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Comparison of the effects of two shortened timed-AI protocols on pregnancy per AI in beef cattle
Theriogenology 142 (2020) 85e91
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Comparison of the effects of two shortened timed-AI protocols on pregnancy per AI in beef cattle K. Macmillan a, M. Gobikrushanth b, A. Sanz c, D. Bignell a, G. Boender d, L. Macrae e, R.J. Mapletoft f, M.G. Colazo a, * a
Livestock Systems Section, Alberta Agriculture and Forestry, Edmonton, T6H 5T6, Canada Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, T6G 2P5, Canada n y Tecnología Agroalimentaria de Arago n (CITA), Zaragoza, 50059, Spain Centro de Investigacio d The Farm Animal Hospital, Leduc, T9E 6Z9, Canada e Agri-Vet Alberta, Westlock, T7P 2N9, Canada f Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, Canada b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2019 Received in revised form 19 September 2019 Accepted 23 September 2019 Available online 25 September 2019
The objective was to compare pregnancy per AI (P/AI) between two shortened timed-AI (TAI) protocols in beef cattle. This study also determined whether administration of eCG in heifers and timing of AI in cows would affect P/AI. Cattle were submitted at random to either a modified 5-d Co-synch protocol (Day 0 ¼ progesterone releasing device (CIDR); Day 5 ¼ CIDR removal and 500 mg of cloprostenol (PGF); Day 8 ¼ 100 mg GnRH concurrent with AI) or J-synch protocol (Day 0 ¼ CIDR insertion and 2 mg of estradiol benzoate i.m.; Day 6 ¼ CIDR removal and 500 mg PGF; Day 9 ¼ 100 mg GnRH concurrent with AI). In Experiment 1, 1135 heifers (13e15 mo of age) received an estrus detection patch (Estrotect™) on Day 5 and 579 were selected at random to receive 300 IU of equine chorionic gonadotropin (eCG) at the time of CIDR removal. Patches were scored from 0 to 3 based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% change, 2 ¼ > 50% change, 3 ¼ missing. Estrus was defined to have occurred when the patch was scored 2 or 3. In Experiment 2, 399 cyclic, non-lactating beef cows from 1 location were submitted to either the modified 5-d Co-synch or J-synch protocol and within each protocol cows were TAI at either 66 ± 1 (n ¼ 199) or 72 ± 1 h (n ¼ 200) following CIDR removal. Transrectal ultrasonography was used in both experiments to determine presence of a corpus luteum (CL) on Day 0, and to diagnose pregnancy 35 d after TAI. In Experiment 1, eCG increased estrus rate only in heifers without a CL on day 0 that were submitted to the modified 5-d Co-synch protocol (41.9 vs. 69.6%). Heifers submitted to the J-synch protocol had greater (P ¼ 0.03) P/AI compared with those in the modified 5-d Co-synch (48.7 vs. 41.1%) and heifers that expressed estrus before AI had increased (P < 0.0001) P/AI compared to those that did not (53.6 vs. 36.5%). Administration of eCG and presence of a CL tended to affect P/AI (P ¼ 0.13). In Experiment 2, cows submitted to the J-synch protocol tended (P ¼ 0.07) to have greater P/AI compared to those in the modified 5-d Co-synch (74.1 vs. 66.5%). There was no association between P/AI and timing of AI. In summary, the J-synch protocol resulted in greater P/AI than the modified 5-day Co-synch protocol in heifers and cows. Administration of eCG increased estrus rate in heifers without a CL at the start of the protocol and tended to improve P/AI in all heifers. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows subjected to either TAI protocol. Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.
Keywords: 5-d Co-Synch J-synch Pregnancy per AI Cyclicity Estrus detection eCG
1. Introduction
* Corresponding author. 303, 7000 e 113 Street NW, Edmonton, Alberta, T6H 5T6, Canada. E-mail address: [email protected] (M.G. Colazo). https://doi.org/10.1016/j.theriogenology.2019.09.038 0093-691X/Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.
Artificial insemination (AI) can benefit beef producers by increasing pregnancy rates at the start of the breeding season, reducing the number of clean-up bulls, increasing access to genetically improved bulls and improving the overall genetic merit of the herd. Despite these benefits, AI use is still low in the Canadian
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beef industry [1]. In order to increase the adoption of AI in the beef industry, timed-AI (TAI) protocols have been developed to synchronize ovulation, eliminating the need for estrus detection, while resulting in a pregnancy per AI (P/AI) of 50% on average in beef cattle [2]. Recently, shortened estradiol- (J-synch [3]) and GnRH-based (5d Co-synch [4]) TAI protocols that provide for a lengthened proestrus have been developed. The length of proestrus is a consistent predictor of P/AI [5,6] and is associated with greater circulating estradiol concentrations pre-ovulation and progesterone (P4) concentrations post-ovulation [7,8]. In comparison to the 7-d Cosynch plus P4-releasing device (CIDR), the 5-d Co-synch plus CIDR protocol increased P/AI by 13% in beef cows [4], while J-synch protocol increased P/AI by 6% compared to a 7-d estradiol-based [3] protocol in beef heifers [9]. However, de la Mata and Bo compared the 5-d Co-synch plus CIDR and J-synch protocols in beef heifers and found no difference in ovulation rate, diameter of the ovulatory follicle and P/AI (50 vs. 57%). Although, both protocols increase P/AI in beef cattle compared to traditional protocols, it should be noted that while estradiol-based protocols are more popular in South America, estradiol benzoate (EB) is registered for use in Canada and many South American countries and can only be used with a veterinary prescription. A downside to the 5-d Co-synch plus CIDR protocol is that it requires additional handling of animals as prostaglandin (PGF) must be administered twice to ensure luteolyis of the developing corpus luteum (CL) when ovulation occurs to the initial GnRH. However, as the ovulatory response to initial GnRH in heifers can be as low as 25% [5], several studies in dairy [5,10] and beef heifers [11,12] have revealed no adverse effects on P/AI in animals that did not receive the initial GnRH. It has been concluded that a modified 5-d Co-synch plus CIDR protocol (no first GnRH and a single PGF) reduces labor while still maintaining acceptable P/AI in heifers. Another consideration for TAI protocols is the use of equine chorionic gonadotropin (eCG) concurrent with P4 device removal. Equine chorionic gonadotropin is a glycoprotein hormone with both FSH and LH activity in cattle [13], stimulating follicle growth and increasing ovulation rate [14]. Studies in beef cattle have indicated that eCG is most effective in increasing P/AI in acyclic cows [14e17], cows with low BCS [15,18] or primiparous cows [19]. Studies in heifers [20] or in beef cows in good BCS and >68 d postpartum [21] have found no effect of eCG on P/AI. However, Menchaca et al. [22] reported a reduction in P/AI in beef heifers submitted to the J-synch protocol when eCG was removed, but only when TAI was performed at 72 h (vs. 60 h). Further studies on the use of eCG in beef heifers subjected to shortened TAI protocols that provide for a lengthened proestrus are required, especially to determine if eCG can increase P/AI in acyclic heifers. There is some discrepancy as to the ideal timing of AI following P4 device removal. While a longer proestrus period between P4 device removal and TAI concurrent with GnRH has been shown to increase fertility in beef cows, Kasimanickam et al. [23] reported that P/AI was increased in beef heifers subjected to the 5-d Cosynch plus CIDR when AI occurred at 56 h compared with 72 h after P4 device removal. Additionally, 24% of dairy heifers in a modified 5-d Co-synch plus CIDR program were detected in estrus between 36 and 48 h after P4 device removal and 74% of those pez-Helguera animals ovulated before TAI at 72 h [24]. Recently, Lo et al. [12] reported no difference in P/AI in beef heifers that were TAI at 66 or 72 h in a modified 5-d Co-synch plus CIDR program, and de la Mata et al. [9] reported no difference in P/AI in the J-synch protocol when heifers were TAI at 60 or 72 h. However, there is still a need to determine if timing of AI affects P/AI in beef cows subjected to either the modified 5-d Co-synch plus CIDR and J-synch protocols.
The objectives of this study were to 1) compare the J-synch and modified 5-d Co-synch plus CIDR protocols in beef heifers and cows, 2) determine the effect of eCG in these protocols in heifers, and 3) determine the effect of timing of AI in these protocols in beef cows. We hypothesized that administration of eCG will increase P/ AI and that the J-synch protocol may result in greater P/AI, considering the lower P/AI obtained in acyclic heifers subjected to the modified 5-d Co-synch protocol previously. We also hypothesized that both protocols will be comparable in cyclic cows and that timing of AI will not affect P/AI. 2. Materials and methods This study was carried out in three commercial beef herds (A, B and C) located near Edmonton, Alberta, Canada (53.54 N, 113.49 W), from April to June 2018 for Experiment 1 (herds A, B and C) and in December 2018 for Experiment 2 (herd A). All procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care [25]. 2.1. Animals and management In Experiment 1, the subjects were Angus and Simmental crossbred beef heifers between 13 and 15 mo of age. Heifers were housed in dry lots and had unrestricted access to water and hay. In all three herds, heifers were fed once daily with a diet composed mainly of barley or corn silage, barley grain, hay, plus a protein, vitamin and mineral supplement, which was designed for a daily gain of approximately 0.7 kg. In Experiment 2, subjects were non-lactating primiparous Angus beef cows that were at least 5 mo postpartum and calves were weaned at least 2 mo prior to the initiation of the study. Cows were housed in a dry lot and had unrestricted access to water and hay. Cows were fed once daily with a diet composed mainly of corn silage, hay, plus a protein, vitamin and mineral supplement. 2.2. Determination of corpus luteum presence and pregnancy diagnosis All animals in both experiments were examined by transrectal ultrasonography (MicroMAXX, color Doppler scanner equipped with a multifrequency 5e10 MHz linear transducer; Sonosite, Bothell, WS, USA) at the initiation of synchronization to determine the presence of a CL, pregnancy status and normalcy of the reproductive tract. Pregnancy diagnosis in both experiments was performed by transrectal ultrasonography (Easi-scan, scanner equipped with a multifrequency 4.5e8.5 MHz linear transducer; BCF Technology LTD, Bellshill, Scotland, UK) 35 d after TAI. Presence of a viable embryo/fetus (positive heartbeat) was used as a determinant of pregnancy. 2.3. Experimental treatments 2.3.1. Experiment 1 A total of 1153 heifers were scanned by transrectal ultrasonography and randomly assigned to either a modified 5-d Co-synch plus CIDR protocol (without initial GnRH and only a single PGF) or a J-synch protocol (Fig. 1). On Day 0, all heifers received a controlled internal drug release device containing 1.38 g of progesterone (CIDR; Zoetis Animal Health, Florham Park, NJ, USA) for either 5 (Co-synch) or 6 (J-synch) d, with heifers on the J-synch protocol also receiving 2.0 mg im of estradiol benzoate (EB; Summit Veterinary Pharmacy Inc., ON, Canada) at CIDR insertion. At CIDR removal, 500 mg im of cloprostenol (PGF; Bioestrovet; Vetoquinal N.-A Inc., Lavaltrie, QC, Canada) was administered to all heifers and
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Fig. 1. Illustrations of synchronization protocols used in Experiment 1. Angus and Simmental crossbred beef heifers from 3 farms were allocated at random to 1 of 4 treatments (2 2 design). Heifers assigned to the modified 5-d Co-synch (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Beef animals assigned to J-Synch (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). Within both protocols, heifers either received (n ¼ 579) or did not receive (n ¼ 574) 300 IU of equine chorionic gonadotropin (eCG). At CIDR removal, all heifers received estrus detection (ED) patches (Estrotect™) that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. All heifers were inseminated by the same technician. Transrectal ultrasonography (U/S) was performed on Day 0 to determine presence of a corpus luteum (CL) and normalcy of the reproductive tracts, and 35 d after AI to determine pregnancy status.
estrus detection (ED) patches (Estrotect™; Rockway Inc., Spring Valley, WI, USA) were applied mid-way between the hip and tail head. Hair was brushed in the area where the patch was to be placed and glue was applied. Three days later (Day 8 e modified 5d Co-synch; Day 9 e J-synch), ED patches were scored from 0 to 3, based on color change from initial application (0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ >50% color change, 3 ¼ missing) and all heifers received 100 mg im of GnRH (Fertiline; Vetoquinol N.-A Inc.) along with TAI (72 ± 1 h after CIDR removal). Estrus was defined to have occurred when an ED patch was scored 2 or 3 at TAI. Estrus response (%) was defined as the number of heifers with an ED patch scored 2 and 3 over the total number of heifers. In total, 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU im of equine chorionic gonoadotropin (eCG; Pregnecol® 6000; Vetoquinol N.-A Inc.) at CIDR removal, on Day 5 for Co-synch and Day 6 for J-synch protocols. 2.3.2. Experiment 2 A total of 399 cows were scanned by transrectal ultrasonography at initiation of synchronization and a CL was detected in all animals. Cows were randomly assigned to either the modified 5d Co-synch plus CIDR protocol (n ¼ 198) or to a J-synch protocol (n ¼ 201), as in Experiment 1. Cows were further sub-divided within TAI protocol to receive TAI and 100 mg of GnRH at 66 ± 1 (n ¼ 199) or 72 ± 1 h (n ¼ 200) following CIDR removal (Fig. 2). Expression of estrus was not determined and no eCG was used in Experiment 2. 2.4. Statistical analyses All data were analyzed using SAS (Statistical Analysis System,
Fig. 2. Illustrations of synchronization protocols used in Experiment 2. Non-lactating Angus beef cows from 1 farm were allocated at random to 1 of 4 treatments (2 2 design). Cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timedAI either 66 or 72 h after CIDR removal. Beef animals assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI either 66 or 72 h after CIDR removal. All cows were inseminated by the same technician. Transrectal ultrasonography (U/S) was performed on Day 0 to determine presence of a corpus luteum (CL) and normalcy of the reproductive tracts, and 35 d after AI to determine pregnancy status.
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Version 9.4 for Windows; SAS Institute Inc., Cary, NC, USA). For Experiment 1, the associations among TAI protocol (J-synch vs. 5-d Co-synch), eCG treatment (yes vs. no), presence of a CL (yes vs. no), and their interactions and estrus rate (ER) were analyzed using the GLIMMIX procedure. As the interaction among TAI protocol, eCG treatment, and CL presence (three-way interaction) was significant, the final model included the TAI protocol, eCG treatment, CL presence and the aforementioned three-way interaction as fixed effects. The effect of sire nested within herd was used as a random effect. The model specifications included a binomial distribution and logit functions, and an option to retrieve odds ratios and their confidence intervals. The associations among TAI protocol (J-synch vs. 5-d Co-synch), eCG treatment (yes vs. no), presence of a CL (yes vs. no), estrus expression (yes vs. no) and their interactions and P/AIwere analyzed using the GLIMMIX procedure. As a majority of the interactions were not significant except for the interaction between eCG treatment and estrus expression, the final model included only TAI protocol, eCG treatment, CL presence, estrus expression and the interaction between eCG treatment and estrus expression as fixed effects. The effect of sire nested within herd was used as a random effect. The model specifications included a binomial distribution and logit function, and an option to retrieve odds ratios and their confidence intervals. For Experiment 2, the associations among TAI protocol (J-synch vs. 5-d Co-synch), AI timing (66 vs. 72 h), sire (A vs. B), and their interactions and P/AI were analyzed using the GLIMMIX procedure. As none of the interactions were significant, the final model included only TAI protocol, AI timing and sire as fixed effects. The model specifications included a binomial distribution and logit functions, and an option to retrieve odds ratios and their confidence intervals. For all statistical analyses, a probability of 0.05 or less was considered statistically significant, and a probability between 0.051 and 0.10 was considered a tendency.
3. Results 3.1. Experiment 1 A total of 1153 beef heifers were inseminated, 14.9% (172/1153) did not have a CL at the initiation of synchronization (Table 1). The percentage of heifers without a CL at day 0 was associated with farm (P < 0.01) and ranged from 7.5 to 23.2%. The overall P/AI was 50.8% (586/1153) and was associated with farm (P < 0.001; Table 1) and sire (P ¼ 0.002). Timed-AI protocol and estrus rate were associated with P/AI (Table 2). Heifers subjected to the modified 5-day Co-synch protocol had lower odds for P/AI (0.74; CI [0.57 to 0.96]) compared to heifers subjected to the Jsynch protocol (P ¼ 0.03). Heifers that did not express estrus by time of TAI had lower odds for P/AI (0.50; CI [0.38 to 0.65]) compared to heifers that did express estrus (P < 0.0001). Presence of a CL and the administration of eCG tended to affect P/AI (Table 2). The interaction between TAI protocol and administration of eCG was not significant (P ¼ 0.30) for P/AI (37.5, 44.9, 48.0 and 49.4% for 5-d Co-synch/-eCG, 5-d Co-synch/þeCG, J-synch/-eCG and J-synch/ þeCG, respectively). The overall estrus rate was 72.0% (830/1153) and was associated with farm (P ¼ 0.005; Table 1). There was an interaction among TAI protocols, eCG treatment and CL presence for estrus rate (P ¼ 0.05). Estrus rate was lower in heifers without a CL compared to heifers with a CL at day 0 in the modified 5-d Co-synch when no eCG was used, but estrus rate was not different based on CL presence when eCG was used in the same protocol (Table 3).
Table 1 Timed-AI protocol, eCG treatment, corpus luteum (CL) presence, estrus rate and pregnancy per AI (P/AI) categorized by farm (Experiment 1).
n 5-d Co-synch, n J-synch, n -eCG, n þeCG, n Without CL, n (%) With CL, n (%) Estrus rate (%) P/AI (%)
Farm A
Farm B
Farm C
Total
406 203 203 202 204 84 (20.7%) 322 (79.3%) 67.0a 58.6a
544 300 244 271 273 41 (7.5%) 503 (92.5%) 76.5b 47.4b
203 101 102 101 102 47 (23.2%) 156 (76.8%) 70.0ab 44.3b
1153 604 549 574 579 172 981 72.0 50.8
a-b
Within a row, values without a common superscript differ significantly (P < 0.05). Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed in all heifers on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
Table 2 Pregnancy per AI (P/AI) based on timed-AI (TAI) protocol, eCG treatment, corpus luteum (CL) presence and estrus rate (Experiment 1). Variable TAI protocol 5-d Co-Synch J-Synch eCG treatment -eCG þeCG CL Presence No Yes Estrus Rate Yes No
P/AI (%)
SEM
P-Value
41.1 48.7
0.14
0.03
42.7 47.1
0.12
0.13
41.6 48.3
0.17
0.12
53.6 36.5
0.14
<0.0001
Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed in all heifers on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
3.2. Experiment 2 Overall P/AI was 69.7% (278/399), with a tendency (P ¼ 0.07) for effect of TAI protocol (Fig. 3). Cows that were inseminated using the modified 5-day Co-synch protocol tended to have lower odds for P/ AI (0.66; CI [0.43 to 1.03]) compared to cows in the J-synch group (66.5 vs. 74.1%). There was no effect of AI timing (P ¼ 0.21) or sire (P ¼ 0.14) on P/AI (Fig. 3). Although the interaction between TAI protocol and AI timing was not significant (P ¼ 0.77), there was a
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Table 3 Estrus rate (ER) and pregnancy per AI (P/AI) outcomes for the interaction between TAI protocol (5-d Co-synch vs. J-synch), eCG treatment (- vs. þ) and corpus luteum presence (NCL vs. CL) in beef heifers (Experiment 1). Variable
5-d Co-synch
J-Synch þeCG
-eCG
ER (%) P/AI (%)
P-Value þeCG
-eCG
NCL
CL
SEM
NCL
CL
SEM
NCL
CL
SEM
NCL
CL
SEM
49.1a 31.6
76.2b 45.9
0.34 0.35
69.6ab 42.6
70.8ab 52.5
0.33 0.31
65.0ab 55.2
69.0ab 52.8
0.34 0.33
71.1ab 46.6
76.5b 57.3
0.45 0.40
0.05 0.29
a-b Within a row, values without a common superscript differ significantly (P < 0.05). Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status in all heifers. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
100 Pregnancy per AI (%)
90 80 70
62.9
67.8
70.8
77.4
60 50
66 h
40
72 h
30 20 10 0
Fig. 3. Pregnancy per AI (%) based on timed-AI protocol, timing of AI and sire (Experiment 2). Angus beef cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along timed-AI either 66 h (n ¼ 90) or 72 h (n ¼ 108) after CIDR removal. Cows assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH GnRH with timed-AI either 66 h (n ¼ 109) or 72 h (n ¼ 92) after CIDR removal. Approximately 50% of the cows were inseminated using sire A and the other 50% using sire B. There was a tendency for association between P/AI and protocol (P ¼ 0.07 [y]), and no association between P/AI for either AI timing (P ¼ 0.21) or sire (P ¼ 0.14).
numerical increase in P/AI in both protocols when cows were TAI at 72 h (Fig. 4). 4. Discussion Finding a TAI protocol that minimizes labor while improving P/ AI may increase adoption of AI in beef herds. Both the J-synch protocol and modified 5-d Co-synch plus CIDR protocol extend the proestrus period, increasing P/AI, while requiring only 3 handlings of animals [3,4]. Results from this study indicate that the J-synch protocol may be preferable in both beef heifers and cows, with an increase and tendency for increase, respectively, in P/AI over the modified 5-d Co-synch plus CIDR protocol. While eCG increased estrus rates in heifers without a CL at the initiation of synchronization, particularly in those subjected to the 5-d Co-synch plus CIDR protocol, administration of eCG only tended to increase the overall P/AI. In beef cows, there was no difference in P/AI based on timing of AI. The overall estrus rate in Experiment 1 was 72%, including the 14.9% of heifers without a CL at day 0, which is similar to previous studies in beef heifers using a modified 5-d Co-synch [26] or a 14d GnRH-based protocol [27]. In the current study there was a 3-way interaction for estrus rate among TAI protocol, eCG treatment and
5-d Co-Synch J-Synch Timed-AI Protocol Fig. 4. Pregnancy per AI (%) based on the interaction between timed-AI protocol and timing of AI (Experiment 2). Angus beef cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI either 66 h (n ¼ 90) or 72 h (n ¼ 108) after CIDR removal. Cows assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI either 66 h (n ¼ 109) or 72 h (n ¼ 92) after CIDR removal. The interaction between TAI protocols and timing of AI was not significant (P ¼ 0.77) for P/AI.
CL presence. Across all variations of treatments/status, the estrus rate ranged from 65 to 76.5%, with the exception of heifers without a CL at day 0 that did not receive eCG in the 5-d Co-synch plus CIDR protocol (49.1%). In a previous study, Colazo et al. [26] also found a lower estrus rate in acyclic heifers compared with cyclic heifers submitted to the modified 5-d Co-synch plus CIDR protocol (44 vs. 56%). In the current study, administering eCG resulted in no difference in estrus rate between heifers with or without a CL at the initiation of the modified 5-d Co-synch protocol (70 vs. 71%). Equine chorionic gonadotropin has FSH and LH like effects in cattle [13] and stimulates the growth of the ovulatory follicle and increases ovulation rate [14]. The use of eCG has also increased estrus rates in both cyclic and acyclic beef cows submitted to a conventional estradiol-based TAI protocol [17]. Whether increased follicle growth could have led to increased estradiol production and expression of estrus in heifers without a CL in the 5-d Co-synch plus CIDR protocol in the current study remains unknown. Additionally, eCG increases the ovulation rate in acyclic cows, particularly when only small and medium follicles were observed at P4 device removal [15]. When the J-synch protocol was used, estrus rate between heifers with or without a CL at day 0 did not differ, regardless of eCG treatment. This suggests that while eCG may not
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be necessary for beef heifers subjected to the J-synch protocol, administration of eCG in heifers without a CL at the initiation of a modified 5-d Co-synch protocol is likely to increase estrus rates. The occurrence of estrus before TAI has been shown to result in increased P/AI in heifers [26,27] and cows [17], with both conventional and sex-selected semen. Expression of estrus also indicates if animals responded to the protocol and can be used to select animals for more valuable semen, i.e. sex-selected semen. In the current study, P/AI was increased by 17.1% points when heifers were classified as in estrus prior to TAI, with no interaction with any other main effects. This is similar to the 19.6% point increase observed previously when cyclic heifers in the modified 5-d Cosynch were observed in estrus prior to TAI [26] and the 21.0% point increase when cyclic heifers were observed in estrus prior to TAI in the J-synch protocol [28]. Expression of estrus before AI results in a shorter and less variable interval from AI to ovulation [24] and was associated with increased circulating estradiol concentrations [29]. Increased estradiol concentrations have been reported to alter the uterine environment in preparation for mating, which includes changing the composition and amount of cervical mucous, enhancing uterine contractions for sperm transport [30], altering uterine pH, increasing lifespan of sperm [31,32] and increasing oviduct secretions all of which improves fertilization rates [29]. Although ovulation is normally induced in a TAI protocol, the expression of estrus before AI and ovulation indicates an optimal timing of sperm deposition in the female reproductive tract. Timed-AI protocol in Experiment 1 also affected P/AI; the Jsynch protocol resulted in greater P/AI than the modified 5-d Cosynch. The 41% P/AI in the modified 5-d Co-synch group is lower than previously reported (55.1% [12]; 54.9% [11]). The 48.6% P/AI for heifers in the J-synch protocol is also lower than previously re [3] compared the J-synch and ported (56.1% [9]). de la Mata and Bo the standard 5-d Co-synch plus CIDR (with initial GnRH and 2 PGF) in beef heifers, and although there was a shorter interval from protocol initiation to new follicle wave emergence with the 5-d Cosynch group, there was no difference in ovulation rate, diameter of the ovulatory follicle, and P/AI (50 vs. 57% for Co-synch and J-synch, respectively). In the current study, P/AI was suboptimal in Farms B and C, which may have been due to the observed reduction in BCS between TAI and pregnancy diagnosis (no data collected). However, the J-synch protocol still outperformed the modified 5-d Co-synch protocol despite this limitation. This may be due to the initial synchronization of follicular wave emergence using EB, which is successful in 91.6% of beef heifers [3] and would not have occurred in the modified 5-d Co-synch plus CIDR protocol. While previous studies in dairy and beef heifers have reported an ovulation rate as low as 25% to initial GnRH in the 5-d Co-synch plus CIDR protocol and no effect on P/AI when using the modified 5-d Co-synch [5,11], some studies have found a decrease in P/AI when the modified 5d Cosynch protocol was used in beef heifers [33], especially when the heifers were acyclic [12]. Optimizing the Co-synch protocol by treating acyclic heifers with the initial GnRH, may improve the P/AI. In the current study, there was a tendency for the administration of eCG to increase P/AI in heifers. While administration of eCG has increased P/AI in beef cows, it has been shown to be most effective in acyclic cows [14e17], cows with low BCS [15,18] and primiparous cows [19,34] which can have reduced fertility to TAI and lower postpartum BCS [34]. Alvarez et al. [21] found no increase in P/AI with eCG use in Bos indicus cows, which the authors attributed to all cows being in good body condition and >68 d postpartum. Similarly, Small et al. [20] found no effect of eCG on fertility of beef heifers, which could be attributed to heifers being in a positive energy balance and good body condition at the time of AI. However, Menchaca et al. [22] did report a reduction in P/AI in beef
heifers submitted to the J-synch protocol when eCG was not used, but only when TAI was done at 72 h (vs. 60 h). There is substantial evidence to suggest that acyclic animals have reduced P/AI [12,17,35]. In the current study, heifers without a CL at day 0 only tended to have lower P/AI, which may be due to only 14.9% of heifers having no CL at initiation of synchronization and/or the Jsynch treatment appearing to result in high P/AI in heifers without a CL, regardless of the use of eCG. There was no significant interaction between protocol and eCG for P/AI; however, there was a numerical increase in P/AI in the modified 5-d Co-synch protocol when eCG is used. This may be due to the increased estrus rate stimulated by eCG in heifers without a CL at day 0, indicating that eCG may be useful in the modified 5-d Co-synch protocol. In Experiment 2, the overall P/AI was exceptional at almost 70%. It is noteworthy that all cows were cyclic and non-lactating, both factors could have been critical for reproductive performance. However, there was still a tendency for cows treated with the Jsynch protocol to have greater P/AI compared with cows treated with the modified 5-d Co-synch protocol. Compared to conventional 7-d GnRH- or estradiol-based protocols, both the 5-d Cosynch and J-synch protocols serve to reduce the length of time the P4 device is in place and extend the length of proestrus. The length of proestrus has been a consistent predictor of P/AI [5,6] and is associated with higher pre-ovulation circulating estradiol concentrations and higher post-ovulation circulating P4 concentrations [7,8]. While P/AI was greater in the 5-d Co-synch compared to the 7-d Co-synch protocol in beef cows [4,36], it also required additional handling of the animals to administer the second PGF. Although ovulation rate to the initial GnRH was around 60% in cows, failure to ovulate did not adversely affect follicular or endocrine characteristics in beef cows submitted to the 5-d Co-synch [7], suggesting that eliminating the initial GnRH and giving only one PGF is possible. While P/AI for the modified 5-d Co-synch plus CIDR protocol was 66.5% in the current study, all animals were cyclic. However, the J-synch protocol still tended to out-perform the modified 5-d Co-synch even though all cows were cyclic. When the J-synch protocol was compared with the 5-d Co-synch, with initial GnRH and two PGF, in beef heifers, there was no difference in P/AI [3]. These findings suggest that synchronizing follicular wave emergence at the beginning of the TAI protocol is beneficial in cows. There was no significant effect of AI timing on P/AI in Experiment 2, despite an overall numerical increase when cows were TAI at 72 h after P4 device removal in both TAI protocols. The 5-d Cosynch plus CIDR protocol, with initial GnRH and two PGF, resulted in increased P/AI compared to the 7-d Co-synch plus CIDR protocol, but the increase was greater when 5-d cows were inseminated at 72 h (13.3%) compared to 60 h (9.1%) after P4 device removal [4]. However, in dairy heifers subjected to a modified 5d Co-synch protocol, 24% were detected in estrus between 36 and 48 h after P4 device removal and 74% of those animals ovulated before TAI at 72 h after P4 device removal [24]. Colazo and Mapletoft [24] also reported a total of 51% of heifers showing estrus at or before 60 h after P4 device removal. Therefore, earlier TAI may optimize P/AI, as these animals are likely to ovulate within 84 h after P4 device removal. Kasimanickam et al. [23] found an increase in P/AI in beef heifers treated with the 5-d Co-synch plus CIDR protocol when TAI was done at 56 h as compared with 72 h after P4 device removal (66 vs. 56%). Based on previous ovulation data in dairy heifers treated with the modified 5-d Co-synch protocol, in which 60% ovulated between 12 and 36 h after TAI at 72 h [24], TAI 66 h was chosen to compare with the standard interval of 72 h in Experiment 2. There was no difference in P/AI between 66 and 72 h in cyclic beef cows for either TAI protocol, which is in agreement pezwith findings from two recent studies [9,12]. In this regard, Lo
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Helguera et al. [12] reported no difference in P/AI in beef heifers that were TAI at 66 or 72 h in a modified 5-d Co-synch, and de la Mata et al. [9] reported no difference when heifers were inseminated at 60 or 72 h in the J-synch protocol. Although results would indicate a large window of time for TAI, the numerical increase does suggest that TAI at 72 h following P4 device removal may still be preferable in beef cows.
[15]
[16]
5. Conclusion [17]
The overall estrus rate and P/AI in beef heifers was 72% and 50.8%, respectively. When compared to the modified 5-d Co-synch plus CIDR protocol, the J-synch protocol resulted in greater P/AI in heifers and a tendency for greater P/AI in cows. The administration of eCG increased estrus rates only in heifers without a CL at the start of the modified 5-d Co-synch protocol and tended to increase overall P/AI. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows when either TAI protocol was used. Acknowledgements Research was supported by Livestock Systems Section, Alberta Agriculture and Forestry. Authors thank Vetoquinol N.-A Inc. (Lavaltrie, QC, Canada) and Rockway Inc. (Spring Valley, WI, USA) for their in-kind support and the Crow families (Thorsby, Alberta, Canada) and Parkland Cattle Co. (Spruce Grove, Alberta, Canada) for their cooperation during the study. References [1] Colazo MG, Mapletoft RJ. A review of current timed-AI (TAI) programs for beef and dairy cattle. Can Vet J 2014;55:772e80. GA, de la Mata JJ, Baruselli PS, Menchaca A. Alternative programs for [2] Bo synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 2016;86:388e96. GA. Estrus synchronization and ovulation using protocols [3] de la Mata JJ, Bo with estradiol benzoate and GnRH and reduced periods of insertion of a progesterone releasing device in beef heifers. Taurus 2012;55:17e23. [4] Bridges GA, Helser LA, Grum DE, Mussard ML, Gasser CL, Day ML. Decreasing the interval between GnRH and PGF2a from 7 to 5 days and lengthening proestrus increases timed-AI pregnancy rates in beef cows. Theriogenology 2008;69:843e51. [5] Colazo MG, Ambrose DJ. Neither duration of progesterone insert nor initial GnRH treatment affect pregnancy per timed-insemination in dairy heifers subjected to a Co-synch protocol. Theriogenology 2011;76:578e88. [6] Day ML. State of the art of GnRH-based timed AI in beef cattle. Anim Reprod 2015;12:473e8. [7] Bridges GA, Mussard ML, Helser LA, Day ML. Comparison of follicular dynamics and hormone concentrations between the 7-day and 5-day COSynch þ CIDR program in primiparous beef cows. Theriogenology 2014;81: 632e8. GA. Treatment with estradiol and progesterone [8] de la Mata JJ, Menchaca A, Bo GA, editors. Proceedings XI interthat prolonged proestrus. In: Caccia M, Bo rdoba, Argentina: Institute of national symposium on animal reproduction. Co rdoba (IRAC); 2015. p. 143e58. Animal Reproduction Co ~ ez-Olivera R, Cuadro F, Bosolasco D, de Brun V, Meikle A, [9] de la Mata JJ, Nún GA, Menchaca A. Effects of extending the length of pro-oestrus in an Bo oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers. Reprod Fertil Dev 2018;30:1541e52. [10] Macmillan K, Loree K, Mapletoft RJ, Colazo MG. Short communication: optimization of a timed artificial insemination program for reproductive management of heifers in Canadian dairy herds. J Dairy Sci 2017;100:4134e8. [11] Cruppe LH, Day ML, Abreu FM, Kruse S, Lake SL, Biehl MV, Cipriano RS, Mussard ML, Bridges GA. The requirement of GnRH at the beginning of the five-day CO-Synch þ controlled internal drug release protocol in beef heifers. J Anim Sci 2014;92:4198e203. pez Helguera I, Whittaker P, Behrouzi A, Mapletoft RJ, Colazo MG. Effect of [12] Lo initial GnRH and time of insemination on reproductive performance in cyclic and acyclic beef heifers subjected to a 5-d Co-synch plus progesterone protocol. Theriogenology 2018;106:39e45. [13] Murphy BD. Equine chorionic gonadotropin: an enigmatic but essential tool. Anim Reprod 2012;9:223e30. ~ ez-Olivera R, de Castro T, García-Pintos C, Bo G, Piaggo J, Menchaca A. [14] Nún Ovulatory response and luteal function after eCG administration at the end of
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a progesterone and estradiol based treatment in postpartum anestrous beef cattle. Anim Reprod Sci 2014;146:111e6. Sales JNS, Crepaldi GA, Girotto RW, Souza AH, Baruselli PS. Fixed-time AI protocols replacing eCG with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows. Anim Reprod Sci 2011;124:12e8. G, Callejas SS. Huguenine E, Peracchia S, Benitez R, Martini H, Cledou G, Bo Effect of the utilization of 5-day CO-Synch protocols combined or not with GA, editors. eCG in suckled cows in postpartum anestrous. In: Caccia M, Bo rdoba, Proceedings X international symposium on animal reproduction. Co rdoba (IRAC); 2013. p. 313 Argentina: Institute of Animal Reproduction Co (abstract). Pessoa GA, Martini AP, Carloto GW, Rodrigues MCC, Claro Júnior I, Baruselli PS, ^a MN, Leivas FG, S Brauner CC, Rubin MIB, Corre a Filho MF. Different doses of equine chorionic gonadotropin on ovarian follicular growth and pregnancy rate of suckled Bos taurus beef cows subjected to timed artificial insemination protocol. Theriogenology 2016;85:792e9. Bilbao MG, Massara N, Ramos S, Zapata LO, Farcey MF, Pesoa J, Turic E, zques MI, Bartolome JA. Extending the duration of treatment with proVa gesterone and equine chorionic gonadotropin improves fertility in suckled beef cows with low body condition score subjected to timed artificial insemination. Theriogenology 2016;86:536e44. Small JA, Colazo MG, Kastelic JP, Mapletoft RJ. Effects of progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI in beef cattle. Theriogenology 2009;71:698e706. Small JA, Colazo MG, Kastelic JP, Erickson NE, Mapletoft RJ. Effects of presynchronization and eCG on pregnancy rates to GnRH-based, fixed-time artificial insemination in beef heifers. Can J Anim Sci 2010;90:23e34. Alvarez RH, Pugliesi G, Natal FLN, Rocha CC, Júnior GAA, Melo AJF, Otzuk IP, de Oliveira CA, Humblot P. Reproductive performance of Bos indicus beef cows treated with different doses of equine chorionic gonadotropin at the end of a progesterone-estrogen based protocol for fixed-time artificial insemination. Theriogenology 2018;118:150e6. ~ ez R, Bo GA. Pregnancy rates in beef heifers synMenchaca A, Cuadro F, Nún chronized with shortened estradiol-based treatment that provides for a prolonged proestrus. Reprod Fertil Dev 2015;27:96 (abstract). Kasimanickam R, Asay M, Firth P, Whittier WD, Hall JB. Artificial insemination at 56 h after intravaginal progesterone device removal improved AI pregnancy rate in beef heifers synchronized with five-day CO-Synch þ controlled internal drug release (CIDR) protocol. Theriogenology 2012:1624e31. Colazo MG, Mapletoft RJ. Pregnancy per AI in Holstein heifers inseminated with sex-selected or conventional semen after estrus detection or timed-AI. Can Vet J 2017;58:365e70. Canadian Council on Animal Care (CCAC). Guide to the Care and use of experimental animals. second ed., vol. 1. Ottawa, Ontario, Canada: CCAC; 2009. Colazo MG, Whittaker P, Macmillan K, Bignell D, Boender G, de Carvalho Guimaraes R, Mapletoft RJ. Evaluation of a modified GnRH-based timed-AI protocol associated with estrus detection in beef heifers inseminated with sex-selected of conventional semen. Theriogenology 2018;118:90e5. Thomas JM, Locke JWC, Vishwanath R, Hall JB, Ellersieck MR, Smith MF, et al. Effective use of SexedULTRA™ sex-sorted semen for timed artificial insemination of beef heifers. Theriogenology 2017;98:88e93. M, Bo GA. Combination of estrus detection and fixed-timed de la Mata JJ, Re artificial insemination in beef heifers following a shortened estradiol-based protocol that provides for a lengthened proestrus. Reprod Fertil Dev 2015;27:96 (abstract). 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;34:333e8. Hawk HW. Sperm survival and transport in the female reproductive tract. J Dairy Sci 1983;66:2645e60. Buhi WC. Characterization and biological roles of oviduct-specific, oestrogendependent glycoprotein. Reproduction 2002;123:355e62. Rodriguez-Martinez H. Role of the oviduct in sperm capacitation. Theriogenology 2007;68:138 (abstract). Kasimanickam RK, Firth P, Schuenemann GM, Whitock BK, Gay JM, Moore DA, Hall JB, Whittier WD. Effect of the first GnRH and two dises of PGF2a in a 5day progesterone-based CO-Synch protocol on heifer pregnancy. Theriogenology 2014;81:797e804. Sales JNS, Bottino MP, Silva LACL, Girotto RW, Massoneto JPM, Souza JC, Baruselli PS. Effects of eCG are more pronounced in primiparous than multiparous Bos indicus cows submitted to a timed artificial insemination protocol. Theriogenology 2016;86:2290e5. Bilbao MG, Zapata LO, Romero Harry H, Perez Wallace S, Farcey MF, Gelid L, Palomares RA, Ferrer MS, Bartolome JA. Comparison between the 5-day cosynch and 7-day estradiol-based protocols for synchronization of ovulation and timed artificial insemination in suckled Bos taurus beef cows. Theriogenology 2019;131:72e8. Whittier WD, Currin JF, Schramm H, Holland S, Kasimanickam RK. Fertility in Angus cross beef cows following 5-day CO-Synch þCIDR or 7-day CO-Synch þ CIDR estrus synchronization and timed artificial insemination. Theriogenology 2013;80:963e9.
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Comparison of the effects of two shortened timed-AI protocols on pregnancy per AI in beef cattle K. Macmillan a, M. Gobikrushanth b, A. Sanz c, D. Bignell a, G. Boender d, L. Macrae e, R.J. Mapletoft f, M.G. Colazo a, * a
Livestock Systems Section, Alberta Agriculture and Forestry, Edmonton, T6H 5T6, Canada Department of Agricultural Food and Nutritional Science, University of Alberta, Edmonton, T6G 2P5, Canada n y Tecnología Agroalimentaria de Arago n (CITA), Zaragoza, 50059, Spain Centro de Investigacio d The Farm Animal Hospital, Leduc, T9E 6Z9, Canada e Agri-Vet Alberta, Westlock, T7P 2N9, Canada f Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, Canada b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 August 2019 Received in revised form 19 September 2019 Accepted 23 September 2019 Available online 25 September 2019
The objective was to compare pregnancy per AI (P/AI) between two shortened timed-AI (TAI) protocols in beef cattle. This study also determined whether administration of eCG in heifers and timing of AI in cows would affect P/AI. Cattle were submitted at random to either a modified 5-d Co-synch protocol (Day 0 ¼ progesterone releasing device (CIDR); Day 5 ¼ CIDR removal and 500 mg of cloprostenol (PGF); Day 8 ¼ 100 mg GnRH concurrent with AI) or J-synch protocol (Day 0 ¼ CIDR insertion and 2 mg of estradiol benzoate i.m.; Day 6 ¼ CIDR removal and 500 mg PGF; Day 9 ¼ 100 mg GnRH concurrent with AI). In Experiment 1, 1135 heifers (13e15 mo of age) received an estrus detection patch (Estrotect™) on Day 5 and 579 were selected at random to receive 300 IU of equine chorionic gonadotropin (eCG) at the time of CIDR removal. Patches were scored from 0 to 3 based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% change, 2 ¼ > 50% change, 3 ¼ missing. Estrus was defined to have occurred when the patch was scored 2 or 3. In Experiment 2, 399 cyclic, non-lactating beef cows from 1 location were submitted to either the modified 5-d Co-synch or J-synch protocol and within each protocol cows were TAI at either 66 ± 1 (n ¼ 199) or 72 ± 1 h (n ¼ 200) following CIDR removal. Transrectal ultrasonography was used in both experiments to determine presence of a corpus luteum (CL) on Day 0, and to diagnose pregnancy 35 d after TAI. In Experiment 1, eCG increased estrus rate only in heifers without a CL on day 0 that were submitted to the modified 5-d Co-synch protocol (41.9 vs. 69.6%). Heifers submitted to the J-synch protocol had greater (P ¼ 0.03) P/AI compared with those in the modified 5-d Co-synch (48.7 vs. 41.1%) and heifers that expressed estrus before AI had increased (P < 0.0001) P/AI compared to those that did not (53.6 vs. 36.5%). Administration of eCG and presence of a CL tended to affect P/AI (P ¼ 0.13). In Experiment 2, cows submitted to the J-synch protocol tended (P ¼ 0.07) to have greater P/AI compared to those in the modified 5-d Co-synch (74.1 vs. 66.5%). There was no association between P/AI and timing of AI. In summary, the J-synch protocol resulted in greater P/AI than the modified 5-day Co-synch protocol in heifers and cows. Administration of eCG increased estrus rate in heifers without a CL at the start of the protocol and tended to improve P/AI in all heifers. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows subjected to either TAI protocol. Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.
Keywords: 5-d Co-Synch J-synch Pregnancy per AI Cyclicity Estrus detection eCG
1. Introduction
* Corresponding author. 303, 7000 e 113 Street NW, Edmonton, Alberta, T6H 5T6, Canada. E-mail address: [email protected] (M.G. Colazo). https://doi.org/10.1016/j.theriogenology.2019.09.038 0093-691X/Crown Copyright © 2019 Published by Elsevier Inc. All rights reserved.
Artificial insemination (AI) can benefit beef producers by increasing pregnancy rates at the start of the breeding season, reducing the number of clean-up bulls, increasing access to genetically improved bulls and improving the overall genetic merit of the herd. Despite these benefits, AI use is still low in the Canadian
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beef industry [1]. In order to increase the adoption of AI in the beef industry, timed-AI (TAI) protocols have been developed to synchronize ovulation, eliminating the need for estrus detection, while resulting in a pregnancy per AI (P/AI) of 50% on average in beef cattle [2]. Recently, shortened estradiol- (J-synch [3]) and GnRH-based (5d Co-synch [4]) TAI protocols that provide for a lengthened proestrus have been developed. The length of proestrus is a consistent predictor of P/AI [5,6] and is associated with greater circulating estradiol concentrations pre-ovulation and progesterone (P4) concentrations post-ovulation [7,8]. In comparison to the 7-d Cosynch plus P4-releasing device (CIDR), the 5-d Co-synch plus CIDR protocol increased P/AI by 13% in beef cows [4], while J-synch protocol increased P/AI by 6% compared to a 7-d estradiol-based [3] protocol in beef heifers [9]. However, de la Mata and Bo compared the 5-d Co-synch plus CIDR and J-synch protocols in beef heifers and found no difference in ovulation rate, diameter of the ovulatory follicle and P/AI (50 vs. 57%). Although, both protocols increase P/AI in beef cattle compared to traditional protocols, it should be noted that while estradiol-based protocols are more popular in South America, estradiol benzoate (EB) is registered for use in Canada and many South American countries and can only be used with a veterinary prescription. A downside to the 5-d Co-synch plus CIDR protocol is that it requires additional handling of animals as prostaglandin (PGF) must be administered twice to ensure luteolyis of the developing corpus luteum (CL) when ovulation occurs to the initial GnRH. However, as the ovulatory response to initial GnRH in heifers can be as low as 25% [5], several studies in dairy [5,10] and beef heifers [11,12] have revealed no adverse effects on P/AI in animals that did not receive the initial GnRH. It has been concluded that a modified 5-d Co-synch plus CIDR protocol (no first GnRH and a single PGF) reduces labor while still maintaining acceptable P/AI in heifers. Another consideration for TAI protocols is the use of equine chorionic gonadotropin (eCG) concurrent with P4 device removal. Equine chorionic gonadotropin is a glycoprotein hormone with both FSH and LH activity in cattle [13], stimulating follicle growth and increasing ovulation rate [14]. Studies in beef cattle have indicated that eCG is most effective in increasing P/AI in acyclic cows [14e17], cows with low BCS [15,18] or primiparous cows [19]. Studies in heifers [20] or in beef cows in good BCS and >68 d postpartum [21] have found no effect of eCG on P/AI. However, Menchaca et al. [22] reported a reduction in P/AI in beef heifers submitted to the J-synch protocol when eCG was removed, but only when TAI was performed at 72 h (vs. 60 h). Further studies on the use of eCG in beef heifers subjected to shortened TAI protocols that provide for a lengthened proestrus are required, especially to determine if eCG can increase P/AI in acyclic heifers. There is some discrepancy as to the ideal timing of AI following P4 device removal. While a longer proestrus period between P4 device removal and TAI concurrent with GnRH has been shown to increase fertility in beef cows, Kasimanickam et al. [23] reported that P/AI was increased in beef heifers subjected to the 5-d Cosynch plus CIDR when AI occurred at 56 h compared with 72 h after P4 device removal. Additionally, 24% of dairy heifers in a modified 5-d Co-synch plus CIDR program were detected in estrus between 36 and 48 h after P4 device removal and 74% of those pez-Helguera animals ovulated before TAI at 72 h [24]. Recently, Lo et al. [12] reported no difference in P/AI in beef heifers that were TAI at 66 or 72 h in a modified 5-d Co-synch plus CIDR program, and de la Mata et al. [9] reported no difference in P/AI in the J-synch protocol when heifers were TAI at 60 or 72 h. However, there is still a need to determine if timing of AI affects P/AI in beef cows subjected to either the modified 5-d Co-synch plus CIDR and J-synch protocols.
The objectives of this study were to 1) compare the J-synch and modified 5-d Co-synch plus CIDR protocols in beef heifers and cows, 2) determine the effect of eCG in these protocols in heifers, and 3) determine the effect of timing of AI in these protocols in beef cows. We hypothesized that administration of eCG will increase P/ AI and that the J-synch protocol may result in greater P/AI, considering the lower P/AI obtained in acyclic heifers subjected to the modified 5-d Co-synch protocol previously. We also hypothesized that both protocols will be comparable in cyclic cows and that timing of AI will not affect P/AI. 2. Materials and methods This study was carried out in three commercial beef herds (A, B and C) located near Edmonton, Alberta, Canada (53.54 N, 113.49 W), from April to June 2018 for Experiment 1 (herds A, B and C) and in December 2018 for Experiment 2 (herd A). All procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care [25]. 2.1. Animals and management In Experiment 1, the subjects were Angus and Simmental crossbred beef heifers between 13 and 15 mo of age. Heifers were housed in dry lots and had unrestricted access to water and hay. In all three herds, heifers were fed once daily with a diet composed mainly of barley or corn silage, barley grain, hay, plus a protein, vitamin and mineral supplement, which was designed for a daily gain of approximately 0.7 kg. In Experiment 2, subjects were non-lactating primiparous Angus beef cows that were at least 5 mo postpartum and calves were weaned at least 2 mo prior to the initiation of the study. Cows were housed in a dry lot and had unrestricted access to water and hay. Cows were fed once daily with a diet composed mainly of corn silage, hay, plus a protein, vitamin and mineral supplement. 2.2. Determination of corpus luteum presence and pregnancy diagnosis All animals in both experiments were examined by transrectal ultrasonography (MicroMAXX, color Doppler scanner equipped with a multifrequency 5e10 MHz linear transducer; Sonosite, Bothell, WS, USA) at the initiation of synchronization to determine the presence of a CL, pregnancy status and normalcy of the reproductive tract. Pregnancy diagnosis in both experiments was performed by transrectal ultrasonography (Easi-scan, scanner equipped with a multifrequency 4.5e8.5 MHz linear transducer; BCF Technology LTD, Bellshill, Scotland, UK) 35 d after TAI. Presence of a viable embryo/fetus (positive heartbeat) was used as a determinant of pregnancy. 2.3. Experimental treatments 2.3.1. Experiment 1 A total of 1153 heifers were scanned by transrectal ultrasonography and randomly assigned to either a modified 5-d Co-synch plus CIDR protocol (without initial GnRH and only a single PGF) or a J-synch protocol (Fig. 1). On Day 0, all heifers received a controlled internal drug release device containing 1.38 g of progesterone (CIDR; Zoetis Animal Health, Florham Park, NJ, USA) for either 5 (Co-synch) or 6 (J-synch) d, with heifers on the J-synch protocol also receiving 2.0 mg im of estradiol benzoate (EB; Summit Veterinary Pharmacy Inc., ON, Canada) at CIDR insertion. At CIDR removal, 500 mg im of cloprostenol (PGF; Bioestrovet; Vetoquinal N.-A Inc., Lavaltrie, QC, Canada) was administered to all heifers and
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Fig. 1. Illustrations of synchronization protocols used in Experiment 1. Angus and Simmental crossbred beef heifers from 3 farms were allocated at random to 1 of 4 treatments (2 2 design). Heifers assigned to the modified 5-d Co-synch (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Beef animals assigned to J-Synch (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). Within both protocols, heifers either received (n ¼ 579) or did not receive (n ¼ 574) 300 IU of equine chorionic gonadotropin (eCG). At CIDR removal, all heifers received estrus detection (ED) patches (Estrotect™) that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. All heifers were inseminated by the same technician. Transrectal ultrasonography (U/S) was performed on Day 0 to determine presence of a corpus luteum (CL) and normalcy of the reproductive tracts, and 35 d after AI to determine pregnancy status.
estrus detection (ED) patches (Estrotect™; Rockway Inc., Spring Valley, WI, USA) were applied mid-way between the hip and tail head. Hair was brushed in the area where the patch was to be placed and glue was applied. Three days later (Day 8 e modified 5d Co-synch; Day 9 e J-synch), ED patches were scored from 0 to 3, based on color change from initial application (0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ >50% color change, 3 ¼ missing) and all heifers received 100 mg im of GnRH (Fertiline; Vetoquinol N.-A Inc.) along with TAI (72 ± 1 h after CIDR removal). Estrus was defined to have occurred when an ED patch was scored 2 or 3 at TAI. Estrus response (%) was defined as the number of heifers with an ED patch scored 2 and 3 over the total number of heifers. In total, 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU im of equine chorionic gonoadotropin (eCG; Pregnecol® 6000; Vetoquinol N.-A Inc.) at CIDR removal, on Day 5 for Co-synch and Day 6 for J-synch protocols. 2.3.2. Experiment 2 A total of 399 cows were scanned by transrectal ultrasonography at initiation of synchronization and a CL was detected in all animals. Cows were randomly assigned to either the modified 5d Co-synch plus CIDR protocol (n ¼ 198) or to a J-synch protocol (n ¼ 201), as in Experiment 1. Cows were further sub-divided within TAI protocol to receive TAI and 100 mg of GnRH at 66 ± 1 (n ¼ 199) or 72 ± 1 h (n ¼ 200) following CIDR removal (Fig. 2). Expression of estrus was not determined and no eCG was used in Experiment 2. 2.4. Statistical analyses All data were analyzed using SAS (Statistical Analysis System,
Fig. 2. Illustrations of synchronization protocols used in Experiment 2. Non-lactating Angus beef cows from 1 farm were allocated at random to 1 of 4 treatments (2 2 design). Cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timedAI either 66 or 72 h after CIDR removal. Beef animals assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI either 66 or 72 h after CIDR removal. All cows were inseminated by the same technician. Transrectal ultrasonography (U/S) was performed on Day 0 to determine presence of a corpus luteum (CL) and normalcy of the reproductive tracts, and 35 d after AI to determine pregnancy status.
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Version 9.4 for Windows; SAS Institute Inc., Cary, NC, USA). For Experiment 1, the associations among TAI protocol (J-synch vs. 5-d Co-synch), eCG treatment (yes vs. no), presence of a CL (yes vs. no), and their interactions and estrus rate (ER) were analyzed using the GLIMMIX procedure. As the interaction among TAI protocol, eCG treatment, and CL presence (three-way interaction) was significant, the final model included the TAI protocol, eCG treatment, CL presence and the aforementioned three-way interaction as fixed effects. The effect of sire nested within herd was used as a random effect. The model specifications included a binomial distribution and logit functions, and an option to retrieve odds ratios and their confidence intervals. The associations among TAI protocol (J-synch vs. 5-d Co-synch), eCG treatment (yes vs. no), presence of a CL (yes vs. no), estrus expression (yes vs. no) and their interactions and P/AIwere analyzed using the GLIMMIX procedure. As a majority of the interactions were not significant except for the interaction between eCG treatment and estrus expression, the final model included only TAI protocol, eCG treatment, CL presence, estrus expression and the interaction between eCG treatment and estrus expression as fixed effects. The effect of sire nested within herd was used as a random effect. The model specifications included a binomial distribution and logit function, and an option to retrieve odds ratios and their confidence intervals. For Experiment 2, the associations among TAI protocol (J-synch vs. 5-d Co-synch), AI timing (66 vs. 72 h), sire (A vs. B), and their interactions and P/AI were analyzed using the GLIMMIX procedure. As none of the interactions were significant, the final model included only TAI protocol, AI timing and sire as fixed effects. The model specifications included a binomial distribution and logit functions, and an option to retrieve odds ratios and their confidence intervals. For all statistical analyses, a probability of 0.05 or less was considered statistically significant, and a probability between 0.051 and 0.10 was considered a tendency.
3. Results 3.1. Experiment 1 A total of 1153 beef heifers were inseminated, 14.9% (172/1153) did not have a CL at the initiation of synchronization (Table 1). The percentage of heifers without a CL at day 0 was associated with farm (P < 0.01) and ranged from 7.5 to 23.2%. The overall P/AI was 50.8% (586/1153) and was associated with farm (P < 0.001; Table 1) and sire (P ¼ 0.002). Timed-AI protocol and estrus rate were associated with P/AI (Table 2). Heifers subjected to the modified 5-day Co-synch protocol had lower odds for P/AI (0.74; CI [0.57 to 0.96]) compared to heifers subjected to the Jsynch protocol (P ¼ 0.03). Heifers that did not express estrus by time of TAI had lower odds for P/AI (0.50; CI [0.38 to 0.65]) compared to heifers that did express estrus (P < 0.0001). Presence of a CL and the administration of eCG tended to affect P/AI (Table 2). The interaction between TAI protocol and administration of eCG was not significant (P ¼ 0.30) for P/AI (37.5, 44.9, 48.0 and 49.4% for 5-d Co-synch/-eCG, 5-d Co-synch/þeCG, J-synch/-eCG and J-synch/ þeCG, respectively). The overall estrus rate was 72.0% (830/1153) and was associated with farm (P ¼ 0.005; Table 1). There was an interaction among TAI protocols, eCG treatment and CL presence for estrus rate (P ¼ 0.05). Estrus rate was lower in heifers without a CL compared to heifers with a CL at day 0 in the modified 5-d Co-synch when no eCG was used, but estrus rate was not different based on CL presence when eCG was used in the same protocol (Table 3).
Table 1 Timed-AI protocol, eCG treatment, corpus luteum (CL) presence, estrus rate and pregnancy per AI (P/AI) categorized by farm (Experiment 1).
n 5-d Co-synch, n J-synch, n -eCG, n þeCG, n Without CL, n (%) With CL, n (%) Estrus rate (%) P/AI (%)
Farm A
Farm B
Farm C
Total
406 203 203 202 204 84 (20.7%) 322 (79.3%) 67.0a 58.6a
544 300 244 271 273 41 (7.5%) 503 (92.5%) 76.5b 47.4b
203 101 102 101 102 47 (23.2%) 156 (76.8%) 70.0ab 44.3b
1153 604 549 574 579 172 981 72.0 50.8
a-b
Within a row, values without a common superscript differ significantly (P < 0.05). Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed in all heifers on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
Table 2 Pregnancy per AI (P/AI) based on timed-AI (TAI) protocol, eCG treatment, corpus luteum (CL) presence and estrus rate (Experiment 1). Variable TAI protocol 5-d Co-Synch J-Synch eCG treatment -eCG þeCG CL Presence No Yes Estrus Rate Yes No
P/AI (%)
SEM
P-Value
41.1 48.7
0.14
0.03
42.7 47.1
0.12
0.13
41.6 48.3
0.17
0.12
53.6 36.5
0.14
<0.0001
Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed in all heifers on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
3.2. Experiment 2 Overall P/AI was 69.7% (278/399), with a tendency (P ¼ 0.07) for effect of TAI protocol (Fig. 3). Cows that were inseminated using the modified 5-day Co-synch protocol tended to have lower odds for P/ AI (0.66; CI [0.43 to 1.03]) compared to cows in the J-synch group (66.5 vs. 74.1%). There was no effect of AI timing (P ¼ 0.21) or sire (P ¼ 0.14) on P/AI (Fig. 3). Although the interaction between TAI protocol and AI timing was not significant (P ¼ 0.77), there was a
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Table 3 Estrus rate (ER) and pregnancy per AI (P/AI) outcomes for the interaction between TAI protocol (5-d Co-synch vs. J-synch), eCG treatment (- vs. þ) and corpus luteum presence (NCL vs. CL) in beef heifers (Experiment 1). Variable
5-d Co-synch
J-Synch þeCG
-eCG
ER (%) P/AI (%)
P-Value þeCG
-eCG
NCL
CL
SEM
NCL
CL
SEM
NCL
CL
SEM
NCL
CL
SEM
49.1a 31.6
76.2b 45.9
0.34 0.35
69.6ab 42.6
70.8ab 52.5
0.33 0.31
65.0ab 55.2
69.0ab 52.8
0.34 0.33
71.1ab 46.6
76.5b 57.3
0.45 0.40
0.05 0.29
a-b Within a row, values without a common superscript differ significantly (P < 0.05). Heifers assigned to the modified 5-d Co-synch protocol (n ¼ 604) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI on Day 8 (72 ± 1 h after CIDR removal). Heifers assigned to the J-synch protocol (n ¼ 549) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI on Day 9 (72 ± 1 h after CIDR removal). A total of 304 heifers in the modified 5-d Co-synch and 275 heifers in the J-synch received 300 IU of equine chorionic gonadotropin (eCG) at CIDR removal. Transrectal ultrasonography was performed on Day 0 to determine presence of a CL and 35 d after TAI to determine pregnancy status in all heifers. All heifers received estrus detection patches (Estrotect™) at CIDR removal that were scored from 0 to 3, based on color change between initial application and AI; 0 ¼ unchanged, 1 ¼ 50% color change, 2 ¼ > 50% color change, 3 ¼ missing. Heifers with patches scored 2 or 3 were considered in estrus.
100 Pregnancy per AI (%)
90 80 70
62.9
67.8
70.8
77.4
60 50
66 h
40
72 h
30 20 10 0
Fig. 3. Pregnancy per AI (%) based on timed-AI protocol, timing of AI and sire (Experiment 2). Angus beef cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along timed-AI either 66 h (n ¼ 90) or 72 h (n ¼ 108) after CIDR removal. Cows assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH GnRH with timed-AI either 66 h (n ¼ 109) or 72 h (n ¼ 92) after CIDR removal. Approximately 50% of the cows were inseminated using sire A and the other 50% using sire B. There was a tendency for association between P/AI and protocol (P ¼ 0.07 [y]), and no association between P/AI for either AI timing (P ¼ 0.21) or sire (P ¼ 0.14).
numerical increase in P/AI in both protocols when cows were TAI at 72 h (Fig. 4). 4. Discussion Finding a TAI protocol that minimizes labor while improving P/ AI may increase adoption of AI in beef herds. Both the J-synch protocol and modified 5-d Co-synch plus CIDR protocol extend the proestrus period, increasing P/AI, while requiring only 3 handlings of animals [3,4]. Results from this study indicate that the J-synch protocol may be preferable in both beef heifers and cows, with an increase and tendency for increase, respectively, in P/AI over the modified 5-d Co-synch plus CIDR protocol. While eCG increased estrus rates in heifers without a CL at the initiation of synchronization, particularly in those subjected to the 5-d Co-synch plus CIDR protocol, administration of eCG only tended to increase the overall P/AI. In beef cows, there was no difference in P/AI based on timing of AI. The overall estrus rate in Experiment 1 was 72%, including the 14.9% of heifers without a CL at day 0, which is similar to previous studies in beef heifers using a modified 5-d Co-synch [26] or a 14d GnRH-based protocol [27]. In the current study there was a 3-way interaction for estrus rate among TAI protocol, eCG treatment and
5-d Co-Synch J-Synch Timed-AI Protocol Fig. 4. Pregnancy per AI (%) based on the interaction between timed-AI protocol and timing of AI (Experiment 2). Angus beef cows assigned to the modified 5-d Co-synch (n ¼ 198) received a progesterone releasing device containing 1.38 g of progesterone (CIDR) on Day 0, CIDR removal and 500 mg cloprostenol (PGF) on Day 5, and 100 mg of Fertiline (GnRH) along with timed-AI either 66 h (n ¼ 90) or 72 h (n ¼ 108) after CIDR removal. Cows assigned to J-Synch (n ¼ 201) received a CIDR and 2 mg estradiol benzoate (EB) on Day 0, CIDR removal and PGF on Day 6, and GnRH with timed-AI either 66 h (n ¼ 109) or 72 h (n ¼ 92) after CIDR removal. The interaction between TAI protocols and timing of AI was not significant (P ¼ 0.77) for P/AI.
CL presence. Across all variations of treatments/status, the estrus rate ranged from 65 to 76.5%, with the exception of heifers without a CL at day 0 that did not receive eCG in the 5-d Co-synch plus CIDR protocol (49.1%). In a previous study, Colazo et al. [26] also found a lower estrus rate in acyclic heifers compared with cyclic heifers submitted to the modified 5-d Co-synch plus CIDR protocol (44 vs. 56%). In the current study, administering eCG resulted in no difference in estrus rate between heifers with or without a CL at the initiation of the modified 5-d Co-synch protocol (70 vs. 71%). Equine chorionic gonadotropin has FSH and LH like effects in cattle [13] and stimulates the growth of the ovulatory follicle and increases ovulation rate [14]. The use of eCG has also increased estrus rates in both cyclic and acyclic beef cows submitted to a conventional estradiol-based TAI protocol [17]. Whether increased follicle growth could have led to increased estradiol production and expression of estrus in heifers without a CL in the 5-d Co-synch plus CIDR protocol in the current study remains unknown. Additionally, eCG increases the ovulation rate in acyclic cows, particularly when only small and medium follicles were observed at P4 device removal [15]. When the J-synch protocol was used, estrus rate between heifers with or without a CL at day 0 did not differ, regardless of eCG treatment. This suggests that while eCG may not
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be necessary for beef heifers subjected to the J-synch protocol, administration of eCG in heifers without a CL at the initiation of a modified 5-d Co-synch protocol is likely to increase estrus rates. The occurrence of estrus before TAI has been shown to result in increased P/AI in heifers [26,27] and cows [17], with both conventional and sex-selected semen. Expression of estrus also indicates if animals responded to the protocol and can be used to select animals for more valuable semen, i.e. sex-selected semen. In the current study, P/AI was increased by 17.1% points when heifers were classified as in estrus prior to TAI, with no interaction with any other main effects. This is similar to the 19.6% point increase observed previously when cyclic heifers in the modified 5-d Cosynch were observed in estrus prior to TAI [26] and the 21.0% point increase when cyclic heifers were observed in estrus prior to TAI in the J-synch protocol [28]. Expression of estrus before AI results in a shorter and less variable interval from AI to ovulation [24] and was associated with increased circulating estradiol concentrations [29]. Increased estradiol concentrations have been reported to alter the uterine environment in preparation for mating, which includes changing the composition and amount of cervical mucous, enhancing uterine contractions for sperm transport [30], altering uterine pH, increasing lifespan of sperm [31,32] and increasing oviduct secretions all of which improves fertilization rates [29]. Although ovulation is normally induced in a TAI protocol, the expression of estrus before AI and ovulation indicates an optimal timing of sperm deposition in the female reproductive tract. Timed-AI protocol in Experiment 1 also affected P/AI; the Jsynch protocol resulted in greater P/AI than the modified 5-d Cosynch. The 41% P/AI in the modified 5-d Co-synch group is lower than previously reported (55.1% [12]; 54.9% [11]). The 48.6% P/AI for heifers in the J-synch protocol is also lower than previously re [3] compared the J-synch and ported (56.1% [9]). de la Mata and Bo the standard 5-d Co-synch plus CIDR (with initial GnRH and 2 PGF) in beef heifers, and although there was a shorter interval from protocol initiation to new follicle wave emergence with the 5-d Cosynch group, there was no difference in ovulation rate, diameter of the ovulatory follicle, and P/AI (50 vs. 57% for Co-synch and J-synch, respectively). In the current study, P/AI was suboptimal in Farms B and C, which may have been due to the observed reduction in BCS between TAI and pregnancy diagnosis (no data collected). However, the J-synch protocol still outperformed the modified 5-d Co-synch protocol despite this limitation. This may be due to the initial synchronization of follicular wave emergence using EB, which is successful in 91.6% of beef heifers [3] and would not have occurred in the modified 5-d Co-synch plus CIDR protocol. While previous studies in dairy and beef heifers have reported an ovulation rate as low as 25% to initial GnRH in the 5-d Co-synch plus CIDR protocol and no effect on P/AI when using the modified 5-d Co-synch [5,11], some studies have found a decrease in P/AI when the modified 5d Cosynch protocol was used in beef heifers [33], especially when the heifers were acyclic [12]. Optimizing the Co-synch protocol by treating acyclic heifers with the initial GnRH, may improve the P/AI. In the current study, there was a tendency for the administration of eCG to increase P/AI in heifers. While administration of eCG has increased P/AI in beef cows, it has been shown to be most effective in acyclic cows [14e17], cows with low BCS [15,18] and primiparous cows [19,34] which can have reduced fertility to TAI and lower postpartum BCS [34]. Alvarez et al. [21] found no increase in P/AI with eCG use in Bos indicus cows, which the authors attributed to all cows being in good body condition and >68 d postpartum. Similarly, Small et al. [20] found no effect of eCG on fertility of beef heifers, which could be attributed to heifers being in a positive energy balance and good body condition at the time of AI. However, Menchaca et al. [22] did report a reduction in P/AI in beef
heifers submitted to the J-synch protocol when eCG was not used, but only when TAI was done at 72 h (vs. 60 h). There is substantial evidence to suggest that acyclic animals have reduced P/AI [12,17,35]. In the current study, heifers without a CL at day 0 only tended to have lower P/AI, which may be due to only 14.9% of heifers having no CL at initiation of synchronization and/or the Jsynch treatment appearing to result in high P/AI in heifers without a CL, regardless of the use of eCG. There was no significant interaction between protocol and eCG for P/AI; however, there was a numerical increase in P/AI in the modified 5-d Co-synch protocol when eCG is used. This may be due to the increased estrus rate stimulated by eCG in heifers without a CL at day 0, indicating that eCG may be useful in the modified 5-d Co-synch protocol. In Experiment 2, the overall P/AI was exceptional at almost 70%. It is noteworthy that all cows were cyclic and non-lactating, both factors could have been critical for reproductive performance. However, there was still a tendency for cows treated with the Jsynch protocol to have greater P/AI compared with cows treated with the modified 5-d Co-synch protocol. Compared to conventional 7-d GnRH- or estradiol-based protocols, both the 5-d Cosynch and J-synch protocols serve to reduce the length of time the P4 device is in place and extend the length of proestrus. The length of proestrus has been a consistent predictor of P/AI [5,6] and is associated with higher pre-ovulation circulating estradiol concentrations and higher post-ovulation circulating P4 concentrations [7,8]. While P/AI was greater in the 5-d Co-synch compared to the 7-d Co-synch protocol in beef cows [4,36], it also required additional handling of the animals to administer the second PGF. Although ovulation rate to the initial GnRH was around 60% in cows, failure to ovulate did not adversely affect follicular or endocrine characteristics in beef cows submitted to the 5-d Co-synch [7], suggesting that eliminating the initial GnRH and giving only one PGF is possible. While P/AI for the modified 5-d Co-synch plus CIDR protocol was 66.5% in the current study, all animals were cyclic. However, the J-synch protocol still tended to out-perform the modified 5-d Co-synch even though all cows were cyclic. When the J-synch protocol was compared with the 5-d Co-synch, with initial GnRH and two PGF, in beef heifers, there was no difference in P/AI [3]. These findings suggest that synchronizing follicular wave emergence at the beginning of the TAI protocol is beneficial in cows. There was no significant effect of AI timing on P/AI in Experiment 2, despite an overall numerical increase when cows were TAI at 72 h after P4 device removal in both TAI protocols. The 5-d Cosynch plus CIDR protocol, with initial GnRH and two PGF, resulted in increased P/AI compared to the 7-d Co-synch plus CIDR protocol, but the increase was greater when 5-d cows were inseminated at 72 h (13.3%) compared to 60 h (9.1%) after P4 device removal [4]. However, in dairy heifers subjected to a modified 5d Co-synch protocol, 24% were detected in estrus between 36 and 48 h after P4 device removal and 74% of those animals ovulated before TAI at 72 h after P4 device removal [24]. Colazo and Mapletoft [24] also reported a total of 51% of heifers showing estrus at or before 60 h after P4 device removal. Therefore, earlier TAI may optimize P/AI, as these animals are likely to ovulate within 84 h after P4 device removal. Kasimanickam et al. [23] found an increase in P/AI in beef heifers treated with the 5-d Co-synch plus CIDR protocol when TAI was done at 56 h as compared with 72 h after P4 device removal (66 vs. 56%). Based on previous ovulation data in dairy heifers treated with the modified 5-d Co-synch protocol, in which 60% ovulated between 12 and 36 h after TAI at 72 h [24], TAI 66 h was chosen to compare with the standard interval of 72 h in Experiment 2. There was no difference in P/AI between 66 and 72 h in cyclic beef cows for either TAI protocol, which is in agreement pezwith findings from two recent studies [9,12]. In this regard, Lo
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Helguera et al. [12] reported no difference in P/AI in beef heifers that were TAI at 66 or 72 h in a modified 5-d Co-synch, and de la Mata et al. [9] reported no difference when heifers were inseminated at 60 or 72 h in the J-synch protocol. Although results would indicate a large window of time for TAI, the numerical increase does suggest that TAI at 72 h following P4 device removal may still be preferable in beef cows.
[15]
[16]
5. Conclusion [17]
The overall estrus rate and P/AI in beef heifers was 72% and 50.8%, respectively. When compared to the modified 5-d Co-synch plus CIDR protocol, the J-synch protocol resulted in greater P/AI in heifers and a tendency for greater P/AI in cows. The administration of eCG increased estrus rates only in heifers without a CL at the start of the modified 5-d Co-synch protocol and tended to increase overall P/AI. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows when either TAI protocol was used. Acknowledgements Research was supported by Livestock Systems Section, Alberta Agriculture and Forestry. Authors thank Vetoquinol N.-A Inc. (Lavaltrie, QC, Canada) and Rockway Inc. (Spring Valley, WI, USA) for their in-kind support and the Crow families (Thorsby, Alberta, Canada) and Parkland Cattle Co. (Spruce Grove, Alberta, Canada) for their cooperation during the study. References [1] Colazo MG, Mapletoft RJ. A review of current timed-AI (TAI) programs for beef and dairy cattle. Can Vet J 2014;55:772e80. GA, de la Mata JJ, Baruselli PS, Menchaca A. Alternative programs for [2] Bo synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 2016;86:388e96. GA. Estrus synchronization and ovulation using protocols [3] de la Mata JJ, Bo with estradiol benzoate and GnRH and reduced periods of insertion of a progesterone releasing device in beef heifers. Taurus 2012;55:17e23. [4] Bridges GA, Helser LA, Grum DE, Mussard ML, Gasser CL, Day ML. Decreasing the interval between GnRH and PGF2a from 7 to 5 days and lengthening proestrus increases timed-AI pregnancy rates in beef cows. Theriogenology 2008;69:843e51. [5] Colazo MG, Ambrose DJ. Neither duration of progesterone insert nor initial GnRH treatment affect pregnancy per timed-insemination in dairy heifers subjected to a Co-synch protocol. Theriogenology 2011;76:578e88. [6] Day ML. State of the art of GnRH-based timed AI in beef cattle. Anim Reprod 2015;12:473e8. [7] Bridges GA, Mussard ML, Helser LA, Day ML. Comparison of follicular dynamics and hormone concentrations between the 7-day and 5-day COSynch þ CIDR program in primiparous beef cows. Theriogenology 2014;81: 632e8. GA. Treatment with estradiol and progesterone [8] de la Mata JJ, Menchaca A, Bo GA, editors. Proceedings XI interthat prolonged proestrus. In: Caccia M, Bo rdoba, Argentina: Institute of national symposium on animal reproduction. Co rdoba (IRAC); 2015. p. 143e58. Animal Reproduction Co ~ ez-Olivera R, Cuadro F, Bosolasco D, de Brun V, Meikle A, [9] de la Mata JJ, Nún GA, Menchaca A. Effects of extending the length of pro-oestrus in an Bo oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers. Reprod Fertil Dev 2018;30:1541e52. [10] Macmillan K, Loree K, Mapletoft RJ, Colazo MG. Short communication: optimization of a timed artificial insemination program for reproductive management of heifers in Canadian dairy herds. J Dairy Sci 2017;100:4134e8. [11] Cruppe LH, Day ML, Abreu FM, Kruse S, Lake SL, Biehl MV, Cipriano RS, Mussard ML, Bridges GA. The requirement of GnRH at the beginning of the five-day CO-Synch þ controlled internal drug release protocol in beef heifers. J Anim Sci 2014;92:4198e203. pez Helguera I, Whittaker P, Behrouzi A, Mapletoft RJ, Colazo MG. Effect of [12] Lo initial GnRH and time of insemination on reproductive performance in cyclic and acyclic beef heifers subjected to a 5-d Co-synch plus progesterone protocol. Theriogenology 2018;106:39e45. [13] Murphy BD. Equine chorionic gonadotropin: an enigmatic but essential tool. Anim Reprod 2012;9:223e30. ~ ez-Olivera R, de Castro T, García-Pintos C, Bo G, Piaggo J, Menchaca A. [14] Nún Ovulatory response and luteal function after eCG administration at the end of
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