147 QUALITY OF PORCINE EMBRYOS CULTURED WITH HYALURONAN

2010 ◽  
Vol 22 (1) ◽  
pp. 232
Author(s):  
B. Gajda ◽  
I. Grad ◽  
E. van der Tuin ◽  
Z. Smorag
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Apoptotic Index ◽  
Total Cell ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Group 2 ◽  
Group 1

Hyaluronan (HA) is a high molecular weight polysaccharide found in the mammalian follicular, oviduct, and uterine fluids. When HA is added in maturation and culture media, it improves the developmental potential of bovine (Stojkovic M et al. 2002 Reproduction 124, 141-153; Palasz AT et al. 2008 Zygote 16, 39-47), and porcine oocytes (Sato E et al. 1990 Mol. Reprod. Dev. 26, 391-397) and embryos (Miyano T et al. 1994 Theriogenology 41, 1299-1305). Physiological concentration of HA in follicular, oviductal, and uterine fluids of pigs range from 0.04 to 1.83 mg mL-1 (Kano K et al. 1998 Biol. Reprod. 58, 1226-1232). The aim of the present study was to investigate the effect of different concentrations of HA on the development and quality of cultured porcine embryos. Zygotes from superovulated pigs were cultured in vitro in NCSU-23 medium supplemented with BSA and 0 mg mL-1 (control group), 0.25 mg mL-1 (Exp. Group 1), and 0.5 mg mL-1 (Exp. Group 2) of HA (Animal Pharma BV). Experiments were replicated 3 times with 30 to 40 embryos per each treatment group. Embryos were cultured up to the blastocyst stage at 39°C in an atmosphere of 5% CO2 in air, in 4-well plastic dishes, which contained approximately 0.8 mL of the NCSU-23 medium. Embryo quality criteria were cleavage (on Day 2 after in vitro culture), morula (on Day 4) and blastocyst (on Days 6 to 8) rates, total cell number per blastocyst, and degree of apoptosis (on Day 7) assessed by TUNEL method. Results were analyzed by ANOVA test. There was no difference in percentage of cleaved embryos between control and treated Group 1 and 2.The proportion of embryos developed to the morula and blastocyst stage was 80.0 and 60.0% for Group 1 (0.25 mg of HA), 73.7 and 44.7% for Group 2 (0.5 mg of HA), and 73.4 and 46.7% for control, respectively (difference NS). Supplementation with HA did not increase the cell number of the blastocysts but significantly reduced number of apoptotic nuclei from 2.0 for control to 0.7 (P < 0.01) and 0.6 (P < 0.01) for Group 1 and 2, respectively, and apoptotic index from 9.70 for control to 3.01 (P < 0.05) and 1.95 (P < 0.05) for Group 1 and 2, respectively. These results indicate that supplementation of culture medium NCSU-23 with HA improves the quality (assessed by apoptotic index) of pig embryos but does not increase the total cell number in pig blastocysts as reported by Kim HS et al. 2005 (Theriogenology 63, 1167-1180). However, further research to test the HA’s effect on cryopreservation of in vitro and in vivo produced pig embryos are needed.

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.

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.

Cryotolerance of porcine in vitro-produced blastocysts relies on blastocyst stage and length of in vitro culture prior to vitrification

2016 ◽  
Vol 28 (7) ◽  
pp. 886 ◽  
Author(s):  
Roser Morató ◽  
Míriam Castillo-Martín ◽  
Marc Yeste ◽  
Sergi Bonet
Keyword(s):  
In Vitro Culture ◽  
Embryo Quality ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Apoptotic Cells ◽  
Total Cell Number ◽  
Developmental Potential ◽  
High Degree

The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24 h. Re-expansion rates were recorded at 3 and 24 h and total cell number and apoptotic cells were determined at 24 h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification–warming procedures and length of in vitro culture, as expanding and hatching–hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.

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.

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.

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

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

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

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
G. Singina ◽  
E. Shedova ◽  
T. Taradajnic ◽  
V. Konnova ◽  
E. Tsyndrina
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Terminal Phase ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
One Step ◽  
Group 2 ◽  
The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

109 INTRAFOLLICULAR GLUCOSE CONCENTRATION HAS AN INFLUENCE ON THE SEX OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 160
Author(s):  
E. Abele ◽  
H. Stinshoff ◽  
A. Hanstedt ◽  
S. Wilkening ◽  
S. Meinecke-Tillmann ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Follicular Fluid ◽  
Glucose Concentration ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Bovine Embryos ◽  
Developmental Rates ◽  
Group 2 ◽  
Group 1

Several factors have been shown to alter the sex ratio of bovine embryos generated in vitro, i.e. the maturity of the oocyte at the time of insemination, the duration of sperm-oocyte co-incubation and the culture conditions after in vitro fertilization. It has been shown that the presence of glucose during in vitro culture reduced the development of female embryos to the blastocyst stage compared with controls cultured in the absence of glucose. The sex ratio of bovine embryos has also been linked with changes in the composition of the follicular fluid in which the oocyte undergoes growth and maturation, i.e. the intrafollicular testosterone concentration. However, no information is available regarding the effect of intrafollicular glucose concentration on the sex ratio of embryos after in vitro production (IVP). The purpose of this study was to determine whether different glucose concentrations in the follicular fluid at the time of cumulus–oocyte complex (COC) collection have an effect on the sex ratio of the resulting blastocysts after IVP. Ovaries from a local abattoir were transported to the laboratory within 2 h of slaughter. Follicles (3–8 mm) were individually dissected and the glucose concentration of each follicle was measured using a blood glucose monitoring system (Freestyle Freedom Lite, Abbott, Germany). Based on a glucose concentration, COC [low glucose: <1.1 mM (group 1) and high glucose: >1.1 mM (group 2)] were pooled in groups and used for blastocyst production employing standard protocols for IVP. Developmental rates were recorded at Day 3 (cleavage) and Day 7/8 (blastocyst stage). Total cell number of blastocysts was determined after Hoechst staining. Sex of the embryos was analysed via PCR using bovine X- and Y-chromosome specific primers. Developmental rates for COC stemming from follicles with different glucose concentrations did not show significant differences (P > 0.05) compared to each other [Cleavage rate: group 1: 81.8 ± 4.7% (93/117); group 2: 79.3 ± 4.9% (94/123); blastocyst rate: group 1: 35.6 ± 5.2% (38/117); group 2: 31.6 ± 5.2% (38/123)]. Total cell numbers were similar in embryos of both groups [Group 1: 117.7 ± 8.1 (n = 18); group 2: 117.2 ± 6.4 (n = 18)]. The overall sex ratio significantly differed (P < 0.05) from 1:1 in favour of females in both groups [Group 1: 85 v. 15% (n = 20); group 2: 63.6 v. 36.4% (n = 22)]. No significant difference (P > 0.05) in the overall sex ratio was detected in blastocysts produced under standard IVP conditions employed in the laboratory [without measurement of follicular glucose concentration, 55.0 v. 45.0%, (n = 20)]. In conclusion, under the conditions used in the present study, the intrafollicular glucose concentration from which the immature COC was collected affects the sex of the resulting embryo after IVP, favouring females. Further studies are needed to confirm these findings in living cows using the ovum pickup technique.

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

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

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

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

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

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

Sign in / Sign up

Export Citation Format

Share Document