Uterokinetic activity of fenprostalene (a prostaglandin F2alpha analog) in vivo and in vitro in the bovine

The luteolytic potency of fenprostalene (a PGF2alpha analog) is about 20-times that of naturally produced PGF2alpha. The objective of this research was to investigate the uterokinetic effects of fenprostalene at a luteolytic dosage (1.0 mg) in the cyclic and early postpartum cow, and in the isolated uterine horn. Uterine motility measurements were conducted on two consecutive days in each cow. Experimental protocol on Day 1 was: spontaneous motility was recorded for 1 h; fenprostalene was injected (1.0 mg i.m.), after which motility was recorded for 2 h; fenprostalene was injected (1.0 mg i.v.) and motility was recorded for 30 min; and oxytocin was injected (40 U i.v.), followed by a 30-min recording period. On Day 2, the treatment sequence was reversed: spontaneous motility was recorded for 1 h; oxytocin was injected (100 U i.m.), after which motility was recorded for 2 h; fenprostalene was injected (1.0 mg i.v.) and motility recorded for 30 min; and oxytocin was injected (40 U i.v.), followed by a 30-min recording period. In the in vitro experiment, different dosages of fenprostalene (5.9, 11.8, 17.6, and 29.4 ng/ml bath solutions) and oxytocin (0.06, 0.12, 0.18, and 0.60 mU/ml bath solutions) were tested in pairs for 1 h. The treatment was then repeated. In a different group, fenprostalene (5.9 ng/ml bath solution) and oxytocin (0.06 mU/ml bath solution) treatments were alternated. Fenprostalene (at luteolytic dosage) was not uterokinetic in either the cyclic or postpartum cow. However, fenprostalene and oxytocin had a significant uterokinetic effect (five- to six-fold pretreatment value) on the isolated uterine horn preparation at all dosages studied. Peak motility occurred between 10 to 15 min, followed by a gradual decrease to 40% at 60 min. When the treatments were repeated at 60 min, oxytocin but not fenprostalene caused a minute, transient contraction. However, fenprostalene-desensitized (by exposure to fenprostalene) uteri reacted significantly to oxytocin, and vice versa.     

Endocrine secretion of prostaglandin F2alpha in cyclic gilts is decreased by intrauterine administration of exogenous oxytocin

When administered systemically, oxytocin (OT) stimulates secretion of uterine prostaglandin F2alpha (PGF2alpha) in swine, but the role of endometrially-derived OT in control of PGF2alpha release is not clear. This study determined the effect of exogenous OT, administered into the uterine lumen of intact cyclic gilts, on PGF2alpha secretion during late diestrus. Intrauterine infusion of 40USP units OT (in 30 ml 0.9% saline) was performed for 30 min (1 ml/min) into each uterine horn between 7:00 and 9:00 h on days 10, 12, 14 and 16 after estrus. Beginning 20 min before infusion, samples of jugular venous blood were drawn at 5-10-min intervals for 140 min for quantification of 13,14-dihydro-15-keto-PGF2alpha (PGFM), the major stable metabolite of PGF2alpha. Progesterone was analyzed in samples collected 0, 60 and 120 min after initiation of OT infusion. Treatment with OT did not alter plasma concentrations of PGFM on days 10 or 12 but decreased (P<0.001) PGFM concentrations for 40 min after onset of infusion on day 16. Concentrations of PGFM also were reduced in the pre-treatment samples on day 14 (P=0.05) and day 16 (P<0.001) in OT-infused gilts. Plasma progesterone declined (P<0.01) between days 10 and 16 in control-infused gilts but did not decline until after day 14 (P<0.001) in gilts infused with OT. These results indicate that when OT is administered into the uterine lumen of pigs during late diestrus, it has an anti-luteolytic effect to reduce endocrine secretion of PGF2alpha and delay the decline in progesterone that occurs during luteolysis.     

Release of oxytocin and prostaglandin f(2alpha) around teasing, natural service and associated events in the mare

Mating has been shown in many species to provoke the release of oxytocin (OT). In our study, various stimuli were applied to mares to study release of OT and prostaglandin F(2alpha) (PGF(2alpha)) associated with mating. Blood samples were collected from mares around the time of teasing both in oestrus and dioestrus and at mating. For comparison, blood samples were also collected at the time of manual manipulation of the genital tract and after intrauterine infusion of 500 ml phosphate buffered saline (PBS). Additional samples were collected 16 to 18 h after mating. Mating caused a significant increase in OT in all mares and teasing caused a significant OT response in 6 of 10 oestrous and 3 of 5 dioestrous mares. However, mating and teasing had no significant effect on concentrations of 15-keto-13,14-dihydro-PGF(2alpha) (PGFM). Manual manipulation of the clitoris, vagina and cervix caused significant OT release in all mares and intrauterine infusion of 500 ml PBS caused significant OT release in three of the five mares. However, only one mare had a significant PGF(2alpha) response during manual manipulation and only one responded positively to intrauterine infusion of 500 ml PBS. We concluded that events around mating, including stimulation of the genital tract and uterine distension, often caused an increase in circulating concentrations of OT but only rarely in PGFM.     

Effect of xylazine on interovulatory interval and serum progesterone concentrations in the horse mare

The objective of this study was to determine the effect of the alpha(2)-adrenergic agonist, xylazine, on interovulatory interval and progesterone concentrations in the horse mare. Mares were assigned to one of four treatments: Group 1 (controls) received an intramuscular injection (i.m.) of 5 cc saline (n=6), Group 2 received 10 mg prostaglandin F(2alpha) (PGF(2alpha)) i.m. (n=5), Group 3 received 500 mg xylazine i.m. (n=6) and group 4 received an intravenous injection (i.v) of 350 mg xylazine (n=6). Treatment was administered on Day 10 of the estrous cycle (Day 0 = Day of detected ovulation). There was no difference in length of interovulatory interval between PGF(2alpha)-treated mares and control mares (mean +/- SEM; 18.8 +/- 1.0 versus 21.7 +/- 1.6 d). When compared with either xylazine-treated group, PGF(2alpha)-treated mares had a shorter interovulatory interval (18.3 +/- 1.0 d versus 22.2 +/- 0.6 and 22.8 +/- 1.3 d, respectively; P < 0.05). There was no difference in the length of interovulatory interval between control mares and either xylazine-treated group. At the time of treatment all mares had progesterone concentrations > 10 ng/ml, therefore the onset of luteolysis was defined as the day of the estrous cycle when progesterone concentrations decreased below 10 ng/ml. In PGF(2alpha)-treated mares, this event occurred earlier than in any other group (Day 11.2 +/- 0.2 of the estrous cycle versus 16.0 +/- 1.3 for control, Day 15.7 +/- 0.2 for Group 3 and Day 15.2 +/- 0.6 for Group 4; P < 0.002). It was concluded that a single treatment with xylazine, either by an intramuscular or intravenous route, had no significant effect on interovulatory interval or progesterone concentrations in horse mares.     

Dynamics of prostaglandin secretion, intrauterine fluid and uterine clearance in reproductively normal mares and mares with delayed uterine clearance

Two experiments were performed to investigate relationships between oxytocin, prostaglandin release, uterine emptying and fluid accumulation in the uterus. In Experiment 1, the effect of oxytocin on the pattern of prostaglandin release during uterine clearance of radiocolloid was measured in 5 normal mares and 5 mares with delayed uterine clearance. Uterine clearance was measured during estrus by scintigraphy at 0, 60 and 120 min after colloid infusion. After the 120-min reading, 20 IU, i.v., oxytocin were given, and the amount of colloid cleared was measured at 135, 150 and 180 min. Plasma was obtained prior to and during scintigraphy at 5- and 15-min intervals to measure concentrations of 15-keto-13,14-dihydro-PGF2 alpha metabolite (PGFM) by RIA. In Experiment 2, plasma PGFM levels were compared after administration of oxytocin in 8 normal mares and 6 mares with delayed uterine clearance to determine if intrauterine fluid stimulated prostaglandin release. Mares received 2 treatments in a cross-over design. Treatment 1 consisted of 20 IU, i.v., oxytocin during estrus. Treatment 2 consisted of an infusion of 10 mL, i.u., saline 15 min prior to oxytocin administration. Treatments were performed 4 to 6 h apart. Blood was collected and PGFM was measured as in experiment 1. Data were analyzed by least squares analysis of variance. In Experiment 1, regression analysis of scintigraphy and PGFM profiles indicated that time response curves differed between groups (P < 0.01). At 120 min, normal mares retained 40.4 +/- 4.9% (mean +/- SEM) of the radiocolloid while mares with delayed clearance retained 88 +/- 5%. Fifteen minutes after oxytocin administration (135 min), all normal mares and 4 of 5 mares with delayed clearance retained only < 6% of the colloid. During the first 120 min, plasma PGFM concentrations did not differ between the 2 groups. After oxytocin was given, plasma PGFM concentrations increased in 4 of 5 mares with delayed uterine clearance (80 to 3,096 pg/mL) but not in normal mares (13 to 46 pg/mL). In Experiment 2, plasma PGFM concentrations did not rise in normal mares but rose in 3 of 6 mares with delayed clearance (135 to 483 pg/mL) independent of treatment or period. The results suggest that intrauterine clearance of radiocolloid after oxytocin administration appears to be independent of PGF2 alpha release in normal mares during estrus. The difference in prostaglandin release response after oxytocin administration between the 2 groups was unrelated to the presence of intrauterine fluid.     

The effects of prostalene and alfaprostol as uterine myotonics, and the effect on postpartum pregnancy rate in the mare following daily treatment with prostalene

The effects of oxytocin and two prostaglandin (PG) F(2)alpha analogues, prostalene and alfaprostol, on uterine pressure in the mare were measured using balloon-tipped catheters connected to pressure transducers. The PGF(2)alpha analogues caused increased uterine pressure beginning 7 to 15 min postinjection and persisting for the duration of each 60 min recording session. Forty postpartum mares of light-horse breed were used to evaluate the effects of prostalene on postpartum pregnancy rate. Eighteen mares were injected by aseptic technique subcutaneously with 1 mg prostalene twice daily, beginning on the day of foaling (Day 0) and continuing for 10 consecutive days (Day 10) or until the mare was first bred at foal heat. Twenty-two postpartum mares were injected with 1.0 ml sterile saline by the same technique as the controls. Of treated mares, 76.9% were diagnosed pregnant after breeding versus 44.4% of the control mares (P = 0.07). Of treated mares, 66.7% bred at their second postpartum estrus became pregnant versus 28.6% of control mares (P = 0.03). Prostalene, given at 1 mg twice daily for 10 d postpartum, produced an increased pregnancy rate after both foal heat and second postpartum estrus breedings in the mare.     

Estrous cycle characteristics, luteal function, secretion of oxytocin (OT) and plasma concentrations of 15-keto-13,14-dihydro-PGF2alpha (PGF2alpha-metabolite) after administration of low doses of prostaglandin F2alpha (PGF2alpha) in pony mares

In the present study, the kinetics of the prostaglandin F2alpha (PGF2alpha)-metabolite 15-keto-13,14-dihydro-PGF2alpha after a single intramuscular application of various doses of the natural PGF2alpha dinoprost at Day 7 of the cycle in the mare were investigated. Effects of low doses on estrous cycle length and life span of corpus luteum were examined, because release of PGF2alpha is still under discussion to have detrimental influence on success rates of transcervical transfer of equine embryos. Eight Shetland pony mares were each randomly assigned to each of four treatments: (a) 0.8 mg/100 kg (group T1), (b) 0.4 mg/100 kg (group T2), (c) 0.2 mg/100 kg BM dinoprost i.m. (group T3), and (d) 1 ml physiological saline i.m. (group CO). Treatments were administered as single doses on Day 7 of the estrous cycle. Administration of dinoprost caused dose-dependent rises of plasma concentrations of PGF2alpha-metabolite, although values of individual mares showed great variation within groups. Prostaglandin treatments resulted in a distinct decrease of plasma progesterone concentrations to values between 1.6 and 7.9 ng/ml within 24 h. Treatment groups had significantly lower progesterone area under the curve (AUC: T1 942.8+/-175.9, T2 1050+/-181.2 and T3 1117+/-179.8 ng/ml/h) when compared with controls (CO 1601.9+/-227.6; t-test, P<0.05 ). There was a small, but significant negative correlation between AUC of progesterone and of PGF2alpha-metabolite ( R=-0.4; P=0.05 ). Administration of PGF2alpha caused secretion of oxytocin in three (T1, T2) and two (T3) mares out of eight ranging from 19.3 to 63.1 pg/ml. The AUC of oxytocin was positively correlated with AUC of PGF2alpha-metabolite ( R=0.4, P<0.05) and negatively correlated with AUC of progesterone ( R=-0.4, P<0.05). Administration of dinoprost yielded significantly shorter intervals from treatment to estrus and ovulation (values in parentheses), respectively, when compared with controls: T1 3.9+/-0.7 days ( 12.1+/-0.7 days), T2 4.5+/-0.6 ( 12.3+/-0.6 ), T3 4.9+/-0.5 ( 12.3+/-0.6 ), and CO 8.9+/-0.6 days ( 16.5+/-0.8 days) (t-test, P<0.01 ) (Fig. 2). Different doses of PGF2alpha caused similar effects. Data suggest that progesterone concentrations at applications influence efficacy of treatments more than doses administered, as demonstrated by their high correlation with estrous cycle patterns. It is important to note that differences we achieved are gradual and that all mares responded to treatment by luteolysis and premature estrus, regardless of doses applied.     

Effect of pregnancy on oxytocin-induced release of prostaglandin F2 alpha in heifers

The effect of pregnancy on the release of prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin (OT) has been examined. Fourteen cyclic heifers received one intravenous injection of 1 IU OT (n = 6) or 100 IU OT (n = 8) 17, 18, or 19 days (Day 17-19) after the onset of estrus (Day 0). Five of these animals also received 100 IU OT at Days 6 and 13 to determine the effect of OT at different times of the cycle. Frequent blood samples were taken for 60 min before and for 90 min after OT injection for the measurement of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) by radioimmunoassay. The experiment was then repeated using the same animals at Day 17-19 of pregnancy (confirmed by the recovery of an embryo the day after OT injection). Following the injection of 1 IU OT, plasma PGFM reached its peak within 30 min with the increase significantly lower (P less than 0.05) in pregnant (1.13 +/- 0.10-fold) than in nonpregnant animals (2.07 +/- 0.27-fold). However, because only 3 of the 6 cyclic animals showed a response to 1 IU OT, the dose was increased to 100 IU in subsequent experiments. The animals that received 100 IU at Days 6 and 13 had no significant increase in PGFM concentrations (1.18 +/- 0.05-fold and 1.01 +/- 0.04-fold, respectively). At Day 17-19 the increase in plasma PGFM reached its peak 5-15 min after 100 IU OT and the increase was significantly greater in nonpregnant (3.23 +/- 0.17-fold) than in pregnant (1.21 +/- 0.02-fold; P = 0.003) heifers. Six of 11 animals injected at Day 17-19 of the cycle showed a decrease in progesterone (P4) the day after OT administration. These data show that the release of PGF2 alpha in response to OT is suppressed in pregnant animals in vivo, suggesting an antiluteolytic role for the embryo in luteostasis.     

Reproductive function of mares given daily injections of prostaglandin F2alpha beginning at day 42 of pregnancy

Mares at Day 42 of pregnancy received daily intramuscular (i.m.) injection of 5 mg of prostaglandin F2alpha (PGF(2alpha)) until the beginning of the first (Group I, n = 3) or second estrous cycle (Group II, n = 2). All mares aborted 3 to 4 d after the first injection; they displayed estrus 2 to 6 d after this injection. As determined by palpation per rectum and serum progesterone levels, each estrus was accompanied by an ovulation. Endometrial cups did not regress after PGF(2alpha) treatment since serum samples from the mares contained pregnant mare serum gonadotropin (PMSG) for at least 30 d after first injection, as determined by mare immunopregnancy test. After the first estrus, two of three mares in Group I displayed a prolonged diestrus (> 25 d). In contrast, the first estrous cycle was short (8 to 12 d) for mares in Group II. Serum progesterone levels in the first 6 d postovulation were lower (P < 0.05) for Group II than for Group I, indicating that formation of the corpus luteum was impaired by daily injections of PGF(2). Results indicate that 1) daily injections of PGF(2alpha) can induce abortion in mares at Day 42 of pregnancy, 2) abortion is followed by estrus and ovulation, 3) the endometrial cups do not regress as a result of this treatment, and 4) daily injections of PGF(2) can impair early corpus luteum development.     

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

Specific PGF-2 alpha binding by the corpus luteum of the pregnant and non-pregnant mare.

The binding of prostaglandin (PG) F-2 alpha to corpora lutea (CL) from pregnant and non-pregnant Pony mares was examined. Studies of the rates of association and dissociation indicated that [3H]PGF was bound specifically and reversibly to a luteal cell membrane preparation (MP) that was isolated by high speed (100,000 g) ultracentrifugation. Various PGs and PG metabolites displaced [3H]PGF from the receptors in the following decreasing order: PGF-2 alpha greater than 13, 14-dihydro-PGF-2 alpha = 13,14-dihydro-15-keto PGF-2 alpha greater than PGD-2 greater than PGF-1 alpha = PGE-2 greater than PGE-2 beta greater than PGE-1. These data implicate the 9 alpha-OH and 5,6 cis double bond as major contributors to PGF receptor recognition. The membrane preparation appeared to contain at least two receptor populations, a high affinity, low capacity and a low affinity, high capacity receptor. The binding of PGF (pg/mg MP protein +/- s.e.m. (n)) to CL of the non-pregnant mare increased from 4.09 +/- 11.6 (4), on Day 4 after ovulation, to reach maximal levels by Day 12, 15.01 +/- 2.5 (4), and declined thereafter. In pregnancy the binding of PGF continued to increase until Day 18, reaching 27.47 +/- 1.7 (3), before it declined on Day 20. The reduction in binding by Day 16 in the non-pregnant mare may reflect the process of luteolysis, while high PGF binding capacity of CL between Days 16 and 18 of pregnancy indicated that luteal maintenance during pregnancy is not associated with a reduction of PGF binding capabilities.     

Oxytocin release and its relationship to dihydro-15-keto PGF2alpha and arginine vasopressin release during parturition and to suckling in postpartum mares

Pituitary blood was collected from the intercavernal sinus in five mares before and during parturition, and in nine mares immediately after parturition to investigate oxytocin patterns during parturition and early lactation, and to determine the relationship between oxytocin, prostaglandin and arginine vasopressin during parturition. In four mares in which sample collection began at least 6 h before rupture of the chorioallantois, a significant increase (P < 0.05) in PGF(2alpha) concentration was detected before a significant increase in oxytocin concentration. Cross-correlation analysis of log-transformed oxytocin and PGF(2alpha) concentrations revealed a significant correlation (P < 0.05) at a 6 min lag period, indicating that in the 2 h before delivery of the foal, an increase in prostaglandin was followed 6 min later by an increase in oxytocin. A significant effect of suckling on oxytocin release by the mare was detected in only two of nine mares, when oxytocin concentrations were evaluated 0-3 min after suckling. When foals were prevented from sucking for 1 h, by being either muzzled (n = 2) or separated from the mare (n = 2), there was no significant association between resumption of suckling and oxytocin release by the mare. The results of these studies show that: (i) oxytocin secretion from the maternal posterior pituitary gland begins before, or in association with, the onset of the second stage of labour, and that prostaglandin increases in the peripheral circulation before oxytocin release; and (ii) suckling is not significantly related to oxytocin release in mares.     

Spontaneous and oxytocin-induced uterine motility in cyclic and postpartum mares

Intrauterine pressure was measured in three cyclic and two postpartum mares. Pressure was recorded using a catheter tip pressure transducer. The transducer was passed transcervically into the uterus.. In cyclic mares recordings were started on Day 1 of estrus and continued daily until ovulation as well as on Days 1 and 8 of diestrus. In postpartum mares recordings were started within 48 h after foaling and continued until the mares ovulated. The intrauterine pressure changes in postpartum mares was also recorded on Days 1 and 8 of diestrus. Spontaneous uterine contractions were recorded in cyclic mares for 30 min and in postpartum mares for 10 min. Induced uterine motilities were recorded for 30 min in both groups after the administration of oxytocin (40 USP, i.v.). Total area under the contraction curve in a 10-min period was used as a uterine motility quantitating unit. All mares demonstrated uterine contractions during estrus and diestrus. All mares demonstrated significant responses to oxytocin during estrus and diestrus. It appears that estrogen priming is not necessary for a significant uterine response to oxytocin.     

Plasma levels of cortisol and oxytocin,and uterine activity after cervical artificial insemination in the ewe

The objective was to compare in the ewe the effects of easy and difficult procedures for artificial insemination (AI) (as related to rapid or poor accessibility of the cervix, respectively) on plasma cortisol (CORT) and oxytocin (OT), and uterine motility. All AI were simulated using a catheter empty of semen to study genital and environmental stimuli only. In experiment 1, 40 ewes were sampled after Al, and whether it was an easy or difficult procedure was reported for each animal. While CORT concentrations rose to a similar amount in all ewes, whatever the Al procedure, a significant OT response occurred after a difficult procedure only (n = 18) (17.4 +/- 1.7 versus 12.7 +/- 0.7 pg x mL(-1) before Al, p < 0.05). In experiment 2, uterine activity was monitored in 4 ewes using an implantable telemetric transmitter equipped with an intrauterine pressure catheter. An increased uterine activity occurred during 2 +/- 1 min after an easy Al (n = 5), whereas the evoked activity lasted for 15 +/- 4 min after a difficult Al (p < 0.001, n = 7). A similar long-lasting response occurred after OT administration (100 mIU, i.v.). We concluded that the increase in uterine motility after a difficult Al resulted from a reflex release of OT, and not to a "stress" effect.     

French field results (1985-2005) on factors affecting fertility of frozen stallion semen

Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.     

Effect of PGF2alpha and 13,14-dihydro-15-keto-PGF2alpha (PGFM) on corpora luteal function in nonpregnant mares

The objective of this study was to determine if the primary circulating metabolite of PGF2alpha, 13,14-dihydro-15-keto-PGF2alpha (PGFM), is biologically active and would induce luteolysis in nonpregnant mares. On Day 9 after ovulation, mares (n = 7/group) were randomly assigned to receive: 1) saline control, 2) 10 mg PGF2alpha or 3) 10 mg PGFM in 5 mL 0.9% sterile saline i.m. On Days 0 through 16, blood was collected for progesterone analysis. In addition, blood was collected immediately prior to treatment, hourly for 6 h, and then at 12 and 24 h after treatment for progesterone and PGFM analysis; PGFM was measured to verify that equivalent amounts of hormone were administered to PGF2alpha- and PGFM-treated mares. Mares were considered to have undergone luteolysis if progesterone decreased to < or = 1.0 ng/mL within 24 h following treatment. Luteolysis was induced in 0/7 control, 7/7 PGF2alpha-treated, and 0/7 PGFM-treated mares. There was no difference (P>0.1) in the occurrence of luteolysis in control and PGFM-treated mares. More (P<0.001) PGF2alpha-treated mares underwent luteolysis than control or PGFM-treated mares. There was no difference (P>0.1) in progesterone concentrations between control and PGFM-treated mares on Days 10 through 16. Progesterone concentrations were lower (P<0.01) on Days 10 through 14 in PGF2alpha-treated compared with control and PGFM-treated mares. There was no difference (P>0.05) in PGFM concentrations between PGF2alpha- and PGFM-treated mares; PGFM concentrations in both groups were higher (P<0.001) than in control mares. These results do not support the hypothesis that PGFM is biologically active in the mare, since there was no difference in corpora luteal function between PGFM-treated and control mares.     

Induction of luteolysis in mares by ultrasound-guided intraluteal treatment with PGF2alpha.

To evaluate the technique of ultrasound-guided luteal injection in mares, PGF2alpha was administered under ultrasound guidance to horse mares (n = 7 to 9 per group) on Day 9 postovulation via either a systemic (i.m.; zero, 0.01, 0.1, or 5 mg/dose) route or a local intraluteal (i.l.; zero, 0.01 or 0.1 mg/dose) route. The luteolytic efficacy of each treatment was determined based on post-treatment decreases in progesterone concentration, interval to uterine edema (IE) and interovulatory interval (IOI). Local administration of PGF2alpha directly into the CL consistently induced luteolysis, at doses up to 50-fold lower than the lowest effective systemic dose. Significant decreases in IOI and IE occurred in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l., but did not occur in mares treated with 0.1 or 0.01 mg PGF2alpha i.m., 0.01 mg PGF i.l., vehicle i.l. or vehicle i.m.. Progesterone concentrations were reduced to less than 10% of pretreatment values by two days post treatment in mares treated with 5 mg PGF2alpha i.m. or 0.1 mg PGF2alpha i.l.. PGF2alpha doses of 0.1 mg i.m. and 0.01 mg i.l. were associated with smaller but significant progesterone decreases (to 66% and 46% of pre-treatment values, respectively) by two days post treatment. Progesterone values after administration of i.l. vehicle did not differ from pre-treatment values by two days post treatment, but were significantly lower (53% of pre-treatment values) by four days post treatment. Intramuscular treatment with vehicle or 0.01 mg of PGF2alpha did not significantly reduce progesterone concentrations below pretreatment values. Overall, the minimum effective luteolytic dose of PGF2alpha given intraluteally was between 0.01 and 0.1 mg. Based on the results of this study, ultrasound-guided i.l. injection appears to be a repeatable method for studying the direct effect of other chemicals on luteal function. However, the current procedure carries some risk, since three i.l. injections were associated with ovarian abscesses.     

The effect of oxytocin and PGF2alpha on the uterine involution and pregnancy rates in postpartum Arabian mares

In this study, the effects of oxytocin and an analog of prostaglandin (cloprostenol) on the uterine involution and pregnancy rates were investigated. Mares received 3 ml of 0.9% NaCl in Group C (n=10), 30 IU/mare of oxytocin in Group O (n=10) and 250 microg/mare of cloprostenol in Group P (n=10) within 12h after parturition. The gravid uterine horn's cross-sectional diameter was measured by ultrasonography. The mean uterine diameters did not differ significantly between the treatment (O and P) and the control (C) groups (p>0.05). The difference between the postpartum ovulation periods (Group C: 12.6+/-0.72 days, Group O: 15+/-1.33 days, Group P: 14.6+/-1.11 days), the pregnancy rates at foal heat (Group C: 60%, Group O: 60%, Group P: 80%) and the embryonic death rates at foal heat (Group C: 33.3%, Group O: 16%, Group P: 25%) were not found to be statistically significant between the treatment and the control groups. The mean progesterone concentrations were similar in all groups and decreased continuously from parturition to until foal heat (Group C: from 2.43+/-0.24 to 0.66 ng/ml, Group O: from 3.07+/-0.6 to 0.27+/-0.27 ng/ml and Group P: from 2.8+/-0.44 to 0 ng/ml) (p>0.05). In conclusion, it was decided that the oxytocin and PGF2alpha treatments performed on the mares with the purpose of stimulating involution had no effect on the duration of parturition-first ovulation, the shrinkage of the uterus diameter, the pregnancy and embryonic death rates.     

Aberrations in uterine contractile patterns in mares with delayed uterine clearance after administration of detomidine and oxytocin

An experiment was conducted to determine whether the uterotonic effects of oxytocin, a drug used to treat mares that have a delay in uterine clearance were affected by the sedative detomidine (an alpha2-agonist), a drug used to treat fractious mares. An additional objective was to identify propagation patterns of uterine contractions and determine whether these patterns differed between normal mares and mares with delayed uterine clearance (DUC). Intrauterine pressure was measured in five reproductively normal mares and four mares with DUC during estrus using an 8-F Milar catheter with two discrete pressure sensors. Mares received one of three treatments in random order: detomidine (0.001 mg/kg; i.v.); detomidine followed in 10 min by oxytocin (10 IU; i.v.); and saline (0.9% NaCl 0.5 ml; i.v.) followed in 10 min by oxytocin. All treatments induced waves of contractions; however, only three mares with DUC exhibited contractions after administration of detomidine. Normal mares experienced more uterine contractions (P < 0.01) that tended to last longer (P < 0.06), and were of greater intensity (P < 0.04) than mares with delayed clearance. Administration of detomidine before oxytocin increased the number of contractions (P < 0.02) and increased the maximum intrauterine pressure in the uterine horn (P < 0.05) in normal mares as compared to response after administration of saline and oxytocin. Detomidine had no effect in mares with delayed clearance. All mares had more propagating than non-propagating uterine contractions (74 +/- 8 versus 25 +/- 8%, respectively). Normal mares exhibited a normal propagation pattern more frequently (P < 0.0001) than mares with DUC. Simultaneous (P < 0.05) and inverted (P < 0.03) contractions occurred more frequently in mares with DUC. Administration of detomidine increased the number (P < 0.01), and tended to increase the percentage (P < 0.07) of normal propagating uterine contractions in normal mares, but did not affect propagation patterns in mares with DUC. In conclusion, detomidine augmented the uterotonic effect of oxytocin in normal mares but not in mares with DUC. Data suggest that mares with DUC have a defect in myoelectrical signaling and a decrease in the contractile strength of the uterine muscle.     

Use of prostaglandin E2 to ripen the cervix of the mare prior to induction of parturition

Eleven light-breed pregnant mares (335 to 347 d gestaton) were used to evaluate the use of prostaglandin E2 as a cervical ripening agent prior to induction of parturition during the months of April and May. Six hours prior to induction, each mare's cervix was examined per vagina for softness and dilation. Each mare was then assigned to 1 of 2 treatment groups: Group PGE mares (n = 7) received 2.0 to 2.5 mg prostaglandin E2 deposited intracervically; Group SAL mares (n = 4) received 0.5 mL of sterile NaCl deposited intracervically. Six hours later, the mares were readied for parturition by wrapping the tail, scrubbing and rinsing the perineum and udder, and examining the cervix as previously described. Each mare was then administered 15 U, i.v. oxytocin at 15-min intervals until the chorioallantois ruptured. Intervals from initial oxytocin injection until rupture of the chorioallantois, from initial oxytocin injection until delivery of the foal, from delivery of the foal until the foal stood unassisted, and from delivery of the foal until the foal suckled were recorded. Mean cervical dilation immediately prior to induction of parturition tended to be greater in Group PGE mares (3.9 +/- 1.7 cm) than in Group SAL mares (1.9 +/- 1.9 cm; P = 0.10). Mean change in cervical dilation over the 6-h period prior to induction (3.4 +/- 1.9 cm vs 1.5 +/- 2.1 cm), mean number of injections of oxytocin required until the chorioallantois ruptured (1.9 +/- 0.7 vs 2.5 +/- 1.0), and mean intervals from initial injection of oxytocin to rupture of the chorioallantois (20 +/- 10 min vs 28 +/- 19 min) and delivery of the foal (28 +/- 7 min vs 34 +/- 22 min) were not different between Group PGE and SAL mares, respectively (P > 0.10). The proportion of foals that stood within 1 h of birth also did not differ between Group PGE foals (6/7; 86%) and Group SAL foals (3/4; 75%; Chi-square = 0.17; P > 0.10). The proportion of foals that nursed within 2 h of birth was higher in Group PGE foals (6/7; 86%) than in Group SAL foals (1/4; 25%; Chi-square = 4.02; P < 0.05). Premature separation requiring manual rupture of the chorioallantois at the vulvar labia occurred in 1 Group PGE mare (cervical dilation of 1.5 cm at time of induction) and 1 Group SAL mare (cervix closed and firm at time of induction). Foals born from the 2 mares with premature placental separation had the longest intervals from initial oxytocin injection to delivery, delivery to ability to stand unassisted, and delivery to suckling within their respective treatment groups. In summary, it appears that cervical ripening prior to induction of parturition favors shorter deliveries and foal vigor. Intracervical administration of prostaglandin E2 may prove useful for ripening the cervix of the mare prior to induction of parturition. Further studies are indicated to determine optimal dosage and method of administration of prostaglandin E2.