84 THE EFFECT OF β-MERCAPTOETHANOL ON CLEAVAGE RATES, DEVELOPMENTAL COMPETENCE AND QUALITY OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 156 ◽  
Author(s):  
E. R. Lliteras ◽  
M. Chong ◽  
S. Andries ◽  
E. Merckx ◽  
E. P. A. Jorssen ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Dapi Staining ◽  
Total Cell Number

The production of excessive levels of reactive oxygen species can be a major problem during in vitro embryo culture. Although studies have shown that supplementation with exogenous antioxidants can improve embryo quality, the results are controversial among researchers. In this study, we examined the effects of different concentrations of β-mercaptoethanol (β-ME) added to the culture media, on cleavage rates, the quality and developmental competence of in vitro-produced bovine embryos. The embryos were produced in vitro as described previously (Van Hoeck et al., 2013). Briefly, in total, 753 grade I cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 EGF for 24 h, fertilized in groups of 100 in 500 μL of fertilization medium for 20 h (5% CO2, 38.5°C). Presumptive zygotes were denuded and randomly assigned to 4 treatments with different concentrations of β-ME: 0 μM (control), 50 μM, 100 μM, and 150 μM. They were cultured in groups of  ±25 in 50 μL of SOF supplemented with ITS (10 μg mL–1 insulin; 5.5 μg mL–1 transferrin; 6.7 ng mL–1 selenium) and 2% BSA and covered with mineral oil (5% O2, 5% CO2, 38.5°C). At 48 h post-insemination (p.i.), cleavage rate was evaluated and expressed as the number of cleaved embryos on total number of oocytes. At Day 7 p.i., blastocyst rate was determined (number of blastocysts on total number of oocytes), blastocysts were fixed in 4% paraformaldehyde, and total cell number was determined by DAPI staining. Data were analysed by ANOVA and post hoc test. Comparable cleavage rates were obtained in treated groups: control (80.8%), 50 μM (77.7%), 100 μM (77.9%), and 150 μM (73.6%; P > 0.05). Also, no significant effect of treatment could be found on blastocyst rates: control (36%), 50 μM (36.5%), 100 μM (38.4%), and 150 μM (30.4%). The total cell number per blastocyst increased significantly (P < 0.05) using 100 μM of β-ME compared with the controls (158.0 ± 24.3 v. 123.2 ± 9.72, respectively). These results suggest that the inclusion of 100 μM β-ME during in vitro embryo culture could be used for production of high quality bovine blastocysts.

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).

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

scholarly journals 95 EFFECT OF ESTROUS COW SERUM ON SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS AFTER SLOW FREEZING OR VITRIFICATION

2005 ◽  
Vol 17 (2) ◽  
pp. 198
Author(s):  
N. Mucci ◽  
J. Aller ◽  
P. Ross ◽  
G. Kaiser ◽  
J. Cabodevila ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
T Test ◽  
Slow Freezing ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Embryo Survival

Until now, the major obstacle associated with the extensive use of in vitro-produced bovine embryos is the lack of suitable methods to cryopreserve them. At least two approaches exist for overcoming this problem. One is to adjust cryopreservation methods to the requirements of these embryos, and the other is to improve embryo quality by using an appropriate in vitro environment for embryo production. The objective of this study was to determine the effect of estrous cow serum (ECS) during in vitro culture on embryo survival after cryopreservation by slow freezing or vitrification. Cumulus-oocytes complexes were in vitro-matured and fertilized as previously described (Ferre et al. 2003 Theriogenology 59, 301 abst). Presumptive zygotes were denuded from cumulus cells and cultured in groups of 50 in 400 μL drops of CR1aa medium. Seventy-two hour post-insemination (PI) embryos were randomly separated into three groups. Each group was then cultured in CR1aa + 5% ECS (without BSA; CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA), or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). Embryos were cultured under 38.5°C, 5% CO2, 5% O2, and 90% N2. At 7.5 days PI, blastocysts from each group were double stained using propidium iodide and bisbenzimide (Hoechst 33342) to determine damaged cells and total cell number. The remaining embryos were randomly cryopreserved by freezing (1.5 M ethylene glycol; cooled at 0.5°C/min to −35°C) or vitrification (open pulled straw, Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). After thawing or warming, embryos were cultured in CR1-ECS-BSA to evaluate embryo survival (hatching rate). Data were analyzed by χ2, ANOVA and Student's t-test (SAS Institute, Inc., Cary, NC, USA). Total cell number was higher in embryos cultured in CR1-ECS than in CR1-BSA or CR1-ECS-BSA (CR1-ECS: 142.1 ± 4.7, n = 23 vs. CR1-BSA 124.7 ± 4.9, n = 21, and CR1-ECS-BSA 125.8 ± 4.5, n = 25; t-test, P < 0.05). No differences were found in percent of damaged cells (CR1-ECS: 0.7%; CR1-BSA: 1.8%; CR1-ECS-BSA: 0.7%). Blastocyst survival after thawing was affected by cryopreservation methods and culture media (P < 0.01, Table 1). No interaction was found between both factors. In conclusion, under our experimental conditions elimination of ECS from CR1aa medium improves embryo cryotolerance. Vitrification allows for higher survival rates, regardless of the presence of serum during embryo culture. Table 1. Effect of cryopreservation method and serum supplementation during embryo culture on survival rate of in vitro-produced bovine embryos

48 DEVELOPMENTAL COMPETENCE OF CLONED OR PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS: EFFECT OF DIAMETER OF PREPUBERTAL GILT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 130
Author(s):  
J. Li ◽  
J. Adamsen ◽  
R. Li ◽  
H. Pedersen ◽  
Y. Liu ◽  
...  
Keyword(s):  
Chemical Activation ◽  
Cell Number ◽  
Visual Observation ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Mean Diameter ◽  
Blastocyst Rate

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al. 1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor's age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120 kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitro matured for 42 to 44 h as described for sow oocytes (Li et al. 2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al. 2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al. 2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium was applied for 4 h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110 μm, from 105 to 116 μm), S group (mean diameter 101 μm, from 93 to 106 μm) and C group (mean diameter 107 μm, from 93 to 116 μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

2009 ◽  
Vol 21 (1) ◽  
pp. 224
Author(s):  
M. M. Pereira ◽  
F. Q. Costa ◽  
P. H. A. Campos ◽  
R. V. Serapiao ◽  
J. Polisseni ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Blastocyst Rate ◽  
Bovine Oocytes

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P > 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P < 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P > 0.05). However, blastocyst rate in G2 (25.0%) was higher (P < 0.05) than in G1 and G3, but was similar to G4 (P > 0.05). Total cell number was similar (P > 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P < 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P < 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P > 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

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.

225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

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.

139 BOVINE PREIMPLANTATION EMBRYOS SECRETE EXTRACELLULAR VESICLES, WHICH MAY INDICATE EMBRYO COMPETENCE

2017 ◽  
Vol 29 (1) ◽  
pp. 178
Author(s):  
E. Mellisho ◽  
A. Velasquez ◽  
M. J. Nuñez ◽  
L. Rodriguez-Alvarez
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Total Cell ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Lower Amount ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Blastulation Rate

Pre-implantation embryos secrete extracellular vesicles (EV) most likely to communicate with the surroundings. The objective of this study was to determine the distribution (size and concentration) of EV secreted by bovine pre-implantation embryos with different developmental competence. The IVF bovine embryos were produced from oocytes recovered from slaughterhouse ovaries. Presumptive zygotes were in vitro cultured (IVC) in groups in 4-well plates (30 zygotes per 500-µL well) using SOFaa medium at 39°C under 5% CO2, 5% O2, and 90% N2 until the morula stage (Day 5 post IVF). Morulae were cultured individually in 96 well at 39°C under until blastulation time (Day 6.5–7.5) in EV-free SOF medium. Culture medium was collected only from embryos that developed to the blastocyst stage that were classified in a group of early (Day 6.5) or late (Day 7.5) blastulation. Blastocysts were kept in culture until Day 11 to assess embryo developmental competence, considering embryo size (>350 µm) and total cell count (>500 blastomeres). For EV analysis, 4 groups were organised a posteriori: G1: Day 6.5-competent; G2: Day 6.5-not competent; G3: Day 7.5-competent; G4: Day 7.5-not competent. The EV in culture media were analysed using a nanoparticle tracking analysis (Nanosight NS300). Statistical analysis was performed using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Early blastulation rate (Day 6.5) was 40.3% (112/278), whereas late blastulation rate (Day 7.5) was 20.5% (57/278), showing a significant difference (P < 0.0001). Embryos derived from Day 6.5 blastocysts have a higher probability (39.3%: 44/112) of posthatching development [until Day 11; Day 7.5, 10.5% (6/57); P = 0.0001]. At Day 11, competent embryos (G1) derived from Day 6.5 blastocysts have a higher diameter and total cell number (447 µm; 688 cells) than those derived from Day 7.5 blastocysts (G3; 405 µm, 598 cells; P < 0.05 for both parameters). It was possible to detect EV from collected medium of individual embryos independent of their competence. Neither the EV size nor the EV concentration was statistically different between Day 6.5 and Day 7.5 blastocysts (without considering their further competence; 2.9 × 108, 147 nm; and 3.0 × 108, 149 nm, respectively). However, independent of the day of blastulation, competent embryos had a significantly lower concentration of EV (2.7 × 108 v. 3.3 × 108; P = 0.03). Moreover, competent embryos from early and late blastocysts (G1 and G3) tend to produce a lower amount of EV (G1: 2.8 × 108; G2: 3 × 108; G3: 2.6 × 108; G4: 3.5 × 108; P = 0.05). Furthermore, EV concentration was statistically different between G3 and G4 (P = 0.002). No differences in EV size were observed among groups (G1: 145 nm; G2: 148 nm; G3: 146 nm; G4: 151 nm). Our results provide an initial approach to study the EV secreted by individual pre-implantation embryos to assess their competence. From these results, we can conclude that blastulation time affects the future development of bovine embryos and a model based on blastulation time and EV secretion could be a simple noninvasive tool to improve embryo selection.

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