243 THE SEX OF THE BOVINE EMBRYO AFFECTS APOPTOTIC RATES AT THE BLASTOCYST STAGE

2015 ◽  
Vol 27 (1) ◽  
pp. 211
Author(s):  
E. Ghys ◽  
D. De Troy ◽  
I. Donnay
Keyword(s):  
Sex Ratio ◽  
Cell Number ◽  
Cell Counting ◽  
Tunel Staining ◽  
Bovine Embryo ◽  
Male And Female ◽  
Tunel Reaction ◽  
Significant Difference ◽  
Sexed Semen

Male and female pre-implantation bovine embryos may differ in several aspects such as kinetics of development, metabolism, or gene expression. These differences vary between culture conditions and may lead to shifts in sex ratio. In a previous study, we showed that female Day 7 blastocysts display a higher apoptotic rate than male ones when cultured in the presence of 5% FCS (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The difference was less important in the presence of BSA. This previous study was performed on in vitro-produced embryos produced with sexed semen and analysed using confocal microscopy. The objective of the present work is to confirm these differences using a) first, the unsexed semen of the same bull (bull 1) as the one used in the previous study in order to exclude any potential bias induced by the use of sexed semen and b) second, the unsexed semen of another bull (bull 2) in order to generalise our findings to the Bos taurus species. Levels of apoptosis were assessed in Day 7 blastocysts using immunohistochemical staining of cleaved caspase-3 and detection of fragmented DNA by TUNEL reaction on Day-7 blastocysts cultured with 5% FCS. Quantification of the number of stained cells was achieved with a fluorescence microscope. After cell counting, embryos were recovered and sexed by PCR. In both experiments, a higher proportion of cells showing caspase staining was observed in female (n = 145) than in male (n = 215) embryos (Bull 1: male: 11.8 ± 0.6%; female: 17.6 ± 1.1%, 2-way ANOVA, P ≤ 0.0001; bull 2: male: 9.5 ± 0.4%; female: 13.3 ± 0.6%, P ≤ 0.0001), whereas the proportion of TUNEL-stained cells was only significantly higher in female embryos produced with the semen of bull 2 (bull 1: male: 10.8 ± 0.4%; female: 11.4 ± 0.6%, P = 0.12; bull 2: male: 7.2 ± 0.3%; female: 9.1 ± 0.4%, P = 0.0009). A significant difference in cell number was observed between male and female blastocysts produced with the semen of bull 2 (male: 172 ± 5; female: 154 ± 5, P = 0.02) and the same tendency was observed for embryos generated with the semen of bull 1 (male: 143 ± 4; female: 132 ± 4, P = 0.07). In conclusion, our study demonstrated that the use of sexed semen does not interfere with the pattern of caspase and TUNEL staining previously observed. Moreover, a similar pattern was observed with 2 different bulls. We can thus conclude that the level of apoptosis of bovine Day 7 blastocysts produced in the presence of FCS is higher in female than male embryos. This could be related to the tendency towards a lower cell number in female blastocysts and to the shift in sex ratio in favour of male embryos often observed in the presence of serum.

92 DOES THE SEX OF THE EMBRYO AFFECT DEVELOPMENTAL AND APOPTOTIC RATES AT THE BLASTOCYST STAGE?

2013 ◽  
Vol 25 (1) ◽  
pp. 194
Author(s):  
E. Ghys ◽  
M. Dallemagne ◽  
C. Sauvegarde ◽  
I. Donnay
Keyword(s):  
Dna Fragmentation ◽  
Caspase 3 ◽  
Culture Media ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Tunel Staining ◽  
Male And Female ◽  
Significant Difference ◽  
Sexed Semen

Several studies have demonstrated differences in developmental rates and metabolism between bovine female and male embryos after IVF. Such differences seem related to the activity of both X chromosomes in female embryos up to the blastocyst stage and can lead to a shift in sex ratio. Developmental differences between male and female embryos are influenced by culture conditions. The objective of this study was to evaluate developmental and apoptotic rates of male and female bovine embryos in 2 SOF-based culture media, one with 5% FCS and the other with 4 mg mL–1 of BSA. Sex-sorted semen of one bull was used to produce cohorts of embryos of the desired sex. In preliminary experiments, IVF procedures were adapted to the use of sexed semen, and the purity of the sexed semen was verified through embryo sexing. The levels of apoptosis were assessed in Day-7 blastocysts using 2 techniques on the same embryos: TUNEL and detection of cleaved caspase-3 by immunostaining (caspase staining). Analysis by confocal microscopy and subsequent 3-D reconstruction allowed a precise cell count. A higher blastocyst rate on cleaved embryos was observed at Day 8 post-insemination for male than for female embryos in both media (BSA: male: 36.7 ± 4.0%, female: 28.6 ± 3.7%; FCS: male: 41.7 ± 2.9%, female: 31.7 ± 4.7%; ANOVA 3, P = 0.01). No significant difference in cell number was observed between male and female blastocysts (BSA: male: 188 ± 9, female: 170 ± 9; FCS: male: 186 ± 6, female: 177 ± 7; ANOVA 3, P = 0.14). In both media a higher proportion of cells showing caspase staining was observed in female than in male embryos (BSA: male: 7.3 ± 1.3%, female: 9.4 ± 2.1%; FCS: male: 9.2 ± 0.6%, female: 14.2 ± 1%; ANOVA 3, P = 0.01), whereas the proportion of stained cells was higher in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.02). The same tendency, although not significant, was obtained for the proportion of cells showing TUNEL staining with higher values in female than in male embryos (BSA: male: 9.3±2.1%, female: 10.5 ± 2.6%; FCS: male: 13.1 ± 0.9%, female: 16.5 ± 1.1%; ANOVA 3, P = 0.07) and higher values in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.05). A tendency for a higher proportion of double-stained cells (TUNEL and caspase-positive) was also observed in female embryos whatever the medium (BSA: male: 3.8 ± 1.1%, female: 5 ± 1.6%; SOF: male: 4.8 ± 0.5%, female: 7.0 ± 0.7%; ANOVA 3, P = 0.08). Intriguingly, only about half of the stained cells showed the double staining (TUNEL and caspase). This could be explained by the fact that caspase-3 activation can appear before DNA fragmentation during the apoptosis process and caspase staining disappear when TUNEL staining is still visible. But both caspase-3 activation and DNA fragmentation can also occur independently of apoptosis. In conclusion, male embryos seem to show a higher developmental rate in both media and could be less affected by apoptosis than female ones, particularly when cultured with FCS. Those experiments have to be repeated with the sexed sperm of another bull to draw final conclusions.

258 FACTORS AFFECTING COMMERCIAL SEXING PROGRAM AND MULTIPLE GENETIC ANALYSIS PERSPECTIVES OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 227
Author(s):  
R. V. Alonso ◽  
J. A. A. Hellú ◽  
S. H. V. Perri ◽  
J. A. Visintin ◽  
J. F. Garcia
Keyword(s):  
Mortality Rate ◽  
Sex Ratio ◽  
Genetic Analysis ◽  
Genetic Material ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Male And Female ◽  
Embryo Stage

The present study aimed to evaluate the interactions among different factors on the viability and sex ratio of in vitro-produced (IVP) bovine embryos, submitted to a large scale sexing program. Additionally, whole genome amplification (WGA) technology was used to amplify genomic DNA from IVP bovine embryo biopsies, in order to perform multiple genetic analyses. The survey was performed in a 4650 IVP bovine sexed embryo database. Embryos were biopsied by a microaspiration technique and sex was determined by PCR of DNA from the biopsy. Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified in accordance with embryo sex (male, female, and indeterminate), five laboratories (A, B, C, D, and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein, and Jersey), embryo stage (MO, EB, BL, XB, and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “nonstandard”). The statistical analysis was carried out by association chi-square test, chi-square for a 1:1 ratio, and logistic regression analysis (PROC LOGISTIC) of SAS. PCR showed 93.3% efficiency, 93.2% accuracy, and male and female rates of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Significant differences were not observed between male and female viability, although indeterminate embryos resulted in more death after micromanipulation. For quality 2 and 3 embryos, the mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryos. For embryos whose biopsies were classified as nonstandard, the embryonic mortality rate was 3.6-fold higher than standard ones. Mortality rate was not affected by embryo stage at biopsy (P > 0.05). Although sex ratio was significantly skewed to male embryos, differences were not observed among laboratories (P > 0.05) and breeds (P > 0.05) on the sex ratio of IVP bovine embryos. To test the feasibility of using WGA method for multiple genetic analysis, biopsies from 28 IVP embryos were submitted to the GenomePlex Single Cell System (Sigma-Aldrich, St. Louis, MO, USA). Aliquots from each DNA sample were purified using column chromatography and submitted to PCR using sexing primers BRY4a, SRY, UMN0920, and S4B. PCR was successful and in agreement among tested DNA aliquots from each single biopsy. The WGA strategy used herein was a useful tool for applications involving restricted amounts of starting genetic material (DNA), such as in preimplantation genetic diagnosis using IVP bovine embryos. To FAPESP and UNESP.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

2018 ◽  
Vol 30 (1) ◽  
pp. 205 ◽  
Author(s):  
R. Emmerstorfer ◽  
K. Radefeld ◽  
V. Havlicek ◽  
U. Besenfelder ◽  
H. Yu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Significant Difference

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

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.

scholarly journals Bioinformatic and experimental findings to indicate anti-cancer targets and mechanisms of calycosin against nasopharyngeal carcinoma

2020 ◽  
Author(s):  
Fangxian Liu ◽  
Qijin Pan ◽  
Liangliang Wang ◽  
Shijiang Yi ◽  
Peng Liu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Network Pharmacology ◽  
Tunel Staining ◽  
Pharmacological Targets ◽  
Experimental Findings

Abstract Background: Calycosin is a naturally-occurring phytoestrogen that reportedly exerts anti- nasopharyngeal carcinoma (NPC) effects. Nevertheless, the molecular mechanisms for anti-NPC using calycosin remain unrevealed. Methods: Thus, a network pharmacology was used to uncover anti-NPC pharmacological targets and mechanisms of calycosin. Additionally, validated experiments were conducted to validate the bioinformatic findings of calycosin for treating NPC. Results: As results, bioinformatic assays showed that the predictive pharmacological targets of calycosin against NPC were TP53, MAPK14, CASP8, MAPK3, CASP3, RIPK1, JUN, ESR1, respectively. And the top 20 biological processes and pharmacological mechanisms of calycosin against NPC were identified accordingly. In clinical data, NPC samples showed positive expression of MAPK14, reduced TP53, CASP8 expressions. In studies in vitro and in vivo, calycosin-dosed NPC cells resulted in reduced cell proliferation, promoted cell apoptosis. In TUNEL staining, calycosin exhibited elevated apoptotic cell number. And immunostaining assays resulted in increased TP53, CASP8 positive cells, and reduced MAPK14 expressions in calycosin-dosed NPC cells and tumor-bearing nude mice. Conclusion: Altogether, these bioinformatic findings reveal optimal pharmacological targets and mechanisms of calycosin against NPC, following with representative identification of human and preclinical experiments. Notably, some of original biotargets may be potentially used to treat NPC.

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.

313 APOPTOSIS IN PARTHENOGENETIC PIG EMBRYOS PRODUCED BY DIFFERENT METHODS

2006 ◽  
Vol 18 (2) ◽  
pp. 264
Author(s):  
J. Hyslop ◽  
Z. Machaty
Keyword(s):  
In Vitro Fertilization ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Total Cell Number ◽  
Vitro Fertilization ◽  
Tunel Reaction ◽  
Group 3 ◽  
Blastocyst Rate ◽  
Group 2

Apoptosis or programmed cell death is a process during which cells die in a controlled fashion in response to a variety of stimuli. Apoptosis has been demonstrated to occur during pre-implantation development both in vivo and in vitro and it is believed to contribute to early embryonic loss. It is also believed that parthenogenetic embryos generally have a lower developmental potential compared to those derived from fertilization. In the present study we investigated the rate of apoptosis in parthenogenetic pig embryos produced by activating oocytes through various methods. Pig oocytes were collected from slaughterhouse ovaries and matured in vitro. Parthenogenetic development was induced by three different methods. In Group 1, oocytes were activated by two consecutive electrical pulses. In Group 2, oocytes were electroporated and then incubated for 4 h in 5 mM butyrolactone I, a specific inhibitor of cyclin dependent kinases. In Group 3, electroporated oocytes were incubated for 5 h in cycloheximide, a protein synthesis inhibitor. Activated oocytes were cultured for 7 days in NCSU-23 medium. At the end of the culture period the embryos were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 with sodium citrate. They were then incubated in a TUNEL reaction medium that specifically identifies apoptotic nuclei by labeling fragmented DNA with a fluorochrome. Blastocysts produced by in vitro fertilization and DNase I-treated embryos were used as controls. The proportions of apoptotic cells were compared using ANOVA. Forty-three blastocysts were analyzed for apoptotic activity in the electroporation group. These embryos had a blastocyst rate of 33.6 ± 8.7%, total cell number of 31.9 ± 13.2, and an average of 2.7 ± 2.2 apoptotic cells per embryo; the rate of apoptosis was 9.1 ± 7.1%. Twenty-eight blastocysts were used for the TUNEL reaction in the group where activation was induced with the combined treatment of electroporation and butyrolactone (Group 2). On average, the blastocyst rate was 54.5 ± 6.4% and blastocysts contained 27.4 ± 9.6 cells of which 2.8 ± 3.9 were apoptotic; the percentage of apoptosis for this group was 10.0 ± 12.1%. In the cycloheximide treated group (Group 3), the onset of apoptosis was investigated using 29 blastocysts. The blastocyst rate was 38.2 ± 15.9% with an average total cell number of 27.2 ± 11.4%. The TUNEL assay revealed that the mean number of apoptotic cells per embryo in these blastocysts was 2.1 ± 1.5, representing 9.0 ± 7.6% apoptotic cells. The blastocysts (n = 14) produced by in vitro fertilization had a blastocyst rate of 18.0 ± 5.1% and 29.9 ± 12.0 cells; 9.2 ± 8.1% of these cells (2.6 ± 2.2 cells per embryo) showed signs of apoptosis. All nuclei in the DNase I-treated embryos showed positive signals following the TUNEL reaction. The results confirm previous findings that apoptosis occurs in blastocyst stage embryos. There was no difference in the percentage of apoptotic cells between embryos whose development was triggered by different oocyte activation methods; the rate of apoptosis in parthenogenetic blastocysts was similar to that in blastocysts produced by in vitro fertilization.

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.

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

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