197 EFFECTS OF HYALURONAN ON OXYGEN CONSUMPTION AND ATP CONTENT IN PIG BLASTOCYSTS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 215
Author(s):  
K. Yoshioka ◽  
M. Yokoo ◽  
T. Ozawa ◽  
C. Suzuki ◽  
H. Abe ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Activity ◽  
Formation Rate ◽  
Defined Medium ◽  
Cell Number ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Atp Content ◽  
Cell Numbers

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization, and embryo development. We have found that exogenous HA improves cell proliferation of porcine embryos cultured in a chemically defined medium (Yoshioka et al. 2004 Reprod. Fertil. Dev. 16, 264–265). Moreover, mitochondrial maturation was clearly more advanced in blastocysts cultured with HA compared to those cultured without HA, as seen by transmission electron microscopy. In the present study, the effects of HA on oxygen consumption and ATP content of blastocysts, produced in a defined system which reflects metabolic activity, were investigated. Porcine immature oocytes were matured for 44 h in porcine oocyte medium (POM) and subsequently fertilized with frozen–thawed ejaculated semen in porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac4). Both POM and PGMtac4 were chemically defined media modified from porcine zygote medium (PZM)-5. After IVF, presumptive zygotes were cultured in PZM-5 containing HA (from the microorganism, Nacalai tesque, Kyoto, Japan) at concentrations of 0 [control], 10 [HA10], or 100 [HA100] �g mL-1 until 5 days after IVF. Blastocyst formation rate and total cell numbers/blastocyst at Day 5 were assessed. In addition, oxygen consumption and ATP content of single Day 5 blastocysts were measured. Blastocyst oxygen consumption was quantified using scanning electrochemical microscopy (HV-403; Research Institute for the Functional Peptides, Yamagata, Japan), and embryonic ATP content was determined using a commercial assay based on the luciferin-luciferase reaction (ATPlite; PerkinElmer, Groningen, The Netherlands). Data were statistically analyzed by ANOVA and Fisher's PLSD test. While the percentage of embryos that developed to the blastocyst stage [30.5% (63/206) to 31.7% (65/206)] did not differ among treatments, blastocyst cell number in the HA100 group [57.9 cells (n = 64)] was greater (P < 0.05) compared to those in the control [48.6 cells (n = 63)] or HA10 [50.0 cells (n = 65)] groups. Blastocyst oxygen consumption rate in the HA100 group [0.629 � 10-14 mol s-1 (n = 15)] was significantly higher than in the control [0.500 � 1-14 mol s-1 (n = 16)] or HA10 [0.464 � 10-14 mol s-1 (n = 14)] groups. ATP content/blastocyst did not differ among treatments [control: 0.645 pmol (n = 38), HA10: 0.727 pmol (n = 42), and HA100: 0.704 pmol (n = 43)]. It is concluded that HA affects the metabolic activity of pig blastocysts developed in a chemically defined medium, enhancing oxygen consumption and their total cell numbers, thus improving the quality of IVP blastocysts.

335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student's t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P < 0.05) after activation with VitE (78.1% and 16.3%, n = 80) than without VitE (66.7% and 11.0%, n = 60). Total cell numbers were also significantly higher (P < 0.05) in blastocysts after activation with VitE (143.0 ± 34.02, n = 21) than in those without VitE (127.63 ± 40.25, n = 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 ± 2.22) and those without VitE (6.76 ± 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

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).

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

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 using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

Replacement of glutamine with the dipeptide derivative alanyl-glutamine enhances in vitro maturation of porcine oocytes and development of embryos

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 286-289 ◽  
Author(s):  
Su Jin Kim ◽  
Ok Jae Koo ◽  
Dae Kee Kwon ◽  
Jung Taek Kang ◽  
Sol Ji Park ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Formation Rate ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Nuclear Maturation ◽  
Porcine Oocyte ◽  
Potent Factor ◽  
Dipeptide Derivative

SummaryThe presence of glutamine (Gln) in in vitro maturation (IVM) and in vitro culture (IVC) medium is a more potent factor for improving porcine oocyte and embryo development than other amino acids. However Gln is inherently unstable and spontaneously breaks down into ammonia, and therefore interferes with proper development. To avoid this adverse effect, Gln was replaced in the present study with its stable dipeptide derivative alanyl-glutamine (Ala-Gln) and the effects of this replacement on porcine IVM and IVC were evaluated. Replacement of Gln with Ala-Gln during IVM did not improve nuclear maturation, however numbers of early cleaved embryos were significantly increased after activation. Blastocyst formation rates were also significantly improved by using Ala-Gln during IVM. Replacement of Gln with Ala-Gln during IVC significantly increased total cell numbers in blastocysts. Blastocyst formation rate was also significantly higher when Ala-Gln was used in both IVM and IVC. In conclusion, the use of Ala-Gln rather than Gln gives better results for development in both porcine IVM and IVC.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

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

scholarly journals 41 EFFECT OF CELL TYPES AND PASSAGES ON DEVELOPMENT AND APOPTOSIS OF PORCINE CLONED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 170
Author(s):  
J.-G. Kim ◽  
Y.-S. Lee ◽  
S.-L. Lee ◽  
S.-A. Ock ◽  
C.-S. Park ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Cell Types ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Female Fetus ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

The purpose of this study was to improve the efficiency of somatic cell nuclear transfer (SCNT) in pigs by assessing the development, cell numbers and apoptosis when using different cell types as nuclear donors and different numbers of passages. Primary cultures of the donor cells, porcine fetal fibroblasts (pFFF) from a female fetus at ∼30 days of gestation and adult female ear skin cells (pAESC), were established in DMEM + 15% FCS. For nuclear donor, cells at different passages were cultured for 5 days until confluent. Cumulus-oocyte complexes were matured and fertilized in vitro as controls by the following methods (2000 Theriogenology 54, 787–797). Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion with two DC pulses (1.4 kV/cm, 50 μs) in 0.28 M mannitol containing 0.01 mM CaCl2 and MgCl2. Eggs were then cultured in NCSU23 + 1.9 mM 6-dimethylaminopurine for 3 h. SCNT and IVF embryos were cultured in NCSU23 for 54 h and subsequently in the same medium with 5.55 mM glucose for 90 h at 38.5°C in 5% CO2 in air. In Experiment 1, when the rates of development between IVF and SCNT embryos constituted with cells at 5–7 passages were compared, no significant (P < 0.05) differences were observed in the cleavage rates. The rates of blastocyst formation were significantly (P < 0.05) higher in IVF than in SCNT embryos with pFFF and pAESC (21% vs. 15% and 10%), but it did not differ between SCNT embryos. Total cell numbers in IVF blastocysts (35.4 ± 12) were significantly (P < 0.05) higher than in SCNT blastocysts with pFFF and pAESC (28.4 ± 8 and 26.2 ± 10, respectively). The apoptosis signal by TUNEL was initiated at Day 3 in IVF and SCNT embryos. Apoptosis rates in SCNT blastocysts with pFFF and pAESC (13.1 ± 2.5 and 16.6 ± 4.3, respectively) were significantly (P < 0.05) higher than in IVF embryos (3.6 ± 1.4). As the embryos developed, the rates of apoptosis were increased. On Day 6, the rates of apoptosis in IVF (4.8%) were significantly (P < 0.05) lower than those in SCNT embryos with pFFF (13.1%) and pAESC (16.6%). However, both total cell number and apoptosis in SCNT embryos with pFFF and pAESC revealed no significant differences. In Experiment 2, SCNT embryos with pFFF in different cell passages were compared for the development and apoptosis. No significant (P < 0.05) differences were observed in the cleavage rates of SCNT embryos among different cell passages. The rates of blastocyst formation were significantly (P < 0.05) higher in SCNT embryos with 5–7 passages than those with other numbers of passages (14% vs. 6–8%, respectively). Although total cell numbers of SCNT blastocysts did not differ among different cell passages, apoptosis rates were significantly (P < 0.05) higher when the number of cell passages was increased. These results suggest that fetal fibroblasts at 5–7 passages are ideal nuclear donor cells for obtaining high-quality porcine SCNT embryos. This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

90 OPEN PULLED STRAW VITRIFICATION OF IN VITRO PORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM

2011 ◽  
Vol 23 (1) ◽  
pp. 150
Author(s):  
J. Sanchez-Osorio ◽  
C. Cuello ◽  
J. Gomis ◽  
C. Maside ◽  
M. A. Gil ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Formation Rate ◽  
Calf Serum ◽  
Defined Medium ◽  
Basic Medium ◽  
Chemically Defined Medium ◽  
Cleavage Rate ◽  
Number Of Cells ◽  
Serum Components

The aim of this study was to design a chemically defined medium for the vitrification of in vitro produced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitro in NCSU-23 for 44 h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess in vitro fertilization (IVF) parameters (N = 200) or for 6 days (N = 600) in order to obtain blastocysts. For chemically delipidation, 10 μM forskolin was added to the culture medium on Day 5 of in vitro culture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n = 65), with 0.1% of polyvinyl alcohol (PVA; n = 64) or without additives (WA; n = 65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al. 2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitro for 24 h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitro culture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. The maturation, penetration, and monospermy rates were 98.5 ± 1.2%, 85.3 ± 3.6%, and 48.8 ± 5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0 ± 4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8 ± 1.4% and 37.3 ± 1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8 ± 5.9) PVA (40.6 ± 6.0), and WA (30.8 ± 5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4 ± 6.8 to 50.8 ± 8.3) among vitrification groups. In conclusion, in vitro derived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitro produced pig blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07).

153 ZINC INSUFFICIENCY DURING PORCINE OOCYTES IN VITRO MATURATION CAUSED MEIOTIC BLOCK AND DEVELOPMENTAL FAILURE

2014 ◽  
Vol 26 (1) ◽  
pp. 190
Author(s):  
E. Kim ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Treated Group ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Total Cell Number ◽  
Nuclear Maturation

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10 μM TPEN was added to the IVM medium for 0, 7, 15, or 22 h. After TPEN treatment, 10 μM Zn were supplemented on IVM medium except in the 0 h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9 ± 3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8 ± 3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0 ± 1.0 and 97.2 ± 1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3 ± 1.4, 42.6 ± 4.8, 2.6 ± 0.1, and 3.0 ± 1.6%; BL formation rate: 29.3 ± 2.8, 9.2 ± 1.5, 0, and 0% for 0, 7, 15, and 22 h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4 ± 4.5) was significantly higher than that in the 7-h-treated group (23.2 ± 1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10 μM TPEN treatment during 22 h of IVM; (3) 10 μM TPEN + 10 μM Zn treatment during 22 h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0 ± 1.2 and 92.7 ± 1.8%, BL formation rate: 42.0 ± 6.7 and 40.0 ± 7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes’ results (MII rate: 0.61 ± 0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 229-236 ◽  
Author(s):  
Zubing Cao ◽  
Liucai Sui ◽  
Yunsheng Li ◽  
Suofei Ji ◽  
Xiaorong Zhang ◽  
...  
Keyword(s):  
Early Development ◽  
Nuclear Transfer ◽  
Defined Medium ◽  
Cell Number ◽  
Early Embryo ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Total Cell Number ◽  
Parthenogenetic Activation ◽  
Blastocyst Rate

SummaryThe present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.

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