The present study was an in vitro analysis of the dynamics of bovine mammary gland neutrophil apop­tosis based on the detection of morphological changes. The neutrophils were isolated from mammary glands of five virgin heifers. The mammary glands were lavaged, the suspensions were then bacteriologically examined, and total and differential cell counts were made. The cells were cultivated in vitro for 4 hours. After 2, 3 and 4 hours of cultivation, they were panoptically stained, and the proportions of apoptotic neutrophils and trypan blue positive neutrophils were determined. Neutrophil apoptosis and impaired cytoplasmic membrane integrity of neutrophils were already observed in the mammary gland lavages (11.9% and 0.8%, respectively). During the cultivation, a progressive increase in the number of apoptotic neutrophils in various stages of apoptosis – karyopyknosis, zeiosis and apoptotic bodies – was observed. Karyopyknotic neutrophils represented a dominant part of the apoptotic neutrophil population in the course of the whole cultivation. The most intensive increase was observed in zeiosis, whereas the levels of apoptotic bodies remained the same. After 4 hours of cultivation, 31.7% apoptotic neutrophils and 9.8% trypan blue positive neutrophils (i.e. Secondary necrotic cells) were found. The results of this work show that spontaneous apoptosis and secondary neutrophil necrosis must be taken into account during in vitro cultivations of bovine mammary gland neutrophils.
Peer Review #3 of "TGF-beta signalling in bovine mammary gland involution and a comparative assessment of MAC-T and BME-UV1 cells as in vitro models for its study (v0.1)"
