Influence of different cell passage numbers on bovine cloned embryo: A systematic review and meta-analysis

Animal cloning remains inefficient, with live birth rates below 5 % in most mammalian species, slightly higher in bovines (20-25 %). Among various factors, nuclear donor cells play crucial roles, and their passage numbers may influence cloning efficiency. However, a definitive association between passage numbers and outcomes in cloning remains inconclusive due to insufficient data and inconsistent results. In the present meta-analytical study, we compared research using high (>6) and low (≤6) cell passage numbers across various types, breeds, and sexes of donor cells to assess their impact on embryo development and associated gene expressions in bovine clones. Our findings revealed that cell passaging influences developmental competence at the cleavage and blastocyst rates, with lower passage numbers yielding better results. Higher acetylation of H3K9 in low-passage cells is consistently associated with improved developmental competence through the blastocyst stage, although the difference was not statistically significant. Donor cells with higher histone acetylation may undergo reprogramming more easily and completely, thus improving cloning efficiency. Further analysis elucidated that the types and breeds of donor cells also affected the blastocyst outcome. Nevertheless, high heterogeneity and meta-bias were identified in the meta-analytical outcomes, particularly in cleavage, 2-cell, 8-cell, blastocyst, the total cell number (TCN), the ratio of inner cell mass and total cell number (ICM/TCN), NANOG, and birth rate, which may contribute to inconsistencies in embryo quality results and hinder comparisons between development-related gene expressions and the embryo transfer outcome.