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 

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

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.

234 HORMONAL FOLLICLE STIMULATION IN HOLSTEIN COWS FOR IN VITRO EMBRYO PRODUCTION USING SPERM SORTED BY FLOW CYTOMETRY

2016 ◽  
Vol 28 (2) ◽  
pp. 248 ◽  
Author(s):  
L. Ferré ◽  
Y. Bogliotti ◽  
J. Chitwood ◽  
M. Kjelland ◽  
P. Ross
Keyword(s):  
Transvaginal Ultrasound ◽  
Prostaglandin F2α ◽  
Holstein Cows ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
Group 2 ◽  
Group 1

Transvaginal ultrasound needle-guided ovum pick-up (OPU) and in vitro embryo production (IVP) offer a reliable alternative to conventional embryo transfer to produce offspring. The success of OPU/IVP greatly depends on the number and quality of retrieved oocytes. The aim of this study was to compare OPU/IVP performance from stimulated Holstein cows. Holstein (Bos taurus) >8-year-old pluriparous open dry cows (n = 28) were used for OPU as oocyte donors. Follicular waves in all groups were synchronized by gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF), and CIDR administrated on Day 0, followed by stimulation treatments 48 h later. No pre-synch was used. Total hormone dosage were administrated as follows: Group 1: pFSH = 180 mg (Folltropin, Bioniche, Belleville, ON, Canada; n = 7), Group 2: pFSH/LH = 500 IU (Pluset, Calier, Barcelona, Spain; n = 7), Group 3: eCG = 1500 IU (eCG, Biogénesis-Bagó, Buenos Aires, Argentina; n = 7) and Group 4: Control (n = 7), no stimulation. All injections were performed intramuscularly (i.m.) twice a day, during three days. OPU was performed 48 (Group 1) or 24 h (Group 2 and 3) after the last injection. The control group received saline solution i.m. Follicles were classified according to diameter in 4 categories: small (2–5 mm); medium (6–9 mm); large (10–14 mm) and extra large (>15 mm). A Mindray DP-30 Vet (Mindray Medical, Shenzhen, China) was equipped with a micro-convex transducer 5.0- to 8.5-MHz probe along with a disposable 21G needle. The OPU flow rate was 15 mL min–1. Retrieved oocytes were classified according to IETS guidelines as viable (grade 1 + 2) and non-viable (grade 3 + 4). The IVP protocol was according to that in Reprod. Fertil. Devel. (2004, 16, 253). Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient (PureSperm, Nidacon, Mölndal, Sweden) and diluted to 1 × 106 sperm mL–1. ANOVA was used for comparisons of mean values and a chi-squared test was used for proportions. Results are presented in the Table 1. In conclusion, pFSH stimulation before ovum pick-up in Holstein cows increased the number of collected and viable oocytes, cleavage, embryo development, and hatching rates in comparison to other follicle stimulation hormones and non-stimulation. A cost-benefit analysis of these methods could be valuable in order to inform whether or not a stimulation protocol is necessary for a commercial IVP operation. Table 1.Numbers of follicles, collected and viable oocytes, cleavage rate, blastocysts and hatching rate

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.

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

2006 ◽  
Vol 18 (2) ◽  
pp. 126 ◽  
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.

232 PARTHENOGENETIC DEVELOPMENT AND PLOIDY OF BOVINE OOCYTES FOLLOWING VARIOUS CHEMICAL ACTIVATION REGIMENS

2008 ◽  
Vol 20 (1) ◽  
pp. 195
Author(s):  
S.-A. Ock ◽  
G.-J. Rho
Keyword(s):  
Polar Body ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Parthenogenetic Development ◽  
Group 5 ◽  
Group 2 ◽  
Ion Treatment ◽  
Bovine Oocytes ◽  
Group 1

Bovine oocytes treated using various combinations of ionomycin (Ion), cycloheximide (CHX), and cytochalasin B (CCB) were evaluated for developmental rates and ploidy status. Metaphase II oocytes were allocated 5 treatment groups, and the groups were treated as follows: Group 1: 5 µm Ion for 5 min; Group 2: Ion + 10 µg mL–1 CHX for 5 h; Group 3: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 1 h + 10 µg mL–1 CHX for 4 h; Group 4: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 3 h + 10 µg mL–1 CHX for 2 h; and Group 5: Ion + 10 µg mL–1 CHX + 5 µg mL–1 CCB for 5. Difference among groups was analyzed using one-way ANOVA by SPSS 10.0 (SPSS, Inc., Chicago, IL, USA). In Experiment 1, 430 oocytes in 4 replicates were compared for the extrusion rate of the second polar body (PB) at 8 h after Ion treatment among groups. Group 5 exhibited significantly (P < 0.05) lower rates of second PB extrusion than did Groups 1–4 (22% v. 53–67%). Experiment 2 compared the rates of cleavage at 48 h and development to the blastocyst stage at 216 h after Ion treatment among groups. A total of 536 oocytes were used in 5 replicates. Parthenotes in Group 1 showed lower rates of cleavage and blastocyst development than those in other groups (20% and 1% v. 53–67% and 6–31%). Among the groups, parthenotes in Group 5 showed significantly (P < 0.05) higher blastocyst development. In Experiment 3, at 8 h after Ion treatment, oocytes from Groups 2, 3, and 5 were divided into two subgroups based on the presence or absence of the second PB, and assessed for cleavage rates and ploidy in 239 2-cell-stage parthenotes in 4 replicates, as described earlier by King et al. (1979 Vet. Sci. Commun. 3, 51–56). The cleavage rates did not differ among activation treatments, or by the presence or absence of the second PB in any activation group. The haploid rate was significantly (P < 0.05) higher in Group 2 than in Groups 3 and 5 (38% v. 19% and 0%, respectively). The diploid rate was significantly (P < 0.05) higher in Group 5 than in Groups 2 and 3 (88% v. 69% and 45%, respectively). In Experiment 4, the diploid rate of Group 2 blastocyst-stage parthenotes was 100% (4/4), whereas the diploid rates of Groups 3 and 5 blastocyst-stage parthenotes were 50% (6/12) and 71% (17/24), respectively, but the rates did not differ among groups. These results indicate that oocyte activation by CHX/CCB for 5 h after Ion treatment could enhance parthenogenetic development in bovines with higher rates of diploidy by preventing the extrusion of the second PB.

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.

scholarly journals Ovarian superstimulation with FSH in Wagyu breed bovine donors impacts folicular dynamics but does not improve the amount of embryos

2021 ◽  
Vol 10 (10) ◽  
pp. e165101018811
Author(s):  
Fabio Marcelo de Queiróz ◽  
Márcia Aparecida Andreazzi ◽  
Fábio Luiz Bim Cavalieri ◽  
Isabele Picada Emanuelli ◽  
Marcelo Marcondes Seneda ◽  
...  
Keyword(s):  
Animal Origin ◽  
World Population ◽  
Livestock Farming ◽  
Oocyte Donor ◽  
The Mean ◽  
Group 2 ◽  
Group 1 ◽  
Donor Cows

The increase of the world population generates the need to raise the production of food of vegetal and animal origin. In Brazil, livestock farming has evolved, demonstrating the important role of the country in the production of food. In this way, researchers of this productive chain have been looking for technologies related to production and reproduction, above all, to the use of reproduction biotechnologies, seeking to increase the production of different bovine breeds. Thus, the objective of this research was to investigate the efficacy of ovarian FSH super stimulation in bovine Wagyu oocyte donor females on follicular dynamics and in vitro embryo production. Twelve Wagyu animals, aged 12 to 24 months, randomly distributed in a crossover design were used in two groups: Group 1= animals not stimulated with FSH and Group 2= animals stimulated with FSH. The follicular, oocyte and embryonic variables were evaluated. It was observed that ovarian overstimulation in Wagyu oocyte donor cows with FSH improved the mean and large follicles but reduced the rate of oocyte recovery and, despite the best percentage of viable oocytes, there was no improvement in the amount of embryos produced in vitro.

scholarly journals In vivo ultrasound guided transvaginal oocyte collection in the cow

2018 ◽  
Vol 49 (3) ◽  
pp. 195
Author(s):  
G. S. AMIRIDIS (Γ.Σ. ΑΜΟΙΡΙΔΗΣ) ◽  
M. SALAHEDDINE ◽  
I. A. JEFFCOATE ◽  
E. VAINAS (Ε. ΒΑΪΝΑΣ) ◽  
L. ROBERTSON
Keyword(s):  
Recovery Rate ◽  
Transvaginal Ultrasound ◽  
Oestrous Cycle ◽  
Ultrasound Guided ◽  
Follicular Aspiration ◽  
Oocyte Recovery ◽  
Oocyte Collection ◽  
Significant Difference

This paper describes the results of the in vivo ultrasound guided follicular aspiration for ovum pick υρ (OPU) in the cow. Twelve non pregnant dry cows aged 4-6 years were used in this experiment. Eight cows underwent OPU during three successive oestrous cycles and another four cows were used as controls having only transvaginal ultrasound scanning of their ovaries. Oocyte collection took place three times during the luteal phase of each natural oestrous cycle (days 3-4,9-11 and 14-17). The content of 326 follicles with a diameter of 4-15mm was aspirated and 104 oocytes were collected (recovery rate 31.9% or 1.55 oocytes per cow and session). The oocyte recovery rate increased after the first three sessions (from 13.04% to 35.0%) and reached levels of υρ to 52.6%. More follicles were aspirated on days 9-11 (133 follicles 40.8%) compared to 111 (34%) follicles on days 14-17 and 82 (25%) on days 3-4) (P<0.05). The evaluation of the collected oocytes revealed that 60 oocytes (57.7%) were suitable for further in vitro manipulation. Neither the origin of the oocyte (left or right ovary) nor the stage of the oestrous cycle affected the recovery rate or the quality of the collected oocytes. There was no significant difference either in the length of the oestrous cycle between the experimental animals and the controls (21.6± 1.4 vs. 22.37±1.0 respectively), or in plasma progesterone concentration in daily collected blood samples from the animals of the two groups. The results of this study are compared to those from the international literature and to the results from endoscopical methods for oocyte recovery. The feasibility of application of this technique to projects designed to improve the genetic merit of cows is discussed.

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