Reproductive performance in anestrous dairy cows following treatment with two protocols and two doses of progesterone

Theriogenology 63 (2005) 1529–1548 www.journals.elsevierhealth.com/periodicals/the

Reproductive performance in anestrous dairy cows following treatment with two protocols and two doses of progesterone S. McDougalla,*, C.W.R. Comptona, D.W. Hanlonb, P.J. Davidsonb, D.J. Sullivana, A.H. Gorea, F.M. Annissa b

a Animal Health Centre, P.O. Box 21, Morrinsville, New Zealand Matamata Veterinary Services, 26 Tainui St., Matamata, New Zealand

Received 8 August 2003; accepted 30 March 2004

Abstract The objectives of this study were to evaluate the effect using two doses of progesterone (P4) releasing devices in two different programs on reproductive performance of anestrous dairy cows. Cows (n = 1555) not detected in estrus by 10 d before the planned start of the seasonal breeding program and in which no CL was palpable were treated with an intravaginal P4-releasing device (‘Single’; 1.56 g of P4) or a modified device with triple the normal P4 dose (‘Triple’; 4.7 g of P4). The devices were in place for either 6 d (‘Short’) or 8 d (‘Long’), with 1 mg estradiol benzoate (EB) given 24 h after device removal. The ‘Long’ program also included treatment with 2 mg EB at device insertion. The Long program resulted in a higher first service conception rate (RR = 1.18 (95% CI = 1.03–1.33); P = 0.02), but had no effect on the 28-d, 56-d or final pregnancy rate compared to the Short program. There were no effects of dose of P4 on any outcome. In conclusion, the Long compared to the Short program, but not the dose of P4, improved first service conception rates in anestrous cows. # 2004 Elsevier Inc. All rights reserved. Keywords: Anestrus; Progesterone; Dairy cattle; Conception rate

* Corresponding author. Tel.: +64 7 889 5159; fax: +64 7 889 3681. E-mail address: [email protected] (S. McDougall). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.03.024

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1. Introduction Anovulatory anestrus is a reproductive problem within the New Zealand dairy industry with 20% of cows not detected in estrus by the start of the seasonal breeding program [1]. Anestrus is associated with lower insemination, conception and pregnancy rates as well as a greater probability of being culled for failure to conceive [1]. The present treatment of anestrus is 5–6 d of intravaginal progesterone (P4) treatment followed 1–2 d later by injection of 0.5–1 mg of estradiol benzoate im (EB) [1–3] which results in approximately 90% of cows undergoing estrus and ovulation and 45% of the inseminated cows conceiving to the first service [1]. However, this conception rate is lower than for cycling herd mates that have an average conception rate of 55% [4]. One reason for this lower conception rate may be insufficient duration or concentration of P4 ‘priming’ following treatment with the currently available intravaginal P4-releasing devices. Peak luteal P4 concentrations increase with sequential luteal phases postpartum [5]. The conception rate to insemination also increases with the number of estrous cycles postpartum [6,7] and with time postpartum [1]. A positive correlation between serum P4 concentration in the previous diestrous phase and the probability of conception has been demonstrated [8–11]. The P4 concentrations in the preceding luteal phase may influence subsequent conception rate either by altering follicle wave dynamics or by effects on the conceptus or uterus. In cycling cows, low plasma P4 concentrations associated with exogenous P4 treatments result in ineffective suppression of LH release, development of abnormally large and persistent dominant follicles, high estradiol production, premature oocyte activation, and depression of subsequent conception rates [12–14]. In contrast, if small follicles are ovulated, the resultant conception rate is lower and plasma P4 concentration in the subsequent luteal phase reduced [15]. Progesterone also has a direct role in modulating uterine secretion, essential for early conceptus development [16]. Progesterone enhances the ability of the fetus to elongate and to produce interferon t, the fetal signal essential for pregnancy recognition, and P4 restricts oxytocin receptor formation in the endometrium, an essential precursor for luteolysis [17]. The oviducts from cows with a persistent dominant follicle have different biosynthetic activity than oviducts from cows without a persistent dominant follicles; this may also affect fertility [18]. Hence, there is evidence that P4 concentrations in the previous luteal phase can affect follicle development and subsequent fertility and that P4 concentration in the luteal phase following insemination may also affect probability of conceptus survival. Plasma or serum P4 concentrations can be increased by increasing the dose of exogenous P4 delivered. For example, increasing the dose of P4 moulded into an intravaginal P4-releasing device or increasing the number of devices inserted results in a curvilinear increase in plasma P4 concentrations [19,20]. Cows not detected in estrus but found to have ovulated (i.e. a corpus luteum (CL) is detected upon palpation) have poorer reproductive performance than herdmates detected in estrus before the start of the seasonal mating program [21]. Treatment of these animals with various combinations of P4, EB and PG has resulted in inconstant responses compared to no treatment [3,22,23].

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It was hypothesized that increasing the P4 dose, or modifying the program by extending the duration of the P4 treatment and adding EB at device insertion, would enhance the first service conception rate in anestrus dairy cattle.

2. Materials and methods Cows (n = 1883 from 17 dairy herds) not detected in estrus but calved >30 d at the planned start of the seasonal breeding program were presented for veterinary examination and assessment of body condition score (on a 1–10 scale; [24]) 10 d before the start of the breeding program. The average interval from calving to the start of the breeding program was 64 d (range 32–121 d). Cows without a corpus luteum and without uterine or ovarian pathology upon rectal palpation (n = 1555; 83% of not detected in estrus cows) were defined as anestrus and assigned to treatment in a 2  2 factorial arrangement. The four treatments were randomly applied with each sequential four cows presented for examination. The treatments were either a Single or Triple P4-releasing device for a period of either 6 or 8 d. The P4-releasing device was a commercially available device (Cue-Mate Cattle Device; Pfizer Animal Health, Auckland, New Zealand). The device consists of a reusable nylon ‘wishbone’ onto which are threaded, two silastic, P4-impregnated ‘pods’ containing a total of 1.56 g of P4. The device was modified by cutting off the closed end of four ‘pods’ and threading two of each of these onto each ‘arm’ of the wishbone before the addition of two intact ‘pods’ to hold the ‘pods’ in place on the wishbone. Thus a total of  4.7 g of P4 was incorporated into the Triple P4-releasing device. Modification of the P4-releasing device resulted in an increase from 1.2  0.2 to 2.9  0.2 ng/mL average (S.E.M.) plasma P4 concentrations over 8 d of insertion (P < 0.01; McDougall et al., unpublished). The Long treatment also included 2 mg EB im at device insertion and all cows were treated with 1 mg of EB im, 24 h after device removal. The insertion of the P4-releaseing devices for the Short treatment occurred 2 d after the Long treatment so that the final EB treatment occurred on the same day (Day 1) for all cows. Day 0 was the start of the breeding program. Those cows not detected in estrus but found to have a CL upon veterinary examination were defined as cycling and alternately assigned at presentation to be treated either with a Single or Triple P4-releasing device for a period of 8 d with injection of 2 mg EB im at device insertion and 1 mg IM, 24 h after device removal. Cows that were detected in estrus for an average 36 d (range = 23–51 among herds) after the start of the breeding program were submitted for artificial insemination (AI). Following this, bulls were placed with the cows in each herd for a further 63 d (range = 39–93 among herds) so that the average herd breeding period was 99 d (range = 69–142 d). Every cow was pregnancy tested by transrectal ultrasonography (Aloka 500 with a 5 MHz linear array transducer; Medtel, Auckland New Zealand) approximately 10 weeks after the commencement of the seasonal breeding program and again 6 weeks after bulls were finally removed from the herd. The stage of gestation was estimated on the first occasion that pregnancy was detected for each cow and from these data conception dates were estimated. Where this estimated conception date was within 7 d of a recorded AI or

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herdowner-recorded bull breeding date, the recorded date was used as the conception date for further calculations. If the estimated conception date was >7 d after the last recorded breeding date, the estimated date was used for subsequent calculations. Only one of the 17 herds did not routinely record milk production and composition every 4–8 weeks. The production data closest to the start of the seasonal breeding program was used for analysis. This was on average 12 d before the start of the breeding program (range 30 d before to 42 d after start of the breeding program among herds). Data collected for each enrolled cow included herd, breed (coded as Friesian (F) if >11/ 16th Friesian, Jersey (J) if >11/16th Jersey or crossbred (XB; all others)), calving date, age (coded as 2, 3, 4–5 and >5 years), milk volume, milk protein percentage, milk fat percentage, date of the first herd test of the season, body condition score at the time of initial examination, every AI and bull breeding date, culling reasons and date (if applicable), and estimated conception date. Data were retrieved from the national data base via the DairyWin or MindaPro programs (Livestock Improvement Corporation, Hamilton, New Zealand) and from herdowner or veterinary practice records. Blood samples were drawn from the tail vein of seven or eight cows randomly selected from each group (i.e. Short Single P4, Short Triple P4, Long Single P4 and Long Triple P4) within each of four herds on Day 13. Samples were drawn into evacuated glass tubes containing lithium heparin anti-coagulant (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA), placed on ice and centrifuged at 1000  g within 4 h of collection. The plasma was stored at 20 8C before analysis. Plasma P4 concentrations were determined using a commercial I125 labeled RIA (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA, USA). Samples were analyzed within one assay and the coefficients of variation were 19.8, 5.7 and 6.4% for quality control sera (n = 8) with mean P4 concentrations of 0.5, 3.0 and 4.2 ng/mL, respectively. The minimum detectable concentration of the assay was 0.09 ng/mL. 2.1. Statistical analyses The specific hypotheses being tested were: (i) that the Triple P4-releasing device would increase first service conception rate compared to the Single P4-releasing device; and (ii) that Long program would increase first service conception rate compared to the Short program. Additionally, a number of other risk factors for reproductive performance were examined including milk composition and body condition score. A number of cows (n = 84) were lost to follow-up or excluded. Exclusion criteria included pregnancy at initial examination (n = 1), death prior to initial pregnancy diagnosis (n = 3), detection in estrus between initial examination and insertion of the Short P4releasing device (n = 18), the loss of the P4-releasing device before due day of removal (n = 53) or not being presented for EB treatment (n = 9). Cows that died or were culled between the start of the breeding program and the first pregnancy diagnosis were only included in the submission rate analysis. The outcome variables examined were:  Submission rate by Day 7 (% of cows; i.e. number of cows inseminated within 7 d of the start of the breeding program/number of enrolled cows),

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 Submission rate by Day 28 (% of cows; i.e. number of cows inseminated within 28 d of the start of the breeding program/number of enrolled cows),  Conception rate to first service (% inseminations; number of cows conceiving within 7 d of the start of the breeding program/number of enrolled cows inseminated within 7 d of start of the breeding program),  Pregnancy rate by Day 28 (% of cows; number of cows conceiving within 28 d of the start of the breeding program/number of enrolled cows),  Pregnancy rate by Day 56 (% of cows; number of cows conceiving within 56 d of the start of the breeding program/number of enrolled cows),  Final pregnancy rate (% of cows; number of cows conceiving/number of enrolled cows), and the  Interval from the start of the breeding program to conception (days; interval from start of the breeding program to conception). The independent (explanatory) variables included:               

Dose of P4 (Categorical; coded as ‘Single’ P4 (reference category) and ‘Triple’ P4), ‘‘Duration’’ of P4 (Categorical; coded as ‘Short’ (reference category) and ‘Long’), Postpartum interval (PPI) (Continuous as days), Postpartum interval (PPI; days) (Categorical ordinal, coded as >76 d = Early, 64–76 d = Early–mid, 50–63 d = Mid–Late, <50 d = Late), Postpartum interval (PPI; d) (Categorical ordinal, coded as 50 d = Early, <50 d = Late), Milk fat % at first herd test (Continuous as a %), Milk volume at first herd test (Continuous as L/cow/d), Protein % at first herd test (Continuous as a %), Protein % at first herd test (Categorical ordinal, coded as >3.73% = High, 3.52–3.73% = Med–High, 3.33–3.51% = Med–Low and <3.33% = Low), Protein % at first herd test (Categorical ordinal, coded as 3.76% = High, <3.76% = Low), Fat to protein ratio (Continuous), Fat to protein ratio (Categorical ordinal, coded as <1.14 = Low, 1.14–1.22 = Med–Low, 1.23–1.34 = Med–High, >1.34 = High), Body condition score (Categorical ordinal, coded as 4 = High, <4 = Low), Breed code (Categorical, coded as Friesian = 1, Crossbred = 2, Jersey = 3), Age code (years; Categorical ordinal, coded as 2 = 2, 3 = 3, 4–5 = 4, >5 = 5).

The lowest value within a categorical variable is the reference category unless stated otherwise. For anestrus cows, logistic regression was performed for each outcome variable (except final pregnancy rate and the start of the breeding program to conception interval). The independent (explanatory) variables included dose (i.e. Single P4 versus Triple P4), program (i.e. Short versus Long), herd, age code, breed, body condition score at the time of enrolment, milk fat to protein ratio, milk protein percentage and postpartum interval.

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Initially, all explanatory variables were screened by univariate analyses (x2 or logistic regression), and then variables associated (P < 0.25) were offered to forward and reverse stepwise logistic regression models using likelihood ratio for variable inclusion/exclusion. The main effects of dose and program were always included in the models, even if not significant. The dose by program interaction was explicitly tested for within each analysis. It was only significant in the Day 56 pregnancy rate model. The validity of the models was assessed using the Hosmer–Lemeshow test and counting the list of outliers (i.e. values >2 standard deviations from expected) to ensure that <5% of values were outliers. The results from the logistic regression analyses were converted from odds ratios to relative risks (RR) using the methodology of Zhang and Kai [25] as the adjusted odds ratio derived from the logistic regression is not a good approximation of relative risk when the outcome of interest (pregnancy in this study) is not rare. A relative risk of >1 and <1 indicates that the risk factor increases or decreases the risk of conception or pregnancy, respectively. Every enrolled cow in two herds finally conceived (Herds 1 and 6) resulting in unstable coefficient estimates with attempts to model the final pregnancy rate of anestrus cows using logistic regression. Consequently, the final pregnancy rate was analyzed using the Mantel– Haenszel technique independently for each of the main effects listed above stratified by herd. This was done as herd was significant in an initial x2 analysis and regarded as a potentially confounding factor. The median interval from the start of the breeding program to conception for each treatment was estimated using Kaplan–Meir survival analysis. The probability of conception was formally tested using forward stepwise Cox’s proportional hazards survival analysis with the same independent variables as above. The data are presented as median days, standard error and 95% confidence intervals (95% CI) (from the Kaplan–Meir analysis) and the hazards ratio (HR) with P-values derived from the final Cox’s model. The validity of the Cox’s model was evaluated by visualizing the Log–Log hazard versus time plots to ensure that the hazards assumption was not violated. The partial residuals and the coefficients with and without each case (i.e. the DfBeta’s) were plotted and no violations of proportional hazards assumptions or extreme outliers were detected. The numbers of not detected in estrus but CL-positive cows enrolled was low. Attempts to model reproductive outcomes using both logistic regression and Mantel–Haenszel techniques resulted in unstable models and a failure in convergence in logistic regression models. Data for these cows were presented as raw data and relative risks from the x2 analysis with dose as the explanatory variable. The Day 13 plasma P4 concentration was analyzed in a general linear model with the main effects of dose (Single P4 versus Triple P4) and program (Short versus Long) as fixed effects, with herd, age code (2, 3, 4–5, and >5 years) and conception to first service within 7 d of the start of the breeding program initially included as random effects and with milk fat percentage, milk protein percentage and fat to protein ratio initially included as covariates. Initially, univariate (i.e. one-way ANOVA and linear regression) analysis was used to analyze the relationship between plasma P4 and the effect in question. Those that were found to be associated (i.e. P < 0.2) and those thought important in the design (i.e. herd, P4 dose and program and conception to first service) were initially included in the model. A manual, backward model building process was used and effects not significant (i.e. P > 0.1) were removed. All first order interactions of remaining effects were tested and

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removed if not significant (P > 0.1). Plasma P4 data are presented as least square means and the standard errors of the difference (S.E.D.). The P4 concentration was arbitrarily defined as low and high for concentrations of <1 ng/mL and >1 ng/mL, respectively. The proportion of cows with low P4 for each dose (Single P4 versus Triple P4) and program (Short versus Long) of treatment as well as conceiving or not conceiving to first insemination was examined using x2. The program SPSS (v11.5; SPSS Inc., Chicago, IL, USA) was used for data analysis.

3. Results A total of 1883 cows were initially presented as not detected in estrus (Table 1). The level of prevalence of cows not detected in estrus averaged 31.2% (range 9.1–52.5% among herds; Table 1). 3.1. Reproductive performance of anestrus cows 3.1.1. 7-d submission rate There was no effect of program or dose of P4 on the 7-d submission rate (Tables 2 and 3). The 7-d submission rate varied among herds (average = 90.4%, range = 74.0–98.7%, P < 0.01) and was lower in cows with high (4) body condition score compared to cows in low (<4) body score (Table 3).

Table 1 Descriptive data for enrolled herds Herd code

Total cows (n)

Enrolled cows (n)

Not detected in estrus (%)a

Anestrus (%)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

340 372 467 523 370 179 253 431 360 160 142 195 469 386 638 343 510

31 80 122 157 103 39 59 182 88 84 67 82 167 101 297 105 119

9.1 21.5 26.1 30.0 27.8 21.8 23.3 42.2 24.4 52.5 47.2 42.1 35.6 26.2 46.6 30.6 23.3

93.5 75.0 86.1 80.3 95.1 69.2 71.2 84.6 94.3 82.1 80.6 92.7 90.4 79.2 67.3 86.7 92.4

a

Percentage of all cows in the herd not detected in estrus. Percentage of not detected in oestrus cows diagnosed as anestrus (i.e. no CL was detected upon palpation of the ovaries). b

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Table 2 Submission (Sub), conception (Con) and pregnancy (Preg) rates (number and %) for anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program) Outcome

Dose

Program

Single

Sub D7 Con S1 Sub D28 Preg D28 Preg D56 Preg Final PSB-cona

Triple

Short

Long

No.

%

No.

%

No.

%

No.

%

707/784 273/707 757/784 467/784 605/753 615/667 21

90.2 38.6 96.6 59.6 80.3 92.2 20–22

700/771 277/700 742/771 450/771 589/726 598/648 21

90.8 39.6 96.2 58.4 81.1 92.3 20–22

699/777 248/699 743/777 447/777 590/739 605/654 21

90.0 35.5 95.6 57.5 79.8 92.5 20–22

708/778 302/708 756/778 470/778 604/740 608/661 20

91.0 42.7 97.2 60.4 81.6 92.0 19–21

a Median interval (and 95% confidence intervals) of the days from the planned start of the seasonal breeding program (PSB) to conception from Kaplan–Meier analysis.

Table 3 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and Pvalue for the final model of 7-d submission rate of anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program) RR P4 dosea Programb BCS 4c a b c

1.01 1.01 0.92

95% CI

P

LCL

UCL

0.97 0.98 0.83

1.03 1.04 0.99

0.66 0.44 0.001

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: BCS <4.

Table 4 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and Pvalues for the final model of first service conception rate for anestrus cows treated with a Single or Triple P4releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program) Category P4 dosea Programb

RR

95% CI

P

LCL

UCL

1.00 1.18

0.85 1.03

1.17 1.33

0.95 0.023

Age group (years)c

2 3 4+5

1.51 1.37 1.52

1.23 1.08 1.22

1.82 1.69 1.83

0.000 0.006 0.000

Fat to protein ratiod

Low Med–low Med–high

1.16 0.99 1.25

0.92 0.77 1.01

1.42 1.23 1.49

0.204 0.902 0.039

a b c d

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: age >5 years. Reference category: milk fat to protein ratio ‘high’ (i.e. >1.34).

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Table 5 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and Pvalues for the final model of 28-d submission rate for anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program) Category P4 dosea Programb Age group (years)c

a b c

3 4+5 >5

RR

95% CI

P

LCL

UCL

1.011 0.997

0.997 0.969

1.018 1.015

0.80 0.11

1.045 1.025 1.034

1.016 0.987 1.005

1.056 1.044 1.048

0.010 0.15 0.027

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: age 2 years.

3.1.2. Conception rate to first service The first service conception rate was higher following the Long than Short program (P = 0.02) but was not affected by the dose of P4 (P = 0.95; Tables 2 and 4). First service conception rate tended to vary among herds (average = 38.8%, range = 26.3–55.4%, P = 0.08), was higher in 2, 3 and 4 to 5-year-old cows relative to cows >5 years (P < 0.001; Table 4). Medium–high fat to protein ratios resulted in higher first service conception rates than high fat to protein ratios (P = 0.04; Table 4). 3.1.3. 28-d submission rate There was no effect of program (P = 0.11) or dose (P = 0.80; Tables 2 and 5) of P4 on the 28-d submission rate. The 28-d submission rate varied among herds (average = 95.7%, range = 81.5–100.0%; P < 0.01) and was higher in 3-year-old and >5-year-old cows compared to 2-year-old cows (Table 5). 3.1.4. 28-d pregnancy rate The 28-d pregnancy rate was unaffected by either the program (P = 0.31) or dose (P = 0.60) of P4 (Tables 2 and 6). Herds varied in 28-d pregnancy rate (average = 59.0%, Table 6 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and Pvalues for the final model of 28-d pregnancy rate for anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program)

P4 dosea Programb BCSc a b c

RR

95% CI LCL

UCL

1.025 1.049 0.828

0.931 0.954 0.719

1.115 1.139 0.938

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: High (4) body condition score.

P

0.60 0.31 0.002

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Table 7 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and P-values for 56-d pregnancy rate for anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program) Category P4 dosea Programb

RR

95% CI

P

LCL

UCL

0.986 1.010

0.926 0.949

1.038 1.060

0.63 0.74

Breedc

Friesian Crossbred

1.128 1.160

0.964 1.024

1.213 1.228

0.107 0.027

Calving to start of breedingd

<50 d 50–63 d 64–76 d

0.892 0.961 1.005

0.791 0.870 0.925

0.976 1.031 1.064

0.008 0.305 0.899

1.086 1.084

1.025 1.016

1.133 1.135

0.008 0.019

BCSe Milk protein %f a b c d e f

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: Jersey. Rerence category: calved >77 d at the start of the breeding program. Reference category: Low (<4) body condition score. Reference category: Low (<3.76) % milk protein.

range = 35.2–86.2%; P < 0.001). The 28-d pregnancy rate increased with increasing interval from calving to start of the breeding program (P < 0.001) and with the protein percentage of milk at herd test (P < 0.01), but decreased in cows with low versus high body condition score (P < 0.01; Table 6). 3.1.5. 56-d pregnancy rate The 56-d pregnancy rate was unaffected by either the program (P = 0.74) or dose (P = 0.63; Tables 2 and 7) of P4. However, there was a dose by program interaction (P = 0.04), whereby the Triple P4-Long group had a higher 56-d pregnancy rate than the Single P4Long group (P = 0.06), while there was no difference (P = 0.2) between the Single or Triple P4-Short groups (81.7, 77.9, 79.0, and 84.3% 56-d pregnancy rates for Single P4-Short, Triple P4-Short, Single P4-Long, Triple P4-Long groups, respectively). Herds varied in 56d pregnancy rate (average = 79.4%, range = 68.5–96.6%; P = 0.01). The 56-d pregnancy rate was lower in cows with short (<50 d) compared with long (>76 d) intervals between calving and the start of the breeding program (Table 7). Crossbred cows had a higher 56-d pregnancy rate than Jersey cows (P = 0.03; Table 7). Cows with a high body condition score had a higher 56-d pregnancy rate than cows in low body condition score (P = 0.01; Table 7). Cows with high milk protein percentage at herd test had a higher 56-d pregnancy rate than cows with low milk protein percentage (P = 0.02; Table 7). 3.1.6. Final pregnancy rate The final pregnancy rate was unaffected by either the program (P = 0.78) or dose (P = 0.89; Tables 2 and 8) of P4. Final pregnancy rate varied among herds (average = 91.9%, range = 83.7–100%; P < 0.05), was higher in cows with high milk protein percentage

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Table 8 Relative risk (RR), 95% confidence intervals (95% CI, lower and upper CI = LCL and UCL, respectively) and P-values for final model of final pregnancy rate for anestrus cows treated with a Single or Triple P4-releasing device (Dose) for either a ‘Short’ or ‘Long’ duration (Program)

P4 dosea Programb Milk protein (%)c Calving to start of breedingd a b c d

RR

95% CI

P

LCL

UCL

1.002 0.995 1.050 0.955

0.966 0.957 1.010 0.896

1.028 1.022 1.068 0.997

0.89 0.78 0.021 0.031

Reference category: Single P4 device. Reference category: Short (6D + EB) program. Reference category: Low (<3.76) % milk protein. Reference category: calved 51 d at the start of the breeding program.

compared to low milk protein percentage (P = 0.02; Table 8) and was lower in late calving cows (P = 0.03; Table 8).

3.1.7. Probability of conception with time (survival analysis) The interval from the start of the seasonal breeding program to conception was unaffected by the program (21  0 versus 20  0 median d (S.E.) from the planned start of the seasonal breeding program to conception for Short versus Long program, respectively; P = 0.56). Dose did not effect the interval from the start of the seasonal breeding program to conception (21  0 versus 21  0 median d (S.E.) from planned start of the seasonal breeding program to conception for Single versus Triple P4, P = 0.77). The interval from the start of the seasonal breeding program to conception varied amongst herds (P < 0.001) and was longer in cows with a short (<51 d) interval from calving to the start of the seasonal breeding program late calving cows (Fig. 1). Cows with high milk

Table 9 Submission (Sub), conception (Con) and pregnancy (Preg) rates (number and %) for cows not detected in estrus by 7 d before the planned start of the seasonal breeding programme which were found to have a corpus luteum upon transrectal ovarian palpation Outcome

P4 dose Single

Sub D7 Con S1 Sub D28 Preg D28 Preg D56 Preg Final

RRa

95% CI

P

1.00 0.96 1.03 1.02 0.96 1.01

0.92–1.08 0.71–1.28 0.99–1.08 0.86–1.21 0.86–1.07 0.95–1.07

0.93 0.76 0.17 0.78 0.43 0.81

Triple

n

%

n

%

141/162 53/141 158/162 101/162 126/162 137/146

87.0 37.6 97.5 62.3 77.8 93.8

145/166 57/145 157/166 101/166 135/166 136/146

87.3 39.3 94.6 60.8 81.3 93.2

Cows were treated with either a Single or Triple P4-releasing device (P4 Dose). a Relative risk and 95% confidence interval around RR estimate.

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Fig. 1. (a) and (b) Median (and upper 95% confidence) interval from the planned start of the seasonal breeding program to conception by quartiles of the interval from calving to the start of the breeding program ((a) upper panel); and by quartiles of the milk protein percentage at first herd test ((b) lower panel). Bars with different superscripts differ at P < 0.05.

protein percentage had a shorter interval from the start of the seasonal breeding program to conception (Fig. 1). 3.2. Reproductive performance of not detected in estrus but CL-positive cows A total of 328 (17.4%) of presented cows were detected with a CL upon examination. The average herd prevalence of these cows was 16.4% but there was a wide range among herds (4.9–32.7%). A total of 162 cows were treated with the Single P4 and 166 with the

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Fig. 2. Frequency histograms of the plasma P4 concentrations (ng/mL) 13 d after the start of the seasonal breeding program for anestrous cows treated with a Short or Long program and using a Single or Triple P4-releasing device. There was no significant difference between the dose of P4 or the program.

Triple P4 dose. There were no significant differences between the Single and the Triple P4 dose for any of the measured reproductive outcomes (Table 9; all P > 0.2). 3.3. Day 13 plasma P4 concentrations The average plasma P4 concentration was 4.3 (S.E. = 0.2) ng/mL (Fig. 2) and the P4 values were normally distributed. The P4 concentration was not affected by either P4 dose (4.69 ng/mL versus 4.18 ng/mL (S.E.D. = 0.34) for Single versus Triple P4, P = 0.14) or program (4.43 ng/mL versus 4.45 ng/mL (S.E.D. = 0.34) for Short versus Long, P = 0.95).

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Fig. 3. Milk protein percentage compared to plasma P4 concentration (ng/mL) 13 d after the start of the seasonal breeding program for anestrous cows treated with a Short or Long program and using a Single or Triple P4-releasing device. The lines are linear regression lines for each herd.

Herds varied in the mean P4 concentration (P < 0.01), P4 concentration was positively associated with milk protein percentage at first herd test (P = 0.003; Fig. 3; univariate R2 = 0.127). The P4 concentration was higher in cows in high body condition score than in lower body condition score (P = 0.05; Fig. 4). Cows conceiving had a higher P4 concentration than those not conceiving to the first insemination (5.2 ng/mL versus

Fig. 4. The estimated marginal mean (and standard errors) plasma P4 concentrations for cows in various body condition scores at 13 d after the start of the seasonal breeding program for anestrous cows treated with a Short or Long program and using a Single or Triple P4-releasing device. Bars with different superscripts differ at P < 0.05.

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4.2 ng/mL (S.E.D. = 0.4) P4 for cows conceiving and not conceiving to first insemination, respectively, P = 0.04). There was no interaction of dose or program with the conception to first service (both P = 0.2). The final model adjusted R2 was 0.353. More of the cows not conceiving had a low P4 concentration at Day 13 (i.e. <1 ng/mL) than those conceiving (12/78 (15.4%) versus 1/41 (2.4%); P = 0.03). There were no differences among dose (6/57 (9.5%) versus 9/54 (14.3%) of Single versus Triple P4, respectively, P = 0.41) or program (8/62 (12.9%) versus 7/64 (10.9%) Short versus Long program, respectively, P = 0.73) in the proportion of cows with low P4 concentrations.

4. Discussion The original hypothesis that a program with a longer duration of P4 treatment and additional EB at device insertion would increase the first service conception rate was accepted. However, the dose of P4 (i.e. Single versus Triple P4) did not effect the first service conception rate. Additionally, neither the dose nor the program affected any of the other outcome variables studied either in anestrus or cycling cows. One explanation of the enhanced conception rate with the Long compared to the Short program may be related to follicle wave dynamics. The Long, but not Short program included EB at device insertion. Treatment with EB or GnRH concurrent with insertion of a P4-releasing device results in atresia or ovulation of the dominant follicle and emergence of a new follicle wave in both anestrus and cycling cows [26–29]. Removal of a dominant follicle present at the commencement of treatment prevents ovulation of a follicle that has had an extended period of dominance. Extended periods of dominance are associated with depressed conception rates [13] related to high estradiol levels within the follicle and structural changes to the oocyte [30]. Treatment with either EB or GnRH at the time of insertion of a P4 device for 7 d or 8 d resulted in an increase in conception rate compared to no EB or GnRH [31,32]. Conversely, an alternative hypothesis would be that the average size of the ovulated follicle will be smaller following the Short than the Long program. Ovulation of small follicles has been associated with smaller CL and a reduced conception rate in cycling cows [15]. However, detailed studies in anestrus cows have suggested that ‘persistence’ of the dominant follicle does not occur following treatment with exogenous P4 in anestrus cows as it does in cycling cows [33]. Thus, the possibility that the increased conception rates seen are related solely to the duration of the P4 treatment, not mediated by changes in follicle dynamics, cannot be ruled out on the basis of the current study. The direct effect of different durations of P4 treatment in the absence of EB at device insertion needs to be undertaken to test this hypothesis directly. Previous descriptive studies have demonstrated a positive correlation between serum P4 concentration in the preceding diestrous phase and the probability of conception [8–11]. No relationship was found between the dose of exogenous P4 preceding insemination and the conception rate in the present study. This may be due to an insufficient dose of P4 delivered or a less than optimal plasma P4 profile over time. The Triple P4-releasing device used in the current study did increase plasma P4 concentrations during the period of treatment, but concentrations were still below those seen in normal cycling cows in the

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luteal phase (McDougall et al.; unpublished). The temporal patterns of concentrations of P4 following exogenous P4 treatment do not mimic those of the natural luteal phase. Manipulation of the P4 profile by use of multiple PG treatments or PG treatment followed by insertion of two intravaginal P4-releasing devices resulted in different P4 profiles and different follicular wave patterns between treatments [34]. This suggests that the shape of the P4 profile as well as the peak concentration of P4 may be important in follicle wave control. The previously reported positive relationship between preceding luteal phase P4 concentration and conception rate may not be a direct causal relationship. If the observed relationship were due to some unrecorded underlying factor that was positively correlated both with serum P4 concentration and conception rate, but that had effects not mediated via P4, ‘confounding’ may have occurred in the previous studies. Hence P4 may be acting as a ‘proxy variable’ for other factors associated with conception rate. For example, LH pulse frequency is influenced by nutritional status [35,36] and P4 concentration is correlated with LH pulse frequency via LH-driven P4 release from the small luteal cells [37]. Progesterone concentration is also correlated with weight gain [10] and reduced in underfed animals [38]. Progesterone production may also be mediated via GH and/or insulin-like growth factor because receptors for these have been found in large luteal cells [39,40] and GH increases secretion of P4 from luteal cells [41]. However, GH, insulin-like growth factor, leptin and other metabolic regulators may also directly effect conception rate and fetal survival independent of P4 [42]. The relationship between preceding P4 and conception rate also appears to be influenced by whether the P4 is of endogenous or exogenous origin. Where cows were treated twice with PG 14 d apart, there was a positive relationship between subsequent conception rate and the P4 concentration 3 d preceding the second PG treatment [11]. However, where the same PG regime was used but with the addition of an intravaginal P4-releasing device 8 d after the initial PG treatment for 7 d, the relationship was reversed [11]. One of the potential mechanisms by which the P4 concentration in the preceding luteal phase could influence subsequent conception rate may be via enhancement of subsequent luteal function and hence enhanced embryo growth and survival. Cows conceiving had higher average P4 concentrations on Day 13 and were less likely to have low (i.e. <1 ng/ mL) P4 concentrations than those not conceiving to first insemination. However, neither the dose nor the program length affected the plasma P4 concentration or the probability of having a low P4 concentration on Day 13 in the subsequent luteal cycle. This is despite the fact that the first service conception rate was higher in anestrus cows treated with the Long versus the Short program before insemination. It appears that the positive effect of the Long program may have been via pathways that were independent of P4 concentrations in the subsequent luteal phase. The dose of P4 and program length were not related to P4 concentration even in the initial univariate models. Inclusion of the other effects (e.g. herd, BCS, and protein percentage) did not lead to colinearity and hence mask some effect of treatment. Additionally, studies which have increased serum/plasma P4 concentrations post insemination, either by providing additional exogenous P4 directly in anestrus cows [43] or indirectly by inducing a second CL by gonadotropin treatment in cycling cows, have also failed to enhance conception or pregnancy rates in some [44,45] but not all

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studies [46]. It appears that factors other than P4 concentration either before or after insemination of P4-treated anestrus cows are required to explain the lower conception rates commonly found in anestrus compared to cycling cows. A number of explanatory variables other than P4 treatment were found to influence the reproductive performance of P4-treated anestrus cows; these included herd, age, body condition score before the planned start of the breeding program, the interval between calving and the start of the breeding program, and milk composition. Herds differed for all reproductive indices examined. Differences in farm-level management including nutritional management, estrus detection and sire and cow genetics may all impact on farm-level performance. Body condition score was negatively associated with the 7-d submission rate, but positively associated with the 28-d, 56-d and final pregnancy rate. That the 7-d submission rate was lower in better conditioned cows is not clear and requires further investigation. Previous studies have demonstrated a positive relationship between body condition around insemination and reproductive performance [47,48] and between the interval from calving to the commencement of the breeding program and reproductive performance [1,3]. Relationships between milk composition and reproductive performance have also previously been demonstrated. Elevated milk fat to protein (i.e. >1.5) ratios have been associated with reduced first service conception rates, increased numbers of inseminations per conception and increased calving to conception intervals [49]. In the current study, cows with high fat to protein ratios (i.e. >1.34) had depressed first service conception rates. Positive relationships have been demonstrated between milk protein percentage and submission and pregnancy rates in studies in Australia [50], Ireland [51] and New Zealand [23]. In common with the previous New Zealand study, the milk protein percentage was correlated with the 28-d and 56-d pregnancy rate, but not the submission rates or first service conception rates. The mechanism for the relationship between milk protein percentage and fertility is unclear but appears to operate via pathways independent of expression of estrus. A novel finding of the current study is that the luteal (Day 13) phase P4 concentration was positively associated with both milk protein percentage and with body condition score. The association between luteal phase P4 concentration, body condition score and milk composition requires further investigation. In conclusion, increasing the dose of P4 used to treat anestrus and not detected in estrus but CL-positive cows did not enhance any measure of reproductive performance. However, a program with a longer duration of progesterone treatment and with addition of EB at P4-releasing device insertion significantly improved the first service conception rate. The increase in conception rate was independent of the dose of P4 and of the concentration of P4 in the subsequent luteal phase.

Acknowledgement The support of the herd owners and staff in completing this project is gratefully acknowledged. Pfizer Animal Health (NZ) LTD kindly provided the P4-releasing devices as well as providing financial support for the project. T. O’Donnell (Dexcel, Hamilton, New Zealand) kindly performed the P4 RIA assays.

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