bluuebunny/crossref_metadata_2025 huggingface dataset:
139 SEX-SPECIFIC GENE EXPRESSION IN PORCINE PRE-IMPLANTATION EMBRYOS
Authors: Kradolfer D. , Knubben J. , Flöter V. , Bick J. , Bauersachs S. , Ulbrich S. E.
Published: December 2015

X-Chromosome inactivation in female mammals starts during early blastocyst stage with expression of the X-inactive specific transcript (XIST), which coats and silences the inactive X chromosome. However, this compensation is not complete in blastocysts, as a large number of X-linked transcripts are more highly expressed in female embryos than in males. Furthermore, the process of X chromosome inactivation is altered in IVF and cloned porcine embryos, possibly explaining problems of embryo survival with these techniques. The aim of this study was to gain more insights into the transcriptional dynamics of the porcine pre-implantation embryo, with a particular focus on sex-specific differences. RNA sequencing (RNA-Seq) was performed for individual blastocysts at 8, 10, and 12 days after ovulation, and the temporal development of sex-specific transcripts was analysed. German Landrace sows were cycle synchronized and inseminated with sperm of the same Pietrain boar. On Days 8, 10, and 12 post-insemination, sows were slaughtered and embryos were removed from the uterus using 10 mL of PBS (pH 7.4) per horn. Single embryos were shock frozen in liquid nitrogen and stored at –80°C until the extraction of RNA and DNA (AllPrep DNA/RNA Micro Kit, Qiagen, Valencia, CA, USA). Using the isolated DNA, the sex of the embryos was determined and 5 female and male embryos, respectively, were analysed per stage. Illumina TruSeq Stranded mRNA libraries (Illumina Inc., San Diego, CA, USA) were sequenced on a HiSEqn 2500 (Illumina Inc.), and 15 to 25 million 100-bp single-end reads were generated per sample. Reads were filtered and processed using Trimmomatic and mapped to the porcine genome assembly Sscrofa10.2 with TopHat2. Mapped reads were counted by the use of QuasR qCount based on the current National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) GFF3 annotation file. Statistical analysis of count data was performed with the BioConductor R (https://www.bioconductor.org/) package DESEqn 2. At all 3 stages, we found 7 Y-linked transcripts that were highly expressed in male embryos (EIF2S3, EIF1AY, LOC100624590, LOC100625207, LOC100624329, LOC102162178, LOC100624937). On the other hand, 47 X-linked transcripts showed increased expression in female blastocysts, most of them at all 3 time points. However, a small number of genes (DDX3X, LAMP2, and RPS6KA3) were more highly expressed in females at Days 8 and 10 but more highly expressed in males at Day 12. Three X-linked genes (OFD1, KAL1, and LOC100525092) were more highly expressed in male embryos, although only at a low fold change of 1.2 to 1.4. Furthermore, expression of 8 transcripts located on autosomes was higher in females. In conclusion, our study expands the current knowledge of sex-specific gene expression in 8- to 12-day-old porcine blastocysts, a critical time period during pre-implantation embryo development.
140 EXPRESSION OF PERILIPIN IN PORCINE OOCYTES AND CUMULUS CELLS AT DIFFERENT STAGES OF IN VITRO MATURATION
Authors: Oh A. , Jin J.-X. , Lee S. , Kim G. A. , Lee B. C.
Published: December 2015

Perilipin, one of the perilipin adipophilin tail-interacting protein of 47 kDa (PAT) family, has been found to coat the surface of intracellular lipid droplets. It limits the interaction of lipases with intracellular lipid droplets and is involved in the formation and regulation of lipids in various kinds of cells. However, little is known about the effect of perilipin on porcine oocytes and cumulus cells. Therefore, this study aimed to detect the expression of perilipin1 (PLIN1), perilipin2 (PLIN2), perilipin3 (PLIN3), and perilipin4 (PLIN4) in porcine oocytes and cumulus cells at 4 stages of in vitro maturation (IVM) by quantitative real-time PCR (RT-qPCR). Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) stage were found to occur predominantly at 18 to 24 h, after 24 h, 30 to 36 h, and 36 to 44 h of IVM, respectively. Cumulus-oocyte complexes (COC) were cultured in IVM medium and oocytes and cumulus cells were isolated after different durations of IVM (20, 26, 32, and 44 h). The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, Sn Diego, CA, USA) and the threshold for statistical significance was set at P < 0.05. Messenger RNA expression of PLIN1 was not detected in either oocytes or cumulus cells during all periods of IVM. PLIN2, on the other hands, showed a significant lower expression in GVBD, MI, and MII oocytes compared with the GV oocytes, but showed no difference in cumulus cells. PLIN3 expression was significantly decreased in oocytes of MI stage, whereas PLIN3 from cumulus cells was expressed significantly lower in GVBD and MII stage compared with GV stage. Expression of PLIN4 was significantly decreased in only cumulus cells of GVBD and MI stage. These findings suggest that PLIN2 may have important roles in lipid metabolism during porcine oocyte maturation, whereas PLIN4 may play a major role in cumulus cells. PLIN3 can be hypothesised as a common lipid droplet-associated protein in both oocytes and cumulus cells. Further studies should be conducted to characterise the expression and distribution of PLIN1, PLIN2, PLIN3, and PLIN4 in porcine oocytes and cumulus cells.
This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#311011-05-4-SB010), Research Institute for Veterinary Science, and the BK21 plus program.
146 SPERM STORAGE: EXPRESSION OF PROGESTERONE RECEPTORS, STRUCTURAL PROTEINS, AND HEAT SHOCK PROTEINS IN THE AVIAN OVIDUCT
Authors: Riou C. , Saint-Dizier M. , Gerard N.
Published: December 2015

In avian species, sperm is stored for a long period in sperm storage tubules (SST) located in the utero-vaginal junction. It has been shown that progesterone (P4) regulates the release of sperm from SST for fertilization [Ito et al. 2011 Endocrinology 152, 3952–3621] in quails. In the same context, heat shock proteins (HSP) such as the HSP70 family activate sperm motility in quails [Hiyama et al. 2013 Reproduction 147, 167–178] and may participate in sperm release. In addition, we can hypothesise that at least some structural proteins from SST cells could play a role during sperm storage and release by interacting with sperm. Our objective was to study the potential effect of P4, HSP, and structural proteins on sperm storage potential, by analysing the expression of P4 receptors (PR and mPRα), HSPA8 and HSPB1, CNN1, TAGLN and DES genes in the avian genital tract. Uterus, vagina, and utero-vaginal junctions were collected from 2 divergent lines of hens. One lines displays a long period of sperm storage (21 days, line F+, n = 5), whereas the second displays a shorter period of sperm storage (10 days, line F–, n = 6). Total RNA was extracted and reverse transcribed, and then mRNA coding for PR, mPRα, HSPA8, HSPB1, CNN1, TAGLN and DES were assessed by qPCR (CFX96 Touch Real-Time PCR Detection System, Bio-Rad Laboratories, Hercules, CA, USA). Quantification was performed by using the relative standard curve method. Results were normalized by the geometric mean value of 2 reference genes, GAPDH and S17. Relative amounts of mRNA in each tissue were compared between lines by a Mann-Whitney test. Differences were considered significant when P < 0.05. No significant difference was observed between lines for both P4 receptors, as well as for HSPA8, regardless of the tissue. Expression of CNN1, TAGLN, and DES was significantly higher in the utero-vaginal junction of line F– than line F+ (P < 0.01). Expression of HSPB1 was significantly higher in utero-vaginal junction of line F– than of line F+ (P < 0.05). In conclusion, our data suggest that HSPB1 and structural proteins CNN1, TAGLN, and DES are involved in the regulation of sperm storage in the utero-vaginal junction in hens.
15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE
Authors: Liu Y. , Lucas-Hahn A. , Petersen B. , Li R. , Hermann D. , Hassel P. , Ziegler M. , Li J. , Larsen K. , Niemann H. , Callesen H.
Published: December 2015

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos.

Table 1.Cell number and H3K18ac level1
16 HISTONE DEACETYLASE INHIBITORS PCI-24781 AND QUISINOSTAT IMPROVE THE IN VITRO DEVELOPMENTAL COMPETENCE OF PIG SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
Authors: Jin L. , Zhu H.-Y. , Guo Q. , Zhang Y.-C. , Li X.-C. , Kang J.-D. , Yin X.-J.
Published: December 2015

The aim of this study was to examine the effects of PCI-24781 and quisinostat, two novel histone deacetylase inhibitors, on the in vitro development of pig somatic cell NT (SCNT) embryos. Pig fetal fibroblasts were used as donor cells for SCNT embryos. In vitro-matured eggs with first polar body were enucleated by aspiration using a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. After 1 h, embryos were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, pig SCNT embryos were treated with various concentrations of PCI-24781 or quisinostat for 24 h. In Experiment 2, NT embryos were treated with 0.5 nM PCI-24781 or 10 nM quisinostat for different durations. The rate of blastocyst formation was significantly higher in the 0.5 nM PCI-24781 group than in the control (25.3 v. 10.5%; P < 0.05; Table 1). Moreover, treatment with 10 nM quisinostat dramatically increased the proportion of embryos that reached the blastocyst stage, in comparison with the control group (18.4 v. 10.7%; P < 0.05). Table 1 shows that SCNT embryos treated with 0.5 nM PCI-24781 for 6 h had higher rates of blastocyst formation than control group (25.2 v. 10.2%; P < 0.05). However, the rate of blastocyst formation was significantly higher in the 10 nM quisinostat for 24 h group than in the control (19.8 v. 10.1%; P < 0.05). We determined the treatment conditions of PCI-24781 (0.5 nM for 6 h) and quisinostat (10 nM for 24 h) that significantly improve the in vitro development of pig SCNT embryos.

Table 1.Concentration-dependent and time-dependent effects of treatment with histone deacetylase inhibitors (HDACi) PCI-24781 or quisinostat on the in vitro development of pig SCNT embryos
160 LIPIDOME SIGNATURES IN EARLY BOVINE EMBRYO DEVELOPMENT
Authors: Sudano M. J. , Rascado T. D. S. , Tata A. , Belaz K. R. A. , Santos V. G. , Valente R. S. , Mesquita F. S. , Ferreira C. R. , Araujo J. P. , Eberlin M. N. , Landim-Alvarenga F. D. C.
Published: December 2015

Mammalian pre-implantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding the membrane lipid profile fluctuation during this crucial period is fundamental to address cellular and molecular mechanisms governing embryogenesis. A full understanding of stage-specific lipid signatures in early bovine embryo development is, however, still lacking. The aim of the present work was to characterise stage-specific changes in lipid profiles during early bovine embryonic development. Immature oocytes were recovered from slaughterhouse-derived bovine ovaries and assigned among 5 in vitro developmental stages for lipid characterisation: immature oocytes, 2-cell embryos (32–40 h post-insemination), 8 to 16-cell embryos (72 h post-insemination), morulas (120 h post-insemination), and blastocysts (168 h post-insemination). Two different culture media were used for in vitro embryo production, SOFaaci medium supplemented with 2.5% of serum and serum-free SOF-BE1 medium. Cytoplasmic lipid droplets content and membrane phospholipids profiles for each development stage were assessed by lipid staining (Nile red; n = 5–9) and matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MS; n = 5–9), respectively. For statistical analysis, univariate and multivariate models were used to compare lipid droplets content and membrane phospholipids profiles. Cytoplasmic lipid droplets content increased from minimum in the immature oocyte stage to maximum at the morula stage, followed by a sharp drop at the blastocyst stage (58.4 ± 10.5ac, 62.5 ± 9.4ac, 85.9 ± 8.2a, 148.3 ± 7.4b, 37.4 ± 9.9c of fluorescence intensity per embryo area, respectively, for immature oocyte, 2-cells, 8 to 16-cells, morulas, and blastocysts; abcP < 0.05). More cytoplasmic lipid droplets were detected in morulas and blastocyts cultured in SOFaaci than in SOF-BE1 (morulas, 162.6 ± 11.3 v. 137.1 ± 9.2 of fluorescence intensity per embryo area, respectively, P < 0.05; blastocysts, 49.9 ± 9.9 v. 20.7 ± 9.9 of fluorescence intensity per embryo area, respectively, P < 0.05). Characteristic dynamic changes of unsaturation level, acyl chain length and class composition (phosphatidylcholines, sphingomyelins, and phosphatidylethanolamines) of phospholipid profiles were observed during early embryo development. This study provides a comprehensive analysis, involving lipid staining and mass spectrometry evaluation, of stage-specific lipid signatures during bovine in vitro embryo development. These results may be useful for assessing the role of specific lipid species during important events of embryogenesis.
Research was supported by CNPq, FAPESP, and FAPERGS.
164 EFFECTS OF KINETICS AND MORPHOLOGY ON EARLY EMBRYONIC DEVELOPMENT IN BOVINE OPU-IVF EMBRYOS
Authors: Takayama M. , Dochi O. , Imai K.
Published: December 2015

In recent years, the use of ovum pick up (OPU) and IVF for embryo production has increased worldwide; however, the conception rate of embryo transfer is lower for OPU-IVF embryos than for in vivo-derived embryos. This study aimed to determine the efficacy of embryo selection by a 3-step observation method by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). In this study, 9 Holstein and 15 Japanese Black cows were used, and the OPU-IVF was conducted from October 2014 to May 2015. The collected cumulus-oocyte complexes (COC) were cultured for 22 h in 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH. Sperm (at a final concentration of 5 × 106 spermatozoa mL–1) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS by using a micro-well culture dish. Kinetics and morphology were observed at 27, 31, and 55 h post-insemination (hpi). The presumptive zygotes were divided to 3 groups (more than 2 cells, 2 cells, and no cleavage) at 27 and 31 hpi. Then, embryos at the 2-cell stage at 31 hpi were divided into 2 groups: 2-cell with normal cleavage and 2-cell embryos with abnormal cleavage (unequal cleavage, 2-cell with fragments, and 2-cell with protrusion). Subsequently, embryos were classified as 8-cell and more than 8 cell, or less than 8 cell at 55 hpi. The blastocyst rate (BL%) was analysed at 7, 8, and 9 days post IVF. Embryos selected by the 3-step observation method were used for fresh embryo transfer. The data were analysed by chi-squared test. In total, 856 oocytes were collected by OPU and 633 oocytes were cultured, of which 39.7% (263/663) developed to the blastocyst stage. The BL% of 2-cell embryos (72.5%, 116/160) was significantly higher (P < 0.01) than that of no cleavage (47.0%, 117/249) at 27 hpi. The BL% of 2-cell (65.4%, 206/315) and more than 2-cell (53.0%, 35/66) was significantly higher (P < 0.01 and P < 0.05) than that of embryos divided as no cleavage (25.9%, 22/85) at 31 hpi. The BL% was not significantly different between 2-cell with normal cleavage (68.5%, 172/251) and abnormal cleavage (53.1%, 34/64). The BL% of 8-cell and more than 8-cell stage (72.8%, 182/250) was significantly higher (P < 0.01) than that of embryos with less than 8 cells (38.8%, 81/209) at 55 hpi. Overall, 2-cell embryos at 27 hpi, 2-cell embryos with normal cleavage at 31 hpi, and 8-cell and more than 8 cell at 55 hpi showed the highest BL% (82.1%, 78/91). The conception rate was higher for following the selected fresh embryo transfer that was 70.6% (12/17) than average of in vitro fertilization embryos transfer that was 40.0%. These results demonstrate that the 3-step observation method used in this study can be effectively applied for the selection of IVF embryos that have a strong ability to develop into blastocysts and high competence for conception.
17 TREATMENT WITH MGCD 0103 IMPROVES THE IN VITRO DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER
Authors: Zhu H.-Y. , Jin L. , Guo Q. , Zhang Y.-C. , Li X.-C. , Kang J.-D. , Yin X.-J.
Published: December 2015

We use MGCD 0103 to test whether the treatment with this novel histone deacetylase inhibitor improves the in vitro development of porcine somatic cell NT (SCNT) embryos. Matured eggs were cultured in medium supplemented with 0.05 M sucrose and 0.4 μg mL–1 demecolcine for 1 h. Treated eggs with a protruding membrane were transferred to medium supplemented with 5 μg mL–1 cytochalasin B and 0.4 μg mL–1 demecolcine. Protrusions were then removed by aspirating with a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. Fused eggs cultured for 1 h were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days at 38.5°C in 5% CO2 humidified air. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, the SCNT embryos were cultured in medium supplemented with 0, 0.2, 2, or 20 μM MGCD 0103 for 24 h and then transferred to medium without MGCD 0103. In Experiment 2, SCNT embryos were cultured in medium supplemented with 0.2 μM MGCD 0103 for 0, 6, 24, or 48 h and then transferred to medium without MGCD 0103. As shown in Table 1, development to the blastocyst stage increased in SCNT embryos treated with 0.2 μM MGCD 0103 compared with the control or groups treated with 2 or 20 μM MGCD 0103 (25.51 v. 10.74, 3.53, 3.20%, respectively; P < 0.05). As shown in Table 1, treatment for 6 h with 0.2 μM MGCD 0103 significantly improved the rate of blastocyst formation compared with the control or groups treated for 24 or 48 h (21.17 v. 10.48, 19.23, 10.20%, respectively; P < 0.05). Our results suggested that 0.2 μM MGCD 0103 treatment for 6 h can improve in vitro developmental competence of porcine SCNT embryos.

Table 1.In vitro development of pig SCNT embryos with different concentrations of MGCD 0103 for 24 h, and with 0.2 μM MGCD 0103 for different durations
173 IN VITRO EMBRYO PRODUCTION FROM OOCYTES FERTILISED WITH UNSORTED OR X-SORTED SPERM AND ISSUED FROM SUBFERTILE HIGH GENETIC MERIT COWS SUBMITTED OR NOT TO A 48-h COASTING PERIOD FOLLOWING FSH STIMULATION
Authors: Gamarra G. , Lacaze S. , Ponsart C. , Mouneyres M. , Le Guienne B.
Published: December 2015

High genetic merit cows may be sidelined from breeding schemes because of reproductive disorders. In vitro production of embryos (IVP) issued from ovum pickup (OPU) can be an alternative to bypass infertility problems as experienced in humans and thus accelerate genetic progress (Duszewska et al. 2012 J. Anim. Feed Sci. 21, 217–233). The aim of this work was to evaluate if IVP from high genetic merit subfertile cows could benefit to the breeding scheme under commercial conditions, at the Biotechnology MIDATEST Station located in Denguin, Southwest, France. Holstein cows (n = 16) from 3.5 to 13 years old with different reproductive pathologic problems (repeated breeding, failure in in vivo embryo production, embryo mortality, permanent cysts, oviduct infection) were used in an OPU-IVP program. Donor cows were stimulated with decreasing doses pFSH twice daily during 3 days (Stimufol®, total dose: 350 µg of pFSH). The OPU was performed 48 h after the last FSH injection in the “coasting” group v. 12 h after the last FSH injection for “no-coasting” group and in vitro matured using a standard IVM protocol. Oocytes were fertilised with frozen-thawed unsorted or X-sorted sperm in TALP medium from different bulls (n = 42) without any previous IVP testing. Presumptive zygotes were cultured in SOF medium (Minitub®) plus 1% cow serum up to Day 7 at 38.5°C in 5% CO2, 5% O2, and 90% N2 atmosphere with maximum humidity. The OPU-IVP was repeated 1 to 6 times (3.75 ± 2.6) for each donor cow. Grade 1 blastocysts and expanded blastocysts (according to IETS classification) were recorded on Days 6.5 and 7. Embryo production was analysed with ANOVA and blastocyst yield was analysed by chi-square. The results in both coasting or no-coasting groups and the effect of fertilization using unsorted or X-sorted sperm are presented in Table 1. The embryonic development rate was significantly higher when using unsorted semen to fertilize the oocytes compared to X-sorted sperm (P < 0.05). On the other hand the coasting period had no significant effect neither on the number of collected oocytes nor on the embryonic development rates. In conclusion, our work confirmed the efficacy of OPU-IVP techniques to produce grade 1 embryos using X-sorted or unsorted sperm in subfertile high genetic merit cows.

Table 1.Oocyte collection and in vitro embryo production, in donor cows submitted or not to a 48-h coasting period and effect of fertilization with unsorted v. X-sorted sperm
177 PERFORMANCE OF Gyr PREPUBERTAL HEIFERS IN IN VITRO EMBRYO PRODUCTION
Authors: Rosa P. M. S. , Camargo A. J. R. , Serapião R. V. , Camargo L. S. A. , Oliveira C. S.
Published: December 2015

Bovine in vitro embryo production is highly relevant for dairy systems in Brazil, and Gyr dams are commonly used as oocyte donors. The aim of this study was to evaluate the use of prepubertal Gyr heifers as oocyte donors, an alternative to anticipate reproduction of those animals. For that, 11 Gyr [4 prepubertal (PP) donors and 7 adult cows © donors] were used in ovum pickup (OPU) sessions. The PP cows presented an average of 282.5 kg and 26.75 months, and had never displayed oestrous. Non-lactating cows presenting an average of 492 kg and 136 months were selected for C. Five replicates were performed, totaling 27 OPU sessions (C-17, PP-10) and 2–3 sessions per animal. Follicular wave was synchronised by aspiration of follicles larger than 8 mm 96 h before OPU. Cumulus-oocyte complexes (COC) were classified accordingly to their quality in viable (G1, G2, and G3) or non-viable (G4). Viable oocytes were matured and fertilized, and the presumptive zygotes were cultured in SOF medium at 38.5°C and 5% CO2 in air. Cleavage rate was assessed 48 to 72 h post-insemination (hpi) and blastocyst rate at 168 hpi. Mean number of structures was analysed by t-test, and percentage of viable, G1, G2, G3, G4, cleavage, and blastocyst rates were compared among groups by Fisher’s exact test (GraphPadInstat, La Jolla, CA, USA; P = 0.05). Results are followed by standard error values. All procedures were approved by a local ethics committee. We found that despite higher (P < 0.05) numbers for both viable oocytes (PP: 15 ± 2.6; C: 6.11 ± 0.76) and total oocytes (PP: 23.70 ± 2.83; C: 8.82 ± 1.19) in the PP group, the rate of viable oocytes was similar (P > 0.05) among PP and C groups (PP: 61.5 ± 6.51%, C: 66.79 ± 3.79%). Mean numbers of G1, G2, G3, and G4 oocytes were higher (P < 0.05) in the PP group (G1 = 7.1 ± 1.18; G2 = 4.9 ± 1.74; G3 = 3.9 ± 1.09; G4 = 7.8 ± 1.38) than in the C group (G1 = 2.70 ± 0.740; G2 = 2.47 ± 0.44; G3 = 1.11 ± 0.31; G4 = 2.52 ± 0.39). However, the proportion was similar (P > 0.05) among PP and C groups (PP: G1 = 29.5 ± 4.21%; G2 = 19.5 ± 2.85%; G3 = 15.9 ± 13.5%; G4 = 35.1 ± 6.33%; and C: G1 = 27.24 ± 4.44%; G2 = 29.60 ± 5.08%; G3 = 12.34 ± 3.01%, G4 = 30.79 ± 4.93%). Cleavage rate (PP: 91.3 ± 17.94%; C: 74.09 ± 4.65%), mean blastocyst number per OPU session (PP: 3.3 ± 1.29; C: 1.76 ± 0.28), and blastocyst rate (PP: 19.74 ± 7.40%; C: 27.03% ± 4.07%) were similar (P > 0.05) among groups. We conclude that prepubertal heifers presented increased numbers of viable oocytes per OPU session, but blastocyst yield was similar to adult cows. This data suggests that prepubertal Gyr heifers can be used as oocyte donors.
Support from FAPERJ and Embrapa is acknowledged.
179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE
Authors: Matoba S. , Kaneda M. , Somfai T. , Nagai T. , Geshi M.
Published: December 2015

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P < 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P < 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P < 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence.
This study was supported by JSPS KAKENHI (26450388).
18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY
Authors: Buemo C. , Gambini A. , Moro L. , Martin R. F. Y. , Salamone D.
Published: December 2015

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.
186 QUANTITATIVE ECHOGENICITY AND ECHOTEXTURE ANALYSIS OF THE ACCESSORY SEX GLANDS OF PUBERTAL AND MATURE DORPER RAMS
Authors: Camela E. S. C. , Nociti R. P. , Santos V. J. C. , Macente B. I. , Maciel G. S. , Feliciano M. A. R. , Vicente W. R. R. , Bartlewski P. M. , Oliveira M. E. F.
Published: December 2015

This study aimed to analyse the ultrasonographic attributes of vesicular, prostate, and bulbourethral glands in pubertal and mature Dorper rams. Forty-five rams were used in the same day (pubertal: 8–11 months, n = 24; mature: ≥12 months, n = 21). The B-mode ultrasounds examinations were performed using MyLab 30Vet equipment (Esaote, Naples, Italy) connected to transrectal linear transducer (frequency of 7.5 MHz). The echogenicity [(mean numerical pixel values (NPV)] and pixel heterogeneity (standard deviation of NPV) of accessory sex glands parenchymas was determined by computerised image analysis using Image ProPlus® software (Media Cybernetics Inc., Rockville, MD, USA). For the pairs organs a mean for each parameter were calculated. Data were analysed by ANOVA with Tukey test (mean ± s.d.; P < 0.05), after the normality and homoscedasticity of residuals were checked with Shapiro-Wilk test and Levene test, respectively, when necessary log-transformation was applied. All statistical procedures was performed with R® software. The echogenicity of parenchymas were different (P < 0.05) between pubertal and mature rams for vesicular glands (181.51 ± 20.80 v. 164.83 ± 26.79) and bulbourethral glands (166.93 ± 16.93 v. 141.80 ± 29.15); however, there was no difference (P > 0.05) for prostate glands (99.39 ± 36.34 v. 87.47 ± 34.24). The pixel heterogeneity did not differ (P > 0.05) between pubertal and mature rams (29.06 ± 2.67 v. 28.09 ± 3.95, 32.14 ± 8.20 v. 30.28 ± 4.71, and 27.82 ± 4.53 v. 28.74 ± 4.29) for vesicular, prostate, and bulbourethral glands, respectively. In conclusion, the sexual maturity only influenced the echogenicity of vesicular and bulbourethral parenchymas of Dorper rams.
193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM
Authors: Nõmm M. , Mark E. , Sarv O. , Kõks S. , Jaakma Ü.
Published: December 2015

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results.
This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.
2 CIRCULATING microRNA SIGNATURES ASSOCIATED WITH EARLY BOVINE PREGNANCY
Authors: Ioannidis J. , Donadeu X.
Published: December 2015

High-producing dairy cattle are characterised by low fertility, which extends calving intervals and reduces annual milk production. Early pregnancy diagnosis before Day 21 of gestation can shorten calving intervals, improve milk production, and increase overall profits from modern dairy herds. At present, accurate diagnosis is only possible after the third week of pregnancy. Circulating microRNA (miRNA) have been proposed as diagnostic biomarkers of numerous human conditions such as cancer and diabetes. Moreover, distinct circulating miRNA profiles have been associated with mid to late stages of human pregnancy. The aim of this study was to determine whether specific circulating miRNA signatures that occur during early pregnancy in cattle could be used for diagnostic purposes. To do this, we have compared miRNA levels in plasma samples from cycling, nonpregnant (n = 24) and cycling, early pregnant (D16, n = 11; D24, n = 11) Holstein heifers using next-generation sequencing and PCR arrays. Using small-RNA sequencing on 46 individual samples, we generated 2.8 million miRNA reads, and up to 328 miRNAs were detected in each sample. There were no significant differences (false discovery rate <0.1) in miRNA levels between nonpregnant and pregnant (D16, D24) heifers. In addition, 12 pooled samples (n = 9 for nonpregnant, n = 3 for D24) were analysed for the expression of 176 miRNA using PCR arrays; 16 miRNA had differentially expressed levels (2- to 3-fold; false discovery rate <0.1) in pregnant heifers. The QPCR validation of miRNA candidates obtained from the high-throughput analyses identified 6 miRNA that were differentially expressed in pregnant heifers. These included bta-miR-26a, bta-miR-29c, bta-miR-138, and bta-miR-204, which were differentially expressed at D16, bta-miR-1249, which was differentially expressed at D24, and hsa-miR-4532, which was differentially expressed at both D16 and D24 of pregnancy (P < 0.05, fold-change ≤2.4). The miRNA miR-26a and miR-204 have been previously associated with early (D20) and late pregnancy in pig and human respectively. Furthermore, miR-1249 has been associated with the regulation of ovarian function, and miR-26a is involved in immune regulation, suggesting a potential role for this miRNA as an immuno-suppressor during early pregnancy. In summary, we identified a miRNA signature associated with early pregnancy in cattle. We are currently using next-generation sequencing to identify differentially expressed miRNA at other stages of pregnancy, including Day-60 heifers, and this data will also be presented.This study was funded by Zoetis and BBSRC.
20 SIALYLTRANSFERASE GENE EXPRESSION IN GGTA1 KNOCKOUT PIGS
Authors: Kim G. A. , Jin J.-X. , Lee S. , Oh A. , Lee B. C.
Published: December 2015

It is considered that GGTA1 knockout (KO) pig production via somatic cell NT would overcome the problem of immune rejection after xenotransplantation. It is reported that although GGTA KO mice showed only a mild increase in sialyltransferase gene expression, GGTA1 deficiency in pig could increase the sialyltransferase activities, non-Gal epitope expression, consequently may raise non-Gal xenoantigenicity. Therefore, in the present study we investigated whether the expression level of Sia-containing glycoconjugate mRNA in transgenic pigs could be affected by knocking out the GGTA1 gene. Besides GGTA1 KO pigs, double genes overexpressing pigs (2TG) and GGTA1 KO with double genes overexpressing (KO+2TG) pigs were produced by somatic cell NT. For the present study, fibroblasts were isolated from wild-type pigs without gene modification, 2TG, GGTA1 KO, and KO+2TG pig. The GAPDH gene was used as an internal standard to normalise the real-time PCR (RT-qPCR) analysis reaction efficiency and to quantify mRNA in pigs-derived fibroblast. The expression levels were compared between them (RT-qPCR) in triplicate for each sample. Oligonucleotide primers for real-time PCR were designed for Hanganutziu-Deicher antigen (ST3Gal1–4, ST6Gal1) and Sialyl-Tn antigen (ST6GalNac1, ST6GalNac2, and ST6GalNac6) analysis. For statistical analysis, one-way ANOVA with Dunn’s multiple comparison test were used. The mRNA expression of GGTA1 KO and KO+2TG pig derived fibroblasts cells genes showed that ST3Gal1, ST3Gal2, ST3Gal3, and ST6Gal1 gene expression were significantly up-regulated compared to the wild and 2TG pigs (P < 0.05). However, ST3Gal4, Sialyl-Tn antigen including ST6GalNac1, ST6GalNac2, and ST6GalNac6 in KO+2TG pigs were not different compared with the wild pigs (P > 0.05), whereas only GGTA1 KO pigs showed significantly higher expressions than wild, 2TG, and KO+2TG pigs (P < 0.05). These results demonstrated that GGTA KO pig-derived cells exhibit a higher Hanganutziu-Deicher antigen on glycoprotein and glycolipid than controls, and KO+2TG pig exhibit no differences when compared with GGTA1 KO pig, indicating that they do not act as an immune antigen in xenograft. Overall, the increase in glycosyltransferase expression suggests a corresponding increase in the cell surface sialyation in GGTA KO pig cells. For xenotransplantation, KO+2TG pigs were more preferable because of absence of immune rejection for Sia-containing glycoconjugate on glycoprotein and glycolipid than GGTA KO pigs.
This study was supported by the Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.
201 ROLE OF THE NITRIC OXIDE SYSTEM IN EFFECTS OF PROLACTIN AND GROWTH HORMONE ON METAPHASE-II CHROMOSOMES IN BOVINE OOCYTES AGING IN VITRO
Authors: Lebedeva I. , Singina G. , Shedova E. , Lopukhov A. , Zinovieva N.
Published: December 2015

Aging of mammalian oocytes is the time-dependent process of cytological and molecular transformations leading to a decline in the ovum quality and developmental capacity. We have previously shown that 2 related pituitary hormones, prolactin (PRL) and growth hormone (GH), may decelerate abnormal changes in the morphology of metaphase II (MII) chromosomes in bovine cumulus-enclosed oocytes (CEO) aging in vitro. The goal of the present research was to examine the involvement of different isoforms of nitric oxide synthase (NOS) in the actions of PRL and GH on MII chromosomes in aging bovine oocytes. Bovine CEO were matured for 20 h in TCM 199 containing 10% FCS, 10 μg mL–1 porcine FSH, and 10 μg mL–1 ovine LH. After IVM, CEO or denuded oocytes (DO) were cultured for 24 h in the aging medium of TCM 199 supplemented with 10% FCS (control). In experimental groups, the medium contained either 50 ng mL–1 bovine PRL or 10 ng mL–1 bovine GH and/or NOS inhibitors. The following inhibitors were applied: (1) N-propyl-l-arginine (NPLA; an inhibitor of neuronal NOS (nNOS), 5 μM) and (2) L-NAME (an effective inhibitor of both endothelial NOS (eNOS) and nNOS, 20 μM). Destructive changes of MII chromosomes in oocytes were assessed by the following morphological signs: decondensation, partial adherence, chromosome clumping into a single mass, and fragmentation. The total activity of NOS in oocytes was determined by NADPH-diaphorase staining. The data from 4–5 replicates were analysed by ANOVA. During CEO aging in the control medium, the rate of MII oocytes with destructive changes of chromosomes rose from 16.8 ± 2.1% to 58.5 ± 1.4% (P < 0.001), whereas both PRL and GH reduced this rate up to 42.0 ± 1.3% and 46.5 ± 1.6%, respectively (P < 0.001). The nNOS inhibitor NPLA abolished (P < 0.001) the inhibitory effect of PRL on abnormal modifications of chromosomes in CEO but did not affect the frequency of these modifications in the control or GH-treated groups. In the absence of the hormones, L-NAME (the eNOS+nNOS inhibitor) decreased the rate of aging CEOs with chromosome abnormalities from 58.5 ± 1.4% to 41.2 ± 2.5% (P < 0.001), acting unidirectionally with PRL and GH. Meanwhile, L-NAME enhanced (P < 0.05) the suppressing effect of PRL on destructive changes of MII chromosomes but did not influence the similar effect of GH. At the same time the chromosome morphology in senescent DOs was unaffected by the hormones or NOS inhibitors. Furthermore, the total activity of NOS in oocytes separated of cumulus after 24 h of aging was similar in the control and experimental groups. Thus, the inhibitory effect of GH on abnormal modifications of MII chromosomes in aging bovine oocytes may be related to a reduction of the eNOS activity in cumulus cells, whereas the respective effect of PRL is likely to be achieved by both inactivation of eNOS and activation of nNOS.
This research was supported by RFBR (No. 13–04–01888).