Effects of oviductal fluid on the development, quality, and gene expression of porcine blastocysts produced in vitro

In mammals, fertilization and early pre-implantation development occur in the oviduct. Previous results obtained in our laboratory have identified specific molecules in the oviduct that affect porcine sperm–egg interactions. The aim of the present study was to determine whether the contact between oocytes and oviductal fluid also affect embryo development, quality, and gene expression. In vitro matured porcine oocytes were exposed to bovine oviductal fluid (bOF) for 30 min prior to fertilization. Cleavage and blastocyst development rates were significantly higher from bOF-treated oocytes than from untreated oocytes. Blastocysts obtained from bOF-treated oocytes had significantly greater total cell numbers than those obtained from untreated oocytes. Using real-time PCR, grade 1 (very good morphological quality) and grade 2 (good morphological quality) blastocysts were analyzed for gene transcripts related to apoptosis (BAX, BCL2L1), mitochondrial DNA (mtDNA) transcription/replication (POLG, POLG2, and TFAM), blastomere connection and morula compaction (GJA1), and blastocyst formation and pluripotency (POU5F1). We found that the entire set of genes analyzed was differentially expressed between grade 1 and 2 blastocysts. Furthermore, bOF treatment reduced the ratio of BAX to BCL2L1 transcripts and enhanced the abundance of TFAM transcripts in grade 2 blastocysts. Not only do these findings demonstrate that factors within the bOF act on porcine oocytes both quickly and positively, but they also suggest that such factors could promote embryo development and quality by protecting them against adverse impacts on mtDNA transcription/replication and apoptosis induced by the culture environment.

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282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab282 ◽

2007 ◽

Vol 19 (1) ◽

pp. 256

Author(s):

W. J. Son ◽

M. K. B. ◽

Y. J. Jeong ◽

S. Balasubramanian ◽

S. Y. Choe ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Expression Patterns ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Blastocyst Development ◽

Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P < 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P < 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P < 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P < 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P < 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

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292 OOCYTES FROM FOLLICLES OF DIFFERENT DIAMETERS: EMBRYO IN VITRO DEVELOPMENTAL POTENTIAL AND QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab292 ◽

2007 ◽

Vol 19 (1) ◽

pp. 261

Author(s):

T. H. C. de Bem ◽

K. R. L. Schwarz ◽

L. G. Mesquita ◽

P. R. Adona ◽

C. L. V. Leal

Keyword(s):

Apoptotic Cell ◽

Total Cell ◽

Sperm Cells ◽

Hatching Rate ◽

Blastocyst Development ◽

Visual Evaluation ◽

Developmental Potential ◽

Cell Numbers ◽

Follicle Diameter

The present study aimed to assess the developmental potential and quality of embryos produced from oocytes originating from follicles of different sizes. Ovaries were collected at a slaughterhouse and follicles of <3, 3 to 6, and >6 mm were aspirated. Follicle diameters were estimated based on the size of their exposed surface on the ovarian cortex using a ruler as reference for the first aspirations and were then based on visual evaluation. Aspirated oocytes, separated by their follicular origin, were matured in vitro in TCM-199 supplemented with 10% FCS, 5.0 �g mL-1 of LH, 0.5 �g mL-1 of FSH, 0.2 mM pyruvate, and 10 �g mL-1 of gentamicin for 22 h. After in vitro maturation, oocytes were in vitro-fertilized (IVF) using frozen–thawed semen prepared by Percoll gradient. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 of heparin. After 20 h, presumptive zygotes were partially denuded and transferred to in vitro culture (IVC) medium (TCM-199 supplemented with 10% FCS, 2.0 mM pyruvate, and 10 mg mL-1 of gentamicin). All cultures were incubated at 38.5�C under 5% CO2 in air and maximum humidity. The cleavage rate was assessed after 48 h of IVC, and blastocyst development was assessed on Day 7 (D7). On Day 9 (D9), the hatching rate was assessed and the hatched embryos were fixed for 1 h (3% paraformaldehyde in PBS), permeabilized for another h (0.5% Triton-X, 0.1% sodium citrate, and 0.1% plyvinyl alcohol in PBS), and evaluated by the TUNEL technique (in situ cell death detection kit). Total cell number and TUNEL-positive cells in embryos were counted under an epifluorescence microcope. Data of 4 replicates were analyzed by ANOVA and comparisons among groups were made by the Tukey test. The level of significance used was 5%. From the oocytes used (<3 mm = 100; 3–6 mm = 99; >6 mm = 88), cleavage rates increased with increasing follicular diameter. Oocytes originating from <3-, 3- to 6-, and >6-mm follicles resulted in 75, 93.6, and 95.5% cleavage rates, respectively (P > 0.05). For blastocyst rates on D7, <3-mm follicles showed 21.5% blastocyt development, which was lower (P < 0.05) than for the other follicle diameter groups (3–6 mm = 35.4% and >6 mm = 41.1%; P > 0.05). Regarding the hatching rate, total cell numbers, and TUNEL-positive cells on D9, <3 mm (20.4%, 268, and 0.37%, respectively), 3–6 mm (29.1%, 248, and 0.32%, respectively), and >6 mm (32.3%, 237, and 0.23%, respectively) were not different (P > 0.05). The results suggest that oocytes from larger follicles are more competent for cleavage and blastocyst development on D7; however, when oocytes reach the blastocyst stage, hatching, total cell numbers, and apoptotic cell numbers are not influenced by follicle diameter. Financial suport for this work was provided by the FAPESP, Brazil.

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

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab188 ◽

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 (<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's exact test and Student'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 < 0.01), Sigma + AlaGln: 12.8% (P < 0.01), and Sigma + Gln: 11.9% (P < 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 � 10) were significantly decreased: 35.5 � 7 (P < 0.001), 34.9 � 9 (P < 0.001), and 29.9 � 9 (P < 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 < 0.01) and 46.6 � 9 vs. 29.4 � 9 P < 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 < 0.01) and 46.9 � 10 vs. 33.6 � 6 (P < 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 < 0.01), and total cell number, 33.6 � 6 vs. 29.4 � 9 (P < 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.

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In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos

International Journal of Molecular Sciences ◽

10.3390/ijms21197067 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7067

Author(s):

Meritxell Vendrell-Flotats ◽

Tania García-Martínez ◽

Iris Martínez-Rodero ◽

Manel Lopez-Bejar ◽

Jonathan LaMarre ◽

...

Keyword(s):

Gene Expression ◽

Embryonic Development ◽

Embryo Development ◽

Leukemia Inhibitory Factor ◽

In Vitro Maturation ◽

Expression Patterns ◽

Inhibitory Factor ◽

Blastocyst Development ◽

Developmental Potential

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

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Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro

Reproduction ◽

10.1530/rep-16-0384 ◽

2017 ◽

Vol 153 (4) ◽

pp. 461-470 ◽

Cited By ~ 47

Author(s):

Ricaurte Lopera-Vasquez ◽

Meriem Hamdi ◽

Veronica Maillo ◽

Alfonso Gutierrez-Adan ◽

Pablo Bermejo-Alvarez ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Extracellular Vesicles ◽

Water Channel ◽

Calf Serum ◽

Bovine Embryos ◽

Analysis System ◽

Oviductal Fluid

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C− group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7–9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C− and OF-EV groups (12.0–14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5–30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C− group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C− being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

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285BIRTH OF PIGLETS AFTER NON-SURGICAL TRANSFER OF PORCINE EMBRYOS CULTURED IN PZM-4 WITH ALTERED CONCENTRATIONS OF AMINO ACIDS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab285 ◽

2004 ◽

Vol 16 (2) ◽

pp. 262

Author(s):

C. Suzuki ◽

K. Yoshioka ◽

S. Iwamura

Keyword(s):

Amino Acids ◽

Embryo Development ◽

Intrauterine Insemination ◽

Blastocyst Stage ◽

Total Cell ◽

Developmental Competence ◽

Double Dose ◽

In Vitro Production ◽

Cell Numbers

We previously developed an in vitro production (IVP) system for porcine embryos and obtained piglets after surgical transfer of blastocysts cultured in Porcine Zygote Medium (PZM)-4. However, the developmental competence of pig IVP embryos to the blastocyst stage is still low and further improvement of IVC medium is needed. In the present study, we evaluated the effects of the addition of glutamine (Gln), hypotaurine (HT), taurine (Tau), BME-essential (EA) and MEM-nonessential (NA) amino acids solutions to PZM-4, and the replacement of polyvinyl alcohol (PVA) with BSA on embryo development to blastocysts. Moreover, the developmental competence of IVP blastocysts after nonsurgical embryo transfer (NS-ET), using a flexible catheter (FC) for deep intrauterine insemination, was investigated. Porcine COC from prepubertal gilts were matured and fertilized in vitro, using frozen-thawed ejaculated boar semen. Presumptive zygotes were cultured in PZM-4, as a basal culture medium, until Day 5 after IVF. Data from six replicates were analyzed by ANOVA. Addition of 0.25 to 4mM Gln to PZM-4 (containing 5mM HT) significantly increased the percentage of embryos that developed to blastocysts (15 to 31%), with addition of 2mM Gln significantly increasing the total cell numbers in blastocysts (43±17 cells) compared with no addition (3% and 20±4 cells, respectively). Addition of 1.25 to 10mM HT to HT-free PZM-4 supplemented with 2mM Gln (named PZM-5) significantly increased the percentage of embryos that developed to blastocysts (22 to 28%) compared with control (no HT;; 4%). In the culture with HT-free PZM-5, addition of 5mM Tau significantly increased blastocyst yield (17%) compared with control (4%). However, Tau addition in the presence of 5mM HT had no effect on development to the blastocyst stage. In combinations of EA and NA added to PZM-5, a single dose of EA significantly increased the percentage of embryos that developed to blastocysts (27%) compared with no dose (19%) or with a double dose of EA (20%), while a double dose of NA significantly increased the total cell numbers in blastocysts (43±16 cells) compared with no NA (37± 6 cells). Replacement of PVA with BSA in PZM-5 had no effect on embryo development to the blastocyst stage. Crossbred sows were used as recipients for NS-ET, and had their estrous cycle synchronized by a described previously method (Yoshioka et al., 2002 Biol. Reprod. 66, 112–119). Five days after hCG injection, a FC was introduced via the cervix into the uterine horn of recipients without sedation. Day-5 blastocysts cultured in PZM-5 were then transferred together with 5ml of TALP-Hepes (45 to 50 blastocysts/recipient). Of 6 recipients, one sow became pregnant and farrowed 7 piglets. Our results indicate that the addition of amino acids to PZM-4 can improve porcine embryo development to the blastocyst stage, and that blastocysts cultured in a chemically defined medium, PZM-5, can develop to full-term following NS-ET.

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Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1)

Reproduction Fertility and Development ◽

10.1071/rd16302 ◽

2017 ◽

Vol 29 (10) ◽

pp. 2011 ◽

Cited By ~ 14

Author(s):

Imran Khan ◽

Sung Woo Kim ◽

Kyung-Lim Lee ◽

Seok-Hwan Song ◽

Ayman Mesalam ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Group Treatment ◽

Sirtuin 1 ◽

Developmental Competence ◽

Control Group ◽

Blastocyst Development ◽

Protein Levels ◽

Development In Vitro

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription–polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0 µM) added to the IVM medium, only 1.0 µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P < 0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P < 0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2 were significantly (P < 0.05) lower in the PD-treated group than in the control group. Treatment with 1.0 µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

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156 IN VITRO DEVELOPMENT OF BOVINE MORULAE PRODUCED AND/OR CULTURED WITH ACTIVIN

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab156 ◽

2010 ◽

Vol 22 (1) ◽

pp. 236 ◽

Cited By ~ 1

Author(s):

B. Trigal ◽

E. Gómez ◽

C. Diez ◽

J. N. Caamaño ◽

I. Molina ◽

...

Keyword(s):

Embryo Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Total Cell ◽

Blastocyst Development ◽

Cell Counts ◽

Morula Stage ◽

Vitro Development ◽

And Control

We reported that the presence of activin during in vitro culture improves embryo development without changing the cell distribution in the blastocyst (Díez et al. 2009 AETE in press). In the present work, we aimed to analyze the morula stage as a putative milestone to activin exert differential effects. Day -5 morulae were produced with IVMFC oocytes from abattoir ovaries, using SOF with amino acids, myo-inositol, and 3 g L-1 of BSA as a culture medium. Embryo culture contained 10 ng mL-1 or 0 ng mL-1 of activin from Day -3 to Day -5. Early morulae (n = 543 out of 1099 cultured oocytes) were selected and subsequently cultured with or without 10 ng mL-1 of activin up to Day -8. Embryo development was daily monitored and cells differentially counted in Day -8 expanded blastocysts. (Thouas et al. 2001 Reprod. Biomed. 2001 3, 25-29). Data were analyzed by general linear model and presented as least squares means ± SEM. Activin from Days 3 to 5 did not change Day -5 morulae rates (P > 0.8). In morulae produced without activin (Days 5 to 8 and control), a treatment with activin from Days 5 to 8 improved total blastocyst rates v. controls, both in Day -7 and Day -8 (50.9 ± 3.6 v. 32.6 ± 3.6 and 60.8 ± 2.9 v. 42.3 ± 2.9, respectively; P < 0.01). Similarly, Day -7 expansion rates with activin (Days 5 to 8) were higher than controls (14.6 ± 1.8 v. 8.6 ± 1.8; P < 0.03). However, the above effects were not the same as those observed in morulae produced with activin (Days 3 to 5 and Days 3 to 8), where blastocyst development between activin treatment and controls only significantly differed in expansion rates on Day -7 (14.9 ± 1.8 v. 5.8 ± 1.8, respectively; P < 0.03). Morulae treated with activin (Days 5 to 8) yielded Day -7, total and expanded blastocyst rates, higher than morulae produced with activin (Days 3 to 5) (50.9 ± 3.6 v. 37.4 ± 3.6 and 14.6 ± 5.8 v. 5.8 ± 1.8, respectively; P < 0.03). Expansion rates on Day -8 were numerically higher within morulae produced and/or treated with activin (Days 3 to 8, Days 5 to 8, and Days 3 to 5) (values between 26.7 ± 2.6 and 27.4 ± 2.6) than in controls without activin at any time (19.2 ± 2.6) (P > 0.05). Trophectoderm (TE) cell numbers were reduced in embryos produced and/or treated with activin (Days 3 to 8, Days 3 to 5, and Days 5 to 8) (values between 109.4 ± 7.6 and 115.3 ± 7.9) as compared with untreated controls (141.2 ± 10.1) (P < 0.05). In morulae produced without activin, total cell counts were lower with activin being present from Day -5 to Day -8 (154.0 ± 8.8 v. 128.4 ± 7.2; P < 0.05). Inner cell mass (ICM) and ICM/total cell ratio were not affected by the presence of activin (P > 0.05). Activin did not change Day -5 morulae rates, although subsequent blastocyst development was in part affected by the presence of activin before the morula stage. Interestingly, improvements in blastocyst development, including expansion rates, triggered by activin led to reduced TE and unaltered ICM cell counts, suggesting that activin inhibits TE differentiation. Support: Cajastur (B. Trigal). MCINN: M. Muñoz (RYC08-03454); D. Martín (PTA2007-0268-I); INIA (I. Molina); Project HF2007-0126.

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218 DEVELOPMENT OF EVENLY AND UNEVENLY CLEAVED TWO-CELL PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab218 ◽

2008 ◽

Vol 20 (1) ◽

pp. 188

Author(s):

D. N. Q. Thanh ◽

K. Kikuchi ◽

T. Somfai ◽

M. Ozawa ◽

M. Nakai ◽

...

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Great Influence ◽

Total Cell ◽

Blastocyst Development ◽

Cell Stage ◽

Cell Numbers ◽

Blastocyst Rate ◽

Further Development

Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 � 0.8% and 91.4 � 3.6%, respectively) or between the 36E and 36U groups (98.2 � 1.1% and 88.0 � 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 � 5.7% and 61.7 � 7.8%, respectively) and total cell numbers (39.1 � 2.1 and 31.7 � 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 � 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 � 5.1%), the total cell number was not different (26.3 � 5.5 and 25.3 � 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 � 6.3%) than did the 36-h embryos (28.2 � 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 � 2.6) were significantly higher than those of the 36-h embryos (26.2 � 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 � 7.0%) and greater cell numbers (40.6 � 2.6) than those not showing a second division during this period (30– group; 46.8 � 5.0% and 19.9 � 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.

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Quality of porcine blastocysts produced in vitro in the presence or absence of GH

Reproduction ◽

10.1530/rep.1.00086 ◽

2004 ◽

Vol 127 (2) ◽

pp. 165-177 ◽

Cited By ~ 24

Author(s):

A Kidson ◽

F J Rubio-Pomar ◽

A Van Knegsel ◽

H T A Van Tol ◽

W Hazeleger ◽

...

Keyword(s):

Embryo Development ◽

Reference Value ◽

Cell Number ◽

Blastocyst Stage ◽

Terminal Deoxynucleotidyl Transferase ◽

Blastocyst Development ◽

Cell Numbers

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

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