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Circadian clock disruption decreased mammary development and impaired lactation in late gestation cows and mice. Transcriptional targets of the circadian clock genes BMAL1 and CLOCK include the cell-cell and cell-ECM proteins e-cadherin (CDH1) and zonula occludens-1 (ZO-1). The polarization and lumen formation of milk-producing acini is dependent on these junctional proteins. We hypothesized that if BMAL1 and CLOCK were disrupted, mammary epithelial cells (HC11) will have a reduced ability to form acini. Our objective was to measure the effect of BMAL1 gene deletion (BMAL1-KO) and CLOCK protein reduction (shCLOCK) in HC11 cells on the formation of acini and expression of CDH1 and ZO-1 in three-dimensional (3D) cultures. Cells were plated on Matrigel, embedded, and cultured to create 3D structures. ImageJ software was used to quantify the acini and found that the BMAL1-KO and shCLOCK lines formed fewer and smaller acini (p < 0.05). Confocal microscopy revealed that CDH1 was located along lateral membranes and ZO-1 was apically located in all three lines. Levels of CDH1 and ZO-1 were greater (p < 0.05) per unit volume of cell in the shCLOCK cell line. Live/dead staining of cells found little to no cell death across three lines. Transmission electron microscopy (TEM) of acini indicated less polarization of cells within BMAL1-KO and shCLOCK acini. Polarization of cells is required for formation of 3D structures and requires coordination of temporal-spatial cues. Disruption of circadian clocks results disturbs temporal organization of cellular processes and causes a reduced ability of cells to polarize and form acini.

Periparturient dairy cattle are susceptible to metabolic disease due to immense physiological stress caused by calving and the onset of lactation. Susceptibility to disease is related to the cow’s ability to maintain metabolic homeostasis and mount a proper immune response, despite high demand for glucose to support fetal growth and milk synthesis, and as a fuel for increased circulating levels of white blood cells (WBC). This study was conducted to determine if there was a correlation among white blood cell percentages, blood glucose, and the levels of the ketone betahydroxybutyrate (BHBA) on the days immediately around parturition, and to determine if the variables exhibited circadian rhythms. We hypothesized that glucose levels would vary inversely with WBC, and BHBA levels would vary directly with glucose levels. Blood was sampled from the tail vein of multiparous Holstein cows (n=12) at 0600 from 4 days prepartum to 4 days postpartum, and every 4 hr over a 24-hr period (n=6) starting at 0600 on postpartum day 3. Blood glucose and BHBA levels were measured using a CentriVet™, and WBC percentage was calculated by measuring packed cell volume. Glucose was higher (p