bluuebunny/crossref_metadata_2025 huggingface dataset:
240 OVUM-PICK UP IN HOLSTEIN-FRIESIAN COWS AT 9 TO 10 MONTHS OF AGE
Authors: Mullaart E. , Dotinga F. , Flapper H. , van de Brink A. , Pietersma N. , Schouten J.
Published: December 2014

Rate of genetic gain can be improved by increasing selection intensity, increasing selection accuracy, creating a higher amount of variation, or shortening the generation interval. A reduction in the generation interval can be realised by collecting embryos from animals at a young age. At CRV it is currently routine practice to start embryo collection (by flushing) at 12 to 15 months of age. The aim of this study was to investigate if the collection of embryos at a younger age by means of ovum pickup (OPU) is beneficial for our breeding program. Healthy 9- to 10-month-old Holstein-Friesian animals were selected at young age based on their maturity, (i.e. OPU could be performed by normal standard procedure/equipment). Animals were not stimulated with hormones. Oocytes were collected by OPU once every week during a period of ~8 weeks. Collected oocytes were matured, fertilized, and cultured for 7 days in SOF culture medium according to standard procedures (Merton et al. 2002). Embryo development was scored at Day 7. Results were analysed by Student's t-test. On average 11.7 oocytes were collected from animals as young as 9 months of age. However, embryo development of the oocytes from young animals was only 0.4 embryo per session at Day 7 (3% embryo development). When only the results of the first OPU session were taken into account, 18.2 oocytes and 0.8 embryo could be collected per animal per session. Whether an animal was observed in oestrus before the first OPU session affected results. Animals that had shown clear signs of oestrus before the first OPU session produced significantly more embryos than animals that did not (Table 1). It is concluded that OPU is possible on animals at 9 to 10 months of age but only when animals have been in oestrus before the first OPU session. Best results were obtained for the first OPU session.
Table 1.Effect of first oestrus before OPU on embryo production
245 EFFECTS OF Gyr (BOS TAURUS INDICUS) DONOR MITOCHONDRIAL DNA AND OVUM PICKUP ORDER ON IN VITRO EMBRYO PRODUCTION
Authors: Pinto T. L. C. , Lopes B. C. , Ferreira M. B. D. , Goncalves T. M. , Souza J. C. , Garcia J. M.
Published: December 2014

Ovum pick (OPU) up is a technique upon which in vitro embryo production (IVP) depends. It permits donor cow maternal ancestry to be assessed by mtDNA analysis. Repetitive ablation of follicles is thought to interfere with the donor cow endocrine profile and influence embryo yield. The objective was to evaluate the effects of mtDNA and OPU order on IVP fertility traits. Gyr donors (85) were submitted to 363 OPU sessions (5 OPU sessions/donor). Donor mtDNA was extracted from leukocytes and sorted by the presence of the HindIII restriction site within the amplified region, indicative of Bos taurus taurus mtDNA (Paneto et al. 2008 Genet. Mol. Res. 7, 592). All animals in the donor pedigree were classified, and the population was divided into two groups according to their maternal genetic grouping: Bos taurus indicus and Bos taurus taurus. For statistical analyses, data from 5 OPU sessions per each donor were submitted to the mixed model procedure of SAS® (SAS Institute Inc., Cary, NC, USA), using the lowest Akaike value to determine the best covariance structure between repeated OPU session results. The model included the effect of OPU session order (1–5) and mtDNA-based maternal grouping (Bos taurus v. Bos indicus) as independent variables. Total and viable oocytes, as well as blastocyst yield per OPU session, were the dependent variables studied. The means of total and viable oocytes and blastocysts produced per donor per OPU were compared by the Tukey test, at 5% significance. The combined OPU sessions resulted in 6084 oocytes, 2537 embryos, which produced 1105 pregnancies. Mean numbers (n = 42 donors/OPU session) of total and viable oocytes between OPU sessions 1 (31.9 ± 4.6 and 19.2 ± 2.9), 2 (35.3 ± 3.5 and 21.2 ± 2.2), 3 (28.9 ± 3.7 and 19.3 ± 2.4), 4 (25.0 ± 5.2 and 18.9 ± 33), and 5 (20.5 ± 6.3 and 13.8 ± 4.0) did not differ. Mean blastocyst production after IVP between OPU sessions 1 (6.5 ± 1.4), 2 (5.15 ± 1.04), 3 (5.5 ± 1.1), 4 (5.8 ± 1.6), and 5 (5.2 ± 1.9) was similar. Mean viable oocyte number was greater (P < 0.0001) for B. t. taurus (21.7 ± 1.4, n = 192 OPU sessions) compared with B. t. indicus (16.1 ± 0.9, n = 138 OPU sessions) maternal genetic groupings. However, in vitro blastocyst yields were similar (P = 0.23) between maternal genetic groupings (7.3 ± 1.6 and 6.2 ± 1.4 for B. taurus and B. indicus, respectively). In conclusion, repeated OPU sessions did not reduce oocyte and embryo yields as expected. Maternal B. taurus genetic origin was associated with higher oocyte quality, although it was not translated into higher embryo yields after in vitro culture. Results warrant further research, which may result in additional selection criteria for OPU Gyr donors considering their maternal genetic background.
249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES
Authors: Douet C. , Parodi O. , Reigner F. , Barrière P. , Goudet G.
Published: December 2014

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.
256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)
Authors: Mellisho E. , Rivas V. , Ruiz J. , Mamani G.
Published: December 2014

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.
262 EFFECT OF SYSTEMIC ANTIOXIDANT TREATMENT IN BOS TAURUS TAURUS BULLS UNDER HEAT STRESS AND SUPPLEMENTED WITH POLYUNSATURATED FATTY ACIDS
Authors: Gualtieri de Andrade Perez E. , Diego di Agostini Losano J. , Dalmazzo A. , Nichi M. , Hyppolito Barnabe V.
Published: December 2014

One reason for lower fertility of European bulls in tropical regions is a higher rate of oxidative stress caused by increased production of reactive oxygen species (ROS) not compensated by antioxidant protection. In that regard, sperm are extremely susceptible to oxidative stress due to a high concentration of polyunsaturated fatty acids (PUFA) in their plasma membranes. However, the presence of these PUFA is fundamental for sperm to be fertile and resistant to cold shock. Thus, treatments that suppress oxidation may increase productivity of these animals. This study aimed to evaluate the most damaging ROS for European bulls subjected to heat stress and to determine a possible antioxidant-targeted treatment. In a second step, we sought to verify the efficiency of the interaction between a diet rich in PUFA and a targeted antioxidant treatment on the quality of ejaculated and epididymal sperm in European bulls subjected to testicular heat stress. Four Bos taurus bulls were subjected to scrotal insulation for 5 days, with semen collection (electroejaculation) 60 days after insulation. Semen from each bull was divided into 4 aliquots and subjected to 4 ROS-generating systems: superoxide anion (xanthine/xanthine oxidase), hydrogen peroxide, hydroxyl radical (ascorbate + ferrous sulfate), and malondialdehyde (MDA; lipid peroxidation product). Samples were incubated for 1 h and assessed by computerized sperm analysis (CASA); eosin/nigrosin (membrane integrity); fast-green/Bengal rose (acrosome integrity); 3,3′ diaminobenzidine (mitochondrial activity); sperm chromatin structure assay (DNA fragmentation); and thiobarbituric acid reactive substances (lipid peroxidation). Overall, MDA had the most deleterious effects on semen quality of Bos taurus bulls subjected to acute heat stress. Thereafter, 16 bulls were subjected to testicular insulation and allocated into 4 groups: control (n = 4; given mineral oil; placebo); vitamin E (n = 4, given 5 mL of Monovin® every 13 days); PUFA (n = 4; given 4 kg day–1 Megalac®); and PUFA+vitamin E (n = 4; combination of PUFA and vitamin E treatment groups). Semen was collected on the day of installation of the insulation, on the day it was removed, and 30 and 60 days later. Overall, vitamin E reduced heat stress-induced damage to sperm DNA and mitochondria, but only in samples collected from the epididymis. Similarly, the combination of vitamin E and PUFA supplementation improved sperm motility patterns. Therefore, a combined antioxidant treatment (vitamin E and PUFA) may reduce damage to sperm caused by acute heat stress in European bulls. However, this treatment may be more effective if instituted before heat stress.
272 EFFECT OF DIETARY RESTRICTION ON SPERM CHARACTERISTICS AND OXIDATIVE STATUS ON TESTICULAR TISSUE IN YOUNG RATS EXPOSED TO LONG-TERM HEAT STRESS
Authors: Aydilek N. , Varisli O. , Kaya M. S. , Kocyigit A. , Taskin A.
Published: December 2014

The objective was to evaluate effects of dietary restriction on oxidative status and sperm parameters in rats exposed to long-term heat stress. Forty healthy Sprague-Dawley rats (2.5 months old) were equally allocated into 4 groups (with respect to diet and temperature): room temperature (22°C)-ad libitum; room temperature-dietary restriction (40%); high temperature (38°C)-ad libitum; and high temperature-dietary restriction. At the end of the 9th week, some oxidants (lipid hydroperoxide, total oxidant status, oxidative stress index) and some antioxidants (total antioxidant status, sulfhydryl groups, ceruloplasmin, paraoxonase and arylesterase activities) were measured in testicular tissues. In addition, concentration, motility, volume, abnormal sperm count, acrosome and membrane integrity of epididymal sperm were also evaluated. All data were analysed by 2-way ANOVA (P < 0.05). High temperature did not significantly affect most oxidative and antioxidative parameters (except for sulfhydryl groups and ceruloplasmin), yet it impaired all sperm values. Neither sperm values nor oxidative status, with the exception of sulfhydryl groups, ceruloplasmin and arylesterase in the testis tissue, were significantly affected by dietary restriction. We concluded that long-term heat stress did not significantly affect testicular oxidative status in young rats, although sperm were sensitive to heat stress. Furthermore, dietary restriction failed to improve sperm quality and oxidative status, except some individual antioxidant parameters in young rats exposed to long-term heat stress.
289 THE EFFECTS OF MEIOSIS BLOCKING BY CDK INHIBITORS ON THE ACTIVITY OF MATURATION PROMOTING FACTOR, ERK1 AND 2, AND THE DISTRIBUTION OF CYTOPLASMIC ORGANELLES IN IN VITRO-MATURED BOVINE OOCYTES
Authors: Maziero R. R. D. , Guaitolini C. R. F. , Paschoal D. M. , Crespilho A. M. , Landim-Alvarenga F. C.
Published: December 2014

Studies have suggested that the prematuration with meiotic blockers can improve oocyte quality promoting embryonic development; however, its exact effects on cytoplasmic characteristics remain unclear. Thus, this study aimed to evaluate the effects of meiosis block of bovine oocytes with the CDK inhibitors roscovitine (ROS) and butyrolactone (BL-I) on nuclear maturation, expression, and localization of ERK 1 and 2, cyclin B1, and p34cdc2 proteins and the ultrastructure of oocytes. Immature oocytes from the slaughterhouse were divided into the following groups: (1) control, in vitro maturated (IVM) in TCM 199 for 24 h; (2) ROS 12.5 µM; (3) BL-I 50 µM; and (4) ROS (6.25 µM) + BL-I (25 µM) treatment for 6 h followed by IVM in CDK inhibitor-free medium for 18 h. Immature oocytes and IVM oocytes in each group were then fixed stained by immunofluorescence for nuclear visualisation (n = 600), localization, and expression of ERK1 and 2 proteins, cyclin B1 and p34cdc2 protein (n = 350), and prepared for evaluation of the ultrastructure by electron microscopy (n = 100). Data were analysed using the ANOVA test (nuclear visualisation), Student-Newman-Keuls test (expression of ERK1 and 2 proteins, cyclin B1 and p43cdc2) by the PROC GLM procedure of SAS (SAS Inst. Inc., Cary, NC, USA). At 6 h of IVM, a lower (P < 0.05) percentage of oocytes were at the metaphase I (MI) stage in the control group (C = 18.2 ± 5.4%) compared with other groups and a higher percentage of oocytes were degenerated in the ROS group (16.3 ± 5.6%) compared with other groups (C = 13.6 ± 4.6%, BL-I = 10.0 ± 4.5%, BL-I+ROS = 8.0 ± 5.6%). At 24 h of IVM, higher (P < 0.05) percentages of oocytes were at the MI stage in the control and ROS group (8.3 ± 5.9% and 6.8 ± 6.4%, respectively). There was no difference (P > 0.05) in percentage of metaphase II (MII) oocytes among the groups. Only the ROS group showed lower fluorescence intensity of ERK1 and 2 proteins in the ooplasma (P < 0.05). Immature oocytes showed higher expression of cyclin B1 and p34cdc2 (P < 0.05). There was no difference in the localization of these proteins in the ooplasm and there was no difference in the oocyte ultrastructure (mitochondria, cortical granules, endoplasmic reticulum, microvilli, zona pellucida, lipid granules) among treatments (P > 0.05). The results suggest that a temporary blocking of oocyte maturation by CDK inhibitors affect neither the expression and distribution of MPF components (cyclin B1/cdc2) nor the distribution of cytoplasmic organelles in IVM oocytes. However, the expression of ERK 1 and 2 in mature oocytes may be reduced by pre-IVM with ROS.
297 EFFECT OF CUMULUS CELL REMOVAL DURING IN VITRO MATURATION OF PORCINE CUMULUS–OOCYTE COMPLEXES ON THE APOPTOTIC STATUS AND MEIOTIC PROGRESSION OF THE OOCYTES
Authors: Ferré P. , Funahashi H.
Published: December 2014

This study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small (SF) and medium follicles (MF) when the oocytes were denuded from cumulus cells (CC) before, during and after culture for in vitro maturation (IVM). Cumulus-oocyte complexes (COC) were aspirated from SF (0.5–2 mm in diameter) or MF (3–6 mm in diameter) of slaughtered prepubertal gilt ovaries. Only COC with a good morphology of the surrounding cumulus cells were cultured for IVM in modified porcine oocyte medium supplemented with 50 µM β-mercaptoethanol, 1 mM dibutyryl c-AMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h at 39°C and 5% CO2 in air and then continued culture in the absence of dibutyryl c-AMP, eCG, and hCG in the same medium for another 24 h. Before and 20 h after the start of IVM culture, some of the oocytes were denuded of CC and the oocytes continued the IVM culture. After IVM culture, oocyte viability and meiotic progression were examined by the annexin V/PI viability assay and DAPI staining. Statistical analyses of 5 replicate data were performed with a 2-way ANOVA and a Tukey's multiple comparisons test. Before IVM culture, there was no significant difference between the viability of SF and MF oocytes, but the incidence of oocytes at the GV0 stage was higher in specimens from SF than MF (24.8 v. 3.3%), and that of oocytes at the GVI stage was the opposite (57.8 in MF v. 22.7% in SF). After IVM culture, apoptotic status of oocytes was only affected by the decumulation timing. The percentage of normal live oocytes was significantly higher when CC were removed after 20 and 44 h of IVM in both SF (39.7 and 39.3 v. 17.7%) and MF (45.4 and 37 v. 22.2%). The incidence of early and late apoptotic oocytes was significantly higher when the CC were removed before IVM culture in both SF (74.3 and 7.4%) and MF (69.4 and 6.7%). The incidence of mature live oocytes was significantly affected by both the origin of COC and the decumulation timing. Although the percentage of mature oocytes was higher in MF, maturation rates were significantly higher when oocytes were denuded at 20 h of IVM culture (SF 65.4%, MF 83.1%) as compared at 0 (SF 27.9%, MF 32.3%) and 44 h (SF 41%, MF 68.5%). However, the percentage of oocytes with normal spindle morphology was significantly higher when oocytes were denuded at 44 h of IVM culture (SF 70.6%, MF 91.5%) than 20 h (SF 66.8%, MF 73%). In summary, regardless of COC from SF and MF, removal of CC at 20 h of IVM culture seems to promote meiotic progression of the oocytes to the MII stage, but factor(s) from or communication with CC during the latter half of IVM culture may be needed to obtain a normal spindle morphology in mature oocytes.
3 SEX INFLUENCES REGULATION OF GENE EXPRESSION BY DICKKOPF 1 IN THE BOVINE MORULA
Authors: Denicol A. C. , Dobbs K. B. , Hansen P. J.
Published: December 2014

Successful embryonic development depends upon molecules secreted by the reproductive tract. Among such molecules is the protein dickkopf 1 (DKK1), an antagonist of canonical WNT signalling that can also activate the noncanonical, planar cell polarity (PCP) pathway. DKK1 increases the proportion of cells that are trophectoderm and hypoblast in the blastocyst and increases competence of embryos to establish pregnancy after transfer to recipients. The objective was to determine whether DKK1 affects cell fate by regulating expression of genes that promote differentiation at the morula stage, possibly by activating the PCP pathway. A second objective was to determine if actions of DKK1 on the embryonic transcriptome were dependent on embryo sex. Bovine oocytes were fertilized in vitro with pools of 3 bulls for which X- and Y-sorted sperm was available. Embryos were treated with 100 ng mL–1 DKK1 or vehicle at Day 5 of development and harvested 24 h later. Embryos were pooled in 5 replicates of 20 embryos each. Following RNA reverse-transcription and amplification, cDNA was used for microarray analysis of global gene expression using the Affymetrix® Bovine Gene 1.0 ST array (Affymetrix, Santa, Clara, CA, USA). Statistical analysis was performed by ANOVA using JMP® Genomics (SAS Institute, Cary, NC, USA). A total of 9931 transcripts were identified as being expressed. Differentially expressed genes (DEG) were considered as those associated with P < 0.05 and fold change ≥1.5 or <0.66. There were 124 DEG between females and males (91 up-regulated in females and 33 up-regulated in males). A total of 68% of the genes up-regulated in females were located in the X chromosome. Treatment with DKK1 resulted in 132 DEG in females (68 up-regulated and 64 down-regulated) and 136 DEG in males (90 up-regulated and 46 down-regulated). Of these, 34 genes were regulated by DKK1 in both sexes: 14 in the same direction and 20 in opposite directions. Analysis by Ingenuity® Pathway software indicated that changes in gene expression caused by DKK1 would increase formation of actin fibers in females and inhibit formation in males. DKK1 inhibited expression of AMOT in male embryos, indicating that DKK1 may inhibit Hippo signalling at this stage of development. Evidence for regulation of the PCP pathway by DKK1 was the finding that DKK1 regulated expression of genes involved in cell polarization and differentiation in both females and males. In both sexes, DKK1 regulated expression of many genes associated with HNF4A, a marker of hypoblast cells that promotes formation of cell junctions. In conclusion, female and male embryos developing in vitro have different transcriptomic profiles at the morula stage. DKK1 regulates cell differentiation and embryonic development in a sex-dependent manner and effects may be mediated, at least in part, by activation of the PCP pathway.Supported by USDA AFRI 2011-67015-30688 and NIH R03 HD080855.
302 EFFECT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION
Authors: Yoon J. D. , Lee E. , Hyun S.-H.
Published: December 2014

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on in vitro porcine oocytes maturation and subsequent embryonic development after pathenogenetic activation (PA) and in vitro fertilization (IVF). We investigated nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, sperm penetration (SP) analysis, and subsequent embryonic development after PA and IVF. Each concentration (0, 1, 10, and 100 ng mL–1) of GDF8 was added in maturation medium during process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± s.e.m. After 44 h of IVM, no significant difference was observed on nuclear maturation from the different concentration (0, 1, 10, and 100 ng mL–1) of GDF8 treatment groups (85.5, 85.9, 89.4, and 87.6%, respectively) compared with the control (P > 0.05). The 10- and 100-ng mL–1 GDF8-treated groups showed a significant (P < 0.05) decrease in intracellular ROS levels compared with other groups. The embryonic developmental competence after PA was affected with GDF8 treatment during IVM. The 10- and 100-ng mL–1 treatment groups showed significantly (P < 0.05) higher cleavage rates (67.5 and 69.1%, respectively) compared with control group (53.7%). The 10- and 100-ng mL–1 treatment groups also showed significantly (P < 0.05) higher blastocyst formation rates (50.5 and 52.7%, respectively) compared with other groups (34.5 and 35.8%). The IVF embryonic developmental competence also was affected with GDF8 treatment during IVM. The 10-ng mL–1 treatment group showed a significantly (P < 0.05) higher blastocyst formation rates and total cell number compared with control (21.5 and 131.3 v. 15.0 and 92.6%, respectively). Also, in the sperm penetration assessment, the 10- and 100-ng mL–1 treatment groups showed higher mono spermy ratio and fertilization efficiency (32.7 and 27.1, 32.0 and 26.5 v. 22.6 and 19.7%, respectively) than control, which was significant (P < 0.05). In conclusion, the treatment with 10 ng mL–1 of GDF8 during IVM improved the PA and IVF porcine embryo developmental competence by decreasing the intracellular ROS levels.This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.
307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING
Authors: Yang M. , Hu S. , Cox L. , Regouski M. , Rutigliano H. , Isom C. , Polejaeva I.
Published: December 2014

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (<3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P < 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P < 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P < 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P < 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P < 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.
326 ASSESSMENT OF PORCINE-INDUCED PLURIPOTENT STEM CELLS BY IN VIVO ASSAYS
Authors: Secher J. , Freude K. , Petkov S. , Ceylan A. , Schmidt M. , Hyttel P.
Published: December 2014

Porcine-induced pluripotent stem cells (piPSC) have been established since 2009, but only 1 report demonstrated contribution to germline chimeras. One well-established in vivo pluripotency assay is the teratoma assay, which has recently been questioned due to the lack of standardized guidelines. In the present study we have characterised GFP-tagged in vitro and in vivo tetracycline-inducible piPSC [porcine MYC, SOX2, KLF4 (pOMSK)] and their capacity to form teratomas. We injected 1.5 million cells in 250 µL of PBS subcutaneous into NOD/SCID mice and followed them up to 6 weeks. The teratomas were analysed by immunohistochemistry for the 3 germlayer markers β3 tubulin, α fetoprotein, and smooth muscle actin. We not only found our teratomas positive for these markers, but also co-positive for GFP, clearly showing that the teratoma was made from porcine cells, which was not sufficiently proven in former studies. Our H&E staining revealed the following structures: cuboidal ephitelium, thyroid-like structure, renal corpuscle, and steroid producing cells. We continued to test the capacity of our venus iPS cells to contribute to in vitro chimeras. To achieve this we used a micromanipulator to inject 15 cells into Day 5 parthenotes, and subsequently cultured them in PZM3 with 10% FCS, cultured with or without doxycycline. These in vitro chimeras were followed until Day 7 in Nikons Biostation IM and used for differential staining. In all groups we saw good survival, hatching, and maintenance of GFP, indicating integration of these cells in our in vitro assay. We only found differences between survivals of the cell lines in the group cultured with doxycycline. Finally, in order to assess if the naïve type venus iPS cells could possibly be a truly naïve piPSC, we tested their capacity to form in vivo germline chimeras. This was tested by injecting 15 piPSC into Day 4 to 5 in vivo embryos. The injected embryos were transferred into 5 surrogate mothers, 3 of them were fed doxycycline before the transfer and 5 days after, and the last 2 recipient sows were not fed doxycycline. The pregnancies were terminated at Day 32 and the embryos were examined for fluorescence and the GFP transgene by PCR. In summary, it appears that both naïve type and primed type venus iPS cells are still strongly dependent on the pOMSK transgene expression, and the ultimate proof for pluripotency, the chimera production, seems to be not achievable under the condition we have chosen.
330 DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS FROM FIBROBLASTS OF A TRANSLOCATED AZOOSPERMIC BOAR
Authors: Congras A. , Barasc H. , Delcros C. , Vignoles F. , Pinton A. , Canale-Tabet K. , Feraud O. , Turhan A. , Afanassieff M. , Yerle-Bouissou M. , Acloque H.
Published: December 2014

Chromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species.
34 COMPARISON OF PROLIFERATION AND TELOMERASE ACTIVITY IN FIBROBLASTS DERIVED FROM GYEONG-JU DONGGYEONG DOGS ACCORDING TO THEIR AGE
Authors: Choi Y. B. , Kim G. A. , Oh H. J. , Kim M. J. , Lee B. C.
Published: December 2014

Donggyeong dog is a breed considered a natural monument in Korea since 2012. Nevertheless, this breed is only found in Gyeong-ju and is classified as endangered. The use of assisted reproductive technologies (ART) could be applied to preserve these endangered dogs. Among various ART, somatic cell nuclear transfer (SCNT) may be a good technique for achieving propagation of genetically identical individuals. For this reason, we investigated the effect of age of the cell donor on several characteristics, including growth pattern, doubling time of cell populations, cell size, viability, and telomerase activity in Gyeong-ju Donggyeong dog fibroblast cultures before SCNT. Primary fibroblast cell cultures were performed using ear biopsies from 1-year-old (D-1yr) and 7-year-old (D-7yr) Donggyeong dogs and cells at passage 2 to 6 from in this study. Cells were plated at 1 × 105 cells well–1 in a 6-well plate. Cells were harvested every 24 h for 6 days, and cell number was determined to measure growth pattern and doubling time. The harvested cells were stained with trypan blue, their size and viability were analysed using a Countess Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Telomerase activity was measured by telomeric repeat amplification protocol using TeloTAGGG Telomerase PCR ELISAPLUS. All experiments were replicated at least 3 times. Statistical analysis was performed using Graphpad Prism (GraphPad Software Inc., San Diego, CA, USA), and t-test (P < 0.05) was used to compare the growth pattern, doubling time, cell size, viability, and telomerase activity between D-1yr and D-7yr groups. Growth curves in both groups showed the typical “S” shape and no significant differences between D-1yr and D-7yr groups. However, doubling times from 2nd to 5th passages of D-1yr (20.0 ± 0.3 h, 38.0 ± 0.5 h, 38.7 ± 0.5 h, and 53.4 ± 1.4 h) were significantly shorter than those of D-7yr (37.0 ± 1.2 h, 54.0 ± 3.6 h, 63.3 ± 1.8 h, and 100.9 ± 4.4 h). Cells from 6th passage of D-7yr and 7th passage of D-1yr did not reach confluence. Cell size of D-1yr (13.6 ± 0.2 μm) was significantly smaller than that of D-7yr (14.9 ± 0.3 μm; P < 0.001). There was no significant difference between D-1yr (89.4 ± 1.4%) and D-7yr (88.9 ± 1.2%) in cell viability but relative telomerase activity was significantly higher in D-1yr (37.8 ± 7.5) compared with D-7yr (19.0 ± 6.2, P < 0.001). In conclusion, these results suggest that fibroblasts derived from young Donggyeong dogs are more suitable for SCNT than those from old donors. For further study, dog cloning using D-1yr and D-7yr should be performed to evaluate the effect of donor age in canine SCNT efficiency.
354 COMPARISON OF OVARIAN CONTENTS OF AKT ISOFORMS AMONG 4 STRAINS OF MICE
Authors: Suzuki O.
Published: December 2014

Strain/individual differences in superovulation efficiency with gonadotropins constitute a serious problem in mouse reproduction (Suzuki et al. 1996 Reprod. Fertil. Dev. 8, 975–980). Because the PI3K/Akt signalling pathway is involved in ovarian folliculogenesis, quantitative difference of protein components in the PI3K/Akt signalling pathway among mouse strains may explain the strain difference in superovulation efficiency. The present study compared ovarian contents of AKT isoforms among 4 strains to examine the involvement of the PI3K/Akt signalling pathway in superovulation. Ovarian protein contents of AKT1, AKT2, and AKT3 isoforms in 4-week-old females of 4 mouse strains were measured by quantitative Western blots with GAPDH as an internal control using whole ovary homogenates (see Suzuki et al. 2011 Exp. Anim. 60, 193–196, for method details). Observed values were compared among strains by 1-way ANOVA after normality (Shapiro-Wilk) and equal variance (Levene) were confirmed. Ovarian protein contents of all 3 AKT isoforms in A/J, C57BL/6N, DBA/2, and C3H/He (arbitrary unit, mean ± s.e.m., n = 4) were significantly different among strains by 1-way ANOVA (P < 0.05). Ovarian AKT1 contents were 1.01 ± 0.10a, 0.98 ± 0.07, 0.89 ± 0.03, and 0.84 ± 0.02b, respectively. Ovarian AKT2 contents were 1.39 ± 0.13a, 0.74 ± 0.10b, 1.03 ± 0.07c, and 0.91 ± 0.19bc, respectively. Ovarian AKT3 contents were 1.24 ± 0.38, 1.55 ± 0.24a, 1.11 ± 0.09, and 0.67 ± 0.18b, respectively. a–cValues with different superscripts in each isoform are significantly different by Tukey test (P < 0.05). Thus, significant quantitative differences in ovarian AKT isoforms among strains suggest that the ovarian PI3K/Akt signalling pathway acts differently among strains. This activity difference of the pathway may explain the strain difference in superovulation efficiency in mice.This work was supported by a grant from the Ministry of Health, Labour and Welfare of Japan.
359 A NONINVASIVE APPROACH TO DIAGNOSE TRANSGENIC CONCEPTI DURING PREGNANCY IN GOATS
Authors: Tavares K. C. S. , Lazzarotto C. R. , Calderon C. M. , Martins L. T. , Neto S. G. , Aguiar L. H. , Miranda A. M. , Bertolini M. , Bertolini L. R.
Published: December 2014

The discovery of cell-free fetal DNA (cffDNA) circulating in the blood of pregnant women, and more recently in cows, ewes, and mares, paves the road towards the development of molecular tools to explore genetic features of embryos and/or fetuses before term. Albeit a wide range of analyses are in current use and development in humans, genetic diagnostic targets other than sex determination are still not described for other mammalian species. The aim of this study was to detect cffDNA from transgenic goat concepti for the human lysozyme (hLZ) gene in the blood of nontransgenic dams. Blood was collected from 3 nontransgenic goats carrying hLZ-transgenic concepti on Days 40–50, 80–90, and 110–120 of gestation. Also, blood was drawn 8 and 12 days after parturition from two other nontransgenic goats that delivered hLZ-transgenic offspring. Blood samples (10 mL) were spun at 1200 rpm for 10 min; resulting serum or plasma were stored at –20°C (serum) or 4°C (plasma). The DNA was extracted by mixing 350 µL of serum or plasma with an equal volume of TE buffer and 5 µL of proteinase K (20 mg mL–1). The mixtures were incubated at 55°C for 3 h, followed by phenol extraction and DNA precipitation by sodium acetate and 100% ethanol, with further incubation at –20°C overnight and centrifugation at 12 000 × g for 10 min. The DNA pellets were washed with 70% ethanol and eluted in 20 µL of ultrapure water. For the PCR, primer sets for the hLZ transgene (hLZ-i1-F 5′ CGGTCCAGGGCAAGGTCTTTGA 3′ and hLZ-i1-R 5′ ACTGCTCCTGGGGTTTTGCC 3′) and for GAPDH as the endogenous control were used. Reactions contained 3 µL of DNA, 200 nM of each primer, and 45 µL of PCR Mastermix (Quatro G Pesquisa & Desenvolvimento, Porto Alegre, Brazil). The DNA from serum and plasma of nontransgenic goats were used as negative controls. The cycling conditions were 95°C for 10 min, followed by 55 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, plus a final extension at 72°C for 10 min. The PCR products were analysed by electrophoresis in 2% agarose gel. As expected, GAPDH was amplified in most of the samples (12/13). The 200-bp PCR product corresponding to hLZ was detected in the dam's serum in all 3 gestational phases, with 2 out of 3 animals being positive on 40 to 50 and 80 to 90 days, and all 3 on 110 to 120 days of pregnancy. Furthermore, the transgene was amplified from dam's plasma in all samples after parturition. Only GAPDH amplification was detected in the blood of nontransgenic goats. These results suggest that cffDNA is present in the goat's blood circulation at the fetal phase during pregnancy and at least during the first 2 weeks after parturition. This method can be safely applied as a useful tool in zygote-DNA microinjection experiments, providing an early and preterm diagnostic of transgenic concepti through the dam's blood.Research was supported by FINEP.
362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS
Authors: De Stefano A. , Gambini A. , Salamone D.
Published: December 2014

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.
43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD
Authors: Caamaño J. N. , Gómez E. , Trigal B. , Muñoz M. , Carrocera S. , Martín D. , Díez C.
Published: December 2014

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions.

Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure

This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.