199 EFFECT OF PRODUCTION SYSTEM AND BREED ON RELATIVE GENE EXPRESSION IN BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 198 ◽  
Author(s):  
S. Wohlres-Viana ◽  
M. C. Boite ◽  
M. M. Pereira ◽  
W. F. Sa ◽  
J. H. M. Viana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Production System ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
In Vitro Production ◽  
System Effect ◽  
Vitro Production

Embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. Nevertheless, there are a few studies showing the effect of in vitro environment on embryos from different bovine subspecies, such as Gyr (Bos indicus) and Holstein (Bos taurus). The aim of this study was to evaluate the relative abundance of aquaporin 3 (AQP3) and ATPase-α1 (Na/K-ATPase alpha 1) transcripts in blastocysts produced in vivo or in vitro from Gyr and Holstein cattle. The production system effect (in vivo × in vitro) for Gyr cattle and the breed effect (Holstein × Gyr) for in vitro-produced embryos were evaluated. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA). The cDNA obtained were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed with REST software© using the pair wise fixed reallocation randomization Test. There was no difference (P > 0.05) in gene expression for AQP3 and ATPase-α1 between in vivo- and in vitro-produced Gyr embryos, although the results suggest that the APQ3 gene was down-regulated (0.81 ± 0.31) and the ATPase-α1 gene was up-regulated (1.20 ± 0.65) in embryos produced in vitro. For breed effect within in vitro production system, ATPase-α1 gene was down-regulated in Holstein (0.56 ± 0.30) when compared with Gyr embryos (P < 0.05). The same trend was observed for AQP3 (0.58 ± 0.25), but with no difference (P > 0.05). In conclusion, the data suggest that embryo production system does not interfere with the transcript amount of the genes studied for Gyr cattle; however, the in vitro production system may have different effects on gene expression according to embryo breed. Other genes should be evaluated for a better understanding of these differences. Financial support: CNPq, Fapemig.

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.

86 Comparison of in vivo and in vitro embryo production in Bonsmara donors

2019 ◽  
Vol 31 (1) ◽  
pp. 168
Author(s):  
B. H. Bernal ◽  
J. L. Barajas ◽  
J. A. Ortega ◽  
A. Cedeño ◽  
S. Andrada ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Gonadotropin Releasing Hormone ◽  
P Value ◽  
In Vitro Production ◽  
Embryo Production ◽  
Releasing Hormone ◽  
Gradient Technique ◽  
Vitro Production

A retrospective analysis of embryo production records from 2013 to 2017 was carried out to evaluate the in vivo and in vitro production (IVP) of embryos in donors of the Bonsmara breed (i.e. tropically adapted Bos taurus). Only donors with production records of both in vivo and in vitro embryos during the same period were used. A total of 127 superovulations and ova/embryo collections of 19 donors were evaluated. The donors were superstimulated with the following protocol: on Day 0 they received a device with 1g of progesterone (DIB, Zoetis, Argentina), 50mg of rogesterone (Progestar, Zoetis), and 5mg of oestradiol-17β (17ßOestradiol, Rio de Janeiro, Argentina) or 2mg of oestradiol benzoate (Gonadiol, Zoetis) intramuscularly (IM) at the same time. Superstimulatory treatments were initiated on the morning of Day 4 with Folltropin-V (Vetoquinol, France; total dose=240 to 340mg IM) in twice-daily decreasing doses over 4 days. All donors received 2 IM injections of 500µg of cloprostenol (Ciclase DL, Zoetis) on the morning and afternoon of Day 6 and; the intravaginal devices were removed on the morning of Day 7 and 100µg of Gonadorelin (gonadotropin-releasing hormone, Gonasyn gdr, Zoetis) was given on the morning of Day 8. Donors were inseminated using semen from 9 Bonsmara bulls, 12 and 24h after gonadotropin-releasing hormone. On Day 15, ova/embryos were collected and classified according to IETS standards. A total of 89 follicular aspirations (ovum pickup) of 19 donors for IVP were evaluated. The ovum pickups were performed at random stages of oestrous cycle, without superstimulation or other hormone treatments. A total of 1109 viable oocytes (12.5±0.9 per ovum pickup) were collected and matured for 24h in 100-µL drops of maturation medium (TCM-199, supplemented with hormones) under mineral oil and incubated at 38.5°C in 5.5% CO2 and humidity at saturation. Fertilization was performed using 3 Bonsmara bulls that were also used for in vivo embryo production. Viable sperm were obtained using the percoll gradient technique (45-90%). The sperm pellet was dissolved in TL-Sperm, centrifuged, and then diluted to a final concentration of 1.5×106 sperm/mL. Zygotes were stripped and placed in drops of 100µL of SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, 7% O2, and humidity at saturation for 7 days. The culture medium was renewed on Days 3 and 5. The data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), a P-value &lt;0.05 was considered significant. The mean (±standard error of the means) number of CL, ova/embryos collected, fertilized ova, and transferable embryos were 12.9±0.6, 8.8±0.6, 6.6±0.5, and 4.7±0.4, respectively. A total of 662 oocytes (66.3±2.4%) cleaved 48h post-IVF. On Day 7, an average of 4.4±0.3 embryos were produced. No differences were detected in the number of transferable embryos produced in vivo v. those produced in vitro. Furthermore, no significant differences were found between the techniques or bulls on the proportion of embryos produced in relation to the ova/embryos or oocytes obtained (in vivo 51.5±3.2% v. in vitro 42.9±2.5%). In conclusion, the in vivo and in vitro production of embryos are both effective alternatives to increase the number of offspring from valuable Bonsmara donors.

A Patient-Derived Xenograft Model for Identifying Therapies and Defining Mechanisms of TSLP-Induced CRLF2 Signals in Ph-like B-ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2633-2633
Author(s):  
Olivia L Francis ◽  
Terry-Ann MIlford ◽  
Ineavely Baez ◽  
Jacqueline Coats ◽  
Christopher L. Morris ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Mtor Pathway ◽  
In Vivo Model ◽  
In Vitro Production ◽  
Pathway Activation ◽  
B Cell Precursors ◽  
Vitro Production

Abstract Philadelphia chromosome (Ph)-like B cell acute lymphoblastic leukemia (B-ALL) is a high-risk leukemia with a gene expression profile similar to BCR-ABL1+ B-ALL. Approximately 50% of all Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. The poor outcomes associated with CRLF2 B-ALL represent a major clinical challenge and an important component of pediatric cancer health disparities. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. Activating JAK mutations are associated with CRLF2 B-ALL, but overall data indicate that JAK mutations are present in 50% or less of CRLF2 B-ALL. Our data show that normal primary human bone marrow (BM) stromal cells express TSLP, suggesting that TSLP-induced CRLF2 signals could play a role in the initiation, maintenance and progression of CRLF2 B-ALL, particularly in cases without JAK mutations. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) to human TSLP and CRLF2. Further, using phospho flow cytometry we show that mouse TSLP is unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP, confirming the species specificity of mouse TSLP. These findings underscore the importance and challenge of developing in vivo systems that can model human TSLP-CRLF2 interactions for evaluating therapies and studying leukemogenesis of CRRLF2 B-ALL. To address this challenge we engineered patient-derived xenograft (PDX) mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (-T) mice were produced by transplanting with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not -T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in CRLF2 B-ALL cells. ELISA assays showed normal serum levels of hTSLP (12-32 pg/ml) in +T mice, while hTSLP was undetectable in -T mice. Since TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and -T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to -T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary leukemia cells. +T and -T PDX mice were produced using primary CRLF2 B-ALL cells. BM was harvested and whole genome microarray was performed on isolated CRLF2 B-ALL cells. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to -T PDX mice, confirming hTSLP activity in the +T PDX mice. Next we tested whether +T PDX mice provide an in vivo model of B-ALL that more closely mirrors patients than -T PDX mice. +T and -T PDX mice were generated from primary high risk B-ALL. RNAseq gene expression profiles from primary patient B-ALL cells were compared to those of the same patient sample expanded in +T and -T PDX mice. The gene expression pattern in +T mice was significantly closer to the primary patient sample than those from -T mice. The +T and -T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of the antral follicle count of Bos taurus × Bos indicus dairy cows on in vitro embryo production

2020 ◽  
pp. 2171-2178
Author(s):  
Sheila Merlo Garcia ◽  
Paula Alvares Lunardelli ◽  
Kleber Luciano Ancioto ◽  
Eduardo Cardoso de Oliveira ◽  
Larissa Zamparone Bergamo ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Antral Follicle ◽  
Estradiol Benzoate ◽  
Antral Follicle Count ◽  
Success Rates ◽  
In Vitro Production ◽  
Follicular Aspiration ◽  
Vitro Production

This study aimed to evaluate the potential of Girolando (Bos taurus × Bos indicus) cows with high and low antral follicle counts (AFC) for the in vitro production of bovine embryos (IVEP), as well as the pregnancy rates of the recipients of these embryos. Girolando cows (Bos taurus × Bos indicus) were classified as high and low AFC when they had 35-52 (n = 13) and 11-17 follicles (n = 15), respectively. All animals were subjected to repeated follicular aspiration [Ovum pick-up (OPU)] and subsequent IVEP sessions. The synchronization protocol of the recipients was performed on a random day of the estrous cycle (Day 0) with the implantation of progesterone, estradiol benzoate, and prostaglandin. The high AFC group had higher aspirated oocyte/OPU (42.6 ± 5.2 vs. 14.6 ± 1.9; p < 0.01) and cultured oocyte/OPU (38.1 ± 6.6 vs. 12.3 ± 2.8; p < 0.01) averages as well as a higher blastocyst percentage on D7 (23.0 ± 1.0% vs. 18.4 ± 1.5%; p < 0.05) and higher pregnancy rate (42.7 ± 2.7% vs. 39.7 ± 4.6%; p < 0.05) than the low AFC group. Thus, we can conclude that animals with high AFC had better IVEP success rates than animals with low AFC.

Comparison of gene expression in Bos indicus and Bos taurus embryos produced in vivo or in vitro

2011 ◽  
Vol 140 (1-3) ◽  
pp. 62-67 ◽  
Author(s):  
Sabine Wohlres-Viana ◽  
Michele Munk Pereira ◽  
Joao Henrique Moreira Viana ◽  
Marco Antonio Machado ◽  
Luiz Sergio de Almeida Camargo
Keyword(s):  
Gene Expression ◽  
Bos Taurus ◽  
Bos Indicus

scholarly journals In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Anita Soares Barbosa GUIMARÃES ◽  
Laiara Fernandes ROCHA ◽  
Ronival Dias Lima de JESUS ◽  
Gisvani Lopes VASCONCELOS ◽  
Gabriela ANGHINONI ◽  
...  
Keyword(s):  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Vitro Production ◽  
In Vitro Performance

ABSTRACT In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P < 0.0001; n = 681 vs. 509 for grade 2 COC, P = 0.010, for zebu and taurine donors, respectively). There were differences in embryo production percentages obtained from OM (0.44% from zebu and 6.42% from taurine, P = 0.017), BL (14.18% from zebu and 3.74% from taurine, P < 0.0001), and BX (81.43% from zebu and 75.13% from taurine, P < 0.0001). No significant difference was observed for embryo production from BI and pregnancy rate (P > 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.

246 INFLUENCE OF BREED AND SEASON ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 255
Author(s):  
B. Bernal ◽  
J. Revol ◽  
J. M. Oviedo ◽  
A. Tribulo ◽  
H. Tribulo ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Follicle Aspiration ◽  
Vitro Production ◽  
Difference Test

A retrospective analysis of in vitro production (IVP) data was done to determine the influence of breed and season on the production of viable oocytes and embryos. Cumulus‐oocyte complexes (COC) were obtained from 1946 ultrasound-guided follicle aspiration (ovum pickup) sessions performed at random stages of the oestrous cycle without superstimulation in Bos taurus and Bos indicus donors in commercial IVP in Argentina. Frozen-thawed conventional semen was used in beef cattle and conventional (n = 139) and sexed-selected (n = 481) semen in dairy cattle. The COC were classified, matured in B-199 medium, fertilized in IVF-SOF medium (Day 0), and cultured in SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, and saturated humidity for 7 days. The number of viable COC and transferable embryos in each breed and season were compared by ANOVA and means were compared by Fisher’s Least Significant Difference test. Proportions were first transformed by arcsin and then analysed by ANOVA. To simplify the interpretation of the results, breeds were grouped as follows: dairy Bos taurus (Holstein, n = 620), beef Bos taurus (Angus and Bonsmara, n = 229), Bos taurus × Bos indicus (Brangus and Braford, n = 1045), and Bos indicus (Brahman, n = 52). There was no interaction between breed and season for any of the end points analysed (P > 0.1). Mean (± standard error of the mean) numbers of viable COC and transferable embryos were higher (P < 0.01) in Bos indicus × Bos taurus (19.3 ± 0.4 and 5.3 ± 0.2, respectively) and Bos indicus (15.8 ± 1.4 and 6.8 ± 0.9, respectively) than in beef (11.6 ± 0.5 and 3.0 ± 0.2, respectively) and dairy (8.0 ± 0.2 and 1.6 ± 0.1, respectively) Bos taurus donors. Cleavage rates were higher (P < 0.01) in Bos indicus (72%) than in the other breeds (57% for Bos indicus × Bos taurus and dairy Bos taurus and 54% for beef). Transferable embryo rates were higher (P < 0.01) in Bos indicus (41%) and Bos indicus × Bos taurus (30%) than in beef Bos taurus (26%). Dairy Bos taurus had the lowest (P < 0.01) embryo rates of all breeds (21%). In dairy Bos taurus, cleavage rates, the number of embryos produced, and transferable embryo production rates were higher (P < 0.01) when conventional semen was used (62%, 2.8 ± 0.15, and 27%, respectively) compared to sexed-selected semen (55%, 1.3 ± 0.1, and 19%, respectively). With regards to season, the number of viable COC was highest (P < 0.01) in the spring (14.3 ± 0.5), lowest in the summer (11.3 ± 1.0), and intermediate in the fall (12.2 ± 1.2) and winter (13.7 ± 1.2), which did not differ. Although not affected significantly by season, the number of embryos produced was numerically lower in the summer (2.8 ± 0.4) than in the spring (4.2 ± 0.2), winter (4.5 ± 0.5), or fall (4.6 ± 0.5). In conclusion, in vitro embryo production was directly influenced by breed and season. Bos indicus influenced cattle and the spring season were preferable for commercial IVP programs that did not include superstimulation.

scholarly journals Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

2011 ◽  
Vol 75 (3) ◽  
pp. 429-433 ◽  
Author(s):  
F.G. Leivas ◽  
D.S. Brum ◽  
S.S. Fialho ◽  
W.P. Saliba ◽  
M.T.T. Alvim ◽  
...  
Keyword(s):  
Fetal Calf Serum ◽  
Bos Taurus ◽  
Calf Serum ◽  
In Vitro Production ◽  
Bos Taurus Indicus ◽  
Vitro Production
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