Changes in prostaglandin secretion by the regressing bovine corpus luteum

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.     

Prostaglandin f(2alpha) induces distinct physiological responses in porcine corpora lutea after acquisition of luteolytic capacity

This study examines differences in intracellular responses to cloprostenol, a prostaglandin (PG)F(2alpha) analog, in porcine corpora lutea (CL) before (Day 9 of estrous cycle) and after (Day 17 of pseudopregnancy) acquisition of luteolytic capacity. Pigs on Day 9 or Day 17 were treated with saline or 500 microgram cloprostenol, and CL were collected 10 h (experiment I) or 0.5 h (experiment III) after treatment. Some CL were cut into small pieces and cultured to measure progesterone and PGF(2alpha) secretion. In experiment I, progesterone remained high and PGF(2alpha) low in luteal incubations from either Day 9 or Day 17 saline-treated pigs. Cloprostenol increased PGF(2alpha) production 465% and decreased progesterone production 87% only from Day 17 luteal tissue. Cloprostenol induced prostaglandin G/H synthase (PGHS)-2 mRNA (0.5 h) and protein (10 h) in both groups. In cell culture, cloprostenol or phorbol 12, 13-didecanoate (PDD) (protein kinase C activator), induced PGHS-2 mRNA in luteal cells from both groups. However, acute cloprostenol treatment (10 min) decreased progesterone production and increased PGF(2alpha) production only from Day 17 luteal cells. Thus, PGF(2alpha) production is induced by cloprostenol in porcine CL with luteolytic capacity (Day 17) but not in CL without luteolytic capacity (Day 9). However, this change in PGF(2alpha) production is not explained by a difference in induction of PGHS-2 mRNA or protein.     

Effect of prostaglandin F2alpha dosage and stage of estrous cycle on the estrous response and corpus luteum function in beef heifers

Ninety-five normal cyclic crossbred beef heifers were used to determine if the proportions of heifers showing estrus, intervals to estrus and corpus luteum (CL) function were influenced by PGF(2alpha) dosage and (or) the stage of luteal phase when PGF(2alpha) was administered. Heifers were assigned randomly to treatments in a 4 x 3 factorial arrangement. Treatments were 5, 10, 25 or 30 mg PGF(2alpha) injected either in early (5 to 9 d), mid (10 to 14 d) or late (15 to 19 d) stages of the luteal phase. Jugular samples were taken at 0 h and at 8 h-intervals for 48 h and again at 60 h after PGF(2alpha) treatment for progesterone assay. Heifers were observed for estrus continuously for 120 h PGF(2alpha) treatment. The proportion of heifers showing estrus was dependent upon (P<0.05) both dosage of PGF(2alpha) and stage of luteal phase. Heifers given 5 mg of PGF(2alpha) showed estrus only if treated during the late stage, while those given 10 mg of PGF(2alpha) showed a progressive increase of heifers in estrus as stage of luteal phase advanced. The proportion of heifers showing estrus after 25 and 30 mg of PGF(2alpha) increased from 56% for the early stage to 100% for the mid and late stages. Interval to estrus in heifers showing estrus within 120 h after PGF(2alpha) treatment did not differ (P>0.05) among dosages but tended (P=0.10) to be longer in heifers treated during the mid luteal stage (67 h) than in heifers treated in the two other stages (56 h). A greater proportion of heifers (P<0.05) showed estrus by 60 h after PGF(2alpha) when treated during the early and late luteal stages (75.5%) than for heifers treated during the mid luteal stage (30.4%). Patterns of progesterone concentrations were influenced (P=0.08) by the three way interaction of dosage, stage and time. In heifers that showed estrus, rate of decline in progesterone tended (P=0.07) to be slower during the mid luteal stage than during the early and late stages. Progesterone did not drop below 1 ng/ml until 32 h in heifers treated during the mid luteal stage; whereas progesterone dropped below 1 ng/ml by 24 h in heifers treated during the early and late stages. These data may be useful in designing more efficient systems for using PGF(2alpha) or its analogues in estrus synchronization of beef cattle.     

Luteinizing hormone receptors and progesterone content in porcine corpora lutea after prostaglandin F2 alpha

The effect of prostaglandin F2 alpha (PGF2 alpha) on luteinizing hormone (LH) receptors, weight and progesterone content of corpora lutea (CL), and serum progesterone concentrations was studied in gilts. Fifteen gilts were hysterectomized between Days 9 to 11 of the estrous cycle. Twelve gilts were injected i.m. with 10 mg of PGF2 alpha and 3 with saline on Day 20. Ovaries were surgically removed from each of 3 gilts at 4, 8, 12 and 24 h following PGF2 alpha treatment and from the 3 control gilts 12 h following saline injection. Jugular blood samples for progesterone analysis were collected from all gilts at 0, 2 and 4 h following treatment and at 8, 12 and 24 h for gilts from which ovaries were removed at 8, 12 and 24 h, respectively. Mean serum progesterone and CL progesterone concentrations decreased within 4 h after PGF2 alpha treatment (P less than 0.05) and remained low through 24 h after treatment. The number of unoccupied LH receptors decreased by 4 h (P less than 0.05) and this trend continued through 24 h. There were no differences in luteal weight or affinity of unoccupied LH receptors of luteal tissue at 4, 8 12 and 24 h after PGF2 alpha when compared to luteal tissue from controls. These data indicate that during PGF2 alpha-induced luteolysis in the pig, luteal progesterone, serum progesterone concentrations and the number of LH receptors decrease simultaneously.     

Positive association, in local release, of luteal oxytocin with endothelin 1 and prostaglandin F2alpha during spontaneous luteolysis in the cow: a possible intermediatory role for luteolytic cascade within the corpus luteum

Luteolysis is caused by a pulsatile release of prostaglandin F(2alpha) (PGF(2alpha)) from the uterus in ruminants, and a positive feedback between endometrial PGF(2alpha) and luteal oxytocin (OXT) has a physiologic role in the promotion of luteolysis. The bovine corpus luteum (CL) produces vasoactive substances, such as endothelin 1 (EDN1) and angiotensin II (Ang II), that mediate and progress luteolysis. We hypothesized that luteal OXT has an additive function to ensure the CL regression with EDN1 and Ang II, and that it has an active role in the luteolytic cascade in the cow. Thus, the aim of the present study was to observe real-time changes in the local secretion of luteal OXT and to determine its relationship with other local mediators of luteolysis. Microdialysis system (MDS) capillary membranes were implanted surgically into each CL of six cyclic Holstein cows (18 lines total among the six cows) on Day 15 (estrus == Day 0) of the estrous cycle. Simultaneously, catheters were implanted to collect ovarian venous plasma ipsilateral to the CL. Although the basal secretion of OXT by luteal tissue was maintained during the experimental period, the intraluteal PGF(2alpha) secretion gradually increased up to 300% from 24 h after the onset of luteolysis (0 h; time in which progesterone started to decrease). In each MDS line (microenvironment) within the CL, the local releasing profiles of OXT were positively associated with PGF(2alpha) and EDN1 within the CL in all 18 MDS lines implanted in the six CLs (OXT vs. PGF(2alpha), 50.0%; OXT vs. EDN1, 72.2%; P < 0.05). On the other hand, the intraluteal OXT was weakly related to Ang II (OXT vs. Ang II, 27.7%). In the ovarian vein, the peak concentration of PGF(2alpha) increased significantly when the peak of PGF(2alpha) coincided with the peak of OXT after the onset of spontaneous luteolysis (P < 0.05). In conclusion, intraluteal OXT may locally modulate secretion of vasoactive substances, particularly EDN1 and PGF(2alpha) within the CL, and thus might be one of the luteal mediators of spontaneous luteolysis in the cow.     

Effect of local interaction of reactive oxygen species with prostaglandin F(2alpha) on the release of progesterone in ovine corpora lutea in vivo

Prostaglandin (PG) F(2alpha) is implicated in the process of luteal regression in many species, and has been shown to increase the generation of reactive oxygen species. In this study, the role of reactive oxygen species in the local regulatory mechanisms of functional luteolysis in the ewe was examined. In Experiment 1, we studied local effects of hydrogen peroxide (H(2)O(2)) and its interaction with PGF(2alpha) on P secretion in ovine corpus luteum (CL) in vivo. For this purpose, a microdialysis system (MDS) was used, where only the cells surrounding the capillary membrane in the microenvironment of the CL are exposed to these factors, and the P secretory ability of the CL is maintained as if intact. The study used a multiple CL model to implant the MDS, enabling us to examine in parallel several experimental infusions into the MDS implanted in different CLs (one MDS line per CL) developed after superovulation in one ewe. On Day 8 after GnRH treatment, the MDS were implanted into multiple CL in both ovaries of six ewes. A 4-h infusion with PGF(2alpha) (10(-6)M) at 8-12 h slightly increased P release during infusion, while a 4-h infusion with H(2)O(2) (10(-3)M) at 20-24 h decreased P release at 27-38 h. A pre-infusion with PGF(2alpha) for 4h at 8-12h, followed by infusion of H(2)O(2) at 20-24 h rapidly decreased the P release at 20-40 h (P<0.05); this decrease occurred 7h earlier than in the CL treated with H(2)O(2) alone. In Experiment 2, by utilizing the MDS we also applied free radical scavengers to examine their possible weakening effect on the inhibition of P secretion in the microenvironment within the regressing CL induced by PGF(2alpha) treatment. On Day 8 after estrus, the MDS were implanted into the CL (single CL model, two MDS lines per CL). Infusion of free radical scavengers, superoxide dismutase (SOD;50mg/ml)+catalase (CAT; 10mg/ml), at 0-28 h first increased P release until 12 h (P<0.05), and consequently delayed the decrease in P release until 30 h after administration of PGF(2alpha) i.m. (P<0.05). The present results support the concept that the leading pathway from PGF(2alpha) induces an increase of reactive oxygen species in luteolysis in the ewe.     

Developmental sensitivity of the bovine corpus luteum to prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1): is ET-1 a mediator of the luteolytic actions of PGF2alpha or a tonic inhibitor of progesterone secretion?

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.     

Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow

One of the postulated main luteolytic actions of prostaglandin (PG) F(2 alpha) is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF(2 alpha). Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF(2 alpha) injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10--12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF(2 alpha) analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5-2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 +/- 0.5 ng/ml (0 h) to 0.6 +/- 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF(2 alpha) induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.     

Evaluation of short estrus synchronization methods in dairy cows

In the present study, two new short estrus synchronization methods have been developed for lactating dairy cows. The study was completed in three consecutive phases. In experiment (Exp) 1, 32 cows, that were not detected in estrus since calving between the 50th and 84th post-partum days, were treated with PGF2alpha (PGF, d-cloprostenol, 0.150 mg), estradiol propionate (EP, 2mg) and GnRH (lecirelina, 50 microg) at 24h intervals, respectively, and timed artificial insemination (TAI) was performed 48 h after PGF. Different from Exp 1, EP and GnRH were given at 48 and 60 h, respectively after PGF in Exp 2 (n=20), instead of 24 and 48 h. Ovulations were investigated by ultrasound for 7 days starting from the day of PGF treatment, and ovulation rates were compared with the ones obtained in Exp 1. In Exp 3, cows were given the same treatments as Exp 2, but treatments started at certain estrus stages. Cows detected in estrus and with a confirmed ovulation (n=27) after the second PGF given 11 days apart were assigned to three treatment groups. Treatment was initiated at Day 3 (group metestrus, n=9), Day 12 (group diestrus, n=9) and Day 18 (group proestrus, n=9) after ovulation. All cows included in Exp 3 were TAI between 16 and 20 h after GnRH treatment. In Exp 2 and 3, blood samples were obtained once every 2 days, starting from Day 0 to the 10th day after GnRH injection, and once every 4 days between the 10th and the 22nd days after GnRH to examine post-treatment luteal development. During the study, animals exhibiting natural estrus were inseminated and served as controls (n=85). The rate of estrus was found to be significantly higher in cows with an active corpus luteum (CL) at the start of Exp 1 (72.7% vs. 30.0%, P<0.05) and the pregnancy rate tended to be higher than cows without an active CL (40.9% vs. 10.0%, P=0.08). Compared to those in Exp 1, cows in Exp 2 had higher rates of synchronized ovulation (94.1% vs. 59.1%, P=0.013). In Exp 3, estrus (P<0.001) and pregnancy rates (P=0.01) were found to be significantly higher in cows in the proestrus group than in those in the metestrus group. Comparable pregnancy rates were obtained from the first and second inseminations in Exp 1 and 3 with results from those inseminated at natural estrus (P>0.05). It was concluded from the study that the treatment in Exp 1 and 3 could result in comparable pregnancy rates after timed AI of lactating dairy cows at random stages of the estrus cycle relating to those inseminated at natural estrus, but the stage of the estrus cycle can have significant effects on pregnancy rates.     

Insulin-like growth factor (IGF)-I, IGF-I receptor, and IGF binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F(2alpha)-treated gilts

Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.     

Role of the DLL4-NOTCH system in PGF2alpha-induced luteolysis in the pregnant rat

We investigated the expression and cell localization of NOTCH1, NOTCH4, and the delta-like ligand DLL4 in corpus luteum (CL) from pregnant rats during prostaglandin F2alpha (PGF2alpha)-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT). Specific staining for NOTCH1 and NOTCH4 receptors was detected predominantly in large and small luteal cells. Furthermore, in line with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression, staining was evident in the nuclei of luteal cells. In addition, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the fact that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4, and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels and increased luteal levels of active caspase 3 and the BAX:BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and they suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.     

Administration of prostaglandin f(2 alpha) during the early bovine luteal phase does not alter the expression of ET-1 and of its type A receptor: a possible cause for corpus luteum refractoriness

Luteal regression is initiated by prostaglandin F(2 alpha) (PGF(2 alpha)). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However, during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF(2 alpha). Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF(2 alpha)-induced luteolysis. Therefore, in this study, we compared the effects of PGF(2 alpha) administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations, and the mRNA levels of PGF(2 alpha) receptors (PGF(2 alpha)-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF(2 alpha) analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF(2 alpha) analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF(2 alpha) injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroidogenic acute regulatory protein and cytochrome P450(scc)) was only affected when PGF(2 alpha) was administered during midcycle. Nevertheless, PGF(2 alpha) elicited certain responses in the early CL: progesterone and oxytocin secretion were elevated, and PGF(2 alpha)-R was transiently affected. Such effects probably result from PGF(2 alpha) acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF(2 alpha), possibly the endothelium, could yet be nonresponsive during the early luteal phase.     

Change in morphology of corpora lutea, central luteal cavities and steroid secretion patterns of postpartum suckled beef cows after melengestrol acetate with or without prostaglandin F2alpha

Change in morphology of the corpus luteum (CL) and patterns of progesterone and estradiol secretion after treatment with melengestrol acetate (MGA) were monitored in postpartum beef cows. Twenty Angus cows were randomly assigned to MGA or MGA + prostaglandin F(2alpha) (PGF) treatments. All cows were fed 0.5 mg of MGA per cow per day for 14 d. The MGA-treated cows (n = 10) were allowed to return to estrus spontaneously at the second estrus after withdrawal of MGA from the feed. The MGA + PGF-treated cows (n = 10) received an injection containing 25 mg of PGF(2alpha) 17 d after the last feeding of MGA. Cycle 1 was defined as the first luteal phase after MGA feeding and Cycle 2 represented the subsequent cycle or luteal phase after PGF. Blood sampling and transrectal ultrasonography of the ovaries was done daily through the completion of 2 estrous cycles upon removal of MGA from the feed. Blood samples were analyzed for plasma progesterone and estradiol concentrations. Area of CL and fluid-filled cavities within each CL were determined by ultrasonography. Concentrations of progesterone and area of CL were similar between cycles and treatments. Estradiol concentrations were higher (P < 0.05) in Cycle 2 than in Cycle 1. Fluid-filled cavities were larger (P < 0.001) in Cycle 1 than in Cycle 2 for both mid-luteal (Days 5 to 9) and late-luteal (Days 10 to 14) phases. Multiple CL (2 or more during 1 cycle) were observed in 5 cows. Progesterone concentrations and total area of luteal tissue did not change with respect to treatment or cycle, but CL morphology was altered in the first cycle after MGA treatment. Of the 19 cows that ovulated after withdrawal of MGA, 3 experienced a short luteal phase. These data characterize changes that occur among cows that are fed melengestrol acetate during the postpartum period and enhance observations from prior studies regarding MGA use.     

Embryotoxic effects adjacent and opposite to the early regressing bovine corpus luteum

Early luteal regression in cattle has an embryotoxic effect that is not overcome by replacement with progesterone, but is prevented by removal of the regressing CL. Two experiments were designed to test the null hypothesis that the luteal component of the embryotoxic effect is delivered by a systemic pathway. Beef heifers and cows (n = 39) received two good quality embryos, one placed into each uterine horn on Day 6 or 7 of the estrous cycle. Treated animals (n = 20) received 15 mg of PGF2alpha three times per day from Day 7 (n = 11; Experiment 1) or 5 (n = 9; Experiment 2) through 8; controls (n = 19) received saline. Progestogen replacement therapy (12 mg flurogestone acetate daily, s.c.) was provided from Day 6 (Experiment 1) or 4 (Experiment 2) until ultrasonographic diagnosis of embryo survival on Day 35 after estrus. The effects of treatment, location of the embryo and location by treatment interaction on embryo survival were tested by Chi square. In Experiment 1, there was no significant difference in embryo survival rate between PGF2alpha-treated and control recipients. In Experiment 2, only 6 of 18 embryos survived to Day 35 when transferred to animals treated with PGF2alpha compared to 12 of 18 in control animals (P< 0.05). The survival of embryos did not differ with location (adjacent or opposite to the regressing CL) or location by treatment interaction. Thus no evidence was obtained to support a local effect of the regressing CL. The embryo mortality associated with luteolytic doses of PGF2alpha in cows receiving replacement therapy with progestogen probably involves compounds that either act systemically or are transported via the uterine lumen to the uterine horn contralateral to the regressing CL.     

Secretion of prostaglandins and progesterone by cells from corpora lutea of mares

Corpora lutea (CL) were collected from mares during early (Day 4-5), mid- (Day 8-9), and late (Day 12-13) dioestrus. Dispersed cell suspensions were obtained by enzymic digestion of tissue. Two distinct luteal cell populations (large and small) were observed. The proportion of small luteal cells significantly increased as age of CL advanced. Cells (2 x 10(6)) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1 alpha (the stable metabolite of prostacyclin). Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those at mid- or late dioestrus. The ratio of PGF:PGE-2 increased from 0.33 in CL of early dioestrus to 1.34 in CL of mid-dioestrus, whereas ratios of PGF:6-keto-PGF-1 alpha remained relatively constant (approximately 0.6). The ratio of PGE-2:6-keto-PGF-1 alpha from CL decreased between early (3.27) and mid-dioestrus (0.43). Addition of LH, dbcAMP, or ionophore to cell cultures did not consistently affect secretion of progesterone or PGs by luteal cells. It is suggested that prostaglandins produced by luteal cells of mares may contribute to control of luteal function and that the changing ratios of prostaglandins may be more important in controlling the lifespan of the CL than absolute concentrations of each.     

In vivo oxytocin release from microdialyzed bovine corpora lutea during spontaneous and prostaglandin-induced regression

The release of luteal oxytocin during spontaneous and prostaglandin-induced luteolysis was investigated in cows. A continuous-flow microdialysis system was used in 11 cows to collect dialysates of the luteal extracellular space between Days 12 and 24 postestrus. Seven cows were untreated and were expected to exhibit spontaneous luteolysis during sampling, whereas 4 cows received prostaglandin F(2alpha) (PGF(2alpha)) systemically between Days 13 and 15 to induce luteolysis during sampling. Oxytocin was detectable in the dialysate of all cows before Day 16 postestrus and occurred as 2 or 3 discrete pulses per 12-h sampling period. For non-PGF(2alpha)-treated cows, dialysate oxytocin content began to decline spontaneously on Day 15 postestrus and was undetectable by Day 17 postestrus. Oxytocin decay curves preceded onset of serum progesterone decline by at least 72 h and were not related temporally with onset of progesterone decline within cow. Exogenous PGF(2alpha) (25 mg, i.m.) produced a 10-fold increase in dialysate oxytocin within 1 h (1.9 +/- 0.3 pg/ml to 20.8 +/- 3.0 pg/ml; P < 0. 01). Dialysate oxytocin then declined to pretreatment concentrations within 2 h and was undetectable within 8 h posttreatment. A second PGF(2alpha) injection given 20 h after the first did not result in a measurable increase in dialysate oxytocin, probably because luteolysis was underway. Although robust luteal oxytocin release was observed after treatment with a pharmacological dose of PGF(2alpha), the lack of detectable oxytocin secretion during spontaneous luteolysis suggests that the contribution of luteal oxytocin in the cow may be less than that proposed for the ewe.     

A progesterone-based timed AI protocol more effectively prevents premature estrus and incomplete luteal regression than an Ovsynch protocol in lactating Holstein cows

The objective of this study was to evaluate pregnancy rates in lactating Holstein cows treated with an Ovsynch protocol (GnRH-PGF(2alpha)-GnRH) or a progesterone-based timed AI (TAI) protocol, and to determine the factors that may influence pregnancy rate following protocol treatment. In experiment 1, lactating Holstein cows were randomly assigned to three treatments: (1) an injection of GnRH (Day 0), an injection of PGF(2alpha) on Day 7, a second injection of GnRH on Day 9, and TAI 16h after the second GnRH injection (GPG group, n = 34); (2) insertion of a CIDR intravaginal progesterone (1.9g) device combined with a capsule containing 10mg estradiol benzoate (Day 0), an injection of PGF(2alpha) and removal of the device on Day 7, an injection of GnRH on Day 9, and TAI 16h after the GnRH injection (CPG group, n = 34); (3) an injection of PGF(2alpha) after confirming the presence of CL by ultrasonographical observation and artificial insemination at estrus (AIE) (P group, n = 75). The pregnancy rate after TAI following the CPG protocol (41.2%) was higher (P<0.05) than that after TAI following the GPG protocol (20.6%) and that after AIE (20.0%). In experiment 2, lactating Holstein cows were randomly assigned to two treatments: a GPG group (n = 31) and a CPG group (n = 31). The GPG and CPG protocols were identical to those used in experiment 1. The proportion of cows with premature estrus prior to injection of PGF(2alpha) and with incomplete luteal regression tended (P = 0.056) to be or were greater (P<0.05) in the GPG group (4/31, 8/31) than in the CPG group (0/31, 2/31), respectively. Average diameters of dominant follicles (1.5+/-0.1mm versus 1.4+/-0.1mm) on Day 7 and preovulatory follicles (1.8+/-0.1mm versus 1.6+/-0.1mm) on Day 9, and the proportion of cows with synchronized ovulation by 40h after the second GnRH injection were not different (81.5% versus 87.1%, P>0.05) between groups, respectively. We conclude that the pregnancy rate after TAI following the CPG protocol was higher than that after TAI following the GPG protocol, probably due to a decreased incidence of premature estrus and incomplete luteal regression.