Emílio Pereira de Brito Neto ¹, Rafael Otero ², Darwin Hernández ², Eduardo Paulino da Costa ¹, Ademir Moraes Ferreira ³

Affiliations
1 Universidade Federal de Viçosa, Viçosa - MG, 36570-900, Brasil
2 Universidad de Sucre, Campus Ciencias Agropecuarias, Sincelejo, Sucre, CO
3 Embrapa Gado de Leite, Juiz de Fora - MG, 36038-330, Brasil

Abstract

Objective: This study was conducted to evaluate the effect of the follicular diameter upon gestation rate of cows submitted to Artificial Insemination at Fixed Time (IATF). Methods: Forty-seven crossbred cows Bos taurus x indicus presenting cyclical luteal ovarian activity and under nursing were used. The cows were given 150μg of PGF_2α via Intra-Muscular (IM). The diameter of the present largest follicle was measured through Ultra-Sound Examination (USE). After 24 hours, the cows were given 2μg of estradiol via IM and they were again evaluated through USE for certification of the follicular dominance. Later, they were divided into two treatments: treatment 1 (cows with dominant follicle <13mm) and treatment 2 (cows with dominant follicle ≥13mm). After 48 hours from the application of E2, the cows were Artificially Inseminated (AI). However, those cows presenting estrus in advance were inseminated at 12 hours after manifestation of the same one. Ten days after AI the concentration of progesterone (P4) was quantified by radioimmunoassay using a commercial kit. The gestation diagnosis was accomplished at 30 and 100 days after IA through USE. Findings: Differences (p<0.05) were observed in the gestation rates, that is 76% and 45.5% for animals of the treatments 1 and 2, respectively. The serum concentrations of P4 were 1.34±0.49 and 2.16±1.17 for the treatments 1 and 2 respectively (p<0.01). A low correlation (r=0.45) was observed between P4 levels and the presentation of pregnancy. Application: Cows with dominant follicles <13 mm in diameter at the time of application E2 have a higher gestation rate even with lower production of P4.

Keywords

Cows, Gestation, Reproductive Biotechnology, Reproduction

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Rafael Otero ¹, Darwin Hernandez ¹, Luiz Sergio de A Camargo ²

Affiliations
1 Universidad de Sucre - Campus Ciencias Agropecuarias, Sincelejo, CO
2 Embrapa Dairy Cattle Research Center, Juiz de Fora, MG, BR

Abstract

Objective: To evaluate the effect of treatment with trichostatin-A (TSA) on the production of bovine embryos, expressing the gene of the green fluorescent protein (GFP) generated by SCNT. Materials: 164 oocytes were distributed in three treatments, NT-GFP: newly reconstructed zygotes with genetically modified cells and not subject to TSA. NTTrico- GFP: newly reconstructed zygotes with genetically modified cells and subjected to TSA. PART: Zygotes generated by parthenogenetic activation, used as a control for the process of oocyte activation and culture of embryos. The rates of cleavage, blastocysts, and embryos that expressed GFP were assessed by contingency tables and chi-square tests. Results: The percentage of cleavage in the zygotes in the NT-GFP treatment was greater but did not vary significantly from the NT-Trico-GFP treatment. However, this last treatment had a higher percentage of blastocyst formation (p=0.077). The percentage of blastocysts from cleaved zygotes, the produced embryos were significantly higher (p<0.05) for the NT-Trico-GFP treatment than for the NT-GFP. In both treatments, all the blastocysts generated expressed the GFP protein. Conclusions: TSA improves the embryonic development of clones of genetically modified cattle that express GFP.

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Rafael Otero ¹, Darwin Hernandez ¹, Luiz Sergio de A Camargo ²

Affiliations
1 Universidad de Sucre - Campus Ciencias Agropecuarias, Sincelejo, CO
2 Embrapa Dairy Cattle Research Center, Juiz de Fora, MG, BR

Abstract

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector with the eGFP gene as a marker. Methods: Four treatments were designed: T1=Control: fertilized in vitro (FIV) with cumulus-oocyte complexes (CCOs), cultivated in CR2 medium with 10% FBS and incubated at 38.5°C in an atmosphere of 95% humidity and 5% CO₂. T2=Control of culture medium: CCOs removed by vortex in the presence of hyaluronidase, FIV, grown in SOF medium in hermetic bag, with a gaseous mixture of 5% CO₂, 5% O₂ and 90% N₂ and humidity saturated at 38.5°C. T3=Microinjection control: CCOs removed microinjected with TALP medium, FIV and cultured under the same treatment conditions T2. T4=Microinjected with the lentivirus: CCOs removed microinjected with the lentiviral vector and FIV and cultured in the same conditions of the T2 and T3 treatments. The rate of development of blastocysts at day eight and the expression of the eGFP gene were evaluated. Findings: No significant statistical differences were found (p> 0.05) in the production of blastocysts at day eight, between treatments T1, T2, and T3. The percentage of blastocysts found in the T4 treatment was significantly lower (p <0.05) than in the other treatments. All embryos obtained in T4 expressed the transgene of interest. Application / Improvements: It is concluded that the culture conditions used were adequate for T1, T2 and T3, added that the microinjection with the lentiviral vector influences in some way the embryonic development, although, the technique was highly efficient for obtaining transgenic embryos.

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Rafael Otero ¹, Darwin Hernandez ¹, Donicer Montes ¹

Affiliations
1 University of Sucre, Sincelejo, CO

Abstract

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector that carries the eGFP gene as a marker in zygotes six hours after fertilization. Methods: 834 oocytes were matured and subjected to one of four treatments designed as follows: CC: Control: IVF with Cumulus-oocyte with (COCs), cultivated in CR2 medium supplemented with 10% FBS and incubated at 38.5°C in atmosphere of 95% humidity and 5% CO₂.CCM: Control culture medium: fertilized in vitro for six hours, cultured in medium SOF aluminum in pouches under the same conditions of CC. MC: Microinjection control: Fertilized under the same treatment conditions CCM. After six hours they were microinjected with TALP medium and cultured in sachets with the same conditions of CCM treatment. ML: Microinjected with the lentivirus: Fertilized in the same conditions of the CCM treatment. After six hours they were microinjected with the lentiviral vector carrying the eGFP transgene and cultured in sachets with the same treatment conditions CCM and MC. Findings: The cleavage rate found in CC was higher (p < 0.05) than that observed in the other treatments. The rates of blastocysts found between CC, CCM and MC did not differ significantly (p > 0.05) in them, but yes, with ML (p < 0.05). On average, 76.4% of the zygotes obtained in ML expressed the green fluorescent protein. Application/Improvements: The cult ure conditions used were suitable for CC, CCM and MC, microinjection with lentiviral vector has some influence on embryo development, it succeeded in obtaining transgenic zygotes.

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