Broiler egg storage induces cell death and influences embryo quality

It is well known that egg storage reduces embryo performance, but the fundamental reasons for reduced embryo quality remain unclear. The objective of this study was to investigate possible cellular and molecular mechanisms that might reduce embryo quality after egg storage. Broiler hatching eggs were obtained from the Ross 308 broiler strain, divided into 2 groups, and stored (4 and 14 d) under the same temperature and humidity conditions. Samples of the eggs were used to assess embryo quality by determining daily embryo weight (wet and dry) from 4 to 21 d of incubation. To understand possible cellular and molecular mechanisms that might affect embryo quality, blastoderms (unincubated embryos) were isolated from a sample of eggs from each storage group, dissociated into single cells, and subjected to flow cytometry analysis to differentiate between viable, apoptotic, and necrotic cell populations. Quantitative real-time PCR analysis was used to compare the expression of selected apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, Bcl-2-related ovarian killer gene, B-cell lymphoma 2 gene, and B-cell lymphoma xL gene) in blastoderms and embryos (6 d old after incubation). Data were analyzed by the MIXED model procedure of SAS (SAS Institute, Cary, NC), with significance set at P ≤ 0.05. After covariance analysis of initial egg weights, the results showed decreased daily embryo weights (wet and dry), an indication of decreased embryo quality that could affect hatch quality. In addition, a decrease in blastodermal cell viability was associated with an increased percentage of apoptotic cell deaths (P < 0.0001). Expression of pro-apoptotic genes (Bcl-2 homologous antagonist/killer gene, Bcl-2-associated X gene, and Bcl-2-related ovarian killer gene) were upregulated at the blastodermal level as the storage duration increased, but all genes were downregulated after 6 d of incubation. This suggests that an increase in egg storage duration could activate mechanisms of apoptotic cell death at the blastodermal level, which may be one of the molecular mechanisms that leads to reduced daily embryonic weight during incubation. Experimental controls capable of reducing the cellular and molecular mechanisms of egg storage should be used to increase embryo quality.