30 Vitrification of Equine Embryos: Application in a Commercial Cloning Program

2018 ◽  
Vol 30 (1) ◽  
pp. 154
Author(s):  
G. Vichera ◽  
R. Jordan ◽  
V. Arnold ◽  
D. Dobler ◽  
R. Olivera
Keyword(s):  
Breeding Season ◽  
Production Efficiency ◽  
Embryo Quality ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Pregnancy Rates ◽  
Embryo Production ◽  
In Vitro Embryo Production ◽  
The One

During a commercial horse cloning program, a critical point is the availability and maintenance of suitable recipient mares for a large quantity of embryo transfers. Usually, pregnancy rates and viable births off the breeding season decrease significantly, whereas the rate of in vitro embryo production remains constant. For this reason, an efficient vitrification system allows continuous embryo production throughout the year with the advantage of doing the transfers only during the breeding season. The vitrification technique evaluated in this study was the one described by Kuwayama et al. (2007 Theriogenology 67, 73-80). By using this method, we compared post-warming recovery efficiency, pregnancy rates, and viable foaling rates in 2 experimental groups: cloned blastocysts vitrified off-season and transferred in breeding season (VC, n = 337), and non-vitrified cloned blastocysts also transferred in breading season (no-VC, n = 516). To achieve this, equine oocytes were collected from slaughterhouse ovaries, matured, enucleated, and fused to a donor cell according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). The reconstructed embryos (RE) were cultured in a well-of-the-well system by adding 3 RE per well for 7 to 8 days to reach the blastocyst stage, at which they were vitrified as mentioned above. During the breeding season, blastocysts were warmed and transferred in couples in a single cycling receptive mare. Pregnancies were confirmed by transrectal ultrasonography 15 days post-transfer. All variables were analysed by Fisher test (P < 0.05). The warming recovery rate was 91% (308/337) for cloned blastocysts. In addition, pregnancy and viable birth rates were similar for the VC and no-VC groups: 15.6% (24/154) v. 16.7% (43/258) for pregnancy rates, respectively, and 37.5% (9/24) v. 37.2% (16/43) for foaling rates, respectively. In summary, 9 viable cloned foals were obtained with off-season embryos warmed and transferred during the breeding season, showing that vitrification did not affect embryo quality. Hence, the proposed strategy provides the ability to maximize production efficiency of equine clones by generating a large number of pregnancies without stopping in vitro embryo production at any time of the year.

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

281 EVALUATION OF IN VITRO EMBRYO PRODUCTION EFFICIENCY USING SEXED BULL SPERM SORTED WITH TWO TYPES OF CELL SORTER

2011 ◽  
Vol 23 (1) ◽  
pp. 238
Author(s):  
H. Hayakawa ◽  
T.-I. Hirata
Keyword(s):  
Production Efficiency ◽  
Bos Taurus ◽  
Digital Processing ◽  
Embryo Production ◽  
Cell Sorter ◽  
Treatment Groups ◽  
Fort Collins ◽  
In Vitro Embryo Production ◽  
Bull Sperm

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitro embryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus (Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo® SX (SX, Dako, Fort Collins, CO, USA) or MoFlo® XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20 h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20 min at 448 × g through an ISolate® (Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5 min at 252 × g. Oocytes were co-incubated with washed sperm (5 to 10 × 106 sperm mL–1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8 h at 38.5°C in 5% CO2 and 95% air (Day 0). Presumptive zygotes were cultured for 90 h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1 350 oocytes used) and 5 times for bull B (total of 1 529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P < 0.001) and XDP (P < 0.002) groups than in control groups for both bulls but not different between SX and XDP (P > 0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P < 0.003) and blastocyst (P < 0.02) rates. MoFlo® SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitro embryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health. Table 1.Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments

scholarly journals Effect of different cryopreservation extenders added with antioxidants on semen quality and in vitro embryo production efficiency in cattle

2021 ◽  
Vol 93 (3) ◽  
Author(s):  
NATALIA C. SILVA ◽  
KAREN M. LEÃO ◽  
JOÃO T. PÁDUA ◽  
THAISA C. MARQUES ◽  
FRANCISCO R.A. NETO ◽  
...  
Keyword(s):  
Production Efficiency ◽  
Semen Quality ◽  
Embryo Production ◽  
In Vitro Embryo Production

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.

241 EFFECT OF PRODUCTION EFFICIENCY IN THE LIKELIHOOD OF PREGNANCY OF IN VITRO-DERIVED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
L. F. Feres ◽  
L. S. A. Camargo ◽  
M. P. Palhao ◽  
F. Z. Brandao ◽  
J. H. M. Viana
Keyword(s):  
Pregnancy Rate ◽  
Production Efficiency ◽  
Production Systems ◽  
Bos Indicus ◽  
Pregnancy Rates ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Chi Square

Improving in vitro culture systems to optimize embryo yield has been a major research goal. The relationship between the efficiency of embryo production systems and the pregnancy outcomes, however, remain controversial. The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced embryos derived from batches with different relative efficiency indexes. Data of 702 ovum pick-up (OPU) and in vitro embryo production (IVEP) sessions, and of 2456 embryo transfers, recorded from 2008 to 2012, were evaluated. All donors were from the same herd, and were of the same breed (Gir, Bos indicus), as well as the semen used for IVF. The cumulus-oocycte complex (COC) recovery and IVEP were performed by the same team, in a single IVF laboratory, and using standard medium and procedures. Only data from embryos transferred as fresh were used, and records from 97 OPU/IVEP sessions in which no embryo was produced, or embryos were frozen or discharged due to lack of recipients, were discharged. The remaining 605 sessions were stratified in quartiles (I to IV, each one corresponding to 25% of total data) according to COC production of the donors, or stratified in ranges (0–25%, 26–50%, 51–75%, and 76–100%) according to COC quality (percentage of viable COC or of grade I COC) and to embryo production efficiency endpoints (cleavage rate, blastocyst rate). Pregnancy rates were compared among quartiles or ranges by the chi-square method. On average, the Gir donors produced 24.8 ± 0.6 COC per OPU, from which 14.4 ± 0.4 were classified as viable (57.8%), and 3.2 ± 0.1 as grade I (12.9%). On average 6.1 ± 0.2 embryos (morulas and blastocysts) were produced per OPU per donor, and mean pregnancy rate was 30.9%. As expected, donors with greater total COC yield (quartile I) also produced more viable oocytes (25.5 ± 0.7 v. 15.7 ± 0.3, 10.5 ± 0.2 and 5.8 ± 0.2), more COC grade I (4.8 ± 0.4 v. 3.9 ± 0.3, 2.6 ± 0.2 and 1.6 ± 0.1), and more embryos (9.0 ± 0.4 v. 6.9 ± 0.3, 5.0 ± 0.2 and 3.3 ± 0.1) than donors from quartiles II, III, or IV, respectively (P < 0.0001). Nevertheless, there was no difference (P > 0.05) in pregnancy rates for embryos produced from donors ranked in the different quartiles (30.9 v. 29.3, 31.5, and 30.5% for quartiles I to IV, respectively). Similarly, there was no difference (P > 0.05) in the pregnancy rate of embryos derived from OPU sessions in which there was a high or low percentage of viable or grade I COC. In vitro production efficiency (cleavage and blastocyst rates) also had no effect (P > 0.05) on further pregnancy rates. In conclusion, these results suggest that there is no relationship among the average number or quality of the COC recovered by OPU, the efficiency of IVEP, and the likelihood of pregnancy of in vitro-derived embryos.Research was supported by Fazendas do Basa, CNPq, and Fapemig.

scholarly journals Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season

Animals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1496
Author(s):  
Anna Rita Piras ◽  
Federica Ariu ◽  
Maria-Teresa Zedda ◽  
Maria-Teresa Paramio ◽  
Luisa Bogliolo
Keyword(s):  
Breeding Season ◽  
Domestic Cat ◽  
Hatching Rate ◽  
Domestic Cats ◽  
Embryo Production ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season.

Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors

2011 ◽  
Vol 75 (9) ◽  
pp. 1640-1646 ◽  
Author(s):  
J.H.F. Pontes ◽  
F.A. Melo Sterza ◽  
A.C. Basso ◽  
C.R. Ferreira ◽  
B.V. Sanches ◽  
...  
Keyword(s):  
Large Scale ◽  
Bos Indicus ◽  
Pregnancy Rates ◽  
Embryo Production ◽  
Commercial Program ◽  
In Vitro Embryo Production ◽  
Nelore Cattle

102 In Vitro Embryo Production and Oocyte Quality in Bos indicus Beef Cows Selected for Fertility Characteristics

2018 ◽  
Vol 30 (1) ◽  
pp. 190
Author(s):  
G. L. Vasconcelos ◽  
R. Maculan ◽  
N. Alves ◽  
A. L. A. P. L. Ribeiro ◽  
A. W. B. Silva ◽  
...  
Keyword(s):  
Production Efficiency ◽  
Fluorescent Microscopy ◽  
Bos Indicus ◽  
Germinal Vesicle ◽  
Antral Follicle ◽  
Beef Cows ◽  
Embryo Production ◽  
Germinal Vesicle Breakdown ◽  
In Vitro Embryo Production

Embryo production may be enhanced when associated with cows selected on the basis of fertility markers, which should be easy to measure, such as antral follicle count (AFC) and genital tract morphometrics. The objective was to evaluate the effects of AFC class on oocyte 24-h outcome and in vitro embryo production in Bos indicus beef cows. Brahman (n = 151) cows (2-13 years old, 344-803 kg of BW, and 7-9 BCS). Low (LAFC), intermediate (IAFC), and high (HAFC) antral follicle classes were defined as follows: LAFC ≤ 30; IAFC 30-49; and HAFC ≥50 AFC. All follicles ≥3 mm in diameter were aspirated by conventional ovum pick-up technique. Only cumulus–oocyte complexes with at least 2 layers of granulosa cells and homogeneous cytoplasm were used for in vitro culture. They were matured in TCM-199 plus supplements for 24 h at 38.7°C in a 5% CO2 humidified atmosphere. After 24 h of maturation, a subset of oocytes (n = 319) was fixed and analysed under fluorescent microscopy and oocyte outcome was evaluated by classification, as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I/anaphase I/telophase I (MIAITI), and metaphase II (MII). The second subset of oocytes (n = 797) was fertilized in Ferti-TALP (10-15 oocytes per 60-µL drop) with frozen–thawed semen (18-22 h at 38.7°C in 5% CO2 after Percoll) from a single bull previously tested for good in vitro fertility. Presumptive zygotes were cultivated in CR2 medium for 48 h at 37.8°C in 5% CO2. For the remaining 96 h, embryos were transferred to 10% FCS-supplemented TCM-199 drops until the final evaluation. Data were analysed by the GENMOD, GLM, and CORR procedures of SAS (SAS Institute Inc., Cary, NC, USA). Viable oocytes, total embryos, and embryo production efficiency (viable oocyte/total embryos produced; P < 0.05) to AFC in various degrees (r2 = 0.87, 0.86, 0.30, respectively). The proportion of oocytes in GV, GVBD, MIAITI, and MII were different (P < 0.05) between LAFC, IAFC, and HAFC classes [GV: 12.3% (13/106)a, 3.1% (3/96)a and 4.3% (5/117)b, respectively]; [GVBD: 32.1% (34/106)a, 8.3% (8/96)a and 6.0 (7/117)b]; [MIAITI: 14.2% (15/106)a, 26.0% (25/96)b and 8.5% (10/117)c, respectively] and [MII: 41.5% (44/106)b, 62.5% (60/96)a and 81.2% (95/117)c, respectively). In conclusion, high AFC is positively related to better in vitro embryo fertility and to 24-h oocyte outcome after in vitro maturation.

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