scholarly journals 258 APOPTOSIS IN FAST- AND SLOW-CLEAVING BOVINE EMBRYOS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 258
Author(s):  
L. Vandaele ◽  
B. Mateusen ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Univariate Analysis ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Tunel Staining ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Random Factor ◽  
Quality Marker

Fast-cleaving embryos have significantly higher chances of reaching advanced developmental stages, and hatched blastocysts developed from fast-cleaving embryos have higher total cell and inner cell mass cell numbers. Few data are available on the prevalence of apoptosis in blastocysts resulting from fast-cleaving bovine embryos. Therefore, it was the aim of this study to search for a correlation between this early quality marker (timing of first cleavage) and a later quality marker (apoptosis in blastocyst stage). A total of 836 immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h after fertilization and cultured in 50 μL droplets of modified SOF medium without serum at 39.0°C in 5% CO2, 5% O2, and 90% N2. At 30 h, 36 h and 48 h post-fertilization (PF), zygotes that had developed to the 2-cell stage or beyond were placed in new droplets and cultured in separate groups. In each of the 3 replicates a control group of 100 presumed zygotes was cultured in SOF medium without manipulation. After 24 h of culture, the SOF medium was supplemented with fetal calf serum (FCS) up to 10%. Blastocysts were evaluated at Days 7 and 8, and fixed in 4% paraformaldehyde. After TUNEL staining, total cell number and TUNEL-positive cells were counted for each group. An univariate analysis of variance was used with % apoptotic cells as the dependent variable, timing of first cleavage as the fixed factor, and replicate as the random factor (mixed model ANOVA). The mean cumulative blastocyst yields in the control group were 20.9% and 27.0% at Days 7 and 8, respectively, which were not different from the total yields from manipulated embryos (Table 1). The cumulative cleavage rates for manipulated embryos were 44.9, 65.1, and 85.7% at 30, 36, and 48 h PF, respectively. The blastocyst yields at Days 7 and 8 PF were declining for embryos which cleaved later, with a distinct drop (P < 0.05) between the 36-h and 48-h groups. The percentage of apoptotic cells in Day 7 blastocysts was significantly lower in the 30-h compared with the 36-h and 48-h groups (P < 0.05). Within the 30-h and 36-h groups, Day 8 blastocysts had significantly more apoptotic cells than Day 7 blastocysts (P < 0.05), but no differences could be detected between groups at day 8. In conclusion, this experiment has confirmed the increased chances of early-cleaving embryos to reach the blastocyst stage. The higher percentages of apoptosis in late cleaving embryos at Day 7 and in all blastocysts at day 8 as determined by TUNEL staining could be an indication of lower blastocyst quality. Table 1. Blastocyst yield (number and percentage) and percentage of apoptotic cells (APC) at Days 7 and 8 PF

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

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

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

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

141 BIOPSY TECHNIQUE IN BOVINE EMBRYOS PRODUCED IN VITRO AT EARLY STAGES OF DEVELOPMENT: EVALUATION OF QUALITY AND DEVELOPMENT CAPACITY

2008 ◽  
Vol 20 (1) ◽  
pp. 151
Author(s):  
J. Polisseni ◽  
M. O. Guerra ◽  
R. V. Serapião ◽  
M. M. Pereira ◽  
I. M. Folhadella ◽  
...  
Keyword(s):  
Preimplantation Genetic Diagnosis ◽  
Embryo Quality ◽  
Genetic Diagnosis ◽  
Blastocyst Stage ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Number Of Cells

One of the causes of embryo mortality is chromosome abnormalities that occur during gametogenesis, fertilization, and embryo early development. Thus, a combination of morphological standards and techniques of molecular analyses could identify abnormal embryos. Preimplantation genetic diagnosis (PGD) is an emergent technology for use with farm animal embryos. With this procedure, blastomeres are removed by the biopsy of embryos at the 8- to 16-cell stage to provide cells for analyses of chromosome abnormalities prior to transfer. The aim of this study was to evaluate the effect of biopsy in bovine 8- to 16-cell embryos fertilized in vitro on embryo quality and subsequent development in vitro. A group of 706 oocytes were obtained from slaughterhouse ovaries, matured, and fertilized in vitro at 38.8�C with 95% humidified air and 5% CO2. The zygotes were semi-denuded and cultured in CR2aa medium under the same conditions as for in vitro fertilization. The rate of cleavage was 78.20%. Three days after fertilization, part of the 8- to 16-cell (298/706) embryos were distributed randomly across two groups: control (n = 103) and biopsy (n = 92) of blastomeres, and then returned to in vitro embryo culture to evaluate development until the blastocyst stage and the capacity to hatch. The amount of cells removed was one-fourth of the embryo. The blastocyst rate was evaluated on Day 8 after fertilization and the hatching rate on Day 10. Embryo morphology and quality were evaluated as previously described in the International Embryo Transfer Society manual (1998). To evaluate overall quality, embryos were stained on the 10th day of culture and the blastomeres were counted with the imaging software AxioVision 3.1 (Carl Zeiss, Feldbach, Switzerland). The blastocyst rate was analyzed by treatment groups with the chi-square test and the number of cells/embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The percentage of 8- to 16-cell embryos that developed to the blastocyst stage was similar (P > 0.05) between the control (66.0%, 68/103) and the biopsied (53.3%, 49/92) groups. Furthermore, no difference was noted in the hatching rates between the control group and the biopsied group (42.6%, 29/42 v. 44.9%, 22/49, respectively). Overall, no impact was detected on embryo quality from embryo biopsy with no difference in mean (�SE) blastocyst cell number between the control group (blastocysts: 67.1 � 3.1; expanded blastocysts: 100.7 � 6.9; hatched blastocysts: 189.9 � 16.1) and the biopsied group (blastocysts: 61.1 � 5.5; expanded blastocysts: 121.87 � 10.6; hatched blastocysts: 187.3 � 18.5). In conclusion, the biopsy used on 8- to 16-cell bovine IVF-derived bovine embryos does not affect the subsequent embryo development and number of cells/embryo or blastocyst, showing that it can be used to provide genetic material for preimplantation genetic diagnosis without affecting embryo quality. This work was supported financially by FAPEMIG.

55 EFFECT OF DIMETHYL SULFOXIDE SUPPLEMENTATION ON BOVINE IN VITRO EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 157 ◽  
Author(s):  
J. Stöhr ◽  
H. Grothmann ◽  
C. Wrenzycki
Keyword(s):  
In Vitro Culture ◽  
Dimethyl Sulfoxide ◽  
Apoptotic Index ◽  
Total Cell ◽  
Control Group ◽  
Apoptotic Cells ◽  
Fat Accumulation ◽  
Developmental Rates

Many techniques for in vitro production of embryos frequently make use of dimethyl sulfoxide (DMSO) as a solvent for compounds that have little or no solubility in water. DMSO is also used as a cryoprotectant. Based on its high glass-forming characteristics, it is also essential for vitrification. Although it is known that high concentrations of DMSO could be embryo toxic, less attention has been focused on whether an effect could be detected from the small concentrations present in culture media when used as a vehicle. Earlier studies deemed concentrations of up to 0.4% in in vitro maturation and 0.1% in in vitro culture (IVC) as safe with regards to morphological criteria. In the present study, bovine in vitro-produced embryos employing standard protocols were exposed to the following DMSO concentrations during IVC: 0% (control), 0.05%, 0.10%, 0.15%, 0.20%, and 0.25%. At Day 8 cleavage and developmental rates were recorded. The morphological quality of expanded Day 8 blastocyst was assessed with determination of the total cell number, live-dead (live-dead ratio), and TUNEL staining (apoptotic index). Fat accumulation was analysed by red-oil staining. Cleavage and developmental rates did not differ (P > 0.05) between embryos of the various groups. Mean cleavage and development rates (%) averaged 58.3 ± 10.6 and 28.4 ± 9.2 (0%), 59.5 ± 11.5 and 26.1 ± 7.4 (0.05%), 57.6 ± 6.6 and 21.7 ± 7.1 (0.10%), 58.1 ± 7.8 and 27.8 ± 5.6 (0.15%), 56.6 ± 7.3 and 24.5 ± 7.0 (0.20%), 56.3 ± 10.9 and 23.5 ± 9.9 (0.25%). Total cell numbers were similar [123.9 ± 26.9 (0%), 115.3 ± 21.5 (0.05%), 114.8 ± 22.3 (0.10%), 125.4 ± 22.4 (0.15%), 122.9 ± 24.7 (0.20%), 115.9 ± 19.7 (0.25%)]; P > 0.05. The live/dead cell ratio was significantly higher (P ≤ 0.05) in those embryos derived from the 0.1% group (40.1 ± 23.1) than that from embryos of the other groups [22.6 ± 13.5 (0%), 23.4 ± 10.4 (0.05%), 24.2 ± 14.6 (0.15%), 22.7 ± 14.0 (0.20%), 20.3 ± 9.9 (0.25%)]. Apoptotic cells were significantly lower in embryos exposed to 0.10% and 0.20% DMSO than in those of other groups, and the number of apoptotic cells in embryos of 0.05% was also slightly lower compared with those of the control group (P = 0.08). Apoptotic index tended to be lower in embryos out of the groups supplemented with 0.1% and 0.2% DMSO compared with those of the control group (0%: 3.8 ± 1.6; 0.05%: 2.6 ± 1.6; 0.10%: 2.3 ± 1.8; 0.15%: 3.2 ± 1.5; 0.20%: 2.2 ± 1.5; 0.25%: 3.1 ± 1.7; P = 0.09; P = 0.06). Fat accumulation was significant higher (P ≤ 0.05) in embryos stemming from the group supplemented with 0.15% DMSO (0%: 6617 ± 2703 µm2; 0.05%: 7346 ± 1981 µm2; 0.10%: 6976 ± 1848 µm2; 0.15%: 9301 ± 1703 µm2; 0.20%: 8675 ± 2271 µm2; 0.25%: 8301 ± 2711 µm2). These findings show that DMSO concentrations of 0.10% and 0.20% used during in vitro culture influence the quality of embryos at the morphological level. However, further analyses to verify these results at the molecular level via RT-qPCR are still needed. The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.

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

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

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

Effects of supplemental progesterone on the development, metabolism and blastocyst cell number of bovine embryos produced in vitro

2011 ◽  
Vol 23 (2) ◽  
pp. 311 ◽  
Author(s):  
Jamie E. Larson ◽  
Rebecca L. Krisher ◽  
G. Cliff Lamb
Keyword(s):  
Present Experiment ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryos ◽  
Morula Stage ◽  
Metabolic Data ◽  
Embryo Metabolism ◽  
Number Of Cells

The objectives of the present experiment were to determine whether supplementation with progesterone (LO, 1 ng mL–1 or HI, 100 ng mL–1) during either the first (Culture-1, Day 1 to 3) or second (Culture-2, Day 4 to 7) phase of culture of in vitro-produced embryos alters embryo development, embryo metabolism or blastocyst cell number. The percentage of oocytes that cleaved, the percentage of cleaved embryos that developed to the morula stage or greater, the blastocyst stage or greater or the hatched blastocyst stage were similar among treatments. Quantities of glucose metabolised per blastocyst per hour were similar, but when metabolic data was normalised for numbers of cells in each blastocyst, the LO treatment during Culture-2 metabolised more glucose (P = 0.03) compared with all other treatments. Embryos receiving LO progesterone tended to have greater (P = 0.085) metabolism of glucose compared with embryos receiving HI progesterone. Quantities of pyruvate oxidised per blastocyst per hour, and per cell, were similar among treatments. The number of cells per blastocyst in the control group was increased (P = 0.039) compared with cells in progesterone-treated groups. In conclusion, supplementation with progesterone during the culture of in vitro-produced embryos does not appear to improve embryo characteristics.

scholarly journals Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

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