Introduction Materials and Methods Experiment 1 Experiment 2

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1 Effects of Administration of Prostaglandin F2α at Initiation of the 7-d CO- Synch+CIDR Ovulation Synchronization Protocol for Suckled Beef Cows and Replacement Beef Heifers V. R. G. Mercadante 1, L. E. Kozicki 2, F. M. Ciriaco 1, D. D. Henry 1, C. R. Dahlen 3, M. R. Crosswhite 3, J. E. Larson 4, B. E. Voelz 4, D. J. Patterson 5, G. A. Perry 6, R. N. Funston 7, T. L. Steckler 8, S. L. Hill 9, J. S. Stevenson 9, G. C. Lamb 1 Syopsis The objective of this study was to determine the effect of administering PGF 2α at the initiation of the 7-d CO-Synch+CIDR fixed-timed AI (TAI) protocol on pregnancy rates of suckled beef cows and replacement heifers. Addition of PGF 2α to the 7-d CO-Synch+CIDR protocol decreased concentrations of progesterone in cows and heifers and increased follicle diameter at CIDR removal in cows, but failed to increase TAI pregnancy rates. Summary Two experiments were conducted to determine the effect of administering PGF 2α at the initiation of the 7- d CO-Synch+CIDR fixed-timed AI (TAI) protocol on pregnancy rates of suckled beef cows and replacement heifers. Within location, cows were stratified by days postpartum (DPP), body condition score (BCS), and parity (Exp. 1; n=1,551) and heifers were stratified by BCS (Exp. 2; n=999) and assigned randomly to 1 of 2 treatments: 1) CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with a 25-mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and TAI on d 0); or 2) PG-CO-Synch+CIDR (a 25-mg injection of PGF 2α on d -10 of the CO- Synch+CIDR protocol). Follicle diameter and corpus luteum (CL) development were assessed on d -10 and -3, and pregnancy status determined on d 30 to 35. Blood was collected on d -20, -10, -3, and 0 relative to TAI to determine concentrations of progesterone (P4). In Exp. 1, TAI pregnancy rates did not differ (P=0.667) between treatments and were affected by BCS (P=0.003) and DPP (P=0.006). Concentrations of P4 were greater (P<0.0001) on d -3 for CO-Synch+CIDR than for PG-CO- Synch+CIDR (4.1±0.2 and 3.4±0.2 ng/ml, respectively). Follicle diameter on d -3 differed (P=0.05) between PG-CO-Synch+CIDR (13.4±0.3 mm) and CO-Synch+CIDR (12.5±0.3 mm) treatments. In Exp. 2, TAI pregnancy rates did not differ (P=0.32) between treatments. Concentrations of P4 differed (P<0.0001) on d -3 with greater concentrations of P4 for CO-Synch+CIDR than PG-CO-Synch+CIDR (3.75±0.20 ng/ml and 3.60±0.21 ng/ml, respectively). Follicle diameter was similar (P=0.75) between treatments on d -10 and d -3. Regardless of treatment, cyclic status tended (P=0.06) to improve pregnancy rates to TAI (55% vs. 45%, for cycling and noncycling heifers, respectively).we concluded that addition of PGF2α to the 7-d CO-Synch+CIDR protocol decreased concentrations of P4 in cows and heifers and increased follicle diameter at CIDR removal in cows, but failed to increase TAI pregnancy rates. 1 University of Florida, North Florida Research and Education Center; Marianna, FL 2 School of Agricultural Sciences and Veterinary Medicine, Pontifical Catholic University, Ponce, PR 3 Department of Animal Sciences, North Dakota State University; Fargo, ND 4 Department of Animal and Dairy Sciences, Mississippi State University; Starkville, MS 5 Division of Animal Science, University of Missouri; Columbia, MO 6 Department of Animal and Range Sciences, South Dakota State University; Brookings, SD 7 University of Nebraska, West Central Research and Extension Center; North Platte, NE 8 Dixon Springs Agricultural Center, University of Illinois; Ubana, IL 9 Department of Animal Sciences and Industry, Kansas State University. Manhattan, KS

2 Introduction Utilization of estrus or ovulation synchronization and fixed-timed artificial insemination (TAI) has facilitated the widespread utilization of AI with proven sires to desirable characteristics such as calving ease and birth weight, which can be especially important when breeding replacement heifers (Lamb et al., 2010). In addition, utilization of TAI protocols can also impact the economic viability of cow-calf systems by enhancing weaning weights per cow exposed (Rodgers et al., 2012). The majority of these TAI protocols depend largely on the use of exogenous progesterone (P4) supplemented by an intravaginal controlled internal drug release (CIDR) insert, GnRH-induced ovulation, and PGF 2α to induce luteolysis (Larson et al., 2006; Lamb et al., 2010). In addition, it has been shown that reduced concentrations of P4 can increase luteinizing hormone (LH) pulse frequency (Kojima et al., 1992) and enhance development and growth rates of ovarian follicles (Fortune, 1994), both of which have improved pregnancy to TAI (Lamb et al., 2001). Therefore, we hypothesized that the addition of an injection of PGF 2α at the initiation of the 7-d CO- Synch+CIDR ovulation synchronization protocol would decrease concentration of P4 and increase follicle diameter at the time of CIDR removal and enhance pregnancy rates to TAI in suckled beef cows and replacement beef heifers. Materials and Methods Experiment 1 A total of 1,551 primiparous and multiparous beef cows were enrolled at 10 different locations in 6 states (Florida, Illinois, Kansas, Mississippi, North Dakota and South Dakota). Within location, cows were stratified by days postpartum (DPP), BCS, and parity, and then assigned randomly to receive one of two treatments: 1) 100-μg injection of GnRH (2 ml Factrel; Zoetis Animal Health) at CIDR (1.38 g P4; Zoetis Animal Health) insertion [d -10] with 25-mg injection of PGF 2α (5 ml Lutalyse; Zoetis Animal Health) at CIDR removal [d -3], followed by injection of 100-μg GnRH and TAI [d 0] by 66 h after CIDR removal (CO-Synch+CIDR; n=773); or 2) same as CO-Synch+CIDR with an additional 25-mg injection of PGF 2α administered at CIDR insertion [d -10] (PG-CO-Synch+CIDR; n=778; Figure 1). Experiment 2 A total of 999 replacement beef heifers were enrolled at nine different locations in six states (Florida, Illinois, Mississippi, North Dakota, Nebraska and South Dakota). Within location heifers were stratified by BCS and then randomly assigned to receive the same treatments (CO-Synch+CIDR, n=498; PG-CO- Synch+CIDR, n=501; Figure 1) previously described in Exp. 1, with the modification of the timing of insemination (54 h after CIDR removal). Blood samples were collected on d -20, -10, -3 and 0 from a subset of cows (n=1,136) and heifers (n=273) randomly select within locations. Concentration of P4 was analyzed by radioimmune assay kits (Coat-A-Count; Siemens Healthcare Diagnostics, Los Angeles, CA, USA). Transrectal ultrasonography (5.0-MHz linear array transducer, Aloka 500V, Instrument of Science and Medicine, Vancouver, BC, Canada) was performed on d -10 and d 0 to determine the diameter of the dominant follicle and corpus luteum (CL) volume in a subset of cows (n=235) at the FL, ND-2 and KS-1 locations in Exp. 1 and in a subset of heifers (n=139) at the FL and ND locations in Exp. 2. The SAS (version 9.3; SAS/STAT, SAS Inst. Inc., Cary, NC, USA) statistical package was used for all analyses. The experiment was designed as a randomized block design. Experiment 1 pregnancy rates per TAI and final pregnancy rates at the end of the breeding season were analyzed using the GLIMMIX procedure. The model included the effects of treatment, and the fixed effects of location, parity (primiparous and multiparous), BCS (<5 5 to 5.75, and 6), DPP (<50 d, 50 d to <70 d, and 70 d) and the respective interactions. The GLIMMIX procedure also was used to analyze follicle diameter and presence and volume of CL on d -10 and -3. Follicle diameter on d -10 was used as a covariate in the analysis of follicle diameter on d - 3. The models included the effects of treatment, location, BCS, DPP, and respective interactions. Experiment 2 pregnancy rates per TAI and final pregnancy rate at the end of the breeding season were analyzed using the GLIMMIX procedure. The model included the effects of

3 treatment and the fixed effects of location, BCS (<5, 5.5 to 6, and 6.5), and the respective interactions. The GLIMMIX procedure was also used to analyze follicle diameter and CL volume on d - 10 and -3. The models included the effects of treatment, location, BCS and respective interactions. Results Experiment 1 Overall pregnancy rates to TAI (54.0±0.5% and 50.8±0.5%, for CO-Synch+CIDR and PG-CO- Synch+CIDR, respectively; Table 1) did not differ (P=0.84) between treatments, and no treatment location interaction was detected (P=0.17). In contrast, a location effect (P<0.0001) was detected for pregnancy rates to TAI being the greatest at the ND-2 location (67.0±0.3%) and the least at the KS-1 location (Table 1). Regardless of treatment, cows with BCS < 5 had the poorest pregnancy rate to TAI (43%) compared with cows with BCS between 5 and 5.75 (49%) and BCS to 6 (56%; P=0.003; Table 1). In addition, cows at less than 50 DPP had the poorest pregnancy rate to TAI (34%) compared with cows at > 50 DPP (46% for 50 to 69 DPP vs. 55% for >70 DPP; P=0.006; Table 1). Where cyclic status was assessed, 53% of cows were cyclic at the beginning of the synchronization protocols and cyclic status did not differ (P=0.88) between treatments. A location effect, however, was detected for cyclic status (P<0.0001; Table 1) with the ND-2 location having the fewest cyclic females (40%), whereas the SD-2 location had the greatest proportion of cyclic females (76%). Nonetheless, incidence of pregnancy to TAI was not associated with cyclic status at the initiation of the treatments (55.5% vs. 49.3%, for cyclic and anestrous cows, respectively; P=0.17) and no treatment cyclicity interaction was detected (P=0.87). Concentrations of P4 did not differ (P>0.10; Figure 2) between treatments on d -10 at CIDR insertion and on d 0 at TAI. Nevertheless, cows in the CO-Synch+CIDR treatment had greater (P<0.0001) concentrations of P4 at CIDR removal on d -3 compared with PG-CO- Synch+CIDR (Figure 2). When present in the ovary, the volume of the CL did not differ between treatments on d -10 (10.7±2.0 mm 3 and 13.9±2.4 mm 3, for CO-Synch+CIDR and PG-CO-Synch+CIDR cows, respectively; P=0.35) or on d -3 (17.5±2.3 mm 3 and 12.9±2.2 mm 3, for CO-Synch+CIDR and PG-CO-Synch+CIDR cows, respectively; P=0.18). Furthermore, diameter of the largest ovarian follicle present on d -10 at CIDR insertion did not differ (P=0.85) between treatments (Figure 3). In contrast, diameter of the largest ovarian follicle on d -3 at CIDR insert removal was greater (P=0.05) in the PG-CO-Synch+CIDR cows than in CO-Synch+CIDR cows (Figure 3). Experiment 2 Overall pregnancy rate by TAI (55.2±0.5% and 52.6±0.6%, for CO-Synch+CIDR and PG-CO- Synch+CIDR, respectively; Table 2) did not differ (P=0.32) between treatments, and no treatment location interaction was detected (P=0.47). However, there was a location effect (P<0.0001) with pregnancy rates to TAI being the greatest at the SD-2 location and the poorest at the MS location (Table 2). Where cyclic status was assessed, 80% of heifers were cyclic at the initiation of TAI protocols. Nonetheless, cyclic status differed (P<0.0001) among locations, with the FL location having the least proportion of cyclic heifers (48%) and SD-1 the most cyclic heifers (100%) at the beginning of the TAI protocols. Regardless of treatment, cyclic status tended (P=0.06) to improve pregnancy rates to TAI (55% vs. 45%, for cyclic and noncyclic heifers, respectively). Concentrations of P4 were similar (P>0.10; Figure 4) between treatments on d -10 at CIDR insertion and on d 0 at TAI. However, heifers in the CO-Synch+CIDR treatment had greater (P<0.0001) concentrations of P4 at CIDR removal on d -3 compared to PG-CO-Synch+CIDR (Figure 4). In addition, when present in the ovary, the volume of the CL did not differ between treatments on d -10 (P=0.75) or on d -3

4 (P=0.64). Also, diameter of the largest ovarian follicle was similar (P=0.75; Figure 5) between treatments on d -10 at CIDR insertion and on d -3 at CIDR removal. Conclusion In the present study, by administering PGF 2α at the initiation of the 7-d CO-Synch+CIDR protocol, we succeeded in reducing concentrations of P4 at the time of CIDR removal in both cows and heifers, and in enhancing follicle size at the time of CIDR removal in cows, but not heifers. Pregnancy rates to TAI, however, were not improved when PGF 2α was administered concurrently with GnRH at CIDR insertion. Further investigation into the ideal concentrations of P4 at CIDR insertion and removal, as well as the effects of follicle size and the presence of a CL at key stages of estrous synchronization protocols and their respective impacts on pregnancy outcomes in beef females need further investigation. Acknowledgements Sincere appreciation is expressed to P. Folsom, M. Foran, O. Helms, D. Jones, C. Nowell, T. Schulmeister, and D. Thomas for their assistance with data collection and laboratory analysis. The authors thank Zoetis Animal Health (Florham Park, NY) for their donation of PGF 2α (Lutalyse), GnRH (Fertagyl), and CIDR inserts (CIDR EAZI-Breed). Literature Cited Fortune, J. E Biol. Reprod. 50: Kojima, N., et al Biol. Reprod. 47: Lamb, G. C., et al J. Anim. Sci. 88:E Lamb, G. C., et al J. Anim. Sci. 79: Larson, J. E., et al J. Anim. Sci. 84: Rodgers, J. C., et al J. Anim. Sci. 10:

5 Table 1. Pregnancy rates to fixed-timed AI in suckled beef cows after treatment with CO-Synch+ CIDR or PG-CO-Synch+CIDR. Treatment 1 Item CO-Synch+CIDR PG-CO-Synch+CIDR Overall no. (%) Location FL 76/122 (62) 55/125 (44) 131/247 (53) xy IL-1 28/51 (55) 32/53 (60) 60/104 (58) xy IL-2 30/54 (55) 36/56 (64) 66/110 (60) xy KS-1 18/92 (19) 22/88 (25) 40/180 (22) z KS-2 71/105 (68) 60/105 (57) 131/210 (62) x MS 27/59 (46) 29/62 (47) 56/121 (46) y ND-1 37/61 (61) 33/60 (55) 70/121 (58) xy ND-2 16/27 (59) 21/28 (75) 37/55 (67) x SD-1 82/147 (56) 78/147 (53) 160/294 (55) xy SD-2 33/55 (60) 29/54 (54) 62/109 (57) xy Overall 418/773 (54) 395/778 (50) 813/1,551 (52) BCS 2 < 5 25/53 (47) 24/62 (39) 49/115 (43) x 5 to /398 (50) 196/409 (48) 395/807 (49) y 6 212/366 (58) 196/356 (55) 408/722 (56) y Days post-partum < 50 24/69 (35) 21/62 (34) 45/131 (34) x 50 to 69 68/141 (48) 57/132 (43) 125/273 (46) y /384 (57) 223/413 (54) 442/797 (55) y 1 CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with a 25-mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and fixed-timed AI on d 0). PG-CO-Synch+CIDR (25-mg injection of PGF 2α administered at CIDR insertion [d -10] of the CO-Synch+CIDR). 2 Body Condition Score in a scale of 1 (emaciate) to 9 (obese). x,y,z Percentages within item and column with differing superscripts differ (P<0.01). Effect of treatment (P=0.84); location (P<0.0001); treatment by location (P=0.17).

6 Table 2. Pregnancy rates to fixed-timed AI in replacement beef heifers after treatment with Co-Synch+CIDR or PG-CO-Synch+CIDR. Treatment 1 Location CO-Synch+CIDR PG-CO-Synch+CIDR Overall no. (%) FL 42/62 (68) 31/62 (50) 73/124 (59) x IL-1 13/32 (41) 10/33 (30) 33/65 (51) y IL-2 11/19 (58) 8/20 (40) 19/39 (49) xy MS 3/14 (21) 5/14 (36) 8/28 (29) z ND 14/25 (56) 14/26 (54) 28/51 (55) xy NE 50/118 (43) 54/115 (47) 104/233 (45) yz SD-1 11/21 (52) 13/20 (65) 24/41 (58) xy SD-2 63/101 (62) 69/108 (64) 132/209 (63) x SD-3 68/106 (64) 60/103 (58) 128/209 (61) x Overall 275/498 (55) 264/501 (53) 539/999 (54) 1 CO-Synch+CIDR (100 μg injection of GnRH at CIDR insertion [d -10] with 25 mg injection of PGF 2α at CIDR removal [d -3], followed by injection of GnRH and fixed-timed AI on d 0). PG-CO-Synch+CIDR (a 25 mg injection of PGF 2α administered at CIDR insertion [d -10] of the CO-Synch+CIDR). x,y,z Percentages within item and column with differing superscripts differ. Effect of Treatment (P=0.61); Location (P<0.0001); Treatment*Location (P=0.47). Figure 1. Schematic of treatments. Blood samples (B) were collected on d -20, -10, -3, and 0. Individual body condition scores (BCS; 1=thin to 9=obese) were assigned at d -20. Ovarian ultrasonography (US) was performed on d -10 and -3. Pregnancy diagnosis was performed by US on d 35 and between d 30 to 45 after the end of the breeding season. The CO-Synch+CIDR females received an injection of GnRH on d -10 and CIDR was inserted, followed by injection of PGF 2α (PGF) and CIDR removal on d -3. All females received fixed-timed AI (TAI) followed by injection of GnRH on d 0 (by 66 h for cows, Exp.1; and by 54 h for heifers, Exp. 2) after CIDR insert removal. The PG-CO-Synch+CIDR females received the same treatment previously described with an additional injection of PGF on d -10.

7 Follicle diameter, mm Progesterone, ng/ml CO-Synch+CIDR ** PG-CO-Synch+CIDR d -10 d -3 d 0 Day relative to TAI Figure 2. Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of suckled beef cows by treatment, Exp. 1. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P<0.0001) CO-Synch+CIDR PG-CO-Synch+CIDR ** d -10 d -3 Treatment Figure 3. Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-timed AI of suckled beef cows by treatment, Exp. 1. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P=0.05).

8 Follicle diameter, mm Progesterone, ng/ml CO-Synch+CIDR PG-CO-Synch+CIDR d -10 d -3 d 0 Day relative to TAI Figure 4. Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of replacement beef heifers by treatment, Exp. 2. CO-Synch+CIDR: an injection of GnRH on d -10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. **Means within day differ between treatments (P<0.0001). 13 CO-Synch+CIDR PG-CO-Synch+CIDR d -10 d -3 Treatment Figure 5. Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-timed AI of replacement beef heifers by treatment, Exp. 2. CO-Synch+CIDR: an injection of GnRH on d - 10 and CIDR insertion, followed by injection of PGF 2α and CIDR removal on d -3. Fixed-timed AI followed by injection of GnRH on d 0. PG-CO-Synch+CIDR: same treatment previously described with an additional injection of PGF 2α on d -10. Effect of treatment (P=0.75).