Fertilization rate after synchronization of the oestrous cycle in heifers with prostaglandin F2alpha.

Oestrus was synchronized in 31 heifers by the intrauterine administration of PGF₂α than salt. Nineteen were given 2 doses of 0.5mg 24 hr apart, and 10 of these received 1500 I.U. of PMSG i.m. 24 hr before the treatment with PGF₂α. The remaining 12 heifers in the experiment were given a single dose of 2mg followed at the beginning of oestrus by 1500 I.U. of HCG i.m. Of 9 heifers which received only the two doses of 0.5mg (Group 1), 7 were observed to have corpora lutea when slaughtered 56–72 hr after the onset of oestrus, and four fertilized eggs were recovered. In those which received PMSG before the double injection of PGF₂α(Group 2), 118 corpora lutea were observed at slaughter and 34 fertilized eggs were recovered. Each heifer which received a single injection of PGF₂α and HCG had a corpus luteum, and 9 fertilized eggs were recovered. Unovulated follicles were most commonly observed in the PMSG-treated heifers but they were also observed in the heifers given the double injection treatment. It was observed that in the two-injection treatments, whether or not given PMSG, time of ovulation relative to the onset of oestrus was variable, and eggs were found in the uterus before the expected time.     

Differential suppression of endometrial prostaglandin F2alpha by the equine conceptus

Prostaglandin F2alpha secretion by the uterine endometrium between Days 13 and 14 postovulation causes luteal regression in mares. A mechanism involving interruption or suppression of this secretion causes pregnancy to be maintained. The present study was designed to determine the age of the conceptus when maximal suppression of PGF2alpha secretion occurs. Mares were examined daily during estrus with ultrasonography (day 0 = day of ovulation). Conceptus tissues were recovered nonsurgically on Days 9 (n = 7), 12 (n = 5), 13 (n = 5), and 16 (n = 7) and uterine biopsies on Day 14. Both uterine and conceptus tissues were washed in phosphate-buffered saline (PBS) with 100 units penicillin G/ml + 100 microg streptomycin/ml, pH 7.4. Endometrial tissue (approximately 200 mg) plus conceptus tissues were incubated in 15 ml of tissue culture medium 199 (M199) + 10% fetal calf serum and 10 units penicillin G/ml and 10 microg streptomycin/ml at 37 degrees C under 5% CO(2): 5% O(2) : 90% N(2). Samples were taken at 4, 8, and 24 h. Two plates that contained only endometrial tissue and two additional plates with 25 mg flunixin meglumine added along with endometrial tissue were also included in the incubations. Concentrations of PGF2alpha were measured in all samples using radioimmunoassay. There was a trend toward suppression of PGF2alpha secretion by conceptus tissues, regardless of age. However, Day 12 concepti significantly suppressed PGF2alpha secretion compared with that of endometrial tissue incubated alone (P = 0.03).     

synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig

The relationship between progesterone (P₄) synthesis by luteal tissue and prostaglandin F (PGF) synthesis by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P₄ and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 0₂:5% CO₂ for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10⁻⁴M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P₄ and PGF by radioimmunoassay. P₄ and PGF release into the medium and total accumulation of P₄ and PGF in the flasks indicated that synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P₄ per flask (53.9 25.0 ng/mg tissue, P<.001) and released more P₄ into the medium (20.8 8.8 ng/mg, P<.001). P₄ synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P₄ synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.     

Effect of pregnancy-specific protein B on prostaglandin F2 alpha and prostaglandin E2 release by day 16-perifused bovine endometrial tissue

Five normal estrous cycling multiparous non-lactating Brahman cows were utilized to determine if pregnancy-specific protein B (PSPB) would alter prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE) synthesis/release by endometrial tissue. The uterine horn ipsilateral to the corpus luteum was excised on Day 16 of the estrous cycle. Endometrial tissue (200 mg wet wt) was cultured in Nutrient Mixture F-10 medium in a perifusion system. The tissue and medium were aerated with 95% O2: 5% CO2 and temperature was maintained at 39 degrees C. The medium flow rate was 100 microliters/min and fractions were collected at 20 min intervals. After a 120 min settling period, tissue culture continued with: 1) control (medium only); 2) 2 micrograms [Asu1,6]-oxytocin/ml medium for 1 h; 3) 4 or 8 micrograms PSPB/ml medium for 2 h; or 4) 4 or 8 micrograms PSPB/ml medium for 2 h plus 2 micrograms oxytocin/ml medium during the second h. Differences in PGF and PGE secretion rate were not found between 4 and 8 micrograms PSPB. Therefore, groups were combined and data were analyzed according to tissue not receiving PSPB (control); receiving PSPB and receiving PSPB plus oxytocin. A nonsignificant rise (p greater than 0.10) in PGF secretion was observed in response to PSPB and PSPB plus oxytocin above the control by the end of the perifusion period (263.7 +/- 41.7, 220.0 +/- 41.7 and 166.1 +/- 41.7 pg/(100 mg tissue/min), respectively). Treatment with PSPB alone elevated (p less than 0.05) PGE secretion rate above control by 100 and 160 min post-removal of PSPB treatment. Treatment with PSPB plus oxytocin elevated (p less than 0.05) PGE release above control by 20 min after starting oxytocin treatment and continued throughout the duration of the perifusion. Pregnancy-specific protein B plus oxytocin-induced PGE release was greater (p less than 0.05) than PSPB alone after initiating the oxytocin treatment until 20 min after removal of the treatments. However, no further differences between PSPB alone and PSPB plus oxytocin treatments were detected throughout the remainder of the perifusion period. It appears that PSPB tends to elevate PGF release and significantly elevates PGE release from Day 16 endometrial tissue.     

Induction of estrus in cattle by intraovarian injection of prostaglandin F2alpha

An effective, reduced dosage (1 10 to 1 20 the systemic dose) method for administering prostaglandin F(2alpha) in heifers to induce estrus is presented in this study. The PGF(2alpha) was injected intraovarially in five heifers at a dose of 2 mg and in another five heifers at a dose of 1 mg. Five additional heifers were injected intraovarially with 0.5 ml of distilled water and served as the controls. Regression of the corpus luteum (CL) occurred in all PGF(2alpha)-treated heifers resulting in marked decline of the peripheral levels of progesterone 24 h after treatment. Estrus was expressed 1 to 3 d later. Regression of the CL, estrus, and decline in the peripheral levels of progesterone were not observed in the control heifers. Conception rates in the heifers given either 2 mg and 1 mg PGF(2alpha) were 60 and 100%, respectively. Seven calves were born at the end of the normal gestation period while one calf was aborted.     

Effect of prostaglandin F2 alpha at different stages of guinea-pig pregnancy

It is believed that suppression of the processes by which prostaglandin F2 alpha is released from the uterus during the estrous cycle is vital to maintenance of pregnancies in guinea-pigs. Prostaglandin F2 alpha was injected into pregnant guinea-pig at four different stages of gestation to investigate the effect increased prostaglandin might have. The study revealed an alteration in the sensitivity of the pregnancy to prostaglandin F2 alpha as pregnancy progressed. Recovery from the prostaglandin insult was more likely if the injection was given after Day 24 than before Day 18. In some animals the serum progesterone levels fell following the injection and then subsequently recovered. It appears that effects which are potentially hazardous to the pregnancy are countered in a variety of ways.     

A two injection schedule with prostaglandin F2alpha for the regulation of the ovulatory cycle of cattle

Thirty randomly cycling Holstein heifers were given two injections of prostaglandin F_2α 10 days apart, to determine whether this treatment would cause most of the animals to show estrus shortly after the second injection. Twenty-five of the 30 heifers (83%) were in estrus and inseminated 2 to 4 days following the second injection and 10 of the 25 (40%) were pregnant at 60 days post insemination. Thirteen of 15 control heifers (87%) were detected in estrus over a 3 week period and seven of the 13 (54%) were pregnant at 60 days. Chi square analysis indicated that the proportion of animals showing estrus and subsequent pregnancy in the treated and in the control groups were not significantly different. The two injection system may have commercial application for the regulation of the ovulatory cycle of cattle.     

Role of phospholipase C in mediating oxytocin-induced release of prostaglandin F2 alpha from ovine endometrial tissue

Two experiments were conducted to determine if the ability of oxytocin to stimulate release of prostaglandin (PG)F2 alpha from ovine uterine tissue involved activation of phospholipase C (PLC). In the first experiment, 9 ewes were injected with progesterone for 11 d (12 mg/d, im). On days 11 and 12, ewes received an injection of estradiol (100 micrograms, im). Caruncular endometrial tissue was collected on d 13 and incubated in the presence or absence of oxytocin (10(-6) M). Concentrations of PGF2 alpha and its metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), in culture media were determined by radioimmunoassay. PLC activity was determined by measuring the intracellular accumulation of 3H-inositol phosphates after preincubation with 3H-inositol. Concentrations of PGF2 alpha and total PGF (PGF2 alpha + PGFM) in culture media were greater for explants treated with oxytocin than for controls (p. less than .02, p less than .06, respectively). A similar effect of oxytocin on intracellular concentrations of 3H-inositol phosphates was observed (p less than .01). A second experiment was conducted to determine if agonists of second messengers, produced by activation of PLC, could stimulate release of PGF2 alpha from ovine endometrial tissue. Seven ewes were treated with progesterone and estradiol as in experiment 1. Explants of caruncular tissue from each ewe were incubated with 1) control medium, 2) A23187 (10(-5) M), 3) oxytocin (10(-6) M), 4) phorbol 12-myristate 13-acetate (PMA, 10(-7) M), 5) PMA + A23187 and 6) PMA + oxytocin. Significant stimulatory effects of oxytocin, PMA and A23187 on concentrations of PGF2 alpha and total PGF in culture media were observed (p. less than .05, p less than .1, p less than .1, respectively). In conclusion, oxytocin stimulated release of PGF2 alpha and activity of PLC in explants of ovine endometrial tissue in vitro. Second messengers associated with activation of PLC enhanced release of PGF2 alpha from ovine endometrial tissue.     

Activity of phospholipase C and release of prostaglandin F2 alpha by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol

Progesterone and estradiol interact to regulate secretion of prostaglandin (PG) F2 alpha from the ovine endometrium in response to oxytocin. Two experiments were conducted to determine if these effects were due to changes in activity of phospholipase C or in the second messenger responsive pathways that regulate production of PGF2 alpha. In both experiments, ovariectomized ewes were assigned to one of four treatment groups (control, estradiol, progesterone, progesterone and estradiol). Steroids were administered, in vivo, to mimic the changes that occur during the estrous cycle. On Day 16 of steroid treatment, endometrial tissue was collected and incubated, in vitro, to measure activity of phospholipase C and release of PGF2 alpha. Treatment with progesterone, in vivo, enhanced basal and oxytocin-induced activity of phospholipase C and release of PGF2 alpha, in vitro. Estradiol suppressed oxytocin-induced activity of phospholipase C, both in the presence and absence of progesterone. In contrast to its effects on phospholipase C, estradiol inhibited basal and oxytocin-induced release of PGF2 alpha when administered alone, but not when administered with progesterone. Steroids had similar effects on the release of PGF2 alpha induced by phorbol 12-myristate 13-acetate and A23187. It was concluded that progesterone and estradiol regulate endometrial release of PGF2 alpha by affecting both the activity of phospholipase C and its associated second messenger responsive pathways that may regulate production of PGF2 alpha.     

Oxytocin stimulates prostaglandin F2alpha secretion and prostaglandin F synthase protein expression in porcine myometrial tissue

The possibility of PGF(2)alpha production and presence of prostaglandin F synthase (PGFS; PGD(2) 11-ketoreductase) was studied in control and oxytocin (OT)-stimulated myometrial slices isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows. Oxytocin (10(-7) M) stimulated (p<0.01) PGF(2)alpha production in both cycling and early pregnant myometrial slices. Prostaglandin F(2)alpha release was higher (p<0.01) in control as well as OT-treated myometrium of early pregnant sows in comparison to cycling myometrium. Prostaglandin F synthase expression at protein level was evident in myometrial slices of cyclic as well as early pregnant sows. The signals of PGFS was stronger (p<0.05) in cycling myometrium exposed to OT compared to that of control. There were no significant differences (p>0.05) in PGFS protein expression between control and OT-stimulated myometrial tissue of early-pregnant sows. The results of this study indicate the local PGF(2)alpha synthesis and the presence of PGFS in porcine cycling and early pregnant myometrial tissue. In addition, OT increased PGD(2) 11-ketoreductase protein expression in myometrium harvested during the porcine estrous cycle. However, the OT-stimulated PGF(2)alpha myometrial secretion was observed in both, cycling and pregnant gilts.     

Immunohistochemical assessment of prostaglandin H-synthase in bovine endometrial biopsy samples collected throughout the oestrous cycle

The study was designed to determine the distribution of prostaglandin H-synthase (PGS) also known as cyclooxygenase in specific uterine cell populations during the oestrous cycle. Endometrial biopsy samples were obtained from a total of 10 clinically healthy cows at days 1 (initiation of behavioural oestrus), 8, 15, and 19 of the oestrous cycle. All animals conceived after biopsy regimen. Data of semiquantitatively scored immunoreactivities were analysed using analyses of variance, t-tests for paired data and correlation analyses. Biotin-streptavidin-peroxidase immunostaining technique was employed to localise PGS. Specific staining was consistently present in endothelial cells of arteries but not capillaries and venules. A gradient of staining intensity was clearly apparent within the endometrium: surface epithelial cells and stromal cells located near the endometrial surface are consistently stained more intensely than glandular epithelial cells and stromal cells lying deeper in the endometrium. Days of oestrous cycle also influenced PGS immunoreactivities. Generally, higher immunoreactivities were recorded in surface epithelium, uterine glands and endometrial stromal cells at cycle days 1 and 19 as compared to cycle days 8 and 15. Minimal scoring values were mainly found at cycle day 8. The results of the present study suggest that the amount of bovine endometrial PGS varies considerably with the day of cycle in the above mentioned cell-type- and location-restricted manner. Therefore, the capacity of the bovine uterine mucosa for prostaglandin production may—amongst other factors—depend on the cycle-restricted availability of the respective enzyme systems.     

Luteal function in sows after unilateral infusion of PGF-2alpha into the anterior uterine vein on different days of the oestrous cycle.

Prostaglandin F-2alpha (1.5 mg over 10 h) was infused into the anterior uterine vein of pigs on Days 6, 8, 10, 12, 14 and 15 of the oestrous cycle. At each stage of the cycle PGF-2alpha suppressed luteal function although the fall in progesterone secretion was much greater and statistically significant when the infusion was performed on Days 12, 14 and 15 of the cycle than on Days 6, 8 and 10. The concentrations of cAMP was depressed on Days 15 and 17 and fatty degeneration of luteal cells on Days 6--8 or 14 was more pronounced in the ovary ipsilateral to the PGF-2alpha infusion than in the contralateral ovary. The results are compatible with the local perfusion of PGF-2alpha from the anterior uterine vein to the ipsilateral ovary, but a systemic effect was also apparent.     

Ultrasound-monitored ovarian responses in normal and superovulated cattle given exogenous progesterone at different stages of the oestrous cycle

In two experiments with female cattle, responses to synchronisation and superovulation were monitored by transrectal ultrasonography and embryo recovery. Each experiment had both a synchronisation phase to establish a reference oestrus and a superovulatory phase with the oestrous cycle controlled by exogenous progesterone commencing at two specific times. The reference oestrus was controlled using a progesterone releasing intravaginal device (PRID) applied for 12 days with prostaglandin F_2α given 1 day before removal. Experiment 1 had two treatments which differed by the absence (A) or presence (P) of a 10mg oestradiol benzoate capsule on the PRID, while in Experiment 2 all animals were on treatment P. In the superovulatory phase of both experiments treatment P commenced on Day 7 (PRID 7 treatment) or Day 14 (PRID 14 treatment) of the oestrous cycle (oestrus designated Day 0). Superovulation, using equine chorionic gonadotrophin in Experiment 1 and oFSH in Experiment 2, commenced 3 days before PRID removal. Treatment P caused rapid regression of the dominant follicle and corpus luteum (CL) irrespective of when treatment commenced. A second wave of follicular growth was detected after 6–8 days and the dominant follicle grew at 1.1 mm day⁻¹ in the 7 days before oestrus. In contrast, in treatment A of Experiment 1, the dominant follicle either grew slowly and eventually ovulated for cows in the mid-luteal phase, or the dominant follicle regressed and a second wave follicle ovulated if cows were early luteal at PRID insertion. In the superovulatory phase of both experiments the dominant follicle of PRID 7 animals increased in size and then regressed, but in PRID 14 cows, the dominant follicle was regressing before PRID insertion. During superovulation, the number of 7–10 mm follicles was significantly (P<0.001) greater in PRID 7 animals in Experiment 2. In both experiments, half the animals on the PRID 14 treatment maintained a large follicle during the superovulatory phase in contrast to the even sized follicles in animals on PRID 7 treatment. In Experiment 1, the number of grade 1 embryos recovered was significantly (P<0.05) higher for PRID 7 than PRID 14 treatments. In Experiment 2, there were significant differences (P<0.001) in the number of corpora lutea, total ova plus embryos and grade 1 embryos in favour of PRID 7 animals following superovulation. We conclude that the initiation of control of the oestrous cycle with a PRID and subsequent superovulating regime should take account of normal follicular wave status for effective superstimulation and production of viable embryos, and that ultrasonography may usefully be applied to the process.     

Control of luteal relaxin release by prostaglandin F2 alpha: differences in the sow cycle and pregnancy

The effect of an in vivo prostaglandin F2 alpha (PGF2 alpha) challenge in pregnant and cyclic sows was compared to determine whether PGF2 alpha-induced release of relaxin (RLX) from the corpus luteum (CL) in late pregnancy is also effective during the cycle. Ovarian venous RLX and progesterone were monitored by radioimmunoassay and RLX localized in the CL by immunohistochemistry. In Day 108 pregnant sows, infusion of PGF2 alpha (100 micrograms) into the ovarian artery resulted in an immediate and sustained rise in ovarian venous RLX with an initial decline in progesterone levels by 30 min which then returned to pretreatment levels. In Day 13 or 15 cyclic sows with functional corpora lutea (i.e., elevated progesterone), RLX was undetectable in ovarian venous blood after 100 micrograms of PGF2 alpha. Administration of PGF2 alpha via either the jugular vein or intramuscular route was also ineffective in releasing RLX from the CL of the cycle. The intensity of RLX immunostaining of the CL was similar in saline and PGF2 alpha-treated sows. These studies indicate that the control of RLX release from the sow CL differs in the estrous cycle and pregnancy.