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Evaluation of three synchrony programs for pasture-based dairy heifers
Theriogenology 79 (2013) 882–889
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Evaluation of three synchrony programs for pasture-based dairy heifers S. McDougall a, *, F.M. Rhodes b, C.W.R. Compton a a b
Cognosco, Anexa Animal Health, Morrinsville, New Zealand Hamilton, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2012 Received in revised form 20 December 2012 Accepted 23 December 2012
The objective was to evaluate the efficacy and economic benefits of three synchrony programs in 1137 heifers from 10 pasture-based dairy herds. Heifers were randomly assigned to one of three treatments within each herd on Day 13 (Day 0 ¼ start of the breeding program). They were treated with: (1) PGF2a on Days 13 and 2, with AI after detection of estrus between Days 0 and 3 (Double PG); (2) GnRH, PGF2a, and GnRH on Days 9, 2, and 0, respectively, with placement of an intravaginal progesterone (P4)-releasing device between Days 9 and 2, and set time AI on Day 1 (GPG þ P4); or (3) same as the GPG þ P4 group but with the set time AI on Day 0 (Cosynch þ P4). Plasma P4 concentrations were determined on Days 20 and 13 to determine pubertal status. The Cosynch þ P4 treatment had a higher (P < 0.05) conception rate to AI (57% vs. 47% vs. 48% for Cosynch þ P4, GPG þ P4, and Double PG, respectively), 21-day in-calf rate (76% vs. 72% vs. 63% for Cosynch þ P4, GPG þ P4, and Double PG), and a shorter median interval from the start of the breeding program to conception (0, 14, and 19 days for Cosynch þ P4, GPG þ P4, and Double PG). Heifers that had reached puberty before breeding, compared with those that had not, had higher (P < 0.05) in-calf rates to AI (53% vs. 47%) at 21 days (74% vs. 64%) and at 42 days (91% vs. 84%). Pubertal status was associated with herd, breed, age, and body condition score at the start of mating (P < 0.05). A partial budget model demonstrated that, compared with the Double PG program, there was an economic benefit from the Cosynch þ P4 (mean, NZ$25.73; 95% confidence interval, 2.99–50.69), but not the GPG þ P4 program (mean, NZ$0.65; 95% confidence interval, 21.87 to 21.58). We concluded that the Cosynch þ P4 program resulted in the highest fertility and economic benefit of the three programs evaluated, and that reproductive response was affected by pubertal status. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Heifer Synchrony Ovsynch Cosynch Progesterone Prostaglandin
1. Introduction Synchronization of estrus and ovulation of dairy heifers, in combination with AI, facilitates improving the rate of genetic gain by shortening the generation interval and obtaining replacements from dams with the highest genetic merit in a herd [1]. However, the use of AI in dairy heifers has historically been limited in New Zealand. For example, in 2006 and 2007 only 118,226 yearlings were mated using AI compared with 2,904,667 cows, from a total * Corresponding author. Tel.: þ64 7 889 5159; fax: þ64 7 889 3681. E-mail address: [email protected] (S. McDougall). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.12.013
population of 5.3 million dairy cattle [2]. The practicalities of inseminating heifers after either daily estrous detection or some form of synchrony might be barriers to uptake of the technology. Various synchrony programs for dairy heifers have previously been evaluated in pasture-based systems [3–6]. However, these programs generally included the use of estradiol, which can no longer be used in food-producing animals because of the European ban, or involved detection of estrus. Therefore evaluation of new programs was required, including those that did not require estrous detection and were practical under the extensive pasturebased management systems of New Zealand.
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Use of fixed-time insemination (FTAI) removes the requirement for estrous detection, but conception rates have been reported to be significantly lower using FTAI after two injections of PGF2a 10 to 14 days apart (“Double PG”) than for dairy heifers inseminated on detection of estrus [7]. These low conception rates were attributed to variations in time to estrus and ovulation depending on the stage of the estrus cycle at PGF2a administration, and the lack of efficacy of PGF2a in prepubertal animals [8,9]. Optimal synchrony of estrus and ovulation requires control of follicle development and luteal function [10]. This can be achieved in dairy cows using programs involving GnRH injections 9 days apart with PGF2a 2 days before the final GnRH treatment, with FTAI 16 hours after the final GnRH treatment (GPG or Ovsynch), but such programs used in heifers do not achieve conception rates comparable with AI after detection of estrus, because of a lack of synchrony of estrus [11,12]. Insemination might also occur coincident with the final GnRH treatment (Cosynch). The Cosynch program reduces the number of handlings of cattle required, but has been reported to result in lower conception rates compared with Ovsynch [13]. However, in beef heifers, a Cosynch program resulted in pregnancy rates superior to a Double PG program [14]. Addition of progesterone (P4) to GPG programs also resulted in significantly increased conception rates after FTAI in beef heifers [15], and improved synchrony of estrus in dairy heifers [16]. Heifers that are prepubertal at the commencement of synchrony programs are less likely to exhibit estrus [17], and have lower conception and pregnancy rates than postpubertal heifers [18]. In prepubertal beef heifers, inclusion of P4 in the synchrony program significantly increased submission and pregnancy rates [17], and conception rates [19]. The aim of the current study was to evaluate the efficacy and economic benefits of three synchrony programs in heifers from pasture-based dairy herds. The programs were GPG or Cosynch, both with the addition (þ) of P4, and Double PG. It was hypothesized that more heifers would conceive to AI and be pregnant by Day 21 of the breeding program after a GPG þ P4 program than after Double PG, and that conception rate to AI would be noninferior after Cosynch þ P4 compared with GPG þ P4. 2. Materials and methods 2.1. Heifers and treatments The study was conducted after approval from the Animal Ethics Committee of AgResearch Ruakura, Hamilton, New Zealand. Dairy heifers (N ¼ 1137) from 10 herds (mean SD, 114 26 per herd) were enrolled on one calendar day for each herd. All herds were spring-calving and located within the Waikato region of New Zealand. Blood samples (10 mL) were drawn from the tail vein into an evacuated glass tube containing lithium heparin as an anticoagulant (Vacutainer; Becton Dickson, Franklin Lakes, NJ, USA) on Days 20 and 13 (where Day 0 ¼ start of the breeding program) for evaluation of P4 concentration in plasma by RIA (Coat-a-Count; DPC Corporation, Los Angeles, CA,
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USA). Heifers were defined as prepubertal if the P4 concentration in both samples was <1.0 ng/mL, and postpubertal if P4 concentration in one or both samples was 1.0 ng/mL. On Day 13, the body condition score (BCS) of each heifer was assessed on a one to 10 scale [20], and tail paint applied to aid estrus detection. Heifers were randomly assigned, within sequentially presented blocks of three heifers, to one of three treatment groups. Different color tail paints were used for identification of each treatment group, but all heifers within a herd were managed as a single management group. The three treatments were: Double PG: Heifers were treated with cloprostenol (Ovuprost 500 mg im; Bomac Laboratories Ltd., Manukau City, New Zealand) on Days 13 and 2, with AI upon estrus detection between Days 0 and 3 (N ¼ 380; 33%); GPG þ P4: Heifers were treated with an intravaginal P4-releasing device (Cue Mate; Bomac Laboratories Ltd.) from Days 9 to 2, and gonadorelin (Ovurelin 100 mg im; Bomac Laboratories Ltd.) on Day 9, 500 mg cloprostenol im on Day 2, and 100 mg gonadorelin on Day 0, with FTAI on Day 1 (N ¼ 383; 34%); and Cosynch þ P4: Heifers were treated in the same manner as for GPG þ P4 but with FTAI coincident with the final gonadorelin treatment (N ¼ 374; 33%). The timing of the treatments was such that the second cloprostenol treatment for the Double PG group coincided with the cloprostenol treatment of the other two groups. Treatments were given between 9:00 AM and 1:00 PM. All heifers were assessed once daily from Days 0 to 3 for loss of tail paint while yarded for drafting for AI. Herd owners decided which of the heifers in the Double PG group were in estrus and were to be inseminated each day based on removal of tail paint or their own observations on pasture. When a heifer had been inseminated, no more estrous observations were conducted. Experienced AI technicians attended each herd at approximately midday on Days 0 to 3. In seven herds, one technician undertook all inseminations, whereas in the remaining three herds, two technicians undertook inseminations. Where two technicians were used, they undertook inseminations on each day of the program. Heifers in the Double PG group detected in estrus between Days 0 and 3 were inseminated on the day of detection. Heifers in the GPG þ P4 group in estrus at the time of the second GnRH injection (Day 0) were recorded as in estrus, but not inseminated until Day 1. Frozen semen from 40 sires was used for AI in the study (one to four sires were used in each treatment group). After the last AI on Day 3, bulls were placed with the heifers for a mean (SD) total duration of 80 10 days. The number of bulls required for each management group was calculated assuming that 50% of the heifers would conceive to first service and that those not conceiving would return to estrus over a 5-day period on average 21 days later. Sufficient bulls were introduced such that no more than three services per bull per day would occur on average.
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Pregnancy diagnosis was conducted in all herds (mean SD) 69 2 days and 109 5 days after the start of the breeding program. In seven herds, additional pregnancy diagnosis took place 139 6 days after the start of the breeding program. The stage of gestation, in days, was estimated at each examination if a heifer was diagnosed pregnant. The date of birth and breed of individual heifers was retrieved from an electronic database (LIC, Hamilton, New Zealand). These data were used to calculate the age at the start of the breeding program (mean SD ¼ 14.3 0.4 months), and to classify heifers as Friesian (N ¼ 575; 51%), Jersey (N ¼ 159; 14%), or other (N ¼ 403; 35%). 2.2. Definitions and statistical methods There were six primary outcome variables in this study. The in-calf to AI rate was defined as the proportion of enrolled heifers that conceived to AI. The conception rate to AI was defined as the proportion of heifers conceiving to AI and was calculated only for heifers that were submitted for AI between Days 0 and 3. It was possible that some heifers in the Double PG group might have conceived to the first natural mating after the introduction of bulls, but natural matings were not recorded, and so these heifers were excluded from this analysis. The 21-day and 42-day in-calf rates were defined as the proportion of enrolled heifers pregnant at 21 or 42 days from the start of the breeding program, respectively. The final in-calf rate was defined as the proportion of heifers present at the final pregnancy test that were pregnant at the final pregnancy test. Finally, the median interval in days from the start of the breeding program to conception was calculated by survival analysis methods. For heifers in the GPG þ P4 group, the reproductive performance measures were calculated using the first day of AI as the start point (Day 1 in the study), so as not to bias the results for this group compared with those in the other two groups that started 1 day earlier (Day 0 in the study). Potential predictor variables evaluated were treatment group, herd of origin, age in months at the start of breeding (categorized into quartiles), pubertal status at commencement of the study, breed category, observation of estrus, and BCS (categorized into quartiles). Balance of treatment groups for age, breed, pubertal status, and BCS of heifers was compared using chi-square tests. Differences between treatment groups in the categorical outcome variables were compared using two-sided Pearson chi-square test with Holm adjustment for multiple testing [21] to control the overall type I error rate. Multivariable logistic regression using a forward stepwise addition method was used to model the effect of treatment group, and other variables associated by univariate analysis (P < 0.2), on the outcomes. Treatment group and herd of origin were fitted as fixed effects and forced into each model. Other variables were tested for significance and as potential confounders and included if their Wald test probability value was P < 0.05, or if their inclusion changed the coefficients for the treatment effects by 15% or more. Interactions between treatment groups and other significant variables were also tested. Preliminary final models
were checked by plotting the deviance residuals against the fitted values and the Cook statistics against the standardized leverages, and if no values were of concern, were declared final. The mean effects of the other nontreatment variables in the final models were also estimated to describe the change in outcome (on a percentage scale) associated with that variable. The noninferiority hypothesis was tested using differences between the means and the confidence intervals of the differences estimated from the final model, with the other included variables set to their median value. The effect on the time to conception of treatment group and potential confounding variables were tested by Kaplan–Meier survival analysis using the log-rank test, and survival curves plotted. A variable of secondary interest was pubertal status as defined by plasma P4 concentrations. Associations were evaluated between pubertal status and herd of origin, breed, and age and BCS at Day 13 using chi-square analysis with adjustment for multiple testing, as previously described. The pubertal status (post vs. prepubertal) of heifers was modeled by multiple logistic regression models and effects of significant variables estimated as previously described. 2.3. Power statistics The first hypothesis was that more heifers would conceive to AI and be pregnant by Day 21 of the breeding program after a GPG þ P4 program than after Double PG. It was assumed that submission rates would be 70% and 100%, and conception to AI rates would be 60% and 55%, for the Double PG and GPG þ P4 groups, respectively. Thus it was expected that approximately 42% and 55% of heifers would be pregnant to AI in the Double PG and GPG þ P4 groups, respectively. To detect this 13% difference, approximately 250 heifers per treatment group were required (a ¼ 0.05; a ¼ 0.2). To allow for loss to follow-up, approximately 300 heifers per treatment group were enrolled. The second hypothesis was that pregnancy rates by Day 21 would be noninferior for the Cosynch þ P4 compared with the GPG þ P4 group. It was assumed that the conception rate of the two groups would be the same (55%) and an acceptable difference between the two groups would be 10% (a ¼ 0.05; b ¼ 0.2). This required 420 heifers per treatment group (Pass 2008; www.ncss.com). 2.4. Partial budget A partial budget model was prepared to estimate the economic effect of synchronization of estrus of heifers using the GPG þ P4 or Cosynch þ P4 programs compared with the Double PG program. The model was built using input data and distributions from the current study, other published data, or the opinions of the authors (Table 1), and the method of stochastic Monte Carlo simulation in the Microsoft Excel add-in @Risk (Version 5; Palisade Corporation, Ithaca, NY, USA). This simulation modeling takes account of the variability of model inputs by assigning each a specific distribution, and generates a distribution for the outcomes from which mean economic effects are estimated.
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Table 1 Assumptions used to develop a stochastic partial budget model (in New Zealand $) in the Microsoft Excel add-in @Risk to compare the economic benefit of estrus synchrony with artificial breeding of pasture-grazed dairy heifers using either GPG þ P4a or Cosynch þ P4b with a Double PGc treatment program. Income variables
Mean
Distribution
Extra days in milk GPG þ P4d Extra days in milk Cosynch þ P4d Mean milk production per day (kg milk solidse) Milk payment next season ($ per kg milk solidse) Percentage in calf to AI, Double PG Percentage in calf to AI, GPG þ P4 Percentage in calf to AI, Cosynch þ P4 Percentage calves female Extra AI heifer calves, GPG þ P4d Extra AI heifer calves, Cosynch þ P4d Male calf value AI calf value (4 days of age)
3.9 6.4 1.2 5.50 31% 47% 57% 50% 8% 13% $20.00 $200.00
RiskNormal(3.9, 1.5) RiskNormal(6.4, 1.5) RiskNormal(1.2, 0.12) RiskUniform(5.65, 5.75) RiskLogistic(0.31, 0.0136) RiskLogistic(0.47, 0.0136) RiskLogistic(0.57, 0.0135) RiskBinomial(100, 0.5)
Cost variables
RiskUniform(15, 25) RiskUniform(150, 250)
Mean
Drug and treatment costs per head Drug costs, Double PG Drug costs, GPG + P4 Drug costs, Cosynch + P4 Veterinary fees, Double PG Veterinary fees, GPG + P4 Veterinary fees, Cosynch + P4
$7.36 $40.00 $40.00 $6.00 $11.00 $11.00
Overhead costs per head
Mean
Average number of heifers per group Number of heifer yardings, Double PG Number of heifer yardings, GPG þ P4 Number of heifer yardings, Cosynch þ P4 Grazier yarding fees Percentage of graziers charging yarding fees Visit and yarding fees plus travel, Double PG Visit and yarding fees plus travel, GPG þ P4 Visit and yarding fees plus travel, Cosynch þ P4 Difference in overhead costs per head, GPG þ P4d Difference in overhead costs per head Cosynch þ P4d
50 5 4 3 $50.00 25% $222.50 $290.00 $277.50 $1.35 $1.10
Distribution
RiskUniform(25, 75) RiskTriang(0.2, 0.25, 0.3)
Distribution is the @Risk formula used to model variability; otherwise the mean value was used. Income variables are shown. a PGF2 on Days 13 and 2 with AI on detection of estrus from Day 0 to 3 of the breeding program. b GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. c GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 0. d Compared with Double PG. e Milk solids ¼ kg of protein plus kg of fat.
In seasonal calving systems with a fixed start and end of lactation, the maximum number of days in milk occurs when an animal calves at the start of the calving program (having conceived at the start of the breeding program) and each day of delay to conception results in one day less in milk. Hence, the additional number of days in milk in the lactation after synchrony for the GPG þ P4 or Cosynch þ P4 heifers, relative to the Double PG heifers, was modeled by subtracting the interval from the start of breeding to the date of conception from the duration of the breeding program for each heifer, and including the mean and SEM of the differences between treatment groups in the model. The economic benefits were assessed in terms of extra milk production, calculated from the estimated value of milk solids (kg of milk protein plus kg of milk fat; the basis of payment in the New Zealand dairy industry), mean daily production of milk solids, and extra days in milk, plus the value of additional AI calves, calculated from the estimated percentage of extra female AI calves born and the value of these calves at 4 days of age. Additional costs were
calculated from estimated treatment costs based on drug costs and vet fees, plus management costs because of yarding of heifers. The means and 95% confidence intervals of the distributions of the economic benefit of each comparison were reported and the treatment comparison declared significantly different if the 95% confidence interval of the difference did not include zero. Spearman rank correlation coefficients were examined to determine the variables with greatest influence on the net economic benefit. Data were managed in a custom-built Microsoft Access database (Microsoft, Redmond, WA, USA), and analysis conducted using R (Version 2.8.1; R Development Core Team, 2008). Statistical significance was declared for test results with P < 0.05. 3. Results Nine heifers were removed from the analysis because they were infertile (freemartins, N ¼ 6), pregnant and
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aborted within 1 day after treatment with PGF2a (N ¼ 1), or were missing at the final treatment with PGF2a (N ¼ 2), leaving data for analysis from 1137 heifers. Six heifers were not present for the final pregnancy diagnosis and were not included in results for the final in-calf rate. Mean BCS of heifers at enrolment was 4.6, with 90% in the range of 4.0 to 5.5 (inclusive). Paired plasma samples were available from 1123 heifers; 671 heifers (60%) had plasma P4 concentrations 1.0 ng/mL in one or both samples. There were no differences between treatment groups in age category (P ¼ 0.71), breed category (P ¼ 0.81), pubertal status (P ¼ 0.87), or BCS category (P ¼ 0.92). 3.1. Detection in estrus A total of 248 of 380 heifers (65%) in the Double PG group were detected in estrus by farmers and submitted for AI over Days 0 to 3. The percentage of heifers detected in estrus in this group was greater for those that were postcompared with prepubertal (87.3% vs. 34.2%; P < 0.01). On Day 0, 107 of 380 (28%), 183 of 383 (48%), and 178 of 374 (48%) of heifers were observed at the time of yarding to have their tail paint disturbed for the Double PG, GPG þ P4 and Cosynch þ P4 groups, respectively (P < 0.001). 3.2. Conception to AI Results for the in-calf to AI rate and conception rate to AI for the three treatment groups are summarized (Table 2). For both outcomes, heifers in the Cosynch þ P4 group achieved higher values than those in the GPG þ P4 group (P < 0.05). In-calf to AI rate was also higher (P < 0.01) and conception to AI tended to be higher (P ¼ 0.069) for heifers in the Cosynch þ P4 group than for heifers in the Double PG group. The Cosynch þ P4 group was declared noninferior to the GPG þ P4 group because the lower 95% confidence interval of the difference in conception rate to AI was greater than 0.10, the a priori defined acceptable difference (difference (Coynsch þ P4) (GPG þ P4) ¼ 0.11; 95% confidence interval, 0.02–0.19). At the univariate level, the in-calf rate to AI
varied among herds and was higher in pubertal compared with prepubertal heifers (53% vs. 47%; P ¼ 0.05), but only treatment group and herd were significant in the final model. Conception rate to AI also varied among herds (range, 37%–63%) and was higher in heifers from which tail paint was removed 1 to 48 hours before AI compared with 0 to 48 hours after AI (266/497 [53.5%] vs. 73/186 [39.2%]; P < 0.001). 3.3. Pregnancy rates Results for the 21-day, 42-day, and final in-calf rates for the three treatment groups are summarized (Table 2). Heifers in the Cosynch þ P4 group had higher 21- and 42-day in-calf rates than heifers in the Double PG group (P < 0.05), but did not differ from those in the GPG þ P4 group. The 21-day in-calf rate varied among herds (range, 53%–80%). In the final models, heifers that had reached puberty before breeding, compared with those that had not, had higher in-calf rates at 21 days (74% vs. 64%; P < 0.001) and 42 days (91% vs. 84%; P ¼ 0.03). The final in-calf rates were not different between treatment groups, but varied among herds. For all final models, there were no significant herd by treatment interactions or pubertal status by treatment interactions. Survival curves for the interval from the start of the breeding program to conception differed between the treatment groups (P < 0.001; Fig. 1). The median interval from the start of breeding to conception was shorter for the heifers in the Cosynch þ P4 than the GPG þ P4 and Double PG groups (P < 0.05), and tended to be shorter for heifers in the Cosynch þ P4 group than the GPG þ P4 group (P ¼ 0.06; Table 2). 3.4. Pubertal status Overall, 671 of 1123 heifers (60%) were categorized as postpubertal, but there was considerable variation among herd of origin (range, 9%–93%). Factors associated with pubertal status were herd of origin (P < 0.001), breed category (P < 0.001), age (P ¼ 0.002), and BCS at the start of the breeding program (P < 0.001; Table 3).
Table 2 Number (%) of dairy heifers conceiving to AI or pregnant after three treatment protocols for synchronization of estrous and ovulation. Treatment group
In-calf to AIa
Conception rate to AIb
21-Day in-calf ratec
42-Day in-calf rated
Final in-calf ratee
Interval to conception (days)f
Double PGg GPG þ P4j Cosynch þ P4k
119/380h (31.3) 180/383i (47.0) 213/374l (57.0)
119/248h,i (48.0) 180/383h (47.0) 213/374i (57.0)
238/380h (62.6) 274/383i (71.5) 284/374i (75.9)
313/380h (82.4) 335/383h,i (87.5) 333/374i (89.0)
347/376 (92.3) 352/381 (92.4) 358/374 (95.7)
19h 14h 0i
a
Proportion of all heifers conceiving to AI. Proportion of heifers artificially bred conceiving to AI. c Proportion of all heifers pregnant after 21 days of the breeding program. d Proportion of all heifers pregnant after 42 days of the breeding program. e Proportion of heifers examined that were pregnant at the end of the breeding program. f Median interval from start of breeding to conception. g PGF2 on Days 13 and 2 with AI on detection of estrus from Day 0 to 3 of the breeding program. h,i, and l Within a column, means without a common superscript differed (P < 0.05). j GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. k GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed-time AI on Day 0. b
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4. Discussion
Fig. 1. Kaplan–Meier survival curves of cumulative time to conception from the start of the breeding program (Day 0) for dairy heifers treated with three synchronization protocols: (1) PGF2a on Days 13 and 2, with AI after detection of estrus between Days 0 and 3 (Double PG); (2) GnRH, PGF2a, and GnRH on Days 9, 2, and 0, respectively, with placement of an intravaginal progesterone (P4)-releasing device between Days 9 and 2, and set time AI on Day 1 (GPG þ P4); or (3) same as the GPG þ P4 group but with the set time AI on Day 0 (Cosynch þ P4). There were differences among treatment groups (P < 0.001).
3.5. Partial budget The Cosynch þ P4 program had an economic benefit compared with the Double PG program, but there was no benefit from the GPG þ P4 program (Table 4). Additional income from greater milk yield because of earlier calving and more AI calves in the Cosynch þ P4 compared with the Double PG program accounted for the difference between the two groups. Costs for the two P4-based treatments were very similar. The variables most highly correlated with the net benefit of the Cosynch þ P4 program were the additional days in milk in the subsequent lactation (rs ¼ 0.83) through earlier conception pattern, daily milk production (rs ¼ 0.35), and value of an artificially-sired calf (rs ¼ 0.3).
Table 3 Percentage of heifers (N ¼ 1123) that were postpubertal, based on progesterone concentrations in two plasma samples, classified by breed, age, and body condition score at the start of the breeding program. Variable
Level
Pubertal (%)
Breed
Friesian Jersey Other 12.2-14.2 14.3 14.5 14.8-15.9 3.0-4.0 4.5 5.0 5.5-7.5
45.1a 86.7b 70.0c 52.5a 59.1a 64.0b,c 65.9b,c 37.9a 66.8b 72.3b 74.3b
Age (mo)
Body condition score
a–c Within an end point, means without a common superscript differed (P < 0.05).
This study demonstrated that synchronization of heifers using a combination of GPG and P4 with FTAI coincident with the final GnRH treatment (Cosynch þ P4) resulted in superior reproductive and economic performance compared with a similar program but with FTAI delayed by approximately 24 hours or to a Double PG program. The conception rate to AI of 57% for heifers in the Cosynch þ P4 group in the current study was comparable with the 53% reported after a previous synchrony program with FTAI in New Zealand [6], and was superior to results for the GPG program in Holstein heifers in the United States when heifers detected in estrus before the final GnRH were inseminated early and overall conception rates were 46% [11]. The 21-day in-calf rate of 76% for the Cosynch þ P4 heifers was also better than the 32-day pregnancy rate of 60% reported for dairy heifers in Florida after an 8-day Cosynch program using P4 inserts from Days 0 to 5 [22]. The higher conception rates to AI in the Cosynch þ P4 group compared with heifers in the GPG þ P4 group in the current study were comparable with the finding of Lima et al. [23], in which a Cosynch þ P4 protocol resulted in improved reproductive outcomes compared with an Ovsynch þ P4 protocol in dairy heifers in Florida. In the current study, heifers observed to have lost tail paint during the 48 hours before AI had higher conception rates to AI than those with loss of tail paint during the 48 hours after AI. The interval from the end of a synchrony program to estrus was affected by the size of the dominant follicle at the end of the program, with a larger dominant follicle associated with a shorter interval to estrus [24]. It is not clear if the shorter interval from the end of the synchrony program to estrus was also associated with a shorter interval to ovulation. After a synchrony program, the interval between onset of estrus and ovulation appeared to be reasonably consistent at 1.3 days in a range of synchrony systems [25]. Thus it is likely that heifers in the current study that were in estrus earlier relative to Day 0, also ovulated sooner. This might partly explain the higher conception rates in the Cosynch þ P4 program compared with the GPG þ P4 program. In the latter program, nearly half of the heifers had been detected in estrus by Day 0, yet by design were not inseminated until 24 hours later. So, for this group of heifers, insemination might have occurred too late for optimum fertility. The interval between estrus and ovulation has been reported as 38.5 3.0 hours (mean SEM) in dairy heifers [26]. Because sperm capacitation takes 4 to 12 hours, logically insemination should occur 12 to 18 hours before ovulation to ensure functional sperm are present at the time of ovulation [27]. In one study, heifers in estrus in the 24 hours preceding FTAI had higher conception rates than those in estrus 25 to 40 hours before FTAI (76% vs. 50%) [28]. In another study, conception rates to FTAI were optimized when insemination occurred 6 to 22 hours before ovulation [29]. Of the heifers in the Double PG group, 65% were detected in estrus between 2 and 6 days after the final treatment with PGF2a. This appeared to be lower than in some previous reports, in which 86% were detected in estrus over 5 days after the final treatment [30]. This lower
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Table 4 Partial budget, in New Zealand dollars per heifer, on the use of two programs for synchronization of estrous compared with a double PGF2 program. Treatment Group
GPG þ P4a Cosynch þ P4b
Additional income
Additional costs
Net benefit
Extra milk production
Value of AI calves
Total
Treatment costs
Management costs
Total
Mean
95% CI
26.68 43.78
14.40 23.40
41.08 67.18
40.36 40.36
1.35 1.10
41.71 41.46
0.65 25.73
21.87 to 21.58 2.99–50.69
Abbreviation: CI, confidence interval. a GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. b GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed-time AI on Day 0.
detection rate might have been related to lower sensitivity of detection of estrus by the observers, a 1-day shorter period of observation, or because only 60% of heifers in the current study had reached puberty when the program started. Pubertal status had a significant effect on reproductive outcomes in the current study; the percentage of heifers that were postpubertal at the start of the study decreased with lower BCS and younger age at commencement of the program. In a previous study, the risk of conception to first service was not associated with withers height, age, BCS, or body weight, but the proportion of heifers that were prepubertal was not defined and it is likely that most heifers were postpubertal, hence the biophysical measures might have been of lesser importance [31]. In the current study, there were significant effects of herd on all outcome variables, likely associated with the percentage of animals that were prepubertal in each herd. At a practical level, our findings demonstrated that optimal outcomes after synchrony programs will be achieved in heifers that are postpubertal, and as such should be 14.5 months old and have a BCS >4.0. In addition to improved reproductive performance, the Cosynch þ P4 program had a significant economic benefit compared with the Double PG program. One potential benefit of use of AI in heifers is a shortening of the generation interval with an associated faster rate of genetic gain. In a previous assessment of the economic benefits of artificially inseminating heifers with or without synchrony, the optimal economic decision was to select high genetic merit heifers and undertake AI on detection of estrus [1]. However, in that analysis, the main benefit modeled was the value of the improved genetics of the calves, and it ignored any potential benefits that synchrony might have in terms of earlier calving of the heifers with resultant increased lactation length. The current study analyzed the returns in terms of extra value associated with AI-sired calves, and additional production in the first lactation. The economic benefit of the Cosynch þ P4 program was because of the 100% submission rate and the higher conception rate that resulted in more AI-sired calves and earlier calving, and thus a longer first lactation length compared with the other programs. Although the Double PG program was less expensive, the poorer pregnancy rates resulted in poorer economic outcomes relative to the Cosynch þ P4 treatment. In populations of heifers with fewer prepubertal animals, the Double PG system might be more effective and less economically disadvantaged.
4.1. Conclusions Synchrony of dairy heifers with GPG þ P4 and with FTAI at the time of the final GnRH injection (Cosynch þ P4) resulted in the highest biological and economic outcomes of the three programs tested. Pubertal status had a significant effect on reproductive performance, irrespective of synchrony program. Acknowledgments The contribution of the herd owners, staff, and the graziers is gratefully acknowledged. The support of the technical team of Laura Clausen, Catherine Sheehan, Aimee Wilson, and Cathy Yanez is also gratefully acknowledged. Progesterone RIA were undertaken by Angela Sheehan of DairyNZ. Treatments and pregnancy testing were undertaken by the authors and by Dr Megan Clements and Dr Geoff Plant of Anexa Animal Health. The funding and product support provided by Bayer Animal Health New Zealand Ltd. and by DairyNZ via an On-farm innovation fund grant (OF811) is acknowledged. Semen was provided by LIC, who were also helpful in organizing inseminators for the study. References [1] Rendel JM, Xu ZZ, Burton LJ. Artificial insemination of yearling heifers: an economic analysis. Proc Soc Dairy Cattle Vet NZVA 1998: 255–62. [2] Anonymous. New Zealand dairy statistics 2010/11. Hamilton, NZ: LIC and DairyNZ; 2011. [3] Macmillan KL, Taufa VK, Day AM. Combination treatments for synchronising oestrus in dairy heifers. Proc NZ Soc Anim Prod 1993; 53:267–70. [4] Hanlon DW, Williamson NB, Wichtel JJ, Steffert IJ, Craigie AL, Pfeiffer DU. The effect of estradiol benzoate administration on estrous response and synchronized pregnancy rate in dairy heifers after treatment with exogenous progesterone. Theriogenology 1996;45:775–85. [5] Hanlon DW, Williamson NB, Wichtel JJ, Steffert IJ, Craigie AL, Pfeiffer DU. Ovulatory responses and plasma luteinizing hormone concentrations in dairy heifers after treatment with exogenous progesterone and estradiol benzoate. Theriogenology 1997;47: 963–75. [6] Xu ZZ, Burton LJ. Reproductive performance of dairy heifers after estrus synchronization and fixed-time artificial insemination. J Dairy Sci 1999;82:910–7. [7] Macmillan KL, Curnow RJ, Morris GR. Oestrus synchronisation with a prostaglandin analogue: 2. Systems in maiden heifers. NZ Vet J 1978;26:93–103. [8] Refsal KR, Seguin BE. Effect of stage of diestrus and number of cloprostenol (ICI 80, 996) injections on intervals to estrus, LH peak and ovulation in heifers. Theriogenology 1980;14:37–48.
S. McDougall et al. / Theriogenology 79 (2013) 882–889 [9] Tanabe TY, Hann RC. Synchronized estrus and subsequent conception in dairy heifers treated with prostaglandin F2a. I. Influence of stage of cycle at treatment. J Anim Sci 1984;58:805–11. [10] Patterson DJ, Kojima FN, Smith MF. A review of methods to synchronize estrus in replacement beef heifers and postpartum cows. J Anim Sci 2003;81:E166–77. [11] Schmitt EJ, Diaz PT, Drost M, Thatcher WW. Use of a gonadotropinreleasing hormone agonist or human chorionic gonadotropin for timed insemination in cattle. J Anim Sci 1996;74:1084–91. [12] Pursley JR, Wiltbank MC, Stevenson JS, Ottobre JS, Garverick HA, Anderson LL. Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. J Dairy Sci 1997;80:295–300. [13] Pursley JR, Mee MO, Wiltbank MC. Synchronisation of ovulation in dairy cows using PGF2a and GnRH. Theriogenology 1995;44:915–23. [14] Dahlen CR, Lamb GC, Zehnder CM, Miller LR, DiCostanzo A. Fixedtime insemination in peripuberal, lightweight replacement beef heifers after estrus synchronization with PGF2alpha and GnRH. Theriogenology 2003;59:1827–37. [15] Martinez MF, Kastelic JP, Adams GP, Cook B, Olson WO, Mapletoft RJ. The use of progestins in regimens for fixed-time artificial insemination in beef cattle. Theriogenology 2002;57:1049–59. [16] Rivera H, Lopez H, Fricke PM. Use of intravaginal progesteronereleasing inserts in a synchronization protocol before timed AI and for synchronizing return to estrus in Holstein heifers. J Dairy Sci 2005;88:957–68. [17] Lucy MC, Billings HJ, Butler WR, Ehnis LR, Fields MJ, Kesler DJ, et al. Efficacy of an intravaginal progesterone insert and an injection of PGF2alpha for synchronizing estrus and shortening the interval to pregnancy in postpartum beef cows, peripubertal beef heifers, and dairy heifers. J Anim Sci 2001;79:982–95. [18] Richardson AM, Hensley BA, Marple TJ, Johnson SK, Stevenson JS. Characteristics of estrus before and after first insemination and fertility of heifers after synchronized estrus using GnRH, PGF2, and progesterone. J Anim Sci 2002;80:2792–800. [19] McGowan MR, Carroll CL, Davies FJ. Fixed-time insemination of Bos indicus heifers following the use of Syncro-Mate-B (Smb) to synchronize estrus. Theriogenology 1992;37:1293–300. [20] Roche JR, Dillon PG, Stockdale CR, Baumgard LH, VanBaale MJ. Relationships among international body condition scoring systems. J Dairy Sci 2004;87:3076–9.
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[21] Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat 1979;6:65–70. [22] Rabaglino MB, Risco CA, Thatcher MJ, Kim IH, Santos JEP, Thatcher WW. Application of one injection of prostaglandin F2a in the five-day Co-Synch þ CIDR protocol for estrous synchronization and resynchronization of dairy heifers. J Dairy Sci 2010;93: 1050–8. [23] Lima FS, Ayres H, Favoreto MG, Bisinotto RS, Greco LF, Ribeiro ES, et al. Effects of gonadotropin-releasing hormone at initiation of the 5-d timed artificial insemination (AI) program and timing of induction of ovulation relative to AI on ovarian dynamics and fertility of dairy heifers. J Dairy Sci 2011;94: 4997–5004. [24] Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfenson D. Regulation of dominant follicle turnover during the oestrous cycle in cows. J Reprod Fertil 1993;97:197–203. [25] Martinez MF, Adams GP, Kastelic JP, Bergfel DR, Mapletoft RJ. Induction of follicular wave emergence for estrus synchronization and artificial insemination in heifers. Theriogenology 2000;54: 757–69. [26] Saumande J, Humblot P. The variability in the interval between estrus and ovulation in cattle and its determinants. Anim Reprod Sci 2005;85:171–82. [27] Vishwanath R, Nebel RL, McMillan WH, Pitt CJ, Macmillan KL. Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers. Theriogenology 1997;48:369–76. [28] Smith RD, Pomerantz AJ, Beal WE, McCann JP, Pilbeam TE, Hansel W. Insemination of Holstein heifers at a preset time after estrous cycle synchronization using progesterone and prostaglandin. J Anim Sci 1984;58:792–800. [29] Tenhagen BA, Kuchenbuch S, Heuwieser W. Timing of ovulation and fertility of heifers after synchronization of estrus with GnRH and prostaglandin F-2 alpha. Reprod Domest Anim 2005; 40:62–7. [30] Kaim M, Rosenberg M, Folman Y. Management of reproduction in dairy heifers based on the synchronisation of estrous cycles. Theriogenology 1990;34:537–47. [31] Donovan GA, Bennett FL, Springer FS. Factors associated with first service conception in artificially inseminated nulliparous Holstein heifers. Theriogenology 2003;60:67–75.
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Evaluation of three synchrony programs for pasture-based dairy heifers S. McDougall a, *, F.M. Rhodes b, C.W.R. Compton a a b
Cognosco, Anexa Animal Health, Morrinsville, New Zealand Hamilton, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2012 Received in revised form 20 December 2012 Accepted 23 December 2012
The objective was to evaluate the efficacy and economic benefits of three synchrony programs in 1137 heifers from 10 pasture-based dairy herds. Heifers were randomly assigned to one of three treatments within each herd on Day 13 (Day 0 ¼ start of the breeding program). They were treated with: (1) PGF2a on Days 13 and 2, with AI after detection of estrus between Days 0 and 3 (Double PG); (2) GnRH, PGF2a, and GnRH on Days 9, 2, and 0, respectively, with placement of an intravaginal progesterone (P4)-releasing device between Days 9 and 2, and set time AI on Day 1 (GPG þ P4); or (3) same as the GPG þ P4 group but with the set time AI on Day 0 (Cosynch þ P4). Plasma P4 concentrations were determined on Days 20 and 13 to determine pubertal status. The Cosynch þ P4 treatment had a higher (P < 0.05) conception rate to AI (57% vs. 47% vs. 48% for Cosynch þ P4, GPG þ P4, and Double PG, respectively), 21-day in-calf rate (76% vs. 72% vs. 63% for Cosynch þ P4, GPG þ P4, and Double PG), and a shorter median interval from the start of the breeding program to conception (0, 14, and 19 days for Cosynch þ P4, GPG þ P4, and Double PG). Heifers that had reached puberty before breeding, compared with those that had not, had higher (P < 0.05) in-calf rates to AI (53% vs. 47%) at 21 days (74% vs. 64%) and at 42 days (91% vs. 84%). Pubertal status was associated with herd, breed, age, and body condition score at the start of mating (P < 0.05). A partial budget model demonstrated that, compared with the Double PG program, there was an economic benefit from the Cosynch þ P4 (mean, NZ$25.73; 95% confidence interval, 2.99–50.69), but not the GPG þ P4 program (mean, NZ$0.65; 95% confidence interval, 21.87 to 21.58). We concluded that the Cosynch þ P4 program resulted in the highest fertility and economic benefit of the three programs evaluated, and that reproductive response was affected by pubertal status. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Heifer Synchrony Ovsynch Cosynch Progesterone Prostaglandin
1. Introduction Synchronization of estrus and ovulation of dairy heifers, in combination with AI, facilitates improving the rate of genetic gain by shortening the generation interval and obtaining replacements from dams with the highest genetic merit in a herd [1]. However, the use of AI in dairy heifers has historically been limited in New Zealand. For example, in 2006 and 2007 only 118,226 yearlings were mated using AI compared with 2,904,667 cows, from a total * Corresponding author. Tel.: þ64 7 889 5159; fax: þ64 7 889 3681. E-mail address: [email protected] (S. McDougall). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2012.12.013
population of 5.3 million dairy cattle [2]. The practicalities of inseminating heifers after either daily estrous detection or some form of synchrony might be barriers to uptake of the technology. Various synchrony programs for dairy heifers have previously been evaluated in pasture-based systems [3–6]. However, these programs generally included the use of estradiol, which can no longer be used in food-producing animals because of the European ban, or involved detection of estrus. Therefore evaluation of new programs was required, including those that did not require estrous detection and were practical under the extensive pasturebased management systems of New Zealand.
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Use of fixed-time insemination (FTAI) removes the requirement for estrous detection, but conception rates have been reported to be significantly lower using FTAI after two injections of PGF2a 10 to 14 days apart (“Double PG”) than for dairy heifers inseminated on detection of estrus [7]. These low conception rates were attributed to variations in time to estrus and ovulation depending on the stage of the estrus cycle at PGF2a administration, and the lack of efficacy of PGF2a in prepubertal animals [8,9]. Optimal synchrony of estrus and ovulation requires control of follicle development and luteal function [10]. This can be achieved in dairy cows using programs involving GnRH injections 9 days apart with PGF2a 2 days before the final GnRH treatment, with FTAI 16 hours after the final GnRH treatment (GPG or Ovsynch), but such programs used in heifers do not achieve conception rates comparable with AI after detection of estrus, because of a lack of synchrony of estrus [11,12]. Insemination might also occur coincident with the final GnRH treatment (Cosynch). The Cosynch program reduces the number of handlings of cattle required, but has been reported to result in lower conception rates compared with Ovsynch [13]. However, in beef heifers, a Cosynch program resulted in pregnancy rates superior to a Double PG program [14]. Addition of progesterone (P4) to GPG programs also resulted in significantly increased conception rates after FTAI in beef heifers [15], and improved synchrony of estrus in dairy heifers [16]. Heifers that are prepubertal at the commencement of synchrony programs are less likely to exhibit estrus [17], and have lower conception and pregnancy rates than postpubertal heifers [18]. In prepubertal beef heifers, inclusion of P4 in the synchrony program significantly increased submission and pregnancy rates [17], and conception rates [19]. The aim of the current study was to evaluate the efficacy and economic benefits of three synchrony programs in heifers from pasture-based dairy herds. The programs were GPG or Cosynch, both with the addition (þ) of P4, and Double PG. It was hypothesized that more heifers would conceive to AI and be pregnant by Day 21 of the breeding program after a GPG þ P4 program than after Double PG, and that conception rate to AI would be noninferior after Cosynch þ P4 compared with GPG þ P4. 2. Materials and methods 2.1. Heifers and treatments The study was conducted after approval from the Animal Ethics Committee of AgResearch Ruakura, Hamilton, New Zealand. Dairy heifers (N ¼ 1137) from 10 herds (mean SD, 114 26 per herd) were enrolled on one calendar day for each herd. All herds were spring-calving and located within the Waikato region of New Zealand. Blood samples (10 mL) were drawn from the tail vein into an evacuated glass tube containing lithium heparin as an anticoagulant (Vacutainer; Becton Dickson, Franklin Lakes, NJ, USA) on Days 20 and 13 (where Day 0 ¼ start of the breeding program) for evaluation of P4 concentration in plasma by RIA (Coat-a-Count; DPC Corporation, Los Angeles, CA,
883
USA). Heifers were defined as prepubertal if the P4 concentration in both samples was <1.0 ng/mL, and postpubertal if P4 concentration in one or both samples was 1.0 ng/mL. On Day 13, the body condition score (BCS) of each heifer was assessed on a one to 10 scale [20], and tail paint applied to aid estrus detection. Heifers were randomly assigned, within sequentially presented blocks of three heifers, to one of three treatment groups. Different color tail paints were used for identification of each treatment group, but all heifers within a herd were managed as a single management group. The three treatments were: Double PG: Heifers were treated with cloprostenol (Ovuprost 500 mg im; Bomac Laboratories Ltd., Manukau City, New Zealand) on Days 13 and 2, with AI upon estrus detection between Days 0 and 3 (N ¼ 380; 33%); GPG þ P4: Heifers were treated with an intravaginal P4-releasing device (Cue Mate; Bomac Laboratories Ltd.) from Days 9 to 2, and gonadorelin (Ovurelin 100 mg im; Bomac Laboratories Ltd.) on Day 9, 500 mg cloprostenol im on Day 2, and 100 mg gonadorelin on Day 0, with FTAI on Day 1 (N ¼ 383; 34%); and Cosynch þ P4: Heifers were treated in the same manner as for GPG þ P4 but with FTAI coincident with the final gonadorelin treatment (N ¼ 374; 33%). The timing of the treatments was such that the second cloprostenol treatment for the Double PG group coincided with the cloprostenol treatment of the other two groups. Treatments were given between 9:00 AM and 1:00 PM. All heifers were assessed once daily from Days 0 to 3 for loss of tail paint while yarded for drafting for AI. Herd owners decided which of the heifers in the Double PG group were in estrus and were to be inseminated each day based on removal of tail paint or their own observations on pasture. When a heifer had been inseminated, no more estrous observations were conducted. Experienced AI technicians attended each herd at approximately midday on Days 0 to 3. In seven herds, one technician undertook all inseminations, whereas in the remaining three herds, two technicians undertook inseminations. Where two technicians were used, they undertook inseminations on each day of the program. Heifers in the Double PG group detected in estrus between Days 0 and 3 were inseminated on the day of detection. Heifers in the GPG þ P4 group in estrus at the time of the second GnRH injection (Day 0) were recorded as in estrus, but not inseminated until Day 1. Frozen semen from 40 sires was used for AI in the study (one to four sires were used in each treatment group). After the last AI on Day 3, bulls were placed with the heifers for a mean (SD) total duration of 80 10 days. The number of bulls required for each management group was calculated assuming that 50% of the heifers would conceive to first service and that those not conceiving would return to estrus over a 5-day period on average 21 days later. Sufficient bulls were introduced such that no more than three services per bull per day would occur on average.
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Pregnancy diagnosis was conducted in all herds (mean SD) 69 2 days and 109 5 days after the start of the breeding program. In seven herds, additional pregnancy diagnosis took place 139 6 days after the start of the breeding program. The stage of gestation, in days, was estimated at each examination if a heifer was diagnosed pregnant. The date of birth and breed of individual heifers was retrieved from an electronic database (LIC, Hamilton, New Zealand). These data were used to calculate the age at the start of the breeding program (mean SD ¼ 14.3 0.4 months), and to classify heifers as Friesian (N ¼ 575; 51%), Jersey (N ¼ 159; 14%), or other (N ¼ 403; 35%). 2.2. Definitions and statistical methods There were six primary outcome variables in this study. The in-calf to AI rate was defined as the proportion of enrolled heifers that conceived to AI. The conception rate to AI was defined as the proportion of heifers conceiving to AI and was calculated only for heifers that were submitted for AI between Days 0 and 3. It was possible that some heifers in the Double PG group might have conceived to the first natural mating after the introduction of bulls, but natural matings were not recorded, and so these heifers were excluded from this analysis. The 21-day and 42-day in-calf rates were defined as the proportion of enrolled heifers pregnant at 21 or 42 days from the start of the breeding program, respectively. The final in-calf rate was defined as the proportion of heifers present at the final pregnancy test that were pregnant at the final pregnancy test. Finally, the median interval in days from the start of the breeding program to conception was calculated by survival analysis methods. For heifers in the GPG þ P4 group, the reproductive performance measures were calculated using the first day of AI as the start point (Day 1 in the study), so as not to bias the results for this group compared with those in the other two groups that started 1 day earlier (Day 0 in the study). Potential predictor variables evaluated were treatment group, herd of origin, age in months at the start of breeding (categorized into quartiles), pubertal status at commencement of the study, breed category, observation of estrus, and BCS (categorized into quartiles). Balance of treatment groups for age, breed, pubertal status, and BCS of heifers was compared using chi-square tests. Differences between treatment groups in the categorical outcome variables were compared using two-sided Pearson chi-square test with Holm adjustment for multiple testing [21] to control the overall type I error rate. Multivariable logistic regression using a forward stepwise addition method was used to model the effect of treatment group, and other variables associated by univariate analysis (P < 0.2), on the outcomes. Treatment group and herd of origin were fitted as fixed effects and forced into each model. Other variables were tested for significance and as potential confounders and included if their Wald test probability value was P < 0.05, or if their inclusion changed the coefficients for the treatment effects by 15% or more. Interactions between treatment groups and other significant variables were also tested. Preliminary final models
were checked by plotting the deviance residuals against the fitted values and the Cook statistics against the standardized leverages, and if no values were of concern, were declared final. The mean effects of the other nontreatment variables in the final models were also estimated to describe the change in outcome (on a percentage scale) associated with that variable. The noninferiority hypothesis was tested using differences between the means and the confidence intervals of the differences estimated from the final model, with the other included variables set to their median value. The effect on the time to conception of treatment group and potential confounding variables were tested by Kaplan–Meier survival analysis using the log-rank test, and survival curves plotted. A variable of secondary interest was pubertal status as defined by plasma P4 concentrations. Associations were evaluated between pubertal status and herd of origin, breed, and age and BCS at Day 13 using chi-square analysis with adjustment for multiple testing, as previously described. The pubertal status (post vs. prepubertal) of heifers was modeled by multiple logistic regression models and effects of significant variables estimated as previously described. 2.3. Power statistics The first hypothesis was that more heifers would conceive to AI and be pregnant by Day 21 of the breeding program after a GPG þ P4 program than after Double PG. It was assumed that submission rates would be 70% and 100%, and conception to AI rates would be 60% and 55%, for the Double PG and GPG þ P4 groups, respectively. Thus it was expected that approximately 42% and 55% of heifers would be pregnant to AI in the Double PG and GPG þ P4 groups, respectively. To detect this 13% difference, approximately 250 heifers per treatment group were required (a ¼ 0.05; a ¼ 0.2). To allow for loss to follow-up, approximately 300 heifers per treatment group were enrolled. The second hypothesis was that pregnancy rates by Day 21 would be noninferior for the Cosynch þ P4 compared with the GPG þ P4 group. It was assumed that the conception rate of the two groups would be the same (55%) and an acceptable difference between the two groups would be 10% (a ¼ 0.05; b ¼ 0.2). This required 420 heifers per treatment group (Pass 2008; www.ncss.com). 2.4. Partial budget A partial budget model was prepared to estimate the economic effect of synchronization of estrus of heifers using the GPG þ P4 or Cosynch þ P4 programs compared with the Double PG program. The model was built using input data and distributions from the current study, other published data, or the opinions of the authors (Table 1), and the method of stochastic Monte Carlo simulation in the Microsoft Excel add-in @Risk (Version 5; Palisade Corporation, Ithaca, NY, USA). This simulation modeling takes account of the variability of model inputs by assigning each a specific distribution, and generates a distribution for the outcomes from which mean economic effects are estimated.
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Table 1 Assumptions used to develop a stochastic partial budget model (in New Zealand $) in the Microsoft Excel add-in @Risk to compare the economic benefit of estrus synchrony with artificial breeding of pasture-grazed dairy heifers using either GPG þ P4a or Cosynch þ P4b with a Double PGc treatment program. Income variables
Mean
Distribution
Extra days in milk GPG þ P4d Extra days in milk Cosynch þ P4d Mean milk production per day (kg milk solidse) Milk payment next season ($ per kg milk solidse) Percentage in calf to AI, Double PG Percentage in calf to AI, GPG þ P4 Percentage in calf to AI, Cosynch þ P4 Percentage calves female Extra AI heifer calves, GPG þ P4d Extra AI heifer calves, Cosynch þ P4d Male calf value AI calf value (4 days of age)
3.9 6.4 1.2 5.50 31% 47% 57% 50% 8% 13% $20.00 $200.00
RiskNormal(3.9, 1.5) RiskNormal(6.4, 1.5) RiskNormal(1.2, 0.12) RiskUniform(5.65, 5.75) RiskLogistic(0.31, 0.0136) RiskLogistic(0.47, 0.0136) RiskLogistic(0.57, 0.0135) RiskBinomial(100, 0.5)
Cost variables
RiskUniform(15, 25) RiskUniform(150, 250)
Mean
Drug and treatment costs per head Drug costs, Double PG Drug costs, GPG + P4 Drug costs, Cosynch + P4 Veterinary fees, Double PG Veterinary fees, GPG + P4 Veterinary fees, Cosynch + P4
$7.36 $40.00 $40.00 $6.00 $11.00 $11.00
Overhead costs per head
Mean
Average number of heifers per group Number of heifer yardings, Double PG Number of heifer yardings, GPG þ P4 Number of heifer yardings, Cosynch þ P4 Grazier yarding fees Percentage of graziers charging yarding fees Visit and yarding fees plus travel, Double PG Visit and yarding fees plus travel, GPG þ P4 Visit and yarding fees plus travel, Cosynch þ P4 Difference in overhead costs per head, GPG þ P4d Difference in overhead costs per head Cosynch þ P4d
50 5 4 3 $50.00 25% $222.50 $290.00 $277.50 $1.35 $1.10
Distribution
RiskUniform(25, 75) RiskTriang(0.2, 0.25, 0.3)
Distribution is the @Risk formula used to model variability; otherwise the mean value was used. Income variables are shown. a PGF2 on Days 13 and 2 with AI on detection of estrus from Day 0 to 3 of the breeding program. b GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. c GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 0. d Compared with Double PG. e Milk solids ¼ kg of protein plus kg of fat.
In seasonal calving systems with a fixed start and end of lactation, the maximum number of days in milk occurs when an animal calves at the start of the calving program (having conceived at the start of the breeding program) and each day of delay to conception results in one day less in milk. Hence, the additional number of days in milk in the lactation after synchrony for the GPG þ P4 or Cosynch þ P4 heifers, relative to the Double PG heifers, was modeled by subtracting the interval from the start of breeding to the date of conception from the duration of the breeding program for each heifer, and including the mean and SEM of the differences between treatment groups in the model. The economic benefits were assessed in terms of extra milk production, calculated from the estimated value of milk solids (kg of milk protein plus kg of milk fat; the basis of payment in the New Zealand dairy industry), mean daily production of milk solids, and extra days in milk, plus the value of additional AI calves, calculated from the estimated percentage of extra female AI calves born and the value of these calves at 4 days of age. Additional costs were
calculated from estimated treatment costs based on drug costs and vet fees, plus management costs because of yarding of heifers. The means and 95% confidence intervals of the distributions of the economic benefit of each comparison were reported and the treatment comparison declared significantly different if the 95% confidence interval of the difference did not include zero. Spearman rank correlation coefficients were examined to determine the variables with greatest influence on the net economic benefit. Data were managed in a custom-built Microsoft Access database (Microsoft, Redmond, WA, USA), and analysis conducted using R (Version 2.8.1; R Development Core Team, 2008). Statistical significance was declared for test results with P < 0.05. 3. Results Nine heifers were removed from the analysis because they were infertile (freemartins, N ¼ 6), pregnant and
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aborted within 1 day after treatment with PGF2a (N ¼ 1), or were missing at the final treatment with PGF2a (N ¼ 2), leaving data for analysis from 1137 heifers. Six heifers were not present for the final pregnancy diagnosis and were not included in results for the final in-calf rate. Mean BCS of heifers at enrolment was 4.6, with 90% in the range of 4.0 to 5.5 (inclusive). Paired plasma samples were available from 1123 heifers; 671 heifers (60%) had plasma P4 concentrations 1.0 ng/mL in one or both samples. There were no differences between treatment groups in age category (P ¼ 0.71), breed category (P ¼ 0.81), pubertal status (P ¼ 0.87), or BCS category (P ¼ 0.92). 3.1. Detection in estrus A total of 248 of 380 heifers (65%) in the Double PG group were detected in estrus by farmers and submitted for AI over Days 0 to 3. The percentage of heifers detected in estrus in this group was greater for those that were postcompared with prepubertal (87.3% vs. 34.2%; P < 0.01). On Day 0, 107 of 380 (28%), 183 of 383 (48%), and 178 of 374 (48%) of heifers were observed at the time of yarding to have their tail paint disturbed for the Double PG, GPG þ P4 and Cosynch þ P4 groups, respectively (P < 0.001). 3.2. Conception to AI Results for the in-calf to AI rate and conception rate to AI for the three treatment groups are summarized (Table 2). For both outcomes, heifers in the Cosynch þ P4 group achieved higher values than those in the GPG þ P4 group (P < 0.05). In-calf to AI rate was also higher (P < 0.01) and conception to AI tended to be higher (P ¼ 0.069) for heifers in the Cosynch þ P4 group than for heifers in the Double PG group. The Cosynch þ P4 group was declared noninferior to the GPG þ P4 group because the lower 95% confidence interval of the difference in conception rate to AI was greater than 0.10, the a priori defined acceptable difference (difference (Coynsch þ P4) (GPG þ P4) ¼ 0.11; 95% confidence interval, 0.02–0.19). At the univariate level, the in-calf rate to AI
varied among herds and was higher in pubertal compared with prepubertal heifers (53% vs. 47%; P ¼ 0.05), but only treatment group and herd were significant in the final model. Conception rate to AI also varied among herds (range, 37%–63%) and was higher in heifers from which tail paint was removed 1 to 48 hours before AI compared with 0 to 48 hours after AI (266/497 [53.5%] vs. 73/186 [39.2%]; P < 0.001). 3.3. Pregnancy rates Results for the 21-day, 42-day, and final in-calf rates for the three treatment groups are summarized (Table 2). Heifers in the Cosynch þ P4 group had higher 21- and 42-day in-calf rates than heifers in the Double PG group (P < 0.05), but did not differ from those in the GPG þ P4 group. The 21-day in-calf rate varied among herds (range, 53%–80%). In the final models, heifers that had reached puberty before breeding, compared with those that had not, had higher in-calf rates at 21 days (74% vs. 64%; P < 0.001) and 42 days (91% vs. 84%; P ¼ 0.03). The final in-calf rates were not different between treatment groups, but varied among herds. For all final models, there were no significant herd by treatment interactions or pubertal status by treatment interactions. Survival curves for the interval from the start of the breeding program to conception differed between the treatment groups (P < 0.001; Fig. 1). The median interval from the start of breeding to conception was shorter for the heifers in the Cosynch þ P4 than the GPG þ P4 and Double PG groups (P < 0.05), and tended to be shorter for heifers in the Cosynch þ P4 group than the GPG þ P4 group (P ¼ 0.06; Table 2). 3.4. Pubertal status Overall, 671 of 1123 heifers (60%) were categorized as postpubertal, but there was considerable variation among herd of origin (range, 9%–93%). Factors associated with pubertal status were herd of origin (P < 0.001), breed category (P < 0.001), age (P ¼ 0.002), and BCS at the start of the breeding program (P < 0.001; Table 3).
Table 2 Number (%) of dairy heifers conceiving to AI or pregnant after three treatment protocols for synchronization of estrous and ovulation. Treatment group
In-calf to AIa
Conception rate to AIb
21-Day in-calf ratec
42-Day in-calf rated
Final in-calf ratee
Interval to conception (days)f
Double PGg GPG þ P4j Cosynch þ P4k
119/380h (31.3) 180/383i (47.0) 213/374l (57.0)
119/248h,i (48.0) 180/383h (47.0) 213/374i (57.0)
238/380h (62.6) 274/383i (71.5) 284/374i (75.9)
313/380h (82.4) 335/383h,i (87.5) 333/374i (89.0)
347/376 (92.3) 352/381 (92.4) 358/374 (95.7)
19h 14h 0i
a
Proportion of all heifers conceiving to AI. Proportion of heifers artificially bred conceiving to AI. c Proportion of all heifers pregnant after 21 days of the breeding program. d Proportion of all heifers pregnant after 42 days of the breeding program. e Proportion of heifers examined that were pregnant at the end of the breeding program. f Median interval from start of breeding to conception. g PGF2 on Days 13 and 2 with AI on detection of estrus from Day 0 to 3 of the breeding program. h,i, and l Within a column, means without a common superscript differed (P < 0.05). j GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. k GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed-time AI on Day 0. b
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4. Discussion
Fig. 1. Kaplan–Meier survival curves of cumulative time to conception from the start of the breeding program (Day 0) for dairy heifers treated with three synchronization protocols: (1) PGF2a on Days 13 and 2, with AI after detection of estrus between Days 0 and 3 (Double PG); (2) GnRH, PGF2a, and GnRH on Days 9, 2, and 0, respectively, with placement of an intravaginal progesterone (P4)-releasing device between Days 9 and 2, and set time AI on Day 1 (GPG þ P4); or (3) same as the GPG þ P4 group but with the set time AI on Day 0 (Cosynch þ P4). There were differences among treatment groups (P < 0.001).
3.5. Partial budget The Cosynch þ P4 program had an economic benefit compared with the Double PG program, but there was no benefit from the GPG þ P4 program (Table 4). Additional income from greater milk yield because of earlier calving and more AI calves in the Cosynch þ P4 compared with the Double PG program accounted for the difference between the two groups. Costs for the two P4-based treatments were very similar. The variables most highly correlated with the net benefit of the Cosynch þ P4 program were the additional days in milk in the subsequent lactation (rs ¼ 0.83) through earlier conception pattern, daily milk production (rs ¼ 0.35), and value of an artificially-sired calf (rs ¼ 0.3).
Table 3 Percentage of heifers (N ¼ 1123) that were postpubertal, based on progesterone concentrations in two plasma samples, classified by breed, age, and body condition score at the start of the breeding program. Variable
Level
Pubertal (%)
Breed
Friesian Jersey Other 12.2-14.2 14.3 14.5 14.8-15.9 3.0-4.0 4.5 5.0 5.5-7.5
45.1a 86.7b 70.0c 52.5a 59.1a 64.0b,c 65.9b,c 37.9a 66.8b 72.3b 74.3b
Age (mo)
Body condition score
a–c Within an end point, means without a common superscript differed (P < 0.05).
This study demonstrated that synchronization of heifers using a combination of GPG and P4 with FTAI coincident with the final GnRH treatment (Cosynch þ P4) resulted in superior reproductive and economic performance compared with a similar program but with FTAI delayed by approximately 24 hours or to a Double PG program. The conception rate to AI of 57% for heifers in the Cosynch þ P4 group in the current study was comparable with the 53% reported after a previous synchrony program with FTAI in New Zealand [6], and was superior to results for the GPG program in Holstein heifers in the United States when heifers detected in estrus before the final GnRH were inseminated early and overall conception rates were 46% [11]. The 21-day in-calf rate of 76% for the Cosynch þ P4 heifers was also better than the 32-day pregnancy rate of 60% reported for dairy heifers in Florida after an 8-day Cosynch program using P4 inserts from Days 0 to 5 [22]. The higher conception rates to AI in the Cosynch þ P4 group compared with heifers in the GPG þ P4 group in the current study were comparable with the finding of Lima et al. [23], in which a Cosynch þ P4 protocol resulted in improved reproductive outcomes compared with an Ovsynch þ P4 protocol in dairy heifers in Florida. In the current study, heifers observed to have lost tail paint during the 48 hours before AI had higher conception rates to AI than those with loss of tail paint during the 48 hours after AI. The interval from the end of a synchrony program to estrus was affected by the size of the dominant follicle at the end of the program, with a larger dominant follicle associated with a shorter interval to estrus [24]. It is not clear if the shorter interval from the end of the synchrony program to estrus was also associated with a shorter interval to ovulation. After a synchrony program, the interval between onset of estrus and ovulation appeared to be reasonably consistent at 1.3 days in a range of synchrony systems [25]. Thus it is likely that heifers in the current study that were in estrus earlier relative to Day 0, also ovulated sooner. This might partly explain the higher conception rates in the Cosynch þ P4 program compared with the GPG þ P4 program. In the latter program, nearly half of the heifers had been detected in estrus by Day 0, yet by design were not inseminated until 24 hours later. So, for this group of heifers, insemination might have occurred too late for optimum fertility. The interval between estrus and ovulation has been reported as 38.5 3.0 hours (mean SEM) in dairy heifers [26]. Because sperm capacitation takes 4 to 12 hours, logically insemination should occur 12 to 18 hours before ovulation to ensure functional sperm are present at the time of ovulation [27]. In one study, heifers in estrus in the 24 hours preceding FTAI had higher conception rates than those in estrus 25 to 40 hours before FTAI (76% vs. 50%) [28]. In another study, conception rates to FTAI were optimized when insemination occurred 6 to 22 hours before ovulation [29]. Of the heifers in the Double PG group, 65% were detected in estrus between 2 and 6 days after the final treatment with PGF2a. This appeared to be lower than in some previous reports, in which 86% were detected in estrus over 5 days after the final treatment [30]. This lower
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Table 4 Partial budget, in New Zealand dollars per heifer, on the use of two programs for synchronization of estrous compared with a double PGF2 program. Treatment Group
GPG þ P4a Cosynch þ P4b
Additional income
Additional costs
Net benefit
Extra milk production
Value of AI calves
Total
Treatment costs
Management costs
Total
Mean
95% CI
26.68 43.78
14.40 23.40
41.08 67.18
40.36 40.36
1.35 1.10
41.71 41.46
0.65 25.73
21.87 to 21.58 2.99–50.69
Abbreviation: CI, confidence interval. a GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed time AI on Day 1. b GnRH, PGF2 , and GnRH on Days 9, 2, and 0 of the breeding program, respectively, with insertion of an intravaginal progesterone-releasing insert for 7 days commencing on Day 9, and fixed-time AI on Day 0.
detection rate might have been related to lower sensitivity of detection of estrus by the observers, a 1-day shorter period of observation, or because only 60% of heifers in the current study had reached puberty when the program started. Pubertal status had a significant effect on reproductive outcomes in the current study; the percentage of heifers that were postpubertal at the start of the study decreased with lower BCS and younger age at commencement of the program. In a previous study, the risk of conception to first service was not associated with withers height, age, BCS, or body weight, but the proportion of heifers that were prepubertal was not defined and it is likely that most heifers were postpubertal, hence the biophysical measures might have been of lesser importance [31]. In the current study, there were significant effects of herd on all outcome variables, likely associated with the percentage of animals that were prepubertal in each herd. At a practical level, our findings demonstrated that optimal outcomes after synchrony programs will be achieved in heifers that are postpubertal, and as such should be 14.5 months old and have a BCS >4.0. In addition to improved reproductive performance, the Cosynch þ P4 program had a significant economic benefit compared with the Double PG program. One potential benefit of use of AI in heifers is a shortening of the generation interval with an associated faster rate of genetic gain. In a previous assessment of the economic benefits of artificially inseminating heifers with or without synchrony, the optimal economic decision was to select high genetic merit heifers and undertake AI on detection of estrus [1]. However, in that analysis, the main benefit modeled was the value of the improved genetics of the calves, and it ignored any potential benefits that synchrony might have in terms of earlier calving of the heifers with resultant increased lactation length. The current study analyzed the returns in terms of extra value associated with AI-sired calves, and additional production in the first lactation. The economic benefit of the Cosynch þ P4 program was because of the 100% submission rate and the higher conception rate that resulted in more AI-sired calves and earlier calving, and thus a longer first lactation length compared with the other programs. Although the Double PG program was less expensive, the poorer pregnancy rates resulted in poorer economic outcomes relative to the Cosynch þ P4 treatment. In populations of heifers with fewer prepubertal animals, the Double PG system might be more effective and less economically disadvantaged.
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