Invasive methods (e.g. histology) limit serial assessment of ovarian structures in rabbits and impede progress in our understanding of factors responsible for inducing ovulation. Serial transrectal or transvaginal ultrasonography of ovarian structures has revolutionized our understanding of reproductive patterns in domestic animals, but the approach is limited in smaller species by the small physical size of the animal and the ovary. The advent of high-resolution ultrasound biomicroscopy (UBM), however, has permitted recent characterisation of ovarian dynamics in species as small as mice. As part of a larger study on factors influencing ovulation in rabbits, the objective of the present study was to validate UBM as a method of assessing ovarian structures in rabbits by comparing ultrasound image attributes with histological attributes. Female New Zealand White rabbits (n = 4; 5 to 5.5 months of age) were given ovulation-inducing treatments and examined daily by conventional transabdominal ultrasonography with a 12-MHz linear-array probe (MyLabTM5). Rabbits were killed with sodium pentobarbital 8 days after treatment. Ovarian UBM was performed ex situ in a water bath. A 25-MHz oscillating sector-array transducer (Visualsonics) was moved mechanically at a constant speed along the long axis of each ovary, and images were recorded (300 frames per 10-s cine-loop). After UBM, ovaries (n = 8) were fixed in 10% formaldehyde, embedded in paraffin, serial-sectioned at 10-μm thickness, and stained with Masson’s trichrome. Digital photomicrographs were taken of every third section of each ovary. For each ovary, follicles ≥0.6 mm and corpora lutea (CL) were counted and measured by scrolling through the UBM cine-loops and the serial micrographs. Ovarian follicles ≥1.5 mm were readily detected by conventional ultrasonography, and such follicles were present in the ovaries of all rabbits on the day of treatment, but CL structures were not clearly discernable during the 8 days after treatment. In vivo transabdominal UBM failed to yield satisfactory images of the ovaries probably because of high acoustic impedance of the body wall. The number (mean ± SEM) of CL detected by UBM ex situ in each pair of ovaries was highly correlated (r = 0.99; P < 0.05) with the number detected by histology (8.5 ± 2.9 v. 8.8 ± 3.0). The diameter of CL was also highly correlated (2.1 ± 0.7 v. 1.8 ± 0.6 mm; r = 0.99; P < 0.05). Similarly, a high correlation was detected between UBM and histology in the number of follicles ≥0.6 mm (17.3 ± 2.3 v. 19.0 ± 1.6; r = 0.96 P < 0.05), and follicle diameter (1.1 ± 0.05 v. 1.1 ± 0.03 mm; r = 0.96; P < 0.05). Interestingly, UBM of each ovary required on average 2 h compared with 30 h for histology. The results validate the accuracy of UBM for assessing rabbit ovaries ex situ, but identify limitations of present ultrasound approaches for examining rabbit ovaries in situ.
Supported by the Natural Sciences and Engineering Research Council of Canada. The authors thank D. Nesbitt for assistance with histological procedures.