248 PRODUCTION OF FLAGGED RECOMBINANT BOVINE BMP15 TO IMPROVE BOVINE IN VITRO EMBRYO PRODUCTION SYSTEMS

2011 ◽  
Vol 23 (1) ◽  
pp. 222
Author(s):  
G. Burns ◽  
P. F. Suchodolski ◽  
A. J. Pearks Wilkerson ◽  
C. Long
Keyword(s):  
Western Blot ◽  
Production Systems ◽  
Protein A ◽  
Bovine Embryo ◽  
Mature Protein ◽  
Embryo Production ◽  
Hek 293 ◽  
Secreted Factors ◽  
Species Specific

Current in vitro systems for bovine embryo production are inefficient and produce embryos with lower viability than their in vivo-derived counterparts. Recent reports demonstrate that in vitro bovine oocyte maturation systems could benefit from the addition of oocyte-secreted factors, specifically GDF9 and BMP15 (Gilchrist et al. 2007 Theriogenology 67, 6–15). The long-term goal of this work is to produce species-specific recombinant oocyte-secreted factors capable of improving bovine embryo production in vitro. In the current project, the objective was to produce a cell line that expresses recombinant bovine BMP15. This protein is first translated as a large precursor peptide consisting of propeptide and mature regions, which are enzymatically cleaved to form the active mature protein. The wild-type BMP15 gene was cloned using reverse transcriptase PCR with RNA obtained from bovine ovarian tissue. For improved detection and purification of the active form of the recombinant protein, a detectable FLAG tag sequence (DYKDDDDK) was incorporated into the wild-type BMP15 gene by PCR and cloned into pCDNA expression vector. The FLAG tag was introduced immediately 3′ of the cleavage site at the N-terminal portion of the mature protein to produce recombinant FLAG-tagged BMP15 (rbFL-BMP15). To ensure efficient production of the mature protein, a Kozak sequence was inserted 5′ of the start ATG and the cleavage site altered to be recognised by PACE/furin enzymes, which are endogenously expressed in most mammalian cells including HEK-293 cells (Li et al. 2009 Mol. Hum. Reprod. 15, 779–788). Following sequencing to verify transcript fidelity, pCDNA-rbFL-BMP15 was transfected into HEK-293 cells, and mature protein production was detected by Western blot analysis. Cells plated at 85% confluency were transfected with Lipofectamine 2000, and lysates were harvested 48 h post-transfection. The presence of bovine rbFL-BMP-15 in cell lysates was confirmed by Western blot using the anti-FLAG antibody. Ongoing experiments will test the bioactivity of the purified rbFL-BMP15 by evaluating activation of the SMAD 1/5 pathway via Western blot for phosphorylated SMAD 1/5. After a biologically active protein is confirmed, purified protein will be collected for testing during in vitro maturation of bovine oocytes. We anticipate the species-specific form of oocyte-secreted factors will further enhance in vitro embryo production systems beyond that reported using heterologous factors.

86 IMPROVED BOVINE EMBRYO PRODUCTION USING NOVEL IN VITRO CULTURE SYSTEMS

2016 ◽  
Vol 28 (2) ◽  
pp. 172
Author(s):  
J. H. Pryor ◽  
J. F. Hasler ◽  
L. Strøbech ◽  
B. Avery ◽  
N. Hashem ◽  
...  
Keyword(s):  
Production Systems ◽  
Uv Light ◽  
Cumulus Cells ◽  
Tween 20 ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Embryo Production ◽  
Cell Counts

Development and testing of new embryo production components is important to improve the outcome following in vitro production of bovine embryos. The objective of this study was to compare media used in two bovine embryo production systems (control and EmbryoTrans Biotech: ETB). In Exp. 1, abattoir-derived cumulus-oocyte complexes were randomly assigned and in vitro matured (IVM) in either control [Medium 199 with Earles salts (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen), 0.2 mM sodium pyruvate, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 5.0 µg mL–1 of Folltropin®-V (Vetoquinol, Pullman, WA, USA)] or ETB BO-IVM medium for 21 to 24 h. IVF was conducted in 500 µL of pre-equilibrated modified Tyrode-lactate medium for control (Pryor et al. 2011 Theriogenology 75, 24–33) or ETB BO-IVF in Nunclon® 4-well multi-dishes (VWR Scientific, Pittsburgh, PA, USA). Seventeen hours post-insemination, presumptive zygotes were cleaned of cumulus cells and cultured in either Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL–1 of Probumin BSA (EMD Millipore, Norcross, GA, USA), under oil (Irvine Scientific, Santa Ana, CA, USA) or ETB BO-IVC medium under BO-oil for 7 days (8 days post-IVF). All cultures were performed at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 using BT37 incubators (Planer Plc, Sunbury, UK). For Exp. 2, all conditions were maintained except a modified ETB BO-IVCA medium was used. On Day 8 of IVC, grade 1 and 2 blastocysts (BL) through hatching blastocysts (HBL) were counted and used to calculate total viable rates. In Exp. 2, these embryos were fixed in cold methanol, washed in PBS/0.1% Tween 20, mounted in 10 μg mL–1 Hoechst 33342/glycerol, and viewed under UV light to count cells (n = 49 and 107 for control and ETB, respectively). Each experiment was replicated 3 times with a total of 425 oocytes in Exp. 1 and 430 in Exp. 2, divided equally between treatments. Percentage data were transformed using arcsine square root function before analysis and means compared using a paired Student’s t-test. For Exp. 1, there were no differences in rates of cleavage or viable embryos between control and ETB systems (81.3% and 42.9% v. 80.5% and 48.4%, respectively). In Exp. 2, ETB was superior to control for percent viable, HBL, and combined HBL/expanded BL (51.9, 23.9, 45.8% v. 29.2, 5.8, 20.5, respectively; P < 0.05). Differences between mean cell counts for viable embryos were significant (control = 127.0 ± 6.7 s.e.m. and ETB = 162.7 ± 5.7; P < 0.0001). Embryo viability decreased in control media between Exp. 1 and 2 (42.9 v. 29.2%; P < 0.05). Seasonal differences may have contributed via heat stress with temperatures ranging from 23.8°C for Exp. 1 to 33.8°C for Exp. 2. Interestingly, embryo development in the ETB media did not decrease under the same conditions. In conclusion, ETB media produced more high-quality embryos than control under varying conditions experienced by commercial IVF companies.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Inadvertent sex selection in a protocol of in vitro bovine embryo production

1997 ◽  
Vol 47 (1) ◽  
pp. 265 ◽  
Author(s):  
E. Behboodi ◽  
A. Gutiérrez-Adán ◽  
G.B. Anderson
Keyword(s):  
Sex Selection ◽  
Bovine Embryo ◽  
Embryo Production

scholarly journals The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation

2014 ◽  
Vol 97 (10) ◽  
pp. 6164-6176 ◽  
Author(s):  
V.A. Absalón-Medina ◽  
S.J. Bedford-Guaus ◽  
R.O. Gilbert ◽  
L.C. Siqueira ◽  
G. Esposito ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Bovine Embryo ◽  
Embryo Production

100 COMPARISON OF PREGNANCY RATES OF FRESH IN VITRO-PRODUCED BOS INDICUS EMBRYOS PRODUCED IN THE SAME LABORATORY BUT COLLECTED AND TRANSFERRED IN PANAMA OR THE DOMINICAN REPUBLIC

2014 ◽  
Vol 26 (1) ◽  
pp. 164
Author(s):  
L. F. Nasser ◽  
S. C. Feliú ◽  
E. Rodríguez ◽  
K. Mojica ◽  
E. G. Oliveira ◽  
...  
Keyword(s):  
Dominican Republic ◽  
Bos Indicus ◽  
Disease Status ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Department Of Agriculture ◽  
Embryo Production ◽  
Essential Variable ◽  
Laboratory Facility

Because of Panama's stricter sanitary status, a specialised protocol was developed with the Department of Agriculture in the Dominican Republic to legalize the exchange of biological materials (oocytes/embryos). This protocol allows the team of specialised technicians, currently working in Born® Animal Biotechnology's Panamanian facility, to operate using the same in vitro bovine embryo production system (IVP, In vitro Brasil®) to service Dominican producers. Because the donors are not located at a specific centre with controlled sanitary management, a special protocol was developed in which blood tests were done to certify that the entirety of the herd at each client's farm was free of infectious bovine rhinotracheitis, DBVD, leptospirosis, leucosis, brucellosis, and tuberculosis. As timing during IVP is an essential variable that can have detrimental effects on the final results, precautions were taken to ensure that the oocytes arrived at the Panamanian laboratory facility within 24 h of aspiration. A portable incubator was used to transport oocytes and embryos during the import and export portions of the procedure. A comparison of pregnancy rates based on oocyte source and recipient transfers from September 2012 until May 2013 was analysed with ?2 (Table 1). The number of embryos produced in Panama was significantly higher than in the Dominican Republic, which was likely due to the larger number of donors and oocytes from the Panama herd. However, pregnancy rate was higher in the Dominican Republic likely because of the health status of the Dominican recipients, which were free of the diseases mentioned above. Recipients were the same type and breed and under similar management conditions in both countries. The disease status aspect will be examined with greater numbers of animals in the future. The data suggest that the present IVP and recipient management protocols could serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.In vitro embryo production and pregnancy rates of Bos indicus embryos transferred in Panama or the Dominican Republic (September 2012 through May 2013)

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas
Keyword(s):  
Lipid Peroxidation ◽  
Embryo Development ◽  
Antioxidant Properties ◽  
Thiobarbituric Acid ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

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.

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