bluuebunny/crossref_metadata_2025 huggingface dataset:
103 OVARIAN STIMULATION OF ANESTROUS CATS WITH FOLLICLE-STIMULATING HORMONE IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL CAPABILITY AFTER PARTHENOGENETIC ACTIVATION
Authors: Veraguas D. , Gallegos P. , Velasquez A. E. , Castro F. O. , Rodriguez-Alvarez L.
Published: December 2015

The domestic cat is a valuable model for developing assisted reproductive technologies in endangered felids. However, the in vitro production of cat embryos is negatively affected during the anestrous season. It seems that a low FSH level or the few FSH receptors in the granulosa cells might affect oocyte quality. The objective of this research was to evaluate the effect of ovarian stimulation of anestrous cats with FSH on oocyte quality and developmental capability after parthenogenetic activation. For this, two experimental groups were made. The first (FSH group) consisted of 9 anestrous cats treated with a subcutaneous dose of 5 mg of porcine FSH, every 24 h, for 4 days. The second (control group) consisted of 10 untreated anestrous cats. The ovaries were recovered by ovariohysterectomy. Cumulus-oocyte complexes (COCs) were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. Grade I and II COCs were matured in vitro during 30 h and matured oocytes were used for parthenogenetic activation. Matured oocytes were activated using 7% ethanol for 5 min, followed by incubation in 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 5 h. Presumed zygotes were in vitro cultured in SOF for 8 days, and blastocyst and hatching blastocyst rates were assessed. The relative expression of pluripotency (OCT4, SOX2, and NANOG) and differentiation markers (CDX2 and GATA6) were evaluated by RT-qPCR in the blastocysts. The results were normalized using the geometric mean of GAPDH and SDHA. Statistical analysis was performed using the Wilcoxon non-parametric test. A total (mean ± s.d.) of 741 COCs (82.33 ± 36.61 per cat) were recovered from the FSH group and 660 COCs (66 ± 20.67 per cat) from the control group (P > 0.05). The FSH group had a higher rate of grade I COCs and a lower rate of grade III and IV COCs (22.28% and 50.62%, respectively) than the control group (13.2% and 60.08%, respectively; P < 0.05). However, no significant differences were found in grade II COCs between FSH (26.21%) and control groups (27.01%; P > 0.05). Thereafter, 190 matured oocytes were activated in the FSH group and 198 in the control group. The FSH group had higher blastocyst and hatching blastocyst rates (30.52% and 13.15%, respectively) than the control group (13.13% and 1.01% respectively; P < 0.05). The blastocysts from the FSH group had a higher cell number (± s.d.) than their counterparts from the control group (199.9 ± 53.6 and 121 ± 34.91, respectively; P < 0.05). Furthermore, the blastocysts from FSH group had an increased relative expression of OCT4 and GATA6 (P < 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improves oocyte quality and embryo developmental capability, which agrees with an increase in relative expression of OCT4 and GATA6.
113 EXPRESSION OF BETA-OXIDATION-RELATED GENES IN PREECLAMPSIA-LIKE MODEL UNDER HYPOXIC CONDITION IN VIVO AND IN VITRO
Authors: Lee M. H. , Shin E.-K. , Kang H. Y. , Hwang J.-U. , Jeung E.-B.
Published: December 2015

Preeclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and large amounts of protein in the urine. Preeclampsia is thought in many cases to be caused by a shallowly implanted placenta that becomes hypoxic. The hypoxic condition during the pregnancy can result from a failure at any stage in the delivery of oxygen to the cells. In peripheral tissues, oxygen diffuses down a pressure gradient into cells and moves into their mitochondria, where it is used to produce energy. As an expression of beta-oxidation-related genes, ACADVL was detected by gene-fishing technology using the placenta of human. We conducted in vitro and in vivo experiments to confirm preliminary study by inducing hypoxic stress in the BeWo cells and mice placenta. BeWo cells were cultured at 37°C under 1% O2, 5% CO2, and balanced with N2. Pregnant mice were maintained from GD 6.5 to 17.5 under 11% O2, 5% CO2, and balanced with N2. The expression of beta-oxidation related genes (ACADVL, EHHADH, HADH, ACAA1) were observed under hypoxic condition at mRNA and protein levels. The expression of genes known as biomarkers for hypoxia, HIF-1α, was increased in BeWo cells and mouse placenta, which induced PE. The beta-oxidation-related genes ACADVL expression was significantly increased by hypoxic stress both BeWo cells and mouse placenta. The elevated level of HIF-1α indicates that our experimental conditions closely mimicked PE. These results indicate that changes of beta-oxidation-related genes are correlated with PE induced hypoxic condition.
12 INITIAL GONADOTROPIN-RELEASING HORMONE TREATMENT INCREASED PREGNANCY PER TIMED INSEMINATION ONLY IN ACYCLIC BEEF HEIFERS SUBJECTED TO A 5-DAY CO-SYNCH PROTOCOL
Authors: López-Helguera I. , Whittaker P. , Behrouzi A. , Colazo M. G.
Published: December 2015

We have shown that inclusion of initial gonadotropin-releasing hormone (GnRH) in a 5-day Co-synch protocol is unnecessary to achieve acceptable pregnancy rates in dairy heifers (2011 Theriogenology 76, 578). This study determined the efficacy of a modified (without initial GnRH) 5-day Co-synch protocol for timed AI (TAI) and the effect of interval from progesterone device removal to TAI on pregnancy per TAI (P/TAI) in acyclic and cyclic beef heifers. Heifers (n = 1062; 302 to 515 kg of body weight, and 13 to 15 months of age) at 2 locations were assigned to either a 5-day Co-synch protocol with (control) or without (modified) an initial injection of 100 μg of GnRH (Fertagyl; Merck Animal Health, Kenilworth, NJ, USA) at progesterone device (CIDR; Zoetis Animal Health, Florham Park, NJ) insertion (Day 0). All heifers received a single dose of 500 μg of cloprostenol (Estrumate; Merck Animal Health) at CIDR removal (Day 5) and were divided into 2 groups to receive GnRH and simultaneously TAI at either 66 or 72 h (Day 8) after CIDR removal. All heifers were inseminated by one technician with frozen-thawed semen from 1 of 4 commercially available sires. Ovarian ultrasonography was performed on Day 0 to determine cyclicity (presence of corpus luteum) and normalcy of the reproductive track, and on Day 35 (27 days after TAI) to determine pregnancy status. Data were analysed using the PROC GLIMMIX in SAS (SAS Institute Inc., Cary, NC, USA). The CIDR retention rate was 98%; P/TAI in heifers that lost the CIDR was 39%. Pregnancy per TAI [least squares means (n)] did not differ (P > 0.05) among sires [48 (235), 51 (318), 52 (274), and 53% (235)], location [57 (330) v. 53% (732)], timing of TAI [52 (533) v. 50% (529) for 66 and 72 h], or treatment groups [48 (525) v. 53% (537) for control and modified groups]. A total of 226 (21%) heifers were acyclic; P/TAI was lower in acyclic heifers compared with that in those cyclic (P < 0.01; 43 v. 59%). Administration of GnRH at CIDR insertion increased P/TAI in acyclic heifers (P < 0.01; 50 v. 35% for control and modified groups) but did not affect P/TAI in those that were cyclic at initiation of protocol (P > 0.05; 57 v. 62% for control and modified groups). In conclusion, timing of TAI did not affect fertility. However, administration of initial GnRH increased P/TAI by 15 percentiles in acyclic females, but as previously shown in Holstein heifers, initial GnRH was unnecessary to achieve acceptable P/TAI in cyclic beef heifers subjected to a 5-days Co-synch protocol.
This study was supported by Alberta Agriculture and Forestry, Intervet Canada Corp., and collaborative beef producers.
137 DYNAMIC SHIFT IN CYTOPLASMIC LIPIDS IN CAT OOCYTES DURING OVARIAN FOLLICLE DEVELOPMENT
Authors: Thongkittidilok C. , Li Y. , Wildt D. , Songsasen N.
Published: December 2015

Cytoplasmic lipids play key roles during oocyte and embryo development. However, there is little information on the changes in lipid types during intraovarian follicular and oocyte growth. Here, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to understand lipid composition in the cat oocyte during differing stages of folliculogenesis. Follicles at different developmental stages were mechanically isolated from cat ovaries within 4 h after routine ovariohysterectomy. Oocytes with granulosa cells were recovered from secondary (preantral stage, n = 387), early antral (<0.5 mm diameter, n = 177), small antral (0.5–1 mm, n = 144), and antral (>1 mm, n = 120) follicles, then subjected to lipid extraction and MALDI-TOF analysis (n = 3 replicates). Resulting mass spectra data were evaluated using MALDIquant in R package to eliminate baseline (background) and to identify peak values. The latter maximal values for each follicle stage were selected and subjected to principal component analysis to identify similarities and differences in mass spectra profile among oocytes from varying developmental stages. Peaks were compared to those calculated molecular formulae available in the Lipid MAP database. Error estimates were calculated using Excel (Microsoft Corp., Redmond, WA), and lipid species identification performed using Lipid MS Predict. Twenty-nine lipid species from six glycerophospholipids groups (glycerophosphates, PA; glycerophosphoserines, PS; glycerophosphoinositols, PI; glycerophosphoglycerols, PG; glycerophosphoinositol monophosphates, PIP; and glycerophosphoethanolamines, PE) were identified, 15 being found in more than 1 developmental stage. Two species in particular, [PI(29:4)+Na]+ and [PIP(23:0)+Na]+, were the most abundant lipids and were identified in oocytes from all developmental stages. There were dynamic shifts in lipid species expressed at different follicle stages. Oocytes from secondary and antral follicles contained more lipid types (15 and 22, respectively) than early (10) and small antral (4) counterparts. Four (PA, PS, PI, and PIP) of the 6 glycerophospholipids were found only in oocytes from secondary and antral follicles. Oocytes from small antral follicles also lacked PA, PS, and PG, whereas PG was not found in early antral stage oocytes. In summary, we showed for the first time in the cat that, similar to goats and rats, there are temporal changes in lipid types within the oocyte during folliculogenesis. We suspect that these changing dynamics, including shifts in presence or absence of lipid species with follicle stage, may be playing key roles in oocyte growth and viability. Our findings also serve as in vivo benchmarks for parallel studies focused on enhancing an in vitro culture system for early-stage ovarian follicles to preserve fertility of genetically valuable domestic and wild felids.
145 OVIDUCTAL EXPRESSION OF GENES INVOLVED IN GROWTH FACTOR, CYTOKINE, HORMONE, AND WNT SIGNALING DURING THE EARLY ESTROUS CYCLE OF THE COW
Authors: Tribulo P. , Siqueira L. G. , Hansen P. J.
Published: December 2015

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the pre-implantation embryo, called embryokines, is largely incomplete. To identify possible candidates for this function, an experiment was conducted to assess expression patterns during the first 7 days after ovulation for 92 genes that could be involved in control of development. Included were genes for 27 growth factors, 11 cytokines, 22 interleukins, 3 hormones, 19 WNT ligands, and 9 WNT regulatory molecules. Cows were slaughtered at Days 0, 3, 5, and 7 relative to predicted ovulation. Reproductive tracts were obtained and transversal sections from the isthmus of oviducts ipsi- and contralateral to the corpus luteum were harvested for gene expression analysis. Abundance of specific mRNA molecules was determined using the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA). Data were normalized against 6 housekeeping genes (ACTB, ERK1, GAPDH, RPL19, SLC30A6, SUZ12) and internal positive controls. Genes were considered expressed if the number of reads was greater than 2 standard deviations above the mean of negative controls. Data were analysed by ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) with day, side, and day × side as fixed effects, and cow as random effect. Side did not have any significant effect so data were analysed without side and day × side in the model. In contrast to what was observed earlier for endometrium (P. Tribulo and P. J. Hansen, unpublished data), we found no difference in gene expression between oviducts ipsi- and contralateral to the corpus luteum. Overall, there was wide variation in the magnitude of gene expression. Among the 20 most expressed genes, average reads varied from 164 to over 10 726. All genes were detected at Days 0, 3, and 7 but only 67 of the 92 genes were expressed at Day 5. The 10 highest-expressed genes were CTGF, CXCL3, CXCL10, CXCL12, GRO1, IGF2, IK, SFRP1, WNT5A, and WNT6. Of these, CTGF, CXCL12, IGF2, IK, HDGF, WNT5A, and CXCL3 were within the 10 highest expressed at all days (P. Tribulo and P. J. Hansen, unpublished data). There were only 6 genes whose expression was significantly affected by day. Expression was highest at oestrus (VEGFA), Day 5 (GRO1, SFRP1) or Day 7 (BMP4, IK, WNT16). This experiment identifies some potential maternal regulators of embryonic development. Expression of most of these putative embryokine genes did not vary with stage of the oestrous cycle, suggesting that expression is either not under endocrine control or varies between cell types within the oviduct. Further studies are needed to determine the effect of these maternally secreted molecules on embryonic development.
Study was supported by the National Institutes of Health (HD080855).
149 THYROID HORMONE MARKER DEIODINASE 1 INDUCED BY 2,4,6-TRIBROMOPHENOL IN GH3 PITUITARY CELLS
Authors: Lee D. , Hwang J.-U. , Kang H. Y. , Jeung E.-B.
Published: December 2015

Numerous medical materials and products in daily use are made of chemicals that are rarely found in the environment. Some of these chemicals may alter reproductive and thyroid function by interfering with endogenous estrogens, progesterone, and thyroid hormones. They are called endocrine-disrupting chemicals (EDC). To inhibit ignition and to reduce flammability of products, brominated flame retardants (BFR) are added to some products. 2,4,6-Tribromophenol (TBP) is an emerging EDC that is produced in some countries. To compare this emerging chemical to traditional EDC, we investigated this chemical in a rat pituitary cell line, GH3 cells. The GH3 cells were starved without phenol red for 7 days to eliminate any steroidogenic effect. Then, GH3 cells were incubated with 1–850 (thyroid receptor antagonist; 5.0 × 10–6 M) for 1 day before treatment with vehicle, triiodothyronine (T3; 1.0 × 10–9 M) and TBP (1.0 × 10–6 M) for 24 h, respectively. The TBP dose was determined from a previous pilot experiment using high, middle, and low doses. Each group of cells was prepared in triplicate and experiments were performed twice. Type I iodothyronine deiodinase (Dio1; thyromimetic marker), thyroid hormone receptor β, isoform 2 (Thrβ2), and growth hormone (Gh) were quantified by RT-qPCR. All data were analysed with a one-way ANOVA and Tukey’s studentized range test. Statistical analyses were performed with SAS software (SAS Institute Inc., Cary, NC, USA); P-values <0.05 were considered significant. RT-qPCR analysis was performed using the 2–ΔΔCT method employing 18S RNA (18S) as endogenous reference gene. Each gene of interest was normalized to vehicle. The mRNA expression of Dio1 and Gh were up-regulated via TBP treatment, but was lower than that of the T3-treated positive control. In pretreatments of 1–850 with T3 or TBP, mRNA levels of Dio1 were diminished through inhibition of thyroid receptor, but Gh mRNA level was not diminished. The mRNA expression of Thrβ2 was decreased in independent treatment of T3 or TBP, and no change in pretreatments of 1–850 with T3 or TBP. Although interfering effects of TBP on thyroid function have been partly studied, the dangers of TBP have been not yet obviously verified. In this study, we confirmed a thyromimetic effect of TBP mediated by thyroid receptor on GH3 cells.
150 IN VITRO MODEL FOR EFFECT OF ENDOCRINE-DISRUPTING CHEMICALS ON PLACENTAL CATION TRANSPORTER CHANNEL
Authors: Lee J.-H. , Lee M. H. , Lee M. J. , Jeung E.-B.
Published: December 2015

Calcium, copper, iron, oxygen, and carbon dioxide are essential factors in fetal growth. These molecules are transferred by specific receptors located on the cell membrane or cytoplasm in placenta. Calcium, copper, and iron transfer genes are regulated by oestrogen, placental lactogen, and vitamin D. During pregnancy, expression of these receptors is controlled by the nutritional status of the maternal and fetal environment. Some synthetic plastics contain endocrine-disrupting chemicals (EDC), which have similar structures to steroid hormones or endogenous hormones related to reproduction. These substances disturb action of hormones (e.g. increasing oestrogen or progesterone) by interacting with their receptors or affecting the expression of transporting genes for cations. We used a BeWo cell line (human trophoblast cell line) to test the effect of EDC during pregnancy. The cells were cultured in phenol red-free DMEM supplemented with 5% charcoal dextran-stripped fetal bovine serum for 48 h to ensure the depletion of steroid hormones in the cells. Ethinyl oestradiol (EE), which activates oestrogen receptors, was used as a positive control. Then, EE (10–9, 10–8, and 10–7 M), octylpehnol (OP; 10–7, 10–6, and 10–5 M), nonylphenol (NP; 10–8, 10–7, and 10–6 M), and bisphenol A (BPA; 10–7, 10–6, and 10–5 M) were treated in BeWo cells for 48 h, and the cells were harvested. The mRNA and protein levels for calcium transporting genes (PMCA1 and TRPV6), copper transporting genes (CTR1 and ATP7A), and iron transporting genes (IREG1 and HEPH) were quantified by RT-qPCR, and Western blotting, respectively. Experiments were carried 3 times, and results were statistically analysed by GraphPad Prism6 (GraphPad Software, San Diego, CA, USA). We observed dose-dependent decreases in mRNA levels of PMCA1, TRPV6, ATP7A, and IREG1 compared with control group in OP-, NP-, or BPA-treated groups. Protein levels showed a similar pattern to mRNA levels. Based on our data, we confirmed that these EDC affect metal ion channels such as calcium, copper, and iron transporters during pregnancy.
153 DIFFERENT MOLECULAR MECHANISMS FOR HISTONE DEACETYLASE INHIBITOR-INDUCED APOPTOSIS IN DOG FIBROBLASTS AND MESENCHYMAL STEM CELLS
Authors: Kim M. J. , Oh H. J. , Kim G. A. , Jo Y. K. , Choi Y. B. , Setyawan E. M. N. , Lee S. H. , Lee B. C.
Published: December 2015

Histone deacetylase (HDAC) inhibitors have been applied to cancer research for a therapeutic purpose or somatic cell nuclear transfer for improvement of embryonic reprogramming. Considering the ubiquitous expression of HDAC in normal cells, effects of HDAC inhibitors on normal cells need to be evaluated in detail. Therefore, we aimed to investigate molecular mechanisms of HDAC inhibitor-induced apoptosis in mesenchymal stem cells and compare with the results from fibroblasts. Beagle skin fibroblasts (BF) and adipose-derived mesenchymal stem cell (MSC) lines were established from a 7-year-old beagle. Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and RCMEp (K-stem Cell Ltd, Seoul, Korea) were used as culture media for BF and MSC, respectively. A Food and Drug Administration-approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), was dissolved in dimethyl sulfoxide (DMSO). On passage 4, cells were subcultured to reach 50% of confluency, and cultured with 5 μM of SAHA. Culture medium containing the same volume of DMSO used in the SAHA group was used for the control. After 24 h, all cells were harvested, RNAs were extracted, and cDNA were synthesised. Transcript expression of anti-apoptotic genes (BFL, MCL, BCLxl, BCL2) and pro-apoptotic genes (BAX, BID, BIM) were analysed using RT-PCR. Two-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) with Bonferroni post-tests was performed for statistical analysis. Expression of MCL and BCLxl was significantly decreased in both groups. However, although BFL expression was remarkably increased only in BF (16.7-fold), BCL2 was significantly increased in MSC (7.2-fold) after SAHA treatment. Also, transcript of BAX was significantly increased in MSC (1.5-fold), and BID was significantly decreased in BF (0.3-fold). These results would be helpful to understand different molecular mechanisms of apoptosis induced by HDAC inhibitor on fibroblasts and mesenchymal stem cells.
This study was supported by RDA (#PJ010928032015), IPET (#311062-04-3SB010), NRF (#2014R1A1A2059928), Research Institute for Veterinary Science, Nestle Purina PetCare, and the BK21 plus program.
158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION
Authors: Nkadimeng M. , van Marle-Koster E. , Lekola K. P. M. , Mphaphathi M. L. , Seshoka M. M. , Raphalalani Z. C. , Kanuya N. L. , Nedambale T. L.
Published: December 2015

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.
176 SERUM ANTIMULLERIAN HORMONE CONCENTRATIONS AND IN VITRO EMBRYO PRODUCTION IN BEEF CATTLE
Authors: Bó G. A. , Anduaga I. , Bernal B. , Moreira G. , Cedeño A. , Barajas J. , Pelizzari M. , Andrada S. , Tribulo A.
Published: December 2015

The collection of high numbers of oocytes for in vitro embryo production in the cow depends on the number of antral follicles available for aspirations. The number of antral follicles varies significantly among cows and can be estimated by ovarian ultrasonography or measurement of serum concentrations of antimullerian hormone (AMH). The AMH is secreted by the granulosa cells of small antral follicles and serum concentrations are independent of the stage of the oestrous cycle. Therefore, it may be useful as a tool to select oocyte donors for in vitro embryo production. An experiment was designed to evaluate the effect of breed of cattle and the correlation of serum AMH concentrations on the number of viable oocytes and embryos produced in vitro. Cumulus-oocyte complex were collected during 44 ultrasound-guided follicle aspiration sessions performed at random stages of the oestrous cycle without superstimulation in 14 Bonsmara, 11 Braford, and 19 Brangus cows that were used for commercial in vitro embryo production using frozen-thawed semen from bulls of the same breed. Blood samples were collected by jugular venipuncture at the time of OPU, centrifuged at 3000 × g for 30 min for serum separation which was frozen at –20°C until AMH analysis. Serum AMH was evaluated using an ELISA (AnshLabs, Webster, TX, USA). The sensitivity of the AMH assay was 0.011 ng mL–1 and intra-assay CV were <5%. The number of viable oocytes collected and embryos produced in each breed were compared by ANOVA and means were compared by the protected l.s.d. test when ANOVA revealed statistically significant differences. Regression analysis and Pearson correlation were used to determine the relationship between the number of viable oocytes aspirated and the number of embryos produced. The mean (± SEM) number of viable oocytes collected was greater (P < 0.05) from Brangus (23.3 ± 4.3) and Braford (22.5 ± 3.6) than from Bonsmara donors (12.2 ± 2.5). Although not statistically different, the numbers of embryos produced followed the same pattern (Brangus, 4.4 ± 0.9; Braford, 3.4 ± 0.6; Bonsmara, 2.9 ± 0.5). Serum AMH concentration was positively correlated with the number of viable oocytes collected in all breeds (R2 = 0.63 for Bonsmara, 0.50 for Braford, 0.32 for Brangus; P < 0.01). Serum AMH concentrations were also correlated with the number of embryos produced in all breeds (R2 = 0.32 for Bonsmara, 0.16 for Braford, 0.39 for Brangus). However, AMH concentrations were not significantly correlated with the proportion of viable oocytes collected and embryos produced. Results demonstrate a breed effect on the number of oocytes collected per session and that circulating AMH concentrations are correlated with the number of viable oocytes collected per session in commercial in vitro embryo production programs.
178 FIRST COMMERCIAL CATTLE IN VITRO EMBRYO PRODUCTION AND PREGNANCY RATES OF BOTH FRESH AND FROZEN IN VITRO EMBRYOS IN THE NORTH COAST OF PERU
Authors: Vivanco-Mackie H. W. , Navarro R. D. , Salazar M. D. P. , Aguirre E. A. , Saldaña G. B. , Lizaraso I. M.
Published: December 2015

The objective of the study was to determine the pregnancy rate of fresh and frozen-thawed in vitro produced embryos transferred into recipients in the north coast of Peru. Artificial insemination results of frozen-thawed semen inseminated to cows in the same herd and season (summer) where the embryo transfers were performed was evaluated as control. For the in vitro embryo production, the rate obtained was 374 oocytes from 21 ovum pickup sessions (15.24 ± 8.91 oocytes/session). Of these oocytes, 246 were matured in bicarbonate buffered TCM-199 supplemented with 10% heat inactivated FCS as well as epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), FSH, LH, oestradiol, and cysteamine for 24 h of incubation at 38.5°C, 5% CO2, and 90% humidity. The oocytes selected post-maturation were fertilised with the frozen-thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugated, and resuspended in a TALP-IVF medium supplemented with 20 μM D-penicillamine, 10 μM hypotaurine, and 1 μM epinephrine. The oocytes were then inseminated with a concentration of 1.5 million sperm mL–1 in TALP IVF fertilization medium and incubated for 24 h at 38.5°C, 5% CO2, and 90% humidity. Subsequently, the presumptive zygotes were transferred to medium of 50 μL drops of SOFaa supplemented with 5% heat-inactivated FCS which was later replaced by SOFaa and 1% heat-inactivated FCS on Day 5 after fertilization. The embryos were inspected and graded on Days 7 and 8 post-fertilization and incubation at 38.5°C, 5% CO2 and 90% humidity. The blastocyst rate was evaluated on Day 7 post-fertilization. The blastocyst rate was 25.3% (21/83) and 4.19 ± 3.37 embryos per ovum pickup session were obtained. The embryo freezing media contained 1.5 M ethylene glycol as a cryo-protectant, and the method of thawing the embryo was the direct method (1 step). The pregnancy rate was compared by chi-squared analysis. The pregnancy rate for artificial insemination was 23.9% (1103/4612), and the pregnancy rate of fresh and frozen-thawed in in vitro embryos was 30% (13/43) and 20% (8/40), respectively (P > 0.05). Overall the pregnancy rates in the herd were relatively low, probably due the high environmental temperature during the season when the embryos were transferred and the semen was inseminated. Under those conditions, pregnancy rate was not affected by the use of fresh and frozen-thawed in vitro embryo transfer in comparison to the conventional artificial insemination of frozen semen in the coast north of Peru.
182 EFFECTS OF IGF-1 OR IGF-1-LongR3 ON CELLULAR AND MOLECULAR ASPECTS OF CUMULUS-OOCYTE COMPLEXES DURING IN VITRO OOCYTE MATURATION IN CATTLE
Authors: Araujo M. S. , Guastali M. D. , Castilho A. C. S. , Landim-Alvarenga F.
Published: December 2015

The insulin-like growth factor-1 recombinant -3 (IGF-1-LongR3), a synthetic analogue of IGF-1 with increased bioavailability has not yet been used in vitro maturation (IVM) medium of bovine oocytes. Therefore, the aim of this study was to evaluate and compare the addition effects of IGF-1-LongR3 or IGF-1 in IVM bovine oocytes on meiotic progression, apoptosis, and profile of oocytes genes (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGFBP4 and IGFBP5) and genes in cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). Bovine ovaries were collected in slaughterhouses, and 739 oocytes with grades 1 or 2 were selected after aspiration of 2- to 8-mm follicles. IVM was carried out in TCM199 with FSH, LH, and antibiotics (BM) supplemented with 100 ng mL–1 IGF-1 or 100 ng mL–1 LongR3-IGF-1. Control oocytes were matured in BM supplemented with 0.1% polyvinyl alcohol (PVA) or 10% FCS. For all groups, maturation was performed during 22–24 h in an incubator at 38.5°C and 5% CO2 in air. Subsequently oocytes were denuded and analysed for apoptosis, nuclear maturation, and gene expression by TUNEL assay, staining Hoechst 33342, and RT-qPCR, respectively. Statistical analysis was performed using a linear mixed effects model, which correlated the change in metaphase stage 1 to 2 and the absence of apoptosis among the experimental groups. ANOVA and Tukey tests were used to analyse the results obtained by RT-qPCR. After 10 replicates of IVM, 339 oocytes were evaluated for meiotic progression and apoptosis and 400 oocytes for gene expression. There was no statistical difference between the experimental groups with respect to meiotic progression and apoptosis. BCL2 and IGFBP4 gene were less expressed in oocytes matured with IGF-1 and LongR3-IGF-1 compared with control groups. GFBP4 was also less expressed in cumulus cell of oocytes from the experimental groups. Moreover COX2 expression was statistically elevated in cumulus cells matured in the presence of IGF-1 and LongR3-IGF-1 It was possible to perform IVM of bovine oocytes in the presence of LongR3-IGF-1, allowing its use in replacement of IGF-1 and FCS. The results of this study will provide more information on the interaction of IGF with the IGFBP and its importance for oocyte maturation.
188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS
Authors: Suvá M. , Canel N. G. , Salamone D. F.
Published: December 2015

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P < 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation.

Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1
197 EFFECTS OF PRE-IN VITRO MATURATION WITH CAFFEINE ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY
Authors: Ulloa S. M. B. , Heinzmann J. , Herrmann D. , Baulain U. , Hadeler K.-G. , Aldag P. , Lucas-Hahn A. , Niemann H.
Published: December 2015

High cyclic adenosine monophosphate (cAMP) concentrations are critical for maintaining oocyte meiotic arrest in vivo. For in vitro maturation (IVM), the oocyte is released mechanically from the follicle, which induces a significant drop in intra-oocyte cAMP levels, triggering non-physiological meiotic resumption. It has been proposed that modulation of cAMP before IVM can increase bovine blastocyst rates in vitro. Caffeine is a nonspecific competitive phosphodiesterases (PDE) inhibitor and can inhibit meiotic resumption of oocytes due to maintenance of cAMP levels. It has been reported that gamete treatment with caffeine can increase developmental potential. The current study evaluated the effects of pre-in vitro maturation culture with different concentrations of caffeine on meiotic progress, developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 6648 cumulus-oocyte complexes were obtained by slicing. Caffeine was used in 5 different concentrations (1, 5, 10, 20, and 30 mM) during slicing, searching, and 2 h pre-IVM culture. A control group, with 2 h pre-IVM without caffeine (0 mM) and a standard control were also included. Oocytes were washed either after standard or pre-IVM treatments and cultured for 24 h in vitro without caffeine. After IVM, oocytes were fertilised in vitro for 19 h, and zygotes were cultured in vitro for 8 days until the blastocyst stage. Subsets of oocytes were fixed in 2% glutaraldehyde at 9, 20, and 24 h after IVM. Hoechst staining was performed to evaluate nuclear status of matured oocytes. Cleavage and blastocyst formation rates were evaluated at Days 3 and 8 after IVF. Expanded blastocysts from all treatments were submitted to differential staining. One-way ANOVA from R software was applied to evaluate differences in cleavage and blastocysts rates and blastocyst cell numbers. Fisher’s exact test complemented by Bonferroni correction was used to determine meiotic progress. Caffeine maintained oocytes in meiotic arrest after 9 h of IVM in a concentration-dependent manner (germinal vesicle: 79.0%, 92.2%, 66.7%, 55.1%, 56.9%, 43.9%, 30.2%, respectively, for 30, 20, 10, 5, 1, 0 mM and standard; P < 0.016). Cleavage rates were similar in all treatments; however 30 mM caffeine decreased blastocyst rates (Table 1; P < 0.05). The number of cells did not differ significantly among in vitro treatments (Table 1; P > 0.05). Developmental competence was not affected by 2 h pre-IVM culture. Caffeine supplementation before IVM delayed resumption of meiosis and affected embryo development.

Table 1.In vitro developmental competence of oocytes treated with different caffeine concentrations before IVM
198 RESVERATROL SUPPLEMENTATION DURING IN VITRO MATURATION AND FERTILISATION ENHANCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES
Authors: Kordowitzki P. , Bernal S. M. , Herrmann D. , Aldag P. , Niemann H.
Published: December 2015

Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin identified in various plant species, particularly in grapevine peel. It is a strong antioxidant, induces mitochondrial biogenesis and enhances Sirtuin 1 (SIRT1) activity by inhibiting phosphodiesterase. SIRT1 belongs to the family of NAD+-dependent histone deacetylates and has been shown to regulate several key cellular processes, including transcriptional silencing, aging, chromatin remodeling, and genomic stability, via deacetylation of p53, FoxO transcription factors, and nuclear factor kappa B (NF-κB). The aim of this study was to determine whether supplementation of the maturation and fertilisation medium with resveratrol influences bovine oocyte maturation and subsequent embryonic development and whether these effects are mediated via SIRT1 pathway. Three different resveratrol concentrations were used during in vitro maturation (IVM) and IVF. Cumulus-oocyte complexes (n = 2878) were collected from slaughterhouse ovaries and subjected to IVM medium supplemented with 0.2 µM, 1 µM, or 20 µM resveratrol® (Sigma-Aldrich, Buchs, Switzerland) for 24 h followed by IVF with the same concentrations of resveratrol for 19 h. The IVM and IVF medium without resveratrol (controls) and dimethyl sulfoxide supplementation as vehicle control were also included. Presumptive zygotes were cultured in vitro until Day 8 to assess embryo development, and maturation rates, cleavage, and blastocyst formation were evaluated. Maturation rates as determined by polar body extrusion (0.2 µM: 64.2% ± 7; 1 µM: 82.3% ± 4; 20 µM: 68.8% ± 2; control: 74.6% ± 5 and vehicle control: 70.2% ± 6, respectively; P ≤ 0.05) did not differ dramatically. Oocytes in 1 µM resveratrol supplemented maturation medium showed distinct detachment of cumulus cells in comparison with those in the other treatment and control groups. Cleavage rates were reduced in the 0.2 µM and 20 µM group compared with controls (0.2 µM: 44.21% ± 2; 1 µM: 58.4% ± 3; 20 µM: 40.9% ± 5; control: 56.6% ± 2 and vehicle control: 55.2% ± 6, respectively; P ≤ 0.05). Blastocyst rates were impaired in the low and high resveratrol concentration groups compared to all other groups (0.2 µM: 11.3% ± 1; 1 µM: 33.4% ± 3; 20 µM: 8.2% ± 4; control: 26.7% ± 4 and vehicle control: 20.8% ± 2, respectively; P ≤ 0.05). Relative mRNA abundance of SIRT1 in matured oocytes from the 1 µM group did not differ significantly compared to the controls. Results so far indicate that very low and high concentrations of resveratrol impair development to the blastocyst stage. In conclusion, a 1 µM resveratrol supplementation during IVM and IVF seems to improve the developmental competence of oocytes, which is reflected not only in the elevated blastocyst rates but also in higher degree of expansion of cumulus cells after IVM and maturation rates.
217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS-OOCYTE COMPLEXES
Authors: Canel N. G. , Suvá M. , Bevacqua R. J. , Salamone D. F.
Published: December 2015

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 106 sperm mL–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos.

Table 1.Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)
231 USE OF SOFT SUBSTRATES TO PROMOTE AND MAINTAIN OCT4 EXPRESSION IN EPIGENETICALLY ERASED FIBROBLASTS
Authors: Pennarossa G. , Santoro R. , Pesce M. , Gandolfi F. , Brevini T. A. L.
Published: December 2015

Pluripotency and commitment are driven by transcription factors that act as molecular switches to activate or repress specific gene expression programs. Among these, the POU transcription factor OCT4 (encoded by POU class 5 homeobox 1, POU5F1) is known to be a regulator in pluripotent cells and it is essential for the maintenance of a high plasticity state in mammalian cells. In recent years, particular attention has been given to the importance of physical extracellular matrix properties in regulating pluripotent cell maintenance and differentiation. In particular, recent studies demonstrated that high plasticity cells are intrinsically soft and respond optimally to physical forces when cultured on a “low stiffness” substrate that matches their intrinsic softness. To investigate these aspects, here we used a protocol where terminally differentiated cells are driven into a high plasticity/OCT4-positive state through the use of the epigenetic eraser, 5-aza-cytidine (5-aza-CR) and we observed the effect of substrate stiffness on the maintenance of OCT4 expression. To this purpose, human skin fibroblasts were plated either on standard plastic dishes (group A) or on polyacrylamide gels (PAA) of low stiffness (1 kPa; group B). Cells were treated with 1 μM 5-aza-CR for 18 h and were subsequently maintained and cultured in embryonic stem cell medium for 14 days. Assessment of OCT4 was carried out both by real-time PCR and immunocytochemical analysis, at different time points: untreated fibroblasts (T0), after 5-aza-CR treatment, and on Days 2, 4, 6, 8, 10, and 14 of culture. The results obtained indicate that untreated fibroblasts (T0) were negative for OCT4, whereas, after 5-aza-CR exposure, cells actively expressed the pluripotency marker, in both the A and B groups. However, whereas group A cells progressively down-regulated OCT4 expression and eliminated its positivity by Day 6, group B cells steadily transcribed and were homogeneously immunopositive for the pluripotency factor until Day 14, when cultures were arrested. Overall, the data obtained suggest that a soft substrate, matching the intrinsic stiffness distinctive of high plasticity cells, may help in promoting their maintenance, via a biophysical mechanism of low-traction/low-stiffness-dependent manner.
Study was supported by Carraresi Foundation.