bluuebunny/crossref_metadata_2025 huggingface dataset:
251 IS THERE A GAP BETWEEN NUCLEAR AND CYTOPLASMIC MATURATION IN IN VITRO-MATURED EQUINE OOCYTES?
Authors: Claes E. , Smits K. , De Schauwer C. , Leemans B. , Wydooghe E. , Favoreel H. , Van Soom A.
Published: December 2014

It is a general belief that as soon as the oocyte is recovered from the follicular environment, the nuclear maturation starts spontaneously in vitro, while specific stimulation for the cytoplasmic maturation is lacking (Gilchrist and Thompson 2007 Theriogenology 67, 6–15; Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). As both nuclear and cytoplasmic maturation are required to prepare the oocyte for successful fertilization and embryonic development, a defective cytoplasmic maturation might be an important cause of low blastocyst rates in vitro (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011). Nuclear and cytoplasmic maturation can be evaluated using fluorescent dyes. Assessment of nuclear maturation is typically based on the visualisation of chromatin, whereas cytoplasmic maturation is evaluated by the localization of cytoplasmic organelles [i.e. the cortical granules (CG)]. Equine oocytes were recovered from ovaries of slaughtered mares. After in vitro maturation (IVM; Smits et al. 2010 Vlaams Diergen. Tijds. 79, 134–138), oocytes were fixed and permeabilized. Subsequently, CG were labelled by incubation in 10 µg mL–1 FITC-labelled lens culinaris agglutinin during 15 min at RT. Chromatin was counterstained to verify the nuclear status with 20 µg mL–1 Hoechst 33342 during 15 min at RT. Stained oocytes with no or dispersed chromatin were classified as degenerated. Based on the absence or presence of the first polar body (PB), non-degenerated oocytes were either classified as nuclear immature (MI, no PB present) or nuclear mature (MII, PB present). The non-degenerated oocytes were further subdivided in 3 categories based on the migration of the CG: 1) cytoplasmic immature oocytes with (clusters of) CG randomly distributed throughout the ooplasm, 2) oocytes in transition stage with progressing CG migration to the oocyte cortex, and 3) cytoplasmic mature oocytes with a clearly visible CG monolayer just underneath the oolemma. The mean and standard deviation of nuclear and cytoplasmic parameters were calculated using Excel (Excel 2007, Microsoft Corp., Redmond, WA, USA). In 3 replicates, 86.6 ± 2.75% of all oocytes (131/151) demonstrated a corresponding nuclear and cytoplasmic maturation pattern (MI corresponding to CG1 and 2; MII corresponding to CG3). Only 12.0 ± 2.82% of the oocytes (16/133) revealed a cytoplasmic maturation pattern (CG 1 or 2) that lagged behind the nuclear maturation (MII). On the other hand, 22.2 ± 9.8% of the oocytes (4/18) were already cytoplasmic (CG3) but not yet nuclear matured (MI). Since most of the equine in vitro matured oocytes exhibited, surprisingly, a corresponding nuclear and cytoplasmic maturation pattern, it can be concluded that the gap between the nuclear and cytoplasmic maturation in vitro is less important than is generally believed. Consequently, the IVM step is not the main obstacle to increase the efficiency of the in vitro production process in horses.
252 BIOCHEMICAL SERUM PROFILE AND LAPAROSCOPIC OVUM PICKUP CORRELATION IN ANGLO NUBIAN GOATS FED WITH DIFFERENT FAT SOURCES
Authors: Nociti R. P. , Uscategui R. A. R. , Padilha L. C. , Barros F. F. P. C. , Motta G. A. , Mariano R. S. G. , Rola L. C. , Coelho C. M. M. , Santos V. J. C. , Feliciano M. A. R. , Vicente W. R. R. , Oliveira M. E. F. , Hossepian de Lima V. F. M.
Published: December 2014

The aim of the present study was to evaluate the correlations between biochemical serum profile with total number of aspirated follicles (AF), oocytes recovered (OR), number of corpus lutea (CL), oocyte morphology quality, and oocyte in vitro maturation (IVM) rate. Eighteen goats were randomly divided into 3 groups based on treatment diets, with different fat sources [soy oil (SG, n = 6), linseed (LG, n = 6), and Megalac® (MG, n = 6)], formulated with 40% concentrate, 60% corn silage, of which 4% was ether extract from fat sources. The animals were submitted to a 15-day adaptation period and then a 70-day experimental period. The laparoscopic ovum pickup (LOPU) was performed after 36 h of an ovarian superstimulation protocol [FSH (80 mg) + eCG (300 IU)] on Day 42 (LOPU1) and Day 70 (LOPU2) of the trial. The number of AF, and the presence and number of CL were evaluated during the LOPU procedure. Blood samples were taken at 14 days, 7 days, and just before each LOPU, and grouped as P1 (mean of blood samples results obtained before LOPU1) and P2 (LOPU2); serum metabolites analysed were total cholesterol, triglycerides, aspartate aminotransferase (AST), creatine kinase, and plasma glucose. The recovered oocytes were classified by morphological appearance, and only G1 (homogenous ooplasma and at least 3 layers of cumulus cells), G2 (homogenous ooplasma and at least 1 complete layers of cumulus cells), and G3 (homogenous ooplasma and incomplete layers or no cumulus cells) were submitted to IVM. The oocytes were incubated in maturation media (TCM 199 supplemented with 10% heat inactivated goat serum) at 38.5°C and 5% CO2 for 27 h. Then, oocytes were subsequently fixed and stained on Hoechst 33342 and analysed using a fluorescent microscope for nuclear maturation. The IVM and analysis were performed for each individual. Pearson correlation test (P < 0.05) was applied in R® software between serum metabolites and LOPU variables. The triglycerides, creatine kinase, and glucose were not correlated with any LOPU variables. The total cholesterol was negatively correlated with G1, G2, and CL, and positively correlated with OR and G3 with determination coefficients (R) of –0.28, –0.38, –0.24, 0.25, and 0.44, respectively. The AST were negatively correlated with AF, G1, G2, and IVM, with R of –0.11, –0.43, –0.13, and –0.16, respectively. In conclusion, these results are suggestive that high serum levels of cholesterol and AST may interfere with oocyte quality, indicating a negative hyperlipidemia effect on reproductive efficiency as reported in cows.Financial support was provided by CNPq and FAPESP.
259 DOES THE SENSITIVITY TO OXIDATIVE STRESS DEPEND ON THE SEX OF THE EMBRYO?
Authors: Dallemagne M. , Ghys E. , De Troy D. , Donnay I.
Published: December 2014

Male and female bovine embryos show several differences as early as the blastocyst stage. For example, differences are observed in metabolism, developmental kinetics, or gene expression that can lead to a shift in the sex ratio. Interestingly, the culture medium differentially affects male and female embryos. We previously showed that male Day 7 blastocysts present lower apoptotic rates than females (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The objective of the present study was to determine if such difference might be related to a differential sensitivity to oxidative stress, known to increase apoptosis in bovine blastocysts. In vitro-produced embryos were cultured in a SOF-based medium containing 0.4% BSA. At Day 5 post-insemination (pi) all the embryos were transferred in drops containing the same culture medium supplemented or not with 1 mM 2,2′-azobis(2-amidinopropane) dihydrochloride, an inducer of reactive oxygen species. Blastocysts were collected at Day 7, and apoptosis was evaluated by an immunofluorescent staining of cleaved caspase-3 (8 replicates, n = 175). Total and apoptotic cells were counted using an epifluorescence microscope. As expected, embryos cultured under stress conditions from Day 5 pi presented a lower blastocyst rate at Day 7 (10.9 ± 1.0% v. 23.1 ± 1.9% for the control group; standard least squares, P < 0.0001). The stressed blastocysts also showed fewer cells (113 ± 3 v. 139 ± 4; P < 0.0001) and higher apoptotic rates (15.3 ± 0.9% v. 9.4 ± 0.6%; P < 0.0001). As previously observed, the mean total cell number of the blastocysts was higher for males than females, whatever the culture condition (stress: males: 119 ± 4, females: 108 ± 4; control: males: 144 ± 5, females: 131 ± 6; sex effect: P = 0.005; interaction of sex × condition: P = 0.9). Interestingly, the sex ratio of the blastocysts was significantly different between control and stress conditions (χ2, P = 0.02); whereas a deviation in favour of the male embryos was observed in the control group (males: n = 57, 61%, females: n = 37, 39%; P = 0.04), it disappeared when embryos were submitted to oxidative stress (males: n = 35, 43%, females: n = 46, 57%; P = 0.22). However, oxidative stress had a similar impact on male and female blastocysts regarding the apoptotic rates (stress: males: 15.3 ± 1.3%, females: 15.4 ± 1.3%; control: males: 8.9 ± 0.7%, females: 10.2 ± 1.1%; standard least squares, sex effect: P = 0.99; interaction of sex × condition: P = 0.3). In conclusion, female embryos seem more resistant to oxidative stress than male ones when the stress is induced from Day 5 pi. Oxidative stress has a similar impact on the apoptotic rates in male and female blastocysts. The higher rate of apoptosis previously observed in female blastocysts can thus not be explained by a higher sensitivity of female embryos to oxidative stress. This is in accordance with the higher level of expression of several X-linked genes related to antioxidant pathways in female blastocysts.
26 HISTONE DEACETYLASE INHIBITOR SCRIPTAID IMPROVES EPIGENETIC REPROGRAMMING AND CLONING EFFICIENCY IN THE PIG
Authors: Liang S. , Kim T. , Kim N.-H. , Cui X.-S.
Published: December 2014

After somatic cell nuclear transfer (SCNT), the epigenetic state of a differentiated donor cell nucleus must be reversed to the embryonic state. Incomplete epigenetic reprogramming and abnormal gene activation of the donor cell nuclei is thought to be the cause of low cloning efficiency. To improve cloning efficiency, we investigated the effect of scriptaid, a novel histone deacetylase inhibitor, on the in vitro development of porcine SCNT embryos were investigated. Cumulus cells collected from cumulus-oocyte complexes (COC) after 44 h of maturation were used for donor cell, and embryos were cultured in porcine zygote medium (PZM)-5 medium for 7 days. We found that treating SCNT embryos with 300 or 500 nM scriptaid for 20 h after activation increased developmental rate to the blastocyst stage (300 nM, 26.2%; 500 nM, 24.6% v. 100 nM, 18.3%; Ctrl, 15.7%; P < 0.05) and total cell numbers (300 nM, 43.5; 500 nM, 40.8 v. 100 nM, 33.8; Ctrl, 32.3; P < 0.05). Additionally, results of the TUNEL assay indicated that scriptaid decreased apoptosis (300 nM, 6.8% v. Ctrl, 11.4%; P < 0.05) in SCNT blastocysts. After the 300 nM scriptaid treatment, the levels of acetylated histone H3 lysine 9 and 5-hydroxymethylcytosines were increased (P < 0.05), and histone H3 lysine 9 trimethylation and 5-methylcytosine were decreased at the 1-cell stage, which might explain the enhanced (P < 0.05) transcript levels of mir-152, Oct4, Cdx2, and Bcl-xL and reduced (P < 0.05) transcription of Dnmt1, Casp3, and Bax in blastocysts. In conclusion, scriptaid enhances the developmental capacity by preventing apoptosis, and improves nuclear reprogramming in porcine SCNT embryos.This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ009601 and PJ009098), Rural Development Administration, Republic of Korea.
263 ADDING RESVERATROL TO THE EXTENDER AFFECTS PROTEIN TYROSINE PHOSPHORYLATION IN BUFFALO SPERM
Authors: Longobardi V. , Bifulco G. , Albero G. , Salzano A. , Zullo G. , Vecchio D. , Gasparrini B.
Published: December 2014

Cryopreservation induces remarkable capacitation- like changes in buffalo sperm (Kadirvel et al. 2011 Theriogenology 75, 1630–1639; Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). The aim of this study was to evaluate the effect of resveratrol, a natural phytoalexin with antioxidant properties, on capacitation status of frozen-thawed buffalo sperm, assessed by protein tyrosine phosphorylation assay. Three ejaculates from four bulls were used for the trial. Each ejaculate was split into two equal aliquots and diluted at 37°C with BioXcell extender containing no supplement (control) or 50 µM resveratrol, to a final concentration of 30 × 106 spermatozoa per mL. After 4 h at 4°C, straws were frozen in an automated system. Immediately after thawing, sperm motility was evaluated by phase-contrast microscopy, sperm viability by Trypan Blue/Giemsa staining and localization of phosphotyrosine proteins by indirect immunofluorescence, as described Kadirvel et al. (2011 Theriogenology 75, 1630–1639). Briefly, after thawing, semen was centrifuged (300 × g, 10 min), fixed in 2% formaldehyde for 1 h at 4°C, and sperm pellets were incubated overnight at 4°C in modified phosphate buffer saline containing 2% BSA. After centrifugation, sperm pellets were resuspended, diluted 1 : 10 in mPBS, smeared onto slides, air-dried, and permeabilized with absolute ethanol for 5 min. Then, spermatozoa were incubated with rabbit anti-phosphotyrosine primary antibody for 1 h at room temperature in a humid chamber. Slides were incubated with secondary antibody, FITC conjugated goat anti-rabbit IgG, for 1 h in a dark humid chamber at room temperature and mounted with 90% glycerol. A total of 100 spermatozoa were screened per slide and classified as described (Luño et al. 2013 Reproduction 146, 315–324): pattern A: uniform fluorescence over the entire acrosome (low capacitation level); pattern E: signal in the equatorial segment (medium capacitation level); and pattern EA: fluorescence at both equatorial and acrosomal areas (high capacitation level). Data were analysed by chi-square. There were no significant differences between control and treated groups for sperm motility (50.0 and 55.0%, respectively) or viability (77.4 and 72.9%). The percentage of sperm cells that did not exhibit fluorescence was very low (2.4 and 4.3% in the control and resveratrol groups, respectively). In resveratrol-treated group, pattern E was higher than the control (4.9 and 2.0%; P < 0.01). More interestingly, in the resveratrol-treated group, an increased percentage of sperm with pattern A (79.6 and 52.5%) and a decreased percentage of sperm with pattern EA (12.2 and 43.1%) were recorded. Based on decreased sperm with a high capacitation level (EA pattern) and increased sperm with low capacitation level (A pattern) at thawing, we concluded that adding resveratrol to semen extender before cryopreservation of buffalo was beneficial.
264 EFFECT OF SEASON ON CRYOCAPACITATION OF BUFFALO (BUBALUS BUBALIS) SEMEN
Authors: Albero G. , Zullo G. , Salzano A. , Brun R. , Longobardi V. , Bifulco G. , Gasparrini B.
Published: December 2014

Buffalo are short-day breeders; at our latitudes, reproductive activity improves during autumn. Although extensive studies have been conducted on the female, seasonal variations were also reported on post-thaw motility and membrane integrity of buffalo sperm (Andrabi 2009 Reprod. Domest. Anim. 44, 552–569). It was reported that cryopreservation induces capacitation-like changes in buffalo spermatozoa, assessed by both chlortetracycline (CTC) fluorescent and protein tyrosine phosphorylation assays (Kadirvel et al. 2011 Theriogenology 75, 1630–1639; Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). The aim of this study was to evaluate the effect of season on cryocapacitation of buffalo semen. At least two ejaculates were collected from 4 bulls during 2 seasons with different daylength: spring (low season) and autumn (peak season). Each ejaculate was diluted at 37°C with BioXcell extender to a final concentration of 30 × 106 spermatozoa per mL. After 4 h at 4°C, straws were frozen in an automated system. Immediately after thawing, sperm motility was evaluated by phase-contrast microscopy and viability, as well as capacitation status, were assessed by CTC fluorescent staining, as reported (Kadirvel et al. 2011 Theriogenology 75, 1630–1639). Briefly, sperm suspensions were first stained with 0.1 µg mL–1 Hoechst 33258 for 2 min. Then, equal volumes of sperm suspension and CTC solution (750 mM CTC, 5 mM cysteine in 130 mM NaCl, and 20 mM Tris-HCl) were mixed at room temperature, and glutaraldehyde (12.5%) was added. Sperm suspensions were mounted on slides and stored at 4°C overnight (in the dark). Each sample was assessed twice under a microscope equipped with phase contrast and epifluorescent optics. At least 100 spermatozoa per slide were evaluated and classified into 3 CTC staining patterns: 1) uniform bright fluorescence over the entire head (uncapacitated spermatozoa, pattern F); 2) fluorescence-free band in the post-acrosomal region (capacitated spermatozoa, pattern B); and 3) dull fluorescence over the entire head, except for a thin punctuate band of fluorescence along the equatorial segment (acrosome-reacted spermatozoa, pattern AR). Data were analysed by chi-square. There were no differences in sperm viability between seasons (78.4 and 76.4%, respectively, in autumn and spring). However, post-thaw motility increased (P < 0.05) in autumn (60.0%) compared with spring (50.0%). The percentage of sperm displaying CTC pattern F increased in autumn compared with spring (40.5 and 27.3%, respectively; P < 0.01), whereas the percentage of sperm with both pattern B (57.9 and 66.6%, respectively; P < 0.01) and AR (1.6 and 6.1%, respectively; P < 0.01) decreased. The number of bulls and ejaculates used in this study was too low to draw definitive conclusions. However, these findings suggested that capacitation-like changes after sperm cryopreservation may be reduced during the favourable season in buffalo.
279 CYTOPLASMIC POLYADENYLATION-REGULATED GENE EXPRESSION DURING BOVINE OOCYTE MATURATION
Authors: Reyes J. M. , Ross P. J.
Published: December 2014

Using RNA-seq of GV and MII oocytes we have described changes in polyadenylated transcript abundance that occur during in vitro bovine oocyte maturation (Reyes J. M., J. L. Chitwood, and P. J. Ross. 2013. Deciphering regulation of transcript abundance in maturing bovine oocytes. Poster session presented at International Plant & Animal Genome XXI. San Diego, CA). These changes can be attributed to transcript degradation, transcription, or transcript polyadenylation levels. The objectives of the present study were to determine the extent of cytoplasmic polyadenylation (CP) by measuring total and polyadenylated transcript abundance and poly(A) tail length in GV and MII oocytes for 8 (CCNB1, CPEB4, DNMT3B, FBXO43, EZH2, GDF9, PRDX2, and PAIP2) genes selected based on RNA-seq results. Oocytes were obtained by aspiration of abattoir-derived ovaries (GV) and in vitro maturation for 24 h (MII). Four pools of 40 oocytes were collected per stage. Enhanced green fluorescent protein (EGFP) cRNA was spiked into each sample before RNA extraction using the PicoPure RNA Isolation Kit. Extracted RNA was equally divided for cDNA synthesis using either random hexamers or anchored oligo(dT) primers to detect total and polyadenylated transcripts, respectively. Quantitative PCR (qPCR) of target genes, EGFP (exogenous control), and PPIA (endogenous control) was performed in duplicate for each replicate and gene. Relative transcript abundance was calculated using the 2[–ΔΔC(T)] method and statistically analysed using the Student's t-test. Transcript poly(A) tail length was determined for all but 2 genes (DNMT3B and EZH2) at the GV and MII stages using rapid amplification of cDNA ends poly(A) test (RACE-PAT). Two replicates of GV (n = 100) and MII (n = 100) pairs were collected to perform RACE-PAT followed by fragment analysis on a Bioanalyzer DNA 1000 chip. Polyadenylated RNA abundance levels matched those of the RNA-seq study for 7/8 genes using both PPIA and EGFP to normalise qPCR data, demonstrating the validity of RNA-seq results. Furthermore, total transcript levels for 6/8 and 7/8 genes remained unchanged when normalized to an endogenous and exogenous control, respectively. Combined, the results suggest a significant role for CP considering changes in polyadenylated transcript levels occur without changes in total RNA abundance. Also, changes in transcript abundance corresponded to differences in poly(A) tail length at the specific stages as determined by RACE-PAT. In conclusion, CP is the predominant mechanism responsible for changes in transcript abundance during oocyte maturation at least for the majority of the examined genes, though more in-depth studies are required to determine the global extent of CP. This study improved the atlas of CP-regulated genes potentially providing researchers with critical knowledge to improve in silico tools that predict genes regulated by CP based on presence, position, and distribution of motifs within the 3′ untranslated region.
291 NOVEL OOCYTE SHIPPING AND MATURATION MEDIUM WITHOUT CO2 GAS PHASE IMPROVES IN VITRO MATURATION AND PREGNANCY OUTCOME IN CATTLE
Authors: Barcelo-Fimbres M. , Campos-Chillon L. F. , Mtango N. R. , Bonilla L. , Verstegen J.
Published: December 2014

The aim of the present work was to evaluate embryonic development after shipping and maturation of bovine cumulus oocyte complexes (COC) collected by ovum pick up (OPU) in medium (SMM) that does not require CO2 gas for transport and maturation. Two experiments were conducted, experiment 1 stimulated nonlactating Holstein (n = 4), Jersey (n = 2), Angus (n = 4), and Wagyu (n = 2) donors with 6 pFSH injections (Pluset, MOFA Global LLC, Verona, WI, USA) were used. From each donor, some OPU sessions were delivered the same day (~3 h after collection) for IVM in conventional gas bicarbonate-equilibrated medium system (control), while COC from the other sessions were placed in a portable incubator at 38.5°C, delivered the next day allowing 24 h of maturation in SMM (BoviPro, Mofa Global, WI, USA). The COC were fertilized using commercial semen for each breed, and embryos were cultured in BBH7 medium (BoviPro, Mofa Global, WI, USA) at 38.5°C in 5% O2, 5% CO2, and 90% N2 atmosphere. Embryonic development was evaluated in this experiment. For experiment 2, Day 7 fresh Holstein and Jersey embryos (n = 610) from SMM (n = 550) and controls (n = 60) were transferred in synchronized virgin heifers and pregnancies were diagnosed by ultrasonography at d 35. Data were analysed by ANOVA using GLM, percentages were transformed using arcsin square root, and pregnancy rates were analysed by GenMod using SAS statistical software (Cary, NC, USA). Similar COC numbers were recovered for maturation treatments (P > 0.1; Table 1). The COC matured in SMM had higher cleavage and blastocyst rates than the control group (P < 0.01; Table 1), and this resulted in more transferable embryos per OPU session (P < 0.05; Table 1). We did not find breeds effects or interactions for any variable (P > 0.1; Table 1). After ET, SMM had similar pregnancy rates than control (53.8 v. 58.3%; P > 0.1); however, as more blastocysts were produced per OPU session in the SMM condition, more pregnancies were obtained per session (4.3 v. 2.1; P < 0.01). We conclude that COC matured in SMM had greater oocyte competence than control in commercial settings. The SMM resulted in greater embryonic development, similar pregnancy rates, but more transferable embryos and pregnancies per OPU session than the conventional maturation system.

Table 1.Least squares means (± SE) of embryonic development of COC matured in SMM or control
299 EFFECT OF ESTRADIOL-17β AND FOLLICLE STIMULATING HORMONE ON THE IN VITRO MATURATION OF PORCINE OOCYTES DERIVED FROM SMALL FOLLICLES
Authors: Li Y. , Moros C. , Izquierdo-Rico M. J. , Romar R. , Funahashi H.
Published: December 2014

In porcine cumulus-oocyte complexes (COC) from middle follicles (MF: 3–6 mm in diameter), FSH is known to induce the resumption of meiosis and accompanied by transactivate of the EGF receptor and activation of MAPK3/1 in the cumulus cells. The aim of the current study was to examine the effect of oestradiol-17β (E2: 0.1 μg mL–1) or FSH on in vitro maturation (IVM) of porcine oocytes derived from small follicles (SF: 1–2 mm in diameter). The COC were aspirated from MF of porcine ovaries obtained at slaughterhouse and cultured for IVM in mPOM (with 1 mM dibutyryl cAMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h and then without those for 24 h in an atmosphere of 5% CO2 in air at 39°C) after washing 3 times. The COC from SF, which were aspirated at the same time with COC from MF, were precultured in the absence or presence of E2 or E2 plus FSH for 6 h before IVM culture. After the culture, oocytes were denuded from cumulus cells with 0.1% (vol/vol) hyaluronidase and the meiotic stage was observed. Relative transcript abundance of FSH and EGF receptors of CC was also examined by real-time RT–PCR just after preincubation for 6 h. Statistical analysis of data from 3 to 5 replicates was analysed by ANOVA and Tukey's multiple comparison tests. Maturation rate of oocytes from SF (40.6 ± 3.1%) was significantly lower than that of oocytes from MF controls (78.8 ± 2.8%, P < 0.01). Preincubation in the presence of E2 alone and E2 plus 0.005 IU of FSH significantly increases the maturation rate of oocytes from SF (56.8 ± 1.5 and 55.7 ± 3.1%, respectively, P < 0.01), although the rate was still lower than MF controls. However, in the presence of E2 plus a higher concentration of FSH (0.05 and 0.5 IU), oocyte maturation rate was similar (36.3 ± 2.4 and 33.7 ± 1.9%, respectively) to SF controls and lower than those of E2 alone and E2 plus 0.005 IU of FSH groups. Relative transcript abundance of FSH receptor of CC increased (P < 0.01) during preincubation in the presence of E2, but decreased in the presence of 0.05 IU of FSH. There were no significant differences in the transcript abundance of EGF receptors among treatments during preincubation (P = 0.09). In conclusion, preincubation of COC from SF in the presence of E2 alone and E2 plus 0.005 IU of FSH improves the maturation rate of the oocytes, whereas the presence of FSH more than 0.05 IU mL–1 concealed the positive effect. These effects may be yielded by change in the relative transcript abundance of FSH receptor of COC through the treatments.
314 MELATONIN ON IN VIVO AND IN VITRO MATURATION OF MOUSE OOCYTES
Authors: Fernandes H. , Schefer L. , Castro F. C. , Leal C. L. V.
Published: December 2014

Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation of oocytes: COC were collected 48 h after 5 IU of eCG and maturated in the presence of 3 different doses of melatonin (10–9, 10–6, and 10–3 M) or 0.5 µg mL–1 of FSH (control) for 16 h. In vitro-maturing oocytes were in incubated at 37°C, 5% CO2, and 95% humidity. Maturation rates were evaluated according to the presence of the first polar body under an inverted microscope. Statistical analyses were performed by ANOVA followed by Tukey's test (4 replicates). In the first treatment, 20 mg kg–1 of melatonin showed the highest in vivo maturation rate, 80.3% (61/76), while 10 mg kg–1 of melatonin was 62.4% (53/85) and the saline control group was 69.4% (77/111), but differences were not significant (P > 0.05). For in vitro maturation of oocytes from animals previously treated with melatonin, the 10 mg kg–1 of melatonin group had the highest maturation rate, 53.2% (99/186), in comparison with the saline and 20 mg kg–1 of melatonin groups, which showed 46.6 (88/189) and 39.0% (85/218), respectively; again, no differences were detected (P > 0.05). In the last treatment, the maturation rates increased from 48.9 (43/88) to 53.7 (51/95) and 56.0% (56/100) as the melatonin concentrations decreased from 10–3, 10–6, and 10–9 M, respectively. The control group had the highest rate of 57.3% (55/96), but no statistical differences were observed (P = 0.706). In conclusion, under the conditions studied, melatonin was unable to improve the maturation rate neither after in vivo nor in vitro treatment. However, during in vitro maturation, melatonin alone was as efficient as FSH in promoting maturation in murine oocytes, indicating its potential effect on stimulating meiosis. Therefore, the role of melatonin in stimulating meiosis needs further investigation.Acknowledgments to FAPESP for fellowship (HF) and funding (CLVL).
320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION
Authors: Arias M. E. , Sanchez R. , Felmer R.
Published: December 2014

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P < 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.
323 ESTROGEN-INDUCED EPITHELIAL-MESENCHYMAL TRANSITION AND EXPRESSION OF PROAPOPTOTIC MARKERS IN PORCINE STEM CELLS
Authors: Kim Y.-S. , Hyun S.-H. , Lee C.-K. , Choi K.-C.
Published: December 2014

In transgenic pig production for generating animal models of human diseases, apoptosis of early implantation embryo disturbs the transgenic pig production. In general, epithelial-mesenchymal transition (EMT) is considered important in embryo development and apoptosis. In addition, it was reported that 17β-oestradiol (E2), among hormones that participate in early implantation of embryo, could induce EMT and neural differentiation in mouse embryonic stem cells. Therefore, in this study, we examined the effects of the steroid hormone, E2, in the changes of EMT and apoptotic markers in porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC). During the study, we cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (b-FGF) and leukemia inhibitory factor (LIF) and performed RT-PCR and an alkaline phophatase (AP) test to measure pluripotent and undifferentiation markers of these porcine stem cells. The RT-PCR results showed that OCT4, NANOG, and SOX2 were expressed in these pESC and piPSC, indicating their pluripotency as stem cells. Also, these porcine stem cells showed positive AP activity, demonstrating undifferentiation. Additionally, we treated pESC and piPSC with E2 to examine effects of steroid hormone on the changes of EMT and apoptotic markers (i.e. bcl-2, bax, E-cad, and vimentin). The E2 treatment increased the expression of vimentin and bcl-2, while decreased the expression of E-cadherin and bax. By using immunocytochemistry (ICC), we examined the protein expression of EMT markers, which are vimentin and E-cadherin at the translational level, and found that expression of vimentin protein was increased while E-cadherin protein level was reduced at periphery of the colonies in pESC and piPSC. In conclusion, these results indicate that E2 can promote EMT process and reverse apoptosis in these pESC and iPSC. In a future study, we will further examine the effects of progesterone on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate the underlying mechanisms of EMT and apoptosis controlled by steroid hormones in porcine stem cells.
324 TESTIS-SPECIFIC EXPRESSION OF Oct4-EGFP TRANSGENE IN PIG
Authors: Nowak-Imialek M. , Lachmann N. , Herrmann D. , Jacob F. , Niemann H.
Published: December 2014

Oct4 is a transcription factor essential for establishment and maintenance of pluripotency in mammalian stem cells. Oct4 expression was found in early embryos and germ cells throughout fetal development. In male mice, Oct4 expression is found in mitotically arrested prospermatogonia until birth. After onset of spermatogenesis, expression is maintained in type A spermatogonia, but is downregulated in type B spermatogonia and in spermatocytes (Pesce et al. 1998 Mech. Dev). Previously, we successfully generated Oct4-EGFP reporter pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (Nowak-Imialek et al. 2011 Stem Cells Dev.). This animal model is unique because it allows in vivo and in vitro visualisation of Oct4-positive cells. Germ line specific Oct4-EGFP expression was analysed in testis isolated from young (<1 week) and adult (>7 months) pigs. Squash preparation of testicular tissue isolated from adult transgenic boars revealed high amounts of EGFP-positive cells compared to young piglets. We confirmed Oct4 and EGFP expression in the testis from young and adult transgenic animals using Northern blot analysis. Specific expression of Oct4 and EGFP in testis could be observed in blots as a single band of 1.5 kb. As a loading control, the blot was rehybridized with a β-actin probe. Mammalian testes contain different cell types, including germ cells, Sertoli cells, Leydig cells, and peritubular cells. To define the cellular origin of EGFP-expressing cells, we isolated these cells from adult transgenic testis using fluorescence-activated cell sorting (FACS)-based techniques. Analysis of isolated EGFP positive cells with qRT-PCR demonstrated the presence of marker genes specific for undifferentiated (Oct4, UTF1, FGFR3, PGP 9.5, THY-1, SALL4, and GFRα1) and differentiated (BOLL and PRM2) germ cells. Markers specific for Sertoli cells (vimentin) and Leydig cells (LHCGR) were not observed. To verify the localization of EGFP-positive cells in seminiferous tubules, we performed immunohistochemical detection of GFP in adult pig testis. Unlike the Oct4-EGFP reporter mouse model, GFP protein was not found in spermatogonia attached to the basement membrane of seminiferous tubules, but instead were found in differentiated germ cells, including spermatocytes and spermatids. These results show that the Oct4-EGFP expression in testis differs between mouse and porcine Oct4-EGFP transgenic models. To verify that the EGFP expression driven by the mouse Oct4 promoter in porcine testis reflects the endogenous Oct4 expression profile, Western blot and histochemical analyses are currently underway.
329 CHONDROGENIC POTENTIAL OF PORCINE ADIPOSE-DERIVED STEM CELLS, CHONDROCYTES, PERIOSTEAL CELLS, AND FIBROBLASTS IN A PELLET CULTURE SYSTEM
Authors: Herzog K. K. , Milner D. J. , Johnson S. J. , Wheeler M. B.
Published: December 2014

Regenerative medicine has long sought to develop therapies for articular cartilage repair and for enhancing endochondral ossification to address complications of long bone healing. The objective of this study was to determine the chondrogenic potential of porcine primary cell cultures for possible utility in orthopedic tissue engineering applications. Adipose-derived mesenchymal stem cell (ASC), chondrocyte (positive control), periosteal cell, and fibroblast (negative control) primary cell cultures from 8- to 12-month-old Yorkshire pigs were plated at 5000 to 10 000 cells cm–2 in 75-cm2 cell culture flasks using high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with NaHCO3, 10% fetal bovine serum (FBS), and antimicrobials (penicillin-streptomycin, gentamicin sulfate, and amphotericin B), then incubated at 37°C, 5% CO2, and 18% O2. Cells were trypsinized at ~80% confluency and transferred into 15-mL conical tubes at 500 000 cells tube–1. Suspensions were washed twice by centrifugation in DPBS then condensed via a final centrifugation in 1.0 mL of negative control media (DMEM + 10% FBS) or chondrogenic base media consisting of high-glucose DMEM with 40 µg mL–1 of proline, 50 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, antimicrobials, and 1× insulin-transferrin-selenium solution added. Chondrogenic additives tested were 2% FBS, Kartogenin (200 nM, 400 nM, or 4 µM), 10 ng mL–1 of BMP-4, or a combination of 10 ng mL–1 of BMP-6 + 10 ng mL–1 of TGFβ-3. Pellets were incubated with media changed every 2 to 3 days for a period of 2 to 4 weeks, fixed with 4% paraformaldehyde in DPBS, and then frozen at –80°C in Neg 50 Frozen Section Medium. Eight-micrometer sections were cut using a cryostat onto charged slides. Histochemical staining was performed using hematoxylin and eosin (H&E) for cell morphology and Safranin O and Alcian Blue for cartilage matrix markers. Immunofluorescent staining was done to detect collagen II and aggrecan with the nuclear marker lamin-C used to assess cell viability. Chondrocyte pellets grown in chondrogenic media, regardless of additive, exhibited cartilage matrix formation with H&E and stained strongly positive with Safranin O, Alcian Blue, and collagen II. The ASC pellets grown in chondrogenic media showed mixed cell morphology and areas of early cartilage matrix formation with H&E and stained faintly positive with Safranin O, Alcian Blue, and collagen II at 3 weeks in culture. Periosteal cell pellets had similar morphology in all conditions and did not stain positive for cartilage matrix markers. Fibroblast pellets did not survive any condition for processing. In conclusion, porcine chondrocytes and ASC were able to form cartilage matrix in a pellet culture system with chondrogenic media regardless of additive, while porcine fibroblasts and periosteal cells showed limited to no chondrogenic potential in the conditions tested.
349 SUPEROVULATORY RESPONSE AND EMBRYO QUALITY RECOVERED FOLLOWING FLUSHING NGUNI HEIFERS AND COWS
Authors: Maqhashu A. , Mphaphathi M. L. , Muchenje V. , Nedambale T. L.
Published: December 2014

Most of the South African indigenous cattle breeds are facing genetic degradation due to unselective crossbreeding and irregular matings especially in small scale farming. The aims of the study were to compare superovulatory (SO) response, fertilization rate and to evaluate embryo quality recovered from superovulated Nguni stud cows and heifers. Nguni stud cows (n = 15) and heifers (n = 10) aged 4–6 and 2 years, respectively, were used as embryo donors for ex situ conservation. Nine days following heat observation for the first oestrus synchronisation protocol, superstimulation of donor cows and heifers were administered with a total of 350 mg of FSH (Follotropin-V®, porcine pituitary glands extract) divided into 2 injections daily 12 h apart (6 a.m. and 6 p.m.) for 4.5 days on a decreasing dosage. Two injections of prostaglandin F2α were administered on Day 3 and 4 of FSH injections. Semen was collected from 2 Nguni bulls (bull 1 and 2) and assessed by computer aided sperm analysis before artificial insemination (AI). The AI was conducted with fresh semen 3 times, 12 h apart, and the first AI was performed at the onset of oestrus. Semen from bull 1 was used to inseminate the cows, and bull 2 was used for heifers. Embryos were flushed 7 days after AI using a nonsurgical technique. Embryos were evaluated under stereo microscope and classified according to IETS standard codes (code 1, good and excellent; code 2, fair). Recovered embryos were then vitrified and stored for future use. Superovulatory response was measured by counting number of corpus lutea in each ovary. Data were analysed using 1-way ANOVA (SAS, 2003, SAS Institute Inc., Cary, NC, USA). There was no statistical difference between bull 1 (93.7%) and bull 2 (83.5%) on total sperm motility rate. Furthermore, no significant differences were recorded on fertilization rate between cows (67.5%) and heifers (53.5%). There was also no significant difference on the proportion of Nguni cows (40%) and heifers (40%) that responded to superovulation treatment. There was a significant difference on the ovary reaction (number of corpus lutea) of cows (11.3 ± 1.41) and heifers (4.0 ± 0.57). There were no significant differences recorded on the embryo quality recovered between Nguni cows (code 1, 2.5 ± 1.00; code 2, 1.3 ± 0.59) and heifers (code 1, 0.8 ± 0.41; code 2, 1.0 ± 0.36). However, cows had higher numbers of unfertilized ova than heifers (5.5 ± 1.05 and 1.8 ± 0.47, respectively) and degenerate embryos (3.7 ± 1.00 and 1.3 ± 0.39, respectively). Although superovulatory response of both Nguni cows and heifers was low, Nguni cows had higher ovarian response than heifers. Moreover, the quality of embryos recovered was similar for both Nguni cows and heifers.
356 SLEEPING BEAUTY TRANSGENESIS IN CATTLE
Authors: Garrels W. , Talluri T. R. , Bevacqua R. , Alessio A. , Fili A. , Forcato D. , Rodriguez N. , Olmos Nicotra M. F. , Ivics Z. , Salamone D. F. , Bosch P. , Kues W. A.
Published: December 2014

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the Sleeping Beauty (SB) transposon system was assessed for stable gene transfer into the cattle genome. The transposon plasmids encoded a ubiquitously active CAGGS promoter-driven Venus reporter and a lens-specific α A-crystallin promoter driven tdTomato fluorophore, respectively. The helper plasmid carried the hyperactive SB100x transposase variant. In total, 50 in vitro-derived zygotes were co-injected (Garrels et al. 2011 PLoS ONE 6; Ivics et al. 2014 Nat. Protoc. 9) and cultured up to blastocyst stage (Day 8). Two blastocysts were Venus-positive and were transferred to synchronized heifers, resulting in one pregnancy. The resulting calf was normally developed and vital; however, it died shortly after cesarean section due to spontaneous bleeding from an undetected aneurism. Phenotypic analysis suggested that the calf was indeed double-transgenic, showing widespread expression of Venus and lens-specific expression of tdTomato. Genotyping and molecular analyses confirmed the integration of both reporter transposons and the faithful promoter-dependent expression patterns. Subdermal tissue of an ear biopsy was used to culture fibroblasts, which were employed in somatic cell nuclear transfer experiments. In total, 39 embryos were reconstructed, of which 34 underwent cleavage, and at the end of culture 12 morulas and 12 blastocysts were obtained. Ten of the blastocysts were Venus positive, and embryo transfer of Venus-positive blastocysts is planned. In summary, we showed that the cytoplasmic injection of SB components is a highly efficient method for transgenesis in cattle. Due to the modular composition of SB plasmids, even double transgenic cattle can be generated in a one-step procedure. Importantly, the SB-catalyzed integration seems to favour transcriptionally permissive loci in the genome, resulting in faithful and robust promoter-dependent expression of the transgenes. The transposon constructs carry heterospecific loxP sites, which will be instrumental for targeted insertion of functional transgenes by Cre recombinase-mediated cassette exchange.Financial support of DFG (Ku 1586/3-1), UNRC, CONICET and Agencia Nacional de Promoción Científica y Tecnológica de la Argentina (ANPCyT) is gratefully acknowledged.
360 DOUBLE KNOCKOUT OF GOAT MYOSTATIN AND PRION PROTEIN GENE USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT (CRISPR)/Cas9 SYSTEMS
Authors: Hu S. , Yang M. , Polejaeva I.
Published: December 2014

Myostatin (MSTN) acts as a negative regulator of skeletal muscle development and growth. Inhibition of MSTN expression may be applied to enhance animal growth performance in livestock production. Prion protein (PrPc) is associated directly with the pathogenesis of the transmissible spongiform encephalopathies occurring in variety of species including human, cattle, sheep, goats and deer. Prion protein-deficient livestock may be a useful model for prion research and producing animal conferring potential disease resistance. The goal of this study was to generate MSTN/PrPc double knockout goat by using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We generated 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc genes, respectively. The CRISPR/Cas9 plasmids targeting each gene were respectively transfected into goat fibroblasts, and the efficiency of gene modification was determined at Day 3 using restriction fragment length polymorphism (RFLP) assay. The RFLP assay showed that CRISPR/Cas9 plasmids targeting MSTN and PrPc induced precise gene mutations with efficiency of 59 and 70%, respectively. Single cell-derived colonies were further isolated by limiting dilution after co-transfection of 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc. The RFLP assay and DNA sequence analysis indicated that 9 out of 45 colonies (20%) carried simultaneous disruption of both target genes. Moreover, 5 of 9 mutant colonies (55%) had mutations in all 4 alleles of 2 genes. These double-gene knockout fibroblast cells will be used as nuclear donors for developing double knockout goat deficient in MSTN and PrPc. The CRISPR/Cas9 system represents a highly effective and facile platform for multiplex editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.This work was supported by the Utah Science Technology and Research Initiative and Utah Agricultural Experiment Station project #31294.