Follicular growth and atresia in the mare

The growth and atresia in equine follicles has received little attention although the mare is a unique model because of the size of the animal, the size of the ovarian follicles and the unusual ovarian anatomy. The size of the animal allows ultrasound scanning to monitor follicle dynamics in vivo and the size of each follicle allows for several parameters to be studied without having to pool follicles together and thereby potentially introducing errors. Several studies in the equine species have focused on follicle dynamics, others on atresia and follicular fluid hormone levels and yet others on the oocyte, but none have combined all of these areas. The aims of this thesis were to establish whether granulosa cells of regressing equine follicles degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells was correlated with other indices of follicle health such as steroid hormone levels and oocyte quality.At first each parameter was investigated in detail at a microscopic level: oocyte chromatin configuration, cumulus morphology, follicle wall atresia, granulosa cell apoptosis and steroid content in follicular fluid. Then followed a whole animal approach with monitoring of follicular growth via ultrasonography. In the end this information was put together when studying ovaries recovered at different stages of the cycle in order to reveal when selection took place and how this was reflected in the parameters of the individual follicle.The temperature at which the ovaries were held did not significantly affect (P > 0.05) chromatin configuration in oocytes (n = 73), but the length of time at which the ovaries were held, did significantly change chromatin configurations (n = 222) after 6 hours (P < 0.001). Health status of the follicle appeared to affect chromatin configuration (n = 118), but this failed to reach significance (P = 0.08). Oocyte chromatin configuration tended to change from even distribution within the germinal vesicle, through different degrees of condensation, to metaphase stages with increasing apoptosis or atresia of the follicle. The puzzling configuration condensed chromatin may be part of the normal development but may also be an end-stage chromatin configuration as it tended to be most frequent in atretic follicles and increased in frequency after long storage in the ovary. The relationship between cumulus morphology and chromatin configuration (n = 149) was evident: compact cumulus morphology was associated with immature oocyte chromatin configurations and fully expanded cumuli were correlated with metaphase chromatin (P < 0.001). Each sequential step of cumulus expansion represented a further step towards meiotic maturation.Evaluation of follicle health based on granulosa cell morphology immediately upon retrieval, follicular wall atresia and apoptosis showed that each of the methods to detect atresia and apoptosis had advantages and limitations and that it was necessary to use the methods in conjunction because they supplemented each other. Histological atresia and ethidium bromide detection of apoptosis (n = 20) was significantly correlated (P < 0.001), and the stereomicroscopic evaluation of freshly isolated granulosa cells were significantly correlated (P < 0.001) with the histological findings (n = 33). Apoptotic granulosa cells and theca cells were detected histologically, a phenomenon which has not previously been described in equine follicles.In a study with 293 follicles from 23 mares, it was found that apoptosis was not a definite measure of death, because growing follicles, as determined by ultrasonography, could be apoptotic and have no other signs of impending death of the follicle, although apoptosis generally was associated with atresia of the follicle (P < 0.001). There was a population of small, atretic follicles with expanded cumuli and oocytes with chromatin progressed towards meiosis, and a population of large, healthy follicles, with young oocyte configurations and no cumulus expansion, and finally a population of follicles larger than 30 mm, which were healthy, but had started to move towards maturation in vivo, i.e. getting ready for ovulation. Size of the follicle appeared to be more important than stage of cycle for predicting chromatin configuration, cumulus morphology and atresia and apoptosis, but size of follicle was correlated with stage of cycle. This led to the theory that despite the larger follicles having stopped growing according to the follicle maps made from ultrasound scans, regression may not initially be related to detectable apoptosis, and that overt apoptosis may be a later event. The fact that most atretic follicles were small suggested that follicles regressed and slowly became overtly atretic and contained low levels of oestradiol and degenerate oocytes surrounded by expanded cumuli.When used in combination with plasma hormone levels, daily mapping of follicles in 7 ponies and 5 mares throughout the cycle made it possible to detect days of changes in follicular growth both of the individual large follicle and smaller follicles in groups in relation to hormone changes. The growth of the largest follicle to a point where it was capable of producing sufficient oestradiol, resulted in decreasing levels of FSH. The IX decreasing levels of FSH caused the smaller follicles to regress. The increased oestradiol concentrations promoted increased LH production as well as partaking in luteolysis, with resulting further increased levels of LH due to lack of negative feed back from progesterone. When the inhibiting effect of oestradiol disappeared just before ovulation, FSH started to increase, resulting in growth of a new wave of follicles, emerging on day 6 (P < 0.01). Divergence in growth rates between the dominant follicle and the largest subordinate follicle happened on day 9 and this became significant on day 12 of the cycle (P < 0.05). At this point FSH levels were high, LH was low and progesterone was still produced. The cause of deviation in this study remains unknown. The subordinate follicles grew until day 17 - 18, at which point they started to regress. The follicle maps were a reliable method of determining growth and regression, when keeping in mind that there are influences from regressing, atretic follicles during periods of general growth and that monitoring of the individual small follicle is not possible when there are many follicles.In conclusion this thesis demonstrated that oocyte chromatin configuration, cumulus morphology, follicular fluid steroid concentration and granulosa cell health are parameters, which change with follicle health, and that ultrasound scanning is an important and necessary tool to use in conjunction with the above measurements when studying follicle growth, selection and regression. Death of follicles happens gradually, and selection for dominance is a gradual process, where one follicle gains advantage over the competitors, resulting in dominance at an early stage during the cycle

Moody mares—Is ovariectomy a solution?

Unwanted behaviour in mares is a commonly presenting problem to the veterinarian. This behaviour may range from the mare being uncooperative or aggressive when handled on the ground, kicking, bucking or rearing when ridden or being aggressive towards other horses. This purpose of the study was to evaluate whether bilateral ovariectomy in mares with unwanted behaviour improved the mare&rsquo;s behaviour and/or rideability from the owner&rsquo;s perspective. The mares were grouped and compared statistically based on their histological classification as having either &ldquo;normal&rdquo; or neoplastic ovaries. This study is a retrospective case series report of twenty-eight ovariectomized mares. A semi-quantitative value (1&ndash;10) pre- and post-ovariectomy for A) behaviour on ground/in stable and B) rideability was given, based on the owner&rsquo;s observations. The horses were grouped based on their histopathologic diagnosis as &ldquo;Normal ovaries&rdquo; or &ldquo;Neoplasia&rdquo;. Following ovariectomy, rideability improved, with a score of &ge;5 in 80% (8/10) of mares with normal ovaries and in 57% (8/14) of mares with ovarian neoplasm. A behavioural improvement of &ge;5 was observed in 40% (4/10) of mares with normal ovaries, and in 43% (6/14) of mares with ovarian neoplasm. A significant difference was observed between the semi-quantitative value pre- and post-ovariectomy in both groups. No difference was observed in change in behaviour and rideability score between the group with normal ovaries and neoplastic ovaries. Mares with unwanted behaviour not obviously related to the oestrus cycle and to painful conditions may benefit from ovariectomy to alter their behaviour and rideability

A Diagnostic Survey of Aborted Equine Fetuses and Stillborn Premature Foals in Denmark

Background: Loss of pregnancy in mares can have many different causes, including both infectious and non-infectious conditions. Extrapolation of findings from other studies is often uncertain as the significance of each cause varies across regions. Causes of pregnancy loss in mares have never been thoroughly studied in Denmark, so a prospective cross-sectional cohort study targeting the entire Danish population of pregnant mares was performed over a period of 13 months to obtain knowledge of the significance of individual causes. Fifty aborted or prematurely delivered stillborn fetuses were submitted for necropsy and examined by a panel of diagnostic laboratory methods.Results: Overall, a cause of fetal loss was established for 72% of the examined cases. Most cases (62%) were lost due to a non-infectious cause, of which obstruction of the feto-placental blood circulation due to severe torsion of the umbilical cord was most prevalent. Pregnancy loss due to a variety of opportunistic bacteria, including bacteria not previously associated with abortion in mares, accounted for 12%, while equid alphaherpesvirus (EHV) type 1 was the cause of pregnancy loss in 8% of the cases. EHV type 4 and Chlamydiaceae species were identified in some cases, but not regarded as the cause of fetal loss.Conclusion: Umbilical cord torsion was found to be the most prevalent cause of fetal loss in Danish mares, while infectious causes such as EHV type 1 and streptococci only accounted for a minor proportion of the losses. The study highlights the need for defined criteria for establishing an abortion diagnosis in mares, particularly in relation to EHV types 1 and 4

Colonization of the bovine uterus by Candida kefyr

Abstract Background While fungal infections of the bovine uterus are well-known diseases in pregnant cattle, very limited knowledge exists on the presence and significance of fungi in the uterus of non-pregnant cows. Presence of fungi in the uterine lumen of postpartum (pp) cows has been reported, but little attention has been paid to this as most studies of the bovine pp uterus have focused on bacteria. Case presentation Microscopy of uterine lavage cytology slides of three cows from one herd revealed the presence of numerous yeast-like organisms, which were located either free in the fluid or within macrophages. Two of the cows were around 30 days pp, while the third was 7 months pp. None of the cows had been treated with antibiotics. Culturing of the flush samples was unsuccessful, but Sanger sequencing of DNA extracted from an endometrial biopsy of one of the cows revealed the presence of Candida kefyr (Kluyveromyces marxianus). Fluorescence in situ hybridization examination of endometrial tissue sections of two cows using probes targeting 18S rRNA of the K. marxianus group was performed and revealed the presence of yeast cells on the endometrium. Histology was performed and demonstrated hyphal and non-hyphal yeast-like organisms on the surface of endometrium and in the crypts. Tissue invasion was restricted to the superficial part of the epithelium and although endometrial inflammation was present, this was mild and considered as not being caused by the fungi. One of the cows became pregnant and delivered a normal calf at term, while the two others were not bred. Conclusions Candida kefyr is commonly isolated from milk of cows with mastitis, but has not been reported in association with other diseases of cattle. The infection was present as a monoculture in all three cows, but the fungi had only colonized the uterine lumen and the endometrial surface. Only a mild non-suppurative endometrial inflammation was present, but within the uterine luminal content, many macrophages having phagocytized yeast cells were present. Re-examination of the cows did not reveal a persistent infection, so the infection probably resolved spontaneously