Use of a small dose of estradiol benzoate during diestrus to synchronize development of the ovulatory follicle in cattle 1

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1 Use of a small dose of estradiol benzoate during diestrus to synchronize development of the ovulatory follicle in cattle 1 C. R. Burke*,2, M. L. Day, C. R. Bunt, and K. L. Macmillan *Dairying Research Corporation, Private Bag 3123, Hamilton, New Zealand; Department of Animal Science, The Ohio State University, Columbus 43210; InterAg, Hamilton, P. O. Box 20055, New Zealand; and Department of Veterinary Science, University of Melbourne, Werribee 3030, Australia ABSTRACT: We tested the hypothesis that a small dose of estradiol benzoate (EB) at the midstage of the estrous cycle in cattle would synchronize the subsequent pattern of ovarian follicular development, estrus, and ovulation. Nonlactating Friesian cows received either 1 mg of EB i.m. on d 13 of the estrous cycle (T; n = 12; estrus = d 0) or served as untreated controls (C; n = 12). Their ovaries were examined daily with transrectal ultrasonography from d 7, and blood samples were collected 0, 2, 4, 8, 24, and 48 h after treatment on d 13. Plasma concentrations of estradiol-17β were elevated to 12 pg/ml during the initial 24 h following treatment, compared with a baseline of 1 pg/ml in untreated controls (P <.001). Progesterone concentrations in cows of the T group declined between 24 and 48 h after treatment ( 3.2 ±.5 ng/ml) compared with little change in concentrations of progesterone in cows of the C group at this time (P <.01). This difference was coincident with an earlier time to regression of the corpus luteum in cows of the T group. Disregarding treatment groups, the second dominant follicle of the estrous cycle (DF2) emerged on d 10.6 ±.3 and was 9.4 ±.4 mm in diameter on d 13. Further growth of the DF2 was halted by EB treatment on d 13. Cessation of growth occurred irrespective of whether the DF2 was in the early or late growth phase, and a new follicular wave emerged 4.5 ±.2 d later. The dominant follicle from this wave (DF3) ovulated 5 d after emergence in most cases. During the estrous cycle of every cow in the T group, there were three waves of follicular development (3-wave), whereas the ratio of 2:3 waves of follicular development in cows of the C group was 1:3. Consequently, the interval from emergence to ovulation of the ovulatory dominant follicle in cows of the C group ranged from 3 to 11 d. The dynamics of ovarian follicular wave development during the estrous cycle can be strategically manipulated by treating with a small dose of EB to synchronize proestrous development of the ovulatory follicle. Key Words: Cattle, Estradiol, Graafian Follicles, Estrus, Synchronization 2000 American Society of Animal Science. All rights reserved. J. Anim. Sci : Introduction Managerial and economic efficiencies of cattle production systems that are seasonal in nature are most likely to benefit from having a compact calving pattern, which can be achieved through estrus synchronization. Synchronization may also be the only practical means for facilitating the use of AI for beef cow operations. 1 Research was supported by funding through the Joint Venture Agreement between InterAg and the Dairying Research Corporation. We thank Bid Clark and Gwyn Verkerk for technical assistance; Trish O Donnell, Eleanor Smith, and Rachel Stevenson for laboratory support; and Rhonda Hooper for statistical analyses. 2 To whom correspondence should be addressed: Department of Animal Science, The Ohio State University, Columbus OH (phone: 614/ ; fax: 614/ ; burke.205@osu. edu). Received March 10, Accepted July 28, The potential of synchronized AI to increase breeding efficiency can be further extended by strategically manipulating return-to-service AI in dairy (Macmillan and Burke, 1996) or beef (Penny et al., 1997) cows. In fact, it may be necessary to consider such an extension when there is an insufficient number of bulls to cope with a large number of cows at return-to-service when estrus synchrony is used. In a study with dairy cattle in commercial herds, Macmillan et al. (1997) reported that a small dose of estradiol benzoate (1 mg EB) injected 12, 13, or 14 d after a synchronized estrus and AI partially synchronized return-to-service to a 9- to 10-d interval from EB treatment. Fertility at the estrus associated with return-to-service was also increased by treatment, and pregnancy rates to the initial AI remained unchanged. Because estradiol has been reported to promote a new wave of ovarian follicular emergence within 4 to 5 d (Bo et al., 1994; Burke et al., 1999), partial synchrony and increased fertility at the estrus associated with 145

2 146 Burke et al. return-to-service are probably a consequence of perturbed patterns of follicular development during the proestrous period. Our objective for this experiment was to verify that partial synchrony at the estrus associated with returnto-service, as reported by Macmillan et al. (1997), was associated with the effect of injecting 1 mg of EB to promote follicular wave turnover as well as synchronous development of ovulatory follicles among cows. Materials and Methods Each of 24 nonlactating Friesian cows aged between 3 and 10 yr and weighing 500 ± 12 kg had a progesteronereleasing device containing 1.9 g of progesterone (Eazi- Breed CIDR, InterAg, Hamilton, New Zealand) inserted into the vagina for 7 d. A PGF 2α analog (500 µg of cloprostenol sodium; Estrumate, Pitman-Moore, Upper Hutt, New Zealand) was injected i.m. on the day before insert removal. Every cow was observed in estrus within a 24-h period (d 0) beginning 48 h after insert removal. Detection of estrus was performed by visual observation of estrous behavior at four hourly intervals during the daytime and aided by the use of the technique of applying paint and an aerosol raddle to the base of the tail (Macmillan et al., 1988). The ovaries of each cow were examined daily with transrectal ultrasonography using a 7.5-MHz probe (Aloka DX210, Medtel, New Zealand) from d 7 to subsequent ovulation. The diameter and location of each corpus luteum (CL) and every follicle 3 mm in diameter were recorded. The largest follicle present in the ovaries at d 7 was assumed to be the first dominant follicle (DF1) of the estrous cycle. The largest follicles that subsequently emerged were defined as second (DF2) or third (DF3) dominant follicles, respectively. The day of new follicle wave emergence was defined as the day on which the dominant follicle was retrospectively traced to be 4 to 5 mm in diameter. Each estrous cycle was described as having two or three waves of follicular development if the DF2 or DF3 ovulated, respectively. On d 13 of the estrous cycle, 12 cows were injected with 1 mg of EB i.m.(t; CIDIROL injection, InterAg, Hamilton, New Zealand). The other 12 cows served as untreated controls (C). Allocation to treatments was balanced for stage of maturity of the DF2, which typically emerged soon after d 10. Blood samples were collected from each cow at 0, 2, 4, 8, 24, and 48 h relative to treatment on d 13. These samples were taken from a coccygeal vessel, collected into evacuated tubes containing anticoagulant, and immediately placed on ice for up to 1 h until centrifugation at 1,500 g for 20 min. The plasma samples were then stored at 20 C until hormone analyses. Concentrations of estradiol-17β in plasma samples were determined in samples collected from all treated cows and from four untreated contemporaries (three with three waves of follicular development and another with two waves of follicular development) using a modified RIA as described by Prendiville et al. (1995). Intraassay CV were 25 and 3.5% for standard concentrations of 1.9 and 12.2 pg/ml, respectively. The interassay CV (n = 3) for the same standards were 23 and 5%. The minimum detectable concentration was.7 pg/ml. Concentrations of progesterone were determined in all plasma samples from all cows with a direct RIA method using commercially prepared, solid phase [ 125 I]progesterone kits (Coat-A-Count, DPC, Los Angeles, CA). Intraassay CV were 12.4, 5.2, and 8.5% for standard concentrations of.4, 2.8, and 4.0 ng/ml, respectively. Interassay CV (n = 2) were 13.6, 5.1, and 1.1% for the same standards, respectively. The minimum detectable concentration was.17 ng/ml. The effects of treatment, time, and the treatment time interaction on diameter of ovarian structures and hormone profiles were analyzed using the MIXED procedure with repeated measures analysis included in the model (SAS, 1996). The repeated measures model was Y ijk = µ + h j + C i + t k + (ht) jk + e ijk, where y ijk is the observation on the i th cow in the j th hour on the k th treatment, µ is the overall mean, C i is a random effect for the i th cow with AR(1) covariance structure, h j is the j th hour, t k is the k th treatment, (ht) jk is the hour treatment interaction term, and e ijk is the residual error term (e ijk N [0, σ 2 ]). Changes in concentrations of estradiol-17β with time were analyzed on a square root scale to stabilize the variance, but mean values are presented on the original scale. Within treatment groups, the DF2 were categorized as less than 10 mm (early growth) or at least 10 mm (late growth) in diameter on d 13 to test the effects of treatment on stage of follicle maturity between d 11 and 17. Effects of treatment on the timing of the emergence of the DF3, the subsequent interval to ovulation, day of ovulation, and interestrous intervals among cows having estrous cycles with three waves of follicular development were tested using the Proc T Test procedure in SAS. This procedure also tested homogeneity of the variances using an F-test. The comparative diameter of the DF3 between cows with three waves of follicular development of the T and C groups was compared on a daily basis by normalizing day of emergence and assigning diameter of the DF3 at this time as a covariate. Data are expressed as the mean ± SEM. Results Plasma Estradiol-17β and Progesterone Maximum elevation in concentrations of estradiol- 17β in plasma in the cows of the T group were observed in plasma collected 4 h after the 1-mg EB injection (12.6 ± 1.6 pg/ml) and remained elevated at approximately 12 pg/ml until 24 h (Figure 1) after the injection. Mean concentrations of estradiol-17β at 48 h (6.3 ±.6 pg/ml) were half that of peak concentrations observed between 4 and 24 h (P <.01) after injection, but they had not returned to basal concentrations of approximately 1

3 Ovarian response to estradiol benzoate 147 Figure 1. Mean concentrations of estradiol-17β in plasma of cows injected with 1 mg of estradiol benzoate immediately after the 0-h sample (T group; n = 12) compared with untreated controls (C group; n = 4). Mean values are greater for the T group (P <.01) at all times beyond the 0-h sample. a,b,c Data points for the T group with uncommon superscript letters differ (P <.05) when compared using repeated measures analysis on a square root scale. Figure 2. Mean concentrations of progesterone in plasma of cows injected with 1 mg of estradiol benzoate immediately after the 0-h sample (T group; n = 12) compared with untreated controls (C group; n = 12). pg/ml as measured in four untreated contemporaries during the 48-h sampling period (P <.01; Figure 1). Average concentrations of progesterone were greater (P <.01) in cows of the T group before the treatments were administered and also during the first 24 h after treatment (Figure 2). Concentrations of progesterone in cows of the T group at 48 h after EB injection (6.7 ±.5 ng/ml) was less (P <.01) than at 24 h (9.9 ±.7 ng/ml). A treatment time interaction (P <.05) was observed as concentrations of progesterone in cows of the C group remained stable between these same time points (Figure 2). Dominant Follicle Development, Follicle Wave Patterns, Estrus, and Ovulation Average diameters of the DF1 were not different between cows of the C and T groups on any day of the estrous cycle. The DF2 emerged on d 10.6 ±.2, and mean diameter of the DF2 was 9.4 ±.4 mm on d 13. Animals were allocated to treatments to balance for timing of emergence of the DF2 as well as its diameter on d 12. There were no significant interactions between treatment, day, or stage of follicle maturity at the time of treatment. Further growth of the DF2 ceased following the injection of EB in cows of the T group, regardless of whether it was less than 10 mm in diameter (early growth ranging from 4 to 9 mm; n = 7) or at least 10 mm in diameter (late growth ranging from 10 to 12 mm; n = 5; Figure 3). Respective ranges for early and Figure 3. Mean diameter of the second dominant follicle (DF2) of cows injected with 1 mg of estradiol benzoate on d 13 of the estrous cycle (T group; n = 12) compared with untreated controls (C group; n = 12). Within each treatment group, development of DF2 that were less than 10 mm in diameter on d 13 are compared to the development of those DF2 that were at least 10 mm in diameter on d 13 of the estrous cycle.

4 148 Burke et al. Table 1. Developmental characteristics of ovarian follicular wave pattern, the dominant follicle of the second (DF2) and third (DF3) waves of development, and the ovulatory dominant follicle during the estrous cycle in cows injected with 1 mg of estradiol benzoate i.m. (T) on d 13 compared with untreated controls (C) C group T group Follicle wave pattern a Two waves Three waves Three waves No. of animals Second dominant follicle (DF2) Day of emergence ± ±.4 Maximum diameter, mm 16.0 ± ± ±.6 Third dominant follicle (DF3) Day of emergence 17.6 ± ±.2 Range in day of emergence x y Maximum diameter, mm 14.2 ± ±.4 Ovulatory dominant follicle b Age at ovulation, d c 10.0 ±.6 x 5.7 ±.5 y 4.8 ±.2 y Range in age at ovulation, d x 4 6 y Day of ovulation 21.0 ±.6 x 23.2 ±.5 y 22.3 ±.3 y Interestrous interval Duration, d 20.7 ±.3 x 23.1 ±.6 y 22.3 ±.3 y Range, d a Number of consecutive waves of ovarian follicular development during the estrous cycle. b The ovulatory dominant follicle was the DF2 in estrous cycles with two waves of follicular development, and the DF3 in those with three waves of follicular development. c Age is the interval from emergence to ovulation. x,y Superscript denotes difference (P <.05) within rows. late growth categories on d 13 among cows of the C group were 7 to 9 mm (n = 6) and 10 to 13 mm (n = 6). The overall divergence in mean diameter of DF2 between the groups was apparent within 24 h (P <.05), and the DF2 of cows in the T group were smaller throughout the remaining period (P <.01). All DF2 in cows of the C group were still visible on d 20 of the estrous cycle, with an average diameter of 11.8 ±.9 mm. In contrast, 7 of 12 DF2 had regressed beyond detection by d 20 in cows of the T group. The DF2 that were still visible in five cows of the latter group ranged from 6 to 11 mm and averaged 7.5 ± 0.6 mm in diameter. Every cow of the T group had an estrous cycle with three waves of follicular development. In the C group, nine cows had estrous cycles with three waves and three cows had estrous cycles with two waves of ovarian follicle development. The mean day of emergence of the DF3 in cows of the T group was not different from that of cows of the C group with three waves of follicular development during the estrous cycle (Table 1). However, DF3 of cows in the T group emerged more synchronously than those of cows in the C group (Table 1; P <.01), and they were larger (P <.05) in diameter on the 4th and 5th d after emerging (Figure 4). The ovulatory follicle was the DF3 of cows with three waves and the DF2 of cows with two waves of ovarian follicular development during the estrous cycle. The age (interval from emergence to ovulation) and diameter of the ovulatory follicle at the time of ovulation was greater (P <.05) in cows with two waves than in those with three waves of follicular development (Table 1). Average age and diameter of the ovulatory follicles at ovulation among cows of the T group (the DF3 in all cases) were not different from those cows of the C group with three follicular waves (Table 1). However, ages of the ovulatory follicle at ovulation among animals with three waves of follicular development were more vari- Figure 4. Mean diameter of the third dominant follicle (DF3) normalized to day of emergence in cows injected with 1 mg of estradiol benzoate on d 13 of the estrous cycle (T group; n = 12) compared with untreated controls (C group; n = 9).

5 Ovarian response to estradiol benzoate 149 Figure 5. Mean diameter of the corpus luteum (CL) of cows injected with 1 mg of estradiol benzoate on d 13 of the estrous cycle (T group; n = 12) compared with untreated controls (C group; n = 12). able (P <.01) among cows of the C group than those of the T group. Average duration of the estrous cycle among cows of the C group with two waves of ovarian follicular development was less (P <.05) than among those with three waves of follicular development in either the C or the T group (Table 1). Mean day of ovulation and mean interestrous intervals were the same among cows with three waves of ovarian follicular development in both treatment groups, and there was no difference in the variability of the two treatment groups in this regard. A single cow (# 10) was not detected in estrus, so the day of ovulation for this cow was assumed as a valid estimation of estrous cycle duration, because this value (22.5 ±.3 d) did not differ from the interestrous interval in contemporaries that had been observed in estrus. Luteal Development Mean daily diameter of CL did not differ between treatment groups from d 7 to 17 (P >.05), but it was less in cows of the T group on each day between d 18 to 21 of the estrous cycle (Figure 5; P <.05). The mean day on which the diameter of CL decreased below 21 mm in diameter was also earlier in this group (d 18.7 ±.3) than in cows of the C group (d 20.3 ±.4; P <.05). Discussion This study confirmed the premise of Macmillan et al. (1997) that altered return-to-estrous intervals in inseminated dairy cows injected with 1 mg of EB on either d 12, 13, or 14 was a consequence of synchronized induction of development of a new wave of follicular development. We found that atresia of the dominant follicle as a result of treatment with EB resulted in emergence of a new wave of follicular development 4 to 5 d after treatment. An additional 4 to 5 d was required for the new dominant follicle to mature and ovulate. The sum of these intervals (i.e., 8 to 10 d) closely correlates with the synchronized return-to-estrous pattern observed in that original study (Macmillan et al., 1997). In close agreement with an earlier study (Burke et al., 1999), growth of the DF2 was attenuated by administration of EB during the luteal phase of the estrous cycle. The cessation in growth of the DF2 occurred irrespective of its diameter or stage of maturity. Successful control of ovarian follicular dynamics requires a treatment that is effective at all stages of maturity, or at least those stages that could otherwise lead to a poor treatment result. Exogenous progesterone alone is able to induce atresia of large follicles that have been induced to persist in dominance beyond the normal time frame (Anderson and Day, 1994; Manikkam and Rajamahendran, 1997), but smaller dominant follicles in the growing phase seem to be resistant to such treatment (Burke et al., 1994; Nation, 1997). This might be due to the different effects of progesterone and estradiol on the secretory pattern of gonadotropins, coupled with possible gonadotropin-dependency differences among dominant follicles in various stages of maturity. In ovariectomized cows, treatment with progesterone provided an effective, but transient, inhibition of LH secretion (i.e., 36 h) but had no directly measurable effect on FSH secretion (Burke et al., 1996). The progesterone-induced suppression of LH was extended several days by additional treatment with EB, and FSH secretion was also depressed. Follicular development is initially supported by FSH, but a greater dependency on LH is acquired as dominant follicles mature (Findlay et al., 1996; Gong et al., 1996). Because progesterone suppresses LH, but not FSH, newly developed FSHdependent follicles may be less affected by progesterone. In contrast, effective suppression of both FSH and LH secretion by administration of progesterone and estradiol in combination probably accounts for induction of atresia regardless of age or diameter of the dominant follicle. The 1-mg EB injection effectively promoted follicle turnover and promoted a synchronized sequence of follicular events leading to ovulation of a newly developed dominant follicle. Emergence of the DF3 in cows of the T group was observed on either d 17, 18, or 19. In contrast, emergence of the DF3 among cows of the untreated control group that had three waves of follicular development ranged from d 14 to 22. Age of the ovulatory follicle was predominantly 4 or 5dincows treated with EB, in contrast to between 3 and 11 d in cows of the control group. The DF3 of cows in the T group were also comparatively larger 4 and 5 d after emerging, implying a faster growth rate during development. Although speculative, this may be a consequence of lower

6 150 Burke et al. circulating progesterone coupled with greater LH pulse frequency following earlier regression of the CL in treated animals (Bergfeld et al., 1996). Such effectiveness and precision are attributes required in designing treatments that aim for a highly fertile synchronized estrus. An alternative method of synchronizing return-toservice is by treatment with a progestogen, ensuring that there is complete luteolysis before progestogen is withdrawn (Macmillan and Peterson, 1993; Penny et al., 1997). This form of treatment is likely to produce a tighter synchrony of estrus depending on the time of progestogen withdrawal but could also promote a persistent ovulatory follicle from which fertilization of the oocyte contained within it will result in reduced fertility (Sirois and Fortune, 1990; Savio et al., 1993). The combination of EB to induce synchrony of follicular wave development among animals and progestin to synchronize the final maturation of the ovulatory dominant follicle has potential to allow set-timed AI with normal fertility. Large-scale field studies in commercial dairy herds are presently underway to compare performance of progesterone alone or in combination with a small dose of EB to synchronize return-to-estrus. Recent evidence has suggested that cattle having estrous cycles with three waves of follicular development are more fertile than those having two waves (Ahmad et al., 1997). One of the major differences between estrous cycles with two and three waves of follicular development is the age of the ovulatory follicle at the time of ovulation (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989). The present study adds to the data showing that an ovulatory DF2 could be twice as old as an ovulatory DF3. The ideal age of an ovulatory follicle is unknown, but fertility is increasingly diminished with prolonged periods of dominance beyond 4 d (Mihm et al., 1994; Kinder et al., 1996). Our results demonstrate that dominant follicles are capable of spontaneous ovulation in as little as 4 d after emergence. If the more aged ovulatory DF2 among animals having two waves of follicular development contributes to reduced fertility, then the demonstrated effect of 1 mg of EB to promote estrous cycles with three waves of follicular development may be considered a fertility treatment. This is a possible explanation for the observations of Macmillan et al. (1997) that fertility at return-to-service increased in cows treated in this manner. Consistent with our earlier report involving intravaginal treatment with 10 mg of EB (Burke et al., 1999), luteal development and function seemed to be perturbed by the small dose of EB. Estrogen is an integral component of the normal luteolytic process (Silvia et al., 1991). However, the effect of exogenous administration of estrogens on luteal function is variable (Lemon, 1975; Knickerbocker et al., 1986; Thatcher et al., 1986; Gyawu et al., 1991; Larson et al., 1991) and should not be considered luteolytic in the same sense as PGF 2α (Seguin, 1979). Unfortunately, in the present study, concentrations of progesterone were higher in cows of the T group before treatments were administered, confounding the interpretation of this response. Further, we are unable to comment on luteal function beyond 48 h after treatment, because blood samples were not collected beyond this time. Future studies are required to better examine the effect of a small dose of EB on luteal function in nonpregnant and pregnant cows. The empirical evidence of Macmillan et al. (1997) suggested that the ability of the CL to maintain support of a pregnancy would not be compromised by the 1-mg dose of EB. Synchrony of follicular wave development was achieved with a small dose of EB during a period of maximal circulating progesterone. Our earlier work with ovariectomized cows, in which LH secretion was suppressed by a combination of EB and progesterone treatment, showed that a precipitous decline in circulating concentrations of progesterone initiated a rapid return of LH secretion to pretreatment levels (Burke et al., 1996). Investigations are currently in progress to determine 1) whether EB can promote ovarian follicle wave turnover during the event of luteolysis in the ovary-intact cow; 2) the duration of time that concentrations of progesterone need to be elevated after EB treatment to facilitate follicle wave turnover; and 3) whether exogenous progesterone can substitute for the effect of endogenous progesterone in the event of luteolysis. In conclusion, we found that a strategically timed injection of a small dose of EB was not only effective in inducing atresia of dominant follicles in a variety of growing states, but also created a high degree of synchrony in the follicle development sequence leading to ovulation of a relatively young dominant follicle. These results complement an earlier field trial (Macmillan et al., 1997) showing that when this treatment was applied to cows approximately 13 d after AI, return-toservice was partially synchronized. Implications Strategic administration of a small dose of estradiol benzoate after a synchronized insemination could be routinely implemented to facilitate a more synchronous return-to-estrus in cows not conceiving to the first synchronized insemination. Literature Cited Ahmad, N., E. C. Townsend, R. A. Dailey, and E. K. 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