128 EFFECT OF OXYGEN TENSION ON INDIVIDUAL IN VITRO BOVINE EMBRYO DEVELOPMENT IN CUMULUS CELL COCULTURE

2008 ◽  
Vol 20 (1) ◽  
pp. 144
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
S. Andries ◽  
J. B. P. De Clercq ◽  
P. E. J. Bols
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Individual Development ◽  
Bovine Embryo ◽  
Group Culture ◽  
Effect Of Oxygen ◽  
Hatching Rates

An in vitro production system, in which a single oocyte can be followed from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to immature oocyte quality and follicular environment. Earlier studies revealed that cumulus cell (CC) coculture during IVC enhances individual development, in contrast to group culture. These studies were performed in 5% O2, whereas generally an atmosphere of 20% oxygen is used for coculture with somatic cells. This study aimed to investigate the effect of oxygen tension on individual embryo development in CC coculture. As a control, the effect of oxygen tension on embryo group culture without CC was assessed simultaneously. Therefore, 692 COC from slaughterhouse ovaries (4 replicates) were routinely matured (TCM-199 + 20% serum) and fertilized in groups and then assigned to 4 culture treatments (SOF + 5% serum under oil). Treatments were T1 = 1 zygote in 20 μL + CC in 5% O2; T2 = 1 zygote in 20 μL + CC in 20% O2; T3 = 20 to 25 zygotes in 50 μL in 5% O2; and T4 = 20 to 25 zygotes in 50 μL in 20% O 2. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days after fertilization, respectively. Possible effects of oxygen tension in individual and group culture were evaluated with binary logistic regression. No interactions between replicate and treatment could be found. Cleavage rates of individual culture showed a tendency (P < 0.1) to be lower in 5% O2 (62.1 v. 71.0% in 20% O2), whereas blastocyst rates were significantly (P < 0.05) higher in 5% O2 (26.6 v. 16.6% in 20% O2). Hatching rates did not differ significantly between the 2 individual treatments (Table 1). Oxygen tension did not have a significant effect on cleavage rates when embryos were cultured in groups, but blastocyst rates were significantly higher in 5% O2 (41.7 v. 27.6% in 20% O2). The group results confirm other studies (Yuan YQ et al. 2003 Theriogenology 59, 1585–1596). In conclusion, higher blastocyst rates can be obtained when an atmosphere of 5% O2 is used for culturing individual zygotes in CC coculture. Because cleavage rates showed a tendency to be higher in 20% O2, an alternating treatment, with 20% O2 until 2 days after fertilization, followed by 5% O2 until the blastocyst stage, should be investigated. Table 1. Cleavage, blastocyst, and hatching rates in 5 and 20% oxygen

48 EFFECTS OF LIPID METABOLIC REGULATORS DURING BOVINE EMBRYO CULTURE ON BLASTOCYST DEVELOPMENT AND CRYOSURVIVAL

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Phenazine Ethosulfate ◽  
Metabolic Regulators ◽  
Hatching Rates

The overall objective was to determine the effects of addition of lipid metabolic regulators during embryo culture on blastocyst development and survival following cryopreservation. For Experiment 1, embryos produced in vitro were cultured in 5% (vol/vol) oxygen in SOF-bovine embryo 1 (SOF-BE1) medium supplemented with or without 100 μM trans-10,cis-12 conjugated linoleic acid (CLA) and 0.3 μM phenazine ethosulfate (PES). Treatment with CLA began at the initiation of culture, whereas treatment with PES began at Day 3 after insemination. At Day 7 after insemination, the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, or hatched) stages was recorded. Blastocysts and expanded blastocyst-stage embryos were harvested and slow frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 medium containing 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. Addition of CLA had no effect on embryo development, whereas PES reduced (P < 0.01) development to the blastocyst (26.0 ± 0.8 v. 22.1 ± 0.8%) and advanced blastocyst (19.2 ± 0.9 v. 14.4 ± 0.9%) stages. Blastocysts cultured in the presence of CLA had higher (P < 0.05) re-expansion rates at 24, 48, and 72 h (50.8 ± 3.7 v. 65.7 ± 3.7%, 57.2 ± 4.0 v. 72.0 ± 4.05%, and 57.2 ± 4.0 v. 72.0 ± 4.0%, respectively). Addition of CLA tended (P < 0.07) to increase the hatching rate at 24 h and did increase (P < 0.05) the hatching rate at 48 h (12.4 ± 1.3 v. 16.2 ± 1.3% and 39.0 ± 3.2 v. 50.0 ± 3.2%, respectively). Treatment with PES had no effect on re-expansion rates but reduced (P < 0.05) hatching rates at 24 and 48 h (18.2 ± 1.3 v. 10.3 ± 1.3 and 50.2 ± 3.2 v. 38.8 ± 3.2%, respectively). There was no interaction between CLA and PES affecting embryo development or cryosurvival. For Experiment 2, embryos were produced in vitro as in Experiment 1 and cultured in SOF-BE1 medium with or without 3.03 mM L-carnitine (LC) and 10 μM forskolin (FK). Treatment with LC began at the initiation of culture and treatment with FK began at Day 6. All other methods were as described for Experiment 1. Addition of LC did not affect development to the blastocyst stage but reduced (P < 0.05) development to the advanced blastocyst stage (21.0 ± 1.2 v. 17.1 ± 1.2%). Treatment with FK had no effect on embryo development to the blastocyst or advanced blastocyst stages. Blastocysts cultured in the presence of LC had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h (60.2 ± 2.0 v. 78.0 ± 2.0%, 62.9 ± 1.2 v. 83.3 ± 1.2%, and 63.0 ± 2.4 v. 82.8 ± 2.4%, respectively) and hatching rates at 48 and 72 h (48.6 ± 4.3 v. 64.1 ± 4.3% and 59.6 ± 3.0 v. 78.5 ± 3.0%, respectively). There was no effect of FK on cryosurvival and no interaction between LC and FK affecting embryo development or cryosurvival. In conclusion, blastocyst yield was not improved by any of the lipid metabolic regulators tested. Cryosurvival was enhanced by addition of CLA and LC but FK reduced survival following freezing. There were no additive effects of either CLA and PES or LC and FK for blastocyst yield or cryosurvival.Support was provided by USDA AFRI Grant 2010-85122-20623.

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.

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.

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.

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
J. Block ◽  
A. M. Zolini ◽  
E. Carrascal-Triana ◽  
A. Ruiz ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Hatching Rates

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

scholarly journals Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium?

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 507-517 ◽  
Author(s):  
M Clemente ◽  
J de La Fuente ◽  
T Fair ◽  
A Al Naib ◽  
A Gutierrez-Adan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Fourfold Increase ◽  
Uterine Environment ◽  
High Concentrations

The steroid hormone progesterone (P4) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P4 in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P4 affects bovine embryo development in vitro and in vivo. mRNA for P4 receptors was present at all stages of embryo development raising the possibility of a direct effect of P4 on the embryo. Exposure to P4 in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P4 in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P4 resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P4-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P4 in order to exhibit advanced elongation.

scholarly journals Culture system and long-term storage of culture media in the in vitro production of bovine embryos

2011 ◽  
Vol 59 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Santiago Varga ◽  
Carmen Diez ◽  
Lina Fernández ◽  
Jenny Álvarez ◽  
Adelino Katchicualula ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Culture Media ◽  
Calf Serum ◽  
Significant Loss ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Fresh Culture Medium ◽  
Hatching Rates

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

scholarly journals 282MATURATION MEDIUM EXCHANGE IS EFFECTIVE ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 261
Author(s):  
Y.S. Park ◽  
S.H. Choi ◽  
H.D. Park ◽  
M.D. Byun
Keyword(s):  
Embryo Development ◽  
Total Protein ◽  
Harmful Effect ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Chi Square ◽  
Medium Exchange ◽  
Chi Square Test

In vitro embryo development is strongly influenced by IVM conditions. Increased duration of IVM may cause aging of the oocytes, which has a harmful effect on the embryo development. Oocyte maturation depends upon the synthesis of several proteins that may play important roles in the cytoplasmic maturation. These experiments were conducted to determine the effect of IVM duration(18-h or 24-h) and medium exchange (at 18h) on embryo development, and to investigate the protein quantities in IVM medium. Korean Native Cow (KNC) ovaries were obtained from a local slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in 50-μL drops of TCM-199 supplemented with 10% fetal calf serum (FBS), 1μgmL−1 MFSH, 10μgmLLH and 1μgmL−1 Estradiol-17β for 18h or 24h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (After Day 3). All types of cultures were carried out in an incubator at 39°C, 5% CO2 in air. The total protein quantity in IVM medium at 18h or 24h were compared by 2-dimensional gel electrophoresis using a 10–15% polyacrylamide gradient gels. Data from three replicates were analyzed by chi-square test. The proportions of oocytes reaching the blastocyst stage was significantly higher in 18h IVM group than 24h IVM group (Table 1). However, there was no difference detected in blastocyst rate between 18h IVM group and 18h medium exchange group. Total protein quantity was reduced between 18h and 24h in IVM medium. There were 299 protein spots identified in IVM medium;; there was an increase at 10 spots in the IVM medium analyzed at 18h and a decrease of 20 spots at 24h. This study suggests that duration of IVM affects subsequent embryo development. The total protein quantity was decreased between 18h and 24h in IVM medium. These proteins may be absorbed into the oocytes and reduce development to the blastocyst stage. However, this may be overcome by IVM medium exchange. Table 1 Effects of duration of IVM and medium exchange on embryo development of KNC oocytes

177 EFFECT OF DIFFERENT FERTILIZATION MEDIA ON IN VITRO BOVINE EMBRYO DEVELOPMENT USING FLOW-CYTOMETRICALLY-SORTED FEMALE SPERM

2014 ◽  
Vol 26 (1) ◽  
pp. 203
Author(s):  
L. B. Ferré ◽  
Y. S. Bogliotti ◽  
J. L. Chitwood ◽  
P. J. Ross
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Cumulus Cell ◽  
Bovine Embryo ◽  
Vitro Fertilization ◽  
Female Sex ◽  
Fetal Bovine

High demand exists for in vitro-derived bovine embryos fertilized with female sex-sorted sperm by seedstock and commercial cattle producers. The aim of this study was to evaluate different fertilization media on in vitro fertilization performance using female sex-sorted semen. Ovaries were collected from a slaughterhouse and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 cumulus-oocyte complexes in 400 μL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 μg mL–1), epidermal growth factor (50 ng mL–1), oFSH (50 ng mL–1), bLH (3 μg mL–1), cysteamine (0.1 mM), and 10% fetal bovine serum for 22 to 24 h. Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm mL–1. Three different fertilization media, M199 (Gibco 11043–023, Grand Island, NY, USA), SOF (Tervit et al. 1972 J. Reprod. Fertil. 30, 493–497), and TALP (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180), were assayed along with 3 female sex-sorted bulls. All fertilization media were supplemented with fructose (90 μg mL–1), penicillamine (3 μg mL–1), hypotaurine (11 μg mL–1), and heparin (20 μg mL–1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-μL drops of KSOM-BSA for 9 days. On Day 3, 3% fetal bovine serum was added. Low oxygen tension (5% O2) was used for the entire culture period. On Days 7 and 9 blastocysts and hatched embryos, respectively, were morphologically evaluated according to IETS standards and recorded. Results are shown in Table 1. Data was compared by chi-squared analysis. Fertilization media affected cleavage rate and subsequent embryo development, quality, and hatching ability. The SOF and TALP fertilization media produced significantly more and higher quality embryos than M199. Table 1.In vitro fertilization performance after oocyte fertilization using sex-sorted sperm

210 CO-CULTURE WITH AUTOLOGOUS CUMULUS CELLS SUPPORTS THE INDIVIDUAL DEVELOPMENT OF SINGLY IN VITRO-MATURED AND FERTILIZED BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 204
Author(s):  
I. G. F. Goovaerts ◽  
J. L. M. R. Leroy ◽  
E. Merckx ◽  
S. Andries ◽  
P. E. J. Bols
Keyword(s):  
Somatic Cells ◽  
Cumulus Cells ◽  
Individual Development ◽  
Control Individual ◽  
In Vitro Production ◽  
Group Culture ◽  
The Individual ◽  
Vitro Production ◽  
Hatching Rates

The ability to produce embryos singly in vitro (in vitro production, IVP) would be a useful tool for many purposes. Without the interfering effects of other developing or degenerating oocytes or embryos, such an individual IVP system is the tool of choice for studies on oocyte quality and oocyte–embryo metabolism. Unfortunately, individual IVP in most cases leads to unsatisfactorily low blastocyst rates. Earlier work showed that individual culture of zygotes on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos, as obtained following regular group culture (Goovaerts et al. 2009 Theriogenology 71, 729–738). However, co-culture with somatic cells is often criticised because of the undefined culture conditions and for sanitary reasons. In the cited study, CC for monolayer production were obtained from a different batch of ovaries. Our specific aim was to use CC from the zygote itself (autologous CC). Grade I COC (n = 660) were collected from slaughterhouse ovaries and randomly assigned to 2 treatments (5 replicates): a completely individual ‘single-oocyte’ IVP protocol, or routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was stripped and cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2), to which the autologous stripped CC were added. Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were determined 2, 8, and 10 days post-fertilization, respectively. Possible effects of the individual and group cultures were evaluated with binary logistic regression (SPSS 15.0, SPSS Inc., Chicago, IL). No interactions between replicate and treatment were found (P > 0.05). Although a blastocyst rate of 15.1% was obtained using single IVP, the general efficacy of the single-embryo production system was lower when compared with group culture (Table 1). In conclusion, although developmental competence was impaired using individual IVP, co-culture with autologous cumulus cells can be useful in specific experimental setups in which the influence of other oocytes or embryos or heterologous somatic cells is unacceptable. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro production (IVP)

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