177 ANTHOCYANIN ISOLATED FROM PURPLE SWEET POTATO IMPROVES DEVELOPMENT AND REDUCES OXIDATIVE STRESS OF BOVINE PRE-IMPLANTATION EMBRYOS EXPOSED TO HEAT SHOCK

Development of cleavage-stage pre-implantation embryos is disrupted by exposure to heat shock. Heat shock also increases intracellular reactive oxygen species (ROS) in pre-implantation embryos. Therefore, reduction of intracellular ROS levels might improve the development of heat-shocked embryos. Recently the antioxidative activities of polyphenols have been widely reported to reduce the oxidative stress. In this study, we investigated the effect of purple sweet potato anthocyanin, a kind of polyphenol that is a strong ROS scavenger, on development and intracellular redox status of bovine pre-implantation embryos exposed to heat shock. Experiment 1: In vitro-produced 8-16-cell-stage embryos on Day 2 after fertilization were exposed to 41.5�C for 6 h in CR1aa containing 0, 0.1, 1, and 10 �g/mL anthocyanin at 5% CO2, 5% O2, and 90% N2. After heat shock, embryos were cultured at 38.5�C at 5% CO2, 5% O2 until Day 8. On Day 8, the proportion of embryos developing to the blastocyst stage was evaluated. Blastocyst total cell number and the ratio between inner cell mass and tropheoderm were evaluated by differential staining. The experiment was replicated five times with more than 70 embryos used in each treatment. Experiment 2: Heat shock treatment of in vitro-produced 8-16-cell-stage embryos was carried out as described in experiment 1. After heat shock, intracellular ROS and glutathione (GSH) levels were measured in individual 8-16 cell stage embryos with fluorescent probes (22,72-dichlorodihydrofluorescein diacetate for ROS and CellTracker" Blue (Invitrogen Japan K. K., Tokyo, Japan) for GSH). The fluorescence emissions of each treatment were normalized to those of 8-16 cell stage embryos cultured at 38.5�C without anthocyanin to obtain the relative fluorescence emission. This experiment was replicated four times. Embryos treated with heat stress without anthocyanin (0 �g/mL) showed low development (14.6 � 3.6%) and blastocyst total cell number (88.2 � 9.4). However, embryos treated with 0.1 �g/mL anthocyanin improved development (31.7 � 4.5%, P < 0.05) and increased the total cell number (96.5 � 11.3). The higher concentrations of anthocyanin (1 and 10 �g/mL) did not affect development and cell number. The intracellular ROS levels in heat-shocked embryos were significantly reduced by all concentrations of anthocyanin (P < 0.05). In addition, anthocyanin increased GSH levels at all doses tested (P < 0.05). These results indicate that an appropriate concentration of anthocyanin improves development by regulating intracellular redox balance in bovine embryos exposed to heat shock.

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82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab82 ◽

2016 ◽

Vol 28 (2) ◽

pp. 170

Author(s):

L. D. Spate ◽

B. K. Redel ◽

R. S. Prather

Keyword(s):

Energy Source ◽

Subsequent Development ◽

Cell Number ◽

Total Cell ◽

Control Medium ◽

Blastocyst Development ◽

Total Cell Number ◽

Cell Stage ◽

Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

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81 HIGH THROUGHPUT VITRIFICATION OF IN VIVO FERTILIZED AND IN VITRO CULTURED PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab81 ◽

2011 ◽

Vol 23 (1) ◽

pp. 146

Author(s):

C. N. Murphy ◽

L. D. Spate ◽

B. K. Bauer ◽

R. S. Prather

Keyword(s):

Ethylene Glycol ◽

Uv Light ◽

Cell Number ◽

Total Cell ◽

Swine Industry ◽

Total Cell Number ◽

Cell Stage ◽

High Osmolarity

One barrier to successfully making embryo transfer viable in the swine industry is an inability to consistently cryopreserve oocytes and embryos. This process is made difficult by the high lipid content of porcine oocytes and embryos. The objective of this study was to test the in vivo fertilized embryo’s sensitivity to vitrification. Gilts were inseminated on the first day of standing oestrus (Day 0) and then again 12 h later. On Day 2 the oviducts and tip of the uterine horns were flushed with PVA-treated TL-HEPES and 2-cell stage embryos were collected and placed into PVA-treated TL-HEPES and centrifuged at 17 000 × g. The treatment groups were 1) 300 mOsmo centrifuged for 6 min, 2) 500 mOsmo centrifuged for 6 min, 3) 500 mOsmo centrifuged for 12 min, and 4) 500 mOsmo centrifuged for 18 min. After centrifugation the embryos were transferred to Porcine Zygote Medium 3 (PZM3) and cultured to Day 6 or 7 at which point blastocysts were vitrified using 10% DMSO, 10% ethylene glycol in M199 supplemented with 20% FBS (holding medium) for 2 min. Embryos were transferred to holding media with 20% DMSO and 20% ethylene glycol and drawn into an open pulled straw via capillary reaction; it was then submerged into LN2. Embryos were thawed using a step down concentration of 0.33 mM and then 0.2 mM sucrose in holding media each for 6–7 min and then were moved to holding medium alone for 6 to 7 min. The embryos were washed in PZM3, then transferred to 500 μL of PZM3 and cultured for 18 h. Re-expanded embryos were observed, and the nuclei of all embryos were stained with Biz-benzimide and visualised with UV light to determine total cell number. After the embryos were centrifuged and cultured, there was no difference in development to blastocyst (SAS Institute, Cary, NC, USA; Proc GLM) with a mean percentage blastocyst of 85.1% and an N of 54, 51, 53, and 51, respectively, for each treatment. After thawing, percentage of embryos re-expanded was 23.5a, 26.4a,b, 43.2a,b, and 45.6b, respectively. Data was analysed using a PROC GLM in SAS (P < 0.05), with 37, 43, 30, and 36 embryos in each group, respectively. No difference in total cell number across treatments was detected after analysis using PROC GLM in SAS (P < 0.05) with a mean cell number of 29.0. These data suggest that in vivo matured and fertilized blastocysts can survive high osmolarity treatment, centrifugation, and vitrification. The data also show that a high osmolarity treatment centrifuged for 18 min leads to a greater number of re-expanded embryos post-thaw, which may be attributed to better separation of the lipid. Funded by the NIH NCRR R21RR025879 and Food for the 21st Century.

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158APOPTOSIS IN IN VITRO PRODUCED BOVINE EMBRYOS ACCORDING TO DEVELOPMENTAL KINETICS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab158 ◽

2004 ◽

Vol 16 (2) ◽

pp. 201 ◽

Cited By ~ 2

Author(s):

F.V. Meirelles ◽

K.L. Schwarz ◽

G.K.F. Merighe ◽

S.F. Carambula ◽

Y.F. Watanabe

Keyword(s):

Rapid Development ◽

Rna Extraction ◽

Cell Number ◽

Total Cell ◽

Differential Staining ◽

Bovine Embryos ◽

Total Cell Number ◽

Cell Stage ◽

Slow Development

Apoptosis has been previously reported in embryos during late pre-implantation development. Fast-developing embryos are known to present higher developmental competence. The aim of the present work was to evaluate the quality of in vitro-produced bovine embryos with fast (8-cells at 48 hours post-insemination (hpi) and slow (8-cells at 90hpi) cleavage and study the correlation of this phenotype with programmed cell death occurrences. Embryos were produced from immature oocytes obtained from slaughtered cow ovaries, after maturation and fertilization, presumed zygotes were cultured in CR2 medium with 10% FCS, together with granulosa cells under 5% CO2 atmosphere. The number of nuclei in the inner cell mass and trophectoderm (ICM/TE), as well as the number of nuclei with fragmented DNA, were estimated by applying differential staining and TUNEL, respectively; data were analyzed by ANOVA (JMP—SAS Institute). To test the expression of apoptosis regulating genes, a pool of fifty 8-cell embryos from each group (fast and slow) were collected. After RNA extraction and reverse transcriptase reaction, cDNA was amplified with Bax and Bcl2 primers, individually. Results indicated, as expected, higher quality in fast-cleaving embryos, estimated by the number of ICM nuclei (20.8±1.4 and 15.6±2.1—P≤0.05); however, the number of TE didn’t show significant differences (54.9±2.4 and 53.2±3.8); the same was observed for total cell number (75.7±2.8 and 68.8±4.4). The frequency of blastocyst TUNEL-positive nuclei as an estimate of total cell number was significantly larger in the slow group when compared to the rapid development group (19.0±2.5% and 8.5±1.4%, respectively, P≤0.05). The greater proportion of morphologic abnormal nuclei in both groups was located in the ICM, and may explain the lower number of ICM nuclei in slow developing embryos. Hence, embryos of slow development show TUNEL-positive blastomeres at the 8-cell stage, but no fragmented nuclei were observed in embryos at 48hpi. Bax and Bcl2 cDNA amplification showed that both mRNAs were constitutively present at the 8-cell stage in both groups. It can be concluded that in vitro-produced bovine blastocysts, with slow development to the 8-cell stage, present lower quality compared with fast development homologues, estimated by mean number of ICM nuclei, as well as nuclei fragmentation in blastomeres (TUNEL-positive). There is a difference in fragmented nuclei proportion between both groups at the 8-cell stage, but this result may be biased by the numbers of hours in culture. It was possible to demonstrate the presence of mRNA for pro (Bax) and anti-apoptotic (Bcl2) genes in slow- and fast-developing embryos at the 8-cell stage, and the future determination of the ratio between these two transcripts may allow the evaluation of the participation of pre-transcriptional regulation of these genes on the induction of DNA fragmentation. Financial support: Grant 99/12351-3 FAPESP São Paulo, Brazil.

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130INSULIN-LIKE GROWTH FACTOR-1 AND INTERLEUKIN-11 AS POSSIBLE SURVIVAL FACTORS FOR THE BOVINE PREIMPLANTATION EMBRYO EXPOSED TO STRESS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab130 ◽

2004 ◽

Vol 16 (2) ◽

pp. 188 ◽

Cited By ~ 2

Author(s):

F. D. Jousan ◽

J. Hernández-Ceron ◽

C. M. Franco ◽

P. J. Hansen

Keyword(s):

Heat Shock ◽

Least Squares ◽

Cell Injury ◽

Cell Number ◽

Total Cell ◽

Bovine Embryos ◽

Total Cell Number ◽

Cell Stage ◽

Survival Factors ◽

Interleukin 11

Both IGF-1 and interleukin-11 (IL-11) are survival factors that modify response to cell injury. Moreover, IGF-1 promotes preimplantation development (Mol. Reprod. Dev. 62, 489) and IL-11 has been reported to reduce effects of heat shock on bovine embryos (Theriogenology 59, 343). For this study, it was hypothesized that IGF-1 and IL-11 improve survival of bovine embryos exposed to lethal stimuli. Embryos were produced in vitro and cultured in KSOM medium. Treatment effects were analyzed using least squares ANOVA with the GLM procedure of SAS (SAS Inst., Inc., Cary, NC, USA). In Exp. 1, 100ngmL−1 IGF-1 increased (P<0.01) the percent of oocytes that became blastocyst at 8 days post-insemination (dpi) (19.0% for control v. 24.5% for IGF-1; SEM=1.3%; 7 replicates; 105 embryos/treatment). For Exp. 2, embryos were cultured±100ngmL−1 IGF-1. At 5 dpi, embryos≥16 cells were cultured at either 38.5°C for 24h or 41°C for 9h and then 38.5°C for 15h followed by TUNEL analysis (7 replicates; 86–100 embryos/treatment). At 38.5°C, IGF-1 did not affect total cell number (60.5 v. 64.4 for control and IGF-1, respectively; SEM=2.3) or percent of blastomeres undergoing apoptosis as determined by TUNEL (5.9% v. 5.7%; SEM=0.6%). Heat shock reduced total cell number (P<0.05) and increased the percent of cells that were TUNEL-positive (P<0.001). For heat-shocked embryos, total cell number was 46.0 for control v. 59.8 for IGF-1 (SEM=2.3) and percent of TUNEL-positive blastomeres was 11.6% for control v. 5.9% for IGF-1 (SEM=0.6%). Effects of heat shock were less for IGF-1-treated embryos (temperature×IGF-1, P=0.07 for cell number and P<0.01 for TUNEL). For Exp. 3 (4 replicates; 111–136 embryos/treatment), embryos were cultured±100ngmL−1 IGF-1 beginning at the 1-cell stage in control medium (KSOM), ethanol (1.0%; v/v) or gossypol (10μgmL−1 in 1% ethanol). Percent of blastocysts at 8 dpi was affected by treatment (P<0.01) and IGF-1 (P<0.04). Without IGF-1, least-squares means were 24.6%, 13.6% and 0.9% for control, ethanol, and gossypol, respectively (SEM=1.7%). With IGF-1, least-squares means were 29.3±3.4%, 26.5±1.6%, and 8.7%±1.7% for control, ethanol, and gossypol (control v. ethanol, NS; control v. gossypol, P<0.01). Thus, IGF-1 blocked the effect of ethanol on development. For Exp. 4, putative zygotes were cultured±10ngmL−1 human IL-11 (4 replicates; 201–214 zygotes/treatment). At 3 dpi, embryos remained at 38.5°C or were cultured at 41°C for 9h and then returned to 38.5°C. Heat shock reduced (P<0.01) the percent of putative zygotes and cleaved embryos that became blastocysts at 8 dpi but IL-11 had no effect at either temperature (percent zygotes to blastocysts=25.3% and 25.6% for control and IL-11 at 38.5°C and 14.0% and 11.8% for control and IL-11 at 41°C; SEM=3.4%). In conclusion, IGF-1 blocked induction of apoptosis caused by heat shock and the reduction in development caused by ethanol. Thus, IGF-1 may play an important role in early development by acting as a survival factor. There was no evidence that IL-11 conferred thermoprotection to bovine embryos. (Support: USDA NRICGP 2002-35203-12664, USDA IFAFS #2001-52101-11318, and USDA TSTAR 2001-34135-11150.)

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48EFFECT OF INSULIN-LIKE GROWTH FACTOR-1 SUPPLEMENT TO NCSU-23 MEDIUM ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM IN VITRO FERTILIZATION AND SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab48 ◽

2004 ◽

Vol 16 (2) ◽

pp. 146

Author(s):

S. Kim ◽

D.H. Nam ◽

Y.W. Jung ◽

H.S. Kim ◽

S.H. Lee ◽

...

Keyword(s):

Growth Factor ◽

Nuclear Transfer ◽

Cell Number ◽

Total Cell ◽

Insulin Like Growth Factor ◽

Total Cell Number ◽

Cell Stage ◽

Vitro Fertilization ◽

Igf I

The developmental potential of in vitro production of embryos is affected by various factors, including the culture system, oocyte quality, the presence of serum, and embryo paracrine and autocrine growth factors. Insulin-like growth factor is a good stimulator of oocyte maturation and embryo development. The present study investigated the effect of insulin-like growth factor-I (IGF-I) supplement on the preimplantation development of porcine embryos derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. The number of total cells and inner cell mass (ICM) cells in blastocysts were counted after differential staining at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). In Experiment 1, a total of 2,462 in vitro-matured oocytes (527, 458, 498, 481 and 498, respectively) were inseminated with frozen-thawed boar semen and subsequently cultured in North Carolina State University (NCSU)-23 medium supplemented with various concentrations of IGF-1 (0, 1, 10, 50 and 100ngmL−1). As a result, significant model effects on the development to the 2-cell stage (P=0.033) and to the blastocyst stage (P=0.0067) were found, and more blastocysts (16.9, 16.6, 17.5, 21.8 and 14.7 %, respectively) were obtained in medium supplemented with 50ngmL−1 of IGF-I. Moreover, increase in the total cell number (56.5, 53.2, 74.0, 76.4 and 58.4) and ICM (6.6, 5.8, 9.3, 9.4 and 6.1) cells was observed in IVF embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 IGF-1. In Experiment 2, porcine cloned embryos were produced by our standard protocol using fetal fibroblasts as donor cells (Hyun SH et al., 2003 Theriogenology 59, 1641–1649) and cultured in NCSU-23 supplemented with the same concentration of IGF-1 as Experiment 1. As a result, a total of 501 reconstructed oocytes (99, 98, 102, 99 and 96, respectively) were cultured and significant model effects on the development to the 2-cell stage (P=0.0179) were found. More blastocysts (10.5, 11.2, 11.8, 20.8 and 10.1%) were produced when embryos were cultured in NCSU-23 medium supplemented with 50ngmL−1, even though no statistical significance was found (P=0.1182). Increases in the total cell number (42.7, 46.0, 45.9, 51.1 and 38.2) and ICM cells (3.8, 3.8, 5.6, 6.6 and 4.8, respectively) were observed in cloned embryos cultured in NCSU-23 medium supplemented with 50ngmL−1 of IGF-I. In conclusion, the present study demonstrated that IGF-1 at the concentration of 50ngmL−1 improves the development of preimplantion embryos derived from IVF and SCNT. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).

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198 BENEFICIAL EFFECT OF GHRELIN ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO-FERTILIZED AND PARTHENOGENETIC EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab198 ◽

2007 ◽

Vol 19 (1) ◽

pp. 215

Author(s):

K. Zhang ◽

H. X. Wei ◽

S. H. Wang ◽

Y. H. Zhang ◽

Y. Li ◽

...

Keyword(s):

Milk Powder ◽

Cell Number ◽

Total Cell ◽

Control Group ◽

Female Reproduction ◽

Total Cell Number ◽

Cell Stage ◽

Treatment Groups ◽

Parthenogenetic Embryos

Accumulating evidence suggests that ghrelin plays an important role in female reproduction. The objective of the present study was to investigate the effect of ghrelin on pre-implantation development of porcine in vitro-fertilized (IVF) and parthenogenetic embryos. Cumulus–oocyte complexes were matured for 44 h in BSA-free NCSU23 supplemented with 10 ng mL-1 epidermal growth factor, 10 ng mL-1 leptin, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, and 10 IU mL-1 hCG. After removal of the cumulus cells, some oocytes were fertilized with fresh boar semen (1 � 105 sperm mL-1) in modified Tween medium B with milk powder (Abeydeera and Day 1997 Theriogenology 48, 537–544) and some oocytes were activated by a single, 100-�s, direct current pulse of 1.4 kV cm-1. Presumptive zygotes (Experiment 1) and parthenogenetic oocytes (Experiment 2) were subsequently cultured in PZM3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) supplemented with ghrelin at 0 (control), 0.5, 5, 50, and 500 ng mL-1 (ghrelin 0.5, 5, 50, 500 groups, respectively) under 5% CO2, 5% O2, 90% N2 and 100% humidity at 39.0�C. Cleavage and blastocyst rates were assessed on Days 2 and 6 (Day 0: the day IVF and activation were conducted). The total cell number in blastocysts was determined by Hoechst 33342 staining on Day 6. All data were analyzed by using SPSS (13.0) with one-way ANOVA. All experiments were done at least 4 times. In Experiment 1, the rate of blasotcyst formation in IVF embryos was significantly (P < 0.05) increased in the ghrelin 500 group compared with that in the control group (26.1 � 1.8 vs. 12.4 � 6.0%, mean � SEM). Furthermore, increased total cell numbers (P < 0.05) were observed in the ghrelin 50 and 500 groups compared with that in the control group (63 � 6.6 and 64 � 5.5 vs. 42 � 6.6). In Experiment 2, we found that the blastocyst rate of parthenogenetic embryos was significantly (P < 0.05) higher in the ghrelin 5 and 500 groups than in the others (24.6 � 4.7 and 25.0 � 3.3 vs. 13.3 � 2.7, 14.9 � 2.4, 18.1 � 2.3% in the control and ghrelin 0.5 and 50 groups, respectively; P < 0.05). The total cell number per blastocyst was significantly increased in the ghrelin 50 group compared with that of the control group (85 � 10.2 vs. 56 � 8.0, P < 0.05). The maximum total cell number in the ghrelin treatment groups of parthenogenetic embryos was higher than in the control group (82, 93, 102, 100 in the ghrelin 0.5, 5, 50, 500 groups, respectively, vs. 69; P < 0.05). We also found that more embryos were developed to the morula stage and fewer embryos died early at the 2- to 4-cell stage in the ghrelin treatment groups than in the control group (data not shown) in both Experiments 1 and 2. The results suggest that supplementation with ghrelin in the embryo culture medium could enhance the pre-implantation development of porcine IVF and parthenogenetic embryos. This study was funded by the Natural Scientific Foundation of Beijing (5030001).

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

Zygote ◽

10.1017/s0967199416000228 ◽

2016 ◽

Vol 24 (6) ◽

pp. 890-899 ◽

Cited By ~ 4

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.

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The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽

10.1242/dev.102.4.793 ◽

1988 ◽

Vol 102 (4) ◽

pp. 793-803 ◽

Cited By ~ 3

Author(s):

V.E. Papaioannou ◽

K.M. Ebert

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Staining Technique ◽

Cell Number ◽

Total Cell ◽

Differential Staining ◽

Morphological Development ◽

Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽

10.1017/s0967199408005005 ◽

2009 ◽

Vol 17 (1) ◽

pp. 57-61 ◽

Cited By ~ 4

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.

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