58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann
Keyword(s):  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

scholarly journals Transvaginal ultrasound-guided oocyte aspiration for production of embryos <i>in vitro</i>

2002 ◽  
Vol 45 (1) ◽  
pp. 99-108
Author(s):  
J. A. Carter ◽  
S. Bellow ◽  
M. Meintjes ◽  
O. Perez ◽  
E. Ferguson ◽  
...  
Keyword(s):  
Transvaginal Ultrasound ◽  
Farm Animals ◽  
Embryo Research ◽  
Ultrasound Guided ◽  
Cyclic Activity ◽  
Embryo Production ◽  
Early Postpartum ◽  
In Vitro Embryo Production ◽  
Oocyte Aspiration

Abstract. reproductive potential in genetically valuable animals (BEAL et al., 1992). Now that repeatable oocyte retrieval methods are being fine-tuned, it is likely these procedures will become routinely used to obtain oocytes for further gamete and embryo research and also by seedstock producers for in vitro embryo production from farm animals in the commercial sector. The use of transvaginal ultrasound-guided oocyte aspiration and IVF procedure does offer an alternative to cattle producers who have genetically valuable cows that for some reason are unable to produce viable embryos through standard embryo collection procedures. This technology can be used on oocytes harvested from older ovulating or nonovulating cows, females with physical injuries (e.g., fractured leg) and problem cows having an abnormal cervix. Good success has been reported using IVF procedures on oocytes obtained from supplemental follicles of cows with cystic ovarian disease. With IVF the potential exists for more embryos to be produced in a shorter period of time, since the procedure can be repeated on the same cow 3 to 4 times or more a month. At this station, we are harvesting oocytes from early postpartum (< 40 days) beef and dairy cattle, before the female begins cyclic activity. The approach allows the opportunity to produce one or more extra calves from the cow before she is mated for a natural pregnancy. Currently, transvaginal ultrasound-guided oocyte aspiration is now being used to harvest valuable oocytes from minor farm animal breeds, from domestic females representing rare bloodlines, clinically infertile females and reproductively senescent cows. Research continues to find applications for this technology, including harvesting oocytes from young prepubertal heifers and early postpartum beef cows for in vitro embryo production. The use of ultrasound-guided oocyte aspiration should not be overlooked to obtain oocytes for in vitro embryo production and to aid in germplasm preservation of endangered exotic species.

Pregnancy obtained in a late gestational mare by in vitro embryo production

2019 ◽  
Vol 31 (12) ◽  
pp. 1926
Author(s):  
Lino Fernando Campos-Chillon ◽  
Jan Martin ◽  
Joy L. Altermatt
Keyword(s):  
Transvaginal Ultrasound ◽  
Experimental Studies ◽  
Late Gestation ◽  
Follicular Growth ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
In Vitro Embryo Production

Recently, the demand for invitro embryo production in the horse has increased worldwide. Most clinical transvaginal ultrasound-guided ovum pick-up (OPU) procedures are performed in non-pregnant donor mares, and few experimental studies have described invitro embryo production from oocytes of pregnant donors 21–150 days in gestation. This report discusses OPU, follicular growth and invitro embryo production in a pregnant mare during late gestation.

scholarly journals In Vitro Embryo Production and Transfer of Bubaline Embryos using Oocytes Derived from Transvaginal Ultrasound-Guided Follicular Aspiration (TUFA)

2014 ◽  
Vol 2 (2) ◽  
pp. 180-184
Author(s):  
FP Aquino ◽  
Eufrocina P. Atabay
Keyword(s):  
Transvaginal Ultrasound ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Follicular Aspiration ◽  
Group 2 ◽  
Group 1 ◽  
Donor Cows

Transvaginal ultrasound-guided follicular aspiration (TUFA) has become a popular tool for embryo production in vitro due to its high degree of repeatability in terms of recovering oocytes from live animals. In Study 1, the quantity and quality of oocytes from Bulgarian Murrah buffalo cows (n=10) of varying ages (Group 1, 8-12; and Group 2, 13-17 years) were assessed. Group 1 buffalo donor cows yielded significantly higher (P<0.05) number of oocytes vs Group 2 buffalo donor cows (71 vs 29 oocytes, respectively), though in terms of oocyte quality, no difference was observed. In Study 2, oocytes collected (n=100) in Study 1 were matured, fertilized in vitro and the resulting zygotes were cultured which developed to blastocyst stage embryos. The maturation, fertilization and blastocyst development rates obtained were 53.0%, 40.0% and 32.5%, respectively. In Study 3, the viability of resulting blastocyst stage embryos was determined by transferring to recipient cows. Of 10 recipients 1 got pregnant and delivered a 35 kg male calf after 310 days gestation period. Overall, the results of the studies conducted demonstrated the potential of TUFA technology in the in vitro production of embryos which eventually could be used in the production of live offspring.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10369Int J Appl Sci Biotechnol, Vol. 2(2): 180-184 

Effects of superovulation on repeated ultrasound guided oocyte collection and in vitro embryo production in pregnant heifers

1997 ◽  
Vol 47 (1) ◽  
pp. 157 ◽  
Author(s):  
C.Guyader Joly ◽  
S. Ponchon ◽  
J.M. Thuard ◽  
M. Durand ◽  
M. Nibart ◽  
...  
Keyword(s):  
Ultrasound Guided ◽  
Embryo Production ◽  
Oocyte Collection ◽  
In Vitro Embryo Production

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P&lt;0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P&lt;0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

scholarly journals 287IN VITRO PRODUCTION OF HOLSTEIN EMBRYOS USING SEX SORTED SEMEN

2004 ◽  
Vol 16 (2) ◽  
pp. 263
Author(s):  
R.D. Wilson ◽  
K.A. Weigel ◽  
P.M. Fricke ◽  
M.L. Leibfried-Rutledge ◽  
D.L. Matthews ◽  
...  
Keyword(s):  
Low Cost ◽  
Laboratory Data ◽  
Blastocyst Stage ◽  
Embryo Production ◽  
Cull Cows ◽  
Sexed Semen ◽  
Dairy Cattle Breeding ◽  
In Vitro Embryo Production ◽  
Freeze Date

Our objective was to explore the synergy between sexed semen and in vitro embryo production and assess benefits of these technologies on commercial farms. Ovaries were collected from high genetic merit Holstein cull cows via colpotomy or at the time of slaughter. Oocytes were aspirated from the ovaries, fertilized 20–24h later, and matured to the morula or blastocyst stage. Embryos were transferred into recipient Holstein cows and heifers on the same farms. Seven Wisconsin herds participated, and 365 embryos were produced from 104 donor cows. Only 272 of these embryos were transferred due to limited availability of recipients. Sexed semen from three Holstein sires was used. On average, 3.5±0.37 transferable embryos were produced per donor, including 1.4±0.18 grade 1 embryos and 1.5±0.20 grade 2 embryos. Individual farms averaged from 1.6 to 5.8 transferable embryos per donor. Laboratory data also revealed interesting results. On average 43.7±4.0 oocytes were collected per donor, and the number of usable oocytes (33.9±3.4), and percent embryos cleaved (52.1±1.9), were significant predicators of the number of blastocysts developed. We divided the usable oocytes and embryos cleaved per donor into quartiles. The fourth quartile for embryos cleaved was significantly greater (P&lt;0.05) than the lower three quartiles, and the usable oocyte quartiles all significantly differed from each other. Semen freeze date was also a significant predicator of the number of blastocysts developed, suggesting significant variation in the quality of sorted semen per ejaculate. To preliminarily test the effect of sorting on the percentage of embryos developing to blastocyst stage, oocytes were recovered from ovaries collected at a slaughterhouse and fertilized using non-sorted semen or sex-sorted semen from the same sires. Oocytes (n=3312) fertilized using non-sorted semen tended (P=0.06) to produce more embryos developing to blastocyst stage than oocytes (n=1577) fertilized using sex-sorted semen (20.1±2.9% v. 12.2±2.3%, respectively). Preliminary pregnancy results show strong farm and sire effects. Overall conception rate was 36% for heifer recipients and 18milking cow recipients. These results suggest that low cost in vitro embryo production may have promise as an early system for utilizing sexed semen in dairy cattle breeding programs.

In vitro embryo production from bovine oocyte donors aspirated at different frequencies or following treatment with FSH

1996 ◽  
Vol 1996 ◽  
pp. 68-68
Author(s):  
K.L. Goodhand ◽  
R.G. Watt ◽  
M.E. Staines ◽  
L.C. Higgins ◽  
P.J. Broadbent ◽  
...  
Keyword(s):  
Transvaginal Ultrasound ◽  
Genetic Change ◽  
Bovine Oocyte ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Follicular Aspiration ◽  
Oocyte Donors ◽  
Pre Treatment

The combination of in vivo recovery of oocytes using transvaginal ultrasound guided aspiration and subsequent in vitro embryo production can be used to increase the rate of genetic change for efficiency of beef production by increasing selection intensity and reducing generation interval. The total number of oocytes recovered by aspiration and embryos produced is directly proportional to the number of aspiration sessions whether recovery takes place once or twice weekly. Pre-treatment of oocyte donors with FSH has been shown to improve the number of follicles available for aspiration but effects on embryo production have been conflicting (Bungartz et al., 1995; Goodhand et al., in press). The objective of this experiment was to compare the effect on embryo production of frequency of follicular aspiration and pre-treatment of donor cattle with FSH.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
J. G. Kim ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. Y. Choe ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P &lt; 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos. Table 1.Development of NT embryos with undifferentiated and differentiated cells This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

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