Effects of presynchronization and ecg on pregnancy rates to GnRH-based, fixed-time artificial insemination in beef heifers

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1 Effects of presynchronization and ecg on pregnancy rates to GnRH-based, fixed-time artificial insemination in beef heifers J. A. Small 1,5, M. G. Colazo 2, J. P. Kastelic 3, N. E. Erickson 4, and R. J. Mapletoft 4 1 Agriculture and Agri-Food Canada, Brandon, Manitoba, Canada R7A 5Y3; 2 Alberta Agriculture and Rural Development, Edmonton, Alberta, Canada T6H 5T6; 3 Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada T1J 4B1; and 4 Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatoon, Canada S7N 5B4. Received 8 July 2009, accepted 22 October Small, J. A., Colazo, M. G., Kastelic, J. P., Erickson, N. E. and Mapletoft, R. J Effects of presynchronization and ecg on pregnancy rates to GnRH-based, fixed-time artificial insemination in beef heifers. Can. J. Anim. Sci. 90: Three experiments were conducted to determine the effects of presynchronization and treatment with equine chorionic gonadotropin (ecg) on corpus luteum (CL) and ovarian follicular development, plasma progesterone concentrations, and pregnancy rates in beef heifers subjected to a gonadotropin releasing hormone (GnRH)-based, fixed-timed AI (TAI) protocol. All heifers were given GnRH on day 0, prostaglandin F 2a (PGF) on day 7, and a second GnRH on day 9 concurrent with TAI (54 h after PGF). In exp. 1(N148), presynchronization with PGF (days 22 and 11) decreased the percentage of heifers with non-luteal plasma progesterone concentrations on day 0 (5.4 vs 29.7%) and day 7 (0 vs 11.6%; PB0.05), but not on day 9 (74.3 vs. 66.2%; P0.20), and reduced the number of heifers in estrus and bred before TAI (PB0.05). Although presynchronization reduced preovulatory follicle diameter ( vs mm; mean9 SEM; PB0.01), it did not affect TAI pregnancy rates (36.5 vs. 29.7%; P0.20). In exp. 2, heifers (N128) were presynchronized with melengestrol acetate (MGA) (days 27 to 12), and received a controlled internal drug release (CIDR) on day 0; on day 7, half were given 300 IU of ecg at CIDR removal. Treatment with ecg tended to increase preovulatory follicle diameter in heifers that did not ovulate to GnRH on day 0 (P0.06), but did not affect the percentage of heifers with non-luteal plasma progesterone concentrations on day 9 (57.8 vs. 57.8%) or TAI pregnancy rates (48.4 vs. 53.1%; P0.20). Experiment 3 was a 22 factorial arrangement of presynchronization (PGF concurrent with a CIDR on day 7) and ecg treatments (on day 7) applied to heifers in three herds (A, N150, B, N260 and C, N40). All heifers had a once-used CIDR from days 0 to 7. Presynchronization increased the percentage of heifers (Herd A) with low-luteal plasma progesterone concentrations on day 0 (70.7 vs. 22.7%) and day 7 (90.7 vs. 53.3%; PB0.01), but did not affect the percentage of heifers with non-luteal concentrations of progesterone on day 9 (97.3 vs. 93.3%; P0.20). Combined for all herds, presynchronization reduced the prevalence of a CL on day 0 (23.5 vs. 73.7%; PB0.01), and increased the prevalence of follicles ]10 mm on day 7 (96.8 vs. 86.7%; PB0.01); however, TAI pregnancy rates (195/ %) were not improved by either presynchronization or ecg treatment (P0.20). Key words: Presynchronization, equine chorionic gonadotropin, GnRH, fixed-time artificial insemination, progesterone Small, J. A., Colazo, M. G., Kastelic, J. P., Erickson, N. E. et Mapletoft, R. J Effets de la pre -synchronisation et de l administration de gonadotrophine chorionique e quine (ecg) sur le taux de conception des génisses de boucherie inséminées artificiellement a` pe riode fixe par administration de GnRH. Can. J. Anim. Sci. 90: Les auteurs ont procéde à trois expe riences pour ve rifier les effets de la pre -synchronisation et de l administration d ecg sur le de veloppement du corps jaune et des follicules ovariens, sur la concentration plasmatique de progeste rone et sur le taux de conception des ge nisses de boucherie inse mine es artificiellement à pe riode fixe (IAPF) apre` s administration de GnRH. Les sujets ont reçu de la GnRH le jour 0, de la PGF le jour 7 et une deuxie` me dose de GnRH le jour 9, paralle` lement a` l IAPF (54 h apre` s la PGF). Dans la premie` re expérience (N 148), la pré-synchronisation avec la PGF (jours 22 et 11) diminue la proportion de ge nisses présentant de la progeste rone lute ale dans le plasma le jour 0 (5,4 c. 29,7 %) et le jour 7 (0 c. 11,6 %; P B 0.05), 5 To whom correspondence should be addressed (current address): Haley Institute, Department ofplant and Animal Sciences, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5B3 ( julie.small@agr.gc.ca, jsmall@nsac.ca). Portions ofthese data were presented at the Annual Meetings ofthe International Embryo Transfer Society, Portland, OR, USA (January 2004), Copenhagen, Denmark (January 2005), and Orlando, FL, USA (January 2006). 23 Abbreviations: CIDR, controlled internal drug release (1.9 g progesterone); CL, corpus luteum; ecg, equine chorionic gonadotropin; GnRH, gonadotropin releasing hormone; LH, lutenizing hormone; MGA, melengestrol acetate; PGF, prostaglandin F 2a ; TAI, fixed-time artificial insemination

2 24 CANADIAN JOURNAL OF ANIMAL SCIENCE mais pas le jour 9 (74,3 c. 66,2 %; P 0,20); la pré-synchronisation réduit aussi le nombre de génisse en æstrus accouple es avant l IAPF (PB0,05). Bien qu elle diminue le diame` tre des follicules pre -ovulatoires (12,990,3 c. 14,990,3 mm; moyenne9e.-t.; PB0,01), la pre -synchronisation ne modifie pas le taux de conception IAPF (36,5 c. 29,7 %; P0,20). Lors de la deuxième expérience, les ge nisses (N128) ont e te pre -synchronise es avec du MGA (jours 27 a` 12) avant de recevoir un me dicament a` libération controˆ le e le jour 0; le jour 7, on a administre a` la moitie 300 UI d ecg au retrait du me dicament a` libe ration controˆ le e. L administration d ecg a tendance à augmenter le diame` tre des follicules pre -ovulatoires chez les ge nisses que la GnRH ne fait pas ovuler le jour 0 (P0,06), mais il ne modifie pas le pourcentage de ge nisses dont le plasma contenait de la progeste rone non-lute ale le jour 9 (57,8 c. 57,8 %) ni le taux de conception IAPF (48,4 c. 53,1%; P0,20). La troisie` me expe rience consistait en un essai en disposition factorielle 2 2 prévoyant la présynchronisation (administration de PGF et d un me dicament a` libération controˆ lée le jour 7) et l administration d ecg (le jour 7) aux ge nisses de trois troupeaux (A, N150, B, N260, et C, N40). Les ge nisses avaient toutes rec uun me dicament à libe ration controˆ le e du jour 0 au jour 7. La pre -synchronisation accroıˆ t la proportion de ge nisses (troupeau A) pre sentant une faible concentration de progeste rone lute ale dans le plasma le jour 0 (70,7 c. 22,7 %) et le jour 7 (90,7 c. 53,3 %; P B 0,01), mais n a aucune incidence sur la concentration de progeste rone non lutéale le jour 9 (97,3 c. 93,3 %; P0,20). Quand on regroupe les résultats des trois troupeaux, on constate que la pre -synchronisation réduit la pre valence d un corps jaune le jour 0 (23,5 c. 73,7 %; PB0,01) et augmente celle de follicules de plus de 10 mm le jour 7 (96,8 c. 86,7 %; PB 0,01); cependant, ni la pre -synchronisation ni l administration d ecg n ame liorent (P 0,20) le taux de conception IAPF (195/43944,4 %). Mots clés: Pre -synchronisation, gonadotrophine chorionique équine, GnRH, insémination artificielle a` pe riode fixe, progeste rone Current gonadotropin releasing hormone (GnRH)- based synchronization protocols for fixed-time AI (TAI) of cattle (Pursley et al. 1995; Small et al. 2001) typically consist of two GnRH treatments 9 d apart, with prostaglandin F 2a (PGF) 7 d after the first GnRH, and TAI either 16 to 18 h after the second GnRH (Ovsynch), or concurrent with the second GnRH (CO- Synch). Pregnancy rates have been lower in heifers than cows subjected to these protocols (Pursley et al. 1997; Wiltbank, 1997), but were generally improved in heifers by inseminating those observed in estrus prior to scheduled TAI (Johnson and Day 2004). Furthermore, exogenous progestin suppressed the expression of estrus between the first GnRH and TAI, and increased pregnancy rates to TAI in heifers (Martinez et al. 2002). Low pregnancy rates in GnRH-based TAI protocols have been attributed to failure to ovulate in response to the first GnRH, premature estrus, anestrus (Wiltbank 1997; Martinez et al. 2000; Thatcher et al. 2001), and GnRH-induced ovulation of small preovulatory follicles (Vasconcelos et al. 2001; Macmillan et al. 2003; Perry et al. 2007). Ovulation to the first GnRH treatment occurred in approximately 85% of lactating dairy cows (Pursley et al. 1995, 1997), but only 55% of heifers (Pursley et al. 1997; Martinez et al. 1999). In Bos taurus cattle, GnRH induced ovulation of ovarian follicles approximately 10 mm in diameter or larger ( Vasconcelos et al. 1999; Sartori et al. 2001). Therefore, the diameter of the largest follicle at the first GnRH treatment influenced the ovulatory response to the subsequent GnRH treatment (Vasconcelos et al. 1999; Martinez et al. 2000; Moreira et al. 2000). Furthermore, the size and maturity of the dominant follicle at the time of PGF treatment influenced the interval from treatment to ovulation (Kastelic et al. 1990). It has also been shown that plasma lutenizing hormone (LH) concentrations and ovulation rate in response to exogenous GnRH were greater in cattle with low- (3.0 ng ml 1 ) than high- (5.7 ng ml 1 ) luteal plasma progesterone concentrations (Colazo et al. 2008). Therefore, presynchronization is often done prior to a GnRH-based TAI protocol. In dairy cows, pregnancy rates to TAI were increased when the first GnRH was given 12 d after the second of two PGF presynchronization treatments (Moreira et al. 2001; Thatcher et al. 2002; El-Zarkouny et al. 2004), when a large GnRHresponsive follicle was expected to be present. Although pregnancy rates in GnRH-based protocols were not influenced by follicle diameter when AI was based on expression of estrus (Perry et al. 2007), GnRHinduced ovulation of small preovulatory follicles (B12 mm diameter) reduced pregnancy rates to TAI (Vasconcelos et al. 2001; Macmillan et al. 2003; Perry et al. 2007). However, treatment with equine chorionic gonadotropin (ecg) increased pregnancy rates in primiparous lactating beef cows subjected to controlled internal drug release (CIDR)-based TAI protocols (Small et al. 2009), presumably by enhancing the diameter of the preovulatory follicle (Bo et al. 2007). The objectives of the present study were to investigate the effects of presynchronization strategies and ecg treatment on corpus luteum (CL) and follicle development, plasma progesterone concentrations, and pregnancy rates in beef heifers subjected to GnRH-based TAI. We hypothesized that pregnancy rates to GnRHbased TAI in heifers would be improved by using presynchronization to increase the probability of a follicle ovulating to the first GnRH, or by giving ecg to increase the growth and development of the preovulatory follicle. MATERIALS AND METHODS Animals, Treatments and Sampling Schedules Three experiments were conducted with beef heifers; treatment and sampling schedules and sources of

3 SMALL ET AL. * PRESYNCHRONIZATION AND ecg 25 pharmaceuticals are shown (Fig. 1). All heifers were given GnRH on day 0, PGF on day 7, and heifers subjected to TAI on day 9 were concurrently given a second GnRH treatment (54 h after PGF). Crossbred beef heifers at the Agriculture and Agri-Food Canada Research Centre, Brandon, MB (Herd A, located 498N and 998W) were used in each of three successive years (i.e., in all experiments). In exp. 1, Gelbvieh-cross beef heifers (N 148) were either presynchronized with PGF treatment on days 22 and 11 (without reference to stage of the estrous cycle), or not presynchronized. Ultrasonographic examinations were done on days 0, 9, and 16, and blood samples were collected on days 1 1,0,7,and 9. In exp. 2, Red Angus-cross heifers (N 128) were presynchronized by feeding melengestrol acetate (MGA; 0.5 mg head 1 d 1 ) for 15 d; 12 d after the last MGA feeding, GnRH was given (day 0) concurrent with insertion of a CIDR. On day 7, CIDR were removed, PGF was given, and half of the heifers received ecg. Ultrasonographic examinations were done on days 0, 7, 9, and 16 and blood samples were collected on days 0, 7, and 9. In exp. 3, Angus-cross heifers (N 150) were randomly assigned to one of four groups in a 22 factorial arrangement of presynchronization and ecg treatments. Half the heifers were presynchronized with a CIDR inserted concurrent with PGF treatment on day 7. On day 0, all heifers received GnRH, CIDR remained in the presynchronized heifers, and the half Exp.group & & & 3.4 PGF z MGA w PGF z PGF z CIDR y that had not been presynchronized were concurrently given a once-used CIDR (used 7 d and autoclaved before reuse) to provide sub-luteal progesterone concentrations, as would be expected in the presynchronized heifers after 7 d of CIDR treatment (Colazo et al. 2004). On day 7, PGF was given and all CIDR were removed; half the heifers in each group were concurrently treated with ecg. Ultrasonographic examinations of the ovaries were done on days 7 (presynchronized heifers only; N 75), 0, and 7. Blood samples were collected from all heifers on days 7, 0, 7, and 9. In addition to Herd A, exp. 3 was conducted with Angus heifers in a commercial herd in Lethbridge, AB, Canada (Herd B, N 260, located 498N and 1128W) and predominantly Hereford-cross heifers at the University of Saskatchewan Goodale Farm in Saskatoon, SK, Canada (Herd C, N40, located 528N and 1068W). Ultrasonographic examinations were done on days 7, 0, and 7 on subsets of heifers in Herd B, and on all heifers in Herd C. In Herd B, examinations on day 7 included heifers assigned to presynchronization (N 123), whereas those examined on day 0 included randomly selected heifers in presynchronized (N 29) and not-presynchronized (N 130) groups, and on day 7 included a subgroup of heifers from each treatment group: Presynchronized, with (N 18) and without (N 11) ecg; and not-presynchronized, with (N 20) and without (N21) ecg that had also been examined on day 0. GnRH y PGF z +/ ecg u GnRH y +TAI x CIDR v u-cidr y +/ ecg u +/ ecg u US PD Day Fig. 1. Treatment days (circles) and sampling schedules are shown for beef heifers in exps. 1, 2, and 3. Indicated, as specified in the text for each experiment, are days of ultrasonographic (US) examinations of ovarian structures for CL and follicle development (open circles), ultrasonographic examinations for pregnancy diagnosis (PD), and blood sampling for progesterone analyses (2). z 500 mg cloprostenol im (PGF 2a ; Estrumate; Schering Plough Animal Health, Pointe Claire, QC, Canada). y 100 mg gonadotropin releasing hormone im (GnRH; Cystorelin; Merial Canada Inc., Baie D Urfe, QC, Canada). x Fixed-time artificial insemination (TAI; 54 h after PGF). w 0.5 mg d 1 melengestrol acetate (MGA; Pfizer Animal Health, Montreal, QC, Canada). v 1.9 g progesterone (CIDR; Pfizer Animal Health), u-cidr; used once for 7 d and autoclaved before reuse. u With () and without () 300 IU equine chorionic gonadotropin im (ecg; Pregnecol, Bioniche Animal Health, Belleville, ON, Canada).

4 26 CANADIAN JOURNAL OF ANIMAL SCIENCE All injectable hormone preparations were given i.m. in the neck. All experiments were reviewed and approved by the respective institutional animal care committees and were conducted in accordance with the guidelines of the Canadian Council on Animal Care (1993). Estrus Detection and Bull Breeding after TAI Each year, Herd A heifers observed in estrus in the morning (0800 to 0830) or afternoon (1600 to 1630) on days 6, 7, or 8 were inseminated the following afternoon (1430) or morning (0830), respectively, and were deemed in premature estrus and not pregnant to TAI. Commercial semen from Angus sires with proven records of fertility was used for all inseminations, and heifers were exposed (36:1) to 2-yr-old Red Angus bulls from days 11 to 53. All bulls had passed a standard breeding soundness evaluation conducted by the herd veterinarian prior to the breeding season. These bulls were equipped with chin-ball marking harnesses (Millennium Genetics, Lethbridge, AB) and observations were made daily to record heifers with substantial marking paint on their tail head. Chin-ball marking paint was checked daily and replenished at least twice weekly. Ultrasonographic Examinations Transrectal ultrasonographic examinations were done with real-time, linear-array ultrasonography with a 7.5 MHz transducer (Aloka 500, Aloka, Tokyo, Japan). Ovaries were visualized and the locations and maximum diameters of the two largest follicles and any detectable CL (Martinez et al. 2002; Colazo et al. 2004, 2008) were recorded. Similarly, ultrasonographic examinations of ovarian structures and uterine contents were done on either days 36, 37 or 38 to determine pregnancy rates to AI or TAI. Cumulative pregnancy rates (overall AI, TAI, and natural service) were determined by transrectal palpation of uterine contents on either days 126 or 127. Bull breeding after TAI was not done for Herds B or C, and ultrasonographic examinations for Herds B and C were as described for Herd A, except that TAI pregnancy rates were determined on day 44 (35 d after TAI) in Herd B and on day 63 (54 d after TAI) in Herd C. The TAI pregnancy rates were calculated as follows: ((number of heifers pregnant to TAI/total number of heifers) 100). Ultrasonography with a 5.0 MHz linear array transducer was used to measure backfat thickness between the 12th and 13th ribs. As part of another experimental protocol for Herd A, body weight and backfat thickness were recorded every 28 d from 6 to 14 mo of age and these data were used to determine body weight and subcutaneous fat on day 12 (Table 1). Blood Sampling and Progesterone Analyses Blood samples (Herd A only) were collected by venipuncture of the caudal coccygeal blood vessels, and plasma progesterone concentrations were determined by enzyme immunoassay (Del Vecchio et al. 1995). The Table 1. Age, body weight, and backfat thickness z of cross-bred beef heifers on day 12, which were subjected to GnRH-based TAI synchronization on (exps. 1 and 2, and Herd A for exp. 3) Experiment Item 12 3 No. heifers Age (mo) a b b Body weight (kg) a b c Backfat thickness (mm) a a b Backfat thickness categories 1to 3.5 mm (%) 63.2a 43.7b 7.0c 3.6 to 5.0 mm (%) 28.1a 39.8b 25.8a 5.1to 13.5 mm (%) 8.6a 16.4a 67.2b z Backfat thickness measured ultrasonically between the 12th and 13th rib. ac Within a row, means (9 SEM) or percentages without a common letter are different (PB0.05). assay was linear to 16 ng ml 1. Values for sensitivity, intra- and inter-assay variation were 0.05 ng ml 1, B10%, and B15%, respectively. Plasma progesterone concentrations were assigned to one of three categories, non-luteal (B1ng ml 1 ), low-luteal (1to 4 ng ml 1 ), and mid-cycle (4 ngml 1 ), as described (Battocchio et al. 1999). Heifers with plasma progesterone concentrations]1ng ml 1 on day 7 andb1ng ml 1 on day 9 were deemed PGF responsive, in that a synchronized decrease from luteal to non-luteal concentrations of plasma progesterone occurred by the time of TAI. Statistical Analyses All statistical analyses were done using SAS software (Release 9.1, SAS Institute, Inc., Cary, NC), and, unless otherwise stated, means are presented as the least squares means9sem. Logistic Regression was used to determine the effects of treatment(s) on rates of estrus and AI before TAI (days 6, 7, and 8), pregnancy to AI (days 6 to 8) and TAI (day 9), rebreeding by bulls (days 11 to 26), pregnancy cumulative to AI, TAI, and bull breeding, ovulation following GnRH on day 0, the occurrence of CL, ovarian follicles ]10 mm, and preovulatory follicles, and the distribution of plasma progesterone concentrations. In exp. 1, the ANOVA for ovarian structures on day 0 included data for all heifers, and on days 9 and 16 included only those subjected to TAI. A second model was used to determine if the diameter of the preovulatory follicle was independent of the effects of PGF presynchronization and TAI pregnancy, or the interaction. A paired Student s t-test was used to compare the diameters of the largest follicle on day 9 and the preovulatory follicle. In exp. 2, a second model was used to determine if the diameter of the preovulatory follicle was independent of the effects of ovulation to the first GnRH, ecg treatment, and TAI pregnancy, or their interactions.

5 SMALL ET AL. * PRESYNCHRONIZATION AND ecg 27 In exp. 3, Herd was the only factor in the ANOVA of data collected on day 7, otherwise the main effects of presynchronization, ecg and Herd, and the twoand three-way interactions were subjected to Logistic Regression (backward selection with probability of 50.20); the exception being the exclusion of herd and three-way interactions in the ANOVA of plasma progesterone concentrations determined for Herd A only. RESULTS Experiment 1: Presynchronization with PGF Presynchronization with PGF reduced the proportion of heifers detected in estrus before TAI and, therefore, increased the number of heifers subjected to TAI (Table 2). The TAI and overall pregnancy rates did not differ between treatments (P 0.20). As rates of estrus within 17 d of TAI were also similar, data for both treatments were combined for presentation (Fig. 2a). Presynchronization influenced CL and ovarian follicular development on days 0 and 9. Although the prevalence of heifers with a CL on day 0 (145/148 98%) did not differ between treatments (P 0.20), the mean diameter of CL, and the prevalence and mean diameter of follicles]10 mm were greater for presynchronized than not-presynchronized groups (Table 2). In contrast, presynchronization reduced the mean diameter of the largest follicle ]10 mm on day 9 (Table 2). At this time, based on high prevalences for follicles ]10 mm (125/12898%), and for CL (127/ 12899%) with relatively small mean diameters ( mm), most heifers were in proestrus, and there was no difference between treatments (P 0.20). It was noteworthy that 97% of heifers subjected to TAI had a CL on day 16; presynchronization had no effect on the mean diameter of CL ( mm; N 124; P 0.20). In retrospect, the mean diameter of preovulatory follicles was smaller for presynchronized than not-presynchronized heifers (Table 2), but not different between pregnant and non-pregnant heifers ( mm; N 108; P 0.20). The proportion of heifers with a retrospectively identified preovulatory follicle did not differ between treatments (108/ %, P 0.20), and the follicle that ovulated following TAI was apparently the largest follicle on day 9 in 89% of heifers (96/108), In the remaining heifers, the diameter of the follicle that ovulated ranged from 1to 8 mm less than the largest follicle on day 9. Table 2. The effects of presynchronization (with PGF on days 22 and 11) in a GnRH-based TAI protocol on premature estrus, pregnancy rates, follicle and CL development and plasma progesterone concentrations in beef heifers (exp. 1) Treatment Item Presynchronized Not-presynchronized Prob. No. heifers Estrus and AI before TAI (%) z 2.7 (2) 24.3 (18) TAI on day 9 (%) 97.3 (72)a 75.7 (56)b B0.01 AI pregnant day 36 (%) 1.3 (1) 16.2 (12) TAI pregnant day 36 (%) 36.5 (27) 29.7 (22) 0.20 Rebred days 11 to 26 (%) 16.6 (12) 21.4 (16) 0.20 Cumulative pregnant day 127 (%) y 83.8 (62) 89.2 (66) 0.20 Ultrasonography Day 0 CL median diameter (mm; quartiles) 20.0; 19,22a 19.0; 12,21b B0.01 Largest follicle ]10 mm diameter (%) 97.3a 81.1b B0.01 Largest follicle diameter (mm) x a b B0.01 Day 9 x,w Largest follicle diameter (mm) a b 0.01 Preovulatory follicle diameter (mm) a b B0.01 Distribution of plasma progesterone concentrations (%) Day 11 Non-luteal 2.7a 24.3b B0.01 Mid-cycle 82.4a 52.7b Day 0 Non-luteal 5.4a 29.7b 0.03 Low-luteal 41.9a 24.3b Day 7 Non-luteal 0.0a 11.6b B0.01 Mid-cycle Day 9 Non-luteal PGF response rate v z Number of heifers in parentheses. y Includes AI, TAI and natural service (days 11 to 51). x Exclusive of follicles B10 mm in diameter. w Includes only heifers subjected to TAI. v PGF response defined as the proportion of heifers with plasma progesterone concentrations]1ng ml 1 on day 7 and B1ng ml 1 on day 9. a, b Within a row, percents, medians or means (9 SEM) without a common letter are different (PB0.05).

6 28 CANADIAN JOURNAL OF ANIMAL SCIENCE (a) 128 Number of heifers (b) 128 Number of heifers (c) 150 Number of heifers Day Day Not-presynchronized Presynchronized Day Fig. 2. Heifers bred by bulls after TAI (day 9) in exps. 1(a), 2 (b) and 3 (c; Herd A). Rebreeding rates were 16, 17 and 7% from days 11 to 26, and were 18, 18 and 65% from days 26 to 34, respectively. The number rebred (vertical bars) did not differ (P0.05) among treatments within experiments. However, in exp. 3, cumulative pregnancy rates were lowest for presynchronized heifers (Table 4) and, therefore, cumulative percent rebred is shown for the not-presynchronized and presynchronized treatments. Presynchronization reduced the prevalence of nonluteal plasma progesterone concentrations from 30% tob5% on days 11, 0 and 7, but had no effect plasma progesterone concentrations on day 9 (Table 2). Conversely, presynchronization increased the prevalence of mid-cycle and low-luteal plasma progesterone concentrations on days 11 and 0, respectively (Table 2). All presynchronized heifers had mid-cycle plasma Cumulative percentage Cumulative percentage Cumulative percentage progesterone concentrations on day 7, and were more likely (P 0.06) to respond to PGF (Table 2). The prevalence of low-luteal plasma progesterone concentrations on day 9 was 25.8% of those subjected to TAI (33/128), 50.0% of those AI early (10/20), and overall 29.0% (43/148), with no difference (P 0.20) between presynchronization treatments. Experiment 2: ecg after MGA Presynchronization and CIDR As no heifers were detected in estrus on days 6, 7, or 8, all were subjected to TAI on day 9. Pregnancy rates and the number of heifers rebred within 17 d of TAI were not affected by ecg treatment (Table 3); therefore, return services were combined for presentation (Fig. 2b). The prevalence of heifers with a CL on day 0 was 87.5% (112/128), ovulation rate to the first GnRH was 61.4% (78/128), and detection of preovulatory follicles on day 9 was 96.0% (123/128). The ecg treatment on day 7 tended to increase the mean diameter of the preovulatory follicle in heifers that did not ovulate after the first GnRH, and the mean diameter of the preovulatory follicle was significantly larger in heifers that subsequently became pregnant to TAI than those that did not (Table 3). Treatment with ecg had no effects on the distribution of plasma progesterone concentrations on day 9 or PGF response rates (Table 3; P 0.20). Although the majority (80%) of heifers had mid-cycle progesterone concentrations on day 7, only 58% were non-luteal on day 9. Experiment 3: ecg with and without Presynchronization with CIDR The proportions of heifers subjected to TAI were 97.3, 97.3, and 100% for Herds A, B, and C, respectively. In Herd A, four heifers were detected in estrus and inseminated 1d before TAI, and in Herd B, seven heifers were excluded on day 0 due to lost CIDR inserts. Presynchronization had no effect on TAI pregnancy rates (overall 44.4%, 195/439), and ecg treatment did not increase TAI pregnancy rates in any herd; on the contrary, ecg decreased TAI pregnancy rates in Herd B (Table 4; PB0.05). Overall, Herd A had the lowest TAI pregnancy rate; within this herd, cumulative pregnancy rates were lower for the presynchronized than potpresynchronized groups (P B0.05). Only 7% (10/150) of heifers in Herd A were rebred by day 26, whereas 65% (98/150) were rebred from days 26 to 34 (Fig. 2c). On day 7 the prevalence and mean diameter of CL, and the mean diameter of follicles ]10 mm were greater for Herd A than Herd B or C. Presynchronization had no effect on the prevalence or mean diameter of follicles ]10 mm on day 0 (274/ % and mm; P0.20, respectively), and increased the prevalence, but not mean diameter, of follicles]10 mm on day 7 (121/ % vs 117/ %, P B0.01and vs mm, P 0.15, for presynchronized

7 SMALL ET AL. * PRESYNCHRONIZATION AND ecg 29 Table 3. The effects of ecg (concurrent with PGF and CIDR removal on day 7) in a GnRH-based TAI protocol on pregnancy rates, follicle development, and plasma progesterone concentrations in beef heifers presynchronized with MGA (days 27 to 12) and given a CIDR insert on days 0 to 7 (exp. 2) Treatment Item ecg ecg Prob. No. heifers TAI pregnant day 37 (%) z 48.4 (31) 53.1 (34) 0.20 Rebred days 11 to 26 (%) 12.5 (8) 23.4 (15) 0.11 Cumulative pregnant day 126 (%) y 93.7 (60) 92.2 (59) 0.20 Ultrasonography: Preovulatory follicle diameter (mm) With ovulation to GnRH on Day Without ovulation to GnRH on Day Preovulatory follicle diameter (mm) TAI pregnant a a 0.03 Not TAI pregnant b b Distribution of plasma progesterone concentrations (%) Day 7 Non-luteal Low-luteal Mid-cycle Day 9 Non-luteal Low-luteal Mid-cycle PGF response rate x z Number of heifers in parentheses. y Includes TAI (all heifers) and natural service (days 11 to 56). x PGF response defined as the proportion of heifers with plasma progesterone concentrations ]1ng ml 1 on day 7 and B1ng ml 1 on day 9. a, b Within a column, follicle diameter means (9 SEM) for TAI pregnant and Not TAI pregnant without a common letter are different (PB0.05). and not-presynchronized, respectively). Presynchronization reduced the prevalence and diameter of CL on day 0 in all herds, but the effect of presynchronization on day 7 CL differed among herds (Table 5). In Herd A, plasma progesterone concentrations were luteal (]1ng ml 1 ) in 75% of heifers (112/150) on day 7. Presynchronization increased the proportion of heifers with low-luteal plasma progesterone concentrations on days 0 and 7, but had no significant effect on PGF response rates or the prevalence of non-luteal plasma progesterone concentrations on day 9 (143/ %; Table 6). DISCUSSION Presynchronization, Progestin and the Prevalence of Premature Estrus Estrus detection and AI before TAI did not significantly improve AI pregnancy rates in these experiments, probably because so few heifers exhibited premature estrus and were inseminated before TAI (days 6, 7, or 8). Table 4. Effects of presynchronization (with PGF at CIDR insertion on day 7) and ecg (concurrent with PGF and CIDR removal on day 7) in a GnRHbased TAI protocol on pregnancy rates in beef heifers in three herds given a once-used CIDR on day 0 concurrent with the first GnRH (exp. 3) Item A B C Prob. No. heifers Estrus and AI before TAI (%) z 2.6 (4) ND y ND TAI on day 9 (%) 97.3 (146) 97.3 (253) TAI pregnant: With () ecg (%) 28.4 (21)a 44.4 (56)bc 55.0 (11)b 0.02 Without () ecg (%) 26.3 (20)a 59.1(75)bd 60.0 (12)b Rebred day 11 to 26 (%) 6.7 (10) ND ND Cumulative pregnant x : Presynchronized (%) 64.0 (48)c 0.03 Not-presynchronized (%) 80.0 (60)d Herd z Number of heifers in parentheses. y Not determined; seven heifers in Herd B were excluded because of lost CIDRs. x Includes AI, TAI and natural service (days 11 to 51) pregnant on day 175; not determined for Herds B or C. a, b Within a row, Herd means with different letters are different (PB0.05). c, d Within a column, within Grouping, Treatment means with different letters are different (PB0.05).

8 30 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 5. Ultrasonographic evaluation of the effects of presynchronization (with PGF at CIDR insertion on day 7) on CL and follicle development in beef heifers, in three herds, given GnRH and a once-used CIDR on day 0 (exp. 3) Item A B C Prob. Herd Day 7 No. heifers CL present (%) 92.7a 79.2b 12.5c B0.01 CL diameter (mm) a ab b 0.05 Largest follicle diameter (mm) a b b B0.01 Day 0 No. heifers CL present (%) z Presynchronized 26.7ad 24.1ad 9.5a B0.01 Not-presynchronized 92.0ae 71.0be 15.8c CL diameter (mm) Presynchronized d d d 0.05 Not-presynchronized e e e Largest follicle ]10 mm (%) Day 7 No. heifers CL present (%) z Presynchronized 73.3a 31.0bd 47.6ab B0.01 Not-presynchronized 61.3a 73.2ae 42.1a CL diameter (mm) z Presynchronized ad ab b B0.01 Not-presynchronized ae a a Largest follicle ]10mm (%) Presynchronized 100.0ad 98.4bd 90.5bd B0.01 Not-presynchronized 97.3ae 89.2be 89.5be B0.01 z Herd by treatment interaction is significant (PB0.05). ac Within a row, Herd means (9 SEM) without a common letter are different (PB0.05). d, e Within a column, Treatment means (9 SEM) without a common letter are different (PB0.05). Approximately 15% of heifers have been reported to exhibit premature estrus when a CIDR was not used in a GnRH-based protocol (Johnson and D ay 2004). In exp. 1, we inferred that most heifers were pubertal, as ]70% of heifers in the not-presynchronized group had luteal plasma progesterone concentrations on days 11 and 0, and 97% had a CL on day 0. Because the response to PGF is dependent on a PGF-responsive CL, PGF presynchronization as a method of suppressing premature estrus may not be suitable for prepubescent heifers. In exp. 2, premature estrus was eliminated with MGA presynchronization and progestin treatment (new CIDR) following the first GnRH. Since MGA suppression of estrus is not dependent on the presence or absence of a CL, this method may be more suited to groups of heifers where the prevalence of puberty is unknown, and it is feasible to feed MGA. In agreement with a previous study (Martinez et al. 2002), progestin treatment between GnRH and TAI suppressed the expression of premature estrus (3% overall in Herd A), but in contrast, pregnancy rates were not improved. Presynchronization and Ovulation to the First GnRH Following GnRH treatment, there was a new wave of ovarian follicular growth due to loss of follicular Table 6. The effects of presynchronization (PGF at CIDR insertion on day 7), and ecg (day 7 concurrent with CIDR removal and PGF on day 7) on GnRH-based synchronization of plasma progesterone concentrations in beef heifers given a once-used CIDR on day 0 (exp. 3: Herd A) Item Treatment Presynchronized Notpresynchronized Prob z No. heifers Day 0 Non-luteal B0.01 Low-luteal 70.7a 22.7b Mid-cycle 29.3a 50.7b Day 7 Non-luteal 0 0 B0.01 Low-luteal 90.7a 53.3b Mid-cycle 9.3a 46.7b Day 9 Non-luteal Low-luteal Mid-cycle 0 0 PGF response rate y z Effects of ecg and interaction with presynchronization were not significant (P0.20). y PGF response defined as the proportion of heifers with plasma progesterone concentrations ]1ng ml 1 on day 7 and B1ng ml 1 on day 9. a, b Within a row, numbers without a common letter are different (PB 0.05).

9 dominance, ascribed to either atresia (Twagiramungu et al. 1995) or ovulation (Twagiramungu et al. 1995; Martinez et al. 1999). Low pregnancy rates in GnRHbased TAI programs have been attributed to failure to ovulate in response to the first GnRH (Wiltbank 1997; Martinez et al. 2000; Thatcher et al. 2001). In the absence of ovulation, synchronous emergence and growth of a dominant follicle that will ovulate in response to the second GnRH does not occur (Vasconcelos et al. 2001; Macmillan et al. 2003; Perry et al. 2007). We anticipated that MGA presynchronization in heifers would yield higher ovulation rates than that expected (50%) when GnRH was given at random stages of the estrous cycle (Pursley et al. 1995; Martinez et al. 2000). However, the ovulation rate following GnRH given 12 d after the end of a 15-d period of feeding MGA in exp. 2 (61%) seemed substantially less than that reported previously (i.e., 100%; Wood et al. 2001). GnRH will only induce ovulation of follicles that have acquired LH receptors (Ireland and Roche 1982, 1983). Stage of the estrous cycle affects the probability of cattle ovulating to the first GnRH (Ginther et al. 2001) because there may only be a few days (depending upon the number of follicular waves per interovulatory interval), when one or more follicles capable of ovulating are present (Ginther et al. 1997). Ovulation and the onset of a new wave of follicle growth may occur up to 6 d following MGA withdrawal and, consequently, stage of follicle development may have varied considerably among heifers on the day of the first GnRH treatment. Furthermore, the stage of the estrous cycle at the time of the first GnRH influenced the size of the dominant follicle at the time of the second GnRH in a CO-Synch protocol (Atkins et al. 2008). In exp. 1, presynchronization with PGF increased the prevalence of follicles]10 mm on day 0 to almost 100%; therefore, we inferred that these heifers were more likely to ovulate to GnRH. In exp. 3, however, CIDR presynchronization had no effect on the prevalence of follicles ]1 0 mm(75%), and reduced the prevalence of a CL on day 0. Although the presence of a CL on day 0 was not expected to influence the effectiveness of GnRH to induce ovulation (Twagiramungu et al. 1995), in a very recent study, plasma LH concentrations and ovulation rate in response to exogenous GnRH were greater in cattle with low (3.0 ng ml 1 ) than high (5.7 ng ml 1 ) plasma progesterone concentrations (Colazo et al. 2008). In Herd A, lowluteal plasma progesterone concentrations on day 0 were attributed to the CIDR, because, only 26.7% of presynchronized heifers had a CL (Table 5), and 100% had luteal-plasma progesterone concentrations (Table 6). Furthermore, 83% of heifers had a follicle]1 0mm on day 0, and low-luteal plasma progesterone concentrations were expected to increase the LH response to GnRH and ovulation rate (Colazo et al. 2008), and SMALL ET AL. * PRESYNCHRONIZATION AND ecg 31 consequently the TAI pregnancy rate; that fertility was not improved was unexpected and disappointing. ecg and the Diameter of the Pre-ovulatory Follicle Although GnRH can induce ovulation of small (B12 mm) preovulatory follicles, this has been associated with reduced pregnancy rates to TAI (Vasconcelos et al. 2001; Macmillan et al. 2003; Perry et al. 2007). A short interestrus interval has also been attributed to GnRH forced ovulation of follicles that were not fully mature and consequently developed into a compromised CL that regressed early (Schmitt et al. 1996). Our hypothesis that ecg would improve the fertility of the GnRHinduced ovulations by promoting growth of the preovulatory follicle was not supported in the current experiments with beef heifers. In lactating beef cattle, ecg increased plasma progesterone concentrations 12 d after TAI, without significantly increasing either preovulatory follicle diameter or CL area; furthermore, the benefit of ecg was more evident as the proportion of anestrous cows (with only small follicles) increased (Bo et al. 2003; Baruselli et al. 2004; Small et al. 2009). In the present study, the prevalence of a small preovulatory follicle at TAI was probably too low to limit fertility. In beef heifers, ovulation of follicles 10.7 to 15.7 mm in diameter had a greater probability of supporting pregnancy than follicles outside this range (Perry et al. 2007). In exps. 1and 2, the mean diameter of the preovulatory follicle was within this size range and was expected to support pregnancy (Perry et al. 2007). In exp. 3, this diameter was achieved 2 d earlier, perhaps resulting in follicles at an advanced stage of maturity (15.7 mm) at the time of TAI, and, consequently, fertility was poor to TAI of heifers in Herd A. If this were true of heifers in Herd B, then the growthpromoting effect of ecg on the preovulatory follicle may have compromised pregnancy rates in the ecg treated heifers. In addition, only 7% of heifers in Herd A exhibited estrus within 17 d after TAI, less than half the rate of short estrous cycles following GnRH in exps. 1and 2, and as reported previously (Schmitt et al. 1996). This was also evidence that Herd A fertility was not compromised by GnRH-induced ovulation of immature preovulatory follicles. Pregnancy Rates The TAI pregnancy rates in these studies seemed generally comparable to those reported previously (Pursley et al. 1997; Small et al. 2001; Johnson and day 2004; Lamb et al. 2006), except for the somewhat lower pregnancy rates in Herd A in exp. 3. Herd effects on TAI pregnancy rates are relatively common (Colazo et al. 2004; DeJarnette et al. 2004), and have been attributed to differences in genetics and management that probably cause complex interactions between nutrition and reproductive function. Indeed, heifers in Herd A in exp. 3, had thicker back-fat than heifers in exps. 1and 2 (Table 1), and overall, had reached puberty

10 32 CANADIAN JOURNAL OF ANIMAL SCIENCE earlier than heifers in Herds B and C. However, heifers in Herd A had lower fertility to TAI, based on the higher percentage of services by bulls (days 26 to 34) 17 to 25 d after TAI (Fig. 2). The lower fertility to TAI may have impacted cumulative pregnancy rates, as the bulls may have been overwhelmed by the number of heifers rebred from days 26 to 34 (N98), which was more than four times higher than in exps. 1(N22) and 2 (N22). The presynchronization strategies employed in these studies were expected to increase the probability that ovulation occurred following the first GnRH treatment. Although PGF presynchronization increased ovulation rate, this did not significantly improve pregnancy rates to TAI, perhaps due to reduced diameter of the preovulatory follicle in many of the presynchronized heifers. The low pregnancy rate in Herd A of exp. 3 is more difficult to explain. Nearly all of these heifers were cycling at the start of the experiment; on days 0 and 7, the proportion with a follicle ]10 mm was 83% and nearly 100%, respectively. We inferred that most of the heifers ovulated soon after GnRH treatment on day 0, and, therefore, the preovulatory follicle may have been dominant for 5 d on day 9, resulting in poor fertility (Austin et al. 1999). In the absence of a CL, the CIDR maintained plasma progesterone concentrations sufficient to suppress the LH surge, but not necessarily LH secretion (Macmillan et al. 1991; Colazo et al. 2004), resulting in rapid growth of the dominant follicle, follicles ] 10 mm on day 7, and inevitably even larger follicles on day 9 (Austin et al. 2002). In other words, increased LH pulse frequency due to low progesterone concentrations may have resulted in a large preovulatory follicle with an aged oocyte, contributing to the poor pregnancy rates in Herd A and ecg-treated heifers in Herd B of exp. 3. In cattle, the developmental competence of fertilized aged oocytes was compromised within 6 d of breeding (Ahmad et al. 1995). Perhaps removal of the CIDR after 5 d would have minimized the prevalence of aged preovulatory follicles and increased fertility. In beef heifers, serum progesterone concentrations declined linearly from luteal (4 ng ml 1 ) to nonluteal (B1ng ml 1 ) concentrations within 36 h following PGF treatment on the 7th or 14th d after estrus (King et al. 1982). In our study, 99% (398/402) of heifers subjected to TAI had luteal plasma progesterone concentrations at the time of PGF on day 7. However, 26% (32/125), 42% (54/127), and 5% (7/146) of heifers in exps. 1, 2, and 3 (Herd A), were in the PGFnonresponse category, as plasma progesterone concentrations were still luteal on day 9, 54 h after PGF. In exps. 1and 2, luteolysis was probably simply delayed, as pregnancy rates for heifers did not differ between PGFresponse and -nonresponse categories (34 vs. 43% and 54 vs. 48%, respectively; P 0.20). However, this was not the case (P B0.05) in exp. 3, where none of the heifers in the PGF-non response category were pregnant to TAI. CONCLUSION Presynchronization treatments eliminated the need for estrus detection before GnRH-based TAI in beef heifers by increasing the prevalence of ovarian follicles ]1 0 mm in diameter on either day 0 (exp. 1) or day 7 (exp. 3). Although treatment with ecg on day 7 tended to increase the diameter of the preovulatory follicle in heifers that failed to ovulate in response to the first GnRH (exp. 2), the effect of ecg on preovulatory follicle diameter was not additive to that of presynchronization, and neither presynchronization nor ecg treatment improved pregnancy rates to TAI significantly. Perhaps ecg treatment was not efficacious because in all experiments, the prevalence of heifers with preovulatory follicles ]10 mm in diameter at TAI was high (85%) and not significantly limiting fertility to TAI. ACKNOWLEDGEMENTS Funding was provided by the Saskatchewan Agriculture Development Fund (ADF Project # ). In-kind contributions of Cystorelin (Merial Canada Inc., Baie D Urfe, QC, Canada), Estrumate (Schering Plough Animal Health, Pointe Claire, QC, Canada), Pregnecol (Bioniche Animal Health, Belleville, ON, Canada), and CIDR (Pfizer Animal Health, Montreal, QC, Canada) are gratefully acknowledged. We also thank D. Ward and T. Lewandoski, Brandon Research Centre, and L. Siqueira, M. Rutledge and H. Davis, University of Saskatchewan, for technical assistance, and the animal care staff at the Brandon Research Centre, the University of Saskatchewan, as well as our private collaborators, for care and management of cattle. Ahmad, N., Schrick, F. N., Butcher, R. L. and Inskeep, E. 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