Animal Reproduction Science 109 (2008) 65–76

Evaluation of short estrus synchronization methodsin dairy cows�

Umut Cirit a,∗, Suleyman Bacinoglu a, Muzaffer Tas b,Kamber Demir a, Ahmet Bas c, Kemal Ak a,

Irfan Kamuran Ileri a

a Istanbul University, Faculty of Veterinary Medicine, Department of Reproductionand Artificial Insemination, 34320 Avcilar, Istanbul, Turkey

b Dicle University, Faculty of Veterinary Medicine, Department of Reproductionand Artificial Insemination, 21280 Diyarbakir, Turkey

c General Directorate of Agricultural Enterprises (TIGEM), Directorate of AgriculturalEnterprises of Turkgeldi, Luleburgaz, Kırklareli, Turkey

Received 20 February 2007; received in revised form 28 November 2007; accepted 12 December 2007Available online 28 December 2007

Abstract

In the present study, two new short estrus synchronization methods have been developed for lactating dairycows. The study was completed in three consecutive phases. In experiment (Exp) 1, 32 cows, that were notdetected in estrus since calving between the 50th and 84th post-partum days, were treated with PGF2� (PGF,d-cloprostenol, 0.150 mg), estradiol propionate (EP, 2 mg) and GnRH (lecirelina, 50 �g) at 24 h intervals,respectively, and timed artificial insemination (TAI) was performed 48 h after PGF. Different from Exp 1,EP and GnRH were given at 48 and 60 h, respectively after PGF in Exp 2 (n = 20), instead of 24 and 48 h.Ovulations were investigated by ultrasound for 7 days starting from the day of PGF treatment, and ovulationrates were compared with the ones obtained in Exp 1. In Exp 3, cows were given the same treatments asExp 2, but treatments started at certain estrus stages. Cows detected in estrus and with a confirmed ovulation(n = 27) after the second PGF given 11 days apart were assigned to three treatment groups. Treatment wasinitiated at Day 3 (group metestrus, n = 9), Day 12 (group diestrus, n = 9) and Day 18 (group proestrus, n = 9)after ovulation. All cows included in Exp 3 were TAI between 16 and 20 h after GnRH treatment. In Exp 2 and3, blood samples were obtained once every 2 days, starting from Day 0 to the 10th day after GnRH injection,and once every 4 days between the 10th and the 22nd days after GnRH to examine post-treatment lutealdevelopment. During the study, animals exhibiting natural estrus were inseminated and served as controls

� The present study was supported by the Research Fund of Istanbul University. Project No. 182/15012004 and UDP-1618/11092007.

∗ Corresponding author. Tel.: +90 212 473 70 70x17264; fax: +90 212 473 72 41.E-mail address: ucirit@istanbul.edu.tr (U. Cirit).

0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.anireprosci.2007.12.004

66 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

(n = 85). The rate of estrus was found to be significantly higher in cows with an active corpus luteum (CL) atthe start of Exp 1 (72.7% vs. 30.0%, P < 0.05) and the pregnancy rate tended to be higher than cows withoutan active CL (40.9% vs. 10.0%, P = 0.08). Compared to those in Exp 1, cows in Exp 2 had higher ratesof synchronized ovulation (94.1% vs. 59.1%, P = 0.013). In Exp 3, estrus (P < 0.001) and pregnancy rates(P = 0.01) were found to be significantly higher in cows in the proestrus group than in those in the metestrusgroup. Comparable pregnancy rates were obtained from the first and second inseminations in Exp 1 and 3with results from those inseminated at natural estrus (P > 0.05).

It was concluded from the study that the treatment in Exp 1 and 3 could result in comparable pregnancyrates after timed AI of lactating dairy cows at random stages of the estrus cycle relating to those inseminatedat natural estrus, but the stage of the estrus cycle can have significant effects on pregnancy rates.© 2007 Elsevier B.V. All rights reserved.

Keywords: Dairy cows; Short synchronization; PGF2�; GnRH; Estradiol propionate

1. Introduction

Failure to detect cows in estrus accurately is a major factor limiting the reproductive perfor-mance of lactating dairy cattle and this is the main reason for the increase in the calving interval inmany dairy herds (Rounsaville et al., 1979). It has been estimated that the mean reduction in thenet revenue from a 1-day increase in adjusted calving interval is $4.7 (Canadian) per cow (Plaizieret al., 1997). A fixed-time AI facilitates precise scheduling of first post-partum AI, while improv-ing reproductive performance during the early post-partum period with no labour investment forestrus detection (DeJarnette et al., 2001). Various estrus synchronization programs allowing timedartificial insemination (TAI) have been developed in cows, but most require several days to sortcows in order to administer the treatment, and it is difficult to reduce the interval between the startof treatment and AI to less than 9 d (Lopez-Gatius, 2000b). Development of short synchronizationmethods that would result in acceptable pregnancy rates as a result of TAI would provide breederswith certain advantages. This would rid the breeders of the burden of determining the estrus, allcows treated following the voluntary waiting period would be inseminated, and early comple-tion of treatment would save breeders time. However, there are only a limited number of studieson the short synchronization programs for TAI, in which cows having a determinable corpusluteum (CL) have been used (Lopez-Gatius, 1989, 2000a; Lopez-Gatius and Vega-Prieto, 1990).In an earlier study, we determined that an additional estradiol propionate (EP) injection (GPEG;Day 0/GnRH, Day 7/PGF2�, Day 8/EP, Day 9/GnRH) on Day 8 of the ovsynch protocol (GPG)shortened the PGF treatment to ovulation interval approximately 4 h (P > 0.05) and increased thepregnancy rate slightly (50.0% vs. 60.0%, P > 0.05) in lactating primiparous Holstein cows (Ciritet al., 2007). These findings encouraged us for the fact that short-term treatment without the firstGnRH injection can allow for TAI.

The simultaneous administration of hCG and estradiol benzoate 12 h after prostaglandin treat-ment in dairy cows with mature CL has been reported to allow TAI 48 h after starting treatment(Lopez-Gatius, 2000a). To our knowledge, there is no study about the effects of short synchroniza-tion methods on random or known different stages of estrus cycles in lactating cows. Additionally,information in related subjects like synchronized ovulation rates and post-treatment luteal devel-opment is absent, except the effects of the treatments over the rates of estrus and pregnancy, sinceall the existing work is field-based studies. In cows, abnormal luteal function, delayed onset ofluteal phase or decreased progesterone concentrations has been shown associated with reducedpregnancy rates (Larson et al., 1997).

U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76 67

The objectives of our study were to develop short estrus synchronization methods allowing TAIin lactating dairy cows. Furthermore, we aimed to determine the effects of the estrus cycle stageat the onset of the treatment on synchrony of ovulation and pregnancy, and to investigate post-treatment luteal development. To evaluate the reproductive performance of such treated cows, thepregnancy rates of the first AI, and the second AI of cows returned to estrus between 8 and 30days after the first AI, was compared with those of cows inseminated at natural estrus.

2. Materials and methods

2.1. Animals and treatments

The experiment was conducted on a commercial dairy farm located in northwest Turkey, wheremultiparous lactating Holstein cows were maintained in free-stall facilities, fed a total mixedration, and milked three times daily. The mean daily milk production of the study cows wasapproximately 28.2 kg at the beginning of the research. The experiment was conducted betweenApril and July. All cows were under daily veterinary supervision. The first day of the treatmentswas considered as Day 0.

2.1.1. Experiment 1In the first experiment, 32 cows, that were not detected in estrus since calving, were given

PGF (PGF2�, d-cloprostenol 0.150 mg, i.m., Dalmazin, Vetas, Turkey) between the 50th and84th post-partum days (Day 0). After 24 h (Day 1), estradiol propionate (2 mg, i.m., Akrofolline,Turfarma, Turkey) was administered. Starting from Day 0, all animals were inspected for signsof estrus in the early morning and late afternoon for 30–45 min. Standing to be mounted for over3 s was considered as the first sign of estrus. Disregarding indications of estrus, all cows wereTAI 48 h after the PGF treatment. Right after TAI, GnRH (lecirelina 50 �g, i.m., Dalmarelin,Vetas, Turkey) was administered. Starting from PGF treatment day, ovaries were scanned bydaily ultrasonography for 7 days and ovulations were detected.

2.1.2. Experiment 2The second experiment was planned during the first week after the ovulation results had been

obtained in Exp 1. In Exp 2, 25 cows that were not detected in estrus since calving were scannedtwice at 10-day intervals by ultrasonography between the 53rd and 88th days post-partum, andtheir blood samples were obtained. Cows determined with abnormal vaginal discharge betweentwo examinations were excluded from the study. The rest of the cows (n = 20) had received PGFon the second blood obtaining day (Day 0) and EP after 48 h (Day 2). GnRH was given after12 h following the administration of EP (60 h after PGF). Cows were examined by ultrasound toconfirm the day of ovulation and estrus detection was accomplished as in Exp 1 for 7 days dailyafter injection of PGF. The objective of the second experiment was to investigate whether or notthe treatments would result in synchronized ovulations, thus the cows were not inseminated.

2.1.3. Experiment 3The third experiment was begun during the first week following confirmation of ovulation of

the second experiment. The same hormonal treatments were administered to all cows both in Exp2 and 3. In contrast to Exp 2, in Exp 3, treatments were initiated at known estrus stages. Thus,32 cows between the 52nd and 90th post-partum days were given PGF twice at 11-day intervals.Detection of estrus was performed as Exp 1 for 7 days after the second injection of PGF. Cows

68 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

were examined daily after injection of PGF by ultrasound to confirm the day of ovulation. Cowsdetected in estrus and with a confirmed ovulation (n = 27) were assigned to three treatment groups.Treatment was initiated at Day 3 (group metestrus (M), n = 9), Day 12 (group diestrus (D), n = 9)and Day 18 (group proestrus (P), n = 9) after ovulation. All cows included to Exp 3 (disregardingindications of estrus) were TAI between 16 and 20 h after GnRH injection.

During the study, animals exhibiting natural estrus within the corresponding 50th–92nd daypost-partum interval were inseminated and served as controls (n = 85). Estruses were detected asin Exp 1 and control cows suspected to be in natural estrus were inseminated 10–12 h later. Estruswas confirmed by rectal examination before AI as (a) uterine tone and vaginal hyperaemia at grade2 or over (1–3 points), (b) cervical mucus discharge or vulval wetness, and (c) a follicle assumedto be ≥1 cm on ovaries. If cows had returned to estrus during Days 8–30 post-insemination, estruswas confirmed by examination per rectum and the animals were re-inseminated without additionaltreatment in Exp1 and Exp 3, and Control groups. Diagnosis of pregnancy was made by ultrasoundon the 35th–40th day post-insemination. In groups M, D and P, abnormal vaginal dischargewas determined in 3, 3 and 1 cows, respectively during the period between AI and pregnancyexamination, and these cows were excluded from the statistical analyses where pregnancy rateswere compared. The pregnancy rate to the first AI was defined as the number of pregnant cowsafter the first AI, as a percentage of the total number of cows inseminated. Pregnancy rate to thesecond AI was defined as the number of pregnant cows after the second AI, as a percentage ofthe total number of cows showing estrus and were inseminated between the 8th and the 30th daysafter the first AI. Ultrasound examinations and artificial inseminations were performed by the firstauthor.

2.2. Blood sampling and progesterone assays

Blood samples were collected by vacuum puncture of a tail blood vessel into silicone plain tubes(Venoject) by 20 G needles (Vacutainer) in all three experiments. Serum was harvested, frozen andstored in plastic tubes at −20 ◦C until the time of analysis. The concentration of progesterone (P4)in the serum was measured in all samples by radioimmunoassay using a commercial kit (DSL-3900, progesterone, DSL, USA). The sensitivity of the assay was 0.12 ng/ml. Intra- and inter-assay coefficients of variation were 5.3% and 8.8%, respectively. Blood samples were obtainedat the start of the treatment (Day 0) in Exp 1. Cows in Exp 1 and 2 were grouped into CL-classes retrospectively according to serum P4 concentrations of the blood samples collectedimmediately before PGF treatments. A concentration higher than 1 ng/ml was considered toindicate the presence of a functional CL. In Exp 2 and 3, blood samples were obtained once everyother day, starting from PGF treatment day to Day 10 after GnRH injection, and once in 4 daysstarting from Day 10 after GnRH injection to Day 22. Corpus luteum regression was defined by acow having a serum P4 concentration of >1 ng/ml, which then declining to a level of <1 ng/ml. Inaddition, blood samples were obtained twice prior to the treatment with 10 days interval in Exp 2(−10th and 0th days). Cows were considered anestrus if the serum progesterone concentrationsof both the Days −10 and 0 samples were <1 ng/ml.

2.3. Ultrasound examinations

2.3.1. Experiments 1 and 2Transrectal ultrasound examinations were performed by a single operator using a real-time, B-

mode scanner with an 8.0-MHz linear-array transducer (Esaote Pie Medical, Falco, Netherlands).

U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76 69

Cows were examined by ultrasound at the start of the study for evaluation of visible CL to aid forclassification of those having active CL or not. Scannings were carried out daily for 7 days aftertreatment to correspond to the day of PGF application in Exp 1 and 2, and all follicles ≥8 mmin diameter and CLs were recorded. Ovulation was considered as the sudden disappearance fromone examination to the next of a previously identified ovarian follicle ≥8.5 mm in diameter, andwas confirmed by the subsequent development of a CL. Synchrony of ovulation was defined asthe proportion of cows ovulating between 24 and 48 h after GnRH out of all cows ovulating in allthe three experiments.

2.3.2. Experiment 3Transrectal ultrasonography of ovarian structures was performed daily on cows from the second

PGF injection to the treatment onset (Day 0) in order to determine the developmental stages ofthe dominant follicle (DF) at treatment onset, and then daily from Days 0 to 21 after the TAI.Ovarian maps were drawn at each examination and the relative positions and sizes of ovarianfollicles greater than 3.0 mm in diameter and CLs were recorded. The dominant follicle of a wavewas defined as the follicle that reached the largest diameter. Wave emergence was retrospectivelydefined as the day when the DF was first detected at a diameter of 4–5 mm. If the DF was notdetected until it reached 6 or 7 mm, the previous day was considered the day of wave emergence(Martinez et al., 1999). For the ovulatory follicles, the duration of dominance was calculated asthe interval between the first day of dominance and the day preceding ovulation. The first day ofdominance defined as (1) the first day that subordinate follicles stopped increasing in diameterbecoming static or regressing, (2) the time when the DF was ≥8.5 mm in diameter, and (3) thedifference in size between the DF and the next largest subordinate follicle was ≥2 mm (Riveraet al., 1998). The age of the ovulatory follicle was defined as the time interval (days) betweenthe day of emergence of the ovulatory follicle and the time of ovulation (Cavalieri et al., 1997).The growing phase of the dominant follicle was defined as the period from the day that thefollicle was first identified until the day that appeared to cease a progressive increase in diameter(day of cessation of growth of the dominant follicle; Rivera et al., 1998). Growing phase wasfurther subdivided into early-growing phase (first 3 days) and late-growing phase, respectively.The static and regressing phases of the DF were defined according to previous reports (Gintheret al., 1989a,b; Rivera et al., 1998).

2.4. Statistical analysis

Categorical data such as pregnancy, ovulation and estrus rates were evaluated by χ2. Quanti-tative data such as interval from GnRH injection to ovulation and size of DFs were evaluated byindependent samples t-test between two groups or subgroups (Exp 1 and 2). In Exp 3, quantitativedata were analysed by one-way ANOVA and means were compared by the Duncan-test to deter-mine differences among groups. The serum progesterone profiles (group × day) were analysedusing Friedman test and differences between the groups were evaluated by the Mann–WhitneyU-test. Data were processed by Jarque–Bera normality test before correlation analysis. Whilethe relation between parametric continuous data was analysed using Pearson correlation test, therelation between non-parametric continuous data was evaluated by Spearman correlation test.Sizes of DFs on GnRH and EP treatment days were ranked from least to greatest and then cowswere classified based on sizes of DFs as having small (≤13.9 mm) or large (≥14.0 mm) DFs.Then, association of the size of DFs on GnRH and EP treatment days with the incidence of earlyluteolysis and estrus, respectively, were evaluated using χ2 analysis.

70 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

Table 1Reproductive traits of cows with or without an active corpus luteum (CL) at the onset of treatment in experiment 1

Cows without CL (n = 10) Cows with CL (n = 22) P-value

Estrus incidence (%) 30.0 72.7 =0.023

Synchronized ovulation rates (%) 57.1 60.0 >0.05Total ovulation rates (%) 70.0 68.2 >0.05Pregnancy rates (%) 10.0 40.9 0.08Size of ovulatory follicles on the GnRH

injection day (mm)13.2 ± 3.80 15.1 ± 3.55 >0.05

EP injection to estrus interval (h) 22.7 ± 1.15 21.9 ± 5.19 >0.05

The values given for continuous variables are mean ± S.D.

3. Results

The total and synchronized ovulation rates of cows with and without an active CL at the onsetof treatment in the first experiment were found to be quite similar (P > 0.05). When comparedwith cows without an active CL, the rate of estrus in cows with an active CL was found to besignificantly higher (72.7% vs. 30.0%, P < 0.05) and the rate of pregnancy tended to be high (40.9%vs. 10.0%, P = 0.08, Table 1). The estrus incidence, the mean EP injection to estrus interval andthe mean size of ovarian follicles on the GnRH treatment day were found to be similar in cowsincluded in Exp 1 and 2; however, synchronized ovulation rate in Exp 2 with respect to Exp 1 wasfound to be significantly higher (94.1% vs. 59.1%, P = 0.013). In addition, in Exp 1, the GnRHinjection-ovulation interval was found to be significantly shorter than that of Exp 2 (P = 0.012,Table 2).

In Exp 2, the PGF treatment did not result in luteolysis in 3 of 10 cows with an active CL at thestart of treatment and high P4 levels were kept constant. However, these cows underwent lutealregression on Days 5, 7 and 13 after GnRH injection and then displayed estrus on subsequentdays. Ovulation did not occur in one of the three cows with failure of luteolysis at the end ofthe PGF treatment. Ovulation was observed in all five cows without an active CL at the onset oftreatment. After GnRH treatment, luteal regression was observed on Day 10 in two of these fivecows, and P4 levels increased again to over 1 ng/ml on Day 18. Treatments resulted in ovulationin three of five anestrous cows, but the serum P4 levels did not exceed 1 ng/ml until Day 21post-ovulation in two of these three cows (Fig. 1). In Exp 2 and 3 and their combination, cowsdisplaying estrus were found to have larger DFs on their ovaries on EP treatment day, whencompared with the ones not displaying estrus (15.2 ± 2.65 mm vs. 14.5 ± 0.99 mm, P < 0.01;

Table 2Some reproductive traits of cows in Exp 1 and 2.

Experiment 1 (n: 32) Experiment 2 (n: 20) P-value

Estrus incidence (%) 59.4 55.0 >0.05Synchronized ovulation rates (%) 59.1 94.1 =0.013Total ovulation rates (%) 68.8 85.0 >0.05Size of ovarian follicles on the GnRH injection day (mm) 14.5 ± 3.68 14.9 ± 2.95 >0.05EP injection to estrus interval (h) 22.0 ± 4.76 22.2 ± 9.18 >0.05GnRH to ovulation interval (h) 38.2 ± 12.08 46.4 ± 5.82 =0.012

The values given for continuous variables are mean ± S.D.

U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76 71

Fig. 1. Mean (±S.E.M.) serum progesterone profiles of cows with (n = 10) or without (n = 5) an active corpus luteum (CL)and at anestrus (n = 5) at the onset of the treatment in experiment 2. Group × day differed significantly for progesteroneon Days 10, 16 and 20 after PGF treatment (P < 0.001).

16.2 ± 2.97 mm vs. 13.8 ± 1.77 mm, P < 0.05; 15.8 ± 2.84 mm vs. 13.2 ± 1.57 mm, P < 0.001,respectively). Similarly, in combination of Exp 1 and 2, cows with large DFs had a significantlyhigher rate of estrus than those of having small DFs on the EP treatment day (76.0%; 19/25 vs.40.9%; 9/22, P = 0.014).

In Exp 3, DFs were at the late growth phase during GnRH injection in all cows in group M. InGroup D, DFs were at the stage of early growth in one cow, early static in one cow, and at the lategrowth stage in the remaining seven cows. In group P, DFs were at the stage of late growth in threecows, early static (first 2 days) in five cows and late static in one cow. When compared with cows ingroups M and D in Exp 3, cows in group P had DFs with longer dominance periods (P < 0.0001)and larger diameter (P = 0.01) on their ovaries during GnRH injection. Estrus (P < 0.001) andpregnancy rates (P = 0.01) in group P cows were found to be significantly higher than in cows ingroup M (Table 3). All cows included in Exp 3, except one cow in group D, displayed ovulation.Synchronized ovulation rates in groups M, D and P were 88.9%, 100% and 100%, respectively(P > 0.05).

Table 3Reproductive traits of cows in groups metestrus (M), diestrus (D) and proestrus (P) in Exp 3.

Group M Group D Group P P-value

Animal number 9 9 9 –Estrus incidence (%) (2/9) 22.2b (6/9) 66.7ab (9/9)100.0a <0.001Synchronized ovulation rates (%) (8/9) 88.9 (9/9) 100.0 (9/9) 100.0 >0.05Total ovulation rates (%) (9/9) 100.0 (8/9) 88.9 (9/9) 100.0 >0.05Pregnancy rates (%)c (0/6) 0.0b (2/6) 33.3ab (5/8) 62.5a 0.01EP injection to estrus interval (h) 18.0 ± 8.48 20.0 ± 4.38 11.6 ± 9.04 >0.05Duration of dominance for OF (day) 3.3 ± 1.00b 3.9 ± 1.00b 6.7 ± 1.58a <0.0001Age of DF on the day of GnRH (day) 5.2 ± 0.43b 5.1 ± 1.33b 9.1 ± 1.67a <0.0001Size of DF on the day of GnRH (mm) 14.8 ± 1.72b 15.1 ± 2.60b 17.8 ± 1.85a 0.01

The values given for continuous variables are mean ± S.D.abWithin rows, means with no common letters are different; OF, ovulatory follicle; DF, dominant follicle.cIn groups M, D and P, abnormal vaginal discharge was determined in 3, 3 and 1 cows, respectively during the periodbetween AI and pregnancy examination, and these cows were excluded from the statistical analyses where pregnancyrates were compared.

72 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

Fig. 2. In experiment 3, mean (±S.E.M.) serum progesterone profiles of cows in groups diestrus (D, n = 9) and proestrus(P, n = 9), and also cows with (LR+, n = 7) or without (LR−, n = 2) luteal regression in group metestrus (M). Group × daydiffered significantly for progesterone on Days 7, 17 and 21 after PGF treatment (P < 0.001).

Table 4Effect of treatment regimen on the pregnancy rates to first and second AI and the percentage of cows returning to estrus.

Experiment 1 Experiment 3 Control P-value

Pregnancy rate to first AI (%) 31.3 (10/32) 35.0 (7/20) 32.9 (28/85) >0.05Cows returning to estrus (%) 53.1 (17/32) 60.0 (12/20) 45.9 (39/85) >0.05Pregnancy rate to second AI (%) 47.1 (8/17) 50.0 (6/12) 48.7 (19/39) >0.05

Luteolysis was determined in seven of nine cows in group M, five on the 9th day and twoon the 13th day post-AI. In four of these cows, obvious symptoms of estrus were observed onthe 11th and 12th days. As a result of ultrasound examinations and P4 analyses, ovulations weredetermined to follow the luteal regressions. In all cows with luteal regression except one, theserum P4 concentrations increased post-ovulation and exceeded 1 ng/ml on the 21st day after AI(Fig. 2). According to this finding, for further analysis, the animals in the M group were dividedinto two subgroups, with luteal regression (LR+) and without luteal regression (LR−). None ofthe cows in groups D and P had luteolysis before the expected time.

In Exp 3, a significant positive correlation was found between the size of DFs on GnRHtreatment day and the maximum diameters of the new CLs formed post-ovulation (r = +0.522,P < 0.01). In the combination of Exp 2 and 3, early luteolysis incidence of cows with small andlarge DFs on the GnRH treatment day was found to be quite similar (22.2%; 4/18 and 20.7%;6/29, respectively).

Comparable pregnancy rates were obtained from Exp 1 and 3 after the first and second AIswith those inseminated at natural estrus (P > 0.05, Table 4).

4. Discussion

The results of our study demonstrate that the treatment in Exp 1 will result in acceptable ratesof pregnancy in cows with an active CL. Although the total and synchronized ovulation rates ofcows with or without active CL at the onset of treatment were found to be quite similar (P > 0.05),the rate of pregnancy of cows with an active CL tended to be high (40.9% vs. 10.0%, P = 0.08).

U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76 73

The low P4 before insemination may have contributed to lower pregnancy rates in these cowsthan in those with an active CL at the start of treatment (Folman et al., 1990).

The critical requirements for any effective estrus cycle control regimen are a predictable andhigh estrus and ovulation responses during a specified 12–24 h period, followed by a high preg-nancy rate to a single pre-programmed AI after treatment (Diskin et al., 2002). Initially the studywas planned to investigate the treatment only in Exp 1. But the total ovulation rate (68.8%) and24-h-synchronized ovulation rate (59.1%) were found to be lower than our expectations. There-fore, Exp 2 was planned immediately after the results of ovulation in Exp 1 had been obtained.Although there are numerous factors affecting pregnancy in cows, occurrence of ovulations ata special time interval between 12 and 24 h is a prerequisite to be able to obtain pregnancy atsufficient levels, especially in TAI programs. Synchronized ovulation rates for 24 h between the24th and the 48th h after GnRH treatment were found to be significantly higher (94.1%, 96.2%and 59.1%, respectively; P = 0.01) in Exp 2 and 3 when compared to Exp 1. Also, the cows in Exp2, had longer GnRH to ovulation intervals than cows in Exp 1 (46.4 ± 5.82 h vs. 38.2 ± 12.08 h,P = 0.012), which is thought favourable for fertility since inseminations are mostly carried out16–24 h after GnRH in TAI protocols. However, no considerable increase in pregnancy rates ofExp 3 versus Exp 1 could be obtained (35.0% and 31.3%, respectively; P > 0.05). It is likely thatstart of treatments in Exp 3 on specific days (3rd, 12th and 18th days after ovulation) differentlyfrom Exp 1 made a contribution on the above result. Despite similar applications, there may bepossible explanations for the methods resulting in synchronized ovulations more tightly in Exp2 than Exp 1. Exogenous estrogens (in a low progesterone environment) and GnRH induce anLH surge approximately 16–24 h (Bo et al., 1994) and 2–3 h after their administration in cows,respectively (Williams et al., 1982), and ovulation occurs between 24 and 32 h after the LH surge(Noakes, 1997). LH concentrations were not measured in our study. But according to the liter-ature, LH surge by estradiol and GnRH will be expected to occur at approximately the 40–48thand 50th h after PGF, respectively, and the majority of the cows will be expected to ovulate atapproximately the 16th and 34th h after GnRH in Exp 1. This is why 28.1% (9/32) of cows(40.9% of total ovulations; 9/22) had ovulated between 0 and 24th h after GnRH in Exp 1. InExp 2, LH secretions after GnRH and EP treatments will be expected to occur at approximatelythe 62nd and 64th–70th h after PGF treatment, and the majority of the cows will be expectedto ovulate at approximately between the 26th and the 42nd h after GnRH injection. Ninety fourpoint one percent (16/17) of total ovulations occurred between the 24th and 48th h after GnRHtreatment in Exp 2 as expected. Other possible explanations for the increase in synchronizedovulation rates in Exp 2 include endogenous and induced LH surges by EP and GnRH prob-ably occurring concurrently (approximately 62–70 h after PGF treatment), and this may havecaused more efficient LH surges. Similarly, the endogenous LH peak was demonstrated to haveoccurred between the 60th and 68th h after PGF in majority of cows with a range of 52–104 hfrom the day of injection (Lucy and Stevenson, 1986). It has been stated that GnRH adminis-tered at the onset of an endogenous LH surge should be highly potent in terms of its ability torelease LH (Lucy and Stevenson, 1986). Only one ovulation was determined between 0 and 24 hafter GnRH treatment in Exp 2, which implied that in a great majority of the cows, the endoge-nous LH surges did not occur before the EP and GnRH induced LH surges. The results of Exp1 and 2 demonstrated that the timing of injections of GnRH, EP or both given after PGF hadcritical significance with respect to ovulation synchronization. Consequently, Exp 3 was plannedimmediately after the ovulation results of Exp 2, in order to be informed about the effects ofthe cycle stage at the onset of treatment on the pregnancy rates. In Exp 3, the synchronizedovulation rates for 24 h were also found to be quite high, similar to Exp 2. When Exp 2 and 3

74 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

are combined (n = 47), it is shown that the success of the treatment in inducing high rate syn-chronized ovulations is not accidental. In Exp 3, both synchronized and total ovulation rates didnot display differences between groups M, D and P. The cows in group P had DFs with largerdiameters (P = 0.01) maintaining extended dominance periods (P < 0.0001) on their ovaries at thetime of GnRH treatment, with respect to cows in groups M and D. Rates of estrus (P < 0.001)and pregnancy (P = 0.01) in group P were found to be significantly higher than those of cows ingroup M.

Ovulation occurred in all cows included in group M, and following this, a second CL wasformed and these CLs continued to develop. But both CLs had regressed between Days 9 and13 post-insemination in 7/9 cows in group M. Though it resembles “early regression of CL”, thepre-treatment estrus cycles and ultrasonographic examination of cows revealed that the CLs werenot exposed to early, but normal regression regarding the previous cycle. The CLs of the cowswere only 3–3.5 days old at treatment onset. Thus, they were rescued from regression as a resultof PGF treatment and maintained their development. Consequently, the CLs of pre-treatmentcycle (and new CLs formed after treatment) naturally regressed at the correct time accordingto previous cycle. In our study, none of the cows showing luteal regression after TAI becomepregnant.

It has been claimed that ovulation of a smaller DF can produce a smaller CL and lowerconcentrations of progesterone (Burke et al., 2001; Vasconcelos et al., 2001), and most likelyshortened cycles (Taponen et al., 2002; Macmillan et al., 2003). However, there was no significantrelation between the size of DFs during GnRH treatment and the incidence of luteolysis before theexpected time. The results of our study suggest that early luteal regression is probably related withthe escape of CLs from lysis by PGF rather than follicle size during GnRH treatment. Althoughthe cows in Exp 2 and 3 had ovulatory follicles ranging from 10.8 to 21.0 mm, the occurrence ofluteolysis prior to the anticipated time in none of the cows, both with large or small follicles, ingroups D and P in Exp 3, and with an active CL (except the ones with unsuccessful luteolysis) inExp 2 has supported our claim. Cordoba and Fricke, have classified the serum samples based on P4concentrations collected on each hormone injection day in ovsynch protocol as low (≤1.0 ng/ml)or high (>1.0 ng/ml). Supporting our results, they have found that the 5/9 (55.6%) cows withunsuccessful luteolysis (HHH and LHH groups) returned to estrus prematurely between the Days0 and 17 after AI while only the 5/56 (8.9%) cows with successful luteolysis (LHL and HHLgroups) had premature estrus. They have also showed that all three cows at metestrus stage (HLHgroup) on the day of the PGF treatment returned to estrus prematurely after AI (Cordoba andFricke, 2002).

The rates of pregnancy obtained in cows included in groups D and P were 33.3% (2/6) and62.5% (5/8), respectively. When the rates of pregnancy (32.9%) of cows inseminated at naturalestrus are concerned, the treatment in Exp 2 and 3 can be said to have resulted in acceptablepregnancy rates in the cows at diestrus or proestrus stages. But it seems that no pregnancy willoccur in cows at metestrus stages at the onset of treatment despite obtaining tightly synchronizedovulations, probably due to taking place of ovulations during early diestrus (which cannot be anappropriate period for fertilization) and/or the occurrence of luteal regression between the 9th and13th days post-AI.

The treatments resulted in ovulation in three of five cows (60.0%) at anestrus in Exp 2, buta functional CL did not develop after ovulation in two of these three cows. These data haveimplied that sufficient rates of pregnancy cannot be obtained after short estrus synchroniza-tion treatments due to insufficient luteal development in anestrous cows even if ovulation isinduced.

U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76 75

One of the best methods in evaluating the success of synchronization treatments is the com-parison of the pregnancy rates with the rates of inseminations in natural estruses. Comparablepregnancy rates were obtained from Exp 1 and 3 after the first and second AIs with thoseinseminated at natural estrus (P > 0.05).

Many researchers agree that follicular development should also be synchronized prior to syn-chronization of luteolysis with PGF. Probably for this reason, only a limited number of studieshave been conducted on short synchronization methods. Although many programs have beendeveloped for timed insemination, the stage of the estrus cycle at the beginning of treatments hassignificant affects on pregnancy rates (Murugavel et al., 2003). Reproductive efficiency in dairycattle has declined over the last five decades. In order to obtain higher pregnancy rates, graduallymore and more researchers consider that different treatments should be used at different periods ofthe cycle, following the determination of the estrus period of cows (Murugavel et al., 2003). Ourresults indicate that cows without an active CL at the onset of the study will render a decrease intotal pregnancy rates of a group of cows subjected to short synchronization treatments. Therefore,in order to obtain better pregnancy rates in total by the short estrus synchronization treatments,a different strategy can be employed. At any day, approximately 60–65% of non-pregnant cycliccows will be expected to have an active CL. These cows can be treated by Exp 3, and the syn-chronization can be initiated with a GnRH injection followed by the Exp 3 treatment 7 days laterin cows without a functional CL. This enables most of a group of cows to be inseminated soonerand no estrus detection. Since a GnRH injection 7 days prior to the treatment synchronizes thefollicular development, Exp 3 treatment can yield higher pregnancy rates in cows without anactive CL (Cirit et al., 2007).

5. Conclusion

It has been concluded from the study that (1) timing of GnRH or estradiol or both to beadministered after PGF has critical significance with respect to ovulation synchronization, (2)treatment in Exp 2 and 3 will result in high rate synchronized ovulations allowing TAI and (3)treatments in Exp 1 and 3 could result in comparable pregnancy rates after TAI of lactating dairycows relating to those inseminated at natural estrus, but the stage of the estrus cycle can havesignificant effects on pregnancy rates.

Acknowledgements

We are very thankful to General Directorate of Agricultural Enterprises (TIGEM) for openingthe doors of the farm for the study and, Directorate of Agricultural Enterprises of Turkgeldi ofTIGEM, General Director Agriculture Engineer Cuma Yıldırım, Head of Department of CattleRearing Agriculture Engineer Aydın Gorgundur and all the employees for their kind assistanceand support during the entire study. The authors also give their thanks to Vetas Veteriner ve TarımIlacları A.S. (Turkey) and general manager Prof. Dr. Huseyin Tan for their valuable contributionsto the study by generously providing the Dalmarelin and Dalmazin.

References

Bo, G.A., Adams, G.P., Pierson, R.A., Tribulo, H.E., Caccia, M., Mapletoft, R.J., 1994. Follicular wave dynamics afterestradiol-17� treatment of heifers with or without a progestogen implant. Theriogenology 41, 1555–1569.

76 U. Cirit et al. / Animal Reproduction Science 109 (2008) 65–76

Burke, C.R., Mussard, M.L., Grum, D.E., Day, M.L., 2001. Effects of maturity of the potential ovulatory follicle oninduction of oestrus and ovulation in cattle with oestradiol benzoate. Anim. Reprod. Sci. 66, 161–174.

Cavalieri, J., Kinder, J.E., De’ath, G., Fitzpatrick, L.A., 1997. Effect of 48 h treatment with 17�-oestradiol or progesteroneon follicular wave emergence and synchrony of ovulation in Bos indicus cows when administered at the end of a periodof progesterone treatment. Anim. Reprod. Sci. 46, 187–201.

Cirit, U., Ak, K., Ileri, I.K., 2007. New strategies to improve the efficiency of the ovsynch protocol in primiparous dairycows. Bull. Vet. Inst. Pulawy 57, 47–51.

Cordoba, M.C., Fricke, P.M., 2002. Initiation of the breeding season in a grazing-based dairy by synchronization ofovulation. J. Dairy Sci. 85, 1752–1763.

DeJarnette, J.M., Salverson, R.R., Marshall, C.E., 2001. Incidence of premature estrus in lactating dairy cows and con-ception rates to standing estrus or fixed-time inseminations after synchronization using GnRH and PGF2�. Anim.Reprod. Sci. 67, 27–35.

Diskin, M.G., Austin, E.J., Roche, J.F., 2002. Exogenous hormonal manipulation of ovarian activity in cattle. Domest.Anim. Endocrinol. 23, 211–228.

Folman, Y., Kaim, M., Herz, Z., Rosemberg, M., 1990. Comparison of methods for synchronization of estrous cycles indairy cows. II. Effects of progesterone and parity on conception. J. Dairy Sci. 73, 2817–2825.

Ginther, O.J., Kastelic, J.P., Knopf, L., 1989a. Composition and characteristics of follicular waves during the bovineestrous cycle. Anim. Reprod. Sci. 20, 187–200.

Ginther, O.J., Knopf, L., Kastelic, J.P., 1989b. Temporal associations among ovarian events in cattle during oestrous cycleswith two and three follicular waves. J. Reprod. Fertil. 87, 223–230.

Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone postbreeding andincreased milk urea N2 in lactating cows. J. Dairy Sci. 80, 1288–1295.

Lopez-Gatius, F., 1989. Effects of cloprostenol, human chorionic gonadotropin and estradiol benzoate treatment on estrussynchronization in dairy cows. Theriogenology 32, 185–195.

Lopez-Gatius, F., 2000a. Reproductive performance of lactating dairy cows treated with cloprostenol, hCG and estradiolbenzoate for synchronization of estrus followed by timed AI. Theriogenology 54, 551–558.

Lopez-Gatius, F., 2000b. Short synchronization system for estrus cycles in dairy heifers: a preliminary report. Theri-ogenology 54, 1185–1190.

Lopez-Gatius, F., Vega-Prieto, B., 1990. Pregnancy rate of dairy cows following synchronization of estrus with clo-prostenol, hCG and estradiol benzoate. J. Vet. Med. A 37, 452–454.

Lucy, M.C., Stevenson, J.S., 1986. Gonadotropin-releasing hormone at estrus: Luteinizing hormone, estradiol, andprogesterone during the periestrual and postinsemination periods in dairy cattle. Biol. Reprod. 35, 300–311.

Macmillan, K.L., Segwagwe, B.V.E., Pino, C.S., 2003. Associations between the manipulation of patterns of folliculardevelopment and fertility in cattle. Anim. Reprod. Sci. 78, 327–344.

Martinez, M.F., Adams, G.P., Bergfelt, D.R., Kastelic, J.P., Mapletoft, R.J., 1999. Effect of LH or GnRH on the dominantfollicle of the first follicular wave in beef heifers. Anim. Reprod. Sci. 57, 23–33.

Murugavel, K., Yaniz, J.L., Santolaria, P., Lopez-Bejar, M., Lopez-Gatius, F., 2003. Luteal activity at the onset of a timedinsemination protocol affects reproductive outcome in early postpartum dairy cows. Theriogenology 60, 583–593.

Noakes, D.E., 1997. Fertility and Obstetrics in Cattle, 2nd ed. Blackwell Science Ltd., Victoria, Australia.Plaizier, J.C.B., King, G.J., Dekkers, J.C.M., Lissemore, K., 1997. Estimation of economic values of indices for repro-

ductive performance in dairy herds using computer simulation. J. Dairy Sci. 80, 2775–2783.Rivera, G.M., Goni, C.G., Chaves, M.A., Ferrero, S.B., Bo, G.A., 1998. Ovarian follicular wave synchronization and

induction of ovulation in postpartum beef cows. Theriogenology 49, 1365–1375.Rounsaville, T.R., Oltenacu, P.A., Milligan, R.A., Foote, R.H., 1979. Effects of heat detection, conception rate, and culling

policy on reproductive performance in dairy herds. J. Dairy Sci. 62, 1435–1442.Taponen, J., Kulcsar, M., Katila, T., Katai, L., Huszenicza, G., Rodrıguez-Martınez, H., 2002. Short estrous cycles and

estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cyclicdairy cows. Theriogenology 58, 1291–1302.

Vasconcelos, J.L., Sartori, R., Oliveira, H.N., Guenther, J.G., Wiltbank, M.C., 2001. Reduction in size of the ovulatoryfollicle reduces subsequent luteal size and pregnancy rate. Theriogenology 56, 307–314.

Williams, G.L., Kotwica, J., Slanger, W.D., Olson, D.K., Tilton, J.E., Johnson, L.J., 1982. Effect of suckling on pituitaryresponsiveness to gonadotropin-releasing hormone throughout the early postpartum period of beef cows. J. Anim. Sci.54, 594–602.