J. Anim. Sci. 2003. 81:1562-1567
© 2003 American Society of Animal Science
The effects of varying the interval from follicular wave emergence to progestin withdrawal on follicular dynamics and the synchrony of estrus in beef cattle
M. D. Utt,
F. D. Jousan and
W. E. Beal1
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306
1 Correspondence:
3200 Litton Reaves Hall (phone: 540-231-4750; fax: 540-231-3010; E-mail:
wbeal{at}vt.edu).
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Abstract
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The objective of this experiment was to examine the effects of varying the interval from follicular wave emergence to progestin (controlled internal drug-releasing insert, CIDR) withdrawal on follicular dynamics and the synchrony of estrus. A secondary objective was to assess the effects of causing the dominant follicle (DF) to develop in the presence or absence of a corpus luteum (CL) on follicular dynamics and the synchrony of estrus and ovulation. The experiment was designed as a 2 x 2 x 2 factorial arrangement of treatments with injection of GnRH or estradiol-17ß and progesterone (E2 + P4) at treatment initiation, duration of CIDR treatment, and injection of PG (prostaglandin F2
) or saline at the time of CIDR insertion as main effects. Estrous cycles (n = 49) in Angus cows were synchronized, and treatments commenced on d 6 to 8 of the estrous cycle. Cows were randomly assigned to receive a CIDR containing 1.9 g of P4 for 7 or 9 d. Approximately half the cows from each CIDR group received either GnRH (100 µg) or E2 + P4 (1 mg of E2 + 100 mg of P4) at CIDR insertion. Cows in GnRH or E2 + P4 groups were divided into those that received PG (37.5 mg) or saline at CIDR insertion. All cows received PG (25 mg) 1 d before CIDR removal. Daily ovarian events were monitored via ultrasound. The intervals from GnRH or E2 + P4 treatment to follicular wave emergence were 1.4 and 3.3 d, respectively (P < 0.05). The interval from follicular wave emergence to CIDR removal was longer (P < 0.05) for cows treated with GnRH (6.6 d) than those treated with E2 + P4 (4.7 d) and longer (P < 0.05) for those fitted with a CIDR for 9 d (6.5 d) than those with a CIDR in place for 7 d (4.8 d). Cows treated with PG or GnRH at CIDR insertion had a larger (P < 0.05) DF at CIDR removal than those treated with saline or E2 + P4. Treatment with a CIDR for 9 d also resulted in a larger (P < 0.07) DF at CIDR removal compared with cows fitted with a CIDR for 7 d. The interval from CIDR removal to estrus was shorter (P < 0.05) in cows treated with PG than those treated with saline. The synchrony of estrus and ovulation was not affected by any of the treatments (P > 0.05). Altering the interval from follicular wave emergence to progestin removal or creating different luteal environments in which the DF developed caused differences in the size of the DF at CIDR removal and the timing of the onset of estrus, but it did not affect the synchrony of estrus or ovulation.
Key Words: Cattle • Estradiol • Follicles • Gonadotropin-Releasing Hormone • Progestogens • Synchronized Females
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Introduction
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Estrus synchronization can improve the efficiency of using AI in a beef cattle breeding program only if heat detection and AI can be confined to a short time following treatment. If estrus and ovulation are closely synchronized, mass mating of cows or heifers at a fixed time may be feasible. Progestin-based estrus synchronization systems have yielded a close synchrony of estrus, but fertility has been impaired by long-term progestin treatments (Hansel et al., 1961; Roche, 1974). Shortening the length of progestin treatment (
9 d) and inducing the development of a new dominant follicle (DF) during progestin treatment resulted in a close synchrony of estrus and normal fertility (Martinez et al., 2000).
To yield a close synchrony of estrus, the development of the DF must be controlled. Controlling the time of emergence of the DF has been facilitated by administering GnRH or an estrogen in conjunction with a progestin. Following administration of an estrogen and progestin, emergence of a new follicular wave (and DF) occurs approximately 3.4 d later, whereas emergence occurs about 1.5 d after GnRH administration (Bentley et al., 1998; Martinez et al., 2000).
The use of GnRH or an estrogen to control emergence of the DF during a progestin-based estrus synchronization system or altering the duration of progestin treatment creates differences in the interval from emergence of the DF to progestin removal. Those differences may affect the timing and synchrony of estrus. The objective of this experiment was to examine the effects of varying the interval from follicular wave emergence to progestin (controlled internal drug-releasing insert, CIDR) withdrawal on follicular dynamics and the synchrony of estrus and ovulation. A secondary objective was to assess the effects of causing the dominant follicle (DF) to develop in the presence or absence of a corpus luteum (CL) on follicular dynamics and the synchrony of estrus and ovulation.
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Materials and Methods
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Pretreatment Synchronization
Before administration of experimental treatments, estrus was synchronized in mature, nonlactating Angus cows with an i.m. injection of 100 µg of GnRH (Cystorelin, Merial, Iselin, NJ) followed by an i.m. injection of 25 mg of PG (Lutalyse, Pharmacia-Upjohn, Kalamazoo, MI) 7 d later. Cows were observed twice daily for signs of behavioral estrus after PG administration. On d 6, 7, or 8 after estrus detection cows were examined via transrectal ultrasonography to verify the presence of a CL and to record the location of the DF. Each cow with a CL detected by ultrasonography was randomly assigned to one of eight experimental treatments. The experiment was conducted in two trials involving 43 cows. Forty-nine estrus periods were detected across both trials. Six cows were used in both trials; however, the estrus periods recorded for each of those cows during each trial were treated as independent events.
Experimental Treatments
The experiment was designed as a 2 x 2 x 2 factorial arrangement of eight treatments with duration of exogenous progestin treatment, the presence or absence of a CL during progestin treatment, and the method of controlling follicular wave emergence as main effects (Figure 1
). On d 6, 7, or 8 after estrus detection, each cow was fitted with an intravaginal insert (CIDR, Vetrepharm Canada Inc., London, Canada) containing 1.9 g of progesterone for 7 or 9 d. One half of the cows in each CIDR treatment group received an i.m. injection of 1 mg of estradiol-17ß (E2 Sigma Chemical Co., St. Louis, MO) and an i.m injection of 100 mg of progesterone (P4; Sigma Chemical Co.) in vegetable oil at the time of CIDR insertion. The remaining half received an i.m. injection of 100 µg of GnRH. The presence or absence of a CL throughout the period of exogenous progestin treatment was controlled by administering saline or a series of luteolytic injections of PG at the beginning of the CIDR treatment. One half of the cows administered the E2 + P4 (subgroups A and C) and one half of the cows administered GnRH (subgroups B and D) received three injections of saline 12 h apart beginning at the time of CIDR insertion. The other half of the animals in each subgroup received three injections of PG (12.5 mg each) at 12-h intervals beginning at the time of CIDR insertion. One day before CIDR removal, all cows in each group received a single injection of PG (25 mg, i.m.). At the time of CIDR removal, cows were fitted with an electronic device (HeatWatch, DDX, Inc., Denver, CO) to detect standing events associated with the onset of estrus. HeatWatch transmitters remained on each cow until ovulation.
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Figure 1. Diagram of the 2 x 2 x 2 factorial arrangement of treatments with a 7- or 9-d controlled internal drug-releasing insert (CIDR), GnRH or estradiol-17ß and progesterone (E2 + P4), and PG or saline as main effects (n = number of estrus periods).
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Ultrasound Examinations
Beginning at the time of CIDR insertion, transrectal ultrasonography was performed once daily using a B-mode ultrasound machine (Sonovet 600, Universal Medical Systems, Inc., Bedford Hills, NY) equipped with a 7.5-MHz linear-array transducer. Daily recording of ovarian ultrasonography continued until the day that each cow exhibited behavioral estrus. Ultrasound observations were recorded on VHS videocassette and were later reviewed to characterize follicular development (Ginther et al., 1989). Emergence of a follicular wave was defined as the appearance of a pool of follicles 
3 mm in diameter following E2 + P4 or GnRH treatment. The growth rate of the DF was calculated from the average daily change in diameter of the DF beginning at treatment initiation and ending at estrus.
Ultrasonography also was used to determine the interval from the onset of estrus to ovulation. The onset of estrus was defined as the time of the first recorded standing event followed by two other standing events within 4 h. Ultrasound examinations commenced 24 h after the onset of estrus and were performed at 3-h intervals until ovulation. Time of ovulation was determined as the average time between the last observation that the DF was present and the time at which the DF had disappeared (ovulation).
Blood Collection and Progesterone RIA
Blood samples were collected once daily via tail venipuncture beginning at the time of CIDR insertion and ending the day the animal displayed behavioral estrus. Samples were allowed to stand at room temperature (20 to 23°C) for 6 h. Serum was separated by centrifugation at 1,678 x g for 20 min, and samples were stored frozen at -20°C. A validated (Holt et al., 1989), solid-phase RIA procedure (Coat-A-Count, Diagnostic Products, Los Angeles, CA) was used to determine serum progesterone concentrations. Samples were analyzed in duplicate in a single assay. The minimal detectable limit of progesterone for the assay was 0.02 ng/mL. The intraassay coefficient of variation was 9%.
Statistical Analysis
The experiment was designed as a 2 x 2 x 2 factorial arrangement of eight treatments with duration of CIDR treatment, E2 + P4 or GnRH treatment, and PG or saline treatment at the time of CIDR insertion as main effects. Because no effect of trial was detected in a preliminary analysis, data from both trials were combined and analyzed using the following procedures. Dependent variables, characteristics of the follicular development and the timing of estrus and ovulation, were analyzed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). All main effects and two-way interactions were examined as fixed sources of variation. In order to confirm the regression of the CL in cows treated with PG at the time of CIDR insertion, serum progesterone concentrations at 1 and 5 d following CIDR insertion in cows that received PG or saline at CIDR insertion were compared using the GLM procedure of SAS. The quadratic regression of interval from CIDR removal to estrus on the diameter of the DF at the time of CIDR removal was formulated using the REG procedure of SAS. The variances in the interval from CIDR removal to estrus or ovulation were used as a measure of the synchrony of estrus and ovulation, respectively, following treatment. Differences in variance associated with the interval from CIDR removal to estrus or ovulation due to main effects were determined using the F-test (Steele and Torrie, 1960).
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Results and Discussion
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Follicular Development
Follicular events were characterized using recordings of ultrasound observations beginning at the time of CIDR insertion and ending at estrus. Least squares means of characteristics of the follicular wave and DF are presented in Table 1. There were no two-way interactions (P > 0.10) among main effects for characteristics of the DF or the timing of estrus or ovulation following treatment. The timing of follicular wave emergence was significantly different between cows treated with E2 + P4 or GnRH at CIDR insertion. Emergence of a new follicular wave occurred an average of 3.3 d following E2 + P4 treatment, but 1.4 d following GnRH treatment. Because a new follicular wave emerged earlier (P < 0.05) in cows treated with GnRH, those animals had a longer (P < 0.05) interval from follicular wave emergence to CIDR removal than cows that received E2 + P4. Likewise, the interval from follicular wave emergence to CIDR removal was longer (P < 0.05) in cows that received a CIDR for 9 d compared with those with a CIDR in place for 7 d. The combined effects of administering different treatments to cause emergence of a new follicular wave and allowing the CIDR to remain in place for either 7 or 9 d led to a range in the interval from follicular wave emergence to CIDR removal of 2 to 9 d among individual cows. Varying the interval from follicular wave emergence to CIDR removal in turn created differences in size of the DF at CIDR removal, estrus, and ovulation.
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Table 1. Main effects of estradiol-17ß and progesterone (E2+ P4) or GnRH administration, 7- or 9-d controlled internal drug-releasing insert (CIDR) treatment, or PG or saline treatment on characteristics of the dominant follicle (DF)a
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Cows treated with GnRH at CIDR insertion had a larger DF at CIDR removal (P < 0.05), at estrus (P < 0.05), and at ovulation (P < 0.07) than those treated with E2 + P4. Treatment with a CIDR for a duration of 9 d also resulted in a longer interval from follicular wave emergence to CIDR removal than cows treated for 7 d and led to a larger (P < 0.07) DF at CIDR removal in cows treated with a CIDR for 9 d than those treated for 7 d. However, no differences existed between CIDR treatment groups in the size of the DF at estrus or at ovulation.
Size of the DF at CIDR removal, estrus, and ovulation was affected by treating cows with PG or saline at CIDR insertion. Treating cows with PG at CIDR insertion induced regression of the CL present at the initiation of treatment (primary CL). Conversely, cows that received saline at CIDR insertion maintained the primary CL until the time at which all cows received a luteolytic dose of PG on the day before CIDR removal. Cows that received PG at CIDR insertion, which regressed the primary CL, had a larger (P < 0.05) DF follicle present at CIDR removal, estrus, and ovulation than those that received saline at CIDR insertion. The greater size attained by the DF of cows treated with PG was the result of an increased (P < 0.05) growth rate of the DF compared to that recorded for cows that received saline at CIDR insertion (1.5 and 1.2 mm/d). The increased growth rate of the DF of cows treated with PG may have been due to the progesterone environment under which the DF developed. Numerous reports have indicated that the DF reached a larger diameter when it developed in the presence of subluteal concentrations of plasma progesterone (Savio et al., 1993; Sirois and Fortune, 1990). Cows treated with PG at CIDR insertion in the experiment had lower (P < 0.05) circulating concentrations of progesterone at 1 and 5 d following treatment initiation when compared to cows that received saline.
Cows that received GnRH at CIDR insertion, but not those that received E2 + P4, were expected to develop a secondary CL that elevated circulating concentrations of progesterone during treatment. Although most cows (22/24) that received GnRH developed a secondary CL that was visible during ultrasound examination, circulating concentrations of progesterone during treatment were not increased (P > 0.05) by the presence of a secondary CL.
The higher growth rate of the DF observed in cows that received PG might have been mediated by increased LH pulse frequency caused by subluteal concentrations of circulating progesterone. It has been reported that increased LH pulse frequency is associated with subluteal concentrations of circulating progesterone (Ireland and Roche, 1982; Roberson et al., 1989). The relationship between circulating concentrations of progesterone, LH pulse frequency, and DF growth rate is represented by events surrounding development of the first (anovulatory) follicular wave during the natural estrous cycle. Ginther et al. (1989) reported the DF of the first follicular wave exhibited a higher growth rate compared with the second (ovulatory) follicular wave. During the period of development of the first follicular wave, in the early luteal period, progesterone concentrations are low and LH pulse frequency is high; however, during development of the second follicular wave, in the midluteal period, progesterone concentrations are higher and LH pulse frequency is lower (Rahe et al., 1980). Although LH pulse frequency was not quantified in the current experiment, it was hypothesized that the increased growth rate of the DF of cows treated with PG was mediated by the effects of lower circulating levels of progesterone and increased LH pulse frequency.
Timing and the Synchrony of Estrus and Ovulation
The purpose of the experimental treatments was to create differences in the interval from follicular wave emergence to CIDR removal that caused differences in size of the DF at CIDR removal. It was hypothesized that cows possessing a larger DF at CIDR removal may have a shorter interval from CIDR removal to the onset of estrus than those with a smaller DF at CIDR removal. There was a linear (P < 0.001) and a quadratic P < 0.001) relationship between follicle diameter at CIDR removal and the interval from CIDR removal to estrus. In general, cows that possessed a larger DF at CIDR removal had a shorter interval to the onset of estrus than those that had a smaller DF, regardless of treatment (Figure 2). Savio et al. (1993) reported a similar relationship, in which cows that had a larger DF at luteolysis or progestin withdrawal exhibited estrus earlier. Despite the general relationhip between size of the DF at CIDR removal and the timing of estrus, the only treatment that significantly affected the timing of the onset of estrus was administration of PG or saline at CIDR insertion (Figure 3). Cows treated with PG at CIDR insertion had a larger (P < 0.05) DF at CIDR removal and a shorter (P < 0.05) interval to the onset of estrus than those that received saline at CIDR insertion.
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Figure 2. The regression of diameter of the dominant follicle at controlled internal drug releasing insert (CIDR) removal on the interval from CIDR removal to the onset of estrus for animals in all treatment groups.
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Figure 3. The mean interval (±SE) from controlled internal drug-releasing insert (CIDR) removal to estrus for cows treated with estradiol-17ß and progesterone (E2 + P4) or GnRH at CIDR insertion, CIDR treatment for 7 or 9 d, and PG or saline at CIDR insertion. Means within each main effect with different superscripts differ (P < 0.05).
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Although differences were created in the timing of estrus, the synchrony of estrus was not affected by experimental treatments. The synchrony of estrus was defined as the variation in the interval from CIDR removal to the onset of estrus. The amount of variation in the timing of the onset of estrus between E2 + P4 (358 h2) or GnRH (278 h2) treatments, between cows treated with a CIDR for 7 (280 h2) or 9 d (359 h2), and between cows receiving saline (309 h2) or PG (281 h2) was not different (P > 0.05). This finding was similar to that reported by Lane et al. (2001). In that experiment, heifers were fitted with a progesterone-releasing intravaginal device (PRID) for 8 d and were administered PG the day before PRID removal. At PRID insertion, heifers received either estradiol benzoate or GnRH. As in the current experiment, treatment with estradiol or GnRH would have led to differences in the time of follicular wave emergence and resulted in a difference in the interval from follicular wave emergence to progestin removal. However, the synchrony of estrus was not affected by treatment with estradiol benzoate or GnRH at PRID insertion. Similarly, Garcia and Salaheddine (2001) administered treatments that may have affected the interval from follicular wave emergence to progestin withdrawal. Cows were fitted with a progestin for a duration of 9 d and received estradiol valerate or estradiol-17ß at the time of or 2 d following progestin insertion. All animals received a luteolytic dose of PG at the time of progestin removal. Their experimental treatments would have caused at least a 2-d difference in the interval from follicular wave emergence to progestin removal. Although the type of estradiol altered the interval from progestin removal to ovulation, altering the time of estradiol-17ß treatment by 2 d did not affect the synchrony of ovulation relative to progestin removal. The synchrony of ovulation also was not affected by experimental treatments in the current experiment.
The synchrony of ovulation was defined as the variation in the interval from CIDR removal to ovulation. The amount of variation in the interval to ovulation between E2 + P4 (352 h2) and GnRH (312 h2) treatments, between cows treated with a CIDR for 7 (310 h2) or 9 d (357 h2), and between cows receiving saline (269 h2) or PG (326 h2) was not different (P > 0.05).
The interval from the onset of estrus to ovulation (32.0 ± 0.63 h) was not affected by experimental treatments. This suggests that timing of ovulation in beef cows used in this experiment was a consistent event relative to the onset of estrus. The timing of ovulation relative to the onset of estrus reported in this study was longer than the mean time (27.6 ± 5.4 [SD] h) in dairy cattle as reported by Walker et al. (1996). The difference in timing of ovulation relative to the onset of estrus recorded during the current study and by Walker et al. (1996) may be a function of differences in management (housing, daily routine) and behavior of dairy and beef cattle.
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Implications
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The results of this experiment indicate that differences in the size of the dominant follicle at the end of progestin treatment caused by altering the length of progestin treatment, differing the times of follicle emergence, or controlling endogenous progesterone levels can affect the timing of the onset of estrus, but they do not affect synchrony of estrus. Interpretation of these results should be limited to the treatments used in this experiment and the time during the estrous cycle when those treatments were applied. Other methods of improving the synchrony of estrus following progestin treatment should be investigated to enhance the potential use of mass mating by artificial insemination at a single fixed time.
Received for publication September 26, 2002.
Accepted for publication January 29, 2003.
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Literature Cited
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Abstract
Introduction
