Placental physiology and metabolism by cordonal canulation in normal, IVF and cloned bovine concepti

Author(s)

Renato Pereira da Costa Gerger

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2010-04-29

Examining board members

Maria Angélica Miglino; Antônio Chaves de Assís Neto; Marcelo Bertolini; Flávio Vieira Meirelles; José Luiz Rigó Rodrigues

• Advisor: Maria Angélica Miglino

Abstract

The in vitro production (IVP) of embryos by in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) in many species, especially in cattle, is usually associated with abnormalities during in vivo development, with the placenta being implicated as the determining factor in the appearance of the disturbances during gestation. The general hypothesis of this study is that alterations in placenta formation, as a consequence of early in vitro embryo manipulations, results in high gestation losses during the first trimester and fetal and placental abnormalities in the last trimester of gestation, including the occurrence of a compensatory fetal growth due to a reduced placental-fetal growth-restricting effect, which increases the total materno-fetal nutrient flux in late pregnancy. This study aimed to evaluate some of the biological and technical effects on the IVP of embryos by SCNT, and also to compare the development of pregnancies established with embryos produced either in vivo (Control), or in vitro by in vitro fertilization (IVF) or by nuclear transfer (SCNT), by the evaluation of morphometric, morphologic and biochemical data collected at distinct time points in gestation. In Chapter 1, the in vitro development of cloned embryos was compared between somatic cells of two genetically different females (Nelore vs. Crioula Lageana), and between three cell confluence intervals, established prior to the cloning procedure of the same animal. The best results were achieved using cells from the Nelore female and also with cells on the highest confluence level on culture. In Chapter 2, the effects of two fusion-activation intervals and the use or not of embryo aggregation during in vitro culture were compared by evaluating the in vitro and in vivo developmental potential of resulting embryos. The embryo aggregation and a higher fusion-activation interval promoted an improvement in blastocyst yield. However, those embryos were neither more advanced in development nor better in morphological quality, having no influence on subsequent pregnancy rates or pregnancy losses. In Chapter 3, Control, IVF and SCNT pregnancies were compared by the evaluation of morphometric and biochemical data collected at distinct moments in gestation. This experiment demonstrated that differences occurred between the three groups of embryos from the beginning of the analyses; the in vitro-produced groups had lower pregnancy rates, with the SCNT group showing the highest pregnancy losses. The analyses of the sonograms on Day 51 of pregnancy revealed a growth retardation pattern for the in vitro concepti, having a compensatory development up to late pregnancy, as in vitro-derived pregnancies, special in the SCNT group, sustained heavier concepti with a wider spectrum of abnormalities on day 225 of gestation. In addition, the SCNT pregnancies demonstrated a greater accumulation of substrates in the fetal system, particularly fructose on the allantoic fluid. Finally, in Chapter 4, results obtained from a retrospective analysis of bovine embryo production by cloning and subsequent pregnancy rates were compared between three equal periods within a range of 28 months. The comparison of the bovine cloning procedures during the three time periods demonstrated the impact of the technical skills and competence on the overall efficiency of in vitro embryo production by SCNT, and subsequent in vivo development. (AU)