In the present study, we analysed the effect of fixative, breed, luteal stage and location on the nuclear density, volume density of connective tissue and vascular tissue/lumina within a bovine luteal gland in view of the development of an in vivo sampling technique to longitudinally monitor luteal histophysiology. The inner zone defined as the zone geometrically closest to the centre of the gland shows a significantly lower nuclear density (for all cell types) and a higher volume density of collagen fibres and vessels when compared with the outer zone (p < 0.001). The nuclear density in luteal glands from Holstein-Friesian cows is not significantly different from that in Belgian Blue cows, nor is it in stage II vs stage III glands. The collagen fibre content was significantly lower in glands of Belgian Blue cows (p = 0.01) and in younger glands (p = 0.003). Hence, it seems that the lower nuclear density in the inner zone was compensated by a higher amount of collagen fibres. As the type of fixative applied has a significant effect on the nuclear density of the different cell types, the present study warrants future research to further optimize the fixation protocol. As a conclusion, we can state that the topographic difference in nuclear distribution for the different cell types in a bovine luteal gland is only significant when comparing the inner vs the outer zone. This implies that if a sample representative for the whole gland has to be taken, for example, when taking an in vivo sample, it is necessary that the biopsy goes through the inner zone and contains the total diameter of the gland.