Identification of bovine embryos cultured in groups by attachment of barcodes to the zona pellucida

2014 ◽  
Vol 26 (5) ◽  
pp. 645 ◽  
Author(s):  
Sergi Novo ◽  
Roser Morató ◽  
Oriol Penon ◽  
Sara Duran ◽  
Leonardo Barrios ◽  
...  
Keyword(s):  
Small Groups ◽  
Zona Pellucida ◽  
Embryo Culture ◽  
Wheat Germ ◽  
Retention Rates ◽  
Bovine Embryos ◽  
Individual Female ◽  
Different Origins ◽  
Tagging System

The low number of oocytes collected from unstimulated donors by ovum pick-up means that embryos produced from each individual female have to be cultured individually or in very small groups. However, it has been demonstrated that single-embryo culture is less efficient than embryo culture in groups. To overcome this limitation, we developed a direct embryo-tagging system, which allows the collective culture of embryos from different origins whilst preserving their pedigree. Presumptive bovine zygotes were tagged with eight wheat-germ agglutinin biofunctionalised polysilicon barcodes attached to the outer surface of the zona pellucida (ZP). Four different barcodes were used to encode groups of 20–25 embryos, which were then cultured in the same drop. Cleavage, Day-7 and Day-8 blastocysts and barcode retention rates were assessed. In addition, Day-7 blastocysts were vitrified and warmed. Barcode attachment to the ZP of bovine embryos affected neither in vitro embryo development nor post-warming survival of the tagged embryos. All the embryos maintained barcodes attached until Day 8 of culture (3.63 ± 0.37 barcodes per embryo) and could be identified. In conclusion, identification of embryos by barcodes attached to the ZP is feasible and will allow the culture of embryos from different donors in the same drop.

120 EFFECT OF INDIVIDUAL CULTURE SYSTEM USING WOW OR CELL-TAK ON DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
S. Matoba ◽  
P. Lonergan
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Basal Medium ◽  
Bovine Embryos ◽  
Paracrine Effects ◽  
Individual Culture ◽  
Cell Adhesive ◽  
Developmental Rates ◽  
One Way Anova

The culture of embryos individually in vitro is generally associated with poorer developmental rates. However, the ability to do this successfully would greatly facilitate studies where identification of individual embryos, or the embryos from a particular donor, is necessary. The objective of this study was to examine the effect of culture system on the development of individual IVP bovine embryos. Presumptive zygotes (n = 1301, 6 replicates), produced by IVM/IVF, were used. The aim of Experiment 1 was to compare development of bovine embryos in SOF or CR1aa supplemented with 5% FCS. Zygotes were cultured in droplets under oil as follows: (i) 20/25 μL, (ii) 20/100 μL or (iii) 20/100 μL individually in the Well of the Well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 254–264). Twenty WOW were prepared in a 100 μL droplet of medium under oil using a sterile rod. The aim of Experiment 2 was to compare development of embryos cultured in groups but individually identifiable on the cell adhesive Cell-Tak (Stokes et al. 2005 Dev. Biol. 284, 62–71) or in the WOW system. Zygotes were cultured as follows: (i) 20/20 μL, (ii) 20/20 μL with Cell-Tak, (iii) 20/100 μL with Cell-Tak or (iv) 20/100 μL in WOW. A drop of Cell-Tak (1 μL/20 μL medium) was placed on the base of the dish, dried for 20 min, washed with sterile water and dried completely. Once dried, the area was covered with 10 μL of FCS-free medium and groups of 20 zygotes were placed on the Cell-Tak in a 5 × 4 grid formation a maximum of 160 μm apart. Then, an additional 10 μL or 90 μL medium supplemented with FCS was added to give a final volume of 20 or 100 μL. Cleavage and blastocyst rates were assessed on Day 2 and Days 7–9, respectively. Data (means ± SE) were analyzed by one way ANOVA. In Experiment 1, there were no differences between SOF and CR1aa with respect to culture of embryos individually in WOW (P > 0.05); therefore, SOF was used as the basal medium for Experiment 2. There were no differences (P > 0.05) between the cleavage and blastocyst rate among drop sizes and individual culture systems; individual culture, irrespective of the system used (Cell-Tak or WOW), resulted in the similar developmental rates to the control. In conclusion, individual embryo culture offers the opportunity to study embryo development in a more powerful manner. Furthermore, the use of the cell adhesive Cell-Tak may be more practical because it removes the potential variability associated with well dimensions in the WOW system and may improve any potential paracrine effects during embryo culture. Further studies are required to establish the viability of such embryos after transfer. Table 1.Effect of individual culture system on development of IVP bovine embryos Supported by Science Foundation Ireland.

154 THE QUANTITY OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VITRO-PRODUCED BOVINE EMBRYOS FOLLOWING ARTIFICIAL EXPOSURE

2008 ◽  
Vol 20 (1) ◽  
pp. 157
Author(s):  
J. A. Gard ◽  
M. D. Givens ◽  
P. K. Galik ◽  
K. P. Riddell ◽  
M. S. D. Marley ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Bovine Viral Diarrhea Virus ◽  
Zona Pellucida ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Viral Diarrhea ◽  
Bovine Embryos ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The primary objective of this study was to determine the percentage of individual, preimplantation, in vitro-produced bovine embryos which maintained association with virus despite washing following artificial exposure to a high affinity strain of bovine viral diarrhea virus (BVDV). Another objective of this study was to determine the quantity of virus associated with these embryos. A total of eighty-seven zona pellucida-intact, Day 7, in vitro-produced bovine embryos were exposed for 1 h to 2 � 106 cell culture infected doses per mL to the 50 percent endpoint (CCID50 mL–1) of a type 1 noncytopathic strain of BVDV (SD-1). Following exposure, the embryos were washed according to International Embryo Transfer Society standards for in vitro-produced bovine embryos; they then underwent sonication, RNA extraction, and freezing at –80�C until assayed for virus. A real-time quantitative polymerase chain reaction (QPCR) was run in duplicate on each of the 87 embryos. Forty-two percent (39/87) of the embryos assayed were determined to be positive for virus. The quantity of virus associated with the embryos averaged 0.55 viral copies per 5 µL (SD = 0.89 copies/5 µL, SEM = 0.14 copies/5 µL). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos were associated with ≤2.40 viral copies per 5 µL while 99% of contaminated embryos were associated with ≤3.44 viral copies per 5 µL. These findings show that there is a low level of virus associated with in vitro-produced embryos but virus is associated with a significant number of exposed embryos. In conclusion, this study indicates that the potential for transmission of BVDV via embryo transfer of in vitro-produced embryos is small given the amount of virus that was found to associate with individual embryos.

Effect of addition of l-carnitine to media for oocyte maturation and embryo culture on development and cryotolerance of bovine embryos produced in vitro

2019 ◽  
Vol 133 ◽  
pp. 135-143 ◽  
Author(s):  
Adriana Moreira Zolini ◽  
Erly Carrascal-Triana ◽  
Antonio Ruiz de King ◽  
Peter J. Hansen ◽  
Ciro A. Alves Torres ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Embryo Culture ◽  
Bovine Embryos

scholarly journals 95 EFFECT OF ESTROUS COW SERUM ON SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS AFTER SLOW FREEZING OR VITRIFICATION

2005 ◽  
Vol 17 (2) ◽  
pp. 198
Author(s):  
N. Mucci ◽  
J. Aller ◽  
P. Ross ◽  
G. Kaiser ◽  
J. Cabodevila ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Total Cell ◽  
T Test ◽  
Slow Freezing ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Embryo Survival

Until now, the major obstacle associated with the extensive use of in vitro-produced bovine embryos is the lack of suitable methods to cryopreserve them. At least two approaches exist for overcoming this problem. One is to adjust cryopreservation methods to the requirements of these embryos, and the other is to improve embryo quality by using an appropriate in vitro environment for embryo production. The objective of this study was to determine the effect of estrous cow serum (ECS) during in vitro culture on embryo survival after cryopreservation by slow freezing or vitrification. Cumulus-oocytes complexes were in vitro-matured and fertilized as previously described (Ferre et al. 2003 Theriogenology 59, 301 abst). Presumptive zygotes were denuded from cumulus cells and cultured in groups of 50 in 400 μL drops of CR1aa medium. Seventy-two hour post-insemination (PI) embryos were randomly separated into three groups. Each group was then cultured in CR1aa + 5% ECS (without BSA; CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA), or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). Embryos were cultured under 38.5°C, 5% CO2, 5% O2, and 90% N2. At 7.5 days PI, blastocysts from each group were double stained using propidium iodide and bisbenzimide (Hoechst 33342) to determine damaged cells and total cell number. The remaining embryos were randomly cryopreserved by freezing (1.5 M ethylene glycol; cooled at 0.5°C/min to −35°C) or vitrification (open pulled straw, Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). After thawing or warming, embryos were cultured in CR1-ECS-BSA to evaluate embryo survival (hatching rate). Data were analyzed by χ2, ANOVA and Student's t-test (SAS Institute, Inc., Cary, NC, USA). Total cell number was higher in embryos cultured in CR1-ECS than in CR1-BSA or CR1-ECS-BSA (CR1-ECS: 142.1 ± 4.7, n = 23 vs. CR1-BSA 124.7 ± 4.9, n = 21, and CR1-ECS-BSA 125.8 ± 4.5, n = 25; t-test, P < 0.05). No differences were found in percent of damaged cells (CR1-ECS: 0.7%; CR1-BSA: 1.8%; CR1-ECS-BSA: 0.7%). Blastocyst survival after thawing was affected by cryopreservation methods and culture media (P < 0.01, Table 1). No interaction was found between both factors. In conclusion, under our experimental conditions elimination of ECS from CR1aa medium improves embryo cryotolerance. Vitrification allows for higher survival rates, regardless of the presence of serum during embryo culture. Table 1. Effect of cryopreservation method and serum supplementation during embryo culture on survival rate of in vitro-produced bovine embryos

214 USE OF VIRUS ISOLATION AND QUANTITATIVE POLYMERASE CHAIN-REACTION TECHNIQUES TO DETECT BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN SINGLE OR SMALL GROUPS OF PRE-IMPLANTATION BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 214
Author(s):  
J. Waldrop ◽  
M. Givens ◽  
K. Riddell ◽  
P. Galik ◽  
D. Stringfellow
Keyword(s):  
Small Groups ◽  
Virus Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Embryos ◽  
Chain Reaction ◽  
Diarrhea Virus ◽  
Polymerase Chain ◽  
Viral Diarrhea Virus

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 106-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche� RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 �L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.

Sign in / Sign up

Export Citation Format

Share Document