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Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares
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Theriogenology 71 (2009) 780–788 www.theriojournal.com
Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares O.J. Ginther a,b,*, M.O. Gastal a, E.L. Gastal b, J.C. Jacob a, M.A. Beg b a
b
Eutheria Foundation, Cross Plains, WI 53528, USA Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706, USA
Received 5 September 2008; received in revised form 26 September 2008; accepted 28 September 2008
Abstract An ovulatory follicular wave was induced by ablation of follicles 6 mm and treatment with prostaglandin F2a (PGF) on Day 10 (ovulation = Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5–6 y, n = 14 waves), intermediate (10–14 y, n = 16), and old (18 y, n = 15). During the common-growth phase of the induced wave (Days 12–17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P < 0.05) follicles that reached 10 mm. The number was correlated (r = +0.7; P < 0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12–17, but were greatest (P < 0.002) in the young group and continued to be greater (P < 0.0001) throughout the ovulatory LH surge. During several days before Day 1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or Bmode echo texture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle. # 2009 Elsevier Inc. All rights reserved. Keywords: Estradiol; Gonadotropins; Progesterone; Reproductive senescence; Ultrasonography
1. Introduction Mares are potentially useful follicle-related research models for women, due to similarities in the number and nature of follicular waves and a constant relative diameter of the largest follicle between the two species at definable events throughout the ovulatory wave [1]. In both species, the ovulatory follicular wave is characterized by emergence of several follicles fol* Corresponding author at: Department of Pathobiological Sciences, 1656 Linden Drive. University of Wisconsin, Madison, WI 53706, USA. Tel.: +1 608 262 1202; fax: +1 608 262 7420. E-mail address: [email protected] (O.J. Ginther). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.09.051
lowed by similar growth rate of the follicles during a common-growth phase. The common-growth phase involves several days and ends at the beginning of diameter deviation, which is characterized by continued growth of a developing dominant follicle and reduced growth and atresia of the remaining follicles (subordinates [2,3]). In mares, deviation begins when the future dominant and largest subordinate follicles are approximately 22.5 and 19.0 mm, respectively. In both species, the emergence of the follicular wave is associated with an FSH surge [2,4]. The declining portion of the surge encompasses the beginning of deviation. The growing dominant follicle near deviation also begins to utilize increasing concentrations of LH [5].
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There are similarities between mares and women in the effects of age on the follicles and hormones during the estrous and menstrual cycles (reviewed in [6]). Additional information on the effects of age on follicle and hormone dynamics during an interovulatory interval (IOI) in reproductively active mares with spontaneous ovulatory waves was reported recently [7]. The oldest mares (18 y) had diminished follicle activity, as indicated by smaller diameter of each of the six largest follicles averaged over the IOI, fewer small follicles (5–15 mm) on certain days of the IOI, fewer 15–25 mm follicles/d averaged over the IOI, and smaller diameter of the preovulatory follicle. Mares of the oldest group (18 y) were not approaching senescence, as indicated by regular lengths of the IOI. The results were consistent with the finding of fewer follicles 2–20 mm in ponies 15 y, based on an abbatoir survey [8] and with fewer follicles 11–20 mm in ponies 20 y [9]; however, some of the mares were approaching senescence. Concentrations of FSH did not differ among age groups, except that the maximum concentration was greater in the 18 y group [7]. The most striking finding was greater concentrations of LH in the young group (5–6 y) throughout the ovulatory LH surge. Concentrations in the young group were an average of 34% and 55% higher than in two older groups, respectively, during the increasing LH concentrations from 6 d before ovulation to the peak of the surge on the day after ovulation. Maximum circulating concentration of estradiol during the preovulatory estradiol surge was greatest in the young group. The effect of age on follicle and hormone dynamics during the identified common-growth phase has not been described in any species. The relationships between age and the ultrasonographic (gray-scale mode) and blood flow (color-Doppler mode) characteristics of the wall of the preovulatory follicle have not been studied in mares. In women, studies with Doppler ultrasonography to assess the effect of age on ovarian stromal blood flow have resulted in contradictory findings [10–12]. A more recent report [13] found a significant negative correlation between age and perifollicular blood flow in women. The present study of induced ovulatory follicular waves in mares compared age groups for follicle development and systemic concentrations of FSH and LH during the common-growth phase and B-mode and color-Doppler characteristics of the follicle wall and concentrations of FSH, LH, and estradiol during the periovulatory period. A follicle-ablation approach was used to initiate an ovulatory wave that was not obscured by regressing and growing follicles of a previous wave.
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2. Materials and methods 2.1. Mares The follicular waves in these mares (n = 23) were used previously for study of the nature of follicle deviation in spontaneous ovulatory waves with one versus two dominant follicles [14] and to compare the hormone and follicle dynamics of a spontaneous versus a subsequent induced ovulatory follicular wave [15]. The mares were mixed breeds of large ponies and apparent pony-horse crosses weighing 250–480 kg. Mares with a docile temperament and no apparent abnormalities of the reproductive tract as determined by ultrasound examinations [16] were used during June– August (northern hemisphere). The mares were kept under natural light in an open shelter and outdoor paddock with ad libitum access to a mixture of alfalfa and grass hay, water, and trace-mineralized salt. All mares remained healthy and in good body condition throughout the experiment. Dental characteristics [17] were used to estimate the age of the mares. Three age groups were separated between groups by 4 y, as follows: young (5–6 y, n = 7), intermediate (10–14 y, n = 8), and old (18 y, n = 8). All mares were reproductively active without signs of approaching senescence, such as late emergence of the future ovulatory follicle [18], a prolonged interval from PGF treatment to ovulation (reported mean, 30 d), and IOIs that were irregular and prolonged (>60 d [19]). 2.2. Experimental protocol The effects of age on induced ovulatory waves were studied for two consecutive IOIs in each mare. The first induced wave in each mare was used previously for comparisons of spontaneous and induced ovulatory waves [15]. One ovulatory wave was omitted, owing to outlying (P < 0.01) high LH concentrations according to a Dixon outlier test [20] and development of luteinized small (20 and 21 mm) follicles, yielding a total of 14, 16, and 15 induced ovulatory follicular waves in the young, intermediate, and old groups, respectively. Examination of the ovaries was done daily from the day of follicle-ablation and treatment with prostaglandin F2a (PGF) 10 d after the pretreatment ovulation to 4 d after the posttreatment ovulation. An ultrasound Bmode instrument (Aloka SSD-900 V; Aloka America, Wallingford, CT, USA) equipped with a 7.5 MHz linear-array transducer was used. Diameters of follicles and cross-sectional area (cm2) of the corpus luteum
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were measured, as described [21]. Ovulation at the beginning and end of the IOI was designated Day 0. Color-Doppler examination was done daily from the day the largest follicle was 30 mm until Day 1, using a duplex ultrasound scanner with B-mode and color-flow mode (Aloka SSD-2000) and a fingermounted convex-array 7.5 MHz transducer. Induction of an ovulatory follicular wave (by ablation of follicles 6 mm on Day 10) was done to allow day-to-day identity of individual growing follicles during the common-growth phase by removing the masking effect of regressing and growing follicles of a previous wave. This approach allowed study of the effect of age on the follicles during the common-growth phase. Follicles were ablated by transvaginal ultrasound-guided aspiration of follicle contents, as described [5,22]. In addition, regression of the corpus luteum was induced on Day 10 by i.m. administration of 5 mg of PGF (Lutalyse; Pfizer Animal Health, New York, NY, USA). The area (cm2) of the maximum cross section of an ultrasonic image of the corpus luteum was determined daily on Days 11–14 as an indicator of luteolysis. The day of emergence of the wave was taken as the day the future ovulatory follicle was 6 mm [22,23]. A follicle was considered part of the ovulatory wave if the interval from emergence of the previous follicle was no more than 2 d [24]. The identity of the four largest follicles was maintained from day to day, as described [22], so that the ovulatory follicle would be available for retrospective identification from emergence to ovulation. Hemorrhagic anovulatory follicles (HAFs [25]) occur frequently after PGF/ablation (reported incidence 24% [15]). The beginning of the formation of an HAF (cloudy follicular fluid) was considered equivalent to the day of ovulation and both ovulation and the beginning of an HAF were designated Day 0. This was based on a previous report [25] that follicles that later form HAFs do not differ in diameter or echotexture from follicles that ovulate, and concentrations of circulating gonadotropins and estradiol on the day before ovulation are similar to the day before the beginning of HAF formation. When multiple ovulations or multiple HAFs or a combination of ovulations and HAFs occurred from multiple dominant follicles of a wave, the day of the first ovulation or the beginning of HAF formation was used to indicate Day 0. The follicle that was the first to ovulate or form an HAF was designated retrospectively as the future ovulatory follicle, whether or not it formed an HAF. The end of the common-growth phase or the day of the expected beginning of deviation between the future
dominant and the largest subordinate follicle was defined as the day that the largest follicle was closest to 22.5 mm, based on a review of previous studies [3]. This was done because the actual day of deviation was not determinable in 35% of the waves, owing to the unavailability of a second- or third-largest follicle for waves with single and multiple dominant follicles, respectively [26]. Determinations were made of the length of intervals from ovulation to ovulation (IOI), ovulation to deviation, ablation to emergence of the future ovulatory follicle, emergence to deviation, deviation to ovulation, and from ablation to ovulation. The interval from maximum diameter of the preovulatory follicle to ovulation was determined to study the effect of age on the reduction in diameter between Days 2 and 1; the reduction has been reported to be more prominent in spontaneous waves than in induced waves [15] and more prominent in spontaneous waves in old mares than in young mares [7]. Growing follicles that met the criteria for being part of the induced follicular wave and reached 10, 15, 20, 25, and 30 mm were counted. The numbers were compared among age groups and repeatability within mares was assessed by correlation analyses between the induced ovulatory waves of consecutive IOIs within mares, as described [27]. The growth rate of the future ovulatory follicle during the common-growth phase was determined from the day of first detection of the follicle (mean, Day 12) to the day it reached 22.5 mm (beginning of deviation; mean, Day 17). The growth rate of the follicle also was determined for Days 5 to 2 and from 2 to 1. The rate for Days 2 to 1 was considered separately to further study the effect of age on the expected reduction in growth rate during the day before ovulation. The diameter of the preovulatory follicle was determined at maximum diameter and on Day 1. The wall characteristics of the preovulatory follicle (Days 4 to 1) were evaluated by B-mode ultrasonography, and the percentage of the follicle wall with blood flow signals was determined by color-Doppler mode. The B-mode characteristics were scored from 1–3 (minimal–maximal) for echogenicity and thickness of the granulosa layer of the follicle wall and prominence of the anechoic band around the granulosa layer. These characteristics and methods of assessment have been described [28]. The blood flow of the follicle wall was estimated by the percentage of follicle circumference with color-Doppler signals of blood flow on the real-time sequential two-dimensional images of the entire follicle. The transducer was held at various angles to obtain maximal overall color signals of the follicle wall; the angle between the ultrasound beam and direction of
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Blood samples were centrifuged (1500 g for 10 min) and decanted, and the plasma was stored at 20 8C until assay. Samples were assayed for FSH and LH by radioimmunoassay [32] and for estradiol [33] by commercial kits, as validated and described for mare plasma in our laboratory. The intra- and interassay coefficients of variation (CV) and mean sensitivity, respectively, were 7.8%, 8.3%, and 0.2 ng/mL for LH; 9.2%, 18.4%, and 1.1 ng/mL for FSH; and 10.0%, 4.9%, and 0.1 pg/mL for estradiol. 2.4. Statistical analyses
Fig. 1. Mean ( SEM) concentrations of LH and FSH on Days 10–17 and Days 5 to 4. Significant main effects of age (A) and day (D) are shown. There were no age-by-day interactions. The asterisk (*) for LH in the young group indicates a decrease (P < 0.02) in concentrations between the indicated days.
blood flow (insonation angle) affects the detectability and extent of the color signals [29]. The technique of estimating the percentage of tissue with color-Doppler signals has been validated by the counting of colored pixels in a still image [30,31]. Data were analyzed among the three age groups for systemic concentrations of LH and FSH on the days extending from ablation (Day 10) to the mean day of the beginning of expected deviation (end of the commongrowth phase; mean, Day 17). A break in continuity of data occurred between Day 17 and Day 5 (Fig. 1). Day 5 was used to begin the preovulatory period so that the average interval from ablation to deviation (7 d) and number of days from Days 5 to 0 (6 d) corresponded to the interval from ablation to ovulation (13 d). Concentrations of LH and FSH for Days 5 to 4 were analyzed separately. Systemic estradiol concentrations were assessed for Days 6, 3, 2, 0, and 2. 2.3. Blood samples and hormone assays Collection of a blood sample was done daily. Jugular blood samples were collected into heparinized tubes.
Data for end points that were not normally distributed, according to Shapiro-Wilk tests, were transformed to natural logarithms or ranked. Data for diameter and number of follicles and concentrations of plasma hormones were examined by the SAS MIXED procedure with a REPEATED statement (9.1.3 Version; SAS, Institute Inc., Cary, NC, USA). If the main effect of age was significant or approached significance, the differences among ages were examined by Duncan’s multiple range test. If an age-by-day interaction was detected, Duncan’s multiple range tests were used to locate differences among ages within days. Single-point data were analyzed by one-way ANOVA or unpaired Student’s t-tests. The correlation between LH concentrations during the common-growth phase and number of follicles that reached 10 mm was examined by Spearman tests. A probability of P 0.05 indicated that a difference was significant, and probabilities between P > 0.05 and P 0.1 indicated that the difference approached significance. Data are given as the mean SEM, unless otherwise stated. 3. Results The ovulatory follicular waves that were induced during two consecutive IOIs were not significantly different for any end point between the first and second IOIs and were combined. The incidence of HAFs was not different among age groups [7]. The area of a cross section of the corpus luteum decreased (P < 0.0001) from 6.9 0.2 cm2 on Day 11 to 3.4 0.3 cm2 on Day 14, averaged over the three age groups; there was no main effect of age or an age-by-day interaction (data not shown). Intervals between defined events during the IOI and the induced ovulatory wave were not significantly different among age groups, except for the longer interval from ablation to ovulation (P < 0.01) and from deviation to ovulation (P < 0.03) in the old mares (Table 1). More (P < 0.03) follicles reached 10 and
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Table 1 Mean (SEM) for intervals; numbers, diameters, and growth rates of follicles; and mean and maximum hormone concentrations for three age groups. End points
Young (5–6 y)
Intermediate (10–14 y)
Old (18 y)
Intervals (d) Ovulation to ovulation Ovulation to deviationa Ablation to emergence b Emergence to deviation Deviation to ovulation Ablation to ovulation Maximum diameter to ovulation
22.7 0.3 c 16.9 0.2 1.4 0.2 5.5 0.1 5.9 0.3c,d 12.8 0.2 c 0.3 0.1
22.4 0.4 c 16.9 0.4 1.7 0.2 5.3 0.3 5.4 0.3 c 12.4 0.4 c 0.1 0.1
23.7 0.3d 17.1 0.4 1.9 0.3 5.2 0.2 6.7 0.4d 13.8 0.3d 0.1 0.1
8.3 0.9 c 4.7 0.5 c 2.1 0.2 1.6 0.2 1.3 0.2
6.5 0.6 d 3.6 0.4 d 2.3 0.3 1.7 0.2 1.4 0.2
6.1 0.9d 3.6 0.5d 2.0 0.1 1.6 0.2 1.1 0.1
No. follicles/ovulatory wave 10 mm 15 mm 20 mm 25 mm 30 mm Ovulatory follicle At maximum (mm) At Day 1 (mm) Growth rate (mm/day) Days 12–17 Days 5 to 2 Days 2 to 1
37.9 1.4 37.4 1.4 3.2 0.1 4.0 0.3c,d 1.2 0.8
39.1 1.2 38.9 1.2
39.8 1.3 38.9 1.4
3.4 0.2 4.1 0.2 c 2.0 0.4
3.1 0.1 3.3 0.3d 1.7 0.7
LH, Days 10–17 At maximum (ng/mL) Day of maximum Mean (ng/mL) LH, Days 5 to 4 At maximum (ng/mL) Day of maximum Mean (ng/mL)
10.3 1.1 c 16.1 0.3 6.3 0.7 c
7.1 0.8 d 16.0 0.2 4.5 0.5 d
5.1 0.7d 15.7 0.7 3.2 0.4d
31.3 5.3 c 1.6 0.2 13.0 1.6 c
12.9 1.9 d 1.4 0.2 7.3 1.0 d
9.7 1.9d 1.2 0.2 5.4 1.1d
FSH At maximum (ng/mL) Day of maximum
23.7 1.3 c 12.4 0.4
24.8 1.7 c 14.1 0.3
28.5 1.7d 14.2 0.4
Estradiol At maximum (pg/mL) Day of maximum
6.2 0.7 2.0 0.0
5.7 0.7 1.7 0.3
6.0 0.7 2.0 0.0
a
Deviation refers to mean Day 17 when the future ovulatory follicle was closest to 22.5 mm. PGF/ablation on Day 10. Emergence is the day the future ovulatory follicle was 6 mm. c, d Means within a row that are different (P < 0.05). n = 14–16 ovulatory waves/age group.
b
15 mm during the common-growth phase in the young group than in the intermediate and old groups (Table 1). The number reaching 20, 25, and 30 mm was not different among groups. The number of follicles reaching 10 mm (r = +0.7; P < 0.0001), 15 mm (r = +0.4; P < 0.03), and 30 mm (r = +0.4; P < 0.03) were correlated between consecutive induced ovulatory waves within mares, indicating measurable repeatability in individual mares. Growth rate of the future ovulatory follicle did not differ among age groups between Days 12 and 17 (Table 1). Growth rate differed (P < 0.01) between
Days 5 and 2. The rate for the old group was less than for the intermediate group, but did not differ significantly from the young group, based on the multiple range test (Table 1). However, the growth rate in the old group was less (P < 0.05) than in the young group when examined by an unpaired t-test. There were no differences among age groups in the day of maximum diameter of the preovulatory follicle, diameter on Day 1, or growth rate between Days 2 and 1 (Table 1). There were no age-related effects for B-mode echotextural changes in the follicle wall or percentage of wall with color-Doppler signals of blood flow in the
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no significant difference between intermediate mares and old mares (Table 1). Concentrations decreased (P < 0.02) between Days 5 and 2 in the young group, approached a decrease (P < 0.1) in the intermediate group, and did not decrease in the old group. The concentrations of FSH on Days 10–17 and also on Days 5 to 4 showed only a day effect with no effect of age or an age-by-day interaction (Fig. 1). However, the maximum concentration of FSH during the commongrowth phase was greater in the old group than in the younger groups, but the day of maximum concentration did not differ among groups (Table 1). Concentrations of estradiol increased (day effect; P < 0.0001) between Days 6 and 2 and then decreased with no effect of age or an age-by-day interaction (data not shown). Maximum concentration of estradiol and day of occurrence of maximum were not different among age groups (Table 1). 4. Discussion
Fig. 2. Means (SEM) for end points involving the preovulatory follicle showing the effects of day. The extent of blood flow was based on the percentage of the follicle wall with color-Doppler signals. The echotextural end points (lower panel) were scored from 1–3 (minimal– maximal). There were significant (P < 0.0001–P < 0.02) main effects of day for each characteristic, but no main effects of age or age-by-day interactions.
preovulatory follicle from Days 4 to 1. The main effects of day are shown (Fig. 2). Concentrations of LH on Days 10–17 showed a main effect of day from an immediate and progressive increase, as previously reported (Fig. 1 [15]). A main effect of age reflected LH decreases from young, to intermediate, to old averaged over Days 10–17 with significantly higher mean concentrations in the young group than in each of the other groups (Table 1). The mean LH concentration in individual waves during the common-growth phase and number of follicles that reached 10 mm were not correlated significantly either for the young group or all groups combined. For LH on Days 5 to 4, the main effects of age and day were significant, but the interaction was not (Fig. 1). The concentration averaged over days and the maximum concentration were highest for young mares, with
During the common-growth phase of an induced ovulatory follicular wave (Days 12–17), about two more follicles reached 10 mm and one more reached 15 mm in the youngest group (5–6 y) than in the older groups (10–14 y and 18 y). In reported results with spontaneous waves, young mares (5–7 y) had a more rapid increase in number of 6–10 mm follicles on Days 12 to 6 [9] and old mares (18 y) had fewer follicles 10–15 mm on Days 11 to 9 [7]. Similarly, the number of follicles in follicular waves is reduced in old cows [34] and women [35]. In the previous studies in mares, all growing and regressing follicles were counted without regard to days of the common-growth phase. The present findings indicate that the reported days likely involved the common-growth phase of the spontaneous ovulatory waves of the previous studies. The absence of a significant correlation between LH concentrations during the common-growth phase and number of follicles indicates that the greater number of follicles in the PGF/ablation-induced waves of young mares was not attributable to the greater concentrations of LH. In addition, the number of LH receptors in equine granulosa cells did not seem to increase until follicle diameters were 15–19 mm [36]. The similarity in the induced FSH surges among ages indicated that differences in FSH concentrations also did not account for the greater number of follicles 10 and 15 mm in the induced waves of young mares. Furthermore, the initial portion of the wave (Days 11–13) develops independently of FSH [37–40]. In this regard, the maximum concentration of FSH during the common-
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growth phase was lower in the young group than in the old group and may have represented a greater FSH inhibitory effect from a greater number of follicles [3]. Dependency on the number of follicles in the underlying reservoir was supported by the significant repeatability within mares in number of follicles 10 and 15 mm between the consecutive induced ovulatory waves. It is concluded that the greater number of 10 and 15 mm follicles in the ovulatory waves of young mares was not a function of differences in concentrations of either gonadotropin and represented a larger reservoir of follicles for recruitment. An increase in LH throughout the common-growth phase following PGF/ablation occurred in all age groups. The LH increase is attributable to the PGF treatment. An immediate (within 5 min) increase in LH occurs after PGF treatment in mares during mid-diestrus, presumably from a direct effect at the hypothalamo-pituitary area [30]. The direct LH effect lasts only a couple of hours but overlaps with an LH increase from the PGF-induced decrease in progesterone and thereby a decrease in the negative effect of progesterone on LH. The negative effect of progesterone on LH is consistent with the close temporal reciprocal relationships between the two hormones at the beginning and end of diestrus [41]. A negative effect of a physiologic dose of progesterone on LH has been demonstrated in experiments that included removal of endogenous progesterone by PGF [5,24,42]. The increase in LH after the loss of the negative effect of progesterone and until after the decrease beginning on Day 1 has been proposed [43] to represent the positive effect of extraovarian factors under the control of the environment, especially day length. Concentrations of LH are low to a similar extent in ovarian-intact and ovariectomized mares during the anovulatory season, but high in ovariectomized mares during the ovulatory season [44,45]. Maximum concentration of LH during the ovulatory LH surge seemed similar to the maximum LH concentration in ovariectomized mares [43], although confirmatory study is needed. In the present study, the LH concentrations during the common-growth phase were a mean of 45% and 55% higher in the young group than in the intermediate and old groups, respectively. The similarity among age groups in the rate of regression of the corpus luteum support the conclusion that the greater LH concentration in the young group represented a stronger response to an enviromental influence in the young group. The posttreatment increase in LH reached a transient plateau at the end of the common-growth phase or beginning of deviation, as previously reported [5,24], but a similar plateau does not occur during spontaneous
ovulatory waves [15,27]. The LH increase in spontaneous waves begins on about Day 15 or near the end of the common-growth phase. In a study of age effects in spontaneous waves, LH concentrations were higher in the youngest group throughout the LH surge, beginning at Day 7 (equivalent to Day 16 [7]). The greater LH concentrations in the young group therefore occurred in both spontaneous waves as previously reported and in induced waves in the present study. Although the results of earlier studies indicated that estradiol has a positive effect on LH concentrations in mares, a more recent report concluded that estradiol has a negative effect on LH throughout the LH surge associated with spontaneous waves [46]. Apparently, the earlier reports involved supradoses of estradiol or a rebound in LH from using a prolonged interval (e.g., 24 h) between estradiol treatment and blood sampling. The estradiol increase from the first day considered (Day 6) to a peak on Day 2, in the present study of induced ovulatory waves is similar to previous reports for spontaneous waves [27,41] and induced waves [15]. The decrease in LH concentrations between Days 5 and 2 (after the LH plateau) in the young group likely represented a negative effect of estradiol. The occurrence of the reduction only in the young group seems related to greater LH concentrations on Day 5, considering that estradiol was not different among age groups. The LH concentrations began to increase on Day 2 in synchrony with the estradiol peak in all age groups. The temporal association between decreasing estradiol and increasing LH has been described and discussed for spontaneous waves [41] and is not affected by age, either for spontaneous waves [7] or induced waves (present results). Growth rate of the ovulatory follicle was reduced in the oldest group, consistent with a longer interval from the end of the common-growth phase (Day 17) to ovulation and the absence of a difference in diameter of the follicle on Day 1. The slower growth rate cannot be attributed to the low LH concentrations; the growth rate was not reduced in the intermediate group despite the low LH. There were no age-related preovulatory effects in induced waves on estradiol concentrations, follicle diameter at maximum and on Day 1, granulosa thickness and echogenicity, prominence of the anechoic layer, percentage of wall with color-Doppler signals of blood flow, and rate of the preovulatory reduction in growth of the follicle at the end of induced waves. These similarities occurred despite the greater LH concentrations during the preovulatory period in the young group. The results suggest that the low concentrations of LH represented by the older groups were adequate for
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these preovulatory changes. However, all mares were reproductively active with no signs of approaching ovarian senescence. The effects of age on these features as senescence approaches will require specific study. Although there were no age-related findings on the ultrasonographic characteristics of the preovulatory follicle on Days 4 to 1, the differences among days are of interest in that they extend upon the earlier findings that were limited to 1.5 d before ovulation [28]. The increase between Days 2 and 1 in thickness and echogenicity of the granulosa layer agrees with the earlier report, but also indicated that the changes in the granulosa were more exaggerated between Days 2 and 1 than between Days 4 and 2. Prominence of the anechoic band did not increase between Days 2 and 1 in agreement with the previous report but did increase between Days 4 and 3. Percentage of follicle wall with blood-flow signals increased after Day 3, extending upon the previous report. In conclusion, induction of an ovulatory wave with PGF treatment and ablation of follicles on Day 10 was used to study the effects of age on the induced wave during the common-growth phase (Days 12–17). The youngest group (5–6 y) had greater concentrations of LH and more follicles 10 mm and 15 mm during the induced wave than the intermediate group (10–14 y) and the oldest group (18 y). In young mares, the greater LH concentrations may have represented a more prominent response to extraovarian factors after the induced luteolysis, and the greater number of follicles likely represented a larger follicle reservoir. Mean FSH concentrations during the common-growth phase were not different among groups. However, the maximum FSH concentration was greater in the old group and may have reflected, at least in part, the reduced inhibitory effect of a smaller number of follicles. Maximum diameter, B-mode echotexture, and color-Doppler signals of blood flow of the preovulatory follicle and plasma estradiol concentrations during the preovulatory period were not different among age groups, even though LH concentrations were greater in the young group. Growth rate of the preovulatory follicle was reduced in the oldest group, consistent with a longer interval from the end of the common-growth phase to ovulation and no difference in diameter of the follicle on Day 1. Acknowledgments Research supported by the Eutheria Foundation. The authors thank Pfizer Animal Health New York, NY, USA for a gift of Lutalyse. J. C. Jacob was on leave
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from the Department of Reproduction and Animal Evaluation, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil. References [1] Ginther OJ, Gastal EL, Gastal MO, Bergfelt DR, Baerwald AR, Pierson RA. Comparative study of the dynamics of follicular waves in mares and women. Biol Reprod 2004;71:1195–201. [2] Ginther OJ, Beg MA, Donadeu FX, Bergfelt DR. Mechanism of follicle deviation in monovular farm species. Anim Reprod Sci 2003;78:239–57. [3] Ginther OJ, Beg MA, Gastal MO, Gastal EL. Follicle dynamics and selection in mares. Anim Reprod 2004;1:45–63. [4] Ginther OJ, Beg MA, Gastal EL, Gastal MO, Baerwald AR, Pierson RA. Systemic concentrations of hormones during development of follicular waves in mares and women: a comparative study. Reproduction 2005;130:379–88. [5] Gastal EL, Bergfelt DR, Nogueira GP, Gastal MO, Ginther OJ. Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares. Biol Reprod 1999;61:1492–8. [6] Carnevale EM. The mare model for follicular maturation and reproductive aging in the woman. Theriogenology 2008;69:23– 30. [7] Ginther OJ, Gastal MO, Gastal EL, Jacob JC, Siddiqui MAR, Beg MA. Age effects on follicle and hormone dynamics during the oestrous cycle in mares. Reprod Fertil Dev 2008;20:955–63. [8] Wesson JA, Ginther OJ. Influence of season and age on reproductive activity in pony mares on the basis of a slaughterhouse survey. J Anim Sci 1981;52:119–29. [9] Carnevale EM, Bergfelt DR, Ginther OJ. Aging effects on follicular activity and concentrations of FSH, LH, and progesterone in mares. Anim Reprod Sci 1993;31:287–99. [10] Pan HA, Cheng YC, Li CH, Wu MH, Chang FM. Ovarian stroma flow intensity decreases by age: a three-dimensional power doppler ultrasonographic study. Ultrasound Med Biol 2002; 28:425–30. [11] Kupesic S, Kurjak A, Bjelos D, Vujisic S. Three-dimensional ultrasonographic ovarian measurements and in vitro fertilization outcome are related to age. Fertil Steril 2003;79:190–7. [12] Ng EH, Chan CC, Yeung WS, Ho PC. Effect of age on ovarian stromal flow measured by three-dimensional ultrasound with power Doppler in Chinese women with proven fertility. Hum Reprod 2004;19:2132–7. [13] Costello MF, Shrestha SM, Sjoblom P, McNally G, Bennett MJ, Steigrad SJ, et al. Power doppler ultrasound assessment of the relationship between age and ovarian perifollicular blood flow in women undergoing in vitro fertilization treatment. J Assisted Reprod Genet 2006;23:359–65. [14] Jacob JC, Gastal EL, Gastal MO, Carvalho GR, Beg MA, Ginther OJ. Follicle deviation in ovulatory follicular waves with one or two dominant follicles in mares. Reprod Domest Anim 2009;44:248–54. [15] Ginther OJ, Jacob JC, Gastal MO, Gastal EL, Beg MA. Follicle and systemic hormone interrelationships during spontaneous and ablation-induced ovulatory waves in mares. Anim Reprod Sci 2008;106:181–7. [16] Ginther OJ. Ultrasonic imaging and animal reproduction: Book 2. Horses. Cross Plains, WI, USA: Equiservices Publishing; 1995.
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[17] American Association of Equine Practitioners. Official Guide for Determining the Age of the Horse. Lexington KY, USA: AAEP, 2002, 1–26. [18] Ginther OJ, Carnevale EM, Bergfelt DR. Delay in emergence of the ovulatory follicular waves in old mares. J Equine Vet Sci 1993;13:75–9. [19] Carnevale EM, Bergfelt DR, Ginther OJ. Follicular activity and concentrations of FSH and LH associated with senescence in mares. Anim Reprod Sci 1994;35:231–46. [20] Dixon WJ, Massey FJ. Introduction to statistical analysis, 4th ed., New York: McGraw-Hill; 1983. [21] Gastal EL, Neves AP, Mattos RC, Petrucci BPL, Gastal MO, Ginther OJ. Miniature ponies: 1. Follicular, luteal, and endometrial dynamics during the oestrous cycle. Reprod Fertil Dev 2008;20:376–85. [22] Gastal EL, Gastal MO, Bergfelt DR, Ginther OJ. Role of diameter differences among follicles in selection of a future dominant follicle in mares. Biol Reprod 1997;57:1320–7. [23] Gastal EL, Gastal MO, Beg MA, Ginther OJ. Interrelationships among follicles during the common-growth phase of a follicular wave and capacity of individual follicles for dominance in mares. Reproduction 2004;128:417–22. [24] Gastal EL, Gastal MO, Nogueira GP, Bergfelt DR, Ginther OJ. Temporal interrelationships among luteolysis. FSH and LH concentrations and follicle deviation in mares. Theriogenology 2000;53:925–40. [25] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Conversion of a viable preovulatory follicle into a hemorrhagic anovulatory follicle in mares. Anim Reprod 2006;3:29–40. [26] Ginther OJ, Jacob JC, Gastal MO, Gastal EL, Beg MA. Development of one versus multiple ovulatory follicles and associated systemic hormone concentrations in mares. Reprod Domest Anim 2008. doi: 10.1111/j.1439-0531.2008.01109.x. [27] Jacob JC, Gastal EL, Gastal MO, Carvalho GR, Beg MA, Ginther OJ. Temporal relationships and repeatability of follicle diameters and hormone concentrations within individuals in mares. Reprod Domest Anim 2009;44:92–9. [28] Gastal EL, Gastal MO, Ginther OJ. Relationships of changes in B-mode echotexture and colour-Doppler signals in the wall of the preovulatory follicle to changes in systemic oestradiol concentrations and the effects of human chorionic gonadotrophin in mares. Reproduction 2006;131:699–709. [29] Ginther OJ. Ultrasonic imaging and animal reproduction: Book 4. Color-Doppler ultrasound. Cross Plains, WI, USA: Equiservices Publishing; 2007. [30] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Effect of prostaglandin F2a on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares. Domest Anim Endocrinol 2007;32:315–28. [31] Araujo RR, Ginther OJ. Vascular perfusion of the reproductive organs during sedation by detomidine and xylazine in mares and heifers. Am J Vet Res 2009;70:141–8.
[32] Donadeu FX, Ginther OJ. Follicular waves and circulating concentrations of gonadotrophins, inhibin and oestradiol during the anovulatory season in mares. Reproduction 2002;124:875–85. [33] Ginther OJ, Beg MA, Gastal EL, Gastal MO, Cooper DA. Ovulation induction by human chorionic gonadotropin (hCG) treatment is associated with an immediate estradiol decrease and a more rapid LH increase in mares. Anim Reprod Sci 2008. doi: 10.1016/j.anireprosci.2008.08.026. [34] Malhi PS, Adams GP, Mapletoft RJ, Singh J. Superovulatory response in a bovine model of reproductive aging. Anim Reprod Sci 2008;109:100–9. [35] Kim SH. Female aging and superovulation induction for IVF. J Obstet Gynecol 1995;21:75–82. [36] Goudet G, Belin F, Be´zard J, Ge´rard N. Intrafollicular content of luteinizing hormone receptor, a-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare. Biol Reprod 1999;60:1120–7. [37] Pedersen HG, Berrocal B, Thompson SRM, Watson ED. Gonadotropic control of follicular growth in the mare. Theriogenology 2002;58:465–8. [38] Donadeu FX, Ginther OJ. Suppression of circulating concentrations of FSH and LH by inhibin and estradiol during the initiation of follicle deviation in mares. Theriogenology 2003;60:1423–34. [39] Imboden I, Janett F, Burger D, Crowe MA, Ha¨ssig M, Thun R. Influence of immunization against GnRH on reproductive cyclicity and estrous behavior in the mare. Theriogenology 2006; 66:1866–75. [40] Checura CM, Beg MA, Gastal EL, Gastal MO, Wiltbank MC, Parrish JJ, et al. Effect of suppression of FSH with a GnRH antagonist (Acyline) before and during follicle deviation in the mare. Reprod Domest Anim 2008. doi: 10.1111/j.14390531.2008.01222.x. [41] Ginther OJ, Utt MD, Bergfelt DR, Beg MA. Controlling interrelationships of progesterone/LH and estradiol/LH in mares. Anim Reprod Sci 2006;95:144–50. [42] Bergfelt DR, Gastal EL, Ginther OJ. Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares. Biol Reprod 2001;65:426–32. [43] Ginther OJ, Gastal EL, Gastal MO, Beg MA. Regulation of circulating gonadotropins by the negative effects of ovarian hormones in mares. Biol Reprod 2005;73:315–23. [44] Garcia MC, Ginther OJ. Effects of ovariectomy and season on plasma luteinizing hormone in mares. Endocrinology 1976;98: 958–62. [45] Freedman LJ, Garcia MC, Ginther OJ. Influence of ovaries and photoperiod on reproductive function in the mare. J Reprod Fertil Suppl 1979;27:79–86. [46] Ginther OJ, Utt MD, Beg MA, Gastal EL, Gastal MO. Negative effect of estradiol on luteinizing hormone throughout the ovulatory luteinizing hormone surge in mares. Biol Reprod 2007; 77:543–50.
Theriogenology 71 (2009) 780–788 www.theriojournal.com
Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares O.J. Ginther a,b,*, M.O. Gastal a, E.L. Gastal b, J.C. Jacob a, M.A. Beg b a
b
Eutheria Foundation, Cross Plains, WI 53528, USA Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706, USA
Received 5 September 2008; received in revised form 26 September 2008; accepted 28 September 2008
Abstract An ovulatory follicular wave was induced by ablation of follicles 6 mm and treatment with prostaglandin F2a (PGF) on Day 10 (ovulation = Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5–6 y, n = 14 waves), intermediate (10–14 y, n = 16), and old (18 y, n = 15). During the common-growth phase of the induced wave (Days 12–17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P < 0.05) follicles that reached 10 mm. The number was correlated (r = +0.7; P < 0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12–17, but were greatest (P < 0.002) in the young group and continued to be greater (P < 0.0001) throughout the ovulatory LH surge. During several days before Day 1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or Bmode echo texture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle. # 2009 Elsevier Inc. All rights reserved. Keywords: Estradiol; Gonadotropins; Progesterone; Reproductive senescence; Ultrasonography
1. Introduction Mares are potentially useful follicle-related research models for women, due to similarities in the number and nature of follicular waves and a constant relative diameter of the largest follicle between the two species at definable events throughout the ovulatory wave [1]. In both species, the ovulatory follicular wave is characterized by emergence of several follicles fol* Corresponding author at: Department of Pathobiological Sciences, 1656 Linden Drive. University of Wisconsin, Madison, WI 53706, USA. Tel.: +1 608 262 1202; fax: +1 608 262 7420. E-mail address: [email protected] (O.J. Ginther). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.09.051
lowed by similar growth rate of the follicles during a common-growth phase. The common-growth phase involves several days and ends at the beginning of diameter deviation, which is characterized by continued growth of a developing dominant follicle and reduced growth and atresia of the remaining follicles (subordinates [2,3]). In mares, deviation begins when the future dominant and largest subordinate follicles are approximately 22.5 and 19.0 mm, respectively. In both species, the emergence of the follicular wave is associated with an FSH surge [2,4]. The declining portion of the surge encompasses the beginning of deviation. The growing dominant follicle near deviation also begins to utilize increasing concentrations of LH [5].
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There are similarities between mares and women in the effects of age on the follicles and hormones during the estrous and menstrual cycles (reviewed in [6]). Additional information on the effects of age on follicle and hormone dynamics during an interovulatory interval (IOI) in reproductively active mares with spontaneous ovulatory waves was reported recently [7]. The oldest mares (18 y) had diminished follicle activity, as indicated by smaller diameter of each of the six largest follicles averaged over the IOI, fewer small follicles (5–15 mm) on certain days of the IOI, fewer 15–25 mm follicles/d averaged over the IOI, and smaller diameter of the preovulatory follicle. Mares of the oldest group (18 y) were not approaching senescence, as indicated by regular lengths of the IOI. The results were consistent with the finding of fewer follicles 2–20 mm in ponies 15 y, based on an abbatoir survey [8] and with fewer follicles 11–20 mm in ponies 20 y [9]; however, some of the mares were approaching senescence. Concentrations of FSH did not differ among age groups, except that the maximum concentration was greater in the 18 y group [7]. The most striking finding was greater concentrations of LH in the young group (5–6 y) throughout the ovulatory LH surge. Concentrations in the young group were an average of 34% and 55% higher than in two older groups, respectively, during the increasing LH concentrations from 6 d before ovulation to the peak of the surge on the day after ovulation. Maximum circulating concentration of estradiol during the preovulatory estradiol surge was greatest in the young group. The effect of age on follicle and hormone dynamics during the identified common-growth phase has not been described in any species. The relationships between age and the ultrasonographic (gray-scale mode) and blood flow (color-Doppler mode) characteristics of the wall of the preovulatory follicle have not been studied in mares. In women, studies with Doppler ultrasonography to assess the effect of age on ovarian stromal blood flow have resulted in contradictory findings [10–12]. A more recent report [13] found a significant negative correlation between age and perifollicular blood flow in women. The present study of induced ovulatory follicular waves in mares compared age groups for follicle development and systemic concentrations of FSH and LH during the common-growth phase and B-mode and color-Doppler characteristics of the follicle wall and concentrations of FSH, LH, and estradiol during the periovulatory period. A follicle-ablation approach was used to initiate an ovulatory wave that was not obscured by regressing and growing follicles of a previous wave.
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2. Materials and methods 2.1. Mares The follicular waves in these mares (n = 23) were used previously for study of the nature of follicle deviation in spontaneous ovulatory waves with one versus two dominant follicles [14] and to compare the hormone and follicle dynamics of a spontaneous versus a subsequent induced ovulatory follicular wave [15]. The mares were mixed breeds of large ponies and apparent pony-horse crosses weighing 250–480 kg. Mares with a docile temperament and no apparent abnormalities of the reproductive tract as determined by ultrasound examinations [16] were used during June– August (northern hemisphere). The mares were kept under natural light in an open shelter and outdoor paddock with ad libitum access to a mixture of alfalfa and grass hay, water, and trace-mineralized salt. All mares remained healthy and in good body condition throughout the experiment. Dental characteristics [17] were used to estimate the age of the mares. Three age groups were separated between groups by 4 y, as follows: young (5–6 y, n = 7), intermediate (10–14 y, n = 8), and old (18 y, n = 8). All mares were reproductively active without signs of approaching senescence, such as late emergence of the future ovulatory follicle [18], a prolonged interval from PGF treatment to ovulation (reported mean, 30 d), and IOIs that were irregular and prolonged (>60 d [19]). 2.2. Experimental protocol The effects of age on induced ovulatory waves were studied for two consecutive IOIs in each mare. The first induced wave in each mare was used previously for comparisons of spontaneous and induced ovulatory waves [15]. One ovulatory wave was omitted, owing to outlying (P < 0.01) high LH concentrations according to a Dixon outlier test [20] and development of luteinized small (20 and 21 mm) follicles, yielding a total of 14, 16, and 15 induced ovulatory follicular waves in the young, intermediate, and old groups, respectively. Examination of the ovaries was done daily from the day of follicle-ablation and treatment with prostaglandin F2a (PGF) 10 d after the pretreatment ovulation to 4 d after the posttreatment ovulation. An ultrasound Bmode instrument (Aloka SSD-900 V; Aloka America, Wallingford, CT, USA) equipped with a 7.5 MHz linear-array transducer was used. Diameters of follicles and cross-sectional area (cm2) of the corpus luteum
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were measured, as described [21]. Ovulation at the beginning and end of the IOI was designated Day 0. Color-Doppler examination was done daily from the day the largest follicle was 30 mm until Day 1, using a duplex ultrasound scanner with B-mode and color-flow mode (Aloka SSD-2000) and a fingermounted convex-array 7.5 MHz transducer. Induction of an ovulatory follicular wave (by ablation of follicles 6 mm on Day 10) was done to allow day-to-day identity of individual growing follicles during the common-growth phase by removing the masking effect of regressing and growing follicles of a previous wave. This approach allowed study of the effect of age on the follicles during the common-growth phase. Follicles were ablated by transvaginal ultrasound-guided aspiration of follicle contents, as described [5,22]. In addition, regression of the corpus luteum was induced on Day 10 by i.m. administration of 5 mg of PGF (Lutalyse; Pfizer Animal Health, New York, NY, USA). The area (cm2) of the maximum cross section of an ultrasonic image of the corpus luteum was determined daily on Days 11–14 as an indicator of luteolysis. The day of emergence of the wave was taken as the day the future ovulatory follicle was 6 mm [22,23]. A follicle was considered part of the ovulatory wave if the interval from emergence of the previous follicle was no more than 2 d [24]. The identity of the four largest follicles was maintained from day to day, as described [22], so that the ovulatory follicle would be available for retrospective identification from emergence to ovulation. Hemorrhagic anovulatory follicles (HAFs [25]) occur frequently after PGF/ablation (reported incidence 24% [15]). The beginning of the formation of an HAF (cloudy follicular fluid) was considered equivalent to the day of ovulation and both ovulation and the beginning of an HAF were designated Day 0. This was based on a previous report [25] that follicles that later form HAFs do not differ in diameter or echotexture from follicles that ovulate, and concentrations of circulating gonadotropins and estradiol on the day before ovulation are similar to the day before the beginning of HAF formation. When multiple ovulations or multiple HAFs or a combination of ovulations and HAFs occurred from multiple dominant follicles of a wave, the day of the first ovulation or the beginning of HAF formation was used to indicate Day 0. The follicle that was the first to ovulate or form an HAF was designated retrospectively as the future ovulatory follicle, whether or not it formed an HAF. The end of the common-growth phase or the day of the expected beginning of deviation between the future
dominant and the largest subordinate follicle was defined as the day that the largest follicle was closest to 22.5 mm, based on a review of previous studies [3]. This was done because the actual day of deviation was not determinable in 35% of the waves, owing to the unavailability of a second- or third-largest follicle for waves with single and multiple dominant follicles, respectively [26]. Determinations were made of the length of intervals from ovulation to ovulation (IOI), ovulation to deviation, ablation to emergence of the future ovulatory follicle, emergence to deviation, deviation to ovulation, and from ablation to ovulation. The interval from maximum diameter of the preovulatory follicle to ovulation was determined to study the effect of age on the reduction in diameter between Days 2 and 1; the reduction has been reported to be more prominent in spontaneous waves than in induced waves [15] and more prominent in spontaneous waves in old mares than in young mares [7]. Growing follicles that met the criteria for being part of the induced follicular wave and reached 10, 15, 20, 25, and 30 mm were counted. The numbers were compared among age groups and repeatability within mares was assessed by correlation analyses between the induced ovulatory waves of consecutive IOIs within mares, as described [27]. The growth rate of the future ovulatory follicle during the common-growth phase was determined from the day of first detection of the follicle (mean, Day 12) to the day it reached 22.5 mm (beginning of deviation; mean, Day 17). The growth rate of the follicle also was determined for Days 5 to 2 and from 2 to 1. The rate for Days 2 to 1 was considered separately to further study the effect of age on the expected reduction in growth rate during the day before ovulation. The diameter of the preovulatory follicle was determined at maximum diameter and on Day 1. The wall characteristics of the preovulatory follicle (Days 4 to 1) were evaluated by B-mode ultrasonography, and the percentage of the follicle wall with blood flow signals was determined by color-Doppler mode. The B-mode characteristics were scored from 1–3 (minimal–maximal) for echogenicity and thickness of the granulosa layer of the follicle wall and prominence of the anechoic band around the granulosa layer. These characteristics and methods of assessment have been described [28]. The blood flow of the follicle wall was estimated by the percentage of follicle circumference with color-Doppler signals of blood flow on the real-time sequential two-dimensional images of the entire follicle. The transducer was held at various angles to obtain maximal overall color signals of the follicle wall; the angle between the ultrasound beam and direction of
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Blood samples were centrifuged (1500 g for 10 min) and decanted, and the plasma was stored at 20 8C until assay. Samples were assayed for FSH and LH by radioimmunoassay [32] and for estradiol [33] by commercial kits, as validated and described for mare plasma in our laboratory. The intra- and interassay coefficients of variation (CV) and mean sensitivity, respectively, were 7.8%, 8.3%, and 0.2 ng/mL for LH; 9.2%, 18.4%, and 1.1 ng/mL for FSH; and 10.0%, 4.9%, and 0.1 pg/mL for estradiol. 2.4. Statistical analyses
Fig. 1. Mean ( SEM) concentrations of LH and FSH on Days 10–17 and Days 5 to 4. Significant main effects of age (A) and day (D) are shown. There were no age-by-day interactions. The asterisk (*) for LH in the young group indicates a decrease (P < 0.02) in concentrations between the indicated days.
blood flow (insonation angle) affects the detectability and extent of the color signals [29]. The technique of estimating the percentage of tissue with color-Doppler signals has been validated by the counting of colored pixels in a still image [30,31]. Data were analyzed among the three age groups for systemic concentrations of LH and FSH on the days extending from ablation (Day 10) to the mean day of the beginning of expected deviation (end of the commongrowth phase; mean, Day 17). A break in continuity of data occurred between Day 17 and Day 5 (Fig. 1). Day 5 was used to begin the preovulatory period so that the average interval from ablation to deviation (7 d) and number of days from Days 5 to 0 (6 d) corresponded to the interval from ablation to ovulation (13 d). Concentrations of LH and FSH for Days 5 to 4 were analyzed separately. Systemic estradiol concentrations were assessed for Days 6, 3, 2, 0, and 2. 2.3. Blood samples and hormone assays Collection of a blood sample was done daily. Jugular blood samples were collected into heparinized tubes.
Data for end points that were not normally distributed, according to Shapiro-Wilk tests, were transformed to natural logarithms or ranked. Data for diameter and number of follicles and concentrations of plasma hormones were examined by the SAS MIXED procedure with a REPEATED statement (9.1.3 Version; SAS, Institute Inc., Cary, NC, USA). If the main effect of age was significant or approached significance, the differences among ages were examined by Duncan’s multiple range test. If an age-by-day interaction was detected, Duncan’s multiple range tests were used to locate differences among ages within days. Single-point data were analyzed by one-way ANOVA or unpaired Student’s t-tests. The correlation between LH concentrations during the common-growth phase and number of follicles that reached 10 mm was examined by Spearman tests. A probability of P 0.05 indicated that a difference was significant, and probabilities between P > 0.05 and P 0.1 indicated that the difference approached significance. Data are given as the mean SEM, unless otherwise stated. 3. Results The ovulatory follicular waves that were induced during two consecutive IOIs were not significantly different for any end point between the first and second IOIs and were combined. The incidence of HAFs was not different among age groups [7]. The area of a cross section of the corpus luteum decreased (P < 0.0001) from 6.9 0.2 cm2 on Day 11 to 3.4 0.3 cm2 on Day 14, averaged over the three age groups; there was no main effect of age or an age-by-day interaction (data not shown). Intervals between defined events during the IOI and the induced ovulatory wave were not significantly different among age groups, except for the longer interval from ablation to ovulation (P < 0.01) and from deviation to ovulation (P < 0.03) in the old mares (Table 1). More (P < 0.03) follicles reached 10 and
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Table 1 Mean (SEM) for intervals; numbers, diameters, and growth rates of follicles; and mean and maximum hormone concentrations for three age groups. End points
Young (5–6 y)
Intermediate (10–14 y)
Old (18 y)
Intervals (d) Ovulation to ovulation Ovulation to deviationa Ablation to emergence b Emergence to deviation Deviation to ovulation Ablation to ovulation Maximum diameter to ovulation
22.7 0.3 c 16.9 0.2 1.4 0.2 5.5 0.1 5.9 0.3c,d 12.8 0.2 c 0.3 0.1
22.4 0.4 c 16.9 0.4 1.7 0.2 5.3 0.3 5.4 0.3 c 12.4 0.4 c 0.1 0.1
23.7 0.3d 17.1 0.4 1.9 0.3 5.2 0.2 6.7 0.4d 13.8 0.3d 0.1 0.1
8.3 0.9 c 4.7 0.5 c 2.1 0.2 1.6 0.2 1.3 0.2
6.5 0.6 d 3.6 0.4 d 2.3 0.3 1.7 0.2 1.4 0.2
6.1 0.9d 3.6 0.5d 2.0 0.1 1.6 0.2 1.1 0.1
No. follicles/ovulatory wave 10 mm 15 mm 20 mm 25 mm 30 mm Ovulatory follicle At maximum (mm) At Day 1 (mm) Growth rate (mm/day) Days 12–17 Days 5 to 2 Days 2 to 1
37.9 1.4 37.4 1.4 3.2 0.1 4.0 0.3c,d 1.2 0.8
39.1 1.2 38.9 1.2
39.8 1.3 38.9 1.4
3.4 0.2 4.1 0.2 c 2.0 0.4
3.1 0.1 3.3 0.3d 1.7 0.7
LH, Days 10–17 At maximum (ng/mL) Day of maximum Mean (ng/mL) LH, Days 5 to 4 At maximum (ng/mL) Day of maximum Mean (ng/mL)
10.3 1.1 c 16.1 0.3 6.3 0.7 c
7.1 0.8 d 16.0 0.2 4.5 0.5 d
5.1 0.7d 15.7 0.7 3.2 0.4d
31.3 5.3 c 1.6 0.2 13.0 1.6 c
12.9 1.9 d 1.4 0.2 7.3 1.0 d
9.7 1.9d 1.2 0.2 5.4 1.1d
FSH At maximum (ng/mL) Day of maximum
23.7 1.3 c 12.4 0.4
24.8 1.7 c 14.1 0.3
28.5 1.7d 14.2 0.4
Estradiol At maximum (pg/mL) Day of maximum
6.2 0.7 2.0 0.0
5.7 0.7 1.7 0.3
6.0 0.7 2.0 0.0
a
Deviation refers to mean Day 17 when the future ovulatory follicle was closest to 22.5 mm. PGF/ablation on Day 10. Emergence is the day the future ovulatory follicle was 6 mm. c, d Means within a row that are different (P < 0.05). n = 14–16 ovulatory waves/age group.
b
15 mm during the common-growth phase in the young group than in the intermediate and old groups (Table 1). The number reaching 20, 25, and 30 mm was not different among groups. The number of follicles reaching 10 mm (r = +0.7; P < 0.0001), 15 mm (r = +0.4; P < 0.03), and 30 mm (r = +0.4; P < 0.03) were correlated between consecutive induced ovulatory waves within mares, indicating measurable repeatability in individual mares. Growth rate of the future ovulatory follicle did not differ among age groups between Days 12 and 17 (Table 1). Growth rate differed (P < 0.01) between
Days 5 and 2. The rate for the old group was less than for the intermediate group, but did not differ significantly from the young group, based on the multiple range test (Table 1). However, the growth rate in the old group was less (P < 0.05) than in the young group when examined by an unpaired t-test. There were no differences among age groups in the day of maximum diameter of the preovulatory follicle, diameter on Day 1, or growth rate between Days 2 and 1 (Table 1). There were no age-related effects for B-mode echotextural changes in the follicle wall or percentage of wall with color-Doppler signals of blood flow in the
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no significant difference between intermediate mares and old mares (Table 1). Concentrations decreased (P < 0.02) between Days 5 and 2 in the young group, approached a decrease (P < 0.1) in the intermediate group, and did not decrease in the old group. The concentrations of FSH on Days 10–17 and also on Days 5 to 4 showed only a day effect with no effect of age or an age-by-day interaction (Fig. 1). However, the maximum concentration of FSH during the commongrowth phase was greater in the old group than in the younger groups, but the day of maximum concentration did not differ among groups (Table 1). Concentrations of estradiol increased (day effect; P < 0.0001) between Days 6 and 2 and then decreased with no effect of age or an age-by-day interaction (data not shown). Maximum concentration of estradiol and day of occurrence of maximum were not different among age groups (Table 1). 4. Discussion
Fig. 2. Means (SEM) for end points involving the preovulatory follicle showing the effects of day. The extent of blood flow was based on the percentage of the follicle wall with color-Doppler signals. The echotextural end points (lower panel) were scored from 1–3 (minimal– maximal). There were significant (P < 0.0001–P < 0.02) main effects of day for each characteristic, but no main effects of age or age-by-day interactions.
preovulatory follicle from Days 4 to 1. The main effects of day are shown (Fig. 2). Concentrations of LH on Days 10–17 showed a main effect of day from an immediate and progressive increase, as previously reported (Fig. 1 [15]). A main effect of age reflected LH decreases from young, to intermediate, to old averaged over Days 10–17 with significantly higher mean concentrations in the young group than in each of the other groups (Table 1). The mean LH concentration in individual waves during the common-growth phase and number of follicles that reached 10 mm were not correlated significantly either for the young group or all groups combined. For LH on Days 5 to 4, the main effects of age and day were significant, but the interaction was not (Fig. 1). The concentration averaged over days and the maximum concentration were highest for young mares, with
During the common-growth phase of an induced ovulatory follicular wave (Days 12–17), about two more follicles reached 10 mm and one more reached 15 mm in the youngest group (5–6 y) than in the older groups (10–14 y and 18 y). In reported results with spontaneous waves, young mares (5–7 y) had a more rapid increase in number of 6–10 mm follicles on Days 12 to 6 [9] and old mares (18 y) had fewer follicles 10–15 mm on Days 11 to 9 [7]. Similarly, the number of follicles in follicular waves is reduced in old cows [34] and women [35]. In the previous studies in mares, all growing and regressing follicles were counted without regard to days of the common-growth phase. The present findings indicate that the reported days likely involved the common-growth phase of the spontaneous ovulatory waves of the previous studies. The absence of a significant correlation between LH concentrations during the common-growth phase and number of follicles indicates that the greater number of follicles in the PGF/ablation-induced waves of young mares was not attributable to the greater concentrations of LH. In addition, the number of LH receptors in equine granulosa cells did not seem to increase until follicle diameters were 15–19 mm [36]. The similarity in the induced FSH surges among ages indicated that differences in FSH concentrations also did not account for the greater number of follicles 10 and 15 mm in the induced waves of young mares. Furthermore, the initial portion of the wave (Days 11–13) develops independently of FSH [37–40]. In this regard, the maximum concentration of FSH during the common-
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growth phase was lower in the young group than in the old group and may have represented a greater FSH inhibitory effect from a greater number of follicles [3]. Dependency on the number of follicles in the underlying reservoir was supported by the significant repeatability within mares in number of follicles 10 and 15 mm between the consecutive induced ovulatory waves. It is concluded that the greater number of 10 and 15 mm follicles in the ovulatory waves of young mares was not a function of differences in concentrations of either gonadotropin and represented a larger reservoir of follicles for recruitment. An increase in LH throughout the common-growth phase following PGF/ablation occurred in all age groups. The LH increase is attributable to the PGF treatment. An immediate (within 5 min) increase in LH occurs after PGF treatment in mares during mid-diestrus, presumably from a direct effect at the hypothalamo-pituitary area [30]. The direct LH effect lasts only a couple of hours but overlaps with an LH increase from the PGF-induced decrease in progesterone and thereby a decrease in the negative effect of progesterone on LH. The negative effect of progesterone on LH is consistent with the close temporal reciprocal relationships between the two hormones at the beginning and end of diestrus [41]. A negative effect of a physiologic dose of progesterone on LH has been demonstrated in experiments that included removal of endogenous progesterone by PGF [5,24,42]. The increase in LH after the loss of the negative effect of progesterone and until after the decrease beginning on Day 1 has been proposed [43] to represent the positive effect of extraovarian factors under the control of the environment, especially day length. Concentrations of LH are low to a similar extent in ovarian-intact and ovariectomized mares during the anovulatory season, but high in ovariectomized mares during the ovulatory season [44,45]. Maximum concentration of LH during the ovulatory LH surge seemed similar to the maximum LH concentration in ovariectomized mares [43], although confirmatory study is needed. In the present study, the LH concentrations during the common-growth phase were a mean of 45% and 55% higher in the young group than in the intermediate and old groups, respectively. The similarity among age groups in the rate of regression of the corpus luteum support the conclusion that the greater LH concentration in the young group represented a stronger response to an enviromental influence in the young group. The posttreatment increase in LH reached a transient plateau at the end of the common-growth phase or beginning of deviation, as previously reported [5,24], but a similar plateau does not occur during spontaneous
ovulatory waves [15,27]. The LH increase in spontaneous waves begins on about Day 15 or near the end of the common-growth phase. In a study of age effects in spontaneous waves, LH concentrations were higher in the youngest group throughout the LH surge, beginning at Day 7 (equivalent to Day 16 [7]). The greater LH concentrations in the young group therefore occurred in both spontaneous waves as previously reported and in induced waves in the present study. Although the results of earlier studies indicated that estradiol has a positive effect on LH concentrations in mares, a more recent report concluded that estradiol has a negative effect on LH throughout the LH surge associated with spontaneous waves [46]. Apparently, the earlier reports involved supradoses of estradiol or a rebound in LH from using a prolonged interval (e.g., 24 h) between estradiol treatment and blood sampling. The estradiol increase from the first day considered (Day 6) to a peak on Day 2, in the present study of induced ovulatory waves is similar to previous reports for spontaneous waves [27,41] and induced waves [15]. The decrease in LH concentrations between Days 5 and 2 (after the LH plateau) in the young group likely represented a negative effect of estradiol. The occurrence of the reduction only in the young group seems related to greater LH concentrations on Day 5, considering that estradiol was not different among age groups. The LH concentrations began to increase on Day 2 in synchrony with the estradiol peak in all age groups. The temporal association between decreasing estradiol and increasing LH has been described and discussed for spontaneous waves [41] and is not affected by age, either for spontaneous waves [7] or induced waves (present results). Growth rate of the ovulatory follicle was reduced in the oldest group, consistent with a longer interval from the end of the common-growth phase (Day 17) to ovulation and the absence of a difference in diameter of the follicle on Day 1. The slower growth rate cannot be attributed to the low LH concentrations; the growth rate was not reduced in the intermediate group despite the low LH. There were no age-related preovulatory effects in induced waves on estradiol concentrations, follicle diameter at maximum and on Day 1, granulosa thickness and echogenicity, prominence of the anechoic layer, percentage of wall with color-Doppler signals of blood flow, and rate of the preovulatory reduction in growth of the follicle at the end of induced waves. These similarities occurred despite the greater LH concentrations during the preovulatory period in the young group. The results suggest that the low concentrations of LH represented by the older groups were adequate for
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these preovulatory changes. However, all mares were reproductively active with no signs of approaching ovarian senescence. The effects of age on these features as senescence approaches will require specific study. Although there were no age-related findings on the ultrasonographic characteristics of the preovulatory follicle on Days 4 to 1, the differences among days are of interest in that they extend upon the earlier findings that were limited to 1.5 d before ovulation [28]. The increase between Days 2 and 1 in thickness and echogenicity of the granulosa layer agrees with the earlier report, but also indicated that the changes in the granulosa were more exaggerated between Days 2 and 1 than between Days 4 and 2. Prominence of the anechoic band did not increase between Days 2 and 1 in agreement with the previous report but did increase between Days 4 and 3. Percentage of follicle wall with blood-flow signals increased after Day 3, extending upon the previous report. In conclusion, induction of an ovulatory wave with PGF treatment and ablation of follicles on Day 10 was used to study the effects of age on the induced wave during the common-growth phase (Days 12–17). The youngest group (5–6 y) had greater concentrations of LH and more follicles 10 mm and 15 mm during the induced wave than the intermediate group (10–14 y) and the oldest group (18 y). In young mares, the greater LH concentrations may have represented a more prominent response to extraovarian factors after the induced luteolysis, and the greater number of follicles likely represented a larger follicle reservoir. Mean FSH concentrations during the common-growth phase were not different among groups. However, the maximum FSH concentration was greater in the old group and may have reflected, at least in part, the reduced inhibitory effect of a smaller number of follicles. Maximum diameter, B-mode echotexture, and color-Doppler signals of blood flow of the preovulatory follicle and plasma estradiol concentrations during the preovulatory period were not different among age groups, even though LH concentrations were greater in the young group. Growth rate of the preovulatory follicle was reduced in the oldest group, consistent with a longer interval from the end of the common-growth phase to ovulation and no difference in diameter of the follicle on Day 1. Acknowledgments Research supported by the Eutheria Foundation. The authors thank Pfizer Animal Health New York, NY, USA for a gift of Lutalyse. J. C. Jacob was on leave
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