Attenuation of PGF2alpha release in ewes infused with the oxytocin antagonist L-368,899

We have investigated the effects of systemic administration of the oxytocin antagonist (OTA) L-368,899 on luteolytic PGF(2alpha) release in ewes. In the first study, carried out in four ovariectomized ewes primed with progesterone to induce responsiveness to oxytocin, 3-h i.v. infusions of 3, 10 and 30 microg/kg/min OTA, carried out on days 12, 14, 16 and 18 in a Latin Square design, resulted in a significant attenuation of the oxytocin induced increase in PGFM concentration at all doses (OTA 139+/-8.3% of pre-oxytocin baseline; control 206.8+/-18.7%; P<0.005). In a further study, continuous infusion of cyclic ewes (n=6) with 10 microg/kg/min OTA from day 13 to day 17 of the cycle resulted in a reduction in both the frequency (OTA 1.0+/-0.4/ewe; control 2.2+/-0.2/ewe; P<0.05) and amplitude (OTA 31.8+/-11.0 pg/ml; control 68.8+/-10.4 pg/ml; P<0.05) of endogenous PGFM episodes compared to control ewes (n=5) measured during daily 8-h sampling windows on days 14-17. This reduction in PGFM concentrations was accompanied by a modest extension in the day of luteolysis (progesterone <0.5 ng/ml) to day 17.5+/-0.4 in the OTA treated group compared with day 16.4+/-0.5 in the control group (P=0.07). The results demonstrate that treatment with OTA caused a significant reduction in episodes of increased PGFM concentration during the period of luteolysis and may provide an approach by which to reduce early pregnancy failure.     

Effect of the administration of PGF2 alpha synchronously with insemination on the pregnancy rate in mares in an insemination program

Investigations in different species including the horse have demonstrated that prostaglandin F2 alpha (PGF2 alpha) is involved in initiating uterine contractions occurring during mating and artificial insemination (A.I.). Uterine contractions play an important role with respect to the sperm transport within the female genital tract. The objective of the present investigation was to evaluate whether the administration of PGF2 alpha (Dinoprost) synchronously to A.I. could have a positive effect on the pregnancy rate in mares. A field study including 346 warmblood-mares (age two to 20 years) belonging to a private studfarm was conducted during the breeding season 1996. The mares were assigned to two groups, group A: mares with spontaneous ovulation, group B: mares in which the ovulation was induced by a GnRH-analog-implant (Deslorelin). PGF2 alpha (Dinoprost) was administered either intramusculary (i.m., 5.0 mg) or intrauterine (i.ut., 0.5 mg diluted in 1.9 ml isotonic NaCl-solution and added to the semen dosis). The study was carried out in a double-blind fashion using isotonic NaCl-solution as a placebo. The mares of each group were randomly assigned to one of the two treatments (i.m. vs. i.ut.). The following first cycle pregnancy rates (day 18) were obtained in mares treated and inseminated once per oestrus: group A1 (PGF2 alpha, i.m.): 54.5% (n = 33); group A2 (placebo, i.m.): 69.7% (n = 33); group A3 (PGF2 alpha, i.ut.): 65.4% (n = 26); group A4 (placebo, i.ut.): 69.8% (n = 32); group B1 (PGF2 alpha, i.m.): 56.5% (n = 46); group B2 (placebo, i.m.): 29.6% (n = 27); group B3 (PGF2 alpha, i.ut.): 66.7% (n = 45); group B4 (placebo, i.ut.): 60.0% (n = 30). The pregnancy rates did not differ between the different groups with the exception of group B2 (p < 0.05). In mares treated repeatedly during the oestrus period (group A, n = 88; group B, n = 23), the pregnancy rates did not differ significantly between treatment and control groups. From the results obtained it is concluded that the PGF2 alpha-application did not show an effect on the pregnancy rate. Further factors influencing the results to a small degree were the stallions, semen age and quality and frequency of insemination per oestrus.     

Plasma concentrations of 13,14-dihydro-15-ketoprostaglandin F2 alpha in mares during uterine involution.

Twelve mares were allowed to foal naturally, after which they were monitored to study uterine involution. Starting on day 3 after parturition, the internal genital tract was examined per rectum manually and ultrasonographically every other day for changes in uterine characteristics and ovarian activity. By day 5, gravid and nongravid uterine horns were similar in size, and by day 7, uterine fluid was absent. On day 7 after parturition, endometrial biopsy samples were obtained for histologic evaluation, and uterine swab specimens were obtained for microbiologic culture. Uterine swab specimens from 10 of 12 mares had slight bacterial growth. The uteri of 8 of the 12 mares were histologically involuted by day 7. All mares ovulated 7 to 12 days after parturition. Concentrations of 13,14-dihydro-15-keto-PGF2 alpha (PGFM) were measured in jugular plasma samples obtained daily for 21 days after parturition. Concentrations of PGFM were low by the day after parturition, and there was no significant correlation between uterine involution and PGFM concentrations in these mares. All 12 mares were bred at the first estrus after parturition, and 9 became pregnant.     

Semen characteristics and libido in boars treated repeatedly with PGF2alpha

The objective was to determine the effects of repeated injections of PGF2alpha (Lutalyse; Pfizer) on semen and libido characteristics in terminal-line boars. Semen was collected once weekly from wk 0 to 15 and on four consecutive days during wk 16. Boars received an i.m. injection of 10 mg of PGF2alpha (n = 11) or 2 mL of vehicle (n = 11) immediately before entering the collection room. For the weekly collections, semen volume (220.3 +/- 3.2 mL; mean +/- SE), gel weight (38.7 +/- 0.7 g), total sperm cells (65.4 +/- 1.2 billion), motile sperm cells (67.4 +/- 0.6 %), and sperm velocity (125.9 +/- 1.2 microm/s), were affected by time (P < 0.01) but not by treatment (P > 0.10). Sperm concentration (0.31 +/- 0.01 billion/mL) was not affected (P > 0.10) by time or treatment. The percentage of morphologically normal sperm cells, assessed at wk 16, did not differ (P = 0.39) between groups (80.8 +/- 1.0). Libido was evaluated from wk 0 to 16. There were no effects of treatment or time (P > 0.10) on the period from injection to the start of ejaculation (225.6 +/- 9.1 s). Duration of ejaculation was affected by treatment (P < 0.01; 472.0 +/- 43.1 s and 280.4 +/- 43.1 s, for PGF2alpha-treated and control boars, respectively) and time (P < 0.01). During the intensive collection period (wk 16), semen volume (200.1 +/- 7.1 mL), gel weight (39.2 +/- 1.5 g), sperm concentration (0.19 +/- 0.01 billion/ mL), total sperm cells (39.5 +/- 3.6 billion), motile sperm cells (65.6 +/- 2.2%), and sperm velocity (117.8 +/- 3.7 microm/ s) were affected by time (P < 0.10) but not by treatment (P > 0.10). The period from injection to the start of ejaculation tended to decrease (by 44%) during the intensive collection period in PGF2alpha-treated boars, but not in controls (treatment x time, P = 0.07). Regardless, the period from injection to the start of ejaculation did not differ (P = 0.63) between groups on d 4 of the intensive collection period. Duration of ejaculation was affected by treatment (P < 0.01; 459.1 +/- 24.1 s for PGF22alpha-treated boars vs. 303.1 +/- 24.1 s for controls) but not by time (P > 0.10). Overall, there were no exceptional positive or negative effects of long-term treatment with PGF2alpha on semen characteristics and libido in boars.     

The effect of estrogen, progesterone and prostaglandin F2 alpha on uterine contractions in seasonally anovulatory mares

Uterine contractions were studied in two experiments utilizing ultrasonography and seasonally anovulatory mares. A one-minute ultrasound scan was done to produce longitudinal real-time images of the uterine body and an overall uterine contractile activity score (0 = no or minimal activity to 4 = maximal activity) was assigned to each scan. In experiment 1, a two-hour uterine activity trial (one score every 10 minutes) was done in mares given a single injection of prostaglandin F2 alpha (PGF2 alpha group; n = 4) and in control mares (n = 4). There was no difference between the two groups over the two-hour trial (mean activity score averaged over the two-hour trial: PGF2 alpha group, 0.2; control group, 0.1). In experiment 2, 16 mares were randomly assigned to one of four groups: 1) controls (corn oil vehicle), 2) 1 mg estradiol 17 beta on days 0 to 9 and 100 mg progesterone on days 10 to 20 (E2--greater than P4 group), 3) 100 mg progesterone on days 0 to 20 (P4 group), and 4) 100 mg progesterone on days 0 to 9 and 1 mg estradiol 17 beta + 100 mg progesterone on days 10 to 20 (P4--greater than E2 + P4 group). Uterine activity was assessed for each mare daily. The day by group interaction was significant. Scores for the E2--greater than P4 group were greater on days 4 to 11 (P less than .05) than for the other three groups. From day 14 to 21, scores did not differ among the three steroid-treated groups (except on day 15), but the scores averaged over each steroid-treated group were greater for each day (P less than .1 or .05) than for the controls (except on day 17).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first postpartum ovulatory cycles

During breeding of mares, ultrasonographic detection of uterine fluid accumulations in the first postpartum ovulatory period was associated with significantly decreased pregnancy rates, when compared with rates in control mares (P less than 0.005). The previously gravid uterine horn was recognized as the larger horn, when assessed for size by ultrasonography, for a mean of 21 days (range, 15 to 25 days) after parturition. On the basis of similar measurements obtained during 3 ultrasonographic scans (5-day period), uterine involution was determined to be completed in a mean of 23 days (range, 13 to 29 days). Progestin treatment did not affect uterine size, fluid accumulation, or rate of involution after parturition. However, delaying the first postpartum ovulation with 8 days of progestin treatment significantly improved pregnancy rates (P less than 0.05). More (P less than 0.05) mares became pregnant (23 of 28, 82%) when ovulation occurred after day 15 in the first postpartum ovulatory period, compared with those mares that ovulated before day 15 (6 of 12, 50%). We concluded that ultrasonographic detection of uterine fluid and postpartum progestin treatment can be used to manipulate breeding strategies and to improve pregnancy rates in mares bred during the first postpartum ovulatory period.     

Nuclear changes in ovine luteal cells in response to PGF2 alpha

Percentages of normal and apoptotic parenchymal cells, fibroblasts and endothelial cells in ovine corpora lutea at 12, 24 and 36 hr following administration of a luteolytic dose of PGF2 alpha were determined and compared to percentages for identical cell types in corpora lutea removed from control ewes on days 10 (n = 5) and 12 (n = 6) postestrus. In corpora lutea obtained from control ewes greater than or equal to 95% of nuclei examined were scored normal for each of the respective cell types with no difference (P greater than .05) observed between luteal tissue obtained on days 10 and 12 postestrus. Following treatment with PGF2 alpha there were significant (P less than .05) reductions in the percentages of nuclei scored normal. Compared to controls the percentage of endothelial cell nuclei scored normal was reduced at 12 hr following PGF2 alpha-treatment; however significant reductions in percentages of parenchymal and fibroblast nuclei scored normal were not evident until 24 and 36 hr, respectively. Consistent with the concept of apoptosis, nuclear condensation and/or margination indicative of apoptosis did not occur synchronously within a given cell type: i.e., irrespective of the time point examined some cells appeared normal, whereas others had undergone nuclear condensation and/or margination. A sequence of events to explain structural and functional changes that occur during luteolysis following the interaction of PGF2 alpha with specific receptors in large steroidogenic luteal cells is discussed.     

A preliminary study of uterine derived polymorphonuclear cell function in mares with chronic uterine infections

From 6 mares with chronic uterine infection, polymorphonuclear neutrophils (PMNs) were obtained from the uterus. In order to recover an adequate number of viable PMNs, 0.1% oyster glycogen was infused into the uterus as a mild irritant 12 h prior to the uterine flushing. Phagocytosis and chemotaxis of the uterine derived PMNs were determined. The supernatant from the uterine flushing was compared with autologous serum for its capacity as an opsonin and chemoattractant. There was a significant increase of both phagocytosis and chemotaxis when autologous serum was used compared with the supernatant from the uterine flushing. This study indicates that autologous serum has a greater opsonizing capacity than uterine secretion from mares with chronic uterine infection. Since all mares conceived following this study, the use of oyster glycogen was not considered to have deleterious effect on the uterine mucosa.     

Carprofen in veterinary medicine. II. Inhibitory effect on the release of PGF2 alpha in the early postpartum cow

Carprofen, a non-steroidal anti-inflammatory drug (NSAID) known to inhibit prostaglandin synthesis, was given intravenously in five cows at a daily dose of 0.7 mg/kg for five days beginning on day 1 postpartum. Blood samples were collected at various times over a period of six days following the first injection. At this dose, carprofen reached highest plasma values of about 45 micrograms/ml after the fifth injection and was well tolerated by all the cows. During the whole experimental period, mean plasma levels of 15-keto-13, 14-dihydro-prostaglandin F2 alpha, the primary metabolite of PGF2 alpha, were significantly (p less than 0.05) lower in treated than in control animals (28-47% vs 64-101% of pretreatment concentrations). The suppressive effect of carprofen on PGF2 alpha-production occurred immediately after its application and was maximal 3-6 h post injection on the first and on the fifth experimental day (60-80% and 40-85%, respectively). We conclude from our results that carprofen in a single dose of 0.7 mg/kg b.w. effectively suppresses PGF2 alpha-release in the postpartum cow. Whether this effect is beneficial in the treatment of uterine inflammatory processes remains to be determined.     

Contractile effect of PGF2alpha and PGE2 on isolated branches of uterine and ovarian artery in different days of estrous cycle and early pregnancy in pigs

The contractile effects of PGF2alpha (3 x 10(-6) to 10(-4) M) and PGE2 (10(-7) to 10(-5) M) were examined on isolated branches of ovarian artery (OA) and extramyometrial branches of uterine artery (UA) collected from pigs in the luteal (day 10-12) and follicular phase (day 17-20) of the estrous cycle, and during early pregnancy (day 10-12). Strong contraction was demonstrated in both arteries during all investigated periods in response to PGF2alpha, which was significantly higher (P < 0.01) than to PGE2, being negligible in the follicular phase. In UA, the effective dose of PGF2alpha (ED50) amounted 7.9 x 10(-6) M and 6.3 x 10(-6) M in the luteal and follicular phase, and 5.0 x 10(-6) M in early pregnancy. ED50 for PGE2 reached 5.0 x 10(-7) M in the luteal phase, and 4.1 x 10(-7) M in early pregnancy. For both prostaglandins, the contraction was much stronger (P < 0.01) in OA than in UA branches. In OA, the ED50 for PGF2alpha was 1.2 x 10(-5) M in the luteal phase and was significantly higher (P < 0.05) than in the follicular phase (3.1 x 10(-6) M) and early pregnancy (2.7 x 10(-6) M). ED50 for PGE2 amounted 7.3 x 10(-7) M in the luteal phase and 1.7 x 10(-7) M in early pregnancy. Studies showed the influence of the estrous cycle and early pregnancy on OA branches sensitivity to the contractile effect of PGF2alpha and the lack of this effect on UA branches, and the influence of the estrous cycle on UA and OA branch contraction in response to PGE2.     

Effects of LH and PGF2alpha in equine dominant follicles observed by MDS

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.     

Role of oxytocin and/or PGF2 alpha on breeding efficiency in buffaloes

A study was conducted on 40 buffalo-cows, assigned randomly, immediately after calving into three groups: group I (n = 10) injected with saline and taken as control; group II (n = 15) received 25 mg PGF2 alpha/animal (Lutalyse); group III (n = 15) received 25 mg PGF2 alpha + 25 i.u. oxytocin/animal (Syntocinon), single i.m. dose. Oxytocin and/or PGF2 alpha significantly (P less than 0.01) shortened the interval from calving to first service (38.33 and 31.53 days for groups II and III respectively, versus 91.60 days for controls). The treatment reduced the service period (38.29 and 35.87 days for groups II and III respectively, versus 45.40 days for controls). Concomitantly a significant (P less than 0.01) decrease in the open-days post partum was achieved (76.62 and 67.40 days for groups II and III respectively, versus 137.00 days for controls). In addition, the treated buffaloes needed significantly (P less than 0.01) fewer services per conception (1.67 and 1.20 S/C for groups II and III respectively) than the untreated ones (2.70 S/C), besides a substantial improvement (P less than 0.01) in their conception rate either at 60 or 85 days post partum. Significantly improved (P less than 0.05) results were obtained in the oxytocin and PGF2 alpha treated animals, than in those receiving PGF2 alpha alone for all the previous parameters, except for the service period. Buffaloes therefore seemed to respond better to such treatment than dairy cows.     

Inhibitory effects of CIDR-based ovulation-synchronization protocols on uterine PGF2alpha secretion at the following luteal phase in early postpartum non-cycling beef cows

We investigated whether CIDR-based ovulation-synchronization protocols inhibit secretion of prostaglandin (PG) F2alpha from the uterus in the following luteal phase in non-cycling beef cows. Ten early (a month) postpartum non-cycling Japanese Black beef cows were treated with (1) Ovsynch (GnRH analogue on Day 0, PGF2alpha analogue on Day 7, and GnRH analogue on Day 9; n=3), (2) Ovsynch+CIDR (Ovsynch protocol plus a CIDR for 7 days from Day 0; n=4), or (3) estradiol benzoate (EB) Ovsynch+CIDR (EB on Day 0 in lieu of the first GnRH treatment followed by the Ovsynch+CIDR protocol; n=3). An oxytocin challenge was administered on Day 24 to examine uterine PGF2alpha secretion. Plasma concentrations of 13,14-dihydro-15-keto- PGF2alpha were lower at 30-120 min after oxytocin administration in the Ovsynch+CIDR group and 75 min after administration in the EB Ovsynch+CIDR group than in the Ovsynch group (P<0.05). Plasma progesterone concentrations were higher from Days 1 to 7 in the Ovsynch+CIDR group and from Days 1 to 5 in the EB Ovsynch+CIDR group than in the Ovsynch group (P<0.05). The progesterone concentrations were higher on Days 27 and 29 in both CIDR-treated groups than in the Ovsynch group (P<0.05). In conclusion, in non-cycling beef cows, CIDR-based ovulation-synchronization protocols inhibit uterine PGF2alpha secretion in the following luteal phase and prevent premature luteolysis as is seen with the Ovsynch protocol.     

Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.     

Lectin binding patterns of uterine glands in mares with chronic endometrial degeneration

OBJECTIVE: To evaluate changes of glycoconjugate in uterine glands of endometrial tissues obtained from mares. ANIMALS: adult mares. PROCEDURE: Uterine biopsy samples were collected during the breeding season and analyzed histologically for signs of chronic endometrial degeneration. Stage of the estrous cycle was established, using clinical examination and determination of hormonal status. Uterine tissue samples were analyzed, using lectin histochemical and immunohistochemical techniques (estrogen and progesterone receptors). Connective tissues were stained to determine alterations of ground substance in periglandular fibrosis. RESULTS: Of 50 mares, 30 (60%) were classified as normal or having modest alterations, and 20 (40%) were classified as having moderate or severe endometrial degeneration. In normal equine endometrium, several lectins (Helix pomatia agglutinin, Lotus tetragonolobus agglutinin, Ricinus communis I agglutinin, Ulex europaeus agglutinin, and wheat germ agglutinin) bound to glycoconjugates of the luminal epithelium and openings of uterine glands. Lectin binding patterns of cystic dilated glands or fibrotic glands in endometrial samples were remarkably strong, whereas normal surrounding cells remained unstained. Lotus tetragonolobus lectin was not suitable for detecting endometrial alterations. Connective tissues stained with Alcian blue and results of Hale colloidal-iron binding revealed acidic ground substance in periglandular fibrosis. Estrogen and progesterone receptors were evenly distributed in healthy and affected endometrial samples. CONCLUSIONS AND CLINICAL RELEVANCE: Glycoconjugate patterns of uterine glands were altered in mares with chronic endometrial degeneration. Therefore, uterine secretions are likely to be altered. These changes are not induced by changes in content of estrogen and progesterone receptors in endometrial tissues.