Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle

R P C Gerger; Rafael Rossetto; E S Ribeiro; Ivens Ortigari Jr; Fabiano Carminatti Zago; L H Aguiar; U M Costa; Rui Fernando Félix Lopes; Carlos Eduardo Ambrósio; Maria Angélica Miglino; José Luiz Rodrigues; Fabiana Forell; Luciana Relly Bertolini; Marcelo Bertolini

doi:10.1016/j.theriogenology.2017.02.025

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle

Theriogenology. 2017 Jun:95:24-32. doi: 10.1016/j.theriogenology.2017.02.025. Epub 2017 Mar 1.

Authors

Affiliations

¹ Center of Agronomy and Veterinary Sciences, Santa Catarina State University, Lages, SC, Brazil; School of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil.
² Molecular and Developmental Biology Lab, University of Fortaleza, Fortaleza, CE, Brazil.
³ Center of Agronomy and Veterinary Sciences, Santa Catarina State University, Lages, SC, Brazil.
⁴ Santa Catarina State Agriculture Research and Extension Center, Lages, SC, Brazil.
⁵ School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
⁶ School of Veterinary Medicine, University of São Paulo, São Paulo, SP, Brazil.
⁷ Pontificial Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil.
⁸ School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. Electronic address: marcelo.bertolini@ufrgs.br.

PMID: 28460676
DOI: 10.1016/j.theriogenology.2017.02.025

Abstract

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

Keywords: Cattle; Handmade cloning; In vitro embryo production; Somatic cell nuclear transfer.

MeSH terms

Animals
Blastocyst / cytology*
Blastocyst / physiology
Cattle
Cell Culture Techniques / veterinary
Cloning, Organism / methods
Cloning, Organism / veterinary*
Efficiency
Embryonic Development
Female
Oocytes / cytology*
Oocytes / physiology
Parthenogenesis
Pregnancy
Pregnancy Outcome / veterinary