Impacts of asymmetry in activity of left and right bovine ovaries on histological characteristics of various vitrified preantral follicle classes

The success of ovarian tissue cryopreservation can be recognized by the live birth in different animal species (mice, rabbit, birds, sheep, and human). In spite of all the advances of cryopreservation of ovarian tissue, it is still a challenge and protocols should be optimized to handle the diversity of cell types and components of this tissue (oocyte, granulosa cells, endothelial cells, extra cellular matrix) and the biological variability among species. It was reported that there was a relationship between the development of corpus luteum (CL) and the development of follicles, which may cause asymmetry in the function of the reproductive organs in dairy cows. Previous studies confirmed asymmetry in the function of reproductive system in dairy cows due to differences in ovarian activity and probably because of physiological differences in the tubular parts of reproductive organs which result from the side of the previous gestation. There are many discrepancies about positive or negative effects of corpus luteum and unequal activity of sides of the reproductive system. Therefore, the aim of this study was to investigate the impacts of CL and side of ovaries (Right vs. Left) on histological characteristics of different types of preantral follicles after bovine ovarian tissue vitrification.  

Ovaries (n=10) were collected from five adult cross-bred cows at a local abattoir. Ovaries were categorized on the basis of the presence or absence of a CL and the side of ovaries and divided into five pools: 1/CL+ (with CL) group, 2/CL -(without CL) group, 3/right ovaries group, 4/left ovaries group and 5/C control group (ovaries which were not selected toward the presence or absence of CL or side of ovaries). From each bovine ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification [CL (˗); CL(+); Right and Left group), either followed by in vitro culture for 1 or 5 days. Samples were fixed in Millonig’s for 2 h, dehydratedin a graded series of ethanol, clarified with xylene,embedded in paraffin wax, and serially sectioned into 7 µmsections. Every fifth section was mounted on a glass slide,stained with Periodic acid–Schiff (PAS), and evaluated usinga light microscope at magnificationof 400×. All procedures for exposure to cryoprotectant agents (CPAs) and vitrification were performed by using the new Ovarian Tissue Cryosystem (OTC). For the in vitro culture, the cortex tissue samples weretransferred to 24-well culture dishes containing 1 mLof the culture medium per well. The culture wasperformed at 38.5 °C in 5 % CO2 in a humidifiedincubator. The ovarian tissue was histologically evaluated before and after culture. The quality of the preantral follicles wasclassified according to the parameters previously described. Briefly, morphologically normal folliclescontained an intact oocyte and granulosa cells, whereasdegenerated follicles contained an oocyte with a pyknoticnucleus, ooplasma shrinkage and/or granulosa cell layers that had disorganized and detached from the basementmembrane. In each treatment, a total of 150 preantral follicles (30 per animal) were examined, which were classified as follicles without an antrum and with an oocyte, surrounded by one layer of flattened (primordial follicle), flattened and cuboidal (transitional follicle) or cuboidal granulosa cells (primary follicle), or with an oocyte surrounded by two or more layers of cuboidal granulosa cells (secondary follicle). In this study, data that were not normally distributed (Kolmogorov-Smirnov test), were submitted to logarithmic transformation. Comparisons of means (morphologically normal follicles) were analyzed by Kruskal-Wallis test and Mann-Whitney test, when appropriate. All statistical tests were performed using Sigma Plot 11 (Systat Software Inc., USA). Differences were considered significant when P< 0.05.

Results of this experiment showed that although, absence of a CL (CL˗) resulted in a greater percentage of normal follicles for transitional (cultured samples on day 1) and primary follicles (post-thawed samples) when compared to that of ovaries bearing a CL (CL+), there was an exception. These results indicate that CL (+) samples also resulted in a greater percentage of normal primordial follicles (cultured samples on day 1) than that of CL (˗) samples (57.6% versus 39/3%, respectively). However, for all classes of preantral follicles during all stages (warming, day 1 or day 5), there were no significant differences in the percentages of normal follicles among Right and Left samples (P> 0.05).

It seems that follicles which are at beginning of their development (Primordial follicles) were not affected by presence of a CL as much as follicles at more advanced stages of development (transitional & primary). According to the results of this study, it can be concluded that, many factors such as presence or absence of a CL, types of preantral follicle classes, stages after vitrification (warming and days after culture) and interactions between these factors could influence the likelihood of a successful vitrification outcome.