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.

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.

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&lt;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&lt;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.)

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

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.

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.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

204 PERIPHERAL PROGESTERONE CONCENTRATION AFFECTS BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2013 ◽  
Vol 25 (1) ◽  
pp. 250
Author(s):  
N. Schlüter ◽  
A. Hanstedt ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Relative Abundance ◽  
Clinical Signs ◽  
Follicular Phase ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Vitro Production

The developmental competence of cumulus–oocyte complexes (COC) used for in vitro production is dependent on several factors including the stage of the oestrus cycle. In a recent study, we were able to show that circulating progesterone (P4) had no effect on follicle number, size, recovery rate, or in vitro production suitability of recovered COC (Schlüter et al. 2012 Reprod. Fertil. Dev. 24, 175–176). The aim of the present study was to determine the influence of circulating P4 concentrations on the molecular quality of bovine COC collected during repeated OPU sessions. The COC were aspirated twice per week for 5 to 6 weeks from 12 Holstein Friesian heifers. The first OPU session took place on Day 7 of the oestrous cycle after spontaneous ovulation (ovulation = Day 0). Blood samples were taken at the time of each OPU session, and P4 concentrations were determined using a radioimmunoassay. All animals showed clinical signs of oestrus and large follicles (≥8.5 mm) during the course of the OPU sessions. Following the aspiration of a large follicle, a CL-like structure (induced CL) could be detected. According to the P4 concentrations, the cycle was divided into 3 phases: CL phase after spontaneous ovulation (oCL; P4: ≥1 ng mL–1), follicle phase 1 (Fp; P4 <1 ng mL–1), and induced CL phase (iCL; P4: ≥1 ng mL–1). The length of the cycle after spontaneous ovulation did not differ significantly from that after induced ovulation (22.4 ± 3.1 days v. 23.8 ± 1.8 days, respectively). During the oCL-phase, blood P4 concentrations were significantly higher than during the iCL-phase (4.9 ± 2.3 ng mL–1 v. 3.0 ± 1.6 ng mL–1). For mRNA analysis, denuded COC were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey’s test. A P-value of ≤0.05 was considered significant. The relative abundance of all transcripts except SCL2A1 was significantly increased in oocytes collected from follicles of the oCL phase compared with that from oocytes that had been aspirated during the iCL phase. A significant increase in the relative amount of PGR, PGRMC1, PGRMC2, and BMP15 transcripts was detected in oocytes stemming from the follicular phase to those from the iCL phase. No differences in the relative abundance of all transcripts were seen comparing oocytes from oCL phase and oocytes from the follicular phase. In summary, circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated OPU session, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system

2003 ◽  
Vol 15 (4) ◽  
pp. 249 ◽  
Author(s):  
J. R. Herrick ◽  
M. L. Conover-Sparman ◽  
R. L. Krisher
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
High Incidence ◽  
Porcine Embryo ◽  
Medium System ◽  
Epidermal Growth ◽  
Vitro Production ◽  
Single Medium

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL−1 hyaluronan, 0.6 mM cysteine, 10 ng mL−1 epidermal growth factor (EGF), 0.1 U mL−1 porcine LH and FSH, and 1 × Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 × 105 sperm mL−1) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mm bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

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