Use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro.

OBJECTIVE: To determine whether specific sugars inhibit adhesion of Streptococcus zooepidemicus, Pseudomonas aeruginosa, and Escherichia coli to equine endometrial epithelial cells in vitro. SAMPLE POPULATION: Endometrial biopsy specimens collected during estrus from 7 healthy mares. PROCEDURE: Endometrial specimens on glass slides were incubated for 30 minutes at 4 C with suspensions of S. zooepidemicus, P. aeruginosa, or E. coli in phosphate-buffered saline solution (PBSS) alone or with various concentrations of D-(+)-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-(+)-glucose, galactose, or N-acetyl-neuraminic acid. Inhibition of bacterial adherence was determined by comparing adhesion of bacteria (i.e., percentage of glandular epithelial cells with adherent bacteria) suspended in each sugar solution with that of bacteria suspended in PBSS. RESULTS: Mannose and N-acetyl-D-galactosamine inhibited adhesion of E. coli and P. aeruginosa to epithelial cells, whereas only mannose inhibited adhesion of S. zooepidemicus. The other sugars did not affect bacterial adherence. CONCLUSIONS AND CLINICAL RELEVANCE: Mannose and N-acetyl-D-galactosamine appear to play a role in adhesion of S. zooepidemicus, P. aeruginosa, and E. coli to equine endometrium. In horses with uterine infections, use of sugars to competitively displace bacteria from attachment sites on cells may provide an adjunct to antibiotic treatment.

Uncoupling of the equine reproductive axes during transition into anoestrus.

The reproductive activity of light horse mares (n=12) was monitored each day from 3 September until 29 January, or until the mares entered anoestrus, by behaviour evaluation, ultrasonography and blood sampling. Follicles, corpora lutea and ovulation, as well as oestradiol, progesterone, and LH and FSH concentrations, were analysed to determine a reproductive profile for the transition into anoestrus. The results of the present study indicate that light horse mares progress through four phases during the autumn transition into anoestrus: (i) normal cycles; (ii) aberrant cycles; (iii) anovulation, with significant follicular activity; and (iv) anoestrus. One of the first changes observed was a progressive decrease in mean progesterone concentrations during normal cycles (summer: 7.1 +/- 0.4 ng ml(-1); last cycle: 3.6 +/- 0.2). Regression analysis indicates that large follicles progressively lose their ability to produce oestradiol in autumn (third last cycle: 8.5 +/- 1.3 pg ml(-1); last cycle: 5.7 +/- 0.5). During the last ovulatory surge, LH concentrations decreased approximately 50% in 6 of 8 mares. The duration of the follicular phase increased with each cycle approaching anovulation. However, the diameters of the follicles ovulated did not differ. The duration of the luteal phase during ovulatory cycles did not change. Four of the 12 mares developed spontaneously prolonged corpora lutea and were eliminated from the analysis. An anovulatory follicular growth phase occurred immediately before anoestrus in 7 of 8 mares. FSH preceded follicular growth during all cycles and persisted throughout the anovulatory period. During anoestrus, plasma oestradiol, progesterone and LH concentrations remained at basal concentrations. FSH concentrations remained high in 3 of 8 mares but did not cause follicular growth. It is concluded that ovarian and pituitary events become uncoupled during the transition into anoestrus.

Morphologic comparisons among equine endometrium categories I, II, and III, using light and transmission electron microscopy.

OBJECTIVE: To evaluate whether the pathologic changes observed by light microscopy in endometrium of categories II and III were reflected by cellular changes and to describe differences in the endometrial cell ultrastructure during estrus and diestrus. ANIMALS: 18 healthy mares. PROCEDURE: Endometrial tissues biopsied during the physiologic breeding season were categorized, using light microscopy, and were studied, using transmission electron microscopy (TEM). RESULTS: Using TEM, glycogen granules were associated with giant mitochondria for all endometrial types during diestrus. Development of rough endoplasmic reticulum (RER) and Golgi apparatus suggested protein synthesis in the endometrial glands during diestrus. TEM did not reveal major ultrastructural differences, between endometrium of categories I and II. This was unlike differences identified by light microscopy. The most extensive pathologic changes were seen in category-III tissue (TEM and light microscopy). Category-III endometria had a large number of light cells with more degenerative structures and fewer organelles, and lacked cilia in the lumen of the glands. This tissue had extensive fibrotic tissue in the lamina propria and many inflammatory cells in most tissue layers. CONCLUSIONS: The severe ultrastructural changes may be one of the many factors decreasing the fertility of mares with category-III, compared with category-1 and -2, endometrium.

Morphologic characteristics of equine endometrium classified as Kenney categories I, II, and III, using light and scanning electron microscopy.

Pathologic changes in the endometrium of mares may be rated according to Kenney's method of classification. Category I endometrium contains healthy tissue with no or few widely scattered pathologic changes. At the opposite end, severe widespread pathologic changes are associated with category III. Uterine biopsy specimens were collected aseptically from 16 mares during the estrous and diestrous stages of the cycle. Pathologic changes were evaluated, using light microscopy, and endometrium was classified as Kenney's category I, II, or III. Endometrial tissue of category I (n = 5 mares in estrus; n = 3 in diestrus); category II (n = 3 in estrus; n = 4 in diestrus), and category III (n = 4 in estrus; n = 4 in diestrus) were processed for scanning electron microscopy (SEM). All specimens were fixed immediately after biopsy because it was found that numerous bleb-like projections were formed when fixation was delayed. Category I endometrium had normal glands, and fibrotic tissue was not observed by light microscopy. Scanning electron microscopy revealed numerous hexagonally shaped cells that were covered with many microvilli. Ciliated cells also were observed, and they contained long healthy cilia. Category II endometrium had 2 to 4 nests surrounded by collagen fibers. Of the 4 specimens, 3 had moderate leukocyte infiltration (59 +/- 14.8 WBC/4 high-power fields [450 x]). Scanning electron microscopy revealed some inflammatory changes with slight swelling of the cell surface. Several cells in category II endometrium lacked microvilli, but they were interdispersed among many healthy hexagonal cells. Many nests were seen in category III tissue, and 2 specimens had severe infiltration of WBC (232,264 cells/4 fields).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of estrous cycle stage on adhesion of Streptococcus zooepidemicus to equine endometrium.

Equine endometria representative of Kenney's categories I, II, and III were incubated in vitro with phosphate buffer, Streptococcus pneumoniae, or S zooepidemicus. Endometrial tissues from mares in estrus and diestrus were first categorized according to Kenney's classification, then were tested for adherence of S pneumoniae and S zooepidemicus to the epithelia. Bacteria were not observed when the endometrial tissue was incubated with phosphate buffer or S pneumoniae. There was no statistical difference in attachment of S zooepidemicus to endometrial tissue from mares in estrus or diestrus if endometrial classification was ignored. However, bacterial attachment was significantly (P < or = 0.05) higher in category III endometrium during estrus.

The influence of photoperiod on gonadotrophin-releasing hormone stimulated luteinising hormone release in the anoestrous mare.

The transition from anoestrus to oestrus in mares is controlled by photoperiod. The present study examined whether additional daylength would accelerate the mares' response to gonadotrophin-releasing-hormone (GnRH). Nine anoestrous mares were placed under ambient or artificial long lighting on 7th January. The four month experimental period was divided into a three-day sequence which was repeated at 21 day intervals. Ovaries were palpated rectally on Day 1; saline was injected (1 ml intravenously [iv]) on Day 2; GnRH was administered (0.59 microgram/kg bodyweight iv) on Day 3. Blood was taken at -60, 0, 15, 30, 60 and 120 mins relative to saline or GnRH treatment. Serum luteinising hormone (LH) was determined by a homologous equine radioimmunoassay (RIA). Several criteria were employed to define a positive response to GnRH and the results were analysed by Fisher's exact probability test. Treatment with artificial light allowed a response to GnRH within six weeks whereas the mares in ambient lighting took 12 weeks to respond to GnRH. The advancement in the time of response to GnRH under the long photoperiod could be related to changes in pituitary LH content, accelerated follicular activity or alterations in other brain-pituitary hormone levels.

Hormonal control of pubertal spermatogenesis.

The spermatogenic process of normal rats at 20, 32, and 44 days of age was characterized. Variations in numbers of degenerating and abnormal cells were noted during the cycle in most age groups, indicating a stage-related vulnerability of these cells. The most advanced cell types that were seen at a particular age were frequently abnormal or degenerating. When the numbers of viable cells available to degenerate were considered, the degeneration rate in normal pubertal animals was about 15, 10, and 2 times greater in 20-, 32-, and 44-day-old animals, respectively, than in 75-day-old animals. In 32-day-old rats, neither hypophysectomy nor hypophysectomy and subsequent hormone supplementation resulted in an alteration in the qualitative pattern of germ cell degeneration during the spermatogenic cycle compared with that in the normal animal; however, the treatments did alter the quantitative response of cellular degeneration. Three days posthypophysectomy there was a marked increase in the numbers of total degenerating germ cells. FSH (60 micrograms) given twice daily (as were all hormones) reduced the numbers of degenerating cells significantly, as did LH (13 micrograms). Low dose LH (0.3 micrograms), representing the approximate contaminating dose of LH in the 60-micrograms FSH preparation, and low dose FSH (30 micrograms) did not elicit a response significantly different from that to hypophysectomy alone. LH (13 micrograms) plus FSH (60 micrograms) reduced the levels of degenerating cells such that there was no significant difference from levels in intact 32-day-old rats. The data indicated, for the cell types studied, a lack of specificity of various hormones or hormone combinations in the survival of specific germ cell types. It emphasizes the importance of FSH in pubertal spermatogenesis as well as the synergistic actions of LH and FSH.

Effects of early treatment with antiserum to ovine follicle-stimulating hormone and luteinizing hormone on gonadal development in the rat.

Neonatal rats secrete considerable amounts of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) between 5 and 15 days of age. Antisera to LH and FSH were injected during this 11-day period in an attempt to study the importance of gonadotropins for functional development of the gonads. Autopsies at 15 days of age revealed normal ovarian follicular histology after either antiserum treatment, but antiserum to LH significantly retarded differentiation of interstitial tissue. Uterine weights were increased, and some of the serum samples showed elevated FSH levels following the antiserum treatments. The testes of antiserum-treated rats were heavier at 15 days of age, while accessory sex organ weights were unchanged. Vaginal opening after antiserum treatment was more variable than in controls, but tended to be earlier. The first appearance of penile serum was not significantly changed. Both males and females were fertile as adults. Important limitations to the use of chronic antiserum injections were found in immature rats. The antisera were present for at least 34 days after injection. The ways in which this pool of antiserum might have influenced the results are discussed.

Alterations in steroid and gonadotropin release resulting from surgical stress during the morning of proestrus in 5-day cyclic rats.

The purpose of this experiment was to determine whether surgical stress on the morning of proestrus would elicit an early release of gonadotropin from the pituitary. Animals exhibiting 5-day estrous cycles underwent bilateral sham-ovariectomy under ether anesthesia at 0800 h of proestrus. These animals had high levels of progesterone and estradiol following the surgery. These steroids were thought to be adrenal in origin, since animals adrenalectomized at 0800 h of proestrus had low progesterone levels and estradiol comparable to unoperated controls. Subsequently, the sham-operated animals showed high FSH but not LH values at 1300 h, prior to the normal critical period for gonadotropin release. By 1400, the LH surge had begun, and progesterone was again being released. Adrenalectomized and unoperated controls showed no increase in any steroid or gonadotropin measured before 1400 h. These findings suggest that stress-induced release of adrenal estradiol and progesterone, rather than some other consequence of the surgical procedure, during the morning of proestrus, can advance the onset of release of FSH, prior to LH. Ovariectomy at 0800 h proestrus led to a rapid and dramatic increase in FSH but not LH secretion by 4 h after surgery. By 6 h after ovariectomy FSH had increased to six times control values and LH had increased to twice control values. Estradiol remained at control values for 6 h following surgery but 20alpha-hydroxypreg-4-en-3-one (20alpha-OHP) dropped quickly to baseline values. It is possible that a reduction in circulating 20 alpha-OHP may be responsible for the increases in FSH prior to LH in this group, but the absence of other negative feedback factors from the ovary or adrenal may also be involved.