperatures using frozen Korean native cattle semen. A total of 5 Hanwoo, Korean Albino, and brindle cattle were used as semen donors. After thawing by using 5°C/2 min or 37°C/40 s with cooling rates, the semen was diluted with the same volume of cryo-media in the first thawing temperature and refrozen. Sperm motilities were determined and compared between animals and groups after rethawing. The mean sperm concentration and motility was 45 × 106 mL–1 (range 2.3 to 89 × 106 mL–1) and 40% (range 13 to 55%). Mean values of motility and viability of sperm that underwent second preservation were significantly higher in 5°C than in 37°C (P < 0.01). However, the activity of viable sperm thawed at 5°C was significantly decreased before refreezing. It is estimated that refreezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa. The higher motility and viability of refrozen semen could be obtained with 5°C thawing procedure for reuse of frozen semen.
44 EFFECT OF CRYOPROTECTANT AGENTS IN EQUINE OVARIAN BIOPSY FRAGMENTS
Authors: Gastal G. D. A. , Alves B. G. , Paiva S. O. , Alves K. A. , de Tarso S. G. S. , Ishak G. , Bashir S. T. , Gastal E. L.
Published: December 2015

Studies on ovarian tissue preservation in horses are scarce, and only one research group (Devireddy et al. 2006 Mol. Reprod. Develop. 73, 330–341) has tested the effect of the cryoprotectant agents in equine ovarian tissue. The purpose of this study was to evaluate (1) the effect of cryoprotectant agents in the morphology of equine preantral follicles (PAF) and structure of the ovarian tissue, (2) the relationship between ovarian stromal cell density and PAF density, and (3) the association of ovarian stromal cell density with PAF morphology after exposure to cryoprotectant agents. Three independent experiments with identical methodologies were performed in this study. Each experiment was composed by one cryoprotectant agent (dimethyl sulfoxide, ethylene glycol, propylene glycol) performed in 3 replicates. Ovarian biopsy fragments (1.5 × 1.5 × 10 mm) were harvested from 6 mares and submitted to 4 times of exposure (0, 10, 15, 20 min) at room temperature in a solution of α-minimal essential medium (MEM)+ and one cryoprotectant (dimethyl sulfoxide, ethylene glycol, or propylene glycol) at a concentration of 1.5 M. The cryoprotectant removal was performed by a 3-step (5 min each) equilibration process using (1) α-MEM + 0.5 M sucrose, (2) α-MEM + 0.25 M sucrose, and (3) α-MEM alone after each time of exposure. All fragments were fixed in Bouin’s solution for histological analysis. Data were analysed by ANOVA, Fisher’s exact test, Spearman’s rank correlation, and power test. The PAF density, stromal cell density, and area of the fragments were not affected (P > 0.05) by any of the cryoprotectant agents throughout the time of exposure. However, the morphology of the PAF was affected (P < 0.05) by the cryoprotectant agents. In the propylene glycol and dimethyl sulfoxide, higher (P < 0.05) percentages of abnormal PAF were observed at 10 and 20 min of exposure, respectively. However, the PAF morphology in the ethylene glycol treatments was not affected (P > 0.05) throughout the times of exposure. Strong correlations (range, 0.57 to 0.77; power, 96 to 99%) were identified between PAF density and stromal cell density in all experiments. When stromal cells were classified into groups of density (i.e. low, 25.67–34.66; medium, 34.67–43.66; and high, 43.67–52.67 cells/2500 μm2), only the high stromal cell density was positively correlated (P < 0.001) with the PAF density; however, no correlation between stromal cell density and PAF morphology was observed. In conclusion, (1) ethylene glycol seems to be a less harmful cryoprotectant agent to equine PAF, (2) exposure to cryoprotectant agents did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAF.
50 ESTIMATION OF CHROMATIN ABNORMALITY OF OGYE ROOSTER SEMEN WITH Diff-Quik STAINING
Authors: Kim S. W. , Choi A. R. , Choe C. Y. , Kim D. K. , Seong H. H. , Kim J. D.
Published: December 2015

The abnormality of Ogye rooster sperm chromatin could be detected by simple sperm staining. In this abstract, a Diff-Quick staining kit was tested for assessment of chicken sperm quality. Using a standard bright-field microscope, Diff-Quik stains can be reproducibly, easily, and routinely monitored with simple staining. The presence of abnormal chromatin staining of rooster sperm was determined by darker stain in head. In the fresh semen, the viabilities of 3 tested Ogye spermatozoa were 93.53, 82.42, and 90.63%, and normal chromatin rates were 87.96, 74.25, and 85.10%, respectively. However, after cryopreservation, the rates of viability of thawed semen were reduced to 69.58, 61.98, and 72.20%, and normal chromatin rate also reduced to 58.91, 48.49, and 63.34%. A significant correlation between live sperm and normal sperm nuclei was 0.875 in fresh semen and 0.513 in frozen semen. After incubation of sperm at 37°C for 5 min, the rates of viability, chromatin normality, and sperm head activity were shown as 90.63 ± 1.28%, 82.44 ± 8.09%, and 66.68 ± 10.29% in fresh semen. However, the rates of thawed semen were reduced to 67.92 ± 7.55%, 56.92 ± 12.15%, and 47.32 ± 5.02%, respectively. The relationship between chromatin normality and sperm head movements in fresh and thawed semen were 0.564 and 0.540, respectively. With these results, the chicken sperm normality could be assessed by the Diff-Quik staining, which could be used for chromatin status of sperm head and activated morphology of live spermatozoa, as a simple and rapid staining method.
61 UNVEILING THE ROLE OF LIPIDS IN ORGANOGESIS: MOLECULAR ANATOMY BY DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY IMAGING MASS SPECTROMETRY
Authors: Pirro V. , Favaron P. O. , Ferreira C. R. , Eberlin L. S. , Barreto R. S. , Cooks R. G. , Miglino M. A.
Published: December 2015

Even though the role of lipids in pandemic diseases such as obesity and diabetes is a focus of increasing research, the role of lipids during organogenesis, when diverse diseases may be triggered, is unexplored. Also, pig embryonic tissues represent an attractive option for organ transplantation. This study introduces a detailed morphological analysis of swine fetal tissues with matching location of lipids acquired by desorption electrospray ionization mass spectrometry (DESI-MS) imaging for the study of differential distribution of free fatty acids (FFA) and phospholipids (PL) in specific organs during fetal development. Samples from a pig fetuses around Day 50 of pregnancy were sectioned at a cryotome and mounted onto glass slides. Fixative agents were not used. DESI-MS images were run with a step size of 300 µm using a morphologically friendly (non-destructive) solvent combination, namely dimethylformamide/acetonitrile 1 : 1 (v/v). Data were acquired in the negative ion mode in the m/z range of 150 to 1000 from different sections representing the whole swine fetus body. Ion images were constructed using BioMAP software. After imaging, the whole-body tissue samples were stained with hematoxylin and eosin (H&E) and were overlaid to the DESI-MS lipid images. Differential distribution of FFA, phosphatidylcholines (PC), phosphatidylserines (PS), sulphatides (ST), and phosphatidylinositols (PI) was observed among organs, especially on nervous and circulatory systems, and digestive glands. Most lipids concentrated in the brain, spinal cord, and digestive glands such as the liver. For example, arachidonic acid was most abundant in neuronal tissue, whereas docosahexaenoic acid predominated in the liver and digestive glands. Distribution of PS (36 : 1) of m/z 788 was observed in all tissues except for the digestive system, but PS (40 : 6) of m/z 834.7 was exclusive of brain and spinal cord. Lipids related to brain and spinal cord were mostly polyunsaturated fatty acids as well as specific PS lipids. Arachidonic and eicosatrienoic acids are more concentrated in hindbrain and spinal cord, whereas PS was more abundant in the brain than in the spinal cord. There is no information on PS chemical composition during brain and spinal cord development, but PS concentration in the nervous tissue membranes varies with age, brain areas, cell type, and subcellular components. Several reports indicate that alteration in PS synthesis might participate in the mechanism of brain damage. Also, PS has been found to be altered in brain tumours. Oleic acid, fatty acid dimers, and the signalling lipid PI (38 : 3) were most significant for the digestive system and liver. Liver is one of the main organs involved in fatty acid metabolism (besides adipose tissue and muscle). By overlying morphological and molecular information, lipids seem to be a major player in the organogenesis process.
66 CUMULUS-OOCYTE-COMPLEX SECRETIONS FROM THE FIRST 8 HOURS OF IN VITRO MATURATION AFFECT OOCYTE DEVELOPMENTAL COMPETENCE
Authors: Uhde K. , van Tol H. T. A. , Stout T. A. E. , Roelen B. A. J.
Published: December 2015

Mammalian oocytes are surrounded by cumulus cells, forming a structure known as the cumulus-oocyte complex (COC). Cumulus cells play important protective functions during oocyte maturation, for example, protecting the oocyte against reactive oxygen species. However, it is not yet fully understood how the cumulus complex modulates the developmental competence of the enclosed oocyte. It was investigated whether direct contact between an oocyte and its cumulus cells is essential throughout the maturation process. To this end, bovine oocytes aspirated from ovarian follicles were matured in vitro. Eight hours after the onset of maturation the cumulus cells were removed, and the oocytes either placed back in the original medium or cultured further in fresh maturation medium. In all experiments, COCs/oocytes were matured for 23 h in M199 supplemented with 0.05 IU FSH and penicillin/streptomycin. All experiments were performed in triplicate, with 35 to 45 COCs per group. Student’s t-test was used for a paired comparison. Denudation after 8 h and return to the same maturation medium had no effect on the cleavage rate (93%) compared with culture without denudation (90.7%). Only if the oocytes were transferred to fresh medium did the cleavage rate decrease slightly (75.4%; P = 0.038). By contrast, blastocyst formation was reduced nearly four times if COCs were denuded before being returned to the medium, compared with controls (14% v. 50.8%; P < 0.001). If the oocytes were transferred to fresh medium after denudation, very few blastocysts resulted (0.9%; P < 0.001). In a second study, oocytes denuded immediately after removal from the follicle were matured in the absence or presence of cumulus cells in a Corning® Transwell® system. Culturing denuded oocytes in the presence of cumulus cells resulted in similar cleavage rates (83.5%) to control conditions (84.8%). However, blastocyst formation was markedly lower (4.3%) than for controls (29.6%; P = 0.003). We conclude that COCs secrete substances during the first 8 h of maturation that are beneficial for oocyte acquisition of developmental competence. Moreover, intimate contact between the cumulus cells and oocyte is essential.
This work was supported by EU FP7 EpiHealthNet, PITN-GA-2012–317146.
77 USE OF SEMINAL PLASMA TO IMPROVE REPRODUCTIVE PERFORMANCE IN ALPACAS (VICUGNA PACOS) FOLLOWING NATURAL MOUNTING
Authors: Huanca W. , Turin J. , Mamani C. , Sanchez R. , Huanca W. F. , Huanca T.
Published: December 2015

Alpacas are classified as induced ovulators, and an external stimulus is required for the occurrence of ovulation. The seminal plasma (SP) of camelids contains a protein identified as β nerve growth factor with the capacity to induce ovulation and corpus luteum formation. Alpacas have a poor reproductive efficiency and high embryo mortality rate. A study was designed to evaluate the use of SP to improve reproductive performance post natural mating (Exp. 1) and with different mounting time (MT; Exp. 2). Experiment 1: Nonpregnant alpacas (n = 117) exhibiting a dominant follicle = 7 mm, detected by transrectal ultrasonography, were bred by natural mating and then assigned randomly to 1 of 3 treatments: T1 (n = 40), 1 mL of SP IM; T2 (n = 39), 1 mL of GnRH analogue (0.0042 mg of buserelin acetate), IM; and T3 (n = 38), control. Experiment 2: Nonpregnant alpacas (n = 180) exhibiting a follicle = 7 mm were assigned randomly to the following treatments: T1 (n = 30): MT 5 min; T2 (n = 30): MT 5 min + 1 mL of SP; T3 (n = 30): MT 10 min; T4 (n = 30): MT 10 min + 1 mL of SP; T5 (n = 30): MT > 15 min; T6 (n = 30): MT > 15 min + 1 mL of SP. The same male was used for mating females in T1 and T2, T3 and T4, and T5 and T6, respectively. Animals were evaluated by ultrasound with an Aloka SSD 500 (Aloka, Tokyo, Japan) and 5.0-MHz linear transducer on Day 25 for pregnancy diagnosis. In Exp. 1 the conception rate was 67.5, 51.3, and 55.3% for T1, T2, and T3, respectively (P > 0.05). In Exp. 2 the conception rate was 46.2, 67.9, 57.1, 73.3, 61.8, and 72.7% for T1, T2, T3, T4, T5, and T6 (P < 0.05) and was significantly different between T1 v. T2 and T3 v. T4. The results suggest that the use of SP could be one method to improve reproductive performance in alpacas.
Research was supported by Project N° 405-PNICP-PIAP-UNMSM.
82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT
Authors: Spate L. D. , Redel B. K. , Prather R. S.
Published: December 2015

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst.
Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).
Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21
Authors: Zhang Cuiyan , Li Xiaotong , Adelmant Guillaume , Dobbins Jessica , Geisen Christoph , Oser Matthew G. , Wucherpfenning Kai W. , Marto Jarrod A. , Kaelin William G.
Published: December 2015

Significance

Many short-lived proteins perform important cellular functions that must be tightly regulated. EID1 is an unstable protein implicated in the control of transcription, differentiation, and genomic integrity. EID1 is particularly unstable in G0 cells. Here we show that an SCF complex containing the orphan F-box only protein FBXO21 is the ubiquitin ligase that recognizes EID1 in both cycling and G0 cells and that it recognizes a modular, peptidic domain within EID1. This degron overlaps with the EID1 region that binds to the retinoblastoma tumor suppressor protein (pRB) and to melanoma-associated antigen (MAGE) proteins, suggesting that multiple proteins compete for binding to EID1 and secondarily influence its ubiquitinylation. This study sheds light on the regulation of EID1, the function of FBXO21, and control of protein turnover by ubiquitin ligases.
A New Interpretation of the Representational Theory of Measurement
Authors: Heilmann Conrad
Published: December 2015

On the received view, the Representational Theory of Measurement reduces measurement to the numerical representation of empirical relations. This account of measurement has been widely criticized. In this article, I provide a new interpretation of the Representational Theory of Measurement that sidesteps these debates. I propose to view the Representational Theory of Measurement as a library of theorems that investigate the numerical representability of qualitative relations. Such theorems are useful tools for concept formation that, in turn, is one crucial aspect of measurement for a broad range of cases in linguistics, rational choice, metaphysics, and the social sciences.
Beyond Fab Four
Authors: Babichev E , Charmousis C , Langlois D , Saito R
Published: December 2015

We show that the two additional Lagrangians that appear in theories beyond Horndeski can be reexpressed in terms of simple generalizations of the ‘John’ and ‘Paul’ terms of the Fab Four theories. We find that these extended Fab Four satisfy the same properties of self-tuning as the original Fab Four.
The irreducible representations of the alternating group which remain irreducible in characteristic 𝑝
Authors: Fayers Matthew
Published: December 2015

Let

p
p

be an odd prime, and

A

n

\mathfrak {A}_n

the alternating group of degree

n
n

. We determine which ordinary irreducible representations of

A

n

\mathfrak {A}_n

remain irreducible in characteristic

p
p

, verifying the author’s conjecture from 2010. Given the preparatory work done in 2010, our task is to determine which self-conjugate partitions label Specht modules for the symmetric group in characteristic

p
p

having exactly two composition factors. This is accomplished through the use of the Robinson–Brundan–Kleshchev ‘

i
i

-restriction’ functors, together with known results on decomposition numbers for the symmetric group and additional results on the Mullineux map and homomorphisms between Specht modules.
Nonnegativity constraints for structured complete systems
Authors: Powell Alexander , Spaeth Anneliese
Published: December 2015

We investigate pointwise nonnegativity as an obstruction to various types of structured completeness in

L
p

(

R

)

L^p(\mathbb {R})

. For example, we prove that if each element of the system

{

f
n

}

n
=
1

∞

⊂

L
p

(

R

)

\{f_n\}_{n=1}^\infty \subset L^p(\mathbb {R})

is pointwise nonnegative, then

{

f
n

}

n
=
1

∞

\{f_n\}_{n=1}^{\infty }

cannot be an unconditional basis or unconditional quasibasis (unconditional Schauder frame) for

L
p

(

R

)

L^p(\mathbb {R})

. In particular, in

L
2

(

R

)

L^2(\mathbb {R})

this precludes the existence of nonnegative Riesz bases and frames. On the other hand, there exist pointwise nonnegative conditional quasibases in

L
p

(

R

)

L^p(\mathbb {R})

, and there also exist pointwise nonnegative exact systems and Markushevich bases in

L
p

(

R

)

L^p(\mathbb {R})

.
Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance
Authors: Turney Stephen G. , Ahmed Mostafa , Chandrasekar Indra , Wysolmerski Robert B. , Goeckeler Zoe M. , Rioux Robert M. , Whitesides George M. , Bridgman Paul C.
Published: December 2015

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation.
LoCuSS: Testing hydrostatic equilibrium in galaxy clusters
Authors: Smith G. P. , Mazzotta P. , Okabe N. , Ziparo F. , Mulroy S. L. , Babul A. , Finoguenov A. , McCarthy I. G. , Lieu M. , Bahé Y. M. , Bourdin H. , Evrard A. E. , Futamase T. , Haines C. P. , Jauzac M. , Marrone D. P. , Martino R. , May P. E. , Taylor J. E. , Umetsu K.
Published: December 2015

We test the assumption of hydrostatic equilibrium in an X-ray luminosity selected sample of 50 galaxy clusters at 0.15 < z < 0.3 from the Local Cluster Substructure Survey (LoCuSS). Our weak-lensing measurements of M500 control systematic biases to sub-4 per cent, and our hydrostatic measurements of the same achieve excellent agreement between XMM–Newton and Chandra. The mean ratio of X-ray to lensing mass for these 50 clusters is $\mathrel {\beta _{\rm X}}\;= 0.95\pm 0.05$, and for the 44 clusters also detected by Planck, the mean ratio of Planck mass estimate to LoCuSS lensing mass is $\mathrel {\beta _{\rm P}}\;= 0.95\pm 0.04$. Based on a careful like-for-like analysis, we find that LoCuSS, the Canadian Cluster Comparison Project, and Weighing the Giants agree on $\mathrel {\beta _{\rm P}}\;\simeq 0.9{\rm -}0.95$ at 0.15 < z < 0.3. This small level of hydrostatic bias disagrees at ∼5σ with the level required to reconcile Planck cosmology results from the cosmic microwave background and galaxy cluster counts.