scholarly journals Individual commitment to a group effect: strengths and weaknesses of bovine embryo group culture

Reproduction ◽  
2014 ◽  
Vol 148 (5) ◽  
pp. 519-529 ◽  
Author(s):  
Eline Wydooghe ◽  
Leen Vandaele ◽  
Sofie Piepers ◽  
Jeroen Dewulf ◽  
Etienne Van den Abbeel ◽  
...  
Keyword(s):  
Classical Group ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Group Culture ◽  
Individual Culture ◽  
Better Than

Recently, new culture devices such as Corral and Primo Vision dishes have been designed for the culture of human embryos to allow the combination of group culture plus follow-up of individual embryos. Bovine inseminated oocytes were allocated to Primo Vision dishes, Corral dishes, individual culture or classical group culture. Blastocyst development in Primo Vision dishes was similar to classical group culture (34.3 and 39.0% respectively), and better than Corral dishes or individual culture (28.9 and 28.5% respectively). In Primo Vision dishes, a higher number of ‘slow’ embryos developed to the blastocyst stage compared with their individually cultured counterparts, while no differences were observed for ‘fast’ embryos. ‘Slow’ embryos in a ‘standard drop’ had a higher chance of becoming a blastocyst compared with individual culture (OR: 2.3), whereas blastulation of ‘fast’ embryos was less efficient in a ‘delayed drop’ than in individual culture (OR: 0.3). The number of non-cleaved embryos in Primo Vision dishes did not negatively influence blastocyst development. Likewise, removing non-cleaved embryos (NC removed) and regrouping the cleaved embryos afterwards (ReGR) did not affect blastocyst development and quality compared with group culture in Primo Vision dishes (CTRL, 31.6%, NC removed, 29.3% and ReGR, 29.6%). The experiments revealed that group culture of bovine embryos in Primo Vision dishes is superior to individual culture, primarily because of the higher blastocyst rate achieved by slow embryos. Non-cleaved or arrested embryos do not hamper the ability of co-cultured bovine embryos to reach the blastocyst stage in group culture.

1 AUTOCRINE COMMUNICATION BETWEEN BOVINE EMBRYOS CULTURED IN Primo Vision DISHES® OUTWEIGHS POSSIBLE NEGATIVE INFLUENCES OF BAD EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 115
Author(s):  
E. Wydooghe ◽  
L. Vandaele ◽  
A. Van Soom
Keyword(s):  
Developmental Stage ◽  
Odds Ratio ◽  
Fixed Effects ◽  
Binary Logistic Regression Model ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Group Culture ◽  
Individual Culture

Group culture is being used extensively for mammalian embryos, but has not been adopted so far in human embryo culture. Some doubts about its possible benefits remain because it has been hypothesised that bad quality embryos might have a negative effect on other embryos. New group culture devices have been designed allowing individual follow-up of embryos, such as Primo Vision dishes® (well of-the-well for 10 embryos in group culture; Cryo-Innovation, Budapest, Hungary). By using Primo Vision dishes®, we investigated the influence of the developmental stage of the neighbours and co-cultured embryos on the outcome at 192 h post-insemination (hpi) of a particular embryo compared with its individually cultured counterparts. Bovine presumed zygotes (n = 789; 4 replicates) produced in vitro were randomly allocated to Primo Vision dishes® or individual culture in SOF supplemented with 0.4% BSA and insulin, transferrin, selenium (Wydooghe et al. 2013 Reprod. Fertil. Dev. Epub). Cleavage rate was checked at 45 hpi: 5- to 8-cell embryos were classified as fast embryos and 2- to 4-cell embryos as slow embryos. Blastocyst development was evaluated at 192 hpi. Moreover, to evaluate which embryos benefited most from being in group (fast or slow embryos), we looked retrospectively at the influence of the developmental stage of the neighbours and the co-cultured embryos on blastocyst development. This was done separately for slow and fast embryos compared with their individually cultured counterparts. Statistical analysis was done using a binary logistic regression model, with group and replicate as fixed effects. Blastocyst development in Primo Vision dishes® was significantly better than individual culture (39.0% v. 28.5%). This beneficial outcome was mainly caused by a higher blastocyst development of slow embryos. A markedly higher percentage of slow embryos developed into a blastocyst at 192 hpi if they were surrounded by many embryos that also developed into a blastocyst, compared with individually cultured slow embryos (odds ratio: 3.0). In this study, we showed that embryos that were not cleaved at 45 hpi did not negatively affect the potential of their neighbours to become a blastocyst at 192 hpi, regardless of whether the embryo in question was a fast or a slow embryo. However, when fast embryos were in a less than favourable environment, meaning that less than 30% of their co-cultured embryos reached the blastocyst stage, blastocyst development was compromised compared with individual culture of fast embryos (odds ratio: 0.3). From our results, we clearly show that Primo Vision dishes® can combine the benefits of group culture (autocrine communication) and individual culture (individual follow-up). Taking fast embryos out of the Primo Vision dish® for further individual culture while slow embryos remain in group is a possible approach to increase total blastocyst rates.

Replacing serum in culture medium with albumin and insulin, transferrin and selenium is the key to successful bovine embryo development in individual culture

2014 ◽  
Vol 26 (5) ◽  
pp. 717 ◽  
Author(s):  
E. Wydooghe ◽  
S. Heras ◽  
J. Dewulf ◽  
S. Piepers ◽  
E. Van den Abbeel ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Individual Factor ◽  
Group Culture ◽  
Serum Free ◽  
Individual Culture ◽  
Negative Effect ◽  
Serum Free Conditions

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

Effect of α-tocopherol on in vitro culture of bovine embryos

2007 ◽  
Vol 87 (4) ◽  
pp. 539-542 ◽  
Author(s):  
A. Marques ◽  
G. Antunes ◽  
P. Santos ◽  
A. Chaveiro ◽  
F. Moreira da Silva
Keyword(s):  
Culture Media ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Media Control ◽  
Oviductal Fluid ◽  
Bovine Oocytes ◽  
Key Words Antioxidant

Bovine oocytes were matured and fertilised under 5% CO2. Presumptive zygotes were co-cultured in synthetic oviductal fluid droplets, supplemented with either 0 (control), 25, 50, 100, or 200 µM of α-tocopherol. Blastocyst development rates were significantly influenced (P < 0.05) by the level of antioxidant in the culture media. Control showed a blastocyst yield of 18.46, 21.11, 27.92, and 31.66%, respectively, at α25; α50 and α100. Blastocyst yield for α200 was severely decreased, to 8.01%. An increase in overall IVF results was also observed as the concentration of α-tocopherol increased (control, 14.21%; α25, 14.35%; α50, 19.52%; α100, 21.11%), decreasing to 5.75% for the concentration of α200. The present study clearly demonstrates that α-tocopherol at a concentration of 100 µM significantly improves the proportion of oocytes that develop to the blastocyst stage. Key words: Antioxidant, α-tocopherol, reactive oxygen species, bovine, embryo

141 BOVINE EMBRYO CULTURE IN THE MODIFIED WELL OF THE WELL SYSTEM INCREASES BLASTOCYST RATE BUT LOWERS BLASTOCYST QUALITY

2013 ◽  
Vol 25 (1) ◽  
pp. 218
Author(s):  
S. Heras ◽  
E. Wydooghe ◽  
A. Van Soom
Keyword(s):  
Embryo Culture ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Group Culture ◽  
Autocrine Factors ◽  
Individual Culture ◽  
Blastocyst Quality ◽  
Negative Effect ◽  
Blastocyst Rate

Individual culture of cattle embryos in serum-containing medium has a detrimental effect on blastocyst development. As we previously demonstrated, this effect is not caused by the individual culture itself but by some component present in serum (Heras et al. AETE Proc. 2012). The high blastocyst rates achieved with group culture in serum-containing medium can be explained by the neutralizing effect of the produced autocrine factors. Autocrine factors produced by individually cultured embryos cannot neutralize the detrimental component due to its low amount and their diffusion in the high culture volume. Using a modified well of the well (mWOW; Vajta et al. 2000 Mol. Rep. Dev.), where embryos are singly cultured in a small well, the effect of the autocrine factors produced by individual embryos can be maximized. We hypothesized that the use of mWOW for individual embryo culture in serum-containing medium, can maximize the neutralizing effect of the autocrine factors, increasing both blastocyst rate and quality. Bovine oocytes (n = 1143, 3 replicates) were matured in TCM-199 with 20% fetal bovine serum (FBS). Presumptive zygotes were cultured in groups (Gr; 25 embryos/50-µL drops), individually (Ind; 20-µL drops), or in mWOW (20-µL drops) in SOFaa with 5% FBS. Blastocyst evaluation occurred 7 and 8 days post-insemination (dpi). Hatching rate is the proportion of hatching/hatched blastocysts out of total 8-dpi blastocysts. Blastocysts were collected at 8 dpi for differential apoptotic staining, evaluating total cell number (TCN), inner cell mass (ICM) ratio, and apoptosis (ACR; Wydooghe et al. 2011 Anal. Biol.). Developmental data were analyzed using binary logistic regression; data concerning blastocyst quality were analyzed using linear mixed model analysis. Since ACR was not normally distributed, a logarithmic transformation was performed. Differences at P < 0.05 were considered significant. Regarding blastocyst development, all groups were significantly different both at 7 dpi (Gr 33.9%, Ind 10.7%, mWOW 19.6%) and at 8 dpi (Gr 37.3%, Ind 14.0%, mWOW 29.1%). Hatching rates were significantly higher in Gr (27.9%) compared to Ind (4.0%) and mWOW (4.1%). Concerning blastocyst quality, TCN was significantly higher (P < 0.001) in Gr (233.23 ± 8.39), no differences between Ind (143.77 ± 6.32) and mWOW (125.11 ± 4.30) were observed. No differences in ICM ratio were observed (Gr 0.41 ± 0.01, Ind 0.30 ± 0.02, mWOW 0.37 ± 0.02). ACR was significantly different between all groups (Gr 0.03 ± 0.002, Ind 0.04 ± 0.003, mWOW 0.08 ± 0.005). In conclusion, the mWOW had a positive effect on blastocyst quantity, increasing blastocyst rates compared to individual culture, but still lower than in group culture. Surprisingly, mWOW had a negative effect on blastocyst quality, producing the highest ACR and the lowest TCN. This negative effect can be caused by toxic products arising from the manufacture (although the wells were rigorously flushed), or by the longer manipulation time needed. This work was funded by FWO G.0210.09N and IWT g111438.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

scholarly journals 153EXPRESSION OF TGF-BETA I AND TYPE I AND TYPE II OF TGF-BETA RECEPTORS IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
B.K. Kim ◽  
H.J. Chung ◽  
B.C. Yang ◽  
D.H. Kim ◽  
J.H. Woo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Type I ◽  
Bovine Embryo ◽  
Type Ii ◽  
Bovine Embryos ◽  
Mrna And Protein Expression ◽  
Bovine Oocytes

Although the effects of TGFβ1, as an important factor in the mice embryo development have been reported, little information relevant to this subject is known in the bovine embryo. The objectives of this study were to investigate the presence and expression patterns of TGFβ1 and TGFβ1 receptors, types I and II, in unfertilized oocytes and fertilized bovine embryos in normal and NT embryo development. We postulated that TGFβ1 may have a beneficial effect on the preimplantation embryo and show different expression patterns at different stages of bovine embryo development. Immature bovine oocytes were aspirated from follicles of ovaries obtained from a local abattoir and they were cultured for up to 24h and fertilized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were used to investigate the presence of TGFβ1 and type I and type II of TGFβ1 receptors (the essential components of the TGFβ1 signaling pathway) in unfertilized oocytes and preimplantation embryos. Also, mRNA and protein expression patterns of TGFβ1 and their receptors at various stages of embryos were examined. It was found that both receptors, as well as TGFβ1, were present in the unfertilized bovine oocytes, indicating that TGFβ1 is a maternally expressed protein. Although the type I TGFβ1 receptor was present at the morulae and blastocyst stages, the type II TGFβ1 receptor was not present at both stages. It was also confirmed that the expression level of TGFβ1 was high at the 8-cell stage, and mRNA and protein expression patterns of TGFβ1 and their receptors were not coincident. Interestingly, TGFβ1 protein was not detected at blastocyst stage of embryos, whereas the mRNA expression level was high at this stage. The results of this experiment indicate that TGFβ1 protein may be needed by embryos after the blastocyst stage and may be expressed in hatched embryos for implantation. These findings support the hypothesis that there may be an interaction between the TGFβ1 and TGFβ1 receptors in the unfertilized oocytes and preimplantation embryos, and that TGFβ1 signaling may be important for the development of the oocytes and the preimplantation embryos.

scholarly journals Characterization and functional roles of paternal RNAs in 2–4 cell bovine embryos

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nicole Gross ◽  
Maria Giuseppina Strillacci ◽  
Francisco Peñagaricano ◽  
Hasan Khatib
Keyword(s):  
Male Fertility ◽  
Expression Patterns ◽  
Low Fertility ◽  
Regulatory Subunit ◽  
Parental Origin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Functional Roles ◽  
Cell Embryo

AbstractEmbryos utilize oocyte-donated RNAs until they become capable of producing RNAs through embryonic genome activation (EGA). The sperm’s influence over pre-EGA RNA content of embryos remains unknown. Recent studies have revealed that sperm donate non-genomic components upon fertilization. Thus, sperm may also contribute to RNA presence in pre-EGA embryos. The first objective of this study was to investigate whether male fertility status is associated with the RNAs present in the bovine embryo prior to EGA. A total of 65 RNAs were found to be differentially expressed between 2–4 cell bovine embryos derived from high and low fertility sires. Expression patterns were confirmed for protein phosphatase 1 regulatory subunit 36 (PPP1R36) and ataxin 2 like (ATXN2L) in three new biological replicates. The knockdown of ATXN2L led to a 22.9% increase in blastocyst development. The second objective of this study was to characterize the parental origin of RNAs present in pre-EGA embryos. Results revealed 472 sperm-derived RNAs, 2575 oocyte-derived RNAs, 2675 RNAs derived from both sperm and oocytes, and 663 embryo-exclusive RNAs. This study uncovers an association of male fertility with developmentally impactful RNAs in 2–4 cell embryos. This study also provides an initial characterization of paternally-contributed RNAs to pre-EGA embryos. Furthermore, a subset of 2–4 cell embryo-specific RNAs was identified.

166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 195
Author(s):  
R. Nishii ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Culture System ◽  
Calf Serum ◽  
Control Culture ◽  
Total Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Culture Systems ◽  
Individual Culture ◽  
Single Culture

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

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