232 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 264
Author(s):  
R. R. D. Maziero ◽  
M. D. Guastali ◽  
M. J. Sudano ◽  
D. M. Paschoal ◽  
P. N. Guasti ◽  
...  
Keyword(s):  
Linear Models ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Negative Effect ◽  
Vitro Production ◽  
Co2 Atmosphere

Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle’s salts + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in the presence of 25 µM BL-I + 6.25 µM ROS. The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO2 atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3 ± 3.74%; BL-I + ROS, 38.0 ± 4.5%. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.

scholarly journals Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

2020 ◽  
Vol 18 (2) ◽  
pp. 249-255
Author(s):  
Nguyen Viet Linh ◽  
Nguyen Thi Hiep
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
Vitro Production ◽  
Normal Fertilization

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

163IMPROVEMENT OF IN VITRO PRODUCTION OF PIG EMBRYOS BY SYNCHRONISATION OF OOCYTE MEIOTIC MATURATION WITH CYCLOHEXIMIDE

2004 ◽  
Vol 16 (2) ◽  
pp. 204 ◽  
Author(s):  
J. Ye ◽  
K.H.S. Campbell ◽  
M.R. Luck
Keyword(s):  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Pre Treatment ◽  
Vitro Production

It is suggested that the relatively high rates of polyspermic fertilization and poor development of pig embryos produced in vitro are caused by asynchronous oocyte maturation. We have recently shown that pre-treatment of pig oocytes with cycloheximide (CHX) is an efficient way of synchronizing their meiotic maturation in vitro. However, it is not known whether this procedure affects fertilization or further development. The present study examined the effects of CHX-synchronised meiotic maturation on subsequent embryo development and the response to FSH. Pig ovaries were collected from a local abattoir. Cumulus-oocyte complexes (COCs) were aspirated from 3–5mm diameter follicles with a translucent appearance and extensive vascularization. COCs were first pre-incubated in defined maturation medium (DM; M199 with Earle’s salts, 25mM HEPES and sodium bicarbonate, 3mM L-glutamine, 0.1% (w/v) BSA, 0.57mM cysteine, 10ngmL−1 EGF, 0.2μgmL−1 pLH, 100μmL−1 penicillin and 0.1mgmL−1 streptomycin) or in DM supplemented with 50ngmL−1 pFSH (DMF) and 5μgmL−1 CHX for 12h. COCs were then further cultured in the same DM without CHX for 24–30h or in DMF for 36h. For controls, COCs were cultured conventionally in DM for 42h or DMF for 48h. After removal of cumulus cells, all cultured oocytes were inseminated with ejaculated sperm at a final concentration of 300000mL−1 for 6h. The IVF medium was modified Tris-buffered medium containing 0.1% BSA, 20μM adenosine and 0.2mM reduced glutathione. Putative embryos were cultured in NCSU23 without glucose but supplemented with 4.5mM Na lactate and 0.33 mM Na pyruvate for 2 days. Cleaved embryos were further cultured in normal NCSU23 for 4 days. IVM and IVF were performed in 5% CO2 in air and IVC in 5% CO2, 5% O2, 90% N2, all at 39°C and 95% RH. Three replicates with DM, with or without CHX, and one with DMF, with or without CHX, were performed with 30–50 oocytes in each replicate. Statistical comparisons were by t-test. The result with DM showed that the rate for normal cleavage at 2 days after insemination of CHX-treated oocytes (40.6±3.8%) was similar to that of controls (40.4±3.5%). However, the proportion developing to healthy blastocysts at Day 6 was significantly higher in the CHX-treated group (16.9±1.2%) than in controls (9.6±1.3%; P<0.05). A significantly higher number of Day 2-cleaved embryos from CHX-treated oocytes developed to the day 6 blastocyst stage compared with controls (44.7±5.0% and 22.3±2.4%, respectively; P<0.05). Supplementation of the basic maturation medium with pFSH increased the rate of cleavage in both CHX-treated oocytes (73.2%) and controls (76.9%) and increased the proportions developing to healthy blastocysts at Day 6 (CHX-treated: 39.0%; control: 11.5%). We conclude that oocytes pre-treated with CHX retain their developmental competence and that meiotic synchronization with CHX improves the efficiency of in vitro production of pig embryos. (Supported by BBSRC 42/S18810.)

In vitro production of bovine embryos: Developmental competence is acquired before maturation

1997 ◽  
Vol 47 (5) ◽  
pp. 1061-1075 ◽  
Author(s):  
P. Blondin ◽  
K. Coenen ◽  
L.A. Guilbault ◽  
M.-A. Sirard
Keyword(s):  
Developmental Competence ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Vitro Production

Effects of lamb age, hormone stimulation and response to hormone stimulation on the yield and in vitro developmental competence of prepubertal lamb oocytes

2005 ◽  
Vol 17 (6) ◽  
pp. 593 ◽  
Author(s):  
Katherine M. Morton ◽  
Sally L. Catt ◽  
W. M. Chis Maxwell ◽  
Gareth Evans
Keyword(s):  
Recovery Rate ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Development ◽  
Blastocyst Formation ◽  
In Vitro Production ◽  
Hormone Stimulation ◽  
Vitro Production ◽  
High Responders

Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3–4 and 6–7 weeks of age. For 3–4-week-old lambs, hormone stimulation increased the number of follicles (29.9 ± 15.3 v. 70.6 ± 8.2), oocytes per ovary (18.3 ± 6.3 v. 39.3 ± 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3–4 v. 6–7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3–4 and 6–7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (<20, 20–50 and >100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3–4-week-old lambs only (P < 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3–4-week-old lambs (15.2–25.6%; P > 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6–7-week-old lambs (P < 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3–4-week-old lambs, although by 6–7 weeks of age a high response to stimulation reduces blastocyst formation.

180 DEVELOPMENTAL CAPACITY OF SINGLE CULTIVATED EMBRYOS IN SMALL AND BIG DROPLETS

2007 ◽  
Vol 19 (1) ◽  
pp. 206
Author(s):  
I. G. F. Goovaerts ◽  
J. B. P. De Clercq ◽  
M. Nichi ◽  
P. E. J. Bols
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Significant Difference ◽  
Group Treatments ◽  
Developmental Capacity ◽  
Droplet Sizes ◽  
The Individual ◽  
Vitro Production

An in vitro production system where a single oocyte can be followed from the ovary to the blastocyst stage would be a useful tool for studies concerning developmental competence or follicular environment. Unfortunately, until now, only low blastocyst rates could be obtained after single embryo production, and there is still discussion about the ideal droplet size. The objective of the present experiment was to compare the developmental competence of single cultivated zygotes in 20- and 500-µL droplets. Cumulus–oocyte complexes were obtained from slaughterhouse ovaries and were matured and fertilized in groups of 100 for 22 h; the presumptive zygotes were divided into 4 groups. In treatment 1, 25 zygotes were transferred into 50 µL of SOF medium supplemented with 5% serum under oil, whereas in treatment 2, 25 zygotes were transferred into 500 µL of medium. Zygotes were cultivated separately in treatments 3 and 4: in treatment 3 in 20 µL of medium under oil and in treatment 4 in 500 µL of medium. Cleavage rates and division stages were assessed after 3 days of cultivation (5% CO2, 5% O2, 90% N2); blastocyst rates were determined after 7 days. Statistical analysis was performed by logistic regression using SAS (PROC LOGISTIC). There was no difference in cleavage rates between the 2 group treatments or between the 2 single treatments. Also, the division stages were not different between the 2 single treatments (16-cell: 2.0 vs. 1.3%; 8-cell: 25.8 vs. 31.6%; 4-cell: 41.2 vs. 38.0%; and 2-cell: 31.0 vs. 29.1% for the 20 µL and the 500 µL droplet sizes, respectively). Group cultivation after 7 days in 50 µL was significantly better than in 500 µL; however, both treatments resulted in significantly higher blastocyst rates compared with the individual cultures in 20 or 500 µL, between which no significant difference could be found. Noteworthy, only 4-cell and 8-cell stages on Day 3 resulted in blastocysts on Day 7 of cultivation. In conclusion, these results indicate that cultivation in groups gives higher blastocyst rates, although the same embryo density is used as in individual cultivation (1 embryo 20 µL in treatments 2 and 3). Moreover, no significant difference could be found between single cultivation in small and big droplets. This is confirmed by the cleavage stages on Day 3, which indicate no difference in timing of cleaving between small and big droplets; time of cleaving is indicative of further developmental capacity. Table 1.Cleavage and blastocyst rates after single and group cultivation

Media and time of oocytes transport influence their developmental competence for in vitro production of bovine embryos

1998 ◽  
Vol 49 (1) ◽  
pp. 299 ◽  
Author(s):  
H. Twagiramungu ◽  
N. Morin ◽  
L.A. Guilbault ◽  
M.A. Sirard ◽  
D. Bousquet
Keyword(s):  
Developmental Competence ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Vitro Production

Polyspermic penetration in porcine IVM - IVF systems

2003 ◽  
Vol 15 (3) ◽  
pp. 167 ◽  
Author(s):  
Hiroaki Funahashi
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Final Maturation ◽  
Morphological Differences ◽  
Vitro Production ◽  
Boar Spermatozoa

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs

2005 ◽  
Vol 17 (7) ◽  
pp. 701 ◽  
Author(s):  
K. M. Morton ◽  
S. L. Catt ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Growth Rate ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Ovarian Weight ◽  
Hormone Stimulation ◽  
Vitro Production ◽  
Stimulation Regimen

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 µg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 ± 0.7 g) and from 1.7 to 54.1 (12.5 ± 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 ± 29.2) and from 13.0 to 455.0 (82.0 ± 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0–93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro, and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 ± 2.8 blastocysts per lamb.

226 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING CHEMICAL PACKETS THAT REGULATE CO2 AND O2

2015 ◽  
Vol 27 (1) ◽  
pp. 202
Author(s):  
K. Saeki ◽  
Y. Fujiki
Keyword(s):  
Gas Phase ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Catalog Number ◽  
Co2 Level ◽  
Vitro Production

Bovine embryos are now routinely produced with oocytes collected from slaughterhouse ovaries or by transvaginal ovum pickup. The oocytes are matured, fertilized, and cultured in a water-jacketed CO2/O2 incubator. Gas phase in incubators is usually maintained at 5% CO2 in air for in vitro maturation (IVM) and IVF of oocytes and at 5% CO2, 5% O2, and 90% N2 for in vitro culture (IVC) of embryos. Here we investigated whether two chemical packets that regulate CO2 and O2 for culturing bacteria (Mitsubishi Gas Chemical, Tokyo, Japan) could be used to control the gas phase in vitro production (IVP) of cattle embryos. One packet (Anaero Pack-CO2) was maintained at a CO2 level of ~5% in a 2.5-L container and the other (Anaero Pack-MicroAnaero) was maintained at a CO2 level of 5–8% and an O2 level of 6 to 12%. Bovine cumulus-oocyte complexes (COC, n = 970) were collected from slaughterhouse ovaries, matured in HEPES-buffered TCM-199 (catalog number 12340–030, Invitrogen) supplemented with 10% FCS, 0.02 Armour unit mL–1 FSH and 1 µg mL–1 E2 for 22 h, and fertilized in medium IVF100 [Research Institute for the Functional Peptides Co. Ltd. (IFP), Yamagata, Japan] with frozen-thawed sperm (4 × 106 cells mL–1) for 6 h. Sperm and cumulus cells were removed from the oocytes. The denuded oocytes were cultured in IVD101 (IFP, 20 to 30 embryos/50 μL) for 8 days (Day 0 = IVF). Culture was carried out at 39°C with maximum humidity. Five different combinations of gas conditions were used for incubation (Table 1). Experiments were repeated 3 times. Cleavage and blastocyst rates were assessed on Day 8. Data were analysed by ANOVA followed by Fisher's PLSD test. In the five conditions, rates of matured oocytes (oocytes at MII, n = 210) were 70 to 73% and rates of normal fertilized oocytes (oocytes with 2 pronuclei, n = 310) were 67 to 75%. Cleavage rates of embryos after 8 days of culture (n = 450) were 68 to 75%, and rates of blastocysts from cleaved embryos were 25 to 40%. None of the above measures were significantly different among the 5 conditions (P > 0.05). These results indicate that gas phase control is not needed for IVM and IVF of bovine oocytes for their subsequent development. Anaero Pack-MicroAnaero (5–8% CO2, 6–12% O2) can be used for IVC of bovine embryos. The CO2-generating and deoxidizing packets can be successfully used to control the gas phase during bovine embryo production. Table 1.Five different combinations of gas conditions used for incubation

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