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Elevated progesterone concentrations enhance prostaglandin F2α synthesis in dairy cows
Animal Reproduction Science 114 (2009) 62–71
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Elevated progesterone concentrations enhance prostaglandin F2␣ synthesis in dairy cows Ricarda Maria dos Santos a, Marcelo Demarchi Goissis b, David Augusto Fantini b, Claudia Maria Bertan c, José Luiz Moraes Vasconcelos d, Mario Binelli b,∗ a
Escola Agrotécnica Federal, Uberlândia, MG, Brazil Department of Animal Reproduction, College of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga, SP, Brazil c College of Animal Sciences, São Paulo State University “Julio de Mesquita Filho”, Campus Dracena, Dracena, SP, Brazil d Department of Animal Production, College of Veterinary Medicine and Animal Science, São Paulo State University “Julio de Mesquita Filho”, Botucatu, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 14 August 2008 Accepted 22 September 2008 Available online 1 October 2008 Keywords: Prostaglandin F2␣ Progesterone Estradiol Oxytocin Dairy cow
a b s t r a c t The objective was to evaluate the influence of varying plasma progesterone (P4 ) concentrations throughout the luteal phase in dairy cows on PGF2␣ production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF2␣ ; PGFM) following treatment with estradiol-17 (E2 ) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n = 9), received 20 mg LH (n = 7), or had ovarian follicles larger than 6 mm aspirated (n = 8). In Experiment 2, cows on Day 6 were untreated (n = 9) or received 5000 IU hCG (n = 10). In Experiments 1 and 2, all cows received 3 mg E2 on Day 17, and blood samples were collected every 30 min from 2 h before to 10 h after E2 . Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n = 4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n = 7) received the alternate sequence. Blood was collected daily for measurement of P4 4 h after concentrate feeding. On Day 17, blood was
∗ Corresponding author at: Centro de Biotecnologia em Reproduc¸ão Animal, Departamento de Reproduc¸ão Animal, Faculdade de Medicina Veterinária e Zootecnia, Avenida Duque de Caxias Norte, 225 Pirassununga, SP, CEP 13635-900, Brazil. Tel.: +55 19 3565 4235; fax: +55 19 3565 4060. E-mail address: [email protected] (M. Binelli). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.09.016
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collected from 1 h before to 1 h after a 100 IU OT injection. In Experiment 1, both plasma P4 and release of PGF2␣ were similar between LH-treated and control cows (P > 0.10). In Experiment 2, plasma P4 was elevated to a greater extent on Day 17 in cows treated with hCG (P < 0.05) and plasma PGFM was also greater in hCG-treated animals (treatment × time interaction; P < 0.05). In Experiment 3, there was a group × period interaction (P < 0.01) for plasma P4 , indicating that less concentrate feeding was associated with greater plasma P4 . Release of PGF2␣ in response to OT was greater for cows receiving less concentrate (group × period interaction; P < 0.05). In conclusion, dairy cows with more elevated blood P4 concentrations released more PGF2␣ in response to E2 or OT. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In cattle, oxytocin (OT), progesterone (P4 ) and estradiol-17 (E2 ) function in concert to regulate episodic secretion of endometrial prostaglandin (PGF2␣ ), which causes luteolysis (Silvia et al., 1991; Goff, 2004). The dynamics of P4 regulation of PGF2␣ synthesis changes throughout the estrous cycle. During the first half of the estrous cycle, P4 inhibits expression of genes for E2 synthesis and OT receptors in the endometrium, thereby preventing premature release of PGF2␣ pulses associated with luteolysis (Vallet et al., 1990; Spencer et al., 2004). Simultaneously, chronic exposure to P4 results in synthesis of the enzyme prostaglandin-endoperoxidase synthase 2 (PTGS2) and accumulation of its substrate, arachidonic acid, in endometrial cells (Goff, 2004). Conversion of arachidonic acid into PGH2 by PTGS2 is a required step for synthesis of PGF2␣ . During the second half of the estrous cycle, there is a gradual loss of suppression of PGF2␣ releasing mechanisms, probably due to down-regulation of P4 receptors, with a concurrent increase in responsiveness to OT (Spencer and Bazer, 1995). Furthermore, timing of P4 production during the estrous cycle controls the onset of luteolysis. In that regard, administration of P4 at the beginning of the estrous cycle promoted premature luteolysis (Woody et al., 1967), whereas administration of a P4 receptor antagonist delayed luteolysis (Morgan et al., 1993). Although P4 primes and prepares the endometrium for PGF2␣ synthesis, actions of E2 and OT are needed for episodic release of PGF2␣ necessary for luteolysis. Indeed, removal of ovarian follicles extended the luteal phase of the estrous cycle (i.e. retarded luteolysis; Villa-Godoy et al., 1985; Hughes et al., 1987), whereas administration of E2 in the second half of the estrous cycle caused release of PGF2␣ and induced premature luteolysis (Thatcher et al., 1986; Salfen et al., 1999). The E2 -stimulated release of PGF2␣ probably involves the stimulation of responsiveness of the endometrium to OT; in that regard, E2 can affect the initiation, magnitude and pattern of PGF2␣ pulses in response to OT in ewes (Beard and Lamming, 1994). Moreover, OT-stimulated PGF2␣ release in ovariectomized cows was stimulated by treatment with P4 and E2 , but not by E2 alone (Lamming and Mann, 1995). This suggests that P4 modulates E2 effects. It remains to be determined whether the magnitude of luteal phase plasma P4 concentrations affects PGF2␣ release in the peri-luteolysis phase of the estrous cycle. In the present study, both pharmacologic and nutritional experimental paradigms were used to manipulate luteal phase plasma P4 concentrations and verify effects on luteolytic mechanisms. The pharmacologic paradigm is based on reports in which plasma P4 concentrations were increased by the strategic administration of human chorionic gonadotropin (hCG) or gonadotropin releasing hormone (GnRH). This resulted in the ovulation from the first-wave dominant follicle and formation of an accessory corpus luteum (CL) which produced supplemental P4 (Price and Webb, 1989; Rajamahendran and Sianangama, 1992; Hariadi et al., 1998; Santos et al., 2001). The nutritional paradigm followed the indication that high-yield cows consumed more dry matter (Harrison et al., 1990) and had lesser plasma P4 concentrations (Vasconcelos et al., 1999). This was attributed to increased hepatic blood flow, and consequent increased rate of P4 metabolism, in response to peaks of intake of the high-energy diet (Sangsritavong et al., 2002; Vasconcelos et al., 2003). In contrast, ewes
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that were fasted during the luteal phase had greater plasma P4 concentrations (Kiyma et al., 2004). Thus, in the present study, we tested the hypothesis that E2 - or OT-stimulated PGF2␣ release was greater in cows treated to increase luteal phase plasma P4 concentrations. The specific objectives were to measure changes in concentrations of 13,14-dihydro-15-keto-PGF2␣ (PGFM, an indirect measure of PGF2␣ ) in response to administration of E2 or OT on Day 17 of a synchronized estrous cycle in cows bearing an accessory CL induced by LH or hCG (Experiments 1 and 2), and in cows with reduced plasma P4 concentrations due to greater intake of concentrate in the diet (Experiment 3). 2. Materials and methods 2.1. Cattle Time of ovulation in multiparous, non-lactating, Holstein cows that had initiated estrous cycling postpartum was synchronized by the “OvSynch” protocol (Pursley et al., 1995); Day 0 of the estrous cycle was considered the day of the second injection of GnRH. Only cows that had ovulations after the second injection of GnRH were included in the experiments. Ovulations were verified by transrectal ultrasonography (Aloka SSD-500 with 7.5 MHz, linear-array transducer; Aloka Ltd., Tokyo, Japan) performed on Day 0 (presence of an ovarian follicle ≥ 8.5 mm) and Day 2 (absence of a follicle ≥ 8.5 mm). 2.2. Experimental designs 2.2.1. Experiment 1 Cattle were allocated randomly on Day 6 to remain untreated (control treatment; n = 9), to receive 20 mg pLH (Lutropin, Bioniche Animal Health, Belleville, ON, Canada) given i.v. (LH treatment; n = 7), or to have all ovarian follicles >6 mm in diameter removed by transvaginal aspiration (Asp treatment; n = 8). The LH was intended to induce ovulation of the dominant follicle from the first wave, thereby inducing an accessory CL. Ovulations were confirmed 48 h after treatment. Transvaginal follicular aspiration, aimed to remove the first wave dominant follicle without inducing an accessory CL. Aspirations were conducted with an Aloka SSD-500 (Aloka, Tokyo, Japan) B-mode ultrasonographic scanner, equipped with a sectorial 5 MHz transducer equipped with a guide and aspiration needle (ovum pick-up aspiration needle, 18 G, 55 cm long, Cook, Brisbane, Australia). The efficacy of follicle aspiration (absence of follicles larger than 6 mm) was confirmed by transrectal ultrasonography (7.5 MHz linear-array transducer). On Day 16, cows received indwelling jugular catheters. On Day 17, all animals received 3 mg E2 (Sigma–Aldrich, St. Louis, USA), dissolved in 1:1 (vol/vol) in ethanol and saline, as a bolus 6 ml injection through the IV catheter. Blood samples were collected every 30 min for 12 h beginning 2 h prior to E2 administration and continuing for 10 h following E2 administration. Blood samples were collected in borosilicate tubes (16 mm × 100 mm) containing sodium citrate. Samples were centrifuged at 2900 × g for 30 min at 4 ◦ C, and plasma was harvested and stored at −20 ◦ C for determination of PGFM concentrations. 2.2.2. Experiment 2 Cattle were allocated randomly on Day 6 of the estrous cycle to remain untreated (control treatment; n = 9) or to receive 5000 IU hCG (Vetecor, Laboratórios Calier do Brasil Ltda, Osasco, SP, Brazil) given i.m. (hCG treatment; n = 10). On Day 6, diameter of the largest follicle was determined for cattle treated with hCG, and 48 h later (Day 8), ovulation was verified by ultrasonography. On Days 16 and 17, experimental procedures were identical to those described in Experiment 1. 2.2.3. Experiment 3 Cattle were submitted to a 28-d adaptation period, receiving 2 kg/d of a soy meal (40%) and cornmeal (60%) based concentrate (TDN 73%; CP 22%; NDF 10.5%), with ad libitum access to cattle mineralized salt, water, and pasture with Brachiaria decumbens (DM 51.3%; TDN 54.3%; CP 4.6%; NDF 78.6%; and ADF 43.2%). On Day 0, cattle were allocated to receive daily allocations of either 2 kg (n = 4) or 8 kg concentrate (n = 7), divided in two meals (in the morning and afternoon). This experiment was replicated
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as a crossover, so that females that received 2 kg/d concentrate during the first period received 8 kg/d concentrate in the second period, and vice versa. This arrangement defined Experimental Groups 2/8 and 8/2, respectively. Starting on the day of second GnRH injection of the OvSynch protocol, and continuing daily for the duration of the experiment, changes in ovarian follicles and corpora lutea were assessed by ultrasonography and blood samples were collected. Blood samples (for determination of plasma P4 concentrations) were collected immediately before and 4 h after the morning concentrate feeding. On Day 16, an IV catheter was installed in a jugular vein. On Day 17, cattle were given 100 IU OT (Ocitocina, Univet, São Paulo, SP, Brazil) iv, and blood samples were collected 60, 30, and 0 min before OT treatment, and 15, 30, 45, and 60 min after treatment. Plasma was harvested and stored as described in Experiment 1. There was a 10-d interval between Day 17 of the first period and the first GnRH injection of the OvSynch protocol of the second period. All animals were fed 4 kg concentrate/d between Day 17 of the first period and Day 0 of the second period. 2.3. Measurement of plasma concentrations of P4 and PGFM 2.3.1. P4 In Experiments 1 and 3, plasma P4 concentrations were measured with a commercial solid-phase radioimmunoassay kit, in accordance with the manufacturer’s instructions (Coat a Count, Diagnostic Products Corporation, Los Angeles, CA, USA). In Experiment 1, samples were measured in a single assay and the intra-assay coefficient of variation was 3.1%. In Experiment 3, the intra-assay coefficient of variation was 5.3%, whereas the inter-assay coefficient of variation was 5.0%. Assay sensitivity was 0.03 ng/ml. In Experiment 2, plasma P4 concentrations were measured by radioimmunoassay, as described by Badinga et al. (1992) and modified by Carrière and Lee (1994). The intra-assay coefficient of variation was 4.0% and the inter-assay coefficient of variation was 0.9%. Assay sensitivity was 0.09 ng/ml. 2.3.2. PGFM Plasma PGFM concentrations were measured by radioimmunoassay, as described (Meyer et al., 1995). Intra- and inter-assay coefficients of variation were 7.5 and 4.2%, respectively. Assay sensitivity was 42.4 pg/ml. 2.4. Statistical analysis Data were tested for normality of residues (Shapiro–Wilk test) and homogeneity of variances (FMax test) for each variable. Variables which did not fulfill assumptions for analysis of variance were transformed by log10 or square-root and re-analyzed. For clarity, only untransformed data are presented. Discrete variables were analyzed by least squares ANOVA, using the GLM procedure of SAS software (SAS, 1988) and repeated measures variables were analyzed using the MIXED procedure of SAS. For the MIXED procedure, fit statistic parameters for unstructured, compound symmetry and autoregressive(1) covariance structures were tested. The autoregressive(1) structure provided the lowest values for fit statistic parameters and was used for all repeated measures analysis. Probability values 0.05 ≤ P ≤ 0.1 were considered as a tendency to significance. 2.4.1. Experiment 1 Plasma PGFM concentrations over time were analyzed after square-root transformation. The MIXED procedure mathematical model contained the fixed effects of treatment, time, and treatment × time interaction and random effect of cow nested within treatment. Plasma P4 concentration on Day 17 was not transformed and the GLM procedure mathematical model contained the fixed effect of treatment. 2.4.2. Experiment 2 Plasma PGFM concentrations over time were analyzed after log10 transformation. Plasma P4 concentrations on Day 17 was not transformed. Mathematical models were as in Experiment 1.
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Fig. 1. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to estradiol-17 injections (3 mg; experimental hour 0 of Day 17 of the estrous cycle) given to cows treated with 0 mg (control; n = 9) or 20 mg LH (LH; n = 7) or submitted to follicle aspiration (ASP; n = 8) on Day 6 of the estrous cycle.
2.4.3. Experiment 3 Daily plasma P4 concentrations, measured 4 h after the morning concentrate feeding, and concentrations of PGFM were square-root transformed. The MIXED procedure mathematical model contained the fixed effects of group, period, day or minute of sampling (for P4 and PGFM concentrations, respectively) and interactions, and the random effect of cow nested within group. 3. Results 3.1. Experiment 1 All cattle treated with LH had an accessory CL on Day 17. On Day 17, plasma P4 concentrations were (mean ± S.E.M.) 4.8 ± 0.54, 5.93 ± 0.58 and 5.5 ± 0.64 ng/ml for the control, LH and aspiration treatments, respectively (P > 0.10). The release of PGFM tended (P < 0.10) to be lower in the aspiration treatment (Fig. 1). 3.2. Experiment 2 Ovulation from the first-wave dominant follicle in response to hCG injection was not detected in two animals and they were excluded from the experiment. On Day 17, all remaining cattle given hCG had an accessory CL and more elevated plasma P4 concentrations than the control treatment (7.1 ± 0.72 and 5.0 ± 0.58 ng/ml, respectively; P < 0.05). Plasma PGFM concentrations were greater for cows treated with hCG than for cows in the control treatment (treatment × time interaction; P < 0.05; Fig. 2); hCG treatment resulted in greater PGFM concentrations between 4 and 5.5 h after E2 . 3.3. Experiment 3 Animals in Group 2/8 had greater plasma P4 concentrations than Group 8/2 in Period 1 (2.8 ± 0.23 compared with 2.2 ± 0.16 ng/ml), while the opposite occurred in Period 2 (1.78 ± 0.21 compared with 2.79 ± 0.17 ng/ml; group × period interaction; P < 0.01; Fig. 3). The significant group × period interaction means that amount of concentrate fed affected plasma P4 . Moreover, differences between groups increased over time (group × period × time interaction; P < 0.01). A significant group × period interaction was detected in each day between Days 9 and 16 (P < 0.02), except for Day 13. Animals in Group 2/8 had greater plasma PGFM concentrations than Group 8/2 in Period 1 (173.11 ± 35.52 compared with 82.69 ± 16.26 pg/ml), while the opposite occurred in Period 2 (75.75 ± 18.77 compared with 102.59 ± 18.77 pg/ml; group × period interaction; P < 0.05; Fig. 4). Con-
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Fig. 2. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to estradiol-17 injections (3 mg; experimental hour 0 of Day 17 of the estrous cycle) given to cows treated with 0 IU (control; n = 9) or 5000 IU (hCG; n = 7) of hCG on Day 6 of the estrous cycle. * Treatment means differed in a given time point (P < 0.05).
centrations of PGFM increased in response to the injection of OT (effect of time; P < 0.01). However, magnitude of increase tended to be greater for animals in Group 2/8 in Period 1 but smaller in Period 2, compared to Group 8/2 (group × period × time interaction; P < 0.1). 4. Discussion In the present study, plasma P4 concentrations in dairy cattle were either increased pharmacologically (Experiment 2) or decreased nutritionally (Experiment 3), and responsiveness to PGF2␣ -releasing
Fig. 3. Least squares means (±S.E.M.) of daily progesterone concentrations in plasma samples colleted during the first 16 d of estrous cycles from cows fed 2 kg/d concentrate in Period 1 and 8 kg/d concentrate in Period 2 (Group 2/8; n = 4) or 8 kg/d concentrate in Period 1 and 2 kg/d concentrate in Period 2 (Group 8/2; n = 7).
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Fig. 4. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to 100 IU oxytocin injected on Day 17 of the estrous cycle in cows fed 2 kg/d concentrate in Period 1 and 8 kg/d concentrate in Period 2 (Group 2/8; n = 4) or 8 kg/d concentrate in Period 1 and 2 kg/d concentrate in Period 2 (Group 8/2; n = 7).
stimuli evaluated on Day 17. In accordance with expectations, PGFM concentrations in response to OT or E2 were greater in cattle with more elevated plasma P4 concentrations. In Experiment 1, 20 mg LH on Day 6 of the estrous cycle induced formation of an accessory CL but did not significantly change plasma P4 concentrations on Day 17. This was unexpected and in variation with other reports in which GnRH was used to induce accessory CLs (Schmitt et al., 1996; Howard et al., 2006), but the reason is unclear to authors. Consistently, E2 -stimulated PGF2␣ release was similar between LH and control treatments. In contrast, plasma PGFM concentrations over time tended to be less with the aspiration treatment. Both LH and aspiration treatments removed the first-wave dominant follicle abruptly, and hastened emergence of the second wave of follicular growth (data not shown). The fact that the pattern of E2 -stimulated PGF2␣ release tended to differ between aspirated and LH-treated animals despite similar effects in reprogramming of follicular growth is intriguing. This finding could be interpreted to mean that removal of the dominant follicle with or without formation of an accessory CL modulates PGF2␣ release differently. Further experimentation would be necessary to elucidate mechanisms controlling this process. In Experiment 2, hCG was given to induce ovulation from the first-wave dominant follicle and to induce an accessory CL, in an attempt to more efficiently increase plasma P4 concentrations relative to the LH treatment used in Experiment 1 (Price and Webb, 1989; Fricke et al., 1993). Indeed, treatment with hCG increased plasma P4 concentrations on Day 17 in comparison to control treatment. Seguin et al. (1977) reported that hCG had a longer circulating lifespan than LH in cows; this apparently results in greater capacity than LH to stimulate P4 synthesis in both the original and accessory CLs. In contrast to Experiment 1, cows with an hCG-induced accessory CL released more PGF2␣ in response to E2 than did control cows; we attributed the greater PGF2␣ release to the more elevated plasma P4 concentrations in these animals.
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In Experiment 3, interpretation of significant period × group and period × group × day interactions indicate that cows given 8 kg/d concentrate had lesser plasma P4 concentrations through the luteal phase. This was due presumably to a greater rate of P4 metabolism, as reported in ewes (Parr et al., 1993) and cows (Vasconcelos et al., 2003). Parr et al. (1993) and Sangsritavong et al. (2002) reported that blood flow in the hepatic portal vein was directly related to the nutrient density of the diet. Because the liver was 96% efficient in clearing circulating P4 , higher blood flow to the digestive system favors more rapid metabolism of P4 by the liver. Thus, females fed more concentrate had reduced plasma P4 concentrations. This rationale is consistent with results in Experiment 3, in which animals fed less concentrate had increased plasma P4 concentrations. Furthermore, less concentrate feeding was associated with a greater release of PGFM in response to OT. One possible explanation for the present findings is that treatments leading to decreased P4 concentrations may have led to a biochemical milieu less favorable to production of PGF2␣ in response to E2 and OT. Indeed, P4 stimulates accumulation of arachidonic acid and PTGS2 in endometrial cells, which is essential for the synthesis of PGF2␣ (Boshier et al., 1987; Silvia et al., 1991; Kombé et al., 2003). In addition, Vallet et al. (1990) showed that P4 stimulated the basal release of PGF2␣ in vitro by stimulating the accumulation of enzymes and substrates for the synthesis of PGF2␣ , such as phospholipids, prostaglandin synthase (Salamonsen et al., 1990) and phospholipase C (Raw and Silvia, 1991). According to McCracken et al. (1999), in the beginning of the estrous cycle, P4 binds to its endometrial receptors and blocks E2 -stimulated synthesis of OT receptors. In the middle of the estrous cycle, P4 down-regulates its own receptors, which results in the release of the P4 block to the action of E2 . Consequently, OT receptor synthesis is stimulated. Therefore, another possible explanation for the lesser plasma PGFM concentrations in cows receiving control treatment (Experiment 2) and in cows that received 8 kg/d concentrate (Experiment 3) is that lesser P4 up to Day 17 of the estrous cycle may have delayed down-regulation of P4 receptors (Spencer et al., 2004). This could have resulted in a prolonged inhibition in E2 - and OT-stimulated PGF2␣ release. Collectively, the three current experiments supported the initial hypothesis that E2 - or OTstimulated PGF2␣ release is greater in cows with elevated P4 plasma concentrations, which agrees with previous reports. For example, Lafrance and Goff (1988) reported that release of PGF2␣ in response to E2 and OT treatments in ovariectomized heifers was less, but such release increased after treatment with P4 for 7, 14, or 21 d. Skarzynski et al. (1999) noted that the endometrial tissue acquired maximum capacity for PGFM synthesis in response to E2 after pre-incubation with P4 or luteal cells. In contrast, present results are in variance to results reported by Mann and Lamming (1995), in studies with ovariectomized cows supplemented with P4 and E2 . These authors observed greater concentrations of PGFM in cows which had lower concentrations of P4 . In addition, in vitro studies reported by Bogacki et al. (2002) showed that, during the late luteal phase, P4 suppressed the ability of OT to induce the secretion of endometrial PGF2␣ by directly interfering with OT receptor binding. Based on the P4 modulation of PGF2␣ verified here in cows that have initiated estrous cycling, it is tempting to speculate about possible implications for pregnant cows. Specifically, the greater production of PGFM in cows with elevated concentrations of P4 demonstrated in the present research seems to contradict several studies which reported greater pregnancy rates in cows treated to have elevated concentrations of P4 (Santos et al., 2001; Marques, 2002). Based on results of the present study, it is speculated that P4 does not act directly in the inhibition of PGF2␣ release. Rather, P4 may favor conceptus development (Garret et al., 1988) and synthesis of interferon-tau (Thatcher et al., 1997), which inhibits PGF2␣ biosynthesis in the endometrium to block luteolysis (i.e. maternal recognition of pregnancy; Mann and Lamming, 2001). However, effects of P4 on endometrial release of PGF2␣ measured in females having estrous cycles may differ from effects in pregnant females due to conceptus signals. Indeed, Gray et al. (2006) reported that P4 modulated expression of endometrial genes both dependent and independent of interferon-tau in the sheep. Specific studies must be conducted to further understand the role of timing and magnitude of P4 increase on E2 - and OT-stimulated PGF2␣ release, both in estrous cycling and pregnant cows. In conclusion, the initial hypothesis for the present research was confirmed as cows treated both hormonally and nutritionally to have elevated P4 concentrations also released more PGF2␣ in response to treatments with OT and E2 .
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Elevated progesterone concentrations enhance prostaglandin F2␣ synthesis in dairy cows Ricarda Maria dos Santos a, Marcelo Demarchi Goissis b, David Augusto Fantini b, Claudia Maria Bertan c, José Luiz Moraes Vasconcelos d, Mario Binelli b,∗ a
Escola Agrotécnica Federal, Uberlândia, MG, Brazil Department of Animal Reproduction, College of Veterinary Medicine and Animal Sciences, University of São Paulo, Pirassununga, SP, Brazil c College of Animal Sciences, São Paulo State University “Julio de Mesquita Filho”, Campus Dracena, Dracena, SP, Brazil d Department of Animal Production, College of Veterinary Medicine and Animal Science, São Paulo State University “Julio de Mesquita Filho”, Botucatu, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 2 May 2008 Received in revised form 14 August 2008 Accepted 22 September 2008 Available online 1 October 2008 Keywords: Prostaglandin F2␣ Progesterone Estradiol Oxytocin Dairy cow
a b s t r a c t The objective was to evaluate the influence of varying plasma progesterone (P4 ) concentrations throughout the luteal phase in dairy cows on PGF2␣ production (assessed as plasma concentrations of 13,14-dihydro-15-keto-PGF2␣ ; PGFM) following treatment with estradiol-17 (E2 ) or oxytocin (OT). In all experiments, time of ovulations was synchronized with the OvSynch protocol and Day 0 corresponded to day of second GnRH injection. In Experiment 1, non-lactating dairy cows on Day 6 remained non-treated (n = 9), received 20 mg LH (n = 7), or had ovarian follicles larger than 6 mm aspirated (n = 8). In Experiment 2, cows on Day 6 were untreated (n = 9) or received 5000 IU hCG (n = 10). In Experiments 1 and 2, all cows received 3 mg E2 on Day 17, and blood samples were collected every 30 min from 2 h before to 10 h after E2 . Experiment 3 was conducted in two periods, each from Days 0 to 17 of the estrous cycle. At the end of Period 1, animals switched treatments in a crossover arrangement. Animals in Group 2/8 (n = 4) received 2 kg/d of concentrate in the first period and 8 kg/d in the second period. Animals in Group 8/2 (n = 7) received the alternate sequence. Blood was collected daily for measurement of P4 4 h after concentrate feeding. On Day 17, blood was
∗ Corresponding author at: Centro de Biotecnologia em Reproduc¸ão Animal, Departamento de Reproduc¸ão Animal, Faculdade de Medicina Veterinária e Zootecnia, Avenida Duque de Caxias Norte, 225 Pirassununga, SP, CEP 13635-900, Brazil. Tel.: +55 19 3565 4235; fax: +55 19 3565 4060. E-mail address: [email protected] (M. Binelli). 0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.09.016
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collected from 1 h before to 1 h after a 100 IU OT injection. In Experiment 1, both plasma P4 and release of PGF2␣ were similar between LH-treated and control cows (P > 0.10). In Experiment 2, plasma P4 was elevated to a greater extent on Day 17 in cows treated with hCG (P < 0.05) and plasma PGFM was also greater in hCG-treated animals (treatment × time interaction; P < 0.05). In Experiment 3, there was a group × period interaction (P < 0.01) for plasma P4 , indicating that less concentrate feeding was associated with greater plasma P4 . Release of PGF2␣ in response to OT was greater for cows receiving less concentrate (group × period interaction; P < 0.05). In conclusion, dairy cows with more elevated blood P4 concentrations released more PGF2␣ in response to E2 or OT. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In cattle, oxytocin (OT), progesterone (P4 ) and estradiol-17 (E2 ) function in concert to regulate episodic secretion of endometrial prostaglandin (PGF2␣ ), which causes luteolysis (Silvia et al., 1991; Goff, 2004). The dynamics of P4 regulation of PGF2␣ synthesis changes throughout the estrous cycle. During the first half of the estrous cycle, P4 inhibits expression of genes for E2 synthesis and OT receptors in the endometrium, thereby preventing premature release of PGF2␣ pulses associated with luteolysis (Vallet et al., 1990; Spencer et al., 2004). Simultaneously, chronic exposure to P4 results in synthesis of the enzyme prostaglandin-endoperoxidase synthase 2 (PTGS2) and accumulation of its substrate, arachidonic acid, in endometrial cells (Goff, 2004). Conversion of arachidonic acid into PGH2 by PTGS2 is a required step for synthesis of PGF2␣ . During the second half of the estrous cycle, there is a gradual loss of suppression of PGF2␣ releasing mechanisms, probably due to down-regulation of P4 receptors, with a concurrent increase in responsiveness to OT (Spencer and Bazer, 1995). Furthermore, timing of P4 production during the estrous cycle controls the onset of luteolysis. In that regard, administration of P4 at the beginning of the estrous cycle promoted premature luteolysis (Woody et al., 1967), whereas administration of a P4 receptor antagonist delayed luteolysis (Morgan et al., 1993). Although P4 primes and prepares the endometrium for PGF2␣ synthesis, actions of E2 and OT are needed for episodic release of PGF2␣ necessary for luteolysis. Indeed, removal of ovarian follicles extended the luteal phase of the estrous cycle (i.e. retarded luteolysis; Villa-Godoy et al., 1985; Hughes et al., 1987), whereas administration of E2 in the second half of the estrous cycle caused release of PGF2␣ and induced premature luteolysis (Thatcher et al., 1986; Salfen et al., 1999). The E2 -stimulated release of PGF2␣ probably involves the stimulation of responsiveness of the endometrium to OT; in that regard, E2 can affect the initiation, magnitude and pattern of PGF2␣ pulses in response to OT in ewes (Beard and Lamming, 1994). Moreover, OT-stimulated PGF2␣ release in ovariectomized cows was stimulated by treatment with P4 and E2 , but not by E2 alone (Lamming and Mann, 1995). This suggests that P4 modulates E2 effects. It remains to be determined whether the magnitude of luteal phase plasma P4 concentrations affects PGF2␣ release in the peri-luteolysis phase of the estrous cycle. In the present study, both pharmacologic and nutritional experimental paradigms were used to manipulate luteal phase plasma P4 concentrations and verify effects on luteolytic mechanisms. The pharmacologic paradigm is based on reports in which plasma P4 concentrations were increased by the strategic administration of human chorionic gonadotropin (hCG) or gonadotropin releasing hormone (GnRH). This resulted in the ovulation from the first-wave dominant follicle and formation of an accessory corpus luteum (CL) which produced supplemental P4 (Price and Webb, 1989; Rajamahendran and Sianangama, 1992; Hariadi et al., 1998; Santos et al., 2001). The nutritional paradigm followed the indication that high-yield cows consumed more dry matter (Harrison et al., 1990) and had lesser plasma P4 concentrations (Vasconcelos et al., 1999). This was attributed to increased hepatic blood flow, and consequent increased rate of P4 metabolism, in response to peaks of intake of the high-energy diet (Sangsritavong et al., 2002; Vasconcelos et al., 2003). In contrast, ewes
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that were fasted during the luteal phase had greater plasma P4 concentrations (Kiyma et al., 2004). Thus, in the present study, we tested the hypothesis that E2 - or OT-stimulated PGF2␣ release was greater in cows treated to increase luteal phase plasma P4 concentrations. The specific objectives were to measure changes in concentrations of 13,14-dihydro-15-keto-PGF2␣ (PGFM, an indirect measure of PGF2␣ ) in response to administration of E2 or OT on Day 17 of a synchronized estrous cycle in cows bearing an accessory CL induced by LH or hCG (Experiments 1 and 2), and in cows with reduced plasma P4 concentrations due to greater intake of concentrate in the diet (Experiment 3). 2. Materials and methods 2.1. Cattle Time of ovulation in multiparous, non-lactating, Holstein cows that had initiated estrous cycling postpartum was synchronized by the “OvSynch” protocol (Pursley et al., 1995); Day 0 of the estrous cycle was considered the day of the second injection of GnRH. Only cows that had ovulations after the second injection of GnRH were included in the experiments. Ovulations were verified by transrectal ultrasonography (Aloka SSD-500 with 7.5 MHz, linear-array transducer; Aloka Ltd., Tokyo, Japan) performed on Day 0 (presence of an ovarian follicle ≥ 8.5 mm) and Day 2 (absence of a follicle ≥ 8.5 mm). 2.2. Experimental designs 2.2.1. Experiment 1 Cattle were allocated randomly on Day 6 to remain untreated (control treatment; n = 9), to receive 20 mg pLH (Lutropin, Bioniche Animal Health, Belleville, ON, Canada) given i.v. (LH treatment; n = 7), or to have all ovarian follicles >6 mm in diameter removed by transvaginal aspiration (Asp treatment; n = 8). The LH was intended to induce ovulation of the dominant follicle from the first wave, thereby inducing an accessory CL. Ovulations were confirmed 48 h after treatment. Transvaginal follicular aspiration, aimed to remove the first wave dominant follicle without inducing an accessory CL. Aspirations were conducted with an Aloka SSD-500 (Aloka, Tokyo, Japan) B-mode ultrasonographic scanner, equipped with a sectorial 5 MHz transducer equipped with a guide and aspiration needle (ovum pick-up aspiration needle, 18 G, 55 cm long, Cook, Brisbane, Australia). The efficacy of follicle aspiration (absence of follicles larger than 6 mm) was confirmed by transrectal ultrasonography (7.5 MHz linear-array transducer). On Day 16, cows received indwelling jugular catheters. On Day 17, all animals received 3 mg E2 (Sigma–Aldrich, St. Louis, USA), dissolved in 1:1 (vol/vol) in ethanol and saline, as a bolus 6 ml injection through the IV catheter. Blood samples were collected every 30 min for 12 h beginning 2 h prior to E2 administration and continuing for 10 h following E2 administration. Blood samples were collected in borosilicate tubes (16 mm × 100 mm) containing sodium citrate. Samples were centrifuged at 2900 × g for 30 min at 4 ◦ C, and plasma was harvested and stored at −20 ◦ C for determination of PGFM concentrations. 2.2.2. Experiment 2 Cattle were allocated randomly on Day 6 of the estrous cycle to remain untreated (control treatment; n = 9) or to receive 5000 IU hCG (Vetecor, Laboratórios Calier do Brasil Ltda, Osasco, SP, Brazil) given i.m. (hCG treatment; n = 10). On Day 6, diameter of the largest follicle was determined for cattle treated with hCG, and 48 h later (Day 8), ovulation was verified by ultrasonography. On Days 16 and 17, experimental procedures were identical to those described in Experiment 1. 2.2.3. Experiment 3 Cattle were submitted to a 28-d adaptation period, receiving 2 kg/d of a soy meal (40%) and cornmeal (60%) based concentrate (TDN 73%; CP 22%; NDF 10.5%), with ad libitum access to cattle mineralized salt, water, and pasture with Brachiaria decumbens (DM 51.3%; TDN 54.3%; CP 4.6%; NDF 78.6%; and ADF 43.2%). On Day 0, cattle were allocated to receive daily allocations of either 2 kg (n = 4) or 8 kg concentrate (n = 7), divided in two meals (in the morning and afternoon). This experiment was replicated
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as a crossover, so that females that received 2 kg/d concentrate during the first period received 8 kg/d concentrate in the second period, and vice versa. This arrangement defined Experimental Groups 2/8 and 8/2, respectively. Starting on the day of second GnRH injection of the OvSynch protocol, and continuing daily for the duration of the experiment, changes in ovarian follicles and corpora lutea were assessed by ultrasonography and blood samples were collected. Blood samples (for determination of plasma P4 concentrations) were collected immediately before and 4 h after the morning concentrate feeding. On Day 16, an IV catheter was installed in a jugular vein. On Day 17, cattle were given 100 IU OT (Ocitocina, Univet, São Paulo, SP, Brazil) iv, and blood samples were collected 60, 30, and 0 min before OT treatment, and 15, 30, 45, and 60 min after treatment. Plasma was harvested and stored as described in Experiment 1. There was a 10-d interval between Day 17 of the first period and the first GnRH injection of the OvSynch protocol of the second period. All animals were fed 4 kg concentrate/d between Day 17 of the first period and Day 0 of the second period. 2.3. Measurement of plasma concentrations of P4 and PGFM 2.3.1. P4 In Experiments 1 and 3, plasma P4 concentrations were measured with a commercial solid-phase radioimmunoassay kit, in accordance with the manufacturer’s instructions (Coat a Count, Diagnostic Products Corporation, Los Angeles, CA, USA). In Experiment 1, samples were measured in a single assay and the intra-assay coefficient of variation was 3.1%. In Experiment 3, the intra-assay coefficient of variation was 5.3%, whereas the inter-assay coefficient of variation was 5.0%. Assay sensitivity was 0.03 ng/ml. In Experiment 2, plasma P4 concentrations were measured by radioimmunoassay, as described by Badinga et al. (1992) and modified by Carrière and Lee (1994). The intra-assay coefficient of variation was 4.0% and the inter-assay coefficient of variation was 0.9%. Assay sensitivity was 0.09 ng/ml. 2.3.2. PGFM Plasma PGFM concentrations were measured by radioimmunoassay, as described (Meyer et al., 1995). Intra- and inter-assay coefficients of variation were 7.5 and 4.2%, respectively. Assay sensitivity was 42.4 pg/ml. 2.4. Statistical analysis Data were tested for normality of residues (Shapiro–Wilk test) and homogeneity of variances (FMax test) for each variable. Variables which did not fulfill assumptions for analysis of variance were transformed by log10 or square-root and re-analyzed. For clarity, only untransformed data are presented. Discrete variables were analyzed by least squares ANOVA, using the GLM procedure of SAS software (SAS, 1988) and repeated measures variables were analyzed using the MIXED procedure of SAS. For the MIXED procedure, fit statistic parameters for unstructured, compound symmetry and autoregressive(1) covariance structures were tested. The autoregressive(1) structure provided the lowest values for fit statistic parameters and was used for all repeated measures analysis. Probability values 0.05 ≤ P ≤ 0.1 were considered as a tendency to significance. 2.4.1. Experiment 1 Plasma PGFM concentrations over time were analyzed after square-root transformation. The MIXED procedure mathematical model contained the fixed effects of treatment, time, and treatment × time interaction and random effect of cow nested within treatment. Plasma P4 concentration on Day 17 was not transformed and the GLM procedure mathematical model contained the fixed effect of treatment. 2.4.2. Experiment 2 Plasma PGFM concentrations over time were analyzed after log10 transformation. Plasma P4 concentrations on Day 17 was not transformed. Mathematical models were as in Experiment 1.
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Fig. 1. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to estradiol-17 injections (3 mg; experimental hour 0 of Day 17 of the estrous cycle) given to cows treated with 0 mg (control; n = 9) or 20 mg LH (LH; n = 7) or submitted to follicle aspiration (ASP; n = 8) on Day 6 of the estrous cycle.
2.4.3. Experiment 3 Daily plasma P4 concentrations, measured 4 h after the morning concentrate feeding, and concentrations of PGFM were square-root transformed. The MIXED procedure mathematical model contained the fixed effects of group, period, day or minute of sampling (for P4 and PGFM concentrations, respectively) and interactions, and the random effect of cow nested within group. 3. Results 3.1. Experiment 1 All cattle treated with LH had an accessory CL on Day 17. On Day 17, plasma P4 concentrations were (mean ± S.E.M.) 4.8 ± 0.54, 5.93 ± 0.58 and 5.5 ± 0.64 ng/ml for the control, LH and aspiration treatments, respectively (P > 0.10). The release of PGFM tended (P < 0.10) to be lower in the aspiration treatment (Fig. 1). 3.2. Experiment 2 Ovulation from the first-wave dominant follicle in response to hCG injection was not detected in two animals and they were excluded from the experiment. On Day 17, all remaining cattle given hCG had an accessory CL and more elevated plasma P4 concentrations than the control treatment (7.1 ± 0.72 and 5.0 ± 0.58 ng/ml, respectively; P < 0.05). Plasma PGFM concentrations were greater for cows treated with hCG than for cows in the control treatment (treatment × time interaction; P < 0.05; Fig. 2); hCG treatment resulted in greater PGFM concentrations between 4 and 5.5 h after E2 . 3.3. Experiment 3 Animals in Group 2/8 had greater plasma P4 concentrations than Group 8/2 in Period 1 (2.8 ± 0.23 compared with 2.2 ± 0.16 ng/ml), while the opposite occurred in Period 2 (1.78 ± 0.21 compared with 2.79 ± 0.17 ng/ml; group × period interaction; P < 0.01; Fig. 3). The significant group × period interaction means that amount of concentrate fed affected plasma P4 . Moreover, differences between groups increased over time (group × period × time interaction; P < 0.01). A significant group × period interaction was detected in each day between Days 9 and 16 (P < 0.02), except for Day 13. Animals in Group 2/8 had greater plasma PGFM concentrations than Group 8/2 in Period 1 (173.11 ± 35.52 compared with 82.69 ± 16.26 pg/ml), while the opposite occurred in Period 2 (75.75 ± 18.77 compared with 102.59 ± 18.77 pg/ml; group × period interaction; P < 0.05; Fig. 4). Con-
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Fig. 2. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to estradiol-17 injections (3 mg; experimental hour 0 of Day 17 of the estrous cycle) given to cows treated with 0 IU (control; n = 9) or 5000 IU (hCG; n = 7) of hCG on Day 6 of the estrous cycle. * Treatment means differed in a given time point (P < 0.05).
centrations of PGFM increased in response to the injection of OT (effect of time; P < 0.01). However, magnitude of increase tended to be greater for animals in Group 2/8 in Period 1 but smaller in Period 2, compared to Group 8/2 (group × period × time interaction; P < 0.1). 4. Discussion In the present study, plasma P4 concentrations in dairy cattle were either increased pharmacologically (Experiment 2) or decreased nutritionally (Experiment 3), and responsiveness to PGF2␣ -releasing
Fig. 3. Least squares means (±S.E.M.) of daily progesterone concentrations in plasma samples colleted during the first 16 d of estrous cycles from cows fed 2 kg/d concentrate in Period 1 and 8 kg/d concentrate in Period 2 (Group 2/8; n = 4) or 8 kg/d concentrate in Period 1 and 2 kg/d concentrate in Period 2 (Group 8/2; n = 7).
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Fig. 4. Least squares means (±S.E.M.) of plasma PGFM concentrations in response to 100 IU oxytocin injected on Day 17 of the estrous cycle in cows fed 2 kg/d concentrate in Period 1 and 8 kg/d concentrate in Period 2 (Group 2/8; n = 4) or 8 kg/d concentrate in Period 1 and 2 kg/d concentrate in Period 2 (Group 8/2; n = 7).
stimuli evaluated on Day 17. In accordance with expectations, PGFM concentrations in response to OT or E2 were greater in cattle with more elevated plasma P4 concentrations. In Experiment 1, 20 mg LH on Day 6 of the estrous cycle induced formation of an accessory CL but did not significantly change plasma P4 concentrations on Day 17. This was unexpected and in variation with other reports in which GnRH was used to induce accessory CLs (Schmitt et al., 1996; Howard et al., 2006), but the reason is unclear to authors. Consistently, E2 -stimulated PGF2␣ release was similar between LH and control treatments. In contrast, plasma PGFM concentrations over time tended to be less with the aspiration treatment. Both LH and aspiration treatments removed the first-wave dominant follicle abruptly, and hastened emergence of the second wave of follicular growth (data not shown). The fact that the pattern of E2 -stimulated PGF2␣ release tended to differ between aspirated and LH-treated animals despite similar effects in reprogramming of follicular growth is intriguing. This finding could be interpreted to mean that removal of the dominant follicle with or without formation of an accessory CL modulates PGF2␣ release differently. Further experimentation would be necessary to elucidate mechanisms controlling this process. In Experiment 2, hCG was given to induce ovulation from the first-wave dominant follicle and to induce an accessory CL, in an attempt to more efficiently increase plasma P4 concentrations relative to the LH treatment used in Experiment 1 (Price and Webb, 1989; Fricke et al., 1993). Indeed, treatment with hCG increased plasma P4 concentrations on Day 17 in comparison to control treatment. Seguin et al. (1977) reported that hCG had a longer circulating lifespan than LH in cows; this apparently results in greater capacity than LH to stimulate P4 synthesis in both the original and accessory CLs. In contrast to Experiment 1, cows with an hCG-induced accessory CL released more PGF2␣ in response to E2 than did control cows; we attributed the greater PGF2␣ release to the more elevated plasma P4 concentrations in these animals.
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In Experiment 3, interpretation of significant period × group and period × group × day interactions indicate that cows given 8 kg/d concentrate had lesser plasma P4 concentrations through the luteal phase. This was due presumably to a greater rate of P4 metabolism, as reported in ewes (Parr et al., 1993) and cows (Vasconcelos et al., 2003). Parr et al. (1993) and Sangsritavong et al. (2002) reported that blood flow in the hepatic portal vein was directly related to the nutrient density of the diet. Because the liver was 96% efficient in clearing circulating P4 , higher blood flow to the digestive system favors more rapid metabolism of P4 by the liver. Thus, females fed more concentrate had reduced plasma P4 concentrations. This rationale is consistent with results in Experiment 3, in which animals fed less concentrate had increased plasma P4 concentrations. Furthermore, less concentrate feeding was associated with a greater release of PGFM in response to OT. One possible explanation for the present findings is that treatments leading to decreased P4 concentrations may have led to a biochemical milieu less favorable to production of PGF2␣ in response to E2 and OT. Indeed, P4 stimulates accumulation of arachidonic acid and PTGS2 in endometrial cells, which is essential for the synthesis of PGF2␣ (Boshier et al., 1987; Silvia et al., 1991; Kombé et al., 2003). In addition, Vallet et al. (1990) showed that P4 stimulated the basal release of PGF2␣ in vitro by stimulating the accumulation of enzymes and substrates for the synthesis of PGF2␣ , such as phospholipids, prostaglandin synthase (Salamonsen et al., 1990) and phospholipase C (Raw and Silvia, 1991). According to McCracken et al. (1999), in the beginning of the estrous cycle, P4 binds to its endometrial receptors and blocks E2 -stimulated synthesis of OT receptors. In the middle of the estrous cycle, P4 down-regulates its own receptors, which results in the release of the P4 block to the action of E2 . Consequently, OT receptor synthesis is stimulated. Therefore, another possible explanation for the lesser plasma PGFM concentrations in cows receiving control treatment (Experiment 2) and in cows that received 8 kg/d concentrate (Experiment 3) is that lesser P4 up to Day 17 of the estrous cycle may have delayed down-regulation of P4 receptors (Spencer et al., 2004). This could have resulted in a prolonged inhibition in E2 - and OT-stimulated PGF2␣ release. Collectively, the three current experiments supported the initial hypothesis that E2 - or OTstimulated PGF2␣ release is greater in cows with elevated P4 plasma concentrations, which agrees with previous reports. For example, Lafrance and Goff (1988) reported that release of PGF2␣ in response to E2 and OT treatments in ovariectomized heifers was less, but such release increased after treatment with P4 for 7, 14, or 21 d. Skarzynski et al. (1999) noted that the endometrial tissue acquired maximum capacity for PGFM synthesis in response to E2 after pre-incubation with P4 or luteal cells. In contrast, present results are in variance to results reported by Mann and Lamming (1995), in studies with ovariectomized cows supplemented with P4 and E2 . These authors observed greater concentrations of PGFM in cows which had lower concentrations of P4 . In addition, in vitro studies reported by Bogacki et al. (2002) showed that, during the late luteal phase, P4 suppressed the ability of OT to induce the secretion of endometrial PGF2␣ by directly interfering with OT receptor binding. Based on the P4 modulation of PGF2␣ verified here in cows that have initiated estrous cycling, it is tempting to speculate about possible implications for pregnant cows. Specifically, the greater production of PGFM in cows with elevated concentrations of P4 demonstrated in the present research seems to contradict several studies which reported greater pregnancy rates in cows treated to have elevated concentrations of P4 (Santos et al., 2001; Marques, 2002). Based on results of the present study, it is speculated that P4 does not act directly in the inhibition of PGF2␣ release. Rather, P4 may favor conceptus development (Garret et al., 1988) and synthesis of interferon-tau (Thatcher et al., 1997), which inhibits PGF2␣ biosynthesis in the endometrium to block luteolysis (i.e. maternal recognition of pregnancy; Mann and Lamming, 2001). However, effects of P4 on endometrial release of PGF2␣ measured in females having estrous cycles may differ from effects in pregnant females due to conceptus signals. Indeed, Gray et al. (2006) reported that P4 modulated expression of endometrial genes both dependent and independent of interferon-tau in the sheep. Specific studies must be conducted to further understand the role of timing and magnitude of P4 increase on E2 - and OT-stimulated PGF2␣ release, both in estrous cycling and pregnant cows. In conclusion, the initial hypothesis for the present research was confirmed as cows treated both hormonally and nutritionally to have elevated P4 concentrations also released more PGF2␣ in response to treatments with OT and E2 .
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