The transfer of egg white into the yolk and consumption of yolk proteins by the embryo are largely unexplored in the pigeon Columba livia domestica. Here, we investigated the route of egg white transfer as well as the degradation and uptake of yolk proteins by the pigeon embryo. Initially, we tested the electrophoretic patterns of proteins in different egg compartments throughout development. Then, we used lysozyme as a reference protein to follow the egg white transfer, and we measured its activity using Micrococcus lysodeikticus as a substrate. Moreover, we determined the general protease activity during different developmental stages in the yolk using casein. Finally, we examined the expression of aminopeptidase-N (APN) and oligopeptide transporter PepT1 genes in the yolk sac membrane (YSM) from incubation day 8 until day 17. Several electrophoretic bands of presumptive egg white proteins appeared in different egg compartments. Also, lysozyme activity was detected chronologically in the egg compartments. It appeared on day 12 in the amniotic and intestinal fluids and on day 14 in the yolk. Moreover, protease activity in the yolk increased significantly on day 14 and thereafter. APN expression was largest on day 8 and reduced generally afterward, whereas PepT1 expression peaked between days 13 and 15 but then reduced substantially. Our results suggest that the egg white proteins move through the amnion and intestine into the yolk where they undergo degradation by the activated proteases. Furthermore, the YSM appears to have a role in protein consumption, and this role decreases toward hatch.

Yolk proteins undergo digestion either inside the egg yolk or in the surrounding yolk sac membrane (YSM) before being consumed by the developing avian embryo. However, the mechanisms underlying the digestion of yolk proteins during embryogenesis are largely unexplored in the pigeon Columba livia domestica. To better understand these mechanisms, the present study examined the classes of activated proteases in the egg yolk and the gene expression patterns of cathepsin B (CTSB) and cathepsin D (CTSD), which encode for lysosomal cysteine and aspartic proteases, respectively, in the YSM. We investigated the activated proteases by applying different types of protease inhibitors to yolk samples taken from incubation day 16. Then, we detected the mRNA levels of CTSB and CTSD in the YSM at incubation days 6, 8, 10, and 12-17. Both cysteine and aspartic proteases appeared to be activated in the egg yolk. Moreover, CTSB expression increased progressively and reached the maximum value on day 13; however, it decreased significantly on days 14 and 15 and further reduced toward hatching (day 17). In contrast, CTSD expression was weak and fluctuated insignificantly during development. Our results suggest that the degradation of yolk proteins at late developmental stages largely occurs in the egg yolk itself, probably by the activated cysteine and aspartic proteases. Furthermore, cathepsin B in the YSM seems to have a primary role in protein digestion, but this role decreases toward hatching.