scholarly journals 260 REPLACEMENT OF PVA WITH FETAL BOVINE SERUM IMPROVES FORMATION AND HATCHING OF PORCINE BLASTOCYSTS PRODUCED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 280 ◽  
Author(s):  
K. Yoshioka ◽  
C. Suzuki ◽  
H. Rodriguez-Martinez
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Significant Difference ◽  
Fetal Bovine ◽  
Hatching Rates

Porcine embryos, derived from in vitro maturation and fertilization, were used to investigate the effects of timing of serum inclusion and PVA replacement in the medium for in vitro culture (IVC) on rates of blastocyst formation and hatching. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) or cleaved embryos obtained by culture in porcine zygote medium (PZM-5) containing 3 mg mL−1 polyvinyl alcohol (PVA) at 48 or 96 hpi were further cultured in either PZM-5 containing PVA or PZM-5 where PVA was replaced by 1%, 5%, or 10% fetal bovine serum (FBS) until Day 6 (Day 0 = the day of in vitro insemination). Supplementation with 1% to 10% FBS at 20 and 48 hpi reduced (P < 0.05; by ANOVA and Fisher's PLSD test) blastocyst rates on Days 5 (0% to 1%) and 6 (3% to 6%) compared with PVA supplementation (4% and 22%, respectively). However, addition of 10% FBS at 96 hpi increased (P < 0.05) blastocyst rates (30%) on Day 5 compared with PVA (11%) and 1% FBS (15%); there was no significant difference among treatments in rates of blastocyst formation on Day 6 (24% to 40%). The total number of blastomeres in Day 6 blastocysts did not differ among treatments at any timing of serum supplementation (26.5 to 48.3 cells). In Experiment 2, presumptive zygotes were cultured from 20 to 96 hpi in PVA medium, and the cleaved embryos were later transferred into PZM-5 containing PVA, or 1%, 5%, or 10% FBS for another 4 days. Hatching rates of embryos on Days 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (15% and 20%, respectively) than those in PZM-5 containing PVA (1% and 5%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (135.1 cells) than that in PVA medium (77.0 cells). In Experiment 3, at 130 hpi, blastocysts derived from IVC with PZM-5 containing PVA were transferred into PZM-5 containing PVA, 3 mg mL−1 bovine serum albumin (BSA) or 10% FBS for another 2 days. Hatching rates of blastocysts on Days 6, 7 and 8 were significantly higher (P < 0.05) in PZM-5 where PVA was replaced with 10% FBS (12%, 56%, and 64%, respectively) than those in PZM-5 containing PVA (0%, 12%, and 20%, respectively) and BSA (0%, 12%, and 20%, respectively). Moreover, the total cell number in hatching/hatched blastocysts on Day 8 were significantly greater (P < 0.05) in medium containing 10% FBS (138.7 cells) than that in PVA (71.7 cells) and BSA medium (70.7 cells). The results indicate that the timing of serum inclusion in the culture medium markedly affects porcine embryo development in vitro and that replacement of PVA with FBS in PZM-5 at 96 hpi or later improves the subsequent development of embryos to the hatching/hatched blastocyst stage. This work was supported by MAFF, Japan, and STINT and FORMAS, Sweden.

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Z. B. Cao ◽  
L. C. Sui ◽  
S. F. Ji ◽  
J. W. Chen ◽  
T. Gui ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Total Cell ◽  
High Oxygen ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Nuclear Maturation ◽  
Air Atmosphere ◽  
Significant Difference

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P < 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

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.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

123 EFFECTS OF TAURO URSODEOXYCHOLIC ACID ON DEVELOPMENT OF BOVINE EMBRYOS FROM IN VITRO FERTILIZATION

2015 ◽  
Vol 27 (1) ◽  
pp. 153 ◽  
Author(s):  
F. Lu ◽  
Z. Li ◽  
Z. Ruan ◽  
X. Liu ◽  
S. Du ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Fetal Bovine Serum ◽  
Cell Number ◽  
Control Group ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Qrt Pcr ◽  
Apoptosis Index ◽  
Fetal Bovine

Endoplasmic reticulum stress (ERS) is a novel apoptotic pathway and plays an important role for embryonic development. Tauro ursodeoxycholic acid (TUDCA) is a specific chemical chaperone that can inhibit ERS. In this study, we investigated the effects of TUDCA on the development and mRNA expression of ERS-related genes in bovine embryos from IVF in order to improve the efficiency of embryo in vitro culture. Bovine oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% fetal bovine serum) for 24 h and fertilized in vitro with bovine sperm. After fertilization, the embryos were respectively placed into the medium (TCM-199 + 3% fetal bovine serum) containing different concentrations of TUDCA (0, 100, 250, 500, 1000 μmol L–1) and cultured in the 5% CO2 at 38.5°C. Blastocyst development was evaluated after 7 days of culture, and then the total cell number and apoptosis index of blastocysts were detected with TUNEL. In addition, X-box binding protein 1 (XBP-1) of embryos at 2-cell, 4-cell, morula, and blastocyst stages was detected with RT-PCR, and the change of the mRNA expression of ERS-related (Grp78, Ire1, Chop) and apoptosis-related (Bax, Bcl-2) genes in blastocyst collected at 7 days of culture were analysed by QRT-PCR. A total of 1336 oocytes were used in this study, and each experimental group comprised 6 replicates. The results revealed that the splicing of XBP-1 was present during the development of bovine embryos, and especially obvious at the 4-cell, morula, and blastocyst stages. When embryos were cultured in medium with different concentrations of TUDCA, compared with the control group (0 μmol L–1), more embryos developed to blastocyst stage with 500 μmol L–1 TUDCA (31.86 ± 7.32% v. 21.11 ± 8.05%; P < 0.05), but the cleavage rate was not significantly different among groups (P > 0.05). The result for TUNEL found that when adding 500 μmol L–1 TUDCA to culture, the bovine embryos significantly improved the total cell number of blastocysts (110. ± 15.21 v. 102.3 ± 8.62; P < 0.05), and the apoptosis index of blastocysts was markedly decreased (3.71 ± 0.91 v. 5.36 ± 1.92; P < 0.05) relative to the control group. Moreover, the result of QRT-PCR analysis showed that treating embryos with 500 μmol L–1 TUDCA significantly reduced the mRNA expression level of Ire1 and Chop genes (P < 0.05) and up-regulated the expression of anti-apoptotic Bcl-2 gene (P < 0.05), while down-regulated the expression of pro-apoptotic Bax gene (P < 0.05). Furthermore, XBP-1 splicing in blastocysts also abated after embryos were treated with 500 μmol L–1 TUDCA. In conclusion, ERS occurs in bovine embryos during in vitro culture, but treating embryos with 500 μmol L–1 TUDCA may reduce ERS to facilitate embryonic development. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2011GXNSFA018084, 2012GXNSFFA060004).

83 IN LOW OXYGEN CULTURE, IS HYPOTAURINE NECESSARY FOR IN VITRO DEVELOPMENT OF PORCINE EMBRYOS?

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
J. A. Benne ◽  
L. D. Spate ◽  
B. M. Elliott ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Free Radical Scavengers ◽  
Total Cell Number ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

For decades it has been known that reactive oxidative species (ROS) form during in vitro embryo culture. A buildup of ROS can be detrimental to individual cells in the embryo and lead to a decrease in development and quality. To overcome oxidative stress in culture systems, additives, such as taurine and/or hypotaurine, have been used. In the pig, taurine or hypotaurine addition is deemed necessary for normal in vitro development. Another commonly used technique to reduce ROS is to culture embryos in a lowered oxygen environment (e.g. 5%). Porcine zygote medium 3 (PZM3) base culture medium is used in the following experiments and contains 5 mM hypotaurine, which is one of the most costly additives in the medium. The objective of this experiment was to determine if hypotaurine is still necessary if the embryos were cultured in 5% O2 from the zygote to the Day 6 blastocyst stage. In Experiment 1, oocytes were matured for 44 h and fertilized in vitro. After fertilization, presumptive zygotes were then transferred to 500 µL of MU-1 medium (PZM3 with 1.69 mM arginine) that either contained or did not contain hypotaurine for overnight culture at 20% O2. On Day 1, the same embryo culture plates were moved to 5% O2, 5% CO2, and 90% N2 and cultured to Day 6. The percent blastocyst stage was determined, and total cell number was counted in 3 of the 5 replicates in order to give us an indication of the embryo quality. The percent blastocyst in the controls (+hypotaurine) was 34.4% ± 2.8 and not different from the no hypotaurine (32.9% ± 2.2; N = 830; 5 replications; P > 0.10). Furthermore, total cell number was not different between the two groups (30.8 ± 1.5 v. 33.6 ± 1.8, respectively, N = 146; 3 replications; P > 0.10). In Experiment 2, the same experiment was repeated in somatic cell nuclear transfer derived embryos, which may be more sensitive to ROS due to the micromanipulation procedure. Wild type fetal fibroblast cells were used as donor cells. There was no significant difference in development to the blastocyst stage due to the presence or absence of hypotaurine (17.7% ± 2.5 v. 11.8% ± 2.3, respectively; N = 454; 4 replications; P = 0.07). All blastocyst data were analysed using the GENMOD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and cell number data were analysed using the PROC GLM also with SAS 9.4. These data show that porcine embryos can be efficiently cultured to the blastocyst stage without adding any oxygen free radical scavengers to the media when culturing in reduced oxygen atmosphere. Further studies include evaluating term development via embryo transfers and measuring ROS production of these embryos. Funding was provided by Food for the 21st Century and the National Institutes of Health (U42 OD011140).

scholarly journals 141 BENEFICIAL EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID COMBINED WITH HEMOGLOBIN ON PRE-IMPLANTATION DEVELOPMENT OF PORCINE IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 221
Author(s):  
J.H. Kim ◽  
G.S. Lee ◽  
H.S. Kim ◽  
S.H. Lee ◽  
D.H. Nam ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Embryo Development ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Porcine Embryo

Developing a porcine embryo culture system is important for increasing the rates of implantation and pregnancy of somatic cell nuclear transfer (SCNT) embryos. Ethylenediaminetetraacetic acid (EDTA) was shown to inhibit glycolytic activity of cleavage stage embryos, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for later-stage embryos as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development. On the other hand, addition of a nitric oxide (NO) scavenger, hemoglobin (Hb), to the culture medium is known to promote embryo development to the blastocyst stage. This study was conducted to evaluate the beneficial effect of EDTA combined with Hb on pre-implantation development of porcine embryos in vitro. Porcine embryos produced by in vitro maturation and fertilization were cultured for 6 days in North Carolina State University (NCSU)-23 medium supplemented with EDTA or/and Hb. All data were subjected to one-way ANOVA and protected least significant difference (LSD) test using the general linear models (GLM) procedure of the statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Statistical significance was determined when the P value was less than 0.05. In Exp. 1, culturing porcine zygotes with 100 mM EDTA (n = 537) significantly increased cleavage rates (85.3%) at 48 h post-insemination compared to supplementing with 0, 1, or 10 mM EDTA (78.9, 79.7, or 78.2%, respectively). However, EDTA at these concentrations did not promote blastocyst formation compared to the control. In addition, no difference was observed in total cell numbers in blastocysts among the experimental groups (41.8, 42.6, 45.8, 44.5, respectively). In Exp. 2, in vitro-fertilized oocytes were cultured with 0, 1, or 10 mg/mL Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the total cell number of blastocysts obtained from 1 mg/mL Hb supplementation (n = 566) compared to that of the control (56.8 vs. 41.6). In Exp. 3, culturing embryos (n = 548) with 100 mM EDTA + 1 mg/mL Hb significantly improved rates of cleavage (84.0% vs. 75.2%) and blastocyst formation (19.2% vs. 12.7%), and the total number of cells in blastocysts compared to those of the control (58.4 vs. 42.3). In conclusion, our results demonstrated that EDTA or Hb have different roles in supporting in vitro pre-implantation development of porcine embryos; EDTA mainly stimulated early cleavage up to the 2- to 4-cell stage, and Hb promoted the total cell number of blastocysts. However, combined supplementation with these two chemicals improved cleavage, blastocyst formation, and total cell number in blastocysts. This study was supported by a grant from Korea Ministry of Science and Technology (Biodiscovery).

158 ASTAXANTHIN EFFECTS ON MATURATION, FERTILIZATION, AND DEVELOPMENT OF PORCINE OOCYTES CULTURED UNDER HEAT STRESS

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
L. T. K. Do ◽  
V. V. Luu ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Heat Stress ◽  
Dna Fragmentation ◽  
Cell Number ◽  
Total Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Maturation Medium ◽  
Significant Difference

Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.

96 GRANULOCYTE–MACROPHAGE COLONY-STIMULATING FACTOR INCREASES DEVELOPMENTAL POTENTIAL OF PORCINE EMBRYOS IN VITRO

2012 ◽  
Vol 24 (1) ◽  
pp. 160
Author(s):  
K. Lee ◽  
J. Teson ◽  
L. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

There have been significant improvements in the culture of porcine embryos in vitro; however, it is still suboptimal. Improvements in porcine embryo culture would benefit utilisation of porcine embryos for a variety of purposes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be expressed in the female reproductive tract and the level of its expression is high between conception and implantation. Previous studies show supplementing GM-CSF in embryo culture promotes embryonic development in human and bovine embryos. The aim of this study was to investigate the effect of GM-CSF on the culture of porcine embryos derived from somatic cell nuclear transfer (SCNT) and IVF. Different concentrations of recombinant porcine GM-CSF (0, 2, 10 ng mL–1) were introduced into Porcine Zygote Medium 3 from Day 1 to 6. Frequencies of cleaved embryos and blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell number in blastocysts from each group were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. A total of 563 SCNT embryos from 6 different donor cell lines on 11 different days were produced for the study. Incubation of SCNT embryos with GM-CSF did not affect the frequency of cleaved embryos. Frequencies of cleaved embryos in control (0 ng mL–1), 2 ng mL–1 GM-CSF and 10 ng mL–1 GM-CSF were 64.2%, 68.1% and 65.0%, respectively. Interestingly, both concentrations of GM-CSF significantly increased the frequency of blastocyst formation as compared with the control. In 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, 30.8% and 32.3% of embryos reached blastocyst respectively, whereas only 22.4% of embryos reached blastocyst in the control group. A significant increase in total cell number in blastocysts was observed when GM-CSF was introduced into embryo culture. An average of 28.8 ± 0.9 cells was recorded in the control group, whereas 31.9 ± 1.1 and 31.8 ± 1.1 were observed in 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, respectively. Similar effects were observed when GM-CSF was introduced to the culture of IVF embryos. For IVF study, 525 embryos were generated on 10 different days and embryos cultured in the presence of GM-CSF tended to show higher blastocyst formation (P = 0.1). Frequencies of blastocyst per cleaved in the 3 groups were 55.7% (control), 65.7% (2 ng mL–1 GM-CSF) and 66.7% (10 ng mL–1 GM-CSF). In addition, culture of IVF embryos with GM-CSF significantly increased total cell number in Day 6 blastocysts. Total cell number in blastocysts in 2 ng mL–1 GM-CSF (34.2 ± 0.8) and 10 ng mL–1 GM-CSF (34.4 ± 1.2) were significantly higher compared with control (27.3 ± 1.2). Our results indicate that introducing GM-CSF into embryo culture media can increase the quality of blastocyst stage embryos. An increase in the frequency of blastocyst formation and total cell number in blastocysts suggests that GM-CSF can be used to produce better-quality embryos in vitro. Currently, effects of GM-CSF on implantation of SCNT embryos are under investigation. Further studies would elucidate the specific mechanism of GM-CSF on porcine embryos.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 222
Author(s):  
S. H. Lee ◽  
E. M. N. Setyawan ◽  
B. C. Lee
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Significant Difference ◽  
Epidermal Growth ◽  
Oviduct Cell

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

213 MATURATION OF OOCYTES WITH FOLLICULAR FLUID FROM GILTS CONSUMING HIGH FAT AND FRUCTOSE AFFECTS SUBSEQUENT EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
R. Poole ◽  
V. McCracken ◽  
M. Rhoads ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Follicular Fluid ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
High Fat ◽  
Total Cell Number ◽  
Treatment Groups ◽  
High Fructose

Infertility among women has become a growing issue in the world requiring a significant number to seek treatment by means of assisted reproductive technologies. One suggested reason for the fertility issue, which is known to specifically affect oocyte quality, is the modern diet. Previously, we have demonstrated that feeding a high-fructose diet to gilts led to poor reproductive tract characteristics and infertility. In this study, pre-pubescent gilts were fed either a high-fructose; high-fat diet (HFHF), with 15% beef tallow and 35% fructose; or an industry control diet (IND). Porcine follicular fluid (pFF) collected from these gilts was introduced into in vitro maturation systems to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. Follicles from ovaries, collected at a local abattoir, were aspirated by an 18 G needle attached to a 10-mL sterile syringe. Then selected cumulus‐oocyte complexes were maturated in vitro in a TCM-199 maturation media with cysteine, glucose, sodium pyruvate, epidermal growth factor (EGF), FSH, LH, and 20% pFF from treatment groups. Additionally, another group of oocytes, labelled follicle fluid free (FFF), were maturated in TCM-199 media without pFF. Three replicate experiments were conducted using a total of 365 oocytes, 124 FFF, 121 IND, and 120 HFHF. Oocytes were denuded by exposure to 0.1% hyaluronidase and oocytes that reached metaphase II (MII) were selected for in vitro fertilisation. After 5 h of co-incubation in modified Tween medium B with milk powder (mTBM)-based IVF media, presumable zygotes were transferred to porcine zygote medium-3 (PZM-3). Blastocyst frequency was recorded on Days 5 and 6. Day 6 blastocysts were stained with Hoechst for total cell number evaluation. The frequencies of blastocyst formation among the treatment groups were compared by a chi-squared test, and total cell numbers were compared by Student's t-test. Statistical significance was defined by P < 0.05. The frequency of oocytes reaching metaphase II (MII) were observed as 77.4% FFF, 72.7% IND, and 71.7% HFHF (P > 0.05), indicating the supplementation of pFF did not affect maturation. Day 5 blastocysts were observed at frequencies of 8.3% FFF, 6.8% IND, and 4.7% HFHF and did not differ. However, frequency of Day 6 blastocysts from HFHF group was tended to be lower compared with that of other groups; 12.5% FFF, 11.4% IND, and 4.7% HFHF (P = 0.06 and P = 0.1). Average total cell number of Day 6 blastocysts observed were 41.0 ± 9.1 FFF, 36.0 ± 8.9 IND, and 48.3 ± 10.6 HFHF. The total cell number from HFHF group tended to be higher than only that of IND group (P = 0.07). Based on these results, we concluded that the follicular fluid of females consuming HFHF diets did not have impact on nuclear maturation of oocytes but might affect oocyte competency, thus resulting in detrimental effects on subsequent development of embryos, especially blastocyst formation. Further studies will help us identify more specific effects of nutrition on oogenesis and subsequent embryo development.

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