Morphometric determination of endometrial leukocyte migration during different stages of the equine oestrous cycle

Abstract

Uterine defences against bacterial challenge are more efficient during oestrus than during dioestrus. The exact reasons and mechanisms responsible for this difference are, however, still incompletely understood. The leukocyte reaction is one of the defence mechanisms that has been cited as being able to respond better to a bacterial challenge during oestrus than during dioestrus. The aim of the present study was to test the hypothesis that the magnitude of endometrial leukocyte migration following the instillation of semen into the uterine lumen is greater during oestrus than during dioestrus. Eight Nooitgedacht mares of normal fertility, aged between 8 and 16 years (11.5 ± 2.7; mean ± SD), were used in the study. Each mare received a different treatment during each of four oestrous cycles, with a rest cycle after each treatment. Two treatments were performed during dioestrus and two during oestrus. One treatment for each stage of the cycle was a control treatment without challenge to the endometrium. At time zero of challenged cycles a single aliquot of 13 ml raw semen, frozen-thawed without addition of any cryoprotectant or extender, was instilled into the uterus. An endometrial biopsy was taken 6 and 48 h after time zero and a swab for cytology and culture (if cytology was positive) was collected 48 and 120 h after time zero. An image analyzer was used to record the total number of cells, round cells, neutrophils and eosinophils per unit surface area of epithelium, stratum compactum (SC) and stratum spongiosum (SS). The relative number of round cells, neutrophils and eosinophils were expressed as proportions of the number of each cell type to the total number of cells. The use of an image analyser made the collection of quantitative data from histologic sections possible. However, the operator still had to make some critical decisions, namely to choose the field of the section for analysis and to assign individual cells to a chosen category. The total numbers of cells in the epithelium and the SS were greater during dioestrus than during oestrus, while no such difference could be demonstrated for the SC. The stage of the oestrous cycle had no meaningful influence on any other (measured or calculated) variable. During challenged cycles, absolute and relative numbers of neutrophils were significantly greater in the epithelium, SC and SS than during control cycles. There was an interaction (not always reaching significance) between treatment and time with regard to the absolute and relative numbers of neutrophils in epithelium and SS and round cells in the epithelium. Numbers of neutrophils and round cells were significantly higher 6 h after treatment than 48 h after treatment in challenged cycles, but did not differ during control cycles. During challenged cycles, the stage of the oestrous cycle when treatment occurred had no effect on the duration of the induced endometritis, the occurrence of positive cytology or culture results, or the type of bacteria that were cultured. Regardless of the stage of their cycles when they were challenged, all mares rid themselves of the opportunistic pathogens placed into the uterine lumen within one oestrous cycle. The hypothesis was rejected and it is therefore concluded that the stage of the oestrous cycle did not influence the magnitude of the endometrial leukocyte response to a standardized challenge with semen in these reproductively sound mares. A similar study will be required to test whether this conclusion also holds true for mares that are susceptible to endometritis.