2019 Prostaglandin F2? induced luteolysis is delayed but not prevented by acute administration of luteotropic drugs in lactating nonpregnant Holstein cows Prostaglandin F2α induced luteolysis is delayed but not prevented by acute administration of luteotropic drugs in lactating nonpregnant Holstein cows Divakar Ambrose,a,b Marcos Colazo,a Mohanathas Gobikrushanthb aLivestock and Crops Research Branch, Alberta Agriculture and Forestry, Edmonton, AB, Canada bDepartment of Agricultural, Food and Nutritional Science University of Alberta, Edmonton, AB, Canada Abstract Inadvertent use of prostaglandin F2α (PGF2α) in pregnant cattle could result in luteolysis and pregnancy loss. Using a nonpregnant cow model, we determined if luteotropic agents could counteract luteolytic effects of PGF2α. Ovarian status of 20 lactating nonpregnant Holstein cows was synchronized using an Ovsynch protocol and ovulation confirmed by transrectal ultrasonography 48 hours after the second gonadotropin releasing hormone (GnRH). Eight days after ovulation, 25 mg of dinoprost (native PGF2α) was administered IM to induce luteolysis. Five minutes after PGF2α, cows were treated IM with gonadorelin (GnRH, 100 μg; n = 5), human chorionic gonadotropin (hCG, 1,000 IU; n = 6), porcine pituitary luteinizing hormone (pLH, 25 mg; n = 5), or saline (control, 2 ml; n = 4). Blood samples were collected before PGF2α (0 hour) and at 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72, and 84 hours after PGF2α to monitor subsequent luteal activity by measuring plasma progesterone concentrations. Although none of the treatments counteracted the luteolytic action of PGF2α, the rate of decline in plasma progesterone within 1 hour after PGF2α was greater (p = 0.04) in control than in GnRH, hCG, and pLH (2.2 versus 0.2, 0.3, and 0.1 ng/ml/hour, respectively). In addition, pLH-treated cows tended (p = 0.07) to have greater overall mean plasma progesterone for up to 84 hours (1.6 ± 0.2 ng/ml) than other treatments combined (1.1 ± 0.1 ng/ml). In the present study, giving GnRH, hCG or pLH 5 minutes after PGF2α administration was not effective in countering luteolytic effects of PGF2α. Nevertheless, pLH treatment tended to delay PGF2α induced luteolysis, which warrants further investigation. Keywords: Bovine pregnancy, abortion, corpus luteum, porcine pituitary LH, progesterone Introduction Corpus luteum (CL), a transient endocrine gland, undergoes dynamic changes to regulate reproductive cyclicity and thereby fertility of mammals. It produces the steroid hormone progesterone (P4) that has a key role in establishment and maintenance of pregnancy. In the absence of a conceptus, CL undergoes luteolysis due to prostaglandin F2α (PGF2α) released from the uterus.1 In case of pregnancy, uterine release of PGF2α is blocked by bovine interferon-tau, the signal of maternal recognition of pregnancy from placental trophoblast cells in ruminants.2 As approved products for reproductive management, both native (dinoprost) and synthetic (cloprostenol) preparations of PGF2α are used routinely in dairy cattle, especially in estrus and ovulation synchronization protocols, integral to herd breeding programs. As pregnancy maintenance in cattle, at least up to 150 days of gestation is dependent on luteal P4,3 inadvertent use of PGF2α, especially during the first 5 months of pregnancy, usually results in abortion. Under such a circumstance, an immediate remedial action that either prevents or delays PGF2α-induced luteolysis would be beneficial, at least until exogenous P4 therapy could be initiated. That bovine luteinizing hormone (LH) and human chorionic gonadotropin (hCG) have antiluteolytic properties in cattle was first demonstrated in Holstein heifers subjected to oxytocin- induced luteolysis.4 In early studies in ewes, ovine LH,5 and gonadotropin releasing hormone (GnRH),6 extended CL lifespan of, possibly by delaying or countering action of PGF2α. In rats, antiluteolytic effect of hCG was evident by pregnancy maintenance in 70% of PGF2α-treated pregnant rats.7 In studies from our laboratory, in cattle treated with exogenous porcine LH (pLH), plasma LH concentrations remained higher than that of controls for up to 20 hours after 599 Clinical Theriogenology • Volume 11, Number 4 • December 2019 treatment.8-10 Higher than basal concentrations of LH for a prolonged interval in pLH-treated cattle may protect the CL from luteolytic action of PGF2α by saturation of luteal membrane LH receptors, as hypothesized.11 All of the above products (hCG, GnRH, and pLH) are approved for use in dairy cattle in Canada. However, efficacy of these products as luteotropic agents to counteract PGF2α-induced luteolysis in cattle has not been adequately investigated. We hypothesized that a luteotropic agent given to cows soon after PGF2α administration prevents or delays luteolysis, as indicated by greater P4 concentrations compared to cows not given a luteotropic treatment. Therefore, our objective was to determine if luteolytic effects of PGF2α in dairy cattle could be counteracted by GnRH, hCG or pLH given 5 minutes after PGF2α administration. Materials and methods Cattle and housing The study was conducted at the Dairy Research and Technology Centre of the University of Alberta. Cattle were handled and cared for in accordance with the Canadian Council on Animal Care guidelines (2009)12 and experimental procedures were approved by the Animal Policy and Welfare Committee, Department of Agricultural, Food and Nutritional Science, University of Alberta. Twenty-four lactating nonpregnant Holstein cows (7 primiparous and 17 multiparous) were enrolled in the study. Cows were individually fed a total mixed ration (primary ingredients were barley silage, alfalfa silage, alfalfa hay, and concentrates) and housed in tie stalls and let out for approximately 2 hours of exercise during weekdays. Diets were formulated according to NRC (2001)13 to meet requirements of a 650 kg lactating cow producing 30 kg of milk/day and delivered individually by a Data Ranger (American Calan Inc., Northwood, NH), once daily at 0800 hour. Cows had ad libitum access to fresh water. Cows averaged (mean ± standard deviation) 3.0 ± 0.4 lactation and 266 ± 25 day postpartum at the beginning of the experiment. Average 305 days milk yield was 9,540 kg. Experimental design and treatments Ovarian status of cows was synchronized using an Ovsynch protocol.14 In brief, the protocol consisted of GnRH (100 μg gonadorelin acetate, IM; Fertiline®, Vetoquinol N. A. Inc. Lavaltrie QC, Canada), followed 7 days later by PGF2α (25 mg dinoprost tromethamine, IM; Lutalyse®, Zoetis Canada Inc. Kirkland, QC, Canada), and a second GnRH (100 μg gonadorelin acetate, IM) given 48 hours after PGF2α. Transrectal ultrasonography (Aloka 500, Aloka Co Ltd., Tokyo, Japan) using a 7.5 MHz linear-array transducer, was first conducted at the time of the second GnRH of Ovsynch to confirm the presence of putative ovulatory follicle(s) (≥ 10 mm in diameter). Ovulation was confirmed 48 hours later by absence of a follicle previously detected. Eight days after ovulation, PGF2α (25 mg dinoprost tromethamine, IM) was administered to induce luteolysis in all cows. Luteotropic treatments were randomly administered IM (n = 6 per treatment), exactly 5 minutes after PGF2α administration. Interval between PGF2α and luteotropic treatments was an arbitrary yet realistic time frame under field conditions, to take remedial action, if PGF2α was administered inadvertently to a pregnant animal and the mistake quickly discovered. Treatments were: GnRH (100 µg gonadorelin acetate, IM; Fertiline®); hCG (1,000 IU human chorionic gonadotropin, IM; Chorulon®, Intervet, Kirkland, QC, Canada); pLH (25 mg porcine pituitary LH, IM, Lutropin-V, Bioniche Animal Health, Belleville, ON, Canada); or control (2 ml sterile saline, IM). Blood sampling and progesterone assay Blood samples were collected into evacuated tubes containing sodium heparin (Vacutainer, Beckton Dickinson and Co., Franklin Lakes, NJ) from an indwelling jugular 600Clinical Theriogenology • Volume 11, Number 4 • December 2019 catheter. Blood samples were collected immediately before PGF2α (0 hour), 1 and 6 hours after PGF2α, then every 6 hours until 48 hours, and thereafter every 12 hours for the next 36 hours, up to 86 hours after PGF2α treatment. Samples were placed on ice immediately after collection and centrifuged at 1500 x g for 20 minutes at 4°C, plasma harvested and frozen at -20 °C until assayed for P4, in duplicate, using a direct enzyme immunoassay (Quanticheck , Faculty of Veterinary Science, Budapest, Hungary). This assay uses an antiP4 monoclonal antibody and horseradish peroxidase as the enzyme label, with a sensitivity (lowest detection limit) of 0.5 ng/ml and has been compared to established assays.15 The intra-assay coefficient of variation was 6.5%. Statistical analyses Differences in plasma P4 concentrations by luteotropic treatment (GnRH, hCG, pLH, or saline), time of sampling (0 hour before PGF2α and 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72, and 84 hours after PGF2α), parity (primiparous and multiparous), and interactions between luteotropic treatment and time were tested by repeated measures analysis using MIXED procedure of SAS. Four cows (GnRH, n = 2; hCG and pLH, n = 1 each) with P4 concentrations < 1 ng/ml at 0 hour (before PGF2α), indicative of either delayed or poor luteal function, were excluded from the analysis. In the remaining 20 cows, P4 concentration at 0 hour was used as a covariate to account for potential influence of P4 at time of PGF2α administration on subsequent P4 concentrations. Time of sampling was used as a repeated measurement nested within cow. Covariance structure of the repeated measurement and appropriate final model were chosen based on the lowest Akaike’s Information Criteria. Since pLH-treated cows had greater mean plasma P4 concentration than GnRH-treated cows, a pairwise comparison between pLH and other groups combined (control, GnRH, and hCG) was also performed using an orthogonal contrast statement. In addition, differences among luteotropic treatments in rate of decline in mean plasma P4 concentrations from 0 to 1 hour after giving PGF2α, were tested using MIXED procedure. Differences in least square means were tested using PDIFF option. Significant differences were reported if p ≤ 0.05 and considered a tendency if p > 0.05 and ≤ 0.10. Results Changes in mean plasma P4 concentrations by luteotropic treatment, time and their interactions are presented in Figure 1. None of the luteotropic treatments countered the luteolytic effect of PGF2α (p = 0.24), as evident from declines in P4 concentrations. The rate of decline in plasma P4 concentrations (ng/ml/hour) within the first hour after PGF2α administration, however, differed (p = 0.04) between luteotropic treatments. In this regard, cows treated with GnRH (0.2 ± 0.2), hCG (0.3 ± 0.2), or pLH (0.1 ± 0.2) had slower rates of decline than cows treated with saline (2.2 ± 0.2 ng/ml/hour; Figure 2). Moreover, orthogonal contrast analysis revealed that pLH-treated cows tended to have greater overall mean P4 concentration from both 0 to 84 hours (1.6 ± 0.2 versus 1.1 ± 0.1 ng/ml; p = 0.07) and from 12 to 84 hours (1.0 ± 0.2 versus 0.5 ± 0.1 ng/ml; p = 0.10) after PGF2α administration, compared to mean P4 concentrations for other treatments combined. Regardless of luteotropic treatment, mean (± SEM) plasma P4 concentrations (ng/ml) differed by time (p < 0.01), declining from 0 to 12 hours after PGF2α (4.1 ± 0.2 [0 hour], 3.6 ± 0.2 [1 hour], 2.3 ± 0.2 [6 hour] and 0.8 ± 0.2 [12 hour]) and then remained unchanged from 12 to 84 hours after PGF2α, ranging from 0.4 to 0.7 ng/ml. Discussion Bovine LH and hCG in heifers,4 ovine LH and GnRH in ewes,5,6 and hCG in rats,7 were reported to have antilutelolytic properties against exogenous oxytocin, endogenous PGF2α, exogenous estradiol and PGF2α, respectively. On the contrary, in the present study, none of the agents, at the administered dose, prevented luteolysis in the nonpregnant cow model, as evident from declines in P4 concentrations from 0 to 84 hours after PGF2α administration. 601 Clinical Theriogenology • Volume 11, Number 4 • December 2019 Few studies have evaluated efficacy of luteotropic agents in preventing or reversing luteolysis in ruminants. In a pioneering study4 in cyclic Holstein heifers, antiluteolytic effects of bovine LH (30 mg daily), hCG (2,000 IU), urea-treated hCG (2,000 IU) and bovine prolactin (60 mg daily) on oxytocin (0.33 USP units/kg BW) induced luteolysis were evaluated. Both bovine LH and hCG when given concurrently with oxytocin treatment not only overcame luteolytic effects of oxytocin, but also increased CL weight and luteal progesterone concentration, indicative of luteotropic effects. When the LH component of hCG was destroyed by urea treatment before administration, the preparation was no longer luteotropic, indicating that LH is the luteotropin in cattle. Likewise, prolactin also had no luteotropic effect. In a contrasting study,16 continuous IV infusions of purified bovine LH (10 μg/minute) for 10 hours, starting 4 hours prior to giving 25 mg PGF2α, at days 10 - 12 of the estrous cycle in beef cattle, failed to prevent PGF2α-induced luteolysis. Intravenous infusions of ovine LH (4 μg/minute), ovine prolactin (42 μg/minute), or LH + prolactin for 12 hours, failed to prevent luteolysis in ewes following IM injections of 6.66 mg PGF2α given 2 and 6 hours after infusions began.17 On the contrary, prolactin had an antiluteolytic effect in pregnant rats when given on day 4 of gestation,18 and maintained pregnancy in 90% of rats when given on day 10 of gestation.7 Prolactin and prolactin receptor mRNA are expressed in bovine CL throughout the estrous cycle, suggesting a role for prolactin in luteal function in cattle.19 However, as pioneering studies in cattle4 and sheep17 reported prolactin not to prevent luteolysis, it may not be a suitable candidate for further consideration as an antiluteolytic agent. Conversely, pLH is a potential candidate for further research as an antiluteolytic agent for two reasons. First, the rate of P4 decline in the first hour after PGF2α administration was the smallest and second, the overall mean plasma P4 concentration up to 84 hours was highest in pLH-treated cows in the present study. Although the overall mean P4 concentration of 1.6 ng/ml in pLH-treated cows was greater than that of all other treatments, such a low P4 concentration during diestrous is likely insufficient to sustain pregnancy, particularly in lactating dairy cows that have lower plasma P4 concentrations, likely due to increased P4 metabolism.20 Small and repeated doses of hCG prevented estradiol-induced luteolysis in macaques (Macaca fascicularis).21 Therefore, using a conceptually similar approach, it would be worth investigating whether repeated doses of pLH treatment would effectively counteract luteolysis in cattle. Inducing formation of a new CL could be another strategy to increase endogenous P4 to maintain pregnancy. However, this process takes time and cannot be implemented quickly when PGF2α is inadvertently administered to pregnant cattle under field conditions. In this regard, when an induced CL formed on the ovary ipsilateral to the pregnant horn after forced lysis of the original CL (while the pregnancy was temporarily sustained by exogenous P4), was capable of maintaining the pregnancy even after exogenous P4 supplementation ended.22,23 More recently, it was reported24 that 72% (18 of 25) of pregnant beef cows and heifers maintained their pregnancies for at least 7 days after induced luteolysis, when exogenous P4 treatments (100 mg SC daily for 7 days) were given, with the first P4 injection starting 2, 6, 10, 12, or 18 hours after PGF2α was given during days 30 - 90 of pregnancy. When an accessory CL was successfully induced using hCG in a subset of those cattle (mean ± SD, 38 ± 5.5 days after PGF2α), 80% carried the pregnancy to term.24 Authors concluded that pregnancies could be maintained following an accidental administration of PGF2α without the need for extended P4 supplementation, when an accessory CL was successfully induced. An accessory CL could be induced with GnRH or pLH, perhaps as effectively as hCG. However, to prevent P4 concentrations from plummeting to basal concentrations after accidental PGF2α administration, a quick remedial measure may be to concurrently insert two intravaginal P4 devices to maintain higher circulating P4 concentration (2.7 ng/ml)25 than attainable with a single P4 device (1.0 ng/ml),26 thereby protecting the embryo from exposure to very low P4 concentrations due to PGF2α-induced decline in luteal P4. As many dairy farms and bovine practitioners are likely to have intravaginal progesterone devices readily available, this approach 602Clinical Theriogenology • Volume 11, Number 4 • December 2019 would be more practical than progesterone injections. Thus, future studies should investigate a combined treatment approach of repeated injections of pLH to maximize LH support to the CL exposed to PGF2α and insertion of two intravaginal progesterone devices to maintain P4 concentrations that could sustain a pregnancy. If a pregnancy could be sustained temporarily with this combined treatment strategy, inducing an accessory CL at the earliest opportunity should be actively considered as a permanent solution to sustain the pregnancy to term, as demonstrated in cattle.24 Although pLH is available in the Canadian market, to the authors’ knowledge there is no FDA approved pLH that is currently available in the United States. The treatment strategies proposed in this paper would be extra label use in many jurisdictions; therefore, any such treatment must occur under the order and discretion of a veterinarian. Conclusion In the present study, giving GnRH, hCG or pLH 5 minutes after PGF2α administration were not effective in countering luteolytic effects of PGF2α in a nonpregnant lactating cow model. Nevertheless, pLH treatment tended to delay PGF2α-induced luteolysis, which warrants further investigation. Annotation Using any of the luteotropic drugs or exogenous progesterone for the purpose described in the manuscript may constitute extra label use and should only be used at the discretion of a practitioner who has an established veterinarian-patient-client-relationship. Acknowledgement Authors acknowledge technical assistance of Govindarajan Thangavelu (University of Alberta) during this study. Product donations by Bioniche Animal Health (Lutropin-V) and Vetoquinol Canada Inc. (Fertiline) are also gratefully acknowledged. Authors’ contribution DA: Study concept, experimental design, data collection, manuscript writing; MC: experimental design, data collection, progesterone assays, manuscript review; MG: data analysis, manuscript writing. Conflict of interest Authors declare no conflicts of interest. References 1. Thatcher WW, Wolfenson D, Curl JS, et al: Prostaglandin dynamics associated with development of the bovine conceptus. Anim Reprod Sci 1984;7:149-176. 2. Bazer FW, Song G, Thatcher WW: Roles of Conceptus secretory proteins in establishment and maintenance of pregnancy in ruminants. Asian Australas J Anim Sci 2012;25:1-16. 3. Gross TS, Williams WF: In vitro steroid synthesis by the placenta of cows in late gestation and parturition. J Reprod Fertil 1988;83:565-573. 4. Donaldson LE, Hansel W, Van Vleck LD: Luteotropic properties of luteinizing hormone and nature of oxytocin induced luteal inhibition in cattle. J Dairy Sci 1965;48:331-337. 5. Karsch FJ, Roche JF, Noveroske JW, et al: Prolonged maintenance of the corpus luteum of the ewe by continuous infusion of luteinizing hormone. Boil Reprod 1971;4:129-136. 6. Adams TE, Kinder JE, Chakraborty PK, et al: Ewe luteal function influenced by pulsatile administration of synthetic LHRH/FSHRH. Endocrinol 1976;97:1460-1467. 7. Chatterjee A: The possible mode of action of prostaglandins: X. Antagonism between prostaglandin F2a and prolactin or human chorionic gonadotropin: a comparative study. Prostaglandins 1976;12:525-534. 8. Ambrose JD, Kastelic JP, Rajamahendran R, et al: Progesterone (CIDR)-based timed AI protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. Theriogenology 2005;64:1457-1474. 603 Clinical Theriogenology • Volume 11, Number 4 • December 2019 9. Ree TO, Colazo MG, Lamont AGA, et al: The effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlacatating cows. Theriogenology 2009;72:120-128. 10. Behrouzi A, Colazo MG, Ambrose DJ: Alterations in bone morphogenetic protein 15, growth differentiation factor 9, and gene expression in granulosa cells in preovulatory follicles of dairy cows given porcine LH. Theriogenology 2016;85:1249-1257. 11. Henderson KM, McNatty KP: A biochemical hypothesis to explain the mechanisms of luteal regression. Prostaglandins 1975;9:779-797. 12. Canadian Council on Animal Care Guidelines on the Care and Use of Farm Animals in Research, Teaching and Testing. 2009. http://ccac.ca/Documents/Standards/Guidelines/Farm_Animals.pdf 13. National Research Council. Nutrient Requirements of Dairy Cattle. 7th edition, National Academic Press, Washington, DC. 2009. 14. Pursley JR, Mee MO, Wiltbank, MC: Synchronization of ovulation in dairy cows using PGF2α and GnRH. Theriogenology 1995;44:915-923. 15. Colazo MG, Ambrose DJ, Kastelic JP, et al: Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk. Can J Vet Res 2008;72:32-36. 16. González-menció F, Murphy BD, Manns J:Failure of exogenous LH to prevent PGF2α-induced luteolysis in beef cows. Prostaglandins 1977;14:535-542. 17. Sasser RG, Niswender GD, Nett TM: Failure of LH and/or prolactin to prevent PGF2α-induced luteolysis of ovine corpora lutea. Prostaglandins 1977;13:1201-1208. 18. Fuchs A, Mok E, Sundaram K: Luteolytic effects of prostaglandins in rat pregnancy, and reversal by luteinizing hormone. Eur J Endocrinol 1974;76:583-596. 19. Shibaya M, Murakami S, Tatsukawa Y, et al: Bovine corpus luteum is an extrapituitary site of prolactin production. Mol Reprod Dev 2006;73:512-519. 20. Sangsritavong S, Combs DK, Sartori RF, et al: High feed intake increases liver blood flow and metabolism of progesterone and estradiol 17b in dairy cattle. J Dairy Sci 2002;85:2831-2842. 21. Westfahl PK, Horton LE, Stadelman HL, et al: Small doses of human chorionic gonadotropin prevent estradiol-induced luteolysis. Endocrinol.Metab 1984;10:E84-E87. 22. Lulai C, Dobrinski I, Kastelic JP, et al: Induction of luteal regression, ovulation and development of new luteal tissue during early pregnancy in heifers. Anim Reprod Sci 1994;35:163-172. 23. Bridges PJ, Wright DJ, Buford WI, et al: Ability of induced corpora lutea to maintain pregnancy in beef cows. J Anim Sci 2000;78:2942-2949. 24. Ferguson CE, Kesler DJ, Godke RA: Maintenance of pregnancy in beef cattle after a luteolytic dose of prostaglandin F2α. Vet Med Anim Sci 2014;2:1. 25. Bisinotto RS, Ribeiro ES, Lima F, et al: Targeted progesterone supplementation improves fertility in lactating dairy cows without a corpus luteum at the initiation of the timed artificial insemination protocol. J Dairy Sci 2013;96:2214-2225. 26. Cerri RL, Rutigliano HM, Chebel RC, et al: Progesterone concentration, follicular development and induction of cyclicity in dairy cows receiving intravaginal progesterone inserts. Anim Reprod Sci 2009;110:56-70. 604Clinical Theriogenology • Volume 11, Number 4 • December 2019 Figure 1. Plasma progesterone (P4) concentrations at administration of PGF2α (25 mg dinoprost; 0 hour) and at 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72 and 84 hours after either a luteotropic or saline (control) treatment given 5 minutes after PGF2α in dairy cows. Luteotropic treatments were gonadotropin releasing hormone (GnRH; 100 μg IM; n = 5), human chorionic gonadotropin (hCG; 1,000 IU; n = 6), porcine pituitary luteinizing hormone (pLH; 25 mg IM; n = 5) or control (sterile saline; 2 ml IM; n = 4). There was no overall counteracting effect of luteotropic treatment on PGF2α- induced luteolysis (p = 0.24); however, orthogonal contrast analysis revealed that mean P4 concentration tended to be greater in pLH treated cows up to 84 hours after PGF2α administration compared to the overall mean for other treatments (1.6 ± 0.2 versus 1.1 ± 0.1 ng/ml; p = 0.07). Irrespective of the luteotropic treatment given, plasma P4 concentrations differed (p < 0.01) by time, with the time effect being most evident between 0 and 12 hours. Concentrations of P4 remained unchanged between 12 and 84 hours after PGF2α treatment. However, there was no interaction between categories of luteotropic treatment and time (p = 0.80). 0 1 2 3 4 5 6 0 1 6 12 18 24 30 36 42 48 60 72 84 Pl as m a pr og es te ro ne (n g/ m l) Hours relative to PGF2α administration Control GnRH hCG pLH Treatment; p = 0.24 Time; p < 0.01 Treatment x time; p = 0.80 605 Clinical Theriogenology • Volume 11, Number 4 • December 2019 Figure 2. Mean (± SEM) rate of decline in plasma progesterone (P4) concentrations between 0 and 1 hour after PGF2α (25 mg dinoprost) administration in lactating dairy cows. A luteotropic agent or control treatment was given 5 minutes after PGF2α (0 hour). Luteotropic treatments were gonadotropin releasing hormone (GnRH; 100 μg IM; n = 5), human chorionic gonadotropin (hCG; 1,000 IU; n = 6), porcine pituitary luteinizing hormone (pLH; 25 mg IM; n = 5) or control (sterile saline; 2 ml IM; n = 4). Cows treated with GnRH (0.2 ± 0.2), hCG (0.3 ± 0.2) or pLH (0.1 ± 0.2) had a smaller rate of decline than those treated with saline (2.2 ± 0.2 ng/ml/hour). a,bColumns without a common superscript differed (p = 0.04). 2.2 0.2 0.3 0.1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Control GnRH hCG pLH Ra te o f d ec lin e in p la sm a P 4 (n g/ m l/h ou r) be tw ee n 0 an d 1 ho ur a fte r P G F 2 α Control / luteotropic treatments p = 0.04 a b b b 606Clinical Theriogenology • Volume 11, Number 4 • December 2019 Untitled