J. Dairy Sci. 90:1281-1288
© American Dairy Science Association, 2007.
Ovarian Traits After Gonadotropin-Releasing Hormone-Induced Ovulation and Subsequent Delay of Induced Luteolysis in an Ovsynch Protocol1
J. S. Stevenson2,
M. A. Portaluppi and
D. E. Tenhouse
Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-0201
2 Corresponding author: jss{at}k-state.edu
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ABSTRACT
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Our objective was to determine whether delaying the PGF2
injection by 24 or 48 h after the first GnRH injection in an Ovsynch protocol (from a standard 7 d) altered ovarian characteristics in lactating dairy cows. Beginning 9 d after removal of a progesterone-releasing controlled internal drug release (CIDR) insert and injection of PGF2 (d 6.4 of the estrous cycle), 36 Holsteins (average body weight = 707 ± 12 kg and body condition score = 2.3 ± 0.1) were administered 100 µg of GnRH (81 ± 2 d in milk) and assigned randomly to receive a treatment injection of PGF2 7, 8, or 9 d later. Timed artificial insemination was performed at 48 h after PGF2 at which time a second injection of GnRH was administered. Ovarian structures were mapped by ultrasonography on d 0 (first GnRH injection); on d 2 to determine responses to the first GnRH injection; at PGF2 injection; and daily thereafter through 72 h after PGF2 to monitor ovulation of preovulatory follicles. Blood was collected on d 0, 2, at PGF2 injection, and at 24 and 48 h after PGF2 to monitor serum changes in estradiol-17ß (E2-17ß) and progesterone (P4). Based on serum P4 and ovarian exams, 2 cows were eliminated because of anestrus and their failure to ovulate a follicle in response to the first GnRH injection. Two other cows in which luteolysis failed to occur after PGF2 treatment also were eliminated. Final numbers of cows per treatment were: 7 d (n = 13), 8 d (n = 9), and 9 d (n = 10). Twenty-nine of 32 cows ovulated (90.6%) in response to the first GnRH injection. Of those cows not ovulating in response to the first GnRH injection, 2 had 1 original corpus luteum and 1 had 2 original corpora lutea. Despite a 24- or 48-h delay between first GnRH and PGF2 injections, the diameter (mm) and volume (mm3) of the ovulatory follicle did not differ among treatments: 14.3 ± 0.6 and 1,526 ± 62 at 7 d; 14.1 ± 0.8 and 1,479 ± 97 at 8 d; and 15.3 ± 0.9 and 1,490 ± 69 at 9 d. In all 32 cows, at least 1 follicle ovulated after treatment, but ovulation rates did not differ: 1.2 ± 0.1, 1.1 ± 0.1, and 1.3 ± 0.2, respectively, for the 7-, 8-, and 9-d treatments. Serum concentrations of E2-17ß did not differ among treatments. Four cows in the 7-d treatment were inseminated 24 h late and were excluded before assessing conception rates, which were 5/9 (55.6%), 5/9 (55.6%), and 1/10 (10%), respectively. We conclude that delaying PGF2 injection by 24 h had no effect on outcomes.
Key Words: Ovsynch • ovulation • follicle • timing of luteolysis
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INTRODUCTION
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Before the advent of the Ovsynch protocol (injection of GnRH 7 d before and 48 h after an injection of PGF2, with 1 timed AI at 12 to 16 h after the second GnRH injection; Pursley et al., 1997), 33% of all dairy operations were using some type of systematic PGF2 program to synchronize estrus (National Animal Health Monitoring System, 1996), with rates of use greater for operations with 200 or more cows (50.2%) than for those with 100 to 199 cows (45%) or less than 100 cows (31.1%). Since then, timed AI (TAI) protocols likely have become even more popular among dairy producers and are used in nearly 10% of all US dairy herds (Lucy, 2001).
Development of synchronized ovulation was based on earlier reports in which a new follicular wave was initiated in response to an injection of GnRH 6 to 7 d before PGF2-induced luteolysis (Thatcher et al., 1989; Twagiramungu et al., 1995; Pursley et al., 1997). Emergence of a new follicular wave in response to GnRH led to greater homogeneity of ovarian follicular inventories among cows at the time of induced luteolysis (Twagiramungu et al., 1995). Improved synchrony of estrus resulting from coordinated follicular maturation and luteal regression (after administering GnRH 7 d before PGF2) was first demonstrated in dairy heifers (Thatcher et al., 1989), and later in lactating dairy (Stevenson et al., 1999) and beef cattle (Twagiramungu et al., 1992a,b). In consequence, GnRH treatment of cows 6 to 7 d before induced luteolysis resulted in 70 to 83% of heifers or cows in estrus during a 4-d period (Thatcher et al., 1989; Twagiramungu et al., 1992a,b). Furthermore, using a GnRH agonist 6 d before PGF2-induced luteal regression improved precision of estrus that occurred without any detrimental effect on fertility of beef cows (Twagiramungu et al., 1995).
Administration of GnRH reduces occurrence of estrus during 6 to 7 d after GnRH injection (Thatcher et al., 1989; Twagiramungu et al., 1992a,b). Reduced estrus during the post-GnRH period occurs because of ovulation of the dominant follicle and formation of a new or ancillary corpus luteum (CL) during the luteal phase (Twagiramungu et al., 1994a,b; Pursley et al., 1995), resulting in decreased peripheral concentrations of serum estra-diol-17ß (E2-17ß; Twagiramungu et al., 1994b). In lactating dairy cows, the average incidence of ovulation is about 64% when cows are injected across various stages of the estrous cycle (Vasconcelos et al., 1999).
Once a new dominant follicle is selected, concentrations of E2-17ß increase, LH pulses increase, and the selected dominant follicle becomes the preovulatory follicle. Maximum concentrations of E2-17ß in serum preceding ovulation, however, are 30% less in lactating dairy cows than in nulliparous heifers, even though ovulatory follicles are 13% greater in diameter (Sartori et al., 2004). Further, maximum concentrations of progesterone (P4) are 30% less in cows than in heifers, despite cows having 53% more luteal tissue (Sartori et al., 2004). Discrepancies between sizes of ovarian structures and serum steroid concentrations may result from greater rates of steroid metabolism in lactating dairy cows than in heifers (Sangsritavong et al., 2002) and in cows of various milk-producing abilities (Lopez et al., 2004).
Reduced serum steroid concentrations may have numerous potential physiologic consequences that compromise fertility in lactating cows. We hypothesized that greater concentrations of serum E2-17ß in lactating cows may occur when using the Ovsynch protocol if PGF2-induced luteolysis is delayed after the first GnRH injection. Our objective was to determine whether lengthening the interval between the first GnRH injection and PGF2 from 7 d to 8 or 9 d might result in greater serum concentrations of E2-17ß and larger follicles, possibly leading to increased fertility after a TAI.
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MATERIALS AND METHODS
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Herd Management
The experiment was conducted at the Kansas State University Dairy Teaching and Research Center, with 36 lactating Holstein cows that calved between August and September 2004 and had an average BCS of 2.3 ± 0.1. Daily test-day milk yield of these cows nearest to the day of first AI averaged 49.6 ± 1.7 kg (3.5% fat and 3.0% protein) and 305-d mature-equivalent yield averaged 15,356 ± 429 kg. Cows were housed in covered freestalls bedded with sand, and were fed thrice daily a TMR that met or exceeded NRC (2001) requirements for lactating cows. The TMR consisted of 30% chopped alfalfa hay, 19% wet corn gluten meal, 15% corn silage, 9.3% whole cottonseed, 4.4% solvent-extracted soybean meal, 3.3% expeller soybean meal, 13% corn grain, 1.3% menhaden fish meal, 1% sugar cane wet molasses, and 3.7% mineral-vitamin premix. Cows had ad libitum access to fresh water. Pens were covered with shade cloth, and water was applied by sprinklers 6 times per hour for 1 min during May to October.
Experimental Design
Beginning at 65 ± 2 DIM, estrous cycles were synchronized in lactating dairy cows (BW = 707 ± 12 kg) by applying a P4-releasing, intravaginally placed, controlled internal drug release (CIDR) insert (Eazi-Breed CIDR, Pfizer Animal Health, New York, NY) for 7 d, plus 25 mg of PGF2 (Lutalyse, Pfizer Animal Health, New York, NY) given 24 h before removal of the CIDR insert (Figure 1
). Nine days after removal of the CIDR insert (approximately d 6 of the estrous cycle), cows received 100 µg of GnRH (Cystorelin, Merial Ltd., Iselin, NJ) and then were allocated randomly to 1 of 3 treatments in which they received 25 mg of PGF2 at d 7, 8, or 9 after the first GnRH injection (d 0; 81 ± 2 DIM).
Inseminations were administered at 48 h after PGF2 (91 ± 2 DIM), at which time the second 100-µg injection of GnRH was administered. Pregnancy was diagnosed 32 to 34 d after TAI by using transrectal ultrasonography (5.0-MHz linear-array transducer, Aloka 500V; Corometrics Medical Systems, Inc., Wallingford, CT). A positive diagnosis included confirmation of a CL, uterine fluid, and an embryonic heart beat.
Ultrasonography was conducted on d 0, d 2, and daily thereafter, beginning with PGF2 treatment through 72 h after PGF2, to monitor ovarian structures. Ovarian follicles were mapped and sized on d 0 and 2 to determine responses to the first GnRH injection. In subsequent scans, all follicles were mapped and sized to monitor the dominant ovarian follicle that developed after the first GnRH injection, and that later became the preovulatory follicle. Follicles were assumed to be spherical. Diameter of structures was determined by averaging their largest cross-sectional width and height measured by ultrasound electronic calipers. Volume of follicles and CL was calculated as follows:
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Blood samples were collected from a coccygeal vessel on d 0, d 2, and then daily beginning with PGF2 treatment through 72 h after PGF2, d 14, and d 21. The samples were stored on ice, and serum concentrations of P4 were later quantified by RIA (Skaggs et al., 1986). Intra- and interassay coefficients of variation for the P4 RIA were 7.7 and 11.4%, respectively, for a sample that averaged 3.2 ± 0.1 ng/mL. In blood samples collected on d 0, d 2, and daily beginning with PGF2 treatment through 48 h after PGF2, serum concentrations of E2-17ß were quantified in a single RIA (Perry et al., 1991). To increase assay sensitivity, modifications included incubating dried serum extracts with E2-17ß antiserum for 24 h at 20°C before adding 125I-labeled E2-17ß and incubating assay tubes for an additional 6 h at 20°C. After that 6-h incubation, bound E2-17ß was separated from free E2-17ß by adding cold (5°C) dextran-coated charcoal. The intraassay coefficient of variation was 7.8% for a sample that averaged 18.9 ± 0.6 pg/mL. Sensitivity of the assay was <0.05 pg/tube.
Statistical Analyses
All analyses were performed by using SAS software (SAS Inst. Inc., Cary, NC). All pretreatment variables were analyzed by ANOVA (GLM procedure) by applying a model that consisted of only lactation number (1, 2, and 3+). Posttreatment continuous variables were analyzed similarly with a model that included treatment (7-, 8-, or 9-d interval between the first GnRH injection and the treatment PGF2 injection), lactation number, and treatment by lactation number interaction. Binomial data (percentages and proportions) were analyzed by logistic regression (GENMOD procedure) using the same model.
Changes in follicular diameters, follicular volumes, and serum concentrations of P4 and E2-17ß, across days of the experiment or standardized to the day of PGF2 treatment, were analyzed by using a mixed model procedure (MIXED procedure) to account for repeated measures. Because lactation number had no effects on treatment outcomes, the model consisted of treatment, cow within treatment (random), day, and treatment by day interaction. The covariance structure varied with model. The final model chosen for each variable produced the smallest Akaike’s information criteria (AIC). Most covariance structures were heterogeneous autoregressive, but a few were heterogeneous compound symmetry. A priori contrasts were constructed to compare treatment on d 7 (control) with each of the other 2 treatments.
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RESULTS
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Pretreatment Responses
Estrus was expressed by 22 of 36 cows after removal of the CIDR insert, as detected by electronic, pressure-sensitive HeatWatch transmitters (CowChips LLC, Denver, CO). Interval to estrus after CIDR insert removal was 52.1 ± 4.6 h (ranged from 24 to 114 h). Average number of standing events was 3.8 ± 0.7, with a total duration of standing time of 9 ± 2 s. Duration of estrus was 6.3 ± 1.1 h. Of those cows that expressed estrus, days from estrus to injection of GnRH was d 6.4 ± 0.2 (range of d 3.7 to 7.4).
Ovarian responses and characteristics of cows before treatments were imposed are summarized in Table 1. Of 36 cows treated, 4 cows were found anestrous by using information from ovarian scans, lack of expressed estrus after CIDR insert removal, and serum concentrations of P4. Two of those 4 cows failed to ovulate in response to GnRH. The CL failed to regress in response to PGF2 treatment in 2 additional cows. Therefore, 4 cows (2 anestrous cows and 2 cows having no luteolysis) were eliminated from the study, leaving a total of 32 cows in the 3 treatments.
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Table 1. Ovarian responses of 36 cows assigned to treatment assessed at 7, 8, or 9 d after the first GnRH injection of a modified Ovsynch protocol
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In the 32 cows studied, pretreatment responses summarized in Table 2 include numbers of original, induced, and total CL; total numbers of follicles 
10 mm in diameter before GnRH; and diameter and volume of the largest follicle and resulting diameter and volume of the CL that formed after ovulation (assessed 7 d after the first injection of GnRH). In addition, in 29 of 32 (90.6%) cows, at least 1 follicle ovulated in response to GnRH. Of the 3 cows not ovulating in response to the first GnRH injection, 1 had 2 original CL and 2 had 1 original CL.
Distribution of CL is shown in Figure 2 based on the number of CL per cow assessed before and 7 d after GnRH. Two anestrous cows had no CL before GnRH, whereas 1 cow had 3 CL before GnRH was administered. One new CL was induced in 23 cows and 2 new CL in 4 cows, so the number of total CL ranged from 1 CL in 4 cows, 2 CL in 17 cows, and 3 or more CL in 11 cows.
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Figure 2. Distribution of original corpus luteum (CL) and those induced by the first GnRH injection, grouped by subsequent treatments of PGF2 administered 7, 8, or 9 d after GnRH.
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Treatment Responses
Changes in average diameter of the largest (putative dominant) follicle, monitored after the first GnRH injection, that subsequently ovulated are illustrated in Figure 3. Small differences were detected among treatments in the diameter of the largest follicle from d 2 to 10. Cows in the 9-d treatment had slightly smaller (P < 0.05) follicle diameters on d 2 to 9. Average diameter or average volume of the resulting preovulatory follicles assessed at 72 h after PGF2, did not differ among treatments (Table 3). When diameters (Figure 4) and volumes (not shown) of the preovulatory follicles were standardized to the day when PGF2 treatment was administered, no differences were detected among treatments. At least 1 follicle ovulated per cow (range of 1 to 2), and ovulation rates did not differ among treatments (Table 3).
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Figure 3. Changes in average diameter of the dominant follicle (DF) that eventually ovulated after each of 3 treatments in which PGF2 was administered either 7 d (n = 13), 8 d (n = 9), or 9 d (n = 10) after the first GnRH injection (d 0).
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Table 3. Largest diameter and volume of preovulatory follicles that ovulated after treatment in response to the second GnRH injection and resulting ovulation rate
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Serum concentrations of E2-17ß differed (P 
0.05) among treatments when plotted by days since the first GnRH injection (Figure 5; upper panel). On d 8, and because of earlier treatment-induced luteolysis, cows in the 7-d treatment (24 h after PGF2) already had greater concentrations of E2-17ß than did cows in the 8- and 9-d treatments; this trend continued to d 9. Once concentrations of E2-17ß were expressed relative to days since PGF2, treatment, differences were no longer evident (Figure 5; bottom panel).
A treatment by day interaction (P < 0.001) was detected for serum concentrations of P4 for d 0 to 9 (Figure 6; upper panel). Concentrations of P4 were greater (P < 0.05) on d 0 to 9 for cows in the 8- and 9-d treatments than for those in the 7-d treatment. When concentrations of P4 were reanalyzed as a percentage of concentrations on d 0, similar patterns of P4 emerged and yielded a significant interaction that was likely caused by differences in designed timing of PGF2-induced luteolysis. Further, patterns of luteal regression, as assessed by decreasing concentrations of P4 at 24 and 48 h after PGF2, were not different when expressed relative to day of PGF2 treatment injection (Figure 6; bottom panel). All 32 cows subsequently ovulated and formed new CL (Table 3
) after treatment as is further evidenced by increased concentrations of P4 in serum at 14 and 21 d after the first GnRH injection (Figure 6; top panel).
Four cows in the 7-d treatment were inseminated 24 h late (i.e., not according to protocol). Of the remaining cows, 5 of 9 cows (55.6%) conceived in the 7-d treatment, and 5 of 9 cows (55.6%) conceived in 8-d treatment, compared with only 1 of 10 cows (10%) in the 9-d treatment. Conception rates of individual 7- and 8-d treatments tended (P = 0.07) to differ from that of the 9-d treatment. Two of the 4 cows (50%) that failed to ovulate after the first GnRH injection conceived, whereas 9 of 24 cows (37.5%) conceived that ovulated at least 1 follicle after the first GnRH injection.
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DISCUSSION
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Our model allowed us to test the hypothesis that delaying luteolysis up to 48 h after a normal 7-d interval between GnRH and PGF2 in an Ovsynch protocol would increase follicle size and E2-17ß secretion. The uniform group of cows tested included 22 cows that were known to be between d 3.7 and 7.4 of the estrous cycle, and 29 of 32 cows had at least 1 follicle ovulate in response to the first GnRH injection. The large incidence (90.6%) of ovulation in response to the first GnRH injection is consistent with earlier reports for cows at similar stages of the estrous cycle (Vasconcelos et al., 1999).
We hypothesized that greater concentrations of serum E2-17ß in lactating cows might occur when using the Ovsynch protocol if PGF2-induced luteolysis was delayed after the first GnRH injection. Our treatments, however, failed to produce larger follicles and did not result in greater concentrations of serum E2-17ß. Neither diameter or volume of the preovulatory follicle nor concentrations of E2-17ß were increased by lengthening the period of dominance for the newly recruited dominant follicle.
In heifers, restricting duration of dominance of the preovulatory follicle to 4 d at the onset of estrus led to more precise estrus and a higher conception rate (Austin et al., 1999). This duration of dominance would be most consistent with cows in our 7- or 8-d treatments. In a recent review (Tenhagen, 2005), it was concluded that intervals of 7 d between GnRH and PGF2 were most effective relative to fertility when the second GnRH followed PGF2 by 48 h and insemination occurred 16 to 20 h later.
Clear differences seem to exist between lactating and nonlactating cows with regard to size of ovarian structures and their steroid-secreting capacity, indicating that lactation per se, age, or both, creates these differences. Lactating cows have a much greater incidence of ovulation failure after luteolysis, more multiple ovulations, lower serum E2-17ß concentrations, and larger follicles and CL than do nulliparous heifers (Sartori et al., 2004). Moreover, expression of estrus (Dransfield et al., 1998) and estrus-detection rates (Stevenson, 2001) are reduced in lactating dairy cows probably as a result of less estrogen exposure during the periestrual period. Among lactating cows, cows producing more milk have shorter durations of estrus, fewer numbers of standing events, and less total standing time than do lower-producing cows (Lopez et al., 2004). It seems clear that lactating cows require a larger follicle and greater E2-17ß production to achieve adequate circulating concentrations of E2-17ß necessary to produce an LH surge and ovulation compared with nonlactating cows (Sartori et al., 2002). In our study, concentrations of E2-17ß were not increased by prolonged growth and dominance of the newly recruited follicles after the first GnRH injection.
Discrepancies between sizes of ovarian structures and serum steroid concentrations may be due to greater rates of steroid metabolism in lactating dairy cows than in heifers (Sangsritavong et al., 2002). Further, it is clear that these differences cannot be explained by differences in patterns of follicular waves (Sartori et al., 2004). Based on those studies (Sangsritavong et al., 2002; Sartori et al., 2004), it seems that the lactating cow metabolizes E2-17ß more quickly, resulting in inadequate peripheral concentrations of E2-17ß to support events associated with normal fertility. It is not known, however, whether E2-17ß biosynthesis by antral follicles may be inferior because of lactation. Further, high-producing cows had lower E2-17ß concentrations than low-producing cows, despite having larger preovulatory follicles (Lopez et al., 2004). Interestingly, E2-17ß concentrations were not correlated with diameter of the preovulatory follicle, but milk production was correlated with both E2-17ß concentrations and diameter of the preovulatory follicle. It is clear that prolonging the life span of the dominant follicle (preovulatory follicle) in our study failed to increase either follicle size or peripheral concentrations of serum E2-17ß.
Other attempts to increase concentrations of estrogen during the periestrual period have been made. Studies in which 0.5 to 1 mg of estradiol cypionate (ECP) was injected in combination with ovulation synchronization indicated that injecting ECP 24 h after PGF2 may not increase conception rates in lactating dairy cows (Pancarci et al., 2002; Stevenson and Tiffany, 2004; Stevenson et al., 2004). Further, when 0.25 mg of ECP was administered at 48 h after PGF2, concomitant with the second GnRH injection of Ovsynch, concentrations of serum E2-17ß were greater at 6 and 12 h after injection than in controls, which only received GnRH, but conception rates did not differ between treatments (Sellars et al., 2006).
Lactating dairy cows were administered 1 mg of E2-17ß (shorter acting and shorter half-life estrogen than ECP; Vynckier et al., 1990) 8 h before the second GnRH injection and compared with cows treated with an Ovsynch-like protocol, in which the second GnRH injection was delayed to 54 h after PGF2, and TAI occurred 16 h later (Souza et al., 2005). Treatment with E2-17ß tended to improve conception rate in cows having lower BCS (2.5), cows in their first lactation, and cows ovulating medium-size (15 to 19 mm) follicles compared with cows treated with Ovsynch alone.
Injection of estrogen or increased exposure of cows to endogenous estrogen increased the incidence of estrus in previous studies (Pancarci et al., 2002; Stevenson et al., 2004), which may benefit fertility in some instances (Souza et al., 2005). Furthermore, other positive benefits of estrogen include induction of normal estrual characteristics, such as mucus secretion, uterine tone, and resulting sexual behavior. These traits are positive indicators of estrus for inseminators because they validate the likelihood of the cow being in estrus (Pancarci et al., 2002).
Although too few cows were inseminated to test differences in conception rates, only 1 of 10 cows in the 9-d treatment conceived when inseminated at 48 h after PGF2 compared with 5 of 9 cows conceiving in each of the 7- or 8-d treatments. In cows whose estrous cycles are presynchronized before Ovsynch, reproductive outcomes may not be reduced when PGF2 injection is delayed by 24 h from the standard 7-d period between GnRH and PGF2 injections, but 48 h may be too long to prevent a potentially reduced conception rate.
In conclusion, prolonging the life span of a newly recruited dominant follicle in the presence of a functional CL by 24 to 48 h, from the standard 7-d interval between the first GnRH injection of the Ovsynch protocol and the injection of PGF2, failed to increase serum concentrations of E2-17ß or the diameter of that dominant follicle. Further study is warranted to verify whether delaying PGF2 injections by 24 h has no effects on fertility and whether PGF2 injections may be given late (d 8), when not given at d 7 as planned.
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ACKNOWLEDGEMENTS
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The authors thank support staff at the Kansas State University Dairy Teaching and Research Center for their able assistance in conducting this study, particularly M. Scheffel and W. Jackson. We thank Irene Vanderwerff for assistance with laboratory procedures.
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FOOTNOTES
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1 Contribution no. 07-004-J, Kansas Agricultural Experiment Station, Manhattan. 
Received for publication July 26, 2006.
Accepted for publication October 11, 2006.
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ABSTRACT
INTRODUCTION
