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

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.

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.

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).

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.

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).

96 GRANULOCYTE–MACROPHAGE COLONY-STIMULATING FACTOR INCREASES DEVELOPMENTAL POTENTIAL OF PORCINE EMBRYOS IN VITRO

2012 ◽  
Vol 24 (1) ◽  
pp. 160
Author(s):  
K. Lee ◽  
J. Teson ◽  
L. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

There have been significant improvements in the culture of porcine embryos in vitro; however, it is still suboptimal. Improvements in porcine embryo culture would benefit utilisation of porcine embryos for a variety of purposes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be expressed in the female reproductive tract and the level of its expression is high between conception and implantation. Previous studies show supplementing GM-CSF in embryo culture promotes embryonic development in human and bovine embryos. The aim of this study was to investigate the effect of GM-CSF on the culture of porcine embryos derived from somatic cell nuclear transfer (SCNT) and IVF. Different concentrations of recombinant porcine GM-CSF (0, 2, 10 ng mL–1) were introduced into Porcine Zygote Medium 3 from Day 1 to 6. Frequencies of cleaved embryos and blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell number in blastocysts from each group were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. A total of 563 SCNT embryos from 6 different donor cell lines on 11 different days were produced for the study. Incubation of SCNT embryos with GM-CSF did not affect the frequency of cleaved embryos. Frequencies of cleaved embryos in control (0 ng mL–1), 2 ng mL–1 GM-CSF and 10 ng mL–1 GM-CSF were 64.2%, 68.1% and 65.0%, respectively. Interestingly, both concentrations of GM-CSF significantly increased the frequency of blastocyst formation as compared with the control. In 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, 30.8% and 32.3% of embryos reached blastocyst respectively, whereas only 22.4% of embryos reached blastocyst in the control group. A significant increase in total cell number in blastocysts was observed when GM-CSF was introduced into embryo culture. An average of 28.8 ± 0.9 cells was recorded in the control group, whereas 31.9 ± 1.1 and 31.8 ± 1.1 were observed in 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, respectively. Similar effects were observed when GM-CSF was introduced to the culture of IVF embryos. For IVF study, 525 embryos were generated on 10 different days and embryos cultured in the presence of GM-CSF tended to show higher blastocyst formation (P = 0.1). Frequencies of blastocyst per cleaved in the 3 groups were 55.7% (control), 65.7% (2 ng mL–1 GM-CSF) and 66.7% (10 ng mL–1 GM-CSF). In addition, culture of IVF embryos with GM-CSF significantly increased total cell number in Day 6 blastocysts. Total cell number in blastocysts in 2 ng mL–1 GM-CSF (34.2 ± 0.8) and 10 ng mL–1 GM-CSF (34.4 ± 1.2) were significantly higher compared with control (27.3 ± 1.2). Our results indicate that introducing GM-CSF into embryo culture media can increase the quality of blastocyst stage embryos. An increase in the frequency of blastocyst formation and total cell number in blastocysts suggests that GM-CSF can be used to produce better-quality embryos in vitro. Currently, effects of GM-CSF on implantation of SCNT embryos are under investigation. Further studies would elucidate the specific mechanism of GM-CSF on porcine embryos.

31 DEVELOPMENT AND APOPTOSIS IN BOVINE CLONED EMBRYOS RECONSTRUCTED WITH OOCYTES COLLECTED BY REPEATED OVUM PICKUP SESSIONS OR FROM SLAUGHTERED COW OVARIES

2011 ◽  
Vol 23 (1) ◽  
pp. 122
Author(s):  
L. S. A. Camargo ◽  
M. M. Pereira ◽  
C. C. R. Quintao ◽  
J. N. S. Sales ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Bos Indicus ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Index ◽  
Sh Groups ◽  
Sh Group

The oocyte has important components for nuclear reprogramming and its cytoplasmic background may influence the somatic cell nuclear transfer success. The current study attempted to evaluate the competence of cytoplasm from oocytes recovered by repeated ovum pickup (OPU) in living cows (OPU group) or obtained from ovaries collected at slaughterhouse from unknown source crossbred cows (SH group) to produce nuclear-transferred bovine embryos. For the OPU group, oocytes were recovered from 4 Bos indicus × Bos taurus crossbred cows in 4 repeated OPU sessions. Oocytes of OPU and SH groups were matured in vitro for 17 to 18 h, denuded and exposed to Hoechst 33342 (Sigma, St. Louis, MO, USA) and cytochalasin (Sigma) before enucleation. Embryos of OPU (n = 100) and SH (n = 105) groups were reconstructed with somatic cells from adult Gyr (Bos indicus) cow, fused with double electric pulse of 2.4 kV cm–1 for 30 μs and activated with ionomycin (Sigma) and 6-DMAP (Sigma). Embryos were cultured in CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell, Campinas, Brazil) under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage and blastocyst rates were evaluated at 72 h and 168 h post-activation, respectively. Blastocysts at 168 h post-activation were fixed and permeabilized for TUNEL assay (DeadEnd™ Fluorimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. IVF bovine blastocysts (IVF group; n = 245) obtained with oocytes of slaughtered cows were used as control group. Fusion, cleavage, and blastocyst rates were analysed by chi-square test and total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analysed by ANOVA. There were no differences (P > 0.05) in fusion (71.0% and 61.0%), cleavage (74.6% and 78.1%) or blastocyst (32.3% and 31.2%) rates between OPU and SH groups, respectively, but both groups presented greater (P < 0.05) blastocyst rates than the IVF group (15.1%). Total cell number (80.66 ± 5.36 and 82.10 ± 4.79), apoptotic cell number (12.66 ± 3.20 and 15.60 ± 3.04), and apoptotic cell index (0.15 ± 0.03 and 0.20 ± 0.04) were also similar (P > 0.05) between OPU and SH groups, respectively. However, apoptotic cell number (7.40 ± 0.93) and apoptotic cell index (0.07 ± 0.01) were lower (P < 0.05) in the IVF group than the SH group and similar (P > 0.05) to the OPU group. In conclusion, oocytes cytoplasm from both groups (OPU and SH) have the same potential to produce nuclear-transferred bovine embryos but only blastocysts from the OPU group present apoptosis levels similar to its in vitro-fertilized counterpart. Financial support: Fapemig and CNPq.

Crucial surviving aspects for vitrified in vitro-produced bovine embryos

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 124-131 ◽  
Author(s):  
Mateus José Sudano ◽  
Daniela Martins Paschoal ◽  
Tatiana da Silva Rascado ◽  
Letícia Ferrari Crocomo ◽  
Luis Carlos Oña Magalhães ◽  
...  
Keyword(s):  
Lipid Content ◽  
Production Quality ◽  
Cell Number ◽  
Total Cell ◽  
Strongly Correlated ◽  
Total Cell Number ◽  
Embryo Survival ◽  
Apoptosis Rate

SummaryThe objective of the present study was to correlate some parameters (cleavage, blastocyst production, quality degree score, total cell number, fresh apoptosis and lipid content) with embryo survival after cryopreservation. A total of 1727 in vitro-produced bovine blastocysts were used to establish the parameters (mean ± standard error of the mean (SEM)) for cleavage (85.6 ± 0.8), blastocyst production (39.9 ± 1.4), quality degree score (1.6 ± 0.1), total cell number (140.1 ± 2.9), fresh apoptosis (20.8 ± 1.1) and lipid content (21.3 ± 0.8 droplets). On the same way 1316 blastocysts were vitrified for the determination of post-cryopreservation embryo survival (49.4 ± 1.9). Fresh apoptosis rate and total lipid droplets value were correlated (P < 0.05) with embryo survival after cryopreservation (r = 0.91 and r = 0.59; respectively). However, cleavage, blastocyst production, quality degree score and total cell number were not correlated (P > 0.05) with embryo cryotolerance (r = 0.23, r = 0.38, r = 0.22 and r = 0.28; respectively). Therefore, the increased lipid content was moderately correlated with apoptosis in vitrified blastocysts. On the other hand, increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts, which indicated that the apoptosis rate in fresh embryos was a better parameter than the lipid content to predict post-vitrification embryo survival.

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