292 EVALUATION OF THREE PROTEIN SOURCES FOR THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 302
Author(s):  
E. A. Ordoñez-Leon ◽  
G. Cancino ◽  
J. Hernandez-Ceron ◽  
J. A. Medrano ◽  
Y. C. Ducolomb ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Embryo Production ◽  
Serum Free ◽  
Serum Replacement ◽  
Knockout Serum Replacement

Bovine embryo development in vitro can be affected by many factors, including protein source, which can cause embryo development failure. The use of in vitro culture media supplemented with serum-free compounds could allow a better understanding of embryo requirements during the preimplantation stages by eliminating a highly variable and undefined compound such as serum. The objective of this study was to evaluate the effect of 3 different protein supplements used during IVM, IVF, and IVC on embryo production. Ovaries were collected from slaughtered cows and then aspirated to obtain oocytes for in vitro embryo production procedures. A total of 2056 oocytes were used, from which 685 were processed with maturation medium supplemented with 10% serum replacement (SR) (Gibco Knockout Serum Replacement, Invitrogen, Carlsbad, CA, USA), a defined serum-free formulation (TCM-199 + SR), fertilization medium with SR (TALP + SR), and culture medium with SR (SOF + SR). These were compared with 675 and 696 oocytes processed with the same IVM, IVF, and IVC media, but supplemented with 10% FCS or 10% heat-inactivated estrous cow serum (ECS), respectively. Data obtained from the variables studied were processed by analysis of variance and means were compared by Tukey’s test. The percentages of embryos produced with FCS (52.4%) and ECS (52.7%) were significantly higher compared with the percentage obtained with SR (41.5%) (P < 0.05). The percentages of morulae were similar in the groups supplemented with FCS (36.5%) and SR (36.7%), but significantly higher than the percentage in the ECS group (26.9%) (P < 0.05). For blastocysts, the percentages of embryos developed with FCS (35.2%) and ECS (35.6%) were significantly higher than that obtained with SR (29.2%) (P < 0.05). When evaluating expanded blastocysts, the percentage obtained in the FCS (45.9%) group was significantly higher than that in the ECS group (33.2%), and this was significantly higher than that obtained in SR (21%), with all these differences being significant (P < 0.05). It is concluded that it is possible to produce bovine embryos in vitro using FCS, ECS, or SR as supplements in IVM, IVF, and IVC media. Significant differences were found in different embryo stages, with the highest proportion of embryos developing with the addition of FCS, whereas supplementation with SR only improved the production of morulae. We thank Consejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico) for the graduate student’s scholarship.

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

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.

Messenger RNA expression patterns in bovine embryos derived from in vitro procedures and their implications for development

2005 ◽  
Vol 17 (2) ◽  
pp. 23 ◽  
Author(s):  
Christine Wrenzycki ◽  
Doris Herrmann ◽  
Andrea Lucas-Hahn ◽  
Karin Korsawe ◽  
Erika Lemme ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Culture Media ◽  
Expression Patterns ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Developmental Abnormalities ◽  
Embryonic Genome

The preimplantation bovine embryo is initially under the control of maternal genomic information that is accumulated during oogenesis. The genetic programme of development soon becomes dependent on new transcripts derived from activation of the embryonic genome. The early steps in development, including the timing of the first cleavage, activation of the embryonic genome, compaction and blastocyst formation, can be affected by the culture media and conditions, as well as the production procedure itself. These perturbations can possibly result in a marked decrease in the quality of the resulting blastocysts and may even affect the viability of offspring born after transfer. In vitro procedures such as in vitro production and somatic nuclear transfer of bovine embryos have been shown to be correlated with significant up- or downregulation, de novo induction or silencing of genes critical for undisturbed fetal and neonatal development. These alterations are likely to be caused by epigenetic modifications, such as DNA methylation and histone modifications. Analysis of perturbed epigenetic reprogramming and of the related phenomena, such as genomic imprinting and X-chromosome inactivation, in bovine embryos is promising for understanding the underlying mechanisms of developmental abnormalities, such as large offspring syndrome.

scholarly journals Urokinase-type plasminogen activator does not affect in vitro bovine embryo development and quality

2015 ◽  
Vol 63 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Fotini Krania ◽  
Eleni Dovolou ◽  
Constantinos A. Rekkas ◽  
Sonia Heras ◽  
Ioannis Pappas ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Embryo Development ◽  
Culture Media ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Serum Free ◽  
Epidermal Growth ◽  
Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P &lt; 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P &gt; 0.05), whereas the combination P4 + EGF was better than P4 alone (P &lt; 0.05). Blastocyst rate was not different (P &gt; 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P &lt; 0.05) blastocyst rate than control but it was lower (P &lt; 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

scholarly journals MATURAÇÃO E FECUNDAÇÃO IN VITRO DE OÓCITOS BOVINOS EM TUBOS PREVIAMENTE GASEIFICADOS E MANTIDOS EM BANHO-MARIA

2002 ◽  
Vol 7 (2) ◽  
Author(s):  
M. KURTZ FILHO ◽  
L. M. SILVA ◽  
B. MOREIRA ◽  
D. S. BRUM ◽  
F. G. LEIVAS ◽  
...  
Keyword(s):  
Water Bath ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Embryo Production ◽  
Vitro Production

A produção in vitro (PIV) de embriões bovinos alcançada com vacas de matadouros ou de aspiração folicular in vivo (OPU) é uma prática cada vez mais difundida e a sua simplificação poderia baixar os custos de produção. O objetivo desta pesquisa foi comparar a produção in vitro de embriões em estufa com temperatura, umidade relativa e atmosfera controlada (controle), com tubos de poliestireno gaseificados e mantidos em banho-maria (tratamento). Oócitos obtidos de ovários de vacas abatidas foram maturados in vitro em TCM- 199 modificado com 25mM de N-2-hidroxietilpiperazina-N -2-ácido etanosulfônico (HEPES); 0,025mg/ml de piruvato de sódio, 0,01UI de rFSHh/ml, 0,5µg/ml de LHs e 10% de soro de vaca em estro. Na fecundação in vitro utilizou-se Talp-Fert com 0,06mg/ml de albumina sérica bovina, 0,022mg/ml de piruvato de sódio e 10µg/ml de heparina. O cultivo foi conduzido em placas de 4 poços em SOF com 5% de soro de vaca em estro, 20µl/ml de aminoácidos essenciais e 10µl/ml de aminoácidos não essenciais, sob óleo mineral, em estufa com atmosfera de 5% de CO2, umidade saturada e 39°C, por 9 dias. Na maturação não houve diferença (P>0,05) entre o tratamento e o controle. Porém, a maturação e a fecundação ou somente a fecundação in vitro em tubos mantidos em banho-maria não demonstrou ser uma alternativa recomendada para a produção de embriões bovinos. In vitro maturation and fertilization of bovine oocytes in tubes previously gasified kept in water bath Abstract In vitro bovine embryo production either obtained from oocytes of slaughtered cows or in vivo follicular aspiration (OPU) is a well-known technique and it’s simplification might reduce the cost of embryo production. The aim of this study was to compare the cleavage rate and embryo development of the in vitro production of bovine embryos using standard culture system (temperature, gas phase and controlled humidity) versus gasified polystyrene tubes kept in water bath. Oocytes obtained from ovaries of slaughtered cows were in vitro maturated in TCM- 199’modified with 25 mM of N-2-hidroxyethylpiperazine-N’-2-ethanosulfonic acid (HEPES); containing 0.01UI rFSHh/ml and 0.5µg/ml LHs, 0.025mg/ml sodium pyruvate and 10% estrous cow serum. The in vitro fertilization was carried out in Talp-Fert containing 0.06mg/ml BSA, 0.022mg/ml sodium pyruvate and 10µg/ml heparin. The culture was performed in SOF medium with 20µl/ml essential aminoacids, 10µl/ml, non-essential aminoacids and 5% estrous cow serum, with oil overlay, in 4 well dishes and incubated with 5% CO2, maximum humidity at 39°C, for 9 days. The results of this study showed no difference (P>0.05) between the treatment and control groups during the maturation process. However, the maturation and fertilization or only the fertilization in tubes do not represent a viable alternative for the in vitro production of bovine embryos.

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Metabolomics ◽  
2016 ◽  
Vol 12 (5) ◽  
Author(s):  
Érika Cristina dos Santos ◽  
Camila Bruna de Lima ◽  
Kelly Annes ◽  
Marcella Pecora Milazzotto
Keyword(s):  
Culture Media ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Noninvasive Characterization ◽  
Vitro Production

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 Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

2021 ◽  
Vol 10 (14) ◽  
pp. e367101422097
Author(s):  
Arianny Rafaela Neto Silva ◽  
Thaisa Campos Marques ◽  
Elisa Caroline Silva Santos ◽  
Tiago Omar Diesel ◽  
Isabelle Matos Macedo ◽  
...  
Keyword(s):  
Culture Media ◽  
Cell Number ◽  
Time Dependent ◽  
Expansion Rate ◽  
Ros Generation ◽  
Hatching Rate ◽  
Protective Effects ◽  
Bovine Embryos ◽  
In Vitro Production

The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.

Sign in / Sign up

Export Citation Format

Share Document