bluuebunny/crossref_metadata_2025 huggingface dataset:
169 MATURE OOCYTES TRIGGER THE RELEASE OF BOVINE SPERM FROM AN IMMOBILIZED OVIDUCT GLYCAN
Authors: Elsokary M. M. , Miller D. J.
Published: December 2016

After mating, sperm move from the site of semen deposition towards the oviduct, where sperm are stored before fertilization. Much of the literature indicates that oviduct glycans bind sperm to retain them in the oviduct reservoir. We have discovered that a specific oviduct glycan on the epithelium (sulfated Lewis A trisaccharide; suLeA) binds bovine sperm and maintains sperm viability. But the way in which sperm are released from oviduct epithelial glycans to fertilize oocytes is enigmatic. In this study, we tested the hypothesis that oocytes signal the release of sperm from an oviduct glycan (suLeA) on a bead and that the released sperm could fertilize the oocytes. Sepharose beads with avidin on their surface were used to immobilize biotinylated suLeA. After coupling, the beads were washed. Frozen bovine semen was thawed, and sperm were washed and allowed to bind to suLeA-coated beads for 1 h in capacitating medium at 39°C. Mature bovine oocytes were then prewashed with the same medium (without heparin) and added to droplets with sperm bound to beads and incubated for 18 h at 5% CO2 at 38.5°C. Bound sperm (mean = 4 per bead) were enumerated before and after oocyte addition. A control using dead (fixed) oocytes was also used. Three independent replicates were tested using 125 oocytes for each treatment. Data were analysed by ANOVA. The addition of live oocytes released 68.1% of the sperm bound to beads, whereas only 18.8% of sperm were released by addition of dead oocytes (control; P < 0.05). If heparin for sperm capacitation (3.125 g mL−1 final concentration) was added concurrently with the addition of live oocytes to sperm bound to beads, the majority of bound sperm were released (87.7%), whereas dead oocytes (control) released fewer sperm (67.3% sperm, P < 0.05). Thus, both heparin and oocytes released sperm from beads independently but, together, they released the greatest number of sperm. Altogether, our results indicate that, in addition to heparin, mature oocytes induce sperm release from immobilized oviduct glycans for fertilization. This is the first demonstration that oocytes can signal sperm release from oviduct glycans in any mammal.
178 TRANSCRIPTIONAL DIFFERENCES IN GENES RELATED TO GLUCOSE METABOLISM BETWEEN COMPETENT AND INCOMPETENT BOVINE IMMATURE CUMULUS-OOCYTE COMPLEXES SELECTED BY BCB
Authors: Lamas-Toranzo I. , Martínez-Corona D. A. , Pericuesta E. , Bermejo-Álvarez P.
Published: December 2016

In vitro maturation is a key step of in vitro embryo production, being the main factor responsible for the low blastocyst yield. In vitro maturation requires the selection of competent immature cumulus-oocytes complexes (COC), which is usually accomplished based on morphological criteria and follicle size. Competent immature COC have finished their growth phase and show decreased G6PDH activity. Brilliant cresol blue (BCB) is a dye that is degraded by G6PDH and, therefore, can be used to distinguish COC that have finished their growth phase (BCB+) from those that are still growing and are less competent (BCB-). The objective of this study was to determine the metabolic differences between BCB- and BCB+ COC by performing a transcriptional analysis of genes related to glucose metabolism. The COC obtained from slaughterhouse ovaries were selected based on BCB staining. The BCB+ and BCB- COC were fertilized and cultured in vitro to determine the differences in developmental ability. For gene expression analysis, BCB+ and BCB- COC were denuded by vortexing and 5 groups of 10 oocytes; their corresponding cumulus cells per group were snap-frozen until analysis. Messenger RNA was extracted by Dynabeads (Dynal Biotech, Lake Success, NY, USA) and relative mRNA abundance was analysed by quantitative PCR using PPIA1 as housekeeping. Statistical differences were determined based on ANOVA (P < 0.05). The genes analysed were G6PDH, its positive regulator SIRT2, 2 glucose transporters SLC2A1 and SLC2A5, 2 genes involved in anaerobic glycolysis GAPDH and LDHA, 2 genes related with Krebs cycle CS and ATP5A1, and 1 gene related to glutathione metabolism GPX1. As expected, the BCB+ group showed a higher cleavage rate (85.6 ± 1.8 v. 74.2 ± 1.3%, BCB+ v. BCB-; P < 0.05) and blastocyst yield (Day 9: 33.3 ± 3.8 v. 16.1 ± 1.4%, BCB+ v. BCB-; P < 0.05) compared with BCB-. Genes SIRT2, GAPDH, and LDHA were significantly up-regulated in BCB- cumulus cells (SIRT2: 1 ± 0.04 v. 1.45 ± 0.21; GAPDH: 1 ± 0.17 v. 1.46 ± 0.15; LDHA: 1 ± 0.22 v. 1.65 ± 0.12; BCB+ v. BCB-; P < 0.05), whereas no significant differences were found in the other genes and in oocytes. In conclusion, the differences in G6PDH activity between BCB+ and BCB- COC are not controlled by G6PDH transcript abundance, but seem to be mediated by SIRT2 regulation of G6PDH activity. The BCB- cumulus cells showed an up-regulation of GAPDH and LDHA, suggesting a higher activity of anaerobic glycolysis in BCB- COC compared with BCB+.
193 ROBUST PROPAGATION OF SELF-RENEWING PORCINE NEURAL STEM CELLS ISOLATED FROM TRANSGENIC PIGS WITH A GFAP-CreERT2 SYSTEM CAPABLE OF CONTROLLING THE EXPRESSION OF EGFP GENES
Authors: Kim E. , Kim H. , Hyun S.-H.
Published: December 2016

Ample evidence has demonstrated the important roles of pigs because their anatomical, immunologic, and physiological characteristics are fairly similar to humans. In particular, their gyrencephalic brain are more comparable to humans than rodents with similar grey and white matter composition and size. In this study, we isolated and propagated the neural stem cells (GFAP-CreERT2-NSCs) from the transgenic piglet with expression of CreERT2, a fusion protein of the DNA recombinase Cre and mutated ligand-binding domain of the human oestrogen receptor, under the control of the GFAP promoter. The primary culture from tissue of porcine CreERT2 brain led to floating spherical masses of cells that revealed similar morphology and size distribution to neurospheres reported by previous studies. Quantitative analysis indicated a yield of 2.50 ± 0.44 primary spheres per 1,000 viable cells from the neocortex, versus 12.92 ± 1.67 primary spheres per 1,000 viable cells from the periventricular region (PVR) including subventricular zone. Secondary spheres (6.67 ± 1.10 spheres from neocortex versus 23.08 ± 1.96 spheres from PVR cells) were formed from primary spheres at 10 days after passage. Tertiary spheres (8.42 ± 0.99 spheres from neocortex versus 23.08 ± 1.91 spheres from PVR cells) could also be obtained after a second passage, indicating that they were proliferating in vitro. The CreERT2-NSCs showed normal 36+XY karyotype and representative NSC markers, such as NESTIN, SOX2, and VIMENTIN. After differentiation, we were able to obtain populations of astrocytes and neurons expressing GFAP and TUJ1, respectively. In summary, we verified and propagated the isolated GFAP promoter-driven CreERT2-NSCs, which would be considered a promising source of cells for treatment of central nervous system diseases.
197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS
Authors: Bertels J. C. , Rubessa M. , Schreiber S.R. , Wheeler M. B.
Published: December 2016

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mM zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mM zinc, and 0.08 mM zinc, respectively. The largest nodule was found in the 0.08 mM zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix.

Table 1.

The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups
201 EFFECT OF A SINGLE SUBCUTANEOUS INJECTION OF FSH AND TIMING OF PROSTAGLANDIN F2α ADMINISTRATION ON SUPEROVULATORY RESPONSE IN JAPANESE BLACK COWS
Authors: Sugawara M. , Kaneda Y. , Miyoshi A. , Sekizawa H. , Dochi O.
Published: December 2016

Superovulation treatment using FSH requires injection twice a day, for 3 to 4 days. This conventional method requires frequent handling of donors and higher labour costs. Therefore, simplification of the superovulation treatment protocol is needed to reduce animal handling and labour costs. The objective of this study was to investigate the effect of a single subcutaneous FSH injection and the timing of prostaglandin F2α (PGF; cloprostenol) administration on the superovulatory response in Japanese Black cows (Hiraizumi et al. 2015 Theriogenology 83, 466–473) and to determine whether the superovulation treatment protocol can be used in on-farm conditions. A total of 270 Japanese Black cows were used in this study. Twenty Armour units of pFSH dissolved in 30 mL of saline was injected subcutaneously in the neck region. In Experiment 1, 32 cows received an intravaginal progesterone device (CIDR) at random stages of the oestrous cycle (Day 0), and 2 mg of oestradiol benzoate on Day 1 (24 h after CIDR insertion). On Day 6, FSH was injected subcutaneously, and 16 cows were simultaneously injected with 0.5 mg of PGF (0-h PGF, Group A); the other 16 cows were injected with 0.5 mg of PGF at 48 h (on Day 8) after FSH injection (48-h PGF, Group B). The CIDR was removed at 60 h after FSH injection and AI was done 42 to 48 h after CIDR removal. Embryo collections were performed 7 days after AI. In Experiment 2, 238 cows were used in farm conditions. The cows were superstimulated using the same protocol as that used for Group A. Data were analysed by ANOVA for the mean numbers of collected ova/embryos and transferrable embryos and chi-square test for the proportion of transferrable embryos. In Experiment 1, there were no differences in the mean numbers of ova/embryos collected (16.9 ± 12.3 v. 16.1 ± 17.1) or transferrable embryos (11.1 ± 9.5 v. 7.2 ± 6.2). However, the proportion of transferrable embryos for Group A was significantly higher than that of Group B (65.9 v. 44.7%; P < 0.01). In Experiment 2, the mean numbers of ova/embryos collected and transferrable embryos were 15.7 ± 13.3 and 6.8 ± 7.8, respectively. These results showed that a superovulation treatment protocol involving a single subcutaneous injection of FSH with simultaneous PGF injection can be effectively used for Japanese Black cows under on-farm conditions.
207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM
Authors: Pinzon C. A. , Snyder M. , Pryor J. , Thompson B. , Golding M. , Long C.
Published: December 2016

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.
41 THE EFFECT OF ULTRARAPID VITRIFICATION FOR SHEEP OOCYTE VIABILITY
Authors: Toishibekov M. , Toishibekov Y. , Yermekova M.
Published: December 2016

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, have stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal’s body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). Ovaries from 19 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% fetal bovine serum, 5 μg mL−1 FSH, 5 μg mL−1 LH, 1 μg mL−1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then, oocytes were placed for 10 min in medium with Hoechst 33342 (3 μg mL−1) and cytochalasin B (7 μg mL−1) to facilitate enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into a cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation, the oocytes were activated for extrusion of the second polar body in 1 mg mL−1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL−1). After activation, the MPII were washed and cultured for 20 h. The control group received the same treatment but were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed that expulsion of the second polar body after activation was observed in more than 59.7% of the MPII that were not vitrified (control group), whereas 37.7% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.
55 SEMINAL PLASMA COMPONENTS AND THEIR RELATIONSHIP WITH STALLION SEMEN FREEZABILITY
Authors: Usuga A. , Restrepo G. , Rojano B.
Published: December 2016

Oxidative stress has been identified as a major cause of low seminal fertility. Among the components of stallion seminal plasma, some enzymatic and non-enzymatic antioxidants have been identified, which protect sperm from injurious effects of reactive oxygen species (ROS); however, the characterisation of these components is still in preliminary stages, as well as their relationship with freezability. The aim of this study was to evaluate the effect of some components of seminal plasma (SP) on stallion semen freezability. Semen of 30 Colombian Creole horses, and a total of 60 ejaculates, were collected. Semen was centrifuged to recover the SP. It was lyophilized and some components were assayed: total protein concentration (TP) by Bradford assay, CRISP3 protein concentration by ELISA, vitamin C (CVIT), vitamin E (EVIT) and vitamin A (AVIT), by HPLC; content of iron (Fe), copper (Cu), magnesium (Mg) and Zinc (Zn) by atomic absorption spectroscopy flame. Semen was supplemented with 10% stallion lyophilized SP and cryopreservation was performed. Post-thaw, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF), were assessed using a computer-assisted sperm analysis (SCA®, Microptic SL, Barcelona, Spain). Sperm viability (SV) was determined by the Live/Dead kit (Molecular Probes Inc., Eugene, OR, USA). Normal sperm morphology (NM) was performed by the supravital technique and plasmatic membrane integrity (MI) was evaluated by the hypo-osmotic test. For statistical analysis, completely randomised mixed models were fitted. Levels according to the concentration of components of SP (high, medium, and low) were established. Comparisons of the means between levels were done with Tukey’s test. The significance level used for all assessments was P < 0.05. Means for TP of 0.35 mg BSA/g, CRISP3 of 55.22 ng/mg, CVIT of 2.66 mg/g, EVIT of 72.36 µg/g, AVIT of 37.37 µg/g, Fe of 17.37 mg/kg, Cu of 33.64 mg/kg, Mg of 109.08 mg/kg, and Zn of 0.49 g/100 g of SP were found. We found that a high level of CRISP3, AVIT, Cu, and Fe had higher results for post-thaw TM, PM, NM; medium levels of TP and Mg showed higher post-thaw TM, PM, NM, and MI; and lower levels of Zn had better results for post-thaw TM, PM, VCL, and VAP. In contrast, high and medium levels of CVIT had a deleterious effect on post-thaw TM, PM, SV, NM, and MI. We concluded that there is a relationship between concentrations of seminal plasma components and stallion semen freezability.
59 EFFECT OF PELLET VOLUME AND THAWING TEMPERATURE ON VITRIFICATION EFFICACY WITH DOMESTIC CAT SEMEN COLLECTED VIA URETHRAL CATHETERIZATION
Authors: Moresco A. , Bateman H. L. , Newsom J. , Swanson W. F.
Published: December 2016

Historically, semen banking in felids has required sample collection via electroejaculation followed by sperm freezing in straws over LN2 vapor. Recent modifications include urethral catheterization of males treated with α-2 agonists for semen recovery and vitrification of cat sperm by suspension in a sucrose-based cryomedium and direct pelleting into LN2. In combination, these latter methods greatly simplify semen cryopreservation in cats but protocols need to be optimized for applied usage. In the present study, our goal was to assess the effect of 2 variables—pellet volume and thawing temperature—on post-thaw sperm motility, acrosome status, and in vitro fertility. Semen was collected from 3 males (3 ejaculates/male) via urethral catheterization under dexmedetomidine-ketamine anaesthesia. Sperm were diluted in Feline Optimized Culture Medium (FOCM), centrifuged (8 min; 300 × g), and resuspended in a soy-lecithin-based vitrification medium (with 0.2 M sucrose). After a 5-min equilibration, sperm was vitrified in 2 volumes (20 or 30 µL) by direct pipetting into LN2. Sperm pellets were thawed in FOCM at 1 of 2 temperatures (37 or 55°C) and the 4 treatment groups (20 µL-37°C, 20–55, 30–37, 30–55) assessed for percentage of progressively motile and acrosome intact sperm. To assess sperm function, additional 30-µL pellets were thawed at 37 or 55°C, and recovered sperm were used to inseminate in vitro-matured domestic cat oocytes (n = 10–25/ejaculate). At 48 h post-insemination, oocytes and embryos were fixed (1% NBF). Hoechst fluorescent stain (#33342) was used to evaluate embryo cleavage and maturation status of unfertilized ova. Sperm motility and acrosomal integrity percentages were analysed by ANOVA, and oocyte cleavage proportions were analysed by chi-squared. Mean (± SEM) progressive sperm motility post-thaw did not differ (P > 0.05) among treatments (38 ± 8, 34 ± 7, 41 ± 7, 32 ± 7% for 20 µL-37°C; 20–55, 30–37, and 30–55, respectively). Similarly, acrosomal integrity did not differ (P > 0.05) among treatments (26 ± 4, 25 ± 4, 17 ± 3, 17 ± 2% for 20 µL-37°C, 20–55, 30–37, and 30–55, respectively). Oocyte cleavage proportions did not differ (P > 0.05) between thawing temperatures for total inseminated oocytes but, after correcting for oocyte maturation status, was higher (P < 0.01) for samples thawed at 55°C (60%, 67/112) compared with 37°C (39%, 52/133). In summary, although variations in pellet volume and thawing temperature had minimal effect on sperm motility or acrosome status immediately post-thaw, sperm function appeared to be enhanced when vitrified pellets were thawed at a higher temperature. In vitro fertility success (~60% embryo cleavage) is comparable to values reported by our laboratory with conventionally collected and frozen cat semen, suggesting these newer methods may be suitable for applied usage in felids.
This study was funded by the Institute of Museums and Library Services.
63 EFFECT OF ENDOMETRIAL BIOPSY ON UTERINE HEALTH OF TROPICALLY ADAPTED BEEF CATTLE
Authors: Ramirez-Garzon O. , Satake N. , Lyons R. E. , Palmieri C. , Hill J. , Gallego-Lopez C. , Holland M. K. , McGowan M.
Published: December 2016

In cattle, endometrial gene expression during the first 2 weeks after fertilization is different in cattle that conceive compared with those that fail to conceive. The potential selection of animals based on endometrial marker expression before the day of embryo transfer may improve the efficiency of bovine embryo transfer. This study aimed to evaluate the morphological and molecular changes induced by endometrial biopsy in cycling tropically adapted beef cattle. In both experiments, a single biopsy was performed on the horn ipsilateral to the corpus luteum using a circular cup biopsy forceps. The uterus was collected after slaughter, and sections were taken adjacent to the biopsy site. Five heifers were not biopsied and acted as controls. In Experiment 1, presynchronised heifers (n = 10) were biopsied on Day 7 post-oestrus (B7) and then resynchronised with cloprostenol (Estromil®, 250 μg IM) to induce oestrus (O), 2 to 3 days after injection. At Day 7 post-oestrus (D7), the reproductive tracts were collected, 10 days after biopsy (B7-O-D7). In Experiment 2, presynchronised heifers (n = 7) were biopsied on Day 4 post-oestrus (B4), and reproductive tracts were collected 3 days later (B4-D7). A quantitative scoring system was used to define histological appearance of the endometrium (1 to 25; above 16 indicates endometritis). The RNA expressions of interleukin-1-β (IL-1β) and transforming growth factor-β (TGF-β) were measured by quantitative real-time PCR (qRT-PCR). Macroscopic examination showed the uterus collected from B7-O-D7 heifers exhibited a focal, well demarcated, dark red discoloration of the endometrium (4/10) with no evidence of the biopsy site found in the remaining heifers (6/10). The biopsied uteri from B4-D7 heifers revealed no macroscopic evidence of the biopsied site (4/7), serosa congestion (2/7), and one case where a severe endometrial haemorrhage was evident (1/7). Histologically, the overall mean score was not indicative of endometritis. The RNA expression showed up-regulation of both IL-1β and TGF-β in both B7-O-D7 and B4-D7 heifers compared with non-biopsied heifers and significantly higher expression of IL-1β in B4-D7 than B7-O-D7 (P = 0.031, P < 0.05). Although in most cases there was little or no evidence of damage to the endometrium within 3 to 10 days after endometrial biopsy, there was still some evidence of up-regulation of pro-inflammatory and tissue repair genes. What effect the latter has on likelihood of conception is yet to be determined.
7 INFLUENCE OF ESTRUS EXPRESSION AND TREATMENT WITH GnRH ON PREGNANCY RATES IN BEEF CATTLE SYNCHRONIZED WITH PROGESTERONE DEVICES AND ESTRADIOL AND INSEMINATED AT A FIXED-TIME
Authors: Bó G. A. , Cedeño A. , Tribulo A. , Andrada S. , Tribulo R. , Barajas J. L. , Ortega J. , Pellizari M. , Mapletoft R. J.
Published: December 2016

We have previously shown that expression of oestrus significantly influences the time of ovulation and pregnancy rates to timed-AI (P/TAI) in beef cattle synchronized with progesterone devices and oestradiol (Bó et al. 2016 Theriogenology 86, 388–396). Furthermore, delaying TAI from 48 h to 54–58 h after device removal improved P/TAI of those animals not showing oestrus by 48 h. The objective of the present study was to determine whether the administration of GnRH to those animals not showing oestrus by 48 h after device removal had an impact on P/TAI. Non-lactating and suckled beef cows and heifers (Bonsmara, Brangus, and Braford; n = 868), with a corpus luteum (CL) or a follicle ≥8 mm in diameter detected by ultrasonography (Mindray DP 30 Vet, 7.5 MHz, China) and body condition score 2 to 4 (1 to 5 scale) were synchronized in 3 replicates. On Day 0, animals received a progesterone device (DIB 0.5 g, Zoetis, Argentina) and 2 mg of oestradiol benzoate (Von Franken, Argentina). On Day 8, DIB were removed and cows received 0.150 mg of D+cloprostenol (Río de Janeiro, Argentina) plus 0.5 mg oestradiol cypionate (Cipiosyn, Zoetis). In addition, all cows were tail-painted on the sacrococcygeal area (CeloTest, Biotay, Argentina) at DIB removal and were observed to determine the expression of oestrus, according to the percentage of paint loss (PL) 48 h later. The animals that showed oestrus (PL > 30%) were recorded and TAI at that time while those with PL ≤ 30% (not in oestrus) were randomly allocated to receive 100 μg of gonadorelin (GnRH; Gonasyn, Zoetis) or no treatment and TAI between 54 to 58 h after DIB removal. Pregnancy was diagnosed by ultrasonography at 45 days after TAI, and data were analysed by logistic regression. There were no significant differences among replicates, inseminators, semen source or animal class (dry cows, suckled cows or heifers; P > 0.11). However, animals showing oestrus by 48 h after DIB removal had higher P/TAI (371/666; 55.7; P < 0.04) than those that did not show oestrus (96/202; 47.5%). Within animals not showing oestrus at 48 h, those that were in oestrus by the TAI at 54 to 58 h had higher P/TAI (71/124, 57.3%; P < 0.01) than those that did not show oestrus within that period (27/78; 34.6%). In addition, P/TAI was higher (P < 0.01) in animals that received GnRH (66/113; 58.4%) than in those that did not receive GnRH (30/89; 33.7%). In conclusion, the expression of oestrus significantly affected P/TAI in beef cattle synchronized with progesterone devices and oestradiol. Furthermore, treatment of animals not showing oestrus by 48 h after DIB removal with GnRH resulted in improved P/TAI performed 6 to 10 h later.
87 EFFECTS OF HUMAN RECOMBINATION GRANULOCYTE–COLONY STIMULATING FACTOR (hrG-CSF) ON IN VITRO CULTURE OF PORCINE CLONED EMBRYOS DERIVED FROM THIN CUMULUS CELL LAYER OF OOCYTES MATURED IN VITRO
Authors: Cai L. , Park E. O. , Jin Y.-X. , Hwang K.-C. , Jeong Y. W. , Hyun S.-H. , Hwang W.-S.
Published: December 2016

Although several cloned pigs have been successfully produced, the developmental competence of cloned embryos in vitro is still very low. Granulocyte colony-stimulating factor receptor (G-CSFR) was founded in the human trophoblastic cell line that is implicated in regulation and proliferation of trophoblast. In the present study, the somatic cell NT embryos derived from oocytes that have more than 3 cumulus cells layer were cultured and supplemented with various concentrations of hrG-CSF (0, 10, 50, and 100 ng mL−1, respectively). Although there were no significant effects on the various concentration of hrG-CSF treatment groups compared with control, the somatic cell NT blastocysts formation tended to increase after 10 ng mL−1 hrG-CSF treatment (24.19 ± 2.90%) compared with control (21.37 ± 2.98%). Moreover, we investigated the effects of 10 ng mL−1 hrG-CSF on in vitro culture of porcine cloned embryos derived from oocytes that were categorized into grade A (cumulus cell layer >10), grade B (10 > cumulus cell layer ≥ 3), and grade C (cumulus cell layer <3). After supplementation of 10 ng mL−1 hrG-CSF on in vitro-culture of different groups, the developmental competence, blastocyst quality, and gene transcript levels were observed. The results showed that 10 ng mL−1 hrG-CSF has no beneficial effects on cloned embryos derived from grade A oocytes (10 ng mL−1 hrG-CSF 25.35 ± 2.53% v. control 25.00 ± 2.66%), but it significantly increased blastocyst formation of embryos derived from grade B oocytes (22.09 ± 2.10%) compared with grade B control (12.09 ± 2.31%, P < 0.05). There were obvious increases in blastocyst formation derived from grade C oocytes after 10 ng mL−1 hrG-SCF treatment (25.74 ± 1.65%) compared with grade C control (16.82 ± 2.30%, P < 0.05). However, there were no significantly differences in cleavage rate and total cell number of blastocysts among each group. Otherwise, the PCNA, POU5F1, Dnmt1, Bcl2, and Bax transcript levels were significantly increased in blastocysts that were derived from grade C oocytes after 10 ng mL−1 hrG-SCF treatment compared with grade C control. In conclusion, supplementation of 10 ng mL−1 hrG-CSF in in vitro-cultured porcine embryos increased blastocyst formation of embryos derived from thin cumulus layer of oocytes by reducing apoptosis while increasing cell proliferation and nuclear reprogramming. These results provide an experimental basis for the use of poor quality oocytes for agricultural production.
This work was supported by a grant from Research Program (No. 307–02) Gyeonggi-province project and the Next-Generation BioGreen21 Program [no. PJ01107702], Rural Developmental Administration (RDA), Republic of Korea.
96 IN VITRO FERTILIZATION IN MOUSE AS A REPROTOXICITY MODEL FOR XENOBIOTICS
Authors: Liu Y. , Pedersen H. S. , Foldager L. , Callesen H. , Sørensen M. T.
Published: December 2016

To assess reprotoxicity of pesticides and other xenobiotics, rodents are often used with natural mating and litter size as end-points. However, because a male mouse can produce normal-size litters even with >50% reduced sperm count (Ecotox. Env. Saf. 73, 1092), this model could approve some chemical substances despite their reprotoxic effect. Thus, we aimed to establish a mouse IVF system to evaluate reprotoxicity of xenobiotics, illustrated by 2 pesticides [vinclozolin (vin), chlormequat (ccc)]. Experimental mice were given 2 pesticide doses by mixing into the feed: low (L) or high (H), equivalent to no or lowest observed adverse effect level, respectively (Env. Health Perspect. 117, 1272). To produce experimental males, mated NMRI females were fed low dose (vinL, cccL), high dose (vinH, cccH), or no pesticide from 1-cell stage through birth and until weaning, from where experimental males continued pesticide feeding. Each experimental male at 8 to 10 weeks old was naturally mated with 2 females having normal feed for 5 days, then continued with pesticide or control feed for at least 35 days before use for IVF. Naturally mated females had normal feed until birth to evaluate litter size. For IVF, oocytes were collected after superovulation of 3- to 4-week-old C57BL/6J females (2–5/male). Based on our previous experiment (Anim. Reprod. 13, in press), 25,000 sperm/mL was within the responsive range and selected for IVF (Theriog. 65, 1716). The IVF embryos were in vitro cultured for 4 days. Pronuclei were evaluated 9 h after IVF start (Day 0), and 2-cell/blastocyst were evaluated on Day 1/4. Mean litter size was estimated by normal linear mixed-effects model, and mean pronuclei, 2-cell, and blastocyst rates were estimated by binomial generalized linear mixed-effects models with identity link function, included a male-subject random intercept accounting for correlation between multiple fertilizations by same male, and a factor defining groups as fixed effect. Preliminary results (Table 1) are based on current data from half of our full experiment. No signs of different litter sizes after natural mating were observed between pesticide groups and control. Compared with control, rates of pronuclei, 2-cell, and blastocyst tended to decrease in vinH and increase in vinL group. No clear differences between cccL, cccH, and control groups were found. These results could indicate a pesticide effect (vinH) on mouse male reproductive system that can be detected in an IVF system but not by natural mating. Our data show a large variation in IVF results between individual males and females, so our full dataset is required before conclusions are made.

Table 1.

Preliminary results
Methodological implications of working memory tasks for L2 processing research
Authors: Leeser Michael J. , Sunderman Gretchen L.
Published: December 2016

A growing body of research within cognitive perspectives of second language acquisition (SLA) has investigated how individual differences in working memory (WM) capacity may impact second language (L2) processes. Many of these studies have employed versions of complex span measures, such as the reading span test (RSPAN). The findings of these studies are mixed, and no standard scoring methods exist for these tasks. Therefore, this study examines the consequences of different scoring methods and data analysis procedures of two common complex span tasks (reading span and operation span) used in L2 sentence processing research. L2 Spanish learners completed two WM span tasks and an online Spanish sentence interpretation task. The results indicate that various scoring methods of the complex span tasks were significantly correlated within tasks. However, in examining the relationship between WM and sentence interpretation, we found that different results emerged, depending on WM task type, scoring method and type of analysis. Implications for research investigating WM resources in SLA are discussed.
An empirical study of working memory, personality, and second language construction learning
Authors: Jackson Daniel O.
Published: December 2016

The study presented in this chapter investigated the role of individual differences in construction learning under different conditions of partial repetition. Ninety participants were exposed to an artificial system of noun-suffix constructions under one of three incidental learning conditions, which either repeated nouns across consecutive trials, repeated suffixes across consecutive trials, or contained no repetition across consecutive trials. It was hypothesized that: (a) working memory (WM) and (b) openness would predict L2 construction learning, and that (c) the influence of WM would be stronger in the absence of repetition, while (d) intellect would be associated with attention to form. The results indicated that participants learned the artificial system, with no differences between conditions. Measures of WM and openness correlated positively and significantly with untrained (i.e. generalization) test scores. Mixed-effects binominal regression models revealed significant effects for WM, but showed no interactions between condition and WM. Intellect was significantly related to a measure of attention. In sum, this study provides additional evidence that WM fosters generalization and further suggests that the distinction between openness and intellect may be useful for understanding novel second language learning.
Working memory and L2 English speakers’ primed and subsequent production of passives
Authors: McDonough Kim , Kim YouJin
Published: December 2016

Although first language researchers have investigated the relationship between individual differences and structural priming (Kaschak, Kutta, & Jones 2011; Kidd 2012a, b), fewer studies have explored this relationship in second language (L2) speech production (McDonough, Kielstra, Crowther, & Smith 2016). This chapter describes an empirical study that tested the relationship between English L2 speakers’ (N = 64) primed and subsequent production of passives and working memory. Results indicated a significant positive correlation between the participants’ primed production and working memory scores, but only when no intervening material occurred between prime and target sentences. Furthermore, there was no relationship between their subsequent production of passives and working memory scores. Implications are discussed in terms of dual-account explanations of structural priming.
The interaction between feedback exposure condition and phonetic coding ability
Authors: Yilmaz Yucel , Koylu Yilmaz
Published: December 2016

This chapter reports on an empirical study that investigated feedback-cognitive ability interactions in three oral negative feedback exposure conditions. In the first condition, learners (i.e. receivers) received feedback on their own errors. In the second condition, learners (i.e. nonreceivers) did not receive feedback on their own errors, but they were allowed to hear the feedback that was provided to the receivers. In the control condition, learners were not exposed to feedback. The cognitive ability investigated was a sub-component of Carroll’s (1962) aptitude model, phonetic coding ability (PCA), or the capacity to recognize and remember previously encountered phonetic material. Results revealed that PCA played a role only in the receivers’ immediate posttest performance, suggesting that the receivers and the nonreceivers might have processed the feedback differently and that higher PCA ability may increase the benefits of receiving feedback directly on one’s own errors but have no effect on feedback that one is merely exposed to.
Morphological structure influences the initial landing position in words during reading Finnish
Authors: Hyönä Jukka , Yan Ming , Vainio Seppo
Published: December 2016

The preferred viewing location in words [Rayner, K. (1979). Eye guidance in reading: Fixation locations within words. Perception, 8, 21–30] during reading is near the word centre. Parafoveal word length information is utilized to guide the eyes toward it. A recent study by Yan and colleagues [Yan, M., Zhou, W., Shu, H., Yusupu, R., Miao, D., Krügel, A., & Kliegl, R. (2014). Eye movements guided by morphological structure: Evidence from the Uighur language. Cognition, 132, 181–215] demonstrated that the word’s morphological structure may also be used in saccadic targeting. The study was conducted in a morphologically rich language, Uighur. The present study aimed at replicating their main findings in another morphologically rich language, Finnish. Similarly to Yan et al., it was found that the initial fixation landed closer to the word beginning for morphologically complex than for monomorphemic words. Word frequency, saccade launch site, and word length were also found to influence the initial landing position. It is concluded that in addition to low-level factors (word length and saccade launch site), also higher level factors related to the word’s morphological structure and frequency may be utilized in saccade programming during reading.
A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes
Authors: Wolfes Anne C. , Ahmed Saheeb , Awasthi Ankit , Stahlberg Markus A. , Rajput Ashish , Magruder Daniel S. , Bonn Stefan , Dean Camin
Published: December 2016

Interactions between astrocytes and neurons rely on the release and uptake of glial and neuronal molecules. But whether astrocytic vesicles exist and exocytose in a regulated or constitutive fashion is under debate. The majority of studies have relied on indirect methods or on astrocyte cultures that do not resemble stellate astrocytes found in vivo. Here, to investigate vesicle-associated proteins and exocytosis in stellate astrocytes specifically, we developed a simple, fast, and economical method for growing stellate astrocyte monocultures. This method is superior to other monocultures in terms of astrocyte morphology, mRNA expression profile, protein expression of cell maturity markers, and Ca2+ fluctuations: In astrocytes transduced with GFAP promoter–driven Lck-GCaMP3, spontaneous Ca2+ events in distinct domains (somata, branchlets, and microdomains) are similar to those in astrocytes co-cultured with other glia and neurons but unlike Ca2+ events in astrocytes prepared using the McCarthy and de Vellis (MD) method and immunopanned (IP) astrocytes. We identify two distinct populations of constitutively recycling vesicles (harboring either VAMP2 or SYT7) specifically in branchlets of cultured stellate astrocytes. SYT7 is developmentally regulated in these astrocytes, and we observe significantly fewer synapses in wild-type mouse neurons grown on Syt7−/− astrocytes. SYT7 may thus be involved in trafficking or releasing synaptogenic factors. In summary, our novel method yields stellate astrocyte monocultures that can be used to study Ca2+ signaling and vesicle recycling and dynamics in astrocytic processes.
Market Institutions and Price Relationships: The Case of Coffee in the Ethiopian Commodity Exchange
Authors: Hernandez Manuel A. , Rashid Shahidur , Lemma Solomon , Kuma Tadesse
Published: December 2016

While the Ethiopian Commodity Exchange (ECX) is widely considered a successful market institution, very little rigorous empirical investigation has been done on it. This paper contributes to filling this knowledge gap. Using a unique set of spatially disaggregated prices for five different coffee varieties, we examine how the ECX has influenced the dynamics of international and domestic prices for coffee, the largest traded commodity in terms of trade value on the exchange floor. We follow a multivariate Generalized Autoregressive Conditional Heteroscedasticity (GARCH) approach to assess the extent of market interdependence (conditional correlations) and volatility transmission. We also evaluate structural changes in price dynamics over time. Our results suggest that, contrary to popular media stories, the ECX's success in strengthening coffee price relationships has been limited. We discuss the underlying reasons and implications of this finding.
The Osmopressor-Induced Angiopoietin-1 Secretion in Plasma and Subsequent Activation of the Tie-2/Akt/eNOS Signaling Pathway in Red Blood Cell
Authors: Chu You-Hsiang , Lu Chih-Cherng , Lin Tso-Chou , Tsou Mei-Yung , Hsu Yu-Juei , Ho Shung-Tai , Tung Che-Se , Tseng Ching-Jiunn , Li Min-Hui , Lee Herng-Sheng
Published: December 2016

BACKGROUND

Water ingestion induces the osmopressor response, which typically presents as increased total peripheral vascular resistance in young healthy subjects. A previous study has suggested that the RBC membrane receptor is involved in osmopressor stress. Recent studies have indicated nitric oxide synthase phosphorylation in RBCs. However, the main process in signaling pathway activation to elicit such a response is unknown. Herein, we hypothesized that hypo-osmotic stress following water ingestion modulates the eNOS/NO pathway, thereby alternating vascular resistance.

METHODS

We included 24 young, healthy subjects. Physiological parameters and blood samples were collected at 5 minutes before and 25 and 50 minutes after 50 ml water, 500 ml water, or 500 ml normal saline ingestion. A human receptor tyrosine kinase (RTK) phosphorylation antibody array was used to simultaneously detect and monitor the biological activation pathways in RBCs.

RESULTS

Of the 71 RTKs assayed during the osmopressor response, several RTKs were significantly upregulated, including Tie-2 and Tie-1. Plasma angiopoietin-1 levels significantly increased at 25 minutes after 500 ml water ingestion compared to those at baseline. Simultaneous phosphorylation of Tie-2, Akt, and eNOS in RBCs occurred. RBCs in vitro were stimulated with angiopoietin-1, Tie-2, or 0.8% saline and showed significant increase in Tie-2, Akt, and eNOS phosphorylation upon angiopoietin-1 treatment and enhanced activation upon cotreatment of angiopoietin-1 and 0.8% saline.

CONCLUSIONS

The hypo-osmotic stimulus of water ingestion increases angiopoietin-1 secretion and subsequently activates the Tie-2/Akt/eNOS signaling pathway in RBCs, thereby revealing a novel biological mechanism simultaneously occurring with the osmopressor response.