148 AN EFFECT OF MELATONIN ON DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN VITRO UNDER OPTIMAL OR ENHANCED OXYGEN TENSIONS

Many different systems of free radical scavengers have been investigated during the last few years for in vitro culture of mammalian embryos. Melatonin is a potent reactive oxygen species scavenger and has been tested in the promotion of mouse embryo development in vitro (Ishizuka et al. 2000 J. Pin. Res. 28, 48–51). An effect of melatonin on bovine embryo development in vitro is described here. Slaughterhouse-derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Presumptive zygotes randomly allocated to experimental groups were cultured for 3 days (Day 1–Day 3) in CR1aaLA medium (Papis et al. 2000 Theriogenology 54, 651–658) supplemented with two different concentrations of melatonin (10−6 M or 10−4 M; Sigma, St. Louis, MO, USA) or without melatonin (control). Culture was performed under two different gas atmospheres containing 4% CO2 and either normal (7%) or enhanced (20%) oxygen concentration (2 × 3 factorial analysis). At the end of Day 3, embryos from each treatment group, developed to at least the 4-cell stage, were collected and cultured without melatonin until Day 10 at optimum 4% CO2 and 7% O2 atmosphere. The numbers of blastocysts at Day 8 and hatching/hatched blastocysts at Day 10 were recorded. Five replicates of each treatment were performed. Blastocyst formation rates of presumptive zygotes and of Day 3, 4-cell embryos were calculated for each group. Differences between groups were analyzed using chi-square and/or Fisher's exact tests where appropriate. P < 0.05 was considered statistically significant. Out of 100, 100, and 101 presumptive zygotes cultured for the first 3 days in 7% oxygen with 10−4 M, 10−6 M, or no melatonin, 31 (31%), 40 (40%), and 44 (43.5%) developed to blastocyst stage and 25 (25%), 33 (33%), and 36 (36%) to hatching/hatched blastocyst stage, respectively. On the other hand, out of 102, 102, and 100 zygotes cultured in the same concentrations of melatonin, but under 20% of oxygen, an opposite tendency was observed, as 42 (41%), 25 (24.5%), and 32 (32%) blastocysts and 26 (25.5%), 21 (20.6%), and 25 (25%) hatching/hatched blastocysts developed, respectively. No statistical significance was reached here. However, out of 4-cell embryos put into in vitro culture after initial treatments in different melatonin concentrations, a decreased ratio of blastocyst formation was observed in the 10−4 M melatonin group (31/65, 47.7%) compared to that of the control (44/65, 67.7%; P = 0.0327) when the lower oxygen concentration was applied. However, a beneficial effect of melatonin was observed in the presence of 20% oxygen. Out of 61 embryos, 42 (68.9%) developed to the blastocyst stage after treatment in 10−4 M melatonin concentrations, vs. 32/63 (50.8%; P = 0.0458) blastocysts developed in control group. In conclusion, a beneficial or a harmful effect of melatonin on bovine embryo in vitro development was observed depending on the oxygen concentration during the treatment. Results presented seem to confirm a potent free radicals scavenging activity of melatonin in a bovine embryo culture system.

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

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab136 ◽

2005 ◽

Vol 17 (2) ◽

pp. 219 ◽

Cited By ~ 5

Author(s):

C.E. Ferguson ◽

T.R. Davidson ◽

M.R.B. Mello ◽

A.S. Lima ◽

D.J. Kesler ◽

...

Keyword(s):

Embryo Development ◽

Direct Effect ◽

Blastocyst Stage ◽

Control Group ◽

Bovine Embryo ◽

Bovine Embryos ◽

Direct Role ◽

Treatment Groups ◽

And Control

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

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27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab27 ◽

2014 ◽

Vol 26 (1) ◽

pp. 128

Author(s):

C. P. Buemo ◽

A. Gambini ◽

I. Hiriart ◽

D. Salamone

Keyword(s):

In Vitro Culture ◽

Somatic Cell ◽

Embryo Development ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Electric Pulse ◽

Blastocyst Stage ◽

Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

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Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa

Reproduction Fertility and Development ◽

10.1071/rd15289 ◽

2017 ◽

Vol 29 (4) ◽

pp. 805 ◽

Cited By ~ 2

Author(s):

Luis B. Ferré ◽

Yanina Bogliotti ◽

James L. Chitwood ◽

Cristóbal Fresno ◽

Hugo H. Ortega ◽

...

Keyword(s):

Embryo Development ◽

X Chromosome ◽

Embryo Quality ◽

Bos Taurus ◽

Control Group ◽

Hatching Rate ◽

Bovine Embryo ◽

Blastocyst Formation ◽

Spermatozoa Motility

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10 mM caffeine (CA), 5 mM theophylline (TH), 10 mM caffeine and 5 mM theophylline (CA + TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA + TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode’s albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte–sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval.

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

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab131 ◽

2018 ◽

Vol 30 (1) ◽

pp. 205 ◽

Cited By ~ 1

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.

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Embryonic disc formation following post-hatching bovine embryo development in vitro

Reproduction ◽

10.1530/rep-20-0243 ◽

2020 ◽

Vol 160 (4) ◽

pp. 579-589 ◽

Cited By ~ 1

Author(s):

Priscila Ramos-Ibeas ◽

Ismael Lamas-Toranzo ◽

Álvaro Martínez-Moro ◽

Celia de Frutos ◽

Alejandra C Quiroga ◽

...

Keyword(s):

Embryo Development ◽

Blastocyst Stage ◽

Embryo Morphology ◽

Bovine Embryo ◽

Pluripotency Marker ◽

Embryonic Disc ◽

Development In Vitro ◽

Apoptosis And Necrosis ◽

Disc Formation

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

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Melatonin effect on bovine embryo development in vitro in relation to oxygen concentration

Journal of Pineal Research ◽

10.1111/j.1600-079x.2007.00479.x ◽

2007 ◽

Vol 43 (4) ◽

pp. 321-326 ◽

Cited By ~ 78

Author(s):

Krzysztof Papis ◽

Olga Poleszczuk ◽

Elzbieta Wenta-Muchalska ◽

Jacek A. Modlinski

Keyword(s):

Oxygen Concentration ◽

Embryo Development ◽

Bovine Embryo ◽

Development In Vitro

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Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽

10.3390/ani11030748 ◽

2021 ◽

Vol 11 (3) ◽

pp. 748

Author(s):

Joanna Kochan ◽

Agnieszka Nowak ◽

Barbara Kij ◽

Sylwia Prochowska ◽

Wojciech Niżański

Keyword(s):

Embryo Development ◽

Embryo Quality ◽

Time Lapse ◽

Blastocyst Stage ◽

Cavity Formation ◽

Non Invasive ◽

Morphokinetic Parameters ◽

Morphological Defects ◽

Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

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35 IN VITRO BOVINE EMBRYO DEVELOPMENT AFTER NUCLEAR TRANSFER BY HANDMADE CLONING USING A MODIFIED WOW CULTURE SYSTEM

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab35 ◽

2006 ◽

Vol 18 (2) ◽

pp. 126 ◽

Cited By ~ 14

Author(s):

C. Feltrin ◽

F. Forell ◽

L. dos Santos ◽

J. L. Rodrigues

Keyword(s):

Embryo Development ◽

Culture System ◽

Nuclear Transfer ◽

Blastocyst Stage ◽

Bovine Embryo ◽

Inner Diameter ◽

Group 2 ◽

Group 1 ◽

Handmade Cloning

The effect of the microenvironment on embryo development during in vitro culture of zona-free embryos after nuclear transfer is still unclear. The aim of this experiment was to determine the effect of the dimensions of the well (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) culture system on the in vitro development of handmade cloned bovine embryos to the blastocyst stage. Appropriately ground steel needles were pressed slightly by hand to the bottom of the well of a polystyrene four-well dish (176740, Nunc, Life Technologies AS, Roskilde, Denmark). Embryos were produced by the handmade cloning (HMC) technique (Vajta et al. 2003 Biol. Reprod. 68, 571-578) with modifications, using primary cultures of skin fibroblast cells from an adult cow as nuclear donors. Cumulus-oocyte complexes were in vitro-matured in M-199 supplemented with 10% estrous cow serum (ECS), FSH, hCG, and estradiol (E2) for 17 h. After maturation, cumulus cells were removed by pipetting. Following zona pellucida removal in 0.5% protease (Sigma, Brazil), zona-free oocytes were incubated for 15 min in 5 mg/mL cytochalasin B (Sigma) and subsequently hand-bisected and screened for nuclear material under UV light after incubation in 10 mg/mL bisbenzimide (Hoechst 33342). Next, two enucleated halves and one donor cell were aggregated after a quick exposure to phytohemagglutinin (PHA) and subsequently fused by two electrical DC pulses of 1 kV/cm for 20 �s, in a BTX 453 chamber coupled to an ECM 2001 Electro Cell Manipulator System (BTX, Inc., San Diego, CA, USA), with additional exposure to brief pre- and post-fusion AC pulses of 15 V. Reconstructed embryos were chemically activated in 5 mM ionomycin (Sigma) for 5 min, followed by 2 mM 6-DMAP (Sigma) for 2.5 h. Finally, activated reconstructed cloned embryos were in vitro-cultured in one of two WOW culture systems (larger vs. smaller micro-wells) in 4-well plates containing 400 mL modified SOF medium supplemented with 10% ECS, under mineral oil, at 5% CO2, 5% O2 and 90% N2, and 39�C for 7 days. In Group 1 (large-size micro-well), embryos were cultured in individual cylindrical micro-wells with an inner diameter and depth of approximately 280 and 250 mm, respectively, whereas in Group 2 (small size micro-well), embryos were cultured in individual conical micro-wells with approximately 130 mm inner diameter and 150 mm depth. Data analysis was performed by the chi-square test. After four replicates, cleavage rates were significantly higher (P < 0.05) in Group 2 (51/63, 80.9%) than in Group 1 (43/67, 64.1%). Embryo development to the blastocyst stage was also greater (P < 0.05) in the small micro-wells (16/63, 25.3%) than in the large ones (8/67, 11.9%). In summary, these results show a significant increase in cleavage and blastocyst developmental rates in handmade cloned embryos cultured in a modified WOW system using individual small size micro-wells, suggesting that a small, tighter micro-well provides favorable in vitro conditions for embryo development.

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22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab22 ◽

2006 ◽

Vol 18 (2) ◽

pp. 119

Author(s):

H. Bagis ◽

S. Arat ◽

H. Odaman ◽

A. Tas

Keyword(s):

Embryo Development ◽

Formation Rate ◽

Cell Number ◽

Blastocyst Formation ◽

Group 4 ◽

Mouse Embryo Development ◽

O2 Concentration ◽

Group 3 ◽

Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

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346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab346 ◽

2007 ◽

Vol 19 (1) ◽

pp. 288

Author(s):

C. Kubota ◽

T. Kojima ◽

T. Nagai ◽

X. Tian ◽

X. Yang

Keyword(s):

Subsequent Development ◽

Industrial Applications ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Bovine Embryo ◽

Becton Dickinson ◽

In Vitro Storage ◽

Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco's PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus–oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL−1 polyvinyl alcohol (PVA). COCs were collected from follicles (3–8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5°C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 µg mL−1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281–286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS–IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM–IVF–IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student's t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM–IVF–IVC (bFF (n = 87): 0&percnt;, 0&percnt;; PBS/FBS (n = 72): 84&percnt;, 1&percnt;; and PBS/PVA (n = 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n = 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS–IVM–IVF (M199A/FBS (n = 97): 82&percnt;, 28&percnt;; and M199A/PVA (n = 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM–IVF–IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

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