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.

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.

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.

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).

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.

37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 166
Author(s):  
S.-S. Kwak ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
S.-A. Cheong ◽  
E. Lee ◽  
...  
Keyword(s):  
Treatment Group ◽  
Embryonic Development ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Class Iii ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Significant Difference

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P < 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P < 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

253 EFFECTS OF ADDITION OF ALPHA-LINOLENIC ACID INTO MATURATION MEDIUM ON IN VITRO DEVELOPMENT AND EXPRESSION OF APOPTOSIS-RELATED GENES OF GOAT EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 216 ◽  
Author(s):  
A. Veshkini ◽  
M. Khajenabi ◽  
A. Mohammadi-Sangcheshmeh
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Apoptotic Cells ◽  
Total Cell Number ◽  
Beneficial Effects ◽  
Maturation Medium ◽  
And Control ◽  
P53 Genes

Fatty acids are important sources of energy for oocytes and embryos. In bovine, an increased level of rumen-inert fatty acids in the diet improved the developmental competence of oocytes to the blastocyst stage and also embryo quality. As described in our previous report, providing appropriate levels of α-linolenic acid (ALA) in maturation medium had beneficial effects on nuclear maturation and embryonic development in the goat. Considering these beneficial effects, here we have conducted experiments to evaluate whether addition of ALA to goat oocyte maturation medium can affect the quality of blastocyst and the transcription of apoptosis-related Bax, Bcl-2, and p53 genes. Ovaries were collected from goats, and cumulus-oocyte complexes (COC) were recovered by the slicing method. The COC were placed in maturation medium supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, oocytes from both the treatment (n = 113) and control (n = 104) groups were subjected to IVF followed by culture in CR1aa medium for 8 days under the conditions stated above. The cleavage and blastocyst rates were recorded at Days 3 and 8 of culture, respectively. To examine the effect of ALA on total cell number and apoptosis of the blastocyst cells, the blastocysts from 50 μM ALA-treated and control oocytes were stained with 4′,6-diamidino-2-phenylindole to count total cell number, and apoptotic cells in these blastocysts were detected with TUNEL assay. Blastocysts derived either from 50 μM ALA-treated oocytes or control oocytes were also evaluated for the expression of Bax, Bcl-2, and p53 genes. The cleavage and blastocyst rates were compared by chi-square analysis. Differentially expressed genes were analysed by 1-way ANOVA. A P-value of less than 0.05 was considered significant. Although cleavage rates after IVF were similar (P > 0.05) between 50 μM ALA-treated (68.1%) and control (55.8%) groups, 50 μM ALA-treated oocytes produced more (25.7%) blastocysts than the control group (13.5%; P < 0.05). Blastocysts derived from oocytes supplemented with 50 μM ALA not only had a greater (P < 0.05) total cell number (115.2), but also a lower (P < 0.05) number of apoptotic cells (3.1) compared with the control blastocysts (110.8 and 4.2, respectively). The relative transcript abundance of Bax and p53 was decreased (P < 0.05) in blastocysts that originated from the 50 μM ALA group compared with control blastocysts. Furthermore, there was an increased (P < 0.05) expression of Bcl-2 transcripts in blastocysts derived from the 50 μM ALA-treated oocytes compared with the control. In conclusion, our findings revealed that ALA-treated medium led to an improvement in blastocyst rate and quality as determined by greater total cell number, lower number of apoptotic cells, and altered expression of apoptosis-related genes.

133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 180 ◽  
Author(s):  
K. Syoji ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Cell Mass ◽  
Calf Serum ◽  
Total Cell ◽  
Differential Staining ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Maturation Medium ◽  
Significant Difference

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

70 Energy metabolites induce differential DNA methylation levels and transcription in trophectoderm and inner cell mass

2021 ◽  
Vol 33 (2) ◽  
pp. 142
Author(s):  
J. Ispada ◽  
C. B. de Lima ◽  
E. C. dos Santos ◽  
A. M. da Fonseca Junior ◽  
J. V. Alcantara da Silva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Significant Difference

DNA methylation/demethylation is one of several epigenetic mechanisms by which metabolism regulates gene expression. More specifically, α-ketoglutarate (αKG) and succinate (Suc) are tricarboxylic acid cycle metabolites that may decrease and increase, respectively, the activity of DNA demethylases. Because pre-implantation embryos undergo reprogramming in both DNA methylation and metabolic pathways, it is possible that metabolic changes influence this epigenetic mark. To test that hypothesis, bovine embryos were invitro produced by using standard protocols and, 8h after fertilization, zygotes were transferred to synthetic oviductal fluid (SOF)-based culture medium (control, CO) or culture medium containing 4mM dimethyl-αKG, or 4mM dimethyl-Suc, where they remained until Day 4. Embryos were collected at Day 4 or remained in culture until Day 7, in control medium. Day 4 embryos were evaluated for DNA methylation levels by immunofluorescence detection of 5-methylcytosine (5mC) and cleavage rate. Day 7 embryos were also assessed for DNA methylation by immunofluorescence of 5mC, total cell number, blastocyst rates, and quantification of ACTB (housekeeping), DNMT1, DNMT3A, and DNMT3B transcript by RT-qPCR in trophectoderm (TE) and inner cell mass (ICM) separated by immunosurgery. The mRNA expression levels of were normalized to internal control ACTB and subsequently calculated using the 2−ΔΔCT method, using the control group for comparisons. All data were submitted to outlier detection using ROUT with Q=1% followed by one-way analysis of variance (ANOVA) and Fisher’s least significant difference (l.s.d.) test in GraphPad Prism. αKG and Suc did not influence cleavage or blastocyst rates, total cell number, or cell allocation. αKG supplementation reduced 5mC fluorescence intensity in embryos assessed at Day 4 (CO: 12.8±0.4 AU; αKG: 9.0±0.2AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; αKG: 23.5±0.4 AU; P&lt;0.0001), whereas Suc incubation increased DNA methylation levels in embryos at Day 4 (CO: 12.8±0.4 AU; Suc: 15.7±0.3 AU; P&lt;0.0001) and Day 7 (CO: 36.5±0.7 AU; Suc: 70.5±0.5 AU; P&lt;0.0001). αKG increased expression of DNMT1 (P=0.0438) in the ICM and led to lower levels of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0013), and DNMT3B (P=0.0015) in TE cells. The culture with Suc increased DNMT1 (P=0.0074), DNMT3A (P=0.0186), and DNMT3B (P=0.0286) in ICM. Regarding TE, Suc resulted in lower expression of DNMT1 (P&lt;0.0001), DNMT3A (P=0.0017), and DNMT3B (P=0.0052). In conclusion, both supplementations resulted in global DNA methylation changes without affecting embryo development rates or morphology. These changes were accompanied by alterations in transcript profiles between ICM and TE, with differences among treatments being more pronounced in transcripts from ICM. This is the first report of DNA demethylation–induced changes by analogues of TCA cycle metabolites during early reprogramming of the bovine embryo with prolonged effects in TE and ICM cells. This research was funded by FAPESP: 2017/18384-0; 2018/11668-6.

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.

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