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Effects of GnRH treatment on initiation of pulses of LH, LH release, and subsequent concentrations of progesterone
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Domestic Animal Endocrinology 37 (2009) 189–195
Effects of GnRH treatment on initiation of pulses of LH, LH release, and subsequent concentrations of progesterone S.D. Fields, B.L. Perry, G.A. Perry ∗ Department of Animal and Range Sciences, South Dakota State University, Brookings, Box 2170, Brookings, South Dakota 57007, USA Received 24 March 2009; received in revised form 30 April 2009; accepted 30 April 2009
Abstract Progesterone is essential for establishment and maintenance of pregnancy. One proposed method to increase progesterone is administering GnRH at insemination. However, this method has resulted in conflicting results. Therefore, 2 experiments were conducted to evaluate how administering GnRH at insemination affected pulses of luteinizing hormone (LH) and subsequent progesterone. In Experiment 1, cows were allotted to 2 treatments: (1) GnRH (100 g) given approximately 12 h after initiation of estrus (n = 5); and (2) Control (n = 5). Blood samples were collected at 15-min intervals for 6 h at 12 (blood sampling period 1), 26 (blood sampling period 2), 40 (blood sampling period 3), 54 (blood sampling period 4), and 68 (blood sampling period 5) h after onset of estrus. Daily blood samples were collected for 17 d. In Experiment 2, cows were allotted into 2 treatments: GnRH administered 10 to 11 h (n = 10) or 14 to 15 h (n = 10) after onset of estrus. Daily blood samples were collected for 17 d. Cows treated with GnRH tended (P ≤ 0.075) to have greater LH release during blood sampling period 1, tended (P = 0.095) to have fewer pulses during blood sampling period 2, tended (P = 0.067) to have greater concentrations of progesterone, and had an earlier (P = 0.05) increase in progesterone than control cows. Cows treated with GnRH 10 to 11 h after onset of estrus had greater (P = 0.01) progesterone and an earlier (P = 0.04) increase in progesterone than cows treated 14 to 15 h. In conclusion, timing of GnRH treatment following onset of estrus influenced pulses of LH and subsequent progesterone. © 2009 Elsevier Inc. All rights reserved. Keywords: GnRH; LH pulses; CL function; Progesterone
1. Introduction Progesterone is essential for the maintenance of pregnancy and embryo development [1]. Therefore, scientists have assessed techniques to increase fertility by increasing corpus luteum (CL) function. One proposed method was to treat with GnRH at time of insemination. However, there have been conflicting results with this method. Treatment with GnRH at the time of insemination resulted in an increase [2], decrease [3,4], or no
∗
Corresponding author. Tel.: +605 688 5456; fax: +605 688 6170. E-mail address: [email protected] (G.A. Perry).
0739-7240/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2009.04.006
change [5] in subsequent concentrations of progesterone compared to controls. Moreover, research has reported that cows detected in standing estrus around the time of the GnRH treatment at fixed-time artificial insemination (FTAI) had increased pregnancy rates compared to cows not detected in estrus [6,7]. The reason for the varying results is not clear. Luteinizing hormone (LH) has been reported to be involved in CL development and function [8–10]. Pituitary LH content returned to normal within 1 d of the ovulatory LH surge [11], and when pulses of LH were blocked around the time of ovulation, cows had decreased subsequent concentrations of progesterone compared to controls [8,9]. Therefore, the objectives of
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these experiments were to determine (1) when pulses of LH resumed following an LH surge, (2) the effect GnRH treatment would have on the initiation of pulses of LH, and (3) the effect of LH pulse initiation on subsequent concentrations of progesterone. 2. Materials and methods Two experiments were conducted at the South Dakota State University’s Beef Breeding Unit. Mature Anguscross, nonpregnant, nonlactating, estrous-cycling beef cows were used in these experiments. All cows were handled according to procedures approved by the South Dakota State University Institutional Animal Care and Use Committee. 2.1. Experiment 1 2.1.1. Experimental design Stage of the estrous cycle was synchronized with the Select Synch + CIDR protocol (n = 32) [12]. Cows were administered GnRH (100 g as 2 mL of OvaCyst i.m.; IVX Animal Health, St. Joseph, MO) and a controlled internal drug releasing device (CIDR) was inserted into the vagina on d -7. An injection of prostaglandin F2␣ (PGF2␣ 25 mg as 5 mL of Prostamate i.m., IVX Animal Health) was given, and the CIDR was removed on d 0. The HeatWatch (DDX, Inc., Denver, CO) electronic estrous detection system was used to determine initiation of standing estrus. Onset of estrus was determined as the first of 3 mounts within a 4-h period that lasted 2 s or longer in duration. After 10 cows were determined to be in standing estrus within a 6-h period of time, indwelling jugular catheters were inserted into each cow. Based on interval from onset of estrus, cows were assigned to 1 of 2 treatment groups: (1) cows were treated with GnRH approximately 12 h after onset of estrus (n = 5), or (2) cows did not receive GnRH treatment and served as controls (n = 5). 2.1.2. Blood collection At the time of blood sample collection, an 8-foot tubular extension was connected to the catheter and was taped along the back of each cow. A 3-way stop plug was connected to the end of the extension. A syringe was connected to the plug, allowing for an airtight connection. Blood samples were collected via jugular catheters every 15 min for 6 h from 12 to 18 (blood sampling period 1), 26 to 32 (blood sampling period 2), 40 to 46 (blood sampling period 3), 54 to 60 (blood sampling period 4), and 68 to 74 (blood sampling period 5) h after onset of estrus. At each collection time, 3 mL of blood was drawn
from the syringe and discarded, 5 mL of fresh blood was collected for the sample, and 5 mL of saline (O.9% saline containing 120 mM Na citrate and 10 cc/L oxytetracycline) was injected back into the cow via the catheter. Daily blood samples were collected by venipuncture of the median caudal vessel into 10 mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) from d 3 to d 17 of the estrous cycle. All blood was allowed to coagulate at room temperature and then stored at 4 ◦ C for 24 h. Samples were centrifuged at 1200 × g for 30 min, and the serum was harvested and frozen at −20 ◦ C until analysis by radioimmunoassay (RIA). 2.1.3. Transrectal ultrasonography Transrectal ultrasonography was performed using an Aloka 500 V ultrasound with a 7.5-MHz transrectal linear probe (Aloka, Wallingford, CT). Both ovaries of each cow were examined 15 to 16 h after onset of estrus. All follicles >8 mm in diameter were recorded. Ovaries were examined again 32 h after onset of estrus to determine if ovulation had occurred. Ovulation was defined as the disappearance of a previously recorded large antral follicle. 2.2. Experiment 2 2.2.1. Experimental design Stage of the estrous cycle was synchronized with the Select Synch + CIDR protocol (n = 32), as previously described in Experiment 1. The HeatWatch electronic detection of estrus system was used to determine initiation of standing estrus, and parameters were set as described in Experiment 1. After cows were determined to be in estrus, they were allotted to 1 of 2 treatments: GnRH treatment given at (1) 10 to 11 h (n = 10); or (2) 14 to 15 h (n = 10) after onset of estrus. 2.2.2. Blood collection Daily blood samples were collected by venipuncture of the jugular vein into 10 mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) from d 0 to d 17 of the estrous cycle. Serum was collected as described in Experiment 1 and frozen at −20 ◦ C until analysis by RIA. 2.3. Radioimmunoassay Serum samples from the intensive blood sampling periods (Experiment 1) were analyzed for concentrations of LH by RIA using methodology described by Perry and Perry [13]. Intra- and interassay coefficients of variation for LH assays were 4.7% and 13.7% for Experiment 1. Assay sensitivity was 0.125 ng/mL. Daily
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blood samples were analyzed for serum concentrations of progesterone by RIA using methodology described by Engel et al. [14]. Intra- and interassay coefficients of variation for progesterone assays were 6.6% and 3.4% for Experiment 1 and 4.2% and 8.7% for Experiment 2. Assay sensitivity was 0.4 ng/mL. 2.4. Statistical analysis Cluster analysis [15] was used to determine area under the LH curve, average concentration of LH, and LH pulse frequency. Effects of GnRH on area under the LH curve, average concentration of LH, LH pulse frequency, and subsequent concentrations of progesterone were determined by analysis of variance (ANOVA) for repeated measures in SAS by PROC MIXED [16]. The statistical model consisted of effects of treatment, time, and treatment × time interactions. All covariance structures were modeled in the initial analysis. The indicated best fit covariance structure, compound symmetry, was used for the final analysis for the LH variables, and heterogeneous compound symmetry was used for the final analysis for progesterone. Differences between treatment groups for the interval from the onset of estrus to treatment (Experiment 1), increase in progesterone (Experiments 1 and 2), and follicle size (Experiment 1) were analyzed by ANOVA using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC; version 9.2; 2007). 3. Results 3.1. Experiment 1 3.1.1. Interval from onset of estrus to GnRH and follicle size The interval from the onset of estrus to blood sampling period 1 was similar (P = 0.82) between GnRH treated (12.5 ± 1.2 h) and control (12.1 ± 1.2 h) cows. All cows had ovulations by 32 h after onset of estrus. There was no difference (P = 0.13) in follicle size between GnRH treated (14.9 ± 0.47 mm) and control (16.0 ± 0.47 mm) cows. 3.1.2. Luteinizing hormone There was an effect of time (P = 0.003); however, there were no effects of treatment (P = 0.74) or treatment × time (P = 0.40) for area under the LH curve (Fig. 1). Blood sampling periods 1 and 5 had a greater (P ≤ 0.05) area under the LH curve than blood sampling periods 2, 3, and 4. Cows treated with GnRH also tended to have greater area under the LH curve (P = 0.075) than control cows during the first sampling period. There was
Fig. 1. Area under the luteinizing hormone curve (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. (a, b) Time intervals with different letters are different (P ≤ 0.05). *Within sampling period, means (± SEM) have a tendency (P = 0.075) to be different between treatments. Effects of time (P = 0.003), treatment (P = 0.74), and treatment × time (P = 0.40).
an effect of time (P = 0.003), but there was no effect of treatment (P = 0.65) or treatment × time (P = 0.40) on average concentration of LH (Fig. 2). Blood sampling period 1 had greater (P < 0.004) average concentrations of LH compared to the second, third, and fourth sampling periods. Blood sampling period 5 had average concentrations of LH that were similar (P = 0.14) to those in the first sampling period, and greater (P ≤ 0.04) average concentrations of LH than those in the third and fourth, but tended (P = 0.07) to have greater average concentrations than only the second sampling period. Treated cows also tended (P = 0.07) to have greater average concentrations of LH than control cows during the first sampling period. There was a tendency (P = 0.07) for an effect of treatment; however, there were no effects of time (P = 0.98)
Fig. 2. Average serum concentration of luteinizing hormone (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. (a, b) Time intervals with different letters are different (P < 0.04). (c, d) Time intervals (± SEM) have a tendency (P = 0.07) to be different. *Within sampling period, means (± SEM) have a tendency (P = 0.07) to be different. Effects of time (P = 0.003), treatment (P = 0.65), and treatment × time (P = 0.40).
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Fig. 3. Luteinizing hormone pulse frequency (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. *Within sampling period, means (± SEM) have a tendency (P = 0.095) to be different. Effects of time (P = 0.98), treatment (P = 0.07), and treatment × time (P = 0.88).
or treatment × time (P = 0.88) for LH pulse frequency (Fig. 3). Cows treated with GnRH tended (P = 0.095) to have fewer pulses per hour than control cows during blood sampling period 2. Furthermore, 2 of the GnRHtreated cows did not have any pulses of LH during blood sampling period 2 (GnRH-no), whereas 3 GnRH-treated cows had pulses of LH (GnRH-pulse). All of the control cows had pulses of LH during the second sampling period. 3.1.3. Subsequent concentrations of progesterone There was an effect of time (P < 0.001) and a tendency for an effect of treatment (P = 0.067), but there was no treatment × time interaction (P = 0.72) for subsequent concentrations of progesterone between GnRH-treated cows and control cows (Fig. 4). On d 6, GnRH cows had greater (P = 0.003) concentrations of progesterone than the control group. Furthermore, progesterone increased over time (P < 0.001) from d 3 to d 17 in
Fig. 4. Subsequent concentrations of progesterone (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. **Within day, means (± SEM) are different (P = 0.003). Effects of time (P < 0.001), treatment (P = 0.067), and treatment × time (P = 0.72).
Fig. 5. Subsequent concentrations of progesterone (means ± standard error of the mean [SEM]) for cows treated with GnRH following detection in standing estrus that had pulses of luteinizing hormone (LH) (GnRH-pulse) and cows treated with GnRH following detection in standing estrus that did not have pulses of LH (GnRH-no) during the second intensive blood sampling period. **Within day, means (± SEM) are different (P ≤ 0.003). *Within day, means (± SEM) have a tendency (P ≤ 0.1) to be different. Effects of time (P < 0.001), treatment (P < 0.001), and treatment × time (P < 0.001).
both groups. There were effects of time (P < 0.001), treatment (P < 0.001), and treatment × time (P < 0.001) for subsequent concentrations of progesterone between GnRH-treated cows that had pulses of LH and those that did not have pulses of LH (Fig. 5). The GnRH-treated cows that had pulses of LH had greater (P ≤ 0.003) concentrations of progesterone on d 6, 7, 8, 9, 16, and 17 and tended (P ≤ 0.1) to have greater concentrations of progesterone on d 5 and d 10 compared to the GnRH-treated cows that did not have pulses of LH. Cows treated with GnRH had an earlier (P = 0.05) increase (indicated by the slope of the lines) in subsequent concentrations of progesterone from d 3 to d 6 compared to control cows (Fig. 6). When GnRH-treated cows were divided into those that had pulses of LH and those that did not during blood sampling period 2, GnRH-treated cows that had pulses of LH tended
Fig. 6. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows treated with GnRH following detection in standing estrus and control cows. Effect of treatment (P = 0.05).
S.D. Fields et al. / Domestic Animal Endocrinology 37 (2009) 189–195
Fig. 7. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows treated with GnRH following detection in standing estrus that had pulses of luteinizing hormone (GnRH-pulse) and cows treated with GnRH following detection in standing estrus that did not have pulses of LH (GnRH-no) during the second intensive blood sampling period. Effect of treatment (P = 0.08).
to have an earlier (P = 0.08) increase in progesterone than GnRH-treated cows that did not have pulses of LH (Fig. 7). 3.2. Experiment 2 There were effects of time (P < 0.001) and treatment (P = 0.01); however, there was no effect of treatment × time (P = 0.95) for subsequent concentrations of progesterone (Fig. 8). Subsequent concentrations of progesterone increased (P < 0.001) over time from d 2 to d 17. Cows receiving GnRH 10 to 11 h after onset of estrus had greater (P = 0.01) subsequent concentrations of progesterone compared to cows receiving GnRH 14 to 15 h after onset of estrus. Compared to cows given GnRH 14 to 15 h after onset of estrus, cows treated with GnRH 10 to 11 h after onset of estrus tended (P = 0.06) to have an earlier increase in progesterone (Fig. 9).
Fig. 8. Subsequent concentrations of progesterone (means ± standard error of the mean) for cows administered GnRH 10 to 11 h or 14 to 15 h after onset of estrus. Effects of time (P < 0.001), treatment (P = 0.01), and treatment × time (P = 0.95).
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Fig. 9. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows administered GnRH 10 to 11 h or 14 to 15 h after onset of estrus. Effect of treatment (P = 0.06).
4. Discussion Treatment with GnRH caused a surge release of LH during the first 6 h after treatment. This finding agrees with previous data in which exogenous GnRH caused a surge release of LH from the pituitary [17–20]. Furthermore, GnRH tended to decrease pulses of LH 26 to 32 h after onset of estrus, which is during the periovulatory period [21,22]. Researchers have reported that when pulses of LH were blocked around the time of ovulation, the CL produced less progesterone [8,9]. However, in the first experiment, GnRH-treated cows tended to have greater subsequent concentrations of progesterone than control cows, even though they tended to have fewer pulses of LH around the time of ovulation. Upon further analysis, GnRH-treated cows could be divided into 2 groups: (1) 2 cows did not have any pulses of LH 26 to 32 h after onset of estrus and (2) 3 cows had pulses of LH. All of the control cows had pulses of LH during that time period (26 to 32 h after onset of estrus). Therefore, when the GnRH-treated cows were divided into those 2 groups, GnRH-treated cows that had pulses of LH had greater subsequent concentrations of progesterone than those that did not have pulses of LH. This finding may explain why some research has reported an increase [2], whereas other studies have reported a decrease [3,4] in subsequent concentrations of progesterone when cows were administered GnRH following the onset of estrus. Increasing progesterone concentrations earlier in the luteal phase could influence pregnancy rates. Cows treated with progesterone on d 1 through d 4 of the estrous cycle had larger embryos than control cows on d 14 [23,24]. Embryos from the progesterone treatment also had the complex of polypeptides forming INF-, whereas control embryos did not have these polypeptides on d 14 [24]. A delayed increase in progesterone also resulted in less developed embryos that produced
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less INF- on d 16 compared to embryos from cows that had an earlier increase in progesterone [25], and cows with normal developing embryos had greater concentrations of progesterone on d 3 and d 6 after insemination compared to cows with degenerating embryos [26]. Shelton et al compared subfertile cows to fertile heifers, and fertile heifers had a faster increase in progesterone than the subfertile cows [27]. Therefore, embryonic mortality in cows with a delayed increase in progesterone may have been a result of decreased early luteal function. Thus, if less INF- is secreted, luteolysis may still occur, even in the presence of an embryo. These studies indicate that an early increase in progesterone influenced embryonic development, which is important for the signal of pregnancy. However, progesterone supplementation too early during the estrous cycle could advance the uterus too quickly, leading to asynchrony between the uterine environment and the fetus [23]. Progesterone treatment early in the estrous cycle can also result in short estrous cycles in cattle [28]. In the first experiment, GnRH administered at time of insemination did not increase progesterone too quickly, but GnRH-treated cows had an earlier increase in progesterone compared to controls. Therefore, GnRH may be able to increase embryo development without affecting the uterine environment. Treatment with GnRH also affected pulses of LH around the time of ovulation. Some GnRH-treated cows had pulses of LH around the time of ovulation and had greater subsequent concentrations of progesterone compared to those without pulses. The only detectable difference between GnRH-treated cows that had pulses of LH and those that did not was the interval from the onset of estrus to the GnRH treatment. Treated cows that had pulses were treated between 8 and 13 h after onset of estrus; whereas cows that did not have pulses of LH had an interval of 14 h from the onset of estrus to the GnRH treatment. Therefore, Experiment 2 was designed to further determine the influence of interval from onset of estrus until GnRH treatment on subsequent concentrations of progesterone. Experiment 2 confirmed the results from Experiment 1 and could explain the increased luteal function. Cows administered GnRH 14 to 15 h after onset of estrus may not have pulses of LH around the time of ovulation, resulting in decreased subsequent concentrations of progesterone and a tendency for a delayed increase in progesterone. Therefore, it seems that the interval from the onset of estrus to the GnRH treatment plays a role in early luteal development. In conclusion, pulses of LH had initiated before the time of ovulation, and the timing of GnRH following initiation of standing estrus influenced LH release, LH pulse frequency, increase in progesterone, and subsequent
concentrations of progesterone. These experiments indicated that pulses of LH around the time of ovulation were important for early luteal function and that the interval from the onset of estrus to treatment may explain why reports have varied on the influence of treatment with GnRH at time of insemination on subsequent concentrations of progesterone. Acknowledgments This research was supported by the South Dakota Research Support Fund and the South Dakota Agricultural Experiment Station and approved for publication as Journal Series No. 3634 by the Director, South Dakota Agricultural Experiment Station, South Dakota State University. Mention of a proprietary product does not constitute a guarantee or warranty of the product by South Dakota Agric. Exp. Stn. or the authors and does not imply its approval to the exclusion of other products that may also be suitable. The authors gratefully acknowledge T. Glaus, A. Knorr, C. Moret, J. Nelson, and A. Schiefelbein for technical assistance and IVX Animal Health for the donation of Prostamate and OvaCyst. References [1] McDonald LE, Nichols RE, McNutt SH. Studies on corpus luteum ablation and progesterone replacement therapy during pregnancy in the cow. Am J Vet Res 1952;13:446–51. [2] Mee MO, Stevenson JS, Alexander BM, Sasser RG. Administration of GnRH at estrus influences pregnancy rates, serum concentrations of LH, FSH, estradiol-17 beta, pregnancy-specific protein B, and progesterone, proportion of luteal cell types, and in vitro production of progesterone in dairy cows. J Anim Sci 1993;71:185–98. [3] Lucy MC, Stevenson JS. Gonadotropin-releasing hormone at estrus: Luteinizing hormone, estradiol, and progesterone during the periestrual and postinsemination periods in dairy cattle. Biol Reprod 1986;35:300–11. [4] Perry GA, Perry BL. Effect of an injection of GnRH at time of insemination following detection in standing estrus on subsequent concentrations of progesterone and pregnancy rates. Theriogenology 2009;71:775–9. [5] Lewis GS, Caldwell DW, Rexroad Jr CE, Dowlen HH, Owen JR. Effects of gonadotropin-releasing hormone and human chorionic gonadotropin on pregnancy rate in dairy cattle. J Dairy Sci 1990;73:66–72. [6] Perry GA, Smith MF, Lucy MC, et al. Relationship between follicle size at insemination and pregnancy success. Proc Natl Acad Sci U S A 2005;102:5268–73. [7] Perry GA, Smith MF, Roberts AJ, MacNeil MD, Geary TW. Relationship between size of the ovulatory follicle and pregnancy success in beef heifers. J Anim Sci 2007;85:684–9. [8] Quintal-Franco JA, Kojima FN, Melvin EJ, et al. Corpus luteum development and function in cattle with episodic release of luteinizing hormone pulses inhibited in the follicular and early luteal phases of the estrous cycle. Biol Reprod 1999;61:921–6.
S.D. Fields et al. / Domestic Animal Endocrinology 37 (2009) 189–195 [9] Peters KE, Bergfeld EG, Cupp AS, et al. Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers. Biol Reprod 1994;51:1248–54. [10] Kaim M, Bloch A, Wolfenson D, et al. Effects of GnRH administered to cows at the onset of estrus on timing of ovulation, endocrine responses, and conception. J Dairy Sci 2003;86:2012–21. [11] Nett TM, Cermak D, Braden T, Manns J, Niswender G. Pituitary receptors for GnRH and estradiol, and pituitary content of gonadotropins in beef cows. I. Changes during the estrous cycle. Domest Anim Endocrinol 1987;4:123–32. [12] Larson JE, Lamb GC, Stevenson JS, et al. Synchronization of estrus in suckled beef cows for detected estrus and artificial insemination and timed artificial insemination using gonadotropin-releasing hormone, prostaglandin F2␣, and progesterone. J Anim Sci 2006;84:332–42. [13] Perry GA, Perry BL. Effect of preovulatory concentrations of estradiol and initiation of standing estrus on uterine ph in beef cows. Domest Anim Endocrinol 2008;34:333–8. [14] Engel CE, Patterson HH, Perry GA. Effect of dried corn distillers grains plus solubles compared to soybean hulls, in late gestation heifer diets, on animal and reproductive performance. J Anim Sci 2008;86:1697–708. [15] Veldhuis JD, Johnson ML. Cluster analysis: A simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol 1986;250:E486–493. [16] Littell RC, Henry PR, Ammerman CB. Statistical analysis of repeated measures data using SAS procedures. J Anim Sci 1998;76:1216–31. [17] Britt JH, Kittok RJ, Harrison DS. Ovulation, estrus and endocrine response after GnRH in early postpartum cows. J Anim Sci 1974;39:915–9. [18] Hausler CL, Malven PV. Interaction of progesterone, GnRH and estradiol in the control of LH release in castrate heifers. J Anim Sci 1976;42:1239–43.
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[19] Chenault JR, Kratzer DD, Rzepkowski RA, Goodwin MC. LH and FSH response of Holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology 1990;34:81– 98. [20] Gazal OS, Leshin LS, Stanko RL, et al. Gonadotropin-releasing hormone secretion into third-ventricle cerebrospinal fluid of cattle: Correspondence with the tonic and surge release of luteinizing hormone and its tonic inhibition by suckling and neuropeptide. Y Biol Reprod 1998;59:676–83. [21] Swanson LV, Hafs HD. LH and prolactin in blood serum from estrus to ovulation in Holstein heifers. J Anim Sci 1971;33:1038–41. [22] Chenault JR, Thatcher WW, Kalra PS, Abrams RM, Wilcox CJ. Transitory changes in plasma progestins, estradiol, and luteinizing hormone approaching ovulation in the bovine. J Dairy Sci 1975;58:709–17. [23] Wilmut I, Sales DI. Effect of an asynchronous environment on embryonic development in sheep. J Reprod Fertil 1981;61:179–84. [24] Garrett JE, Geisert RD, Zavy MT, Morgan GL. Evidence for maternal regulation of early conceptus growth and development in beef cattle. J Reprod Fertil 1988;84:437–46. [25] Mann GE, Lamming GE. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 2001;121:175–80. [26] Maurer RR, Echternkemp SE. Hormonal asynchrony and embryonic development. Theriogenology 1982;17:11–22. [27] Shelton K, Gayerie De Abreu MF, Hunter MG, Parkinson TJ, Lamming GE. Luteal inadequacy during the early luteal phase of subfertile cows. J Reprod Fertil 1990;90:1–10. [28] Van Cleeff J, Macmillan KL, Drost M, Thatcher WW. Effects of administering progesterone at selected intervals after insemination of synchronized heifers on pregnancy rates and resynchronization of returns to estrus. Theriogenology 1996;46:1117–30.
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Effects of GnRH treatment on initiation of pulses of LH, LH release, and subsequent concentrations of progesterone S.D. Fields, B.L. Perry, G.A. Perry ∗ Department of Animal and Range Sciences, South Dakota State University, Brookings, Box 2170, Brookings, South Dakota 57007, USA Received 24 March 2009; received in revised form 30 April 2009; accepted 30 April 2009
Abstract Progesterone is essential for establishment and maintenance of pregnancy. One proposed method to increase progesterone is administering GnRH at insemination. However, this method has resulted in conflicting results. Therefore, 2 experiments were conducted to evaluate how administering GnRH at insemination affected pulses of luteinizing hormone (LH) and subsequent progesterone. In Experiment 1, cows were allotted to 2 treatments: (1) GnRH (100 g) given approximately 12 h after initiation of estrus (n = 5); and (2) Control (n = 5). Blood samples were collected at 15-min intervals for 6 h at 12 (blood sampling period 1), 26 (blood sampling period 2), 40 (blood sampling period 3), 54 (blood sampling period 4), and 68 (blood sampling period 5) h after onset of estrus. Daily blood samples were collected for 17 d. In Experiment 2, cows were allotted into 2 treatments: GnRH administered 10 to 11 h (n = 10) or 14 to 15 h (n = 10) after onset of estrus. Daily blood samples were collected for 17 d. Cows treated with GnRH tended (P ≤ 0.075) to have greater LH release during blood sampling period 1, tended (P = 0.095) to have fewer pulses during blood sampling period 2, tended (P = 0.067) to have greater concentrations of progesterone, and had an earlier (P = 0.05) increase in progesterone than control cows. Cows treated with GnRH 10 to 11 h after onset of estrus had greater (P = 0.01) progesterone and an earlier (P = 0.04) increase in progesterone than cows treated 14 to 15 h. In conclusion, timing of GnRH treatment following onset of estrus influenced pulses of LH and subsequent progesterone. © 2009 Elsevier Inc. All rights reserved. Keywords: GnRH; LH pulses; CL function; Progesterone
1. Introduction Progesterone is essential for the maintenance of pregnancy and embryo development [1]. Therefore, scientists have assessed techniques to increase fertility by increasing corpus luteum (CL) function. One proposed method was to treat with GnRH at time of insemination. However, there have been conflicting results with this method. Treatment with GnRH at the time of insemination resulted in an increase [2], decrease [3,4], or no
∗
Corresponding author. Tel.: +605 688 5456; fax: +605 688 6170. E-mail address: [email protected] (G.A. Perry).
0739-7240/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2009.04.006
change [5] in subsequent concentrations of progesterone compared to controls. Moreover, research has reported that cows detected in standing estrus around the time of the GnRH treatment at fixed-time artificial insemination (FTAI) had increased pregnancy rates compared to cows not detected in estrus [6,7]. The reason for the varying results is not clear. Luteinizing hormone (LH) has been reported to be involved in CL development and function [8–10]. Pituitary LH content returned to normal within 1 d of the ovulatory LH surge [11], and when pulses of LH were blocked around the time of ovulation, cows had decreased subsequent concentrations of progesterone compared to controls [8,9]. Therefore, the objectives of
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these experiments were to determine (1) when pulses of LH resumed following an LH surge, (2) the effect GnRH treatment would have on the initiation of pulses of LH, and (3) the effect of LH pulse initiation on subsequent concentrations of progesterone. 2. Materials and methods Two experiments were conducted at the South Dakota State University’s Beef Breeding Unit. Mature Anguscross, nonpregnant, nonlactating, estrous-cycling beef cows were used in these experiments. All cows were handled according to procedures approved by the South Dakota State University Institutional Animal Care and Use Committee. 2.1. Experiment 1 2.1.1. Experimental design Stage of the estrous cycle was synchronized with the Select Synch + CIDR protocol (n = 32) [12]. Cows were administered GnRH (100 g as 2 mL of OvaCyst i.m.; IVX Animal Health, St. Joseph, MO) and a controlled internal drug releasing device (CIDR) was inserted into the vagina on d -7. An injection of prostaglandin F2␣ (PGF2␣ 25 mg as 5 mL of Prostamate i.m., IVX Animal Health) was given, and the CIDR was removed on d 0. The HeatWatch (DDX, Inc., Denver, CO) electronic estrous detection system was used to determine initiation of standing estrus. Onset of estrus was determined as the first of 3 mounts within a 4-h period that lasted 2 s or longer in duration. After 10 cows were determined to be in standing estrus within a 6-h period of time, indwelling jugular catheters were inserted into each cow. Based on interval from onset of estrus, cows were assigned to 1 of 2 treatment groups: (1) cows were treated with GnRH approximately 12 h after onset of estrus (n = 5), or (2) cows did not receive GnRH treatment and served as controls (n = 5). 2.1.2. Blood collection At the time of blood sample collection, an 8-foot tubular extension was connected to the catheter and was taped along the back of each cow. A 3-way stop plug was connected to the end of the extension. A syringe was connected to the plug, allowing for an airtight connection. Blood samples were collected via jugular catheters every 15 min for 6 h from 12 to 18 (blood sampling period 1), 26 to 32 (blood sampling period 2), 40 to 46 (blood sampling period 3), 54 to 60 (blood sampling period 4), and 68 to 74 (blood sampling period 5) h after onset of estrus. At each collection time, 3 mL of blood was drawn
from the syringe and discarded, 5 mL of fresh blood was collected for the sample, and 5 mL of saline (O.9% saline containing 120 mM Na citrate and 10 cc/L oxytetracycline) was injected back into the cow via the catheter. Daily blood samples were collected by venipuncture of the median caudal vessel into 10 mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) from d 3 to d 17 of the estrous cycle. All blood was allowed to coagulate at room temperature and then stored at 4 ◦ C for 24 h. Samples were centrifuged at 1200 × g for 30 min, and the serum was harvested and frozen at −20 ◦ C until analysis by radioimmunoassay (RIA). 2.1.3. Transrectal ultrasonography Transrectal ultrasonography was performed using an Aloka 500 V ultrasound with a 7.5-MHz transrectal linear probe (Aloka, Wallingford, CT). Both ovaries of each cow were examined 15 to 16 h after onset of estrus. All follicles >8 mm in diameter were recorded. Ovaries were examined again 32 h after onset of estrus to determine if ovulation had occurred. Ovulation was defined as the disappearance of a previously recorded large antral follicle. 2.2. Experiment 2 2.2.1. Experimental design Stage of the estrous cycle was synchronized with the Select Synch + CIDR protocol (n = 32), as previously described in Experiment 1. The HeatWatch electronic detection of estrus system was used to determine initiation of standing estrus, and parameters were set as described in Experiment 1. After cows were determined to be in estrus, they were allotted to 1 of 2 treatments: GnRH treatment given at (1) 10 to 11 h (n = 10); or (2) 14 to 15 h (n = 10) after onset of estrus. 2.2.2. Blood collection Daily blood samples were collected by venipuncture of the jugular vein into 10 mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) from d 0 to d 17 of the estrous cycle. Serum was collected as described in Experiment 1 and frozen at −20 ◦ C until analysis by RIA. 2.3. Radioimmunoassay Serum samples from the intensive blood sampling periods (Experiment 1) were analyzed for concentrations of LH by RIA using methodology described by Perry and Perry [13]. Intra- and interassay coefficients of variation for LH assays were 4.7% and 13.7% for Experiment 1. Assay sensitivity was 0.125 ng/mL. Daily
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blood samples were analyzed for serum concentrations of progesterone by RIA using methodology described by Engel et al. [14]. Intra- and interassay coefficients of variation for progesterone assays were 6.6% and 3.4% for Experiment 1 and 4.2% and 8.7% for Experiment 2. Assay sensitivity was 0.4 ng/mL. 2.4. Statistical analysis Cluster analysis [15] was used to determine area under the LH curve, average concentration of LH, and LH pulse frequency. Effects of GnRH on area under the LH curve, average concentration of LH, LH pulse frequency, and subsequent concentrations of progesterone were determined by analysis of variance (ANOVA) for repeated measures in SAS by PROC MIXED [16]. The statistical model consisted of effects of treatment, time, and treatment × time interactions. All covariance structures were modeled in the initial analysis. The indicated best fit covariance structure, compound symmetry, was used for the final analysis for the LH variables, and heterogeneous compound symmetry was used for the final analysis for progesterone. Differences between treatment groups for the interval from the onset of estrus to treatment (Experiment 1), increase in progesterone (Experiments 1 and 2), and follicle size (Experiment 1) were analyzed by ANOVA using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC; version 9.2; 2007). 3. Results 3.1. Experiment 1 3.1.1. Interval from onset of estrus to GnRH and follicle size The interval from the onset of estrus to blood sampling period 1 was similar (P = 0.82) between GnRH treated (12.5 ± 1.2 h) and control (12.1 ± 1.2 h) cows. All cows had ovulations by 32 h after onset of estrus. There was no difference (P = 0.13) in follicle size between GnRH treated (14.9 ± 0.47 mm) and control (16.0 ± 0.47 mm) cows. 3.1.2. Luteinizing hormone There was an effect of time (P = 0.003); however, there were no effects of treatment (P = 0.74) or treatment × time (P = 0.40) for area under the LH curve (Fig. 1). Blood sampling periods 1 and 5 had a greater (P ≤ 0.05) area under the LH curve than blood sampling periods 2, 3, and 4. Cows treated with GnRH also tended to have greater area under the LH curve (P = 0.075) than control cows during the first sampling period. There was
Fig. 1. Area under the luteinizing hormone curve (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. (a, b) Time intervals with different letters are different (P ≤ 0.05). *Within sampling period, means (± SEM) have a tendency (P = 0.075) to be different between treatments. Effects of time (P = 0.003), treatment (P = 0.74), and treatment × time (P = 0.40).
an effect of time (P = 0.003), but there was no effect of treatment (P = 0.65) or treatment × time (P = 0.40) on average concentration of LH (Fig. 2). Blood sampling period 1 had greater (P < 0.004) average concentrations of LH compared to the second, third, and fourth sampling periods. Blood sampling period 5 had average concentrations of LH that were similar (P = 0.14) to those in the first sampling period, and greater (P ≤ 0.04) average concentrations of LH than those in the third and fourth, but tended (P = 0.07) to have greater average concentrations than only the second sampling period. Treated cows also tended (P = 0.07) to have greater average concentrations of LH than control cows during the first sampling period. There was a tendency (P = 0.07) for an effect of treatment; however, there were no effects of time (P = 0.98)
Fig. 2. Average serum concentration of luteinizing hormone (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. (a, b) Time intervals with different letters are different (P < 0.04). (c, d) Time intervals (± SEM) have a tendency (P = 0.07) to be different. *Within sampling period, means (± SEM) have a tendency (P = 0.07) to be different. Effects of time (P = 0.003), treatment (P = 0.65), and treatment × time (P = 0.40).
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Fig. 3. Luteinizing hormone pulse frequency (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. *Within sampling period, means (± SEM) have a tendency (P = 0.095) to be different. Effects of time (P = 0.98), treatment (P = 0.07), and treatment × time (P = 0.88).
or treatment × time (P = 0.88) for LH pulse frequency (Fig. 3). Cows treated with GnRH tended (P = 0.095) to have fewer pulses per hour than control cows during blood sampling period 2. Furthermore, 2 of the GnRHtreated cows did not have any pulses of LH during blood sampling period 2 (GnRH-no), whereas 3 GnRH-treated cows had pulses of LH (GnRH-pulse). All of the control cows had pulses of LH during the second sampling period. 3.1.3. Subsequent concentrations of progesterone There was an effect of time (P < 0.001) and a tendency for an effect of treatment (P = 0.067), but there was no treatment × time interaction (P = 0.72) for subsequent concentrations of progesterone between GnRH-treated cows and control cows (Fig. 4). On d 6, GnRH cows had greater (P = 0.003) concentrations of progesterone than the control group. Furthermore, progesterone increased over time (P < 0.001) from d 3 to d 17 in
Fig. 4. Subsequent concentrations of progesterone (means ± standard error of the mean [SEM]) for cows treated with GnRH and control cows following detection in standing estrus. **Within day, means (± SEM) are different (P = 0.003). Effects of time (P < 0.001), treatment (P = 0.067), and treatment × time (P = 0.72).
Fig. 5. Subsequent concentrations of progesterone (means ± standard error of the mean [SEM]) for cows treated with GnRH following detection in standing estrus that had pulses of luteinizing hormone (LH) (GnRH-pulse) and cows treated with GnRH following detection in standing estrus that did not have pulses of LH (GnRH-no) during the second intensive blood sampling period. **Within day, means (± SEM) are different (P ≤ 0.003). *Within day, means (± SEM) have a tendency (P ≤ 0.1) to be different. Effects of time (P < 0.001), treatment (P < 0.001), and treatment × time (P < 0.001).
both groups. There were effects of time (P < 0.001), treatment (P < 0.001), and treatment × time (P < 0.001) for subsequent concentrations of progesterone between GnRH-treated cows that had pulses of LH and those that did not have pulses of LH (Fig. 5). The GnRH-treated cows that had pulses of LH had greater (P ≤ 0.003) concentrations of progesterone on d 6, 7, 8, 9, 16, and 17 and tended (P ≤ 0.1) to have greater concentrations of progesterone on d 5 and d 10 compared to the GnRH-treated cows that did not have pulses of LH. Cows treated with GnRH had an earlier (P = 0.05) increase (indicated by the slope of the lines) in subsequent concentrations of progesterone from d 3 to d 6 compared to control cows (Fig. 6). When GnRH-treated cows were divided into those that had pulses of LH and those that did not during blood sampling period 2, GnRH-treated cows that had pulses of LH tended
Fig. 6. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows treated with GnRH following detection in standing estrus and control cows. Effect of treatment (P = 0.05).
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Fig. 7. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows treated with GnRH following detection in standing estrus that had pulses of luteinizing hormone (GnRH-pulse) and cows treated with GnRH following detection in standing estrus that did not have pulses of LH (GnRH-no) during the second intensive blood sampling period. Effect of treatment (P = 0.08).
to have an earlier (P = 0.08) increase in progesterone than GnRH-treated cows that did not have pulses of LH (Fig. 7). 3.2. Experiment 2 There were effects of time (P < 0.001) and treatment (P = 0.01); however, there was no effect of treatment × time (P = 0.95) for subsequent concentrations of progesterone (Fig. 8). Subsequent concentrations of progesterone increased (P < 0.001) over time from d 2 to d 17. Cows receiving GnRH 10 to 11 h after onset of estrus had greater (P = 0.01) subsequent concentrations of progesterone compared to cows receiving GnRH 14 to 15 h after onset of estrus. Compared to cows given GnRH 14 to 15 h after onset of estrus, cows treated with GnRH 10 to 11 h after onset of estrus tended (P = 0.06) to have an earlier increase in progesterone (Fig. 9).
Fig. 8. Subsequent concentrations of progesterone (means ± standard error of the mean) for cows administered GnRH 10 to 11 h or 14 to 15 h after onset of estrus. Effects of time (P < 0.001), treatment (P = 0.01), and treatment × time (P = 0.95).
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Fig. 9. Increase in subsequent concentrations of progesterone (slope of the line for means ± standard error of the mean) for cows administered GnRH 10 to 11 h or 14 to 15 h after onset of estrus. Effect of treatment (P = 0.06).
4. Discussion Treatment with GnRH caused a surge release of LH during the first 6 h after treatment. This finding agrees with previous data in which exogenous GnRH caused a surge release of LH from the pituitary [17–20]. Furthermore, GnRH tended to decrease pulses of LH 26 to 32 h after onset of estrus, which is during the periovulatory period [21,22]. Researchers have reported that when pulses of LH were blocked around the time of ovulation, the CL produced less progesterone [8,9]. However, in the first experiment, GnRH-treated cows tended to have greater subsequent concentrations of progesterone than control cows, even though they tended to have fewer pulses of LH around the time of ovulation. Upon further analysis, GnRH-treated cows could be divided into 2 groups: (1) 2 cows did not have any pulses of LH 26 to 32 h after onset of estrus and (2) 3 cows had pulses of LH. All of the control cows had pulses of LH during that time period (26 to 32 h after onset of estrus). Therefore, when the GnRH-treated cows were divided into those 2 groups, GnRH-treated cows that had pulses of LH had greater subsequent concentrations of progesterone than those that did not have pulses of LH. This finding may explain why some research has reported an increase [2], whereas other studies have reported a decrease [3,4] in subsequent concentrations of progesterone when cows were administered GnRH following the onset of estrus. Increasing progesterone concentrations earlier in the luteal phase could influence pregnancy rates. Cows treated with progesterone on d 1 through d 4 of the estrous cycle had larger embryos than control cows on d 14 [23,24]. Embryos from the progesterone treatment also had the complex of polypeptides forming INF-, whereas control embryos did not have these polypeptides on d 14 [24]. A delayed increase in progesterone also resulted in less developed embryos that produced
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less INF- on d 16 compared to embryos from cows that had an earlier increase in progesterone [25], and cows with normal developing embryos had greater concentrations of progesterone on d 3 and d 6 after insemination compared to cows with degenerating embryos [26]. Shelton et al compared subfertile cows to fertile heifers, and fertile heifers had a faster increase in progesterone than the subfertile cows [27]. Therefore, embryonic mortality in cows with a delayed increase in progesterone may have been a result of decreased early luteal function. Thus, if less INF- is secreted, luteolysis may still occur, even in the presence of an embryo. These studies indicate that an early increase in progesterone influenced embryonic development, which is important for the signal of pregnancy. However, progesterone supplementation too early during the estrous cycle could advance the uterus too quickly, leading to asynchrony between the uterine environment and the fetus [23]. Progesterone treatment early in the estrous cycle can also result in short estrous cycles in cattle [28]. In the first experiment, GnRH administered at time of insemination did not increase progesterone too quickly, but GnRH-treated cows had an earlier increase in progesterone compared to controls. Therefore, GnRH may be able to increase embryo development without affecting the uterine environment. Treatment with GnRH also affected pulses of LH around the time of ovulation. Some GnRH-treated cows had pulses of LH around the time of ovulation and had greater subsequent concentrations of progesterone compared to those without pulses. The only detectable difference between GnRH-treated cows that had pulses of LH and those that did not was the interval from the onset of estrus to the GnRH treatment. Treated cows that had pulses were treated between 8 and 13 h after onset of estrus; whereas cows that did not have pulses of LH had an interval of 14 h from the onset of estrus to the GnRH treatment. Therefore, Experiment 2 was designed to further determine the influence of interval from onset of estrus until GnRH treatment on subsequent concentrations of progesterone. Experiment 2 confirmed the results from Experiment 1 and could explain the increased luteal function. Cows administered GnRH 14 to 15 h after onset of estrus may not have pulses of LH around the time of ovulation, resulting in decreased subsequent concentrations of progesterone and a tendency for a delayed increase in progesterone. Therefore, it seems that the interval from the onset of estrus to the GnRH treatment plays a role in early luteal development. In conclusion, pulses of LH had initiated before the time of ovulation, and the timing of GnRH following initiation of standing estrus influenced LH release, LH pulse frequency, increase in progesterone, and subsequent
concentrations of progesterone. These experiments indicated that pulses of LH around the time of ovulation were important for early luteal function and that the interval from the onset of estrus to treatment may explain why reports have varied on the influence of treatment with GnRH at time of insemination on subsequent concentrations of progesterone. Acknowledgments This research was supported by the South Dakota Research Support Fund and the South Dakota Agricultural Experiment Station and approved for publication as Journal Series No. 3634 by the Director, South Dakota Agricultural Experiment Station, South Dakota State University. Mention of a proprietary product does not constitute a guarantee or warranty of the product by South Dakota Agric. Exp. Stn. or the authors and does not imply its approval to the exclusion of other products that may also be suitable. The authors gratefully acknowledge T. Glaus, A. Knorr, C. Moret, J. Nelson, and A. Schiefelbein for technical assistance and IVX Animal Health for the donation of Prostamate and OvaCyst. References [1] McDonald LE, Nichols RE, McNutt SH. Studies on corpus luteum ablation and progesterone replacement therapy during pregnancy in the cow. Am J Vet Res 1952;13:446–51. [2] Mee MO, Stevenson JS, Alexander BM, Sasser RG. Administration of GnRH at estrus influences pregnancy rates, serum concentrations of LH, FSH, estradiol-17 beta, pregnancy-specific protein B, and progesterone, proportion of luteal cell types, and in vitro production of progesterone in dairy cows. J Anim Sci 1993;71:185–98. [3] Lucy MC, Stevenson JS. Gonadotropin-releasing hormone at estrus: Luteinizing hormone, estradiol, and progesterone during the periestrual and postinsemination periods in dairy cattle. Biol Reprod 1986;35:300–11. [4] Perry GA, Perry BL. Effect of an injection of GnRH at time of insemination following detection in standing estrus on subsequent concentrations of progesterone and pregnancy rates. Theriogenology 2009;71:775–9. [5] Lewis GS, Caldwell DW, Rexroad Jr CE, Dowlen HH, Owen JR. Effects of gonadotropin-releasing hormone and human chorionic gonadotropin on pregnancy rate in dairy cattle. J Dairy Sci 1990;73:66–72. [6] Perry GA, Smith MF, Lucy MC, et al. Relationship between follicle size at insemination and pregnancy success. Proc Natl Acad Sci U S A 2005;102:5268–73. [7] Perry GA, Smith MF, Roberts AJ, MacNeil MD, Geary TW. Relationship between size of the ovulatory follicle and pregnancy success in beef heifers. J Anim Sci 2007;85:684–9. [8] Quintal-Franco JA, Kojima FN, Melvin EJ, et al. Corpus luteum development and function in cattle with episodic release of luteinizing hormone pulses inhibited in the follicular and early luteal phases of the estrous cycle. Biol Reprod 1999;61:921–6.
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