73 LIPID CONTENT AND CRYOTOLERANCE OF PORCINE EMBRYOS CULTURED WITH PHENAZINE ETHOSULFATE

2011 ◽  
Vol 23 (1) ◽  
pp. 142 ◽  
Author(s):  
B. Gajda ◽  
M. Romek ◽  
I. Grad ◽  
E. Krzysztofowicz ◽  
M. Bryla ◽  
...  
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Cell Number ◽  
Fluorescent Dye ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Phenazine Ethosulfate ◽  
Experimental Group

In this study, the addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on cytoplasmic lipid content in cultured pig embryo and survival after open pulled straw (OPS) vitrification (Vajta et al. 1997 Acta Vet. Scand. 38, 363–366). In addition, in cultured blastocysts, the total cell number per blastocyst and the degree of apoptosis were assessed. Porcine zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 0 (control, n = 146) or 0.05 μM PES (n = 150). To evaluate the lipid content in embryos, we employed Nile Red (NR), a fluorescent dye specific for intracellular lipids (Genicot et al. 2005 Theriogenology 63, 1181–1194). We measured the amount of fluorescence originating from NR using LSM 510 Meta Zeiss confocal microscope and ImageJ version 1.38x software (National Institutes of Health, Bethesda, MD, USA) and an Integrated Density (ID) parameter. The total amount of fluorescence per embryo (TF), proportional to the amount of lipids, was calculated as the sum of ID measured for all optical slices in each individual z-stack. Blastocysts that were cultured with (n = 48) or without PES (n = 34) were vitrified using OPS technology. Results were analysed using chi-square, Fisher, and Student’s t-tests. The total number of cells and the percentage of TUNEL-positive nuclei of PES-treated blastocysts were significantly different than for the control group (43.6 v. 37.6; P < 0.05 and 1.6 v. 2.9; P < 0.01, respectively). Blastocysts stained with Nile Red fluorescent dye showed intracellular lipid mainly localised to the lipid droplets. They were present both in the embryoblast and trophoblast cells. Mean values of TF estimated for the experimental group was lower by ∼23% than those of the control group. Thereby, blastocysts of the control group possess a higher content of lipids then those found in the experimental group cultured in medium with 0.05 μM PES (P < 0.001). The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (44.8 and 37.1%, respectively). These results showed that culturing porcine embryos in medium with phenazine ethosulfate supplementation increased the total cell number per blastocyst and reduced the index of DNA fragmentation of cultured blastocysts. Use of PES in porcine culture medium reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. However, the use of PES during in vitro culture had a limited effect on porcine blastocyst survival after vitrification. This study was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.

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

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

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

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

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

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

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

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

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

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.

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium

2014 ◽  
Vol 26 (4) ◽  
pp. 570 ◽  
Author(s):  
Eva Torner ◽  
Eva Bussalleu ◽  
M. Dolors Briz ◽  
Marc Yeste ◽  
Sergi Bonet
Keyword(s):  
Hyaluronic Acid ◽  
Sex Ratio ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Porcine Embryo ◽  
Preferential Loss

In the present study, the effects of replacing glucose with pyruvate–lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0 mg mL–1 HA, and with either 5.55 mM glucose (IVC-Glu) or pyruvate (0.17 mM)–lactate (2.73 mM) from 0 to 48 h post insemination (h.p.i.) and then with glucose from 48 to 168 h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7 ± 1.5%) than those cultured with IVC-Glu (14.27 ± 2.75%). At 1.0 mg mL–1, HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0 mg mL–1 HA and IVC-Glu (4.28 ± 0.28% vs 11.01 ± 1.42% and 10.14 ± 2.77%, respectively) and IVC-PL (14.37 ± 1.35% vs 20.96 ± 2.85% and 22.99 ± 1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0 mg mL–1 HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL.

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

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

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

97 SUPPLEMENTATION WITH FOLATE IN VITRO INCREASES TROPHECTODERM AND TOTAL CELL NUMBER IN IN VITRO-DERIVED PORCINE BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 161 ◽  
Author(s):  
B. K. Redel ◽  
L. D. Spate ◽  
A. N. Brown ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Transcriptional Profiling ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Total Cell Number ◽  
Culture Systems

It is vital that improvements are made to current culture environments because in vitro culture systems are suboptimal compared with in vivo. A previous transcriptional profiling endeavour conducted by Bauer et al. (2010 Biol. Reprod. 83, 791–798) identified hundreds of mRNA transcripts that were mis-expressed in porcine embryos fertilized in vivo and then cultured in vitro to Day 6 compared with in vivo Day-6 embryos. Enriched in the downregulated transcripts were 4 genes involved with the one carbon pool by folate KEGG pathway. This downregulation of genes involved with folate metabolism may illustrate an impaired folate homeostasis in embryos cultured in the current culture environment. The objective of this study was to determine the effects folate had on embryo development of in vitro fertilized embryos. Porcine cumulus–oocyte complexes were matured for 44 h in M199 supplemented with epidermal growth factor (EGF), FSH and LH. Oocytes with a visible polar body were selected and fertilized in modified tris buffered medium for 5 h and then placed into porcine zygote medium 3 with 0 mM, 0.2 mM, 0.4 mM and 0.8 mM folate to find the optimal concentration of folate. Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25-μL drops of respective culture medium and cultured to Day 6 in a water-saturated atmosphere of 5% CO2, 5% O2, 90% N2, at 38.5°C. To determine the effect folate had on development, the blastocyst rate for each treatment group was measured. Results were log-transformed and analysed by using PROC GLM in SAS (SAS Institute Inc., Cary, NC). A least-significant difference post-test comparison was completed to determine if significant differences existed between treatment groups. The percentage of cleaved embryos on Day 6 that developed to blastocyst was 56.2%, 55.9%, 66.9% and 61.8% (n = 133, 149, 135 and 135) in 0 mM, 0.2 mM folate, 0.4 mM folate and 0.8 mM, respectively. The 0.4 mM folate group tended (P = 0.07) to have a higher number of cleaved embryos that developed to the blastocyst stage. Consequently, this concentration was used for all further embryo culture experiments. Differential staining was completed to compare the number of trophectoderm and inner cell mass nuclei for embryos cultured in 0 mM or 0.4 mM folate concentrations. Staining revealed that embryos cultured with folate had an increase in number of trophectoderm (29.7 ± 1.5 vs 24.4 ± 1.4 cells; P = 0.0058) and total cell (36.9 ± 1.0 vs 31.7 ± 1.0; P = 0.0007) numbers compared with embryos cultured without folate. These results illustrate that the addition of folate to current culture medium doesn't hinder development to blastocyst and by increasing trophectoderm and total cell number may give rise to better-quality in vitro-derived embryos. It is evident that using transcriptional profiling can be a great method of identifying ways to improve embryo culture systems and, in this case, supplementing with folate. Funded by Food for the 21st Century.

152 Activation of Bovine Oocytes Using the Zinc Chelator TPEN

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

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

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