Effects of changing culture medium on preimplantation embryo development in rabbit

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

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

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

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

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

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.

188 THE IMPACT OF OIL SOURCE AND TYPE OF GLUTAMINE ON MEDIA EQUILIBRATION TIMES AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. W. Linck ◽  
D. K. Gardner
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Oil Source ◽  
The Impact

Recently, there has been much debate involving the time necessary to effectively equilibrate an embryo culture system, i.e. dishes, media, and oil. Glutamine present in the culture medium spontaneously deaminates at 37�C to release embryo-toxic ammonium. Additionally, the source of the oil overlay can impact the culture environment. A sub-optimal oil overlay, combined with free glutamine (Gln), could effectively become embryo-toxic over a short time period (&lt;48 h). Therefore, the aim of this study was to determine how times of media equilibration, using various combinations of oil source and type of Gln, affect embryo development. Zygotes were collected from 4-week-old CF1 outbred female mice following superovulation and mating. Embryos were cultured in groups of 10 in 20-�L drops of medium G1.2. Initially, embryos were cultured in one of 4 treatment media. In this 2 � 2 factorial design, the culture medium was pre-equilibrated 18 h prior to embryo retrieval and contained free Gln or the heat-stable dipeptide alanyl-glutamine (AlaGln), combined with an oil source of either Sigma mineral oil (Sigma-Aldrich Corp., St Louis, MO, USA) or Ovoil paraffin oil (Vitrolife, Inc., Englewood, CO, USA). The initial study was then repeated using only the best and worst case groups to determine the effect of incubation time as a variable (either 2 h or 18 h). Blastocyst development and total cell numbers were analyzed after 72 h of culture, and differences between treatments were assessed using Fisher&apos;s exact test and Student&apos;s t-test. After 18 h of pre-equilibration (n e 300 embryos/treatment), blastocyst development in Ovoil + AlaGln (38.6%) was significantly greater when compared to: Ovoil + Gln: 25.5% (P &lt; 0.01), Sigma + AlaGln: 12.8% (P &lt; 0.01), and Sigma + Gln: 11.9% (P &lt; 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 � 10) were significantly decreased: 35.5 � 7 (P &lt; 0.001), 34.9 � 9 (P &lt; 0.001), and 29.9 � 9 (P &lt; 0.001), respectively. In the second experiment, blastocyst development and total cell number between Ovoil + AlaGln (n = 224) and Sigma + Gln (n = 264) after 18 h of pre-equilibration were: 40.4% vs. 9.9% (P &lt; 0.01) and 46.6 � 9 vs. 29.4 � 9 P &lt; 0.001), respectively. However, after 2 h of pre-equilibration, the results between Ovoil + AlaGln (n = 260) and Sigma + Gln (n = 284) were: 42.3% vs. 18.3% (P &lt; 0.01) and 46.9 � 10 vs. 33.6 � 6 (P &lt; 0.001), respectively. Therefore, when comparing blastocyst development and total cell number between pre-equilibration times (2 h vs. 18 h), the Ovoil + AlaGln group, 42.3% vs. 40.4% and 46.9 � 10 vs. 46.6 � 9, showed no significant differences, respectively. In contrast, the Sigma + Gln group produced significant differences for both blastocyst development, 18.3% vs. 9.9% (P &lt; 0.01), and total cell number, 33.6 � 6 vs. 29.4 � 9 (P &lt; 0.05), between pre-equilibration times (2 h vs. 18 h), respectively. Data presented confirm the need for an alternative source of glutamine in embryo culture media. The data also indicate that the source of oil has a profound effect on the experimental outcome. Using the appropriate oil and form of Gln means that media can be safely equilibrated for 18 h.

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).

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 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 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).

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.

213 MATURATION OF OOCYTES WITH FOLLICULAR FLUID FROM GILTS CONSUMING HIGH FAT AND FRUCTOSE AFFECTS SUBSEQUENT EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
R. Poole ◽  
V. McCracken ◽  
M. Rhoads ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Follicular Fluid ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
High Fat ◽  
Total Cell Number ◽  
Treatment Groups ◽  
High Fructose

Infertility among women has become a growing issue in the world requiring a significant number to seek treatment by means of assisted reproductive technologies. One suggested reason for the fertility issue, which is known to specifically affect oocyte quality, is the modern diet. Previously, we have demonstrated that feeding a high-fructose diet to gilts led to poor reproductive tract characteristics and infertility. In this study, pre-pubescent gilts were fed either a high-fructose; high-fat diet (HFHF), with 15% beef tallow and 35% fructose; or an industry control diet (IND). Porcine follicular fluid (pFF) collected from these gilts was introduced into in vitro maturation systems to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. Follicles from ovaries, collected at a local abattoir, were aspirated by an 18 G needle attached to a 10-mL sterile syringe. Then selected cumulus‐oocyte complexes were maturated in vitro in a TCM-199 maturation media with cysteine, glucose, sodium pyruvate, epidermal growth factor (EGF), FSH, LH, and 20% pFF from treatment groups. Additionally, another group of oocytes, labelled follicle fluid free (FFF), were maturated in TCM-199 media without pFF. Three replicate experiments were conducted using a total of 365 oocytes, 124 FFF, 121 IND, and 120 HFHF. Oocytes were denuded by exposure to 0.1% hyaluronidase and oocytes that reached metaphase II (MII) were selected for in vitro fertilisation. After 5 h of co-incubation in modified Tween medium B with milk powder (mTBM)-based IVF media, presumable zygotes were transferred to porcine zygote medium-3 (PZM-3). Blastocyst frequency was recorded on Days 5 and 6. Day 6 blastocysts were stained with Hoechst for total cell number evaluation. The frequencies of blastocyst formation among the treatment groups were compared by a chi-squared test, and total cell numbers were compared by Student's t-test. Statistical significance was defined by P < 0.05. The frequency of oocytes reaching metaphase II (MII) were observed as 77.4% FFF, 72.7% IND, and 71.7% HFHF (P > 0.05), indicating the supplementation of pFF did not affect maturation. Day 5 blastocysts were observed at frequencies of 8.3% FFF, 6.8% IND, and 4.7% HFHF and did not differ. However, frequency of Day 6 blastocysts from HFHF group was tended to be lower compared with that of other groups; 12.5% FFF, 11.4% IND, and 4.7% HFHF (P = 0.06 and P = 0.1). Average total cell number of Day 6 blastocysts observed were 41.0 ± 9.1 FFF, 36.0 ± 8.9 IND, and 48.3 ± 10.6 HFHF. The total cell number from HFHF group tended to be higher than only that of IND group (P = 0.07). Based on these results, we concluded that the follicular fluid of females consuming HFHF diets did not have impact on nuclear maturation of oocytes but might affect oocyte competency, thus resulting in detrimental effects on subsequent development of embryos, especially blastocyst formation. Further studies will help us identify more specific effects of nutrition on oogenesis and subsequent embryo development.

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.

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