Exogenous platelet-activating factor stimulates cell proliferation in mouse pre-implantation embryos prior to the fourth cell cycle and shows isoform-specific stimulatory effects

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 261-268 ◽  
Author(s):  
Neil R. Stoddart ◽  
William E. Roudebush ◽  
Steven D. Fleming
Keyword(s):  
Cell Proliferation ◽  
Biological Activities ◽  
Platelet Activating Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Embryonic Cell ◽  
Cell Stage ◽  
Cell Numbers ◽  
Paf Receptor

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 μM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 μM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embyronic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE

2006 ◽  
Vol 18 (2) ◽  
pp. 248
Author(s):  
S.-G. Lee ◽  
C.-H. Park ◽  
D.-H. Choi ◽  
H.-Y. Son ◽  
C.-K. Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Transgenic Animals ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Vitro Fertilization

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos. Table 1. Development, cell number, and ICM ratio of aggregated porcine embryos

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

scholarly journals 276INFLUENCE OF SPERM TREATMENTS ON BLASTOCYST DEVELOPMENT IN VITRO AND CELL NUMBER IN CATTLE

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
B.G. Jeon ◽  
J.S. Moon ◽  
K.C. Kim ◽  
G.J. Rho
Keyword(s):  
Silicone Oil ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Glass Wool ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Serum Free ◽  
Cell Numbers ◽  
Bull Sperm

Experiments were designed to examine the effects of developmental rate and cell numbers in embryos produced by in vitro fertilization (IVF) using sperm from 2 bulls (sperm A and B purchased from commercial sale) isolated by three methods. Cumulus-oocyte-complexes collected from ovaries harvested from a local slaughter house were matured in 50μL droplets of serum-free M199 medium supplemented with 1μgmL−1 estradiol-17β, 10μgmL−1 LH and FSH under silicone oil at 39°C in a humidified atmosphere of 5% CO2 in air. After 24h of culture, oocytes were fertilized with the sperm treated by three different methods of isolation;; percoll gradient, swim-up and glass wool filtration;; a final concentration of 2×106 cells mL−1 was used. At 16h after fertilization, presumptive zygotes were co-cultured in serum-free M199 with BOEC for up to 192h post-insemination. At 48h and 120h post-insemination, the cultures were fed with 25μL of serum-free M199. The embryos were compared for their rates of cleavage at 48h post-insemination, development to the blastocyst stage, and hatching, and also the cell number at 192h post-insemination. Differences between treatments were analyzed using one-way ANOVA after arc-sine transformation of the proportional data of cleavage, development into blastocyst stage and hatching. Comparisons of means among treatments were performed using Tukey-Kramer multiple comparisons test. The results are summarized below. The rates of cleavage in embryos produced by IVF using sperm from 2 bulls isolated by percoll, swim-up and glass wool were not significantly different. The blastocyst development and hatching rates between sperm treatment were not significant within bull sperm A and within sperm B. However, although the hatching rate in percoll treatment of bull sperm A was higher than in bull sperm B, the difference was not statistically significant. The mean cell numbers in percoll treatment of bull sperm A (176.5±7.1) were significantly higher (P&lt;0.001) than the others. In bull sperm B the cell numbers in percoll treatment were higher than the other two treatments, but the differences were not statistically significant. In conclusion, these results support the concept that sperm preparation using percoll has beneficial effects on blastocyst cell number. Table 1 Developmental rates of in vitro embryos using sperm from 2 bulls isolated by three methods with 4 replicates

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals Controlled spermatozoa–oocyte interaction improves embryo quality in sheep

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Debora Agata Anzalone ◽  
Luca Palazzese ◽  
Marta Czernik ◽  
Annalaura Sabatucci ◽  
Luca Valbonetti ◽  
...  
Keyword(s):  
Embryo Quality ◽  
Time Frame ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Free Oxygen Radicals ◽  
High Concentration ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Set Up

AbstractThe current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture.

75 Culture of isolated blastomeres supplemented with L-ascorbic acid 2-phosphate in a well-of-the-well culture dish

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
Y. Hasiyada ◽  
H. Matsuda ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
T. Yamanouchi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage ◽  
Quality Grade

We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P&lt;0.05) and 500 µM (150v. 158 hpi; P&lt;0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.

scholarly journals 194 BIRTH OF KITS AFTER STORAGE IN CULTURE AND TRANSFER OF IN VIVO EMBRYOS IN THE FARMED EUROPEAN POLECAT MUSTELA PUTORIUS)

2005 ◽  
Vol 17 (2) ◽  
pp. 247 ◽  
Author(s):  
H. Lindeberg ◽  
K. Kananen-Anttila ◽  
M. Eronen ◽  
E. Reinikainen ◽  
A. Helin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mustela Putorius ◽  
Cell Stage ◽  
Cell Numbers ◽  
Embryo Recovery ◽  
Morula Stage

The effect of in vitro culture on viability of pre-implantation stage embryos in the farmed European polecat was studied, aimed at developing assisted reproductive technology for conservation of endangered mustelids, particularly the European mink (Mustela lutreola). Embryo storage in culture would enable embryo recovery and transfer in different locations. Ferret (Mustela putorius furo) kits have been produced from embryos that were cultured for 3 days in serum-containing medium (Li et al. 2001 Reproduction 122, 611–618). In our earlier studies, polecat embryos were maintained for 24 h in culture conditions (Lindeberg et al. 2003 Theriogenology 60, 965–970). Fourteen estrous donors were kept in the same cage with a fertile male overnight and sacrificed 3 days after the start of mating for recovery of embryos from the oviducts. Embryos were flushed with Emcare™ Complete ultra flushing medium (ICPBio, Auckland, New Zealand), washed twice in it, washed once in Emcare™ embryo holding solution and transported in the holding solution at room temperature for 1 h to the laboratory. Embryos of seven donors were pooled and cultured in 30-μL drops of TCM199 + glutamax I (GIBCO™) supplemented with fatty acid-free albumin (FAFBSA, Sigma-Aldrech, Helsinki, Finland) under a cover of paraffin oil (Medicult) for 3 days in a humidified atmosphere (39°C) and in 5% of O2. At the end of the culture, the embryos were evaluated and the ones that had developed at least to morula stage were chosen for transfers. The selected embryos were transported at room temperature in Emcare™ embryo holding solution for 1 h to the farm where they were surgically transferred under general anesthesia into seven recipients. The recipients had been mated the same way as the donors but with vasectomized males either on the same day as the donors (the first set: 7 donors, 3 recipients) or one day later than the donors (the second set: 7 donors, 4 recipients). Five embryos were cultured a total of 6 days to the blastocyst stage and stained for a count of cell numbers. A total number of 169 one- to 16-cell-stage embryos were recovered. At the end of the 3-day culture period, a total of 139 (139/169, 82%) had developed to morula (56.6%), compact morula (9.8%), early blastocyst (30.3%), or blastocyst stage (3.3%). Of these 139 embryos, a total of 102 were surgically transferred. Five of the 7 recipients delivered one to 5 kits each 43 to 45 days after the mating. Altogether 21 kits were born and the success rate was 21% (21 kits/102 transferred embryos). Cell numbers of the five Day 6 blastocysts varied from 130 to 430. In conclusion, this preliminary trial confirms that polecat embryos can be stored in culture for 3 days. In this study polecat embryos were cultured in 5% oxygen and without addition of serum which resulted in considerably better cell numbers for Day 6 blastocysts than in our earlier studies (90 to 165 cells; Lindeberg et al. 2003 Theriogenology 60, 965–970).

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