Difference In Total Cell Number Research Articles

#193 : Comparison of the Different Expression of OCT-4, GATA-3 and Clinical Pregnancy Outcome Between the Day 5 and Day 6 Blastocysts

Background and Aims: In common experience of in vitro-fertilization (IVF) cycle, blastocysts formed at Day 5 (D5) are regarded as higher quality and are more desirable for embryo transfer compared with those formed at Day 6 (D6). How those delayed D6 embryo differs from D5 embryo is less discussed. This study aimed to elucidate the cell content distinguished by immunofluorescent (IF) staining between the same grade of D5 and D6 blastocysts that might link to the clinical pregnancy outcome in frozen–thawed embryo from different day of blastocyst formation. Method: The retrospective study included 1327 frozen-thawed single blastocyst transfer (SBT) cycles in the Taipei Fertility Center during March 2020 to December 2022. All D5 (D5-SBT) and D6 (D6-SBT) frozen-thawed SET cycles (n=1327) were separated to non-PGS (n=671) and PGS-euploid (n=656) groups, then assigned to high grade (AA, AB, BA, BB) and low grade (BC, CB, CC). Aneuploidy and mosaicism embryo are excluded. For cell content analysis, pluripotent marker OCT-4 initially expressed in all blastomeres but restricted to the inner cell mass (ICM) of the blastocyst and downregulated in the trophectoderm (TE). GATA-3 is a TE specific marker which increases during pre-implantation embryo development. The cell number, OCT-4 and GATA-3 expression of same grade frozen-thawed D5 (n=14) and D6 (n=14) blastocysts were analyzed with IF staining. D5 extensively cultured to D6 (n=10) were used as the control group of regularly development. Cell numbers of TE and ICM region were examined by OCT-4 and GATA-3 IF detection. The total cell number was visualized with DAPI staining. Results: According to staining results, there were no significant differences in total cell number, TE and ICM cell number between the same grade D5 and D6 blastocysts. However, TE cell number significantly increased in D5 extensively cultured blastocysts, indicating higher proliferative capacity in TE cells from D5 blastocysts. In the clinical data of the non-PGS group, chemical pregnancy rate (HCG+) and clinical pregnancy rate (SAC+) of high grade D5-SBT were significantly increased compared with high grade D6-SBT (HCG+: 69.1% versus 47.0%, P<0.001; SAC+: 63.0% versus 43.4%, P=0.001). In the PGS-euploid group, the chemical pregnancy rate (HCG+) showed significantly increased in high grade D5-SBT compared with high grade D6-SBT (69.5% versus 60.0%, P=0.047). The clinical pregnancy rate (SAC+) was significantly higher in high grade D5-SBT compared with high grade D6-SBT (65.5% versus 52.5%, P=0.009). The ongoing pregnancy rate was significantly higher in high grade D5-SBT compared with high grade D6-SBT (57.6% versus 43.3%, P=0.005). Conclusion: According to the IF data in this study, we demonstrated that the number of GATA3-positive TE cells were significantly decreased in D6 blastocyst compared with D5 extensively cultured blastocysts. Due to decreased proliferative capacity in high grade D6 blastocyst, transfer of high grade D5 blastocyst would exhibit higher ongoing pregnancy rate than D6 blastocyst.

KSOM-R supports both mouse and rat preimplantation embryo development in vitro

A modified KSOM for rat embryo culture (KSOM-R), which has enriched taurine, glycine, glutamic acid, and alanine, promoted rat embryo development in vitro. Since mice and rats share similar amino acid profiles in their female reproductive tracts, this study explored whether KSOM-R would also have a positive effect on mouse embryo development and if KSOM-R modifications could extend its shelf time at 2–8 °C for consistency. We first examined the effects of newly made (≤1 month at 2–8 °C) antibiotics-free KSOM-R (mKSOM-R), antibiotics-free KSOM (mKSOM) and KSOM on the development of in vivo or in vitro derived C57BL/6NJ zygotes. We then investigated the effect of extended shelf life (6 months at 2–8 °C) of mKSOM-R and mKOSM on the development of C57BL/6NJ mouse and Sprague Dawley (SD) rat embryos. The results showed that there were no significant differences in cleavage, blastocyst, and hatching rates of C57BL/6NJ embryos among the three freshly made media. After 6 months of storage at 2–8 °C, mKSOM-R and mKSOM were still able to support the development of in vivo C57BL/6NJ zygotes at comparable rates seen with newly made (≤1 month at 2–8 °C) KSOM (control) in terms of cleavage, blastocyst formation and hatching. There were also no significant differences in total cell numbers in day 4 blastocysts among the three groups. After surgical embryo transfers, C57BL/6NJ blastocysts cultured in mKSOM-R (6 months at 2–8 °C) and newly made (≤1 month at 2–8 °C) KSOM culture developed into live pups. These pups had no gross abnormalities in animal morphology and growth. SD zygotes cultured in mKSOM-R stored at 2–8 °C for 6 months developed at comparable rates in cleavage, blastocyst and hatching rates when compared to those cultured in newly made mKSOM-R (≤1 month at 2–8 °C). The data showed that, although no significant beneficial effects were observed on mouse embryo development, mKSOM-R was able to support both mouse and rat embryo development in vitro. Additionally, mKSOM-R and mKSOM can be stored at 2–8 °C for at least 6 months without significantly compromising quality. This study suggests that it is possible to reduce the media inventory by using only mKSOM-R to culture both mouse and rat embryos, and quality media with extended shelf life can be made through modifications.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Mutation of the RelA(p65) Thr505 phosphosite disrupts the DNA replication stress response leading to CHK1 inhibitor resistance.

Novel allele elh of the UBP14 gene affects plant organ size via cell expansion in Arabidopsis thaliana.

Plant organ size control is an essential process of plant growth and development. The regulation of plant organ size involves a complicated network of genetic, molecular interactions, as well as the interplay of environmental factors. Here, we report a temperature-sensitive hypocotyl elongation EMS-generated mutant, hereby referred to as elongated hypocotyl under high-temperature (elh). The elongated hypocotyl phenotype was prominent when the elh seedlings were grown at high temperature, 28°C, but not under the growth temperature of 21°C. We observed significantly larger organ sizes in elh plants, including cotyledons, petals and seeds. In elh plants, the cell sizes in cotyledons and petals were significantly larger than wild type. By measuring the cell density and organ area of cotyledons, petals and mature dissected embryos, we found no differences in total cell numbers in any organ indicating that cell expansion rather than cell proliferation was perturbed in elh. elh plants produced leaves at a slower rate than wild type plants, suggesting that perturbing the balance between cell division and cell expansion is linked to the developmental rate at which leaves are produced.

EXPOSURE OF HUMAN BLASTOCYSTS TO SPECIFIC GROWTH FACTORS BASED ON RECEPTOR PRESENCE IMPROVES EPIBLAST FORMATION IN EXTENDED EMBRYO CULTURE

Embryos are exposed to multiple growth factors during pre-implantation development in vivo. However, inclusion of a variety of growth factors in embryo culture medium has resulted in disparate results between studies and across species, making it difficult to ascertain the potential efficacy of growth factors to improve human embryo culture. Our objective was to identify the presence or absence of specific growth factor receptors in human blastocysts using western blot analysis, and then to determine the effect of exposing embryos to the growth factors for which receptors are present on embryo development and viability. Research Study Western blots were conducted using the Protein-Simple Jess system, with detection of each putative growth factor receptor assessed in a pool of two human blastocysts as well as a positive control (human granulosa cells). Due to the paucity of human zygotes that are available for such research, we used vitrified human blastocysts donated for research, exposing blastocysts to growth factors for 48 hrs prior to placement into an extended embryo culture system (WIRB study # 1179872). After warming, day (D) 5 human blastocysts (n=35) were cultured in the presence (GF) or absence (control) of selected growth factors. After 48 hrs (D7), the zona pellucidae were removed prior to placing blastocysts on fibronectin coated 8 well chambered microslides for extended culture. Embryo outgrowth area was measured on D10, D11, and D12, at which time embryos were fixed and stained with DAPI and antibodies against F-actin and the pluripotency marker POU5F1 to determine the number of epiblast cells and total cells using 3D spinning disk confocal microscopy. Data was analyzed using t-test. Of the eleven growth factor receptors analyzed, six were found to be present in human blastocysts. Receptors that were not detected included EGFR, ACVR1, ACVR2, IGF1R, and TGFβR2. There were no differences in embryo outgrowth area on D10, D11, or D12 (control, 0.206 ± 0.053 mm2; GF, 0.234 ± 0.046 mm2). More embryos cultured with chosen growth factors formed an epiblast (control, 35.7%; GF, 84.6%; p=0.03). However, there were no differences in the number of epiblast cells as an average of all embryos (control, 27.8 ± 15.4; GF, 38.8 ± 9.4) or as an average of only those embryos with epiblast cells (control, n=5, 77.8 ± 34.3; GF, n=11, 45.9 ± 9.6). There was also no difference in total cell number (control, 344.1 ± 116.0; GF, 413.4 ± 116.8) in the D12 embryo outgrowths. In summary, inclusion of growth factors in embryo culture medium at the blastocyst stage, chosen based upon receptor presence, increased the ability of the embryos to form an epiblast suggesting that the presence of these growth factors increased embryo viability. Future work will examine the effect of these growth factors during preimplantation embryo culture on blastocyst development and quality prior to embryo transfer.

Tauroursodeoxycholic acid acts via TGR5 receptor to facilitate DNA damage repair and improve early porcine embryo development

DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNA-mediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.

152 Activation of Bovine Oocytes Using the Zinc Chelator TPEN

Traditionally, artificial oocyte activation has been induced by stimulating intracellular calcium increase in the oocyte. Recently, the use of zinc chelators has also shown to be effective in inducing activation by decreasing intracellular concentration of zinc, mimicking events during fertilization; however, this has not been demonstrated in bovine oocytes. The use of artificial activation in bovine has potential for overcoming subfertility-related production loss and aid in livestock cloning. In this study, we determined whether bovine oocytes could be artificially activated in the presence of the zinc chelator TPEN [N,N,N’,N’-tetrakis(2-pyridylmethyl) ethane-1,2-diamine]. Bovine cumulus–oocyte complexes (COC) were collected from abattoir-derived ovaries and incubated for 24 h in TCM-199 maturation medium supplemented with fetal bovine serum, sodium pyruvate, Glutamax, oestradiol, and FSH. The COC were denuded by vortexing in denuding medium containing 0.1% hyaluronidase, and individual oocytes were selected based on presence of a visible polar body. Matured oocytes were then incubated in TL-HEPES medium supplemented with 1 of 5 treatments for parthenogenetic activation: (1) DMSO for 2 h (control, n = 116), (2) 100 µM TPEN for 45 min (100-45, n = 103), (3) 100 µM TPEN for 120 min (100-120, n = 102), (4) 200 µM TPEN for 45 min (200-45, n = 63), or (5) 200 µM TPEN for 120 min (200-120, n = 142). After treatment, oocytes were washed with culture media and incubated in droplets of SOF-Be1 medium under oil to monitor subsequent development. The number of blastocysts was recorded on Day 10 of culture. Blastocysts were stained with Hoechst for 15 min to evaluate total cell number. The frequencies of blastocyst formation were compared using the Chi-squared test, and differences in total cell number were compared using the Student’s t-test. All TPEN treatments significantly increased the number of oocytes developed to the blastocyst stage relative to the control group, which was unable to form blastocysts (P < 0.01). The 100-45 treatment had a greater % blastocysts compared with the 200-120 treatment (16.5% vs 7.77%; P < 0.05), tended to be greater than 100-120 treatment (16.5% vs 7.8%, P = 0.058), and was numerically greater than the 200-45 treatment (16.5% vs 7.94%; P = 0.114). Three treatments that resulted in blastocysts were analyzed for cell counting: 200-120 (n = 5), 100-120 (n = 4), and 100-45 (n = 7). Average total cell number was 119.20 ± 52.28 for the 200-120 group, 83.75 ± 51.06 for the 100-120 group, and 111.71 ± 59.06 for the 100-45 group. There was no difference in total cell number among groups (P ≥ 0.341). Here, we demonstrated that mature bovine oocytes can successfully be parthenogenetically activated by incubating with the zinc chelator TPEN. Oocytes incubated with 100 µM TPEN for 45 min provided the greatest blastocyst yield. Total cell number did not differ between treatments, but all groups analyzed showed blastocysts containing over 100 cells, demonstrating the effectiveness of the oocyte activation approach. Further studies will focus on optimizing the use of TPEN to activate bovine oocytes.

51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73–81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651–3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008–16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n = 753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n = 60) or absence (n = 54) of 20 µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347–55). Blastocysts were warmed in decreasing sucrose solutions (0.25 M for 1 min and 0.15 M for 5 min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48 h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Student’s t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6 v. 55.0%; P < 0.01) and hatching rates (40.7 v. 23.3%; P < 0.05) after 48 h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1 ± 0.6 v. 4.2 ± 0.3; P < 0.01) and the percentage (6.3 ± 0.6 v. 3.0 ± 0.2; P < 0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90 ± 1.6). These results suggest that addition of 20 µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.

Effect of short-term exposure of cumulus-oocyte complex to 3-morpholinosydnonimine on in vitro embryo development and gene expression in cattle.

Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100μM SIN-1) for 1hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10μM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1μM; SOD1 at 0.1 and 100μM; PRDX5 at 0.1, 10 and 100μM; and NANOG at 10 and 100μM; and an upregulation of CDX2 expression was observed at 100μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.

Developmental competence and cryotolerance of caprine parthenogenetic embryos cultured in chemically defined media

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 μM, Ca2+ ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.

84 PORCINE EMBRYOS UTILIZE SMALL AMOUNTS OF PYRUVATE, LACTATE, AND GLUCOSE IN VITRO

Porcine embryo culture systems are suboptimal to the in vivo environment, and significant effort has been made to improve development to the blastocyst stage in vitro. Since metabolism of the early embryo has many similarities to the Warburg effect, our goal was to determine the role of glucose on development, gene expression, and metabolism of other energy substrates in the blastocyst stage embryo. Pig embryos were in vitro produced and cultured in MU1 containing pyruvate, lactate, amino acids, and either 0, 7.5, 15, or 250 µM glucose, N = 1164, 4 replications. There was no difference in blastocyst percentage between the 0 µM and 7.5 µM glucose (34% ± 6.5 v. 29% ± 8.2), but there was a decrease in development in response to 15 and 250 µM compared with 0 µM glucose (25% ± 8.5, 23% ± 8.7 v. 34% ± 6.5; P ≤ 0.01). Glucose transporters (SLC2A1 and SLC2A2) and hexokinases (HK1 and HK2) were analysed by qPCR to detect differences in gene expression, 3 replicates containing 10 blastocyst pools. The abundance of both HK1 and HK2 was decreased in blastocysts cultured with 7.5 µM glucose compared with 0 µM (P ≤ 0.04). Glucose transporters were not affected by glucose supplementation (P ≥ 0.5). Metabolic data were collected to determine if embryos were adjusting their energy substrate use in response to glucose. Two assays were completed to determine lactate and pyruvate consumption or release into the media by embryos, in comparison with media without embryos. In vitro-produced embryos were cultured in MU1 with 0 or 7.5 µM glucose N = 360, 4 replications. Both treatments consumed lactate, but there were no differences between treatments (6.8 ± 9.4 pmol/blastocyst/h v. 12.5 ± 1.6 pmol/blastocyst/h; P = 0.6). Blastocysts cultured in 7.5 µM glucose consumed pyruvate, whereas blastocysts without glucose produced pyruvate (–0.34 ± 0.3 pmol/blastocyst/h v. 0.73 ± 0.2 pmol/blastocyst/h; P < 0.01). It has been suggested that fructose is a more efficient replacement for glucose in pig embryo culture. Therefore, we produced pig embryos in vitro and cultured these embryos in MU1, MU1 + 2 mM glucose, or MU1 + 2 mM fructose to the blastocyst stage, 4 replications, N = 389. Again, there was a decrease in embryos that developed to the blastocyst stage in 2 mM glucose compared with MU1 control blastocysts (26% ± 5.8 v. 11% ± 2.5; P = 0.001), but there was only a trend for a decrease in development in response to 2 mM fructose (17 ± 2.3%; P = 0.06). There was no difference in total cell number between MU1, 2 mM glucose, and 2 mM fructose (30.6 ± 2.2, 30.5 ± 3.7, and 32.6 ± 3.0, respectively; P ≥ 0.9) 3 replications, N = 32. Because there is very little consumption of lactate and very low levels of pyruvate are being consumed when glucose is present, it does not appear that any of these energy substrates are major players for the developing pig embryo. Future experiments should be conducted to determine other means of energy production and metabolism in these embryos. The research was funded by Food for the 21st Century.

134 SERIAL TREATMENT OF RESVERATROL-TROLOX IMPROVED EMBRYONIC DEVELOPMENT OF PORCINE PARTHENOTES

Antioxidants are widely used for in vitro production of embryos due to their activity as reactive oxygen species scavengers. Among various antioxidants, resveratrol supplementation in in vitro-maturation (IVM) media and trolox supplementation in in vitro-culture (IVC) media improves oocyte maturation and embryonic development in other species, such as cattle and sheep. Limited information is available, however, on the effect of resveratrol and/or trolox on porcine embryos produced in vitro. In this study, we evaluated the effect of resveratrol supplemented to the media of IVM and trolox treatment during IVC on porcine parthenotes. We used TCM-199 as IVM media and porcine zygote medium (PZM)-5 as IVC media. For activation, matured oocytes after 44 h of IVM were electrically activated with 280 mM mannitol and cultured in IVC medium (PZM-5). Statistical analyses of all data were carried out using SPSS 17.0 (one-way ANOVA, followed by Duncan's multiple range test). In the experiment 1, a total of 618 oocytes were used in 4 independent replicates to evaluate the effect of 4 different concentrations (0, 1, 2, or 4 μM) of resveratrol during IVM on parthenotes. Oocytes treated with 2 μM resveratrol during IVM had significantly higher cleavage rates and blastocyst formation rates (73.0 and 34.4% v. 64.0 and 18.3%, respectively) than the control group. Experiment 2 involved supplementation with trolox (0 μM, 100 μM, 200 μM, 400 μM) to 957 parthenotes during IVC for 7 days (4 replicates). Cleavage rates significantly increased in the 100 μM group (75.6 v. 69.1%), and blastocyst formation rates in the 200 μM group were significantly higher compared to the control group (33.7 v. 23.8%). To determine the combined effects of resveratrol treatment during IVM and trolox treatment during IVC, in the experiment 3 we selected an optimized concentration (2 μM of resveratrol and 200 μM of trolox) from each experiment and evaluated the combined effects (3 times replicated). We designed 4 groups: (1) control, (2) resveratrol only (R), (3) trolox only (T), and (4) resveratrol-trolox (R-T). The R group and R-T group showed significantly higher cleavage rates than the control group (81.8 and 83.1% v. 72.3%). All treatment groups showed significantly increased blastocyst formation rates compared with the control group (39.2, 37.8, and 38.4% v. 23.7%). There is no significant difference in total cell numbers of blastocyst among the control, R, and T groups (47.8 v. 54.2 v. 54.7). However, the R-T group had significantly more cells than the control group (67.1 v. 47.8). Our results suggest that 2 μM resveratrol treatment during IVM, followed by 200 μM trolox treatment during IVC, improves developmental potential of the parthenotes. For a further study, we will apply this condition to somatic cell nuclear transfer, and we also will verify quantitative PCR analysis of apoptosis-related mRNA expression of PA and somatic cell nuclear transfer embryos. This study was supported by the MOTIE (#10033839), IPET (#311011-05-3-SB010), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Effects of flunixin meglumine and prostaglandin F2 α treatments on the development and quality of bovine embryos in vitro.

Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F2 -alpha (PGF2 α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC-II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF2 α (23.9%) and FM+PGF2 α groups (24.5%). The percentage of hatched blastocysts was also higher (p<0.05) in the FM group (41.2%) than in the control (27.8%) and PGF2 α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF2 α group (8.2±6.6) than in the control (4.7±3.2), FM (4.7±2.5) and FM+PGF2 α (4.9±3.4) groups. Detected by real-time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p<0.05) in the PGF2 α group. However, SSLP1 and PTGS2 gene expression in the FM+PGF2 α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF2 α group compared with other groups (p<0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF2 α.

Laser-assisted hatching of cleavage-stage embryos impairs developmental potential and increases DNA damage in blastocysts.

This study aims to investigate the influence of two- (day 2) and six-to-eight-cell-stage (day 3) laser-assisted hatchings on the developmental potential and genetic integrity of the embryos. In this prospective experimental study, two- and six-to-eight-cell-stage mouse embryos were subjected to laser hatching using 1,480 nm diode laser, and then assessed for the developmental potential and DNA integrity in blastocysts. Similarly, four-cell-stage human embryos from 20 patients were also subjected to laser hatching, and then assessed for the developmental competence. Laser-assisted hatching in mouse embryos significantly enhanced the blastocyst hatching potential on day 4.5 (P < 0.0001). However, a significant decline in blastocyst total cell number (TCN) was observed in six-to-eight-cell-stage laser-hatched embryos (P < 0.001). Conversely, no significant difference in TCN was observed between laser-hatched and unhatched human four-cell-stage embryos after 24 h. Attempt to understand the genetic integrity in laser-hatched mouse blastocysts revealed significantly higher labeling index when hatching was done at two- (P < 0.01) and six-to-eight-cell stage (P < 0.05). DNA damage induced by the laser manipulation may affect implantation and postimplantation developmental potential of the embryos. However, further studies are required to elucidate the impact of laser-induced DNA damage on the reproductive outcome.

Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos

The aim of the present study was to determine the effect of L-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification-warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without L-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without L-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with L-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with L-ascorbic acid. Thus, supplementing culture and/or vitrification media with L-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.

Development of large and small blastomeres from 2‐cell embryos produced in vitro in pigs

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P<0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P<0.05) than that of large blastomeres and blastomeres from evenly cleaved 2-cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2-cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.

Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level

There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of vitrified embryos to those frozen conventionally solely showed significant differences in the relative abundance of IFNT2. These results indicate that although not visible in gross morphology, but in the relative amount of gene transcripts, vitrification may be the more suitable method to cryopreserve in vitro produced bovine embryos cultured in SOF media.

Nicotinic acetylcholine receptor activation completely blocks osteoclastogenesis by interfering with osteoclast precursor differentiation in vitro

Background and objectiveIn the last few years, the discovery of various neurotransmitter receptors on bone cells suggested that the nervous system may participate in the control of bone metabolism. However...

Differential developmental requirements for individual histone H3K9 methyltransferases in cleavage-stage porcine embryos

Dimethylated H3K9 is a heritable epigenetic mark that is closely linked with transcriptional silencing and known to undergo global remodelling during cleavage development. Five mammalian histone methyltransferases (HMTases), namely Suv39H1, Suv39H2, SetDB1, EHMT1 and EHMT2, have been shown to mediate the methylation of H3K9. The aim of the present study was to determine the developmental requirements of these HMTases during cleavage development in porcine embryos. We hypothesised that knockdown of the abovementioned HMTases would differentially affect porcine cleavage development. To test this hypothesis, IVM and IVF porcine oocytes were divided into one of three treatment groups, including non-injected controls, oocytes injected with a double-stranded interfering RNA molecule specific for one of the HMTases and oocytes injected with a corresponding mutated (control) double-stranded RNA molecule. Nuclei were counted in all embryos 6 days after fertilisation. Although no significant difference in total cell number was detected in embryos injected with EHMT1 and EHMT2 interfering RNAs (compared with their respective control groups), embryos injected with interfering RNAs that targeted Suv39H1, Suv39H2 and SetDB1had significantly lower cell numbers than their respective control groups (P<0.05). This suggests that individual HMTases differentially affect in vitro developmental potential.

81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P &lt; 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P &lt; 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.