152 Activation of Bovine Oocytes Using the Zinc Chelator TPEN

2018 ◽  
Vol 30 (1) ◽  
pp. 216
Author(s):  
C. L. Timlin ◽  
K. Uh ◽  
V. R. G. Mercadante ◽  
K. Lee
Keyword(s):  
Polar Body ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Cell Counting ◽  
Control Group ◽  
Oocyte Activation ◽  
Total Cell Number ◽  
Zinc Chelator ◽  
Bovine Oocytes

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.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

34 Effect of polysaccharide from Flammulina velutipes on the vitrification of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 143
Author(s):  
Y. Ihara ◽  
K. Tatakura ◽  
Y. Wada ◽  
H. Kawahara ◽  
K. Yamanaka
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Flammulina Velutipes ◽  
Control Group ◽  
Cleavage Pattern ◽  
Total Cell Number ◽  
Bovine Oocytes

The developmental competence of oocytes after cryopreservation is compromised by the physical injury due to the ice crystallisation. Recent studies have reported that polysaccharide (xylomannan) derived from the mycelium and fruit body of the basidiomycete Flammulina velutipes inhibits the ice recrystallisation in the cryopreserved Chinese hamster ovary cells. In this study, we aimed to clarify the effect of xylomannan from Flammulina velutipes on the developmental competence of bovine vitrified oocytes. Bovine ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) were aspirated from follicles (2-6mm in diameter) using a 19-gauge needle attached to a syringe. The COCs were matured for 22h in tissue culture medium-199 supplemented with 5% fetal bovine serum (FBS), 0.02IUmL−1 FSH, and 10μgmL−1 gentamycin. After maturation, COCs were incubated in base solution (BS: 10% FBS-tissue culture medium-199, control group; n=149) or BS supplemented with 100μgmL−1 xylomannan (xylomannan group; n=175) for 1h before vitrification. All vitrification procedures were performed at room temperature. The COCs were equilibrated in BS with 3% ethylene glycol for 12min and then in vitrification solution (BS with 30% ethylene glycol, 1.0M sucrose) for 1min. The COCs were loaded on a Cryotop (Kitazato) and transferred into liquid nitrogen. The warming procedure was performed on a warm plate (42°C). The COCs were placed into BS supplemented with 0.5, 0.25, 0.125, and 0M sucrose for 5min each. After washing with IVF100 solution (Research Institute for the Functional Peptide), COCs were applied for IVF. The viability of putative zygotes was morphologically evaluated following IVF, and ones that survived were cultured in CR1aa supplemented with 5% FBS. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the 1-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The rates of cleavage and blastocyst formation were calculated on 2 and 8 days after culture, respectively. Total cell number and apoptosis of blastocysts were measured by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. All data were obtained from more than four replicates. Viability and invitro development data were analysed using the chi-squared test. Total cell number and apoptosis data were analysed by a Student's t-test. Although no significant differences in viability, cleavage pattern, and cleavage rate (85.8 vs. 80.3%, 17.2 vs. 14.8%, and 35.4 vs. 36.7%, respectively) were observed, the developmental rate to blastocysts in the xylomannan group was significantly higher than that in the control group (68.6 vs. 42.2%; P&lt;0.01). The present results suggest that co-incubation with xylomannan before vitrification is an effective method to improve the vitrification outcome in bovine oocytes.

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

187 Effect of cytokines during invitro maturation of bovine oocytes on the development potential of parthenogenetic embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 222
Author(s):  
G. N. Singina ◽  
E. N. Shedova ◽  
I. A. Polejaeva ◽  
T. E. Taradajnic
Keyword(s):  
Growth Factor ◽  
Polar Body ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Maturation Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The efficiency of assisted reproductive technologies is critically dependent on the quality of the oocytes used to produce the embryos. The aim of the present research was to study dose-dependent effects of three cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) individually on IVM in bovine oocytes and their consecutive development to blastocysts after artificial activation. Slaughterhouse-derived cumulus-oocyte complexes (COC) (n=2052 COC) were cultured for 22h in either standard maturation medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1porcine FSH, and 10μgmL−1 ovine LH; control] or maturation medium supplemented with different concentrations (5-160ngmL−1) of FGF2, LIF, and IGF1. After IVM, matured oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, cleavage and blastocyst rates were determined. In addition, obtained blastocysts were fixed with 4% paraformaldehyde, and the total cell number was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. The data from 4 to 6 replicates (77-140 oocytes per treatment) were analysed by ANOVA. After 22h of culture, the rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and ranged from 74.4 to 90.7%. No statistical differences were found in the cleavage rate between oocytes matured in cytokine-treated groups compared with the control. Cleavage rates for the LIF experimental groups was 63.5 to 77.2%, for the IGF1 experimental groups was 68.1 to 80.7%, and for the FGF experimental groups 63.7 to 77.0%. Optimal concentrations of LIF (5 and 20ngmL−1), and FGF2 (20 and 40ngmL−1) increased (P&lt;0.05) the blastocyst rate from 21.7±1.5 (control for LIF-treated groups) to 32.7±7.1 and 27.1±3.4 and from 19.6±1.8% (control for FGF-treated groups) to 29.8±1.9 and 31.1±2.1%, respectively. Furthermore, the addition of FGF2 in IVM medium (except at 5ngmL−1) led to an increase in the total cell number in embryos that developed to the blastocyst stage, whereas LIF did not have this effect. The maturation of COC in the presence of IGF1 had no effect on the yield of parthenogenetic blastocysts or on the total cell number in blastocysts compared with the control medium. However, the blastocyst rate was lower in groups with IGF1 at 40 to 80ngmL−1 compared with those with IGF1 at 5 to 20ngmL−1 (P&lt;0.05). Thus, both LIF and FGF2 (each individually) (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro, and FGF2 additionally can improve parthenogenetic blastocyst quality. This research was supported by RFBR (Project no. 18-29-07089).

The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 139-150 ◽  
Author(s):  
Inchul Choi ◽  
Jie Zhu ◽  
Keith H. S. Campbell
Keyword(s):  
Treatment Group ◽  
Combined Treatment ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Oocyte Activation ◽  
Total Cell Number

SummaryPoor embryo quality is a major problem that contributes to the failure of pregnancy in somatic cell nuclear transfer (SCNT). The aims of this study were to improve the quality of ovine SCNT embryos by modifying the conventional activation protocol with the addition of SrCl2. In order to achieve this objective we conducted a series of experiments with in vitro-matured oocytes to optimize conditions for oocyte activation with strontium, and subsequently applied the protocol to SCNT embryos. The results showed that in vitro-matured oocytes could be activated effectively by 10 mM SrCl2 + 5 mg/ml cytochalasin B (CB) for 5 h in the absence of Ca2+ and that the blastocyst rate on day 7 (33.2%) was similar to that in the control group (31.0%) (5 M calcium ionophore [IP] A23187 for 5 min and cultured in CB/cycloheximide [CHX] for 5 h; P > 0.05). In SCNT experiments, the total cell number/blastocyst (104.12 ± 6.86) in the IP + SrCl2/CB-treatment group was, however, significantly higher than that in the control group (81.07 ± 3.39; P < 0.05). Apoptotic index (12.29 ± 1.22%) was significantly lower than the control (17.60 ± 1.39%; P < 0.05) when a combination of IP and SrCl2/CB was applied to SCNT embryos. In addition, karyotyping of the SCNT embryos showed that the percentage of diploid blastocysts in the IP + SrCl2/CB-treatment group was slightly higher than that in the control (P > 0.05). We conclude that the modified activation protocol with IP + SrCl2/CB can improve significantly the quality of ovine SCNT embryos in terms of total cell number, apoptosis and ploidy.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

95 DEVELOPMENT OF PORCINE TWO-CELL EMBRYOS WITH OR WITHOUT ZONA PELLUCIDA AND SINGLE BLASTOMERES

2009 ◽  
Vol 21 (1) ◽  
pp. 148
Author(s):  
D. N. Q. Thanh ◽  
K. Matsukawa ◽  
M. Kaneda ◽  
S. Akagi ◽  
Y. Kanai ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Monozygotic Twins ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
First Polar Body ◽  
Blastocyst Quality ◽  
Single Blastomeres

In the mouse, single blastomeres of the 2-cell embryos can develop into adult mice and occasionally both separated blastomeres can give rise to twin animals (reviewed by Tarkowski AK et al. 2001 Int. J. Dev. Biol. 45, 591–596). As a preliminary study for production of monozygotic twins from porcine 2-cell embryos, we investigated the effects of removal of zona pellucida and blastomere isolation at the 2-cell stage on subsequent development of parthenogenetic embryos. Oocytes with the first polar body were parthenogenetically activated after 44 h of in vitro maturation. Stimulated oocytes were then incubated in IVC-PyrLac (IVC medium with pyruvate and lactose) according to the method reported by Kikuchi K et al. (2002 Biol. Reprod. 66, 1033–1041). After 24 to 30 h of parthenogenetic activation, equally cleaved 2-cell embryos were selected and used for the experiments. Some 2-cell embryos were then treated with pronase to remove the zona pellucida and cultured individually as zona-free 2-cell embryos having 2 blastomeres in pair (ZF group), and single blastomeres were split from ZF group and cultured separately (SB group) in V-shaped microwells. In addition, intact 2-cell embryos were cultured individually without pronase treatment as a control group. After 24 h of in vitro culture, IVC-PyrLac was replaced by IVC-Glu (IVC with glucose). The blastocyst rates on Day 6 (Day 0 was defined as the day of electrical stimulation) in control, ZF, and SB groups did not differ (47.6, 50.0, and 42.1%, respectively). Nevertheless, blastocysts derived from the ZF (28.6 ± 3.0) and SB groups (25.9 ± 1.3) had a significantly lower total cell number than that of the control group (41.7 ± 3.2; P < 0.01 by ANOVA). Although the total cell number of blastocysts originating from single blastomeres was significantly lower than that in the intact embryos, the blastocyst formation rates were not different between them. This indicated the possibility of production of monozygotic twins from porcine 2-cell embryos divided into 2 single blastomeres. However, further research is needed to improve blastocyst quality descended from single blastomeres. In conclusion, the removal of the zona pellucida had a negative influence on blastocyst quality but did not affect the development of porcine embryos to the blastocyst stage.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

158 EFFECTS OF AMINO ACIDS IN EMBRYO TRANSPORT MEDIA ON PORCINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 227
Author(s):  
E. J. Park ◽  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
G. A. Kim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Number ◽  
Total Cell ◽  
Cell Counting ◽  
Buffer System ◽  
Total Cell Number ◽  
Transport Medium ◽  
Significant Difference ◽  
Embryo Transport ◽  
Parthenogenetic Embryos

Due to the distance from the laboratory to the recipient farm, several laboratories, including ours, carry somatic cell nuclear transfer (SCNT)-derived porcine embryos to the farm using a portable incubator for a few hours. If the embryos are nourished well during the transport, viability of embryos might be increased and cloning efficiency can be improved. TALP, which is widely used as a porcine embryo transport medium, lacks amino acids (AA). Proper supply of AA in the uterus is important for the development of pre-implantation embryos because AA have functions as osmolytes, metabolic regulators, or substrates and buffers of intracellular pH. Thus, supplementation of AA could affect the embryonic viability during the transport of SCNT-derived porcine embryos. The aim of this study is to determine whether the transport medium containing AAs affects the in vitro development of parthenogenetic embryos compared to TALP. Porcine zygote medium-5 (PZM-5) was chosen as transport medium containing AA due to its similarity in constituents with TALP except for the AA. Because PZM-5 contains sodium bicarbonate as a buffer system which can not cover wide variation of pH, 10 mM HEPES was added into PZM-5 (PZM+H) as it was normally done with TALP. Porcine cumulus–oocyte complexes (COC) were collected from ovaries of slaughtered pigs and cultured for 44 h using a two-step culture protocol. After denuded, matured oocytes were activated by thimerosal for 10 min followed by dithiothreitol for 30 min. The parthenogenetic embryos were cultured in PZM-5 for 2 days, monitored for cleavage, and loaded in a straw with TALP or PZM+H, respectively. Embryos were stored in a portable incubator (MTG, Bruckberg, Germany; no CO2) at 37°C for three hours and moved to PZM-5 drop for additional 5 days culture. The development was monitored on Day 7 after activation and blastocysts (BL) were collected for total cell number counts and RNA extraction. Ten BL from the TALP group and 11 BL from the PZM+H group were stained with 10 µg mL–1 bisbenzimide (Hoechst 33342) and were visualized for cell counting under fluorescence microscopy. Messenger RNA was extracted from 7 BL of the TALP and PZM+H groups and cDNA were synthesized. Quantitative real-time PCR were done to detect expression levels of apoptosis-related genes using the cDNA. The Bax/Bcl2 ratio was investigated as expression level of apoptosis-related genes and GAPDH was used as control. Each experiment was repeated at least 3 times. Data were analyzed by paired Student’s t-test using Graphpad Prism (version 5, Graphpad Software Inc., La Jolla, CA, USA). No difference was observed between the TALP and PZM+H groups with respect to blastocyst formation rate (22.46 ± 1.47% and 23.17 ± 2.13%, respectively) and total cell number (32.9 ± 2.22 and 37.09 ± 2.18, respectively). There was no significant difference between groups in the Bax/Bcl2 ratio. The use of PZM-5 media, which contains AA, did not affect the development and apoptosis of parthenogenetic embryos. This study was supported by MKE (#10033839-2012-21), IPET (#311011-05-1-SB010), the Research Institute for Veterinary Science, and TS Corporation.

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