Induction of ovulation in nonlactating dairy cows and heifers using different doses of a deslorelin implant

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1 Theriogenology 61 (4) Induction of ovulation in nonlactating dairy cows and heifers using different doses of a deslorelin implant J.A. Bartolome a,b, J.E.P. Santos c, S.M. Pancarci a, P. Melendez b, A.C.M. Arteche a, O. Hernandez a, L.F. Archbald b, T. Trigg d, W.W. Thatcher a,* a Department of Animal Sciences, University of Florida, P.O. Box 119, Gainesville, FL 3611, USA b Department of LACS, CVM, University of Florida, Gainesville, FL 361, USA c Veterinary Medicine Teaching and Research Center, University of California-Davis, Tulare, CA 9374, USA d Peptech Animal Health, North Ryde, Australia Received 17 January 3; accepted 15 May 3 Abstract The objective of this study was to evaluate ovarian function after inducing ovulation with a deslorelin implant in nonlactating dairy cows and heifers. Cattle received GnRH on Day 9, and PGF a on Day. On Day, in Experiment 1, cows received either 1 mg GnRH (Control), a 75 mg (DESLOR- ELIN 75) or 1 mg (DESLORELIN 1) deslorelin implant. On Day, in Experiment, cows received 1 mg of GnRH or a 45 mg (DESLORELIN 45) deslorelin implant. In Experiments 1 and, cows received PGF a on Day 16. Ultrasonography and blood sampling for plasma progesterone (P 4 ) were used to monitor ovarian activity. On Day, in Experiment 3, heifers received either 1 mg of GnRH or 75 mg (DESLORELIN 75) deslorelin implant. On Day 16, all heifers received PGF a. Blood samples were collected on Days 7, 13 and 16. In Experiments 1 3, deslorelin implants did not elevate plasma concentrations of P 4 in a systematic manner during the late luteal phase. In Experiments 1 and, deslorelin implants decreased the size of the largest follicle and the number of Class II and III follicles. In Experiments 1 and, deslorelin-treated cows failed to ovulate by Day 8. In conclusion, deslorelin implants induced ovulation, stimulated development of a normal CL, and delayed follicular growth during the subsequent diestrus period. For future applications, the dose of the deslorelin implant will have to be adjusted, and if used for timed-inseminations, nonpregnant cows will have to be resynchronized to minimize delayed returns to estrus and ovulation. # 3 Elsevier Inc. All rights reserved. Keywords: GnRH; Ovulation; Progesterone; Cattle * Corresponding author. Tel.: þ ; fax: þ address: thatcher@animal.ufl.edu (W.W. Thatcher) X/$ see front matter # 3 Elsevier Inc. All rights reserved. doi:1.116/s93-691x(3)41-3

2 48 J.A. Bartolome et al. / Theriogenology 61 (4) Introduction Timed-artificial insemination is a common practice in dairy herds to improve reproductive efficiency, since all cows are inseminated independent of estrus detection [1,]. Timed-insemination requires synchronization of follicular development, regression of the corpus luteum (CL), and synchronous induction of ovulation [3]. Human chorionic gonadototrophin (hcg) [4], gonadotropin releasing hormone (GnRH) [3], GnRH agonists [4], and estradiol [5] all have been used to induce ovulation. A synthetic GnRH-agonist (deslorelin) implant was used to synchronize ovulation, enhance CL function, and delay the first-wave dominant follicle [6]. Enhanced CL function was associated with high blood concentrations of P 4 early in the estrous cycle which stimulated embryo development, increased interferon-t production [7], and may contribute to improved fertility [8]. In addition, suppression of follicular development may also extend CL lifespan [9]. The hypothesis of this study was that induction of ovulation with a deslorelin implant would increase plasma P 4 concentrations (due to enhanced development of the CL) and suppress follicular development, without extending the interestrus interval. The objective was to evaluate ovarian function after inducing ovulation with different doses (45, 75 and 1 mg) of a deslorelin implant (GnRH agonist) in comparison to gonadorelin diacetate (GnRH, 1 mg) in nonlactating dairy cows and heifers.. Materials and methods.1. Experiment 1 Nonlactating dairy cows (n ¼ )weregivenani.m.injectionof1mg GnRH (Cystorelin TM ; Merial Ltd., Iselin, NJ, USA) on Day 9, and two i.m. doses of 5 mg PGF a (Lutalyse TM ; Pharmacia, Kalamazoo, MI, USA) on Day. On Day, cows were assigned randomly to three treatment groups and received either 1 mgi.m.gnrh (Control; n ¼ 6), a subcutaneous (s.c.) biodegradable implant (Peptech Animal Health, North Ryde, Australia) containing 75 mg deslorelin (D-Trp 6 -Pro 6 -des-gly 1 -LHRH ethylamide, DESLORELIN 75; n ¼ 7) or a s.c. implant containing 1 mg deslorelin (DESLORELIN 1; n ¼ 7). On Day 16, cows were given PGF a (two, 5-mg doses given 8 h apart) to induce luteolysis. Ovaries were evaluated by ultrasonography daily from Day to ovulation and every other day to Day 8. From Day to administration of PGF a (Day 16), daily blood samples were collected to determine plasma P 4 concentrations. The Heat Watch 1 system (DDx Inc., Denver, CO, USA) was used to monitor estrus. After Day 16, cows that did not display signs of estrus and ovulate received 1 mg i.m. GnRH when the largest follicle reached mm in diameter, 5 mg i.m. PGF a wasgiven7dayslater,and1mg i.m. GnRH was given days after PGF a (Ovsynch protocol) [3]. The outcome variables were plasma P 4 concentrations (ng/ml), first-wave largest follicle (mm; the term largest will be used since deslorelin-implanted cows did not have a dominant follicle), second-wave largest follicle until Day 16 (mm), number of Class II (5 9 mm) and Class III (1 mm) follicles [1] between Days

3 and 16, size of the largest follicle between Days 16 and (mm), rate of growth for largest follicle (mm per day) between Days 16 and 8 for DESLORELIN 75 and DESLORELIN 1 Groups, expression of estrus after Day 16 (yes/no), number of Class II and Class III follicles between Days 16 and 8 for DESLORELIN 75 and DESLORELIN 1 Groups, and response to Ovsynch for cows that did not ovulate after Day Experiment Fourteen nonlactating dairy cows were synchronized using GnRH on Day 9 and two doses of PGF a 1 h apart on Day. On Day, cows received either 1 mg of GnRH (Control, n ¼ 8) or a 45 mg deslorelin implant (DESLORELIN 45, n ¼ 6). On Day 16, all cows received PGF a. Ovaries were evaluated by ultrasonography daily from Day to the day of ovulation and then every other day to Day 8. Daily blood samples (to determine plasma P 4 concentrations) were collected from Day to PGF a administration on Day 16. The Heat Watch 1 (DDx Inc., Boulder, CO, USA) system was used to monitor estrus. After Day 16, cows that did not show estrus and did not ovulate (based upon ultrasonography), received 1 mg i.m. GnRH at the time the largest follicle reached 1 mm in diameter, 5 mg i.m. PGF a 7 days later, and 1 mg i.m. GnRH days after PGF a (Ovsynch protocol). The outcome variables were plasma P 4 concentrations (ng/ml), first-wave largest follicle (mm), second-wave largest follicle to Day 16 (mm), number of Class II and Class III follicles between Days and 16, size of the largest follicle (mm) between Days 16 and 8, expression of estrus after Day 16 (yes/no), and response to Ovsynch for cows that did not ovulate after Day Experiment 3 J.A. Bartolome et al. / Theriogenology 61 (4) Forty-four dairy heifers were synchronized using GnRH on Day 9 and PGF a on Day. On Day, heifers received either 1 mg of Cystorelin TM (Control, n ¼ ) or a 75 mg deslorelin implant (DESLORELIN 75, n ¼ ). On Day 16, all heifers received PGF a and estrus was monitored for 6 days. Heifers that had not displayed signs of estrus within 1 days after PGF a (i.e. Day 6) were evaluated by ultrasonography to determine ovarian structures. Blood samples were collected on Days 7, 13, and 16 after either GnRH injection or insertion of the deslorelin implant (i.e. Day ), to evaluate plasma P 4 concentrations. Outcome variables were plasma P 4 concentration (ng/ml) and estrus expression after PGF a on Day 16 (yes/no)..4. Collection of blood samples and radioimmunoassay Blood samples for P 4 analysis were collected by puncture of the coccygeal vein or artery into evacuated tubes containing ethylene diamine tetra acetic acid (EDTA) as an anticoagulant (1.5 mg, Monoject, Sherwood Medical, St. Louis, MO, USA). Plasma P 4 concentrations were determined with a single antibody radioimmunoassay procedure [11]. Inter- and intra-assay coefficients of variation were 6.7 and 1.3 %, respectively. Sensitivity of the assay was.15 ng/ml.

4 41 J.A. Bartolome et al. / Theriogenology 61 (4) Statistical analyses Plasma P 4 concentrations were analyzed using the repeated measures procedure (Proc Mixed, SAS) for Mixed Models [1], adjusting for the number of CL as a covariate. Differences between means for specific days were examined by the PDIFF option. Follicular growth was analyzed using the repeated measures procedure and by the homogeneity of regression procedure (Proc GLM, SAS) [13]. Number of Class II and III follicles was compared using the repeated measures procedure [1] and the repeated procedure for counted data (Proc Genmod, SAS) [14]. The expression of estrus was analyzed by using Chi Square (Proc Freq, SAS). Differences were considered significant when P :5 and tendency when P :1. 3. Results 3.1. Experiment 1 All cows ovulated and formed a CL after treatments on Day. Average plasma P 4 concentrations from Day to Day 16 were not different among cows in the Control (5:9 :6 ng/ml), DESLORELIN 75 (6:4 :6 ng/ml), and DESLORELIN 1 (6:3 :6 ng/ml) Groups. However, sporadic elevations in plasma P 4 concentrations were detected on Day 11 for cows in the DESLORELIN 1 (11:1 1: ng/ml) compared to cows in the Control (8: 1:1 ng/ml; P < :5) Groups, and on Day 1 for cows in the DESLORELIN 75 (1:4 1: ng/ml) compared to cows in the Control (9: 1:1 ng/ml, P < :5; Fig. 1) Groups. Overall, a deslorelin-induced increase in plasma progesterone concentrations was minimal. The first-wave largest follicle was larger (P < :) for cows in the Control Group (9:48 1:1 mm) compared to cows in DESLORELIN 1 Group (4:9 1: mm), but not different from that of cows in DESLORELIN 75 Group (7:8 1: mm). The secondwave largest follicle, evaluated until Day 16, was larger (P < :1) for cows in Control Group (7:63 :6 mm) compared to cows in DESLORELIN 75 (5:5 :6 mm) and DESLORELIN 1 (4:5 :6 mm; Fig. ) Groups. The number of Class II follicles from Day to Day 16 was higher (P < :1) for cows in Control Group (:1 :3) compared to cows in DESLORELIN 75 Group (1: :3) and DESLORELIN 1 Group (:5 :3; Fig. 3A). The number of Class III follicles from Day to Day 16 was higher (P < :5) for cows in Control Group (1:1 :) compared to cows in DESLORELIN 1 Group (:7 :; Fig. 3B). From Day 16 to Day, the average diameter of the largest follicle was greater (P < :1) for cows in Control Group (16:1 :9 mm) compared to cows in DESLOR- ELIN 75 Group (6:3 :8 mm) and DESLORELIN 1 Group (4:7 :8 mm; Fig. 4). The number of cows expressing estrus and ovulating after PGF a on Day 16 until Day was higher (P < :1) in Control Group (6/6) compared to cows in DESLORELIN 75 Group (1/7) and DESLORELIN 1 Group (/7). The rate of follicular growth from Day 16 to Day 8 was greater in cows in DESLORELIN 75 Group (.67 mm per day) than in cows in DESLORELIN 1 Group (.4 mm per day; P < :1; Fig. 4). Twelve of

5 J.A. Bartolome et al. / Theriogenology 61 (4) Progesterone (ng/ml) * * Fig. 1. Least squares means and S.E. for plasma P 4 concentration between Days and 16 for Control (&), DESLORELIN 75 (*) and DESLORELIN 1 (~) Groups in Experiment 1. DESLORELIN 1 vs. Control on Day 11 (P :5), DESLORELIN 75 vs. Control on Day 1 (P :5). 14 (86%) cows in DESLORELIN 75 and DESLORELIN 1 Groups expressed estrus between Days 4 and 3 (one in estrus on Day 19 and one did not show estrus) but failed to ovulate. The 1 cows that failed to ovulate were subjected to the Ovsynch protocol, and 9/ 1 (75%) ovulated synchronously in response to the second GnRH injection. Fig.. Least squares means and S.E. for the first-wave largest follicles from Days to 16 for Control (&), DESLORELIN 75 (*) and DESLORELIN 1 (~) Groups (P : Control vs. DESLORELIN 1) and second-wave largest follicle for Control (&), DESLORELIN 75 (*), and DESLORELIN 1 (~) Groups (P :1) in Experiment 1.

6 41 J.A. Bartolome et al. / Theriogenology 61 (4) No. follicles (A) No. follicles (B) Fig. 3. (A) Least squares means and S.E. for the number of Class II follicles after treatment for from Days to 16 for Control (&), DESLORELIN 75 (*) and DESLORELIN 1 (~) Groups in Experiment 1 (P :1). (B) Least squares means and S.E. for the number of Class III follicles after treatment for from Day to Day 16 for Control (&), DESLORELIN 75 (*) and DESLORELIN 1 (~) Groups in Experiment 1 (P :1). The number of Class II follicles from Day 16 to Day 8 was not different (P ¼ :36) between cows in DESLORELIN 75 Group (1: :4) compared to those in DESLOR- ELIN 1 Group (:6 :3). Likewise, the number of Class III follicles from Day 16 to Day 8 was not different (P ¼ :57) between cows in DESLORELIN 75 Group (:5 :) compared to cows in DESLORELIN 1 Group (:4 :). 3.. Experiment Overall plasma P 4 concentration was not different among cows in the Control Group (5:9 :8 ng/ml) and DESLORELIN 45 Group (7:4 1: ng/ml). However, plasma P 4 concentration tended to be higher (P < :1) on Day 9 for cows in the DESLORELIN 45 Group (11:4 1:5 ng/ml) compared to cows in Control Group (8: 1: ng/ml), and was

7 J.A. Bartolome et al. / Theriogenology 61 (4) Largest follicle (mm) Fig. 4. Least squares means and S.E. for the largest follicle after PGF a on Day 16 for cows in Control (&), DESLORELIN 75 (*) and DESLORELIN 1 (~) Groups in Experiment 1 (P :1). higher (P < :5) on Day 1 for cows in DESLORELIN 45 Group (13: 1:5 ng/ml) compared to cows in Control Group (8: 1: ng/ml; Fig. 5). The first-wave largest follicle was larger (P < :1) for cows in Control Group (11:4 1: mm) compared to that for cows in DESLORELIN 45 Group (6: 1:1 mm). The Progesterone (ng/ml) Fig. 5. Least squares means and S.E. for plasma P 4 concentration between Days and 16 for Control (&) and DESLORELIN 45 (*) Groups in Experiment. DESLORELIN 45 vs. Control on Day 9 (P :1), and DESLORELIN 45 vs. Control on Day 1 (P :5).

8 414 J.A. Bartolome et al. / Theriogenology 61 (4) Fig. 6. Least squares means and S.E. for the first-wave largest follicles from Day to Day 16 for Control (&) and DESLORELIN 45 (*) Groups (P :1), and second-wave largest follicle for control (&), DESLORELIN 45 (*) Groups (P :1) in Experiment. No. follicles (A) No. follicles (B) Fig. 7. (A) Least squares means and S.E. for the number of Class II follicles after treatment for from Day to Day 16 for Control (&), DESLORELIN 45 (*) Groups (P :1) in Experiment. (B) LSM and S.E. for the number of Class III follicles after treatment for from Day to Day 16 for Control (&) and DESLORELIN 45 (*) Groups (P :1) in Experiment.

9 J.A. Bartolome et al. / Theriogenology 61 (4) Largest follicle (mm) Fig. 8. Least squares means and S.E. for the largest follicle after PGF a on Day 16 for cows in Control (&) and DESLORELIN 45 (*) Groups (P :1) in Experiment. second-wave largest follicle monitored until Day 16 was larger (P < :1) for cows in Control Group (8:5 :4 mm) compared to cows in DESLORELIN 45 Group (3:3 :5 mm; Fig. 6). The number of Class II follicles from Day to Day 16 was higher (P < :1) for cows in Control Group (:5 :) compared to cows in DESLORELIN 45 Group (:9 :; Fig. 7A). The number of Class III follicles from Day to Day 16 was higher (P < :1) for cows in Control Group (1:3 :1) compared to cows in DESLORELIN 45 Group (:4 :1; Fig. 7B). The average diameter of the largest follicles from Day 16 to Day 8 was larger (P < :1) in cows in Control Group (16:5 1:85 mm) compared to cows in DESLOR- ELIN 45 Group (9:7 1:85 mm; Fig. 8). The number of cows expressing estrus and ovulating after PGF a on Day 16 until Day was higher (P < :1) in Control Group (8/ 8) compared to cows in DESLORELIN 45 Group (1/6). One cow in the DESLORELIN 45 Group showed estrus on Day 8, but failed to ovulate. Therefore, five cows in DESLORELIN 45 Group where subjected to the Ovsynch protocol and three of them ovulated in response to the second GnRH. Progesterone (ng/ml) Fig. 9. Least squares means and S.E. for plasma P 4 concentration on Days 7, 13 and 16 for Control (&) and DESLORELIN 75 (*) Groups (P :7) in Experiment 3.

10 416 J.A. Bartolome et al. / Theriogenology 61 (4) Experiment 3 Overall plasma P 4 concentration tended (P < :7) to be higher for heifers in DESLOR- ELIN 75 Group (1:9 :7 ng/ml) compared to that of heifers in the Control Group (11:1 :7 ng/ml; Fig. 9). Within the 6-day period after PGF a, estrus expression was higher (P < :1) for heifers in Control Group (/) compared to that for heifers in DESLORELIN 75 Group (1/). Among the 1 heifers that did not show estrus, 11 of them did not have follicles >1 mm in diameter. 4. Discussion In the present study, ovulation was synchronized with either gonadorelin diacetate (GnRH) or a deslorelin implant (GnRH agonist) in nonlactating dairy cows (Experiments 1 and ) and dairy heifers (Experiment 3). Clearly, deslorelin implants did not suppress plasma P 4 concentrations; rather it increased P 4 concentrations on certain days in late diestrus. However, these increases were sporadic and of minimal magnitude for all three experiments. The results are in agreement with a previous report using a 7 mg deslorelin implant to induce ovulation in nonlactating dairy cows [6]. Size of the CL was not evaluated and there was no significant effect of treatment on the number of CL. In a previous study, it was suggested that a deslorelin implant may increase differentiation and development of the CL, since the number and size of the CL had no effect on plasma P 4 concentration [6]. Luteinizing hormone (LH) surge and pulsatile secretion from the pituitary gland are responsible for ovulation, CL development and CL differentiation [15]. Pulsatile secretion of LH is necessary to maintain luteal activity between Days and 1, but not between Days 1 and 17 [15]. Administration of deslorelin induces a normal LH surge and elevates basal LH during the following [16] or 7 [17] days. High basal concentrations of LH during early stages of the estrus cycle may stimulate CL development and differentiation in cows induced to ovulate with a deslorelin implant. Follicular development was decreased after induction of ovulation with a deslorelin implant. The first-wave largest follicle was inhibited by the deslorelin implant; however, there was variability among doses in the degree of inhibition. In addition, deslorelin implants reduced the number of Class II and III follicles from Days to 16, but there was variability among doses. The differences could be explained due to cow-to-cow sensitivity to different doses of the implant. Substantial variation among cattle has been detected previously using deslorelin implants [6,17]. The second-wave largest follicle was clearly suppressed using 1, 75 or 45 mg deslorelin implants. Follicular growth and return to estrus after PGF a on Day 16 were clearly delayed in cows and heifers induced to ovulate with a deslorelin implant (1, 75 or 45 mg) and the rate of follicular growth was decreased in a dose-dependent manner. In cows that failed to ovulate, the Ovsynch protocol had to be used in order to induce ovulation. Clearly cattle that experience follicular suppression and delayed return to estrus, will grow follicles in the absence of P 4 and therefore, they will need to be resynchronized to avoid development of anovulatory follicles. The deslorelin implants used in the present series of experiments

11 J.A. Bartolome et al. / Theriogenology 61 (4) were biodegradable and therefore not removed. Although the release pattern of deslorelin in vitro is over a 4-day period [18], there appeared to be a greater period of deslorelin release in vivo or an extended period of deslorelin-induced pituitary desensitization to GnRH. Continuous administration of GnRH [19 1] or a single administration of a deslorelin implant [18] desensitizes the pituitary gland to the stimulatory effect of GnRH. Chronic administration of 1 mg of GnRH and 5 mg of buserelin (GnRH agonist) twice daily for 1 days in beef heifers suppressed pulsatile secretion of LH, but not FSH, and inhibited the growth of follicles >9 mm [19]. A more prolonged buserelin administration (using a minipump) suppressed LH and FSH concentrations, and follicles did not grow >5 mm in diameter []. Administration of different doses of a deslorelin implant during the postpartum period and at various stages of the estrous cycle suppressed follicular development and delayed first estrus or extended the interestrus interval [16 18]. Induction of ovulation with a 7 mg deslorelin implant reduced the number of Class III follicles but did not extend CL lifespan, although some cows failed to ovulate by Day 36 [6]. The luteolytic effect of free and conjugated estrogens in cattle has been reported previously [ 4]. Administration of estradiol-17b on Day 13 [5] or Day 17 [6] of the estrous cycle induces PGF a secretion associated with luteolysis. In addition, follicular development and estradiol concentrations have a negative effect on CL lifespan [9] and fertility [7,8]. Electrocauterization and X-ray radiation of ovarian follicles on Day 1 of the cycle reduced estradiol concentrations and extended CL lifespan [9]. Pregnant heifers had a lower estradiol/progesterone ratio compared to nonpregnant heifers [7]. High concentrations of estradiol on Days of the cycle may reduce fertility [9]. Interestingly, a higher conception rate was reported in cows with three follicular waves than those with two waves [3]. In addition, cows with follicles <15 mm in diameter between Days 1 and 14 of the cycle had a higher conception rate compared to cows with follicles >15 mm [31]. The present experiment indicated that administration of a deslorelin implant, as part of a timed-insemination protocol, reduced follicular development during this critical period when the CL is maintained for pregnancy. Indeed a deslorelin implant appeared to reduce pregnancy losses from Day 7 to Day 45, compared to GnRH, when used to induce ovulation in the Ovsynch protocol. However, pregnancy rates at Days 7 and 45 were not different [3]. In conclusion, deslorelin implants induced ovulation, stimulated development of a normal CL, and delayed follicular growth during the subsequent diestrus period. The effect of a slight increase in plasma concentrations of P 4 and the attenuation of follicular development on embryo survival warrant further investigation. However, the dose will have to be adjusted and nonpregnant cows will have to be resynchronized (e.g. at the time of pregnancy diagnosis) to minimize abnormal returns to estrus and ovulation. Acknowledgements The studies received partial support from USDA-BARD Grant No. IS-3-98R. Appreciation is also extended to Pharmacia Corp. (Kalamazoo, MI, USA) for providing Lutalyse TM. This is Florida Agricultural Experiment Station Journal Series No. R-947.

12 418 J.A. Bartolome et al. / Theriogenology 61 (4) Portions of the results presented in this manuscript were reported at The Fourth International Conference on Farm Animal Endocrinology, 7 1 October, 1, Parma, Italy (Domestic Animal Endocrinology vol. 3; 3. p ). References [1] Pursley JR, Kosorok MR, Wiltbank MC. Reproductive management of lactating dairy cows using synchronization of ovulation. J Dairy Sci 1997;8:31 6. [] Momcilovic D, Archbald LF, Walters A, Tran T, Kelbert D, Risco CA, et al. Reproductive performance of lactating dairy cows treated with gonadotrophin-releasing hormone (GnRH) and/or prostaglandin Fa (PGF a ) for synchronization of estrus and ovulation. Theriogenology 1998;5: [3] Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF a and GnRH. Theriogenology 1995;44: [4] Schmitt EJ-P, Diaz T, Drost M, Thatcher WW. Use of a gonadotropin-releasing hormone agonist or human chorionic gonadotropin for timed insemination in cattle. J Anim Sci 1996;74: [5] Dailey RA, Price JC, Simmons KR, Meisterling EM, Quinn PA, Washburn SP. Synchronization of estrus in dairy cows with prostaglandin Fa injection. J Dairy Sci 1986;69: [6] Ambrose JD, Pires MFA, Moreira F, Diaz T, Binelli M, Thatcher WW. Influence of deslorelin (GnRHagonist) implant on plasma progesterone, first wave dominant follicle and pregnancy in dairy cattle. Theriogenology 1998;5: [7] Mann GE, Lamming GE. Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction 1;11: [8] Butler WR, Calaman JJ, Beam SW. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J Anim Sci 1996;74: [9] Fogwell RL, Cowley JL, Wortman A, Ames NK, Ireland JJ. Luteal function in cows following destruction of ovarian follicles at mydcycle. Theriogenology 1985;3: [1] Macmillan KL, Thatcher WW. Effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. Biol Reprod 1991;45: [11] Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB, et al. Proteins secreted by day-16 to -18 bovine conceptuses extend corpus luteum function in cows. J Reprod Fertil 1986;77: [1] Littell RC, Henry PR, Ammerman CB. Statistical analysis of repeated measures data using SAS procedures. J Anim Sci 1997;76: [13] Wilcox CJ, Thatcher WW, Martin FG. Statistical analysis of repeated measurements in physiological experiments. In: Proceedings of the Final Research Co-ordination Meeting of the FAO/IAEA/ARACAL III Regional Network for Improving the Reproductive Management of Meat- and Milk-Producing Livestock in Latin America with the Aid of Radioimmunoassay. Vienna: International Atomic Agency; 199. p [14] Agresti A. An introduction to categorical data analysis. 1st ed. New York: Wiley; [15] Peters KE, Bergfeld EG, Cupp AS, Kojima FN, Mariascal V, Sanchez T, 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: [16] Rajamahendran R, Ambrose JD, Schmitt EJP, Thatcher MJ, Thatcher WW. Effects of buserelin injection and deslorelin (GnRH agonist) implants on plasma progesterone, LH, accessory CL formation and corpus luteum dynamics in Holstein cows. Theriogenology 1998;5: [17] D Occhio MJ, Aspden WJ, Whyte TR. Controlled, reversible, suppression of oestrous cycle in beef heifers and cows using agonists of luteinizing hormone-releasing hormone. J Anim Sci 1996;74:18 5. [18] Mattos R, Orlandi C, Williams J, Staples CR, Trigg T, Thatcher WW. Effect of an implant containing the GnRH agonist deslorelin on secretion of LH. Theriogenology 1;56: [19] Gong JG, Bramley TA, Gutierrez CG, Peters AR, Webb R. Effects of chronic treatment with gonadotropin-releasing hormone agonist on peripheral concentrations of FSH and LH, and ovarian function in heifers. J Reprod Fertil 1995;15:63 7.

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