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The effects of dose and duration of administration of pFSH during the first follicular wave on the ovulation rate of beef heifers
ELSEVIER
THE EFFECTS OF DOSE AND DURATION OF ADMINISTRATION OF pFSH DURING THE FIRST FOLLICULAR WAVE ON THE OVULATION RATE OF BEEF HEIFERS M.G. Murphy 1, M.P. Boland2 and J.F. Roche 1 Faculties of 1Veterinary Medicine and 2Agriculture University College Dublin, Dublin 4, Ireland Received for publication: September 6, 1996 Accepted: November 2, 1997 ABSTRACT Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n=ll to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n=l 1 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline. © 1998 by ElsevierScience Inc.
Key words: pFSH, twin ovulations, heifers, dominant follicle
Acknowledgments We thank Vetrepharm, Canada, for Folltropin; Goodman International, South Eastern Breeding Society, and Kildangan Stud for use of animals and facilities. Theriogenology 49:557-569, 1998 © 1998 by Elsevier Science Inc.
O093-691X/98f$19.00 PII S0093-691X(98)00007-7
Theriogenology
558 ~TRODUCTION
The incidence of twin ovulations is low (1 to 4%) in cattle (21). This is a major limitation to increasing the calf crop in beef cows since most beef cows produce a single calf per year. Previous attempts to induce twin ovulations and twin pregnancies in cattle have focused on immunization against a number of ovarian steroids (dehydroepiandrostenedione: 25; androstenedione: 31; testosterone and estradiol: 30), or follicular fluid, which presumably contains inhibin (20), and on the use of exogenous gonadotropins such as PMSG (5, 7). Although much work has been done with varying doses of PMSG (750 to 1000 IU; 3, 7, 16, 28), the use of FSH to induce twin pregnancies has not received the same attention. Administration of 12 mg pFSH over 5 d in the mid-luteal phase increased the ovulation rate and the percentage of twin ovulations (4), it decreased the fertilization rate and increased the degree of embryo mortality compared with PMSG administration at the same stage of the cycle (15). The variation in ovarian responsiveness may be due to differences in FSH potencies (16) or to the stage of follicular development at the time of treatment. A previous study (8) has shown definitively that the stage of the estrous cycle at which exogenous gonadotropins are administered has a significant effect on ovulation rate and that in general the mean ovulation rate is lower at the beginning of the cycle than at mid-cycle. However, no earlier study has specifically examined the effect of exogenous pFSH given early in the estrous cycle on the pattern of development of dominant follicles, particularly with regard to the effects on the induction of twin ovulations. Follicular development during the estrous cycle in cattle is characterized by the successive development of a number of dominant follicles (> 10 mm in diameter), usually 2 to 3 per cycle (13, 23, 24). Recent data (1, 26) have clearly shown that there are cyclical fluctuations in FSH concentrations during the estrous cycle of heifers. Concentrations of FSH increase prior to the emergence of each new wave of follicular growth, and, as a follicle becomes dominant, there is an associated suppression of FSH concentrations (1, 26). Thus, the decline in FSH concentrations occurs during a period when a single follicle is selected to continue growing, and this evidence suggests that the decline in FSH may be responsible for dominant follicle selection. Accordingly, the aim of these experiments was to give extra pFSH during the selection phase in order to have 2 rather than a single dominant follicle selected and to then ovulate the follicles following induction of luteolysis with prostaglandin F2ct (PG). The first wave of follicular growth was chosen in this series of experiments because it is the most predictable in terms of time of occurrence. MATERIALS AND METHODS Heifer Management and pFSH Administration Nulliparous Sirnmental x Friesian heifers, 1.5 to 2.0 yrs of age, were used in the studies. The estrous cycle of each heifer was synchronized using 2 intramuscular injections of a synthetic PG analogue (cloprostenol, Estrumate, Cooper's Animal Health, Berks, UK) 11 d apart. After the second injection of PG, the heifers were observed 4 times daily (30 rain per observation) for signs of estrus. If housed, the animals were given access to a solid concrete floor to aid in estrus
Theriogenology
559
detection. In these studies, the FSH used was lyophilized porcine FSH (Folltropin, Vetrepharm Inc., London, Ontario, Canada). The administration of pFSH began on Day 3 (estrus = Day 0) and continued twice daily until Day 5 or until estrus, as outlined in each of the experiments. Heifers were treated with PG on the evening of Day 5 to induce luteolysis, estrus and ovulation. Detection of estrus began at approximately 36 h and continued until approximately 120 h post PG administration. Ultrasound Scanning The ovaries of the heifers were examined once daily by the same operator, from Day 3 until estrus, using a 7.5 MHz transducer probe and a real-time ultrasound scanner (Dynamic Imaging, Livingston, UK), as described previously (23). The diameter (mm) and location relative to other ovarian structures of all follicles > 5mm were recorded daily. Ovulation rate was determined either 9 d after the administration of PG by counting the number of corpora lutea (CL) on each ovary (Experiments 1, 2 and 4) or at slaughter (Experiments 3 and 4). Experiment 1 Forty-seven heifers were allocated to 1 of 4 treatments using a randomized incomplete block design: a) control (n = 11); b) 1.5 mg pFSH until estrus (n = 12); c) 2.0 mg pFSH until estrus (n = 12); or d) 2.5 mg pFSH until estrus (n = 12). This experiment was carried out when the heifers were at pasture. Experiment 2 Forty-five heifers were assigned to 1 of 5 treatments using a randomized incomplete block design: a) control (n = 8); b) 1.0 mg pFSH until PG (n = 8); c) 1.0 mg pFSH until estrus (n = 10); d) 1.5 mg pFSH until PG (n = I0); e) 1.5 mg pFSH until estrus (n = 9); . Experiment 2 was carried out when the heifers were housed in a straw-bedded shed and were fed grass silage ad libitum. Experiment 3 Forty-three heifers were allocated to 1 of 3 treatment groups using a randomized incomplete block design: a) control (n = 11); b) 1.0 mg pFSH until estrus (n = 16); or c) 1.5 mg pFSH until estrus (n --- 16). This experiment was carried out when the heifers were housed on a slatted floor system and were fed grass silage ad libitum. The ovaries of the heifers in this experiment were not scanned during treatment with pFSH. Heifers were slaughtered 14 d after PG and the number of CL was counted. Experiment 4 Sixty-five heifers were allocated to 1 of 3 of the following treatments using a randomized incomplete design: a) pFSH from Day 3 (estrus = Day 0) to coincide with the development of the first dominant follicle (n = 19); b) pFSH to coincide with the plateau phase of development (Day
Theriogenology
560
6 to Day 8 of the cycle) of the first dominant follicle (n = 17); c) pFSH to coincide with the decline phase of the first dominant follicle (n = 17), which was based on the dominant follicle maintaining its maximum diameter for 2 d, after which it was assumed that it would start to become atretic. The dose ofpFSH for each of the treatment groups was 1.5 mg twice daily until estrus. Control heifers (n = 4) were assigned to each treatment group and received PG at the same time as the pFSH-treated heifers. The ovaries of all heifers were scanned dally from Day 3 until the first dominant follicle was identified. Subsequently, the pattern of growth of the first dominant follicle was monitored in all heifers until PG was administered. The heifers were slaughtered 5 to 7 d after the administration of PG. The ovulation rate was determined by counting the number of CL on both ovaries. These data were compared with assessments of the ovulation rate made by using the ultrasound scanner; the correlation coefficient was 0.92 (P < 0.01). The heifers were housed on a slatted floor system and were fed an ad libitum diet of raisins, beet pulp and grass silage. Statistical Analysis Differences between means were determined after one-way analysis of variance and using Fischer least significant difference to indicate significant differences between treatment means. Statistical significance was inferred at P < 0.05. Chi-square analysis (27) was used to determine if there were significant differences between treatments in the proportion of heifers with twin or multiple ovulations or that were detected in estrus after administration of PG. RESULTS Experiment 1 The pattern of emergence of the first follicular wave in control heifers was similar to that previously reported (23). There was no overall effect (P > 0.05) of dose of pFSH on the number of medium (5 to 9 mm) follicles (data not presented). However, on the first day post PG, heifers assigned to 2.5 mg pFSH had increased (P < 0,05) numbers of medium follicles compared with controls or with heifers assigned to 1.5 mg pFSH. Injection of 2.0 or 2.5 mg pFSH from Day 3 until estrus increased the mean number of large follicles on Day 5 and on the first and second day post PG compared with that of the control heifers, while 1.5 mg pFSH until estrus increased the number of large follicles on the first and second day post PG (Table 1). All control heifers had single ovulations. The ovulation rate of heifers injected with pFSH between Day 3 and estrus was higher (P < 0.05) than in control heifers. However, there was wide variation in the ovulation rate among pFSH-treated heifers. Although pFSH-treated heifers had multiple o~axlations (Table 1), there was no significant difference (P > 0.05) between pFSH treatments in the proportion of heifers that had multiple ovulations (> 3). Some of the large follicles identified on Day 2 post PG did not ovulate. The estrous response in control, 1.5 mg pFSH, 2.0 mg pFSH and 2.5 mg pFSH-treated heifers was 7/11 (64 %), 9/12 (75 %), 11/12 (92 %) and 9/12 (75 %) respectively (P > 0.05). Treatment with pFSH did not affect (P > 0.10) the interval from PG to the onset of estrus (Table 1).
12
12
12
1.5mgFSHp
2.0mg FSHp
2.5mg FSHp
2.1+0.4 b
2,0_+0.4 b
1.8+0.3 "b
0.9+0.2 =
5
Day of estrous cycle
2.9+0.6 b
3.7+0.5 b
2,7+0,3 b
1.2+0.2 a
1
6,3+0.9 ~
4 . 3 + 0 . 7 b¢
3.2+0.7 b
1.0+0.1"
2
Day after PG
3.4+0.7 b
3.3+0.6 b
2.6+0.4 ~
1.0_+0.0 ~
Mean OR
5
4
4
11
1
1
1
2
2
3
3
1
4
1
4
No. of CL
2
2
5
3
6
2
7
42 + 8"
51 + 8 a
39 + 7"
53 + 14"
Interval from PG to estrus (hours)
Data are not presented from Days 3 and 4 of the estrous cycle since there were no significant differences between treatments on these days.
abc Means in the same column with different superscripts are significantly different (P<0.05).
11
n
Control
Treatment
No. of large (>10mm) follicles
Table 1. The effect of 1.5, 2.0 or 2.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until estrus on the mean (+ SEM) number of large (_>10 mm) follicles on Day 5 of the estrous cycle, and on the first and second day after PG; on the mean ovulation rate (OR), on the number of corpora lutea (CL); and on the interval from PG to onset of estrus in beef heifers. (Experiment 1)
.-L
03
ol
2
562
Theriogenology
Experiment 2 The pattern of emergence of the first follicular wave was typical of untreated control heifers, similar to Experiment 1. There was no overall significant effect ofpFSH between Days 3 and 5, or until estrus on the mean number of medium follicles (data not presented). The mean number of large follicles was increased (P < 0.05) from Day 5 until the second day post PG by 1.0 and 1.5 mg pFSH compared with that of the untreated controls. The ovulation rate of heifers injected with pFSH between Day 3 and estrus was higher (P < 0.05) than that of the controls (Table 2). The mean ovulation rate for all pFSH-treated heifers was lower than the mean number of large follicles detected on the second day post PG. Multiple ovulations were detected in all pFSHtreated groups (Table 2). However, there was no significant difference between pFSH treatments in the proportion of heifers which had twin or multiple ovulations. There was also no significant difference between treatments in the interval to estrus after PG (Table 2) or in the estrous response after PG (control: 6/8 (75 %); 1.0 mg pFSH until PG: 7/8 (88 %); 1.0 mg pFSH until estrus: 10/10 (100 %); 1,5 mg pFSH until PG: 9/10 (90 %); and 1.5 mg pFSH until estrus: 9/9
(100%). Experiment 3 Four control heifers did not ovulate (based on the absence o f a CL at ultrasound examination), and their data were excluded from analysis. The mean ovulation rate was higher (P < 0.05) in pFSH-treated heifers than in controls, but there was no difference in ovulation rate between heifers given 1.0 (2.0 + 0.2) or 1.5 mg pFSH (2.2 + 0.4) until estrus. The proportion of heifers with twin ovulations following 1.0 mg or 1.5 mg pFSH was greater (P = 0.011) than in untreated controls (Table 3). However, there was no difference in the percentage of heifers with 2 or more ovulations between the 2 pFSH treatments. Although the percentage of control heifers that exhibited estrus after PG was 64 % (7/11), this was not different (P > 0.05) compared with pFSH-treated heifers (1.0 mg pFSH until estrus: 13/16, 81% vs 1.5 mg pFSH until estrus: 15/16, 94 %). Treatment with pFSH did not affect (P > 0.10) the interval between PG and estrus. Experiment 4 The mean ( + SEM) diameter (mm) of the first dominant follicle at the time of initiation of pFSH for the 3 treatments (pFSH-incline, pFSH-plateau and pFSH-decline, respectively) was 9.0 + 0.3, 13.2 + 0.4 and 13.8 + 0.6. The mean diameter of the dominant follicle was significantly smaller (P < 0.05) for heifers given pFSH at the incline (growth) stage of development of the first dominant follicle than at the plateau (static) and decline phases. There was no difference (P > 0.05) between the pFSH-treated heifers and their respective controls in the mean diameter of the first dominant follicle at the initiation of pFSH treatment. All of the control heifers had a single ovulation after PG. There was no difference (P > 0.05) in the mean ( + SEM) ovulation rate of heifers assigned to the pFSH-incline or pFSH-plateau treatments (Table 4). However, the mean ovulation rate in the pFSH-deeline treatment was higher (P < 0.05) than for either of the other pFSH treatment groups. The proportion of heifers with multiple ovulations (>_ 3 ovulations) in the pFSH-decline treatment group (7/17) was greater (P < 0.05) than in the pFSH-incline
8
1.0 mg FSHp
10
9
1.5 mg FSHp --> PG
1.5 mg FSHp --> Estrus
2.3 + 0.3 b
1.6 +0.3b
2.1 +0.3 b
2.0+0.3 b
1.0 + 0 . 2 a
5
Day of estrous cycle
-+ 0.0 a
3.1 +0.5 ~
22+0.3 b
2.5 + 0.4 b
2.1 +0.4 b
1.0
1
3.3_+0.5 bc
2.2 + 0.3 b
2.2+0.4 b
2.3+0.4 =
1.0 + 0.0"
2
Day after PG
3.1 +0.4 c
1.6+0.3 ab
2.1 +0.3 b
1.3+0.3 ab
1.0 -+ 0 . 0 a
Mean OR
1
6
3
7
8
1
2
2
2
1
3
3
1
-
4
1
5
8a
5 2 + 5a
47_+ 6 a
52-+ 5"
53+
61 -+ 14a
Interval from PG to estrus (hours)
Data are not presented from Days 3 and 4 of the estrous cycle since there were no significant differences between treatments on these days.
2
2
4
2
No. of CL
abc Means in the same column with different superscripts are significantly different (P<0.05).
10
1.0 mg FSHp --> Estrus
--> PG
8
n
Control
Treatment
No. of large (_>10mm) follicles
Table 2. The effect of 1.0 and 1.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until Day 5 or until estrus (induced by a single injection of Cloprostenol [PG] on Day 5) on the mean (_+SEM) number of large (>10mm) follicles on Day 5 of the estrous cycle and on the first and second day after PG; on the mean (_+SEM) ovulation rate (OR); on the number of corpora lutea (CL); and on the interval from PG to onset of estrus (hours _+SEM) in beef heifers. (Experiment 2)
O1 6a
55+
6a
44 + 6"
16
16
38 + 13a
11
Hours from PG to Estrus
2.2+0.4 ~
2.0 +_0.2 b
1.0 + 0.0a
Mean OR
5
5
7
1
ab Means in the same column with different superscripts are significantly different (P
--> Estrus
1.5 mg FSHp
--> Estrus
1.0 mg FSHp
Control
Treatment
slaughter in beef heifers. (Experiment 3)
8
7
2
3
3
2
1
4
No. of CL
Table 3. The effect of 1.0 or 1.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until estrus (induced by a single injection of cloprostenol [PG] on Day 5) on the interval (hours + SEM) from PG to onset to estrus; on the mean (+ SEM) ovulation rate (OR); and on the number of corpora lutea (CL) detected at
¢)
ol o')
17
17
Static Phase
Decline Phase
58 -+ 9=
43_+ 4 a
45_+ 7a
54 +_ 10 a
Hours from PG to Estrus
3.9 -+ 0.8c
1.9+_0.3 b
1.9+0.3 b
1.0 + 0.0 a
Mean OR
5
8
8
12
1
3
5
8
2
2
2
2
3
Means in the same column with different superscripts are sign ficantly different (P<0.05).
19
Growth Phase
abc
12
Control
Treatment 4
5
6
No. of CL 7
8
9
Table 4. The effect of 1.5 mg pFSH given twice daily to coincide with the growth phase (Day 3), static phase (Day 6 - 8) or decline phase (Day 8 +) of the development of the first dominant follicle, followed by a single injection of cloprostenol (PG) 2 days after the initiation of treatment with FSH, on the interval (hours + SEM) from PG to onset of estrus; on the mean (+ SEM) ovulation rate (OR); and on the number of corpora lutea (CL) in beef heifers. (Experiment 4)
10
11
O"1
O1 O~
(b (b
Theriogenology
566
treatment group (1/19). Although there were numerically more heifers with double ovulations in the pFSH-incline treatment (8/19), this was not significant (P = 0.12) compared with the pFSHdecline treatment (3/17). The estrous response of heifers assigned to the pFSH-incline treatment group was 68% (13/19). However, this was not statistically significant (P > 0.10) compared with that of the pFSH-plateau (15/17; 88 %) or the pFSH-decline (14/17; 82 %) treatment groups. Heifers assigned to the pFSH-decline treatmefit group tended to have a longer interval to estrus after PG (57.8 h) than after the pFSH-incline (44.8 h) and the pFSH-plateau (43.3 h) treatments. However, this effect was also not statistically significant (P > 0.10). DISCUSSION The results of our 4 experiments indicate that low dosages of pFSH administered twice daily from Day 3 of the estrous cycle result in the selection and ovulation of 1 to 2 extra follicles. The administration of pFSH until estrus appears to be essential for the ovulation of these extra selected follicles. Treatment with pFSH did not significantly increase the number of medium follicles, however, the number of large follicles increased following injections of 1.5, 2.0 or 2.5 mg pFSH twice daily in all the experiments. Although variation in the ovulation rate was observed in pFSH-treated heifers, the incidence of twin ovulations was low in Experiments 1 and 2. In Experiments 3 and 4, the incidence of twin ovulations was 47% and 30% respectively. The results from Experiment 4 suggest that the stage of development of the first dominant follicle may influence the ovulation rate and the occurrence of twin and multiple ovulations. In cattle (12), FSH is the most important hormone to stimulate follicular growth to the dominant follicle stage. Recent data have shown that the secretion of FSH during the estrous cycle follows a recurrent pattern of elevation and plateau (1, 26). One day after the elevation in FSH concentration, a new follicular wave emerges. Selection of the dominant follicle occurs as FSH concentrations decline, and dominance is associated with plateau concentrations of FSH. The precise temporal relationship between the decline in FSH concentration and the selection of the dominant follicle remains to be elucidated. Recent data suggest that selection of a single dominant follicle can be delayed by 4 dally injections of pFSH which slows the rate of FSH decline after follicular wave emergence (2). The results of the four experiments do not fully support a previous study (14) which suggested that the fate of subordinate follicles was determined by Day 3. Clearly, the presence of an active dominant follicle when treatment with pFSH was initiated, lowered the ovulatory response compared to other stages of the estrous cycle (9, 10), and in this regard, the lower ovulation rates obtained in our study support this observation. The results from Experiment 4, in which the effects of pFSH at various stages of development of the first dominant follicle were examined, showed significant differences in the ovulation rate between the decline phase and the growth and plateau phases of development. This is most likely due to the loss/lack of physiological dominance of the first dominant follicle from Day 8 forward and supports the hypothesis of higher ovulation rates associated with the absence of a dominant follicle. The administration of pFSH from Day 3 did not increase the number of follicles present during the first wave but appeared to result in more medium follicles attaining large diameters. Since data were not recorded for small (< 5 mm) follicles, it is not possible to say if that
Theriogenology
567
follicular population was influenced by pFSH. The current data suggest that exogenous FSH from Day 3 onward probably delays the onset of atresia in cohort subordinate follicles observed on Day 6 rather than rescuing atretic follicles, since treatment with FSH was initiated before atresia had begun. Previously, it has been demonstrated that PMSG can "rescue" follicles from atresia (17). It has also been postulated that preselection superovulatory treatments (pFSH) may be acting to delay onset of atresia (2). Data which were generated in this laboratory (22) have shown that a single injection of 7 mg pFSH on Day 1 of the estrous cycle increased the number of medium follicles, decreased the number of small follicles but did not affect the number of large follicles or the total follicle population. Similarly, in prepubertal heifers which received FSH, a shift from small to meditma and large follicles without any effect on the total number of follicles was observed (19). The failure of the FSH-stimulated follicles to ovulate, which was noted in Experiments 1 and 2, may be due to the initiation of atresia as a consequence of reduced FSH concentrations in association with luteal phase LH pulse frequency. In Experiments 3 and 4, data were not recorded on the number of large follicles that were present 2 d after the administration of PG, and on the number of large follicles that subsequently ovulated. Increased FSH concentrations appear to be important in the maintenance of multiple dominant follicles from PG to estrus, suggesting that FSH may play a role in estrogen activity of these follicles, as termination of FSH at the time of initiation of luteolysis resulted in a decreased ovulation rate (1, 26); it may also be that the decline in FSH is responsible for follicle selection. The interaction of insulin-like growth factor-I (IGF-I) and FSH in the induction of the aromatase system is important for estradiol production within the follicle. However, it is possible that if FSH support is withdrawn in the presence of elevated concentrations of progesterone, androgen production declines as a consequence of reduced LH pulse frequencies, and this leads to decreased intrafollicular production of estradiol. In turn, IGF-I concentrations decrease due to effects of IGF binding proteins (IGFBPs; 11). For example, IGFBP 3 inhibits the binding of FSH to its receptor (29), and FSH inhibits the production of IGFBPs within the follicle (11). Therefore, a fall in FSH concentrations may lead to increased IGFBPs, which decrease IGF production (11) and thus follicular atresia may ensue. The development of practical regimens to induce twin ovulations and twin pregnancies in cattle has been an ongoing concern for many years. Regardless of the approaches used, with the exception of embryo transfer, variability in ovulatory response has been a consistent finding (3, 5, 6). The results of our present experiments suggest that a low dose of pFSH (1.0 to 1.5 mg twice daily from Day 3 until estrus) together with the presence of an active dominant follicle resulted in a significant increase in the occurrence of twin ovulations. Although multiple ovulations were detected, some heifers failed to respond to ovarian stimulation by pFSH. However, our data show that a 50% twin ovulation rate can be achieved when treatment is initiated during the first wave of follicular growth in conjunction with low doses ofpFSH. In summary, administration of pFSH during the putative phase of selection of the first dominant follicle can increase the number of follicles detected by 1 or 2. The successful ovulation of most of the selected dominant follicles is dependent on the continued administration
568
Theriogenology
of pFSH after PG until estrus. Variability in ovulation rate remains a problem even with the low dosages of pFSH used in these studies. REFERENCES 1.
2. 3.
4. 5.
6. 7. 8.
9.
10.
11. 12. 13.
14.
15. 16.
Adams, GP, Matted, RL, Kastelic, JP, Ko, JCH, Ginther, OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992; 94: 177-188. Adams, GP, Kot, K, Smith, CA, Ginther, OJ. Selection of a dominant follicle and suppression of follicular growth in heifers. Anim Reprod Sci 1993; 30: 259-271. Anderson, GB, Bondurant, RH. Multiple ovulation resulting from low level administration of PMSG to cattle at different stages of the oestrous cycle. Anim Reprod Sci 1982; 5: 8591. Bellows RA, Anderson DC, Short RE. Dose-response relationship in synchronised beef heifers treated with follicle stimulating hormone. J Anim Sci 1969; 28: 628-644. Chupin D, Cognie Y, Combarnous R, Procureur R, Saumande J. Effect of purified LH and FSH on ovulation in the cow and ewe. In: Roche JF and O'Callaghan D (eds), Follicular Growth and Ovulation Rate in Farm Animals. Dordecht: Martinus Nijhoff, 1987; 63-72. Gordon I. Controlled Breeding in Farm Animals. Oxford: Pergamon Press, 1983. Gordon I, Williams G, Edwards J. The use of serum gonadotrophin (PMS) in the induction of twin pregnancy in the cow. J Agric Sci 1962; 59: 143-198. Goulding D, Williams DH, Duffy P, Boland MP, Roche JF. Superovulation in heifers given FSH initiated either at Day 2 or at Day 10 of the estrous cycle. Theriogenology 1990; 34: 767-778. Grasso F, Guilbault LA, Roy GL, Lussier JG. Ultrasonographic determination of ovarian follicular development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle. Theriogenology 1989; 31: 1209-1219. Guilbault LA, Grasso F, Lussier JG, Roulllier P, Matton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 1991; 91: 81-89. Hammond JM, Mondschein JS, Samaras SE, Smith JD, Hagen DR. The ovarian insulinlike growth factor system. J Reprod Fertil 1991; 43 (Suppl): 199-208. Ireland JJ. Control of follicular growth and development. J Reprod Fertil 1987; 34 (Suppl): 39-54. Knopf, L, Kastelic, JP, Schallenberger, E, Ginther, OJ. Ovarian follicular dynamics in heifers: test of two-wave hypothesis by ultrasonically monitoring individual follicles. Dom Anita Endocrinol 1989; 6:111-119. Ko JHC, Kastelic JP, Del Campo MR, Ginther OJ. Effects of a dominant follicle on ovarian follicular dynamics during the oestrous cycle in heifers. J Reprod Fertil 1991; 91: 511-519. Laster DB. Ovulation, fertility and prenatal mortality in heifers treated with PMSG or porcine FSH. J Reprod Fertil 1973; 33: 275-282. Laster DB, Turman EJ, Stephens DF, Renbarger RE. Ovulation rates of beef cows and heifers treated with equine gonadotropin (PMS) and chorionic gonadotropin (hCG). J Anita Sci 1971; 33: 433-439.
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17. Monniaux D, Mariana JC, Gibson WR. Action of PMSG on follicular populations in the heifer. J Reprod Fertil 1984; 70: 243-253. 18. Morris DG, McDermott MG, Diskin MG, Morrison CA, Swift PJ, Sreenan JM. Effect of immunisation against synthetic peptide sequences of bovine inhibin ~-subunit on ovulation rate and twin-calving rate in heifers. J Reprod Fertil 1993; 97: 255-261. 19. Moser, MT, Garverick, HA, Smith, M.F, Youngquist, RS. Follicular and endocrine patterns of prepubertal heifers administered bovine follicular fluid and (or) follicle stimulating hormone. Anim Reprod Sci 1989; 18: 227-242. 20. Price CA, Morris BA, O'Shea T, Webb R. Active immunisation of cattle against partly purified follicular fluid from sheep. J Reprod Fertil 1987; 81: 161-168. 21. Rutledge JJ. Twinning in cattle. J Anim Sci 1975; 40: 803-815. 22. Savio JD. A study of the dominant follicle in cattle using ultrasound. Ph.D. Thesis. National University of Ireland, 1988. 23. Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Fertil 1988; 83: 663-671. 24. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Bio 1Reprod 1988; 39:308 -317. 25. Sreenan JM, Morris D, Tait A, Diskin MG. Manipulation of the immune system to increase ovulation rate in cows. In: Roche JF and O'Callaghan D (eds), Follicular Growth and Ovulation Rate in Farm Animals. Dordecht: Martinus Nijhoff, 1987; 73-86. 26. Sunderland SJ, Crowe MA, Boland MP, Roche JF, Ireland JJ. Selection, dominance and atresia of follicles during the oestrous cycle of heifers. J Reprod Fertil 1994; 101: 547-555. 27. Swinscow TDV. Statistics at Square One. Bath, England, Dawson and Goodall, 1977; 4353. 28. Turman EJ, Laster DB, Renbarger RE, Stephens DF. Multiple births in beef cows treated with equine gonadotropin (PMS) and chorionic gonadotropin (HCG). J Anim Sci 1971; 32: 962-969. 29. Ui M, Shimonaka M, Shimasaki S, Ling N. An insulin-like growth factor-binding protein in ovarian follicular fluid blocks follicle stimulating hormone-stimulated steroid production by ovarian granulosa cells. Endocrinology 1989; 125: 912-916. 30. Webb R, Land RB, Pathiraja N, Morris BA. Passive immunisation against steroid hormones in the female. In: Crighton DB (ed). Immunological Aspects of Reproduction in Mammals. London: England: Butterworth, 1984; 475-499. 31. Wise TH, Schanbacher BD. Reproductive effects of immunizing heifers against androstenedione and oestradiol 17-13. J Reprod Fertil 1983; 69: 605-612.
THE EFFECTS OF DOSE AND DURATION OF ADMINISTRATION OF pFSH DURING THE FIRST FOLLICULAR WAVE ON THE OVULATION RATE OF BEEF HEIFERS M.G. Murphy 1, M.P. Boland2 and J.F. Roche 1 Faculties of 1Veterinary Medicine and 2Agriculture University College Dublin, Dublin 4, Ireland Received for publication: September 6, 1996 Accepted: November 2, 1997 ABSTRACT Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n=ll to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n=l 1 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline. © 1998 by ElsevierScience Inc.
Key words: pFSH, twin ovulations, heifers, dominant follicle
Acknowledgments We thank Vetrepharm, Canada, for Folltropin; Goodman International, South Eastern Breeding Society, and Kildangan Stud for use of animals and facilities. Theriogenology 49:557-569, 1998 © 1998 by Elsevier Science Inc.
O093-691X/98f$19.00 PII S0093-691X(98)00007-7
Theriogenology
558 ~TRODUCTION
The incidence of twin ovulations is low (1 to 4%) in cattle (21). This is a major limitation to increasing the calf crop in beef cows since most beef cows produce a single calf per year. Previous attempts to induce twin ovulations and twin pregnancies in cattle have focused on immunization against a number of ovarian steroids (dehydroepiandrostenedione: 25; androstenedione: 31; testosterone and estradiol: 30), or follicular fluid, which presumably contains inhibin (20), and on the use of exogenous gonadotropins such as PMSG (5, 7). Although much work has been done with varying doses of PMSG (750 to 1000 IU; 3, 7, 16, 28), the use of FSH to induce twin pregnancies has not received the same attention. Administration of 12 mg pFSH over 5 d in the mid-luteal phase increased the ovulation rate and the percentage of twin ovulations (4), it decreased the fertilization rate and increased the degree of embryo mortality compared with PMSG administration at the same stage of the cycle (15). The variation in ovarian responsiveness may be due to differences in FSH potencies (16) or to the stage of follicular development at the time of treatment. A previous study (8) has shown definitively that the stage of the estrous cycle at which exogenous gonadotropins are administered has a significant effect on ovulation rate and that in general the mean ovulation rate is lower at the beginning of the cycle than at mid-cycle. However, no earlier study has specifically examined the effect of exogenous pFSH given early in the estrous cycle on the pattern of development of dominant follicles, particularly with regard to the effects on the induction of twin ovulations. Follicular development during the estrous cycle in cattle is characterized by the successive development of a number of dominant follicles (> 10 mm in diameter), usually 2 to 3 per cycle (13, 23, 24). Recent data (1, 26) have clearly shown that there are cyclical fluctuations in FSH concentrations during the estrous cycle of heifers. Concentrations of FSH increase prior to the emergence of each new wave of follicular growth, and, as a follicle becomes dominant, there is an associated suppression of FSH concentrations (1, 26). Thus, the decline in FSH concentrations occurs during a period when a single follicle is selected to continue growing, and this evidence suggests that the decline in FSH may be responsible for dominant follicle selection. Accordingly, the aim of these experiments was to give extra pFSH during the selection phase in order to have 2 rather than a single dominant follicle selected and to then ovulate the follicles following induction of luteolysis with prostaglandin F2ct (PG). The first wave of follicular growth was chosen in this series of experiments because it is the most predictable in terms of time of occurrence. MATERIALS AND METHODS Heifer Management and pFSH Administration Nulliparous Sirnmental x Friesian heifers, 1.5 to 2.0 yrs of age, were used in the studies. The estrous cycle of each heifer was synchronized using 2 intramuscular injections of a synthetic PG analogue (cloprostenol, Estrumate, Cooper's Animal Health, Berks, UK) 11 d apart. After the second injection of PG, the heifers were observed 4 times daily (30 rain per observation) for signs of estrus. If housed, the animals were given access to a solid concrete floor to aid in estrus
Theriogenology
559
detection. In these studies, the FSH used was lyophilized porcine FSH (Folltropin, Vetrepharm Inc., London, Ontario, Canada). The administration of pFSH began on Day 3 (estrus = Day 0) and continued twice daily until Day 5 or until estrus, as outlined in each of the experiments. Heifers were treated with PG on the evening of Day 5 to induce luteolysis, estrus and ovulation. Detection of estrus began at approximately 36 h and continued until approximately 120 h post PG administration. Ultrasound Scanning The ovaries of the heifers were examined once daily by the same operator, from Day 3 until estrus, using a 7.5 MHz transducer probe and a real-time ultrasound scanner (Dynamic Imaging, Livingston, UK), as described previously (23). The diameter (mm) and location relative to other ovarian structures of all follicles > 5mm were recorded daily. Ovulation rate was determined either 9 d after the administration of PG by counting the number of corpora lutea (CL) on each ovary (Experiments 1, 2 and 4) or at slaughter (Experiments 3 and 4). Experiment 1 Forty-seven heifers were allocated to 1 of 4 treatments using a randomized incomplete block design: a) control (n = 11); b) 1.5 mg pFSH until estrus (n = 12); c) 2.0 mg pFSH until estrus (n = 12); or d) 2.5 mg pFSH until estrus (n = 12). This experiment was carried out when the heifers were at pasture. Experiment 2 Forty-five heifers were assigned to 1 of 5 treatments using a randomized incomplete block design: a) control (n = 8); b) 1.0 mg pFSH until PG (n = 8); c) 1.0 mg pFSH until estrus (n = 10); d) 1.5 mg pFSH until PG (n = I0); e) 1.5 mg pFSH until estrus (n = 9); . Experiment 2 was carried out when the heifers were housed in a straw-bedded shed and were fed grass silage ad libitum. Experiment 3 Forty-three heifers were allocated to 1 of 3 treatment groups using a randomized incomplete block design: a) control (n = 11); b) 1.0 mg pFSH until estrus (n = 16); or c) 1.5 mg pFSH until estrus (n --- 16). This experiment was carried out when the heifers were housed on a slatted floor system and were fed grass silage ad libitum. The ovaries of the heifers in this experiment were not scanned during treatment with pFSH. Heifers were slaughtered 14 d after PG and the number of CL was counted. Experiment 4 Sixty-five heifers were allocated to 1 of 3 of the following treatments using a randomized incomplete design: a) pFSH from Day 3 (estrus = Day 0) to coincide with the development of the first dominant follicle (n = 19); b) pFSH to coincide with the plateau phase of development (Day
Theriogenology
560
6 to Day 8 of the cycle) of the first dominant follicle (n = 17); c) pFSH to coincide with the decline phase of the first dominant follicle (n = 17), which was based on the dominant follicle maintaining its maximum diameter for 2 d, after which it was assumed that it would start to become atretic. The dose ofpFSH for each of the treatment groups was 1.5 mg twice daily until estrus. Control heifers (n = 4) were assigned to each treatment group and received PG at the same time as the pFSH-treated heifers. The ovaries of all heifers were scanned dally from Day 3 until the first dominant follicle was identified. Subsequently, the pattern of growth of the first dominant follicle was monitored in all heifers until PG was administered. The heifers were slaughtered 5 to 7 d after the administration of PG. The ovulation rate was determined by counting the number of CL on both ovaries. These data were compared with assessments of the ovulation rate made by using the ultrasound scanner; the correlation coefficient was 0.92 (P < 0.01). The heifers were housed on a slatted floor system and were fed an ad libitum diet of raisins, beet pulp and grass silage. Statistical Analysis Differences between means were determined after one-way analysis of variance and using Fischer least significant difference to indicate significant differences between treatment means. Statistical significance was inferred at P < 0.05. Chi-square analysis (27) was used to determine if there were significant differences between treatments in the proportion of heifers with twin or multiple ovulations or that were detected in estrus after administration of PG. RESULTS Experiment 1 The pattern of emergence of the first follicular wave in control heifers was similar to that previously reported (23). There was no overall effect (P > 0.05) of dose of pFSH on the number of medium (5 to 9 mm) follicles (data not presented). However, on the first day post PG, heifers assigned to 2.5 mg pFSH had increased (P < 0,05) numbers of medium follicles compared with controls or with heifers assigned to 1.5 mg pFSH. Injection of 2.0 or 2.5 mg pFSH from Day 3 until estrus increased the mean number of large follicles on Day 5 and on the first and second day post PG compared with that of the control heifers, while 1.5 mg pFSH until estrus increased the number of large follicles on the first and second day post PG (Table 1). All control heifers had single ovulations. The ovulation rate of heifers injected with pFSH between Day 3 and estrus was higher (P < 0.05) than in control heifers. However, there was wide variation in the ovulation rate among pFSH-treated heifers. Although pFSH-treated heifers had multiple o~axlations (Table 1), there was no significant difference (P > 0.05) between pFSH treatments in the proportion of heifers that had multiple ovulations (> 3). Some of the large follicles identified on Day 2 post PG did not ovulate. The estrous response in control, 1.5 mg pFSH, 2.0 mg pFSH and 2.5 mg pFSH-treated heifers was 7/11 (64 %), 9/12 (75 %), 11/12 (92 %) and 9/12 (75 %) respectively (P > 0.05). Treatment with pFSH did not affect (P > 0.10) the interval from PG to the onset of estrus (Table 1).
12
12
12
1.5mgFSHp
2.0mg FSHp
2.5mg FSHp
2.1+0.4 b
2,0_+0.4 b
1.8+0.3 "b
0.9+0.2 =
5
Day of estrous cycle
2.9+0.6 b
3.7+0.5 b
2,7+0,3 b
1.2+0.2 a
1
6,3+0.9 ~
4 . 3 + 0 . 7 b¢
3.2+0.7 b
1.0+0.1"
2
Day after PG
3.4+0.7 b
3.3+0.6 b
2.6+0.4 ~
1.0_+0.0 ~
Mean OR
5
4
4
11
1
1
1
2
2
3
3
1
4
1
4
No. of CL
2
2
5
3
6
2
7
42 + 8"
51 + 8 a
39 + 7"
53 + 14"
Interval from PG to estrus (hours)
Data are not presented from Days 3 and 4 of the estrous cycle since there were no significant differences between treatments on these days.
abc Means in the same column with different superscripts are significantly different (P<0.05).
11
n
Control
Treatment
No. of large (>10mm) follicles
Table 1. The effect of 1.5, 2.0 or 2.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until estrus on the mean (+ SEM) number of large (_>10 mm) follicles on Day 5 of the estrous cycle, and on the first and second day after PG; on the mean ovulation rate (OR), on the number of corpora lutea (CL); and on the interval from PG to onset of estrus in beef heifers. (Experiment 1)
.-L
03
ol
2
562
Theriogenology
Experiment 2 The pattern of emergence of the first follicular wave was typical of untreated control heifers, similar to Experiment 1. There was no overall significant effect ofpFSH between Days 3 and 5, or until estrus on the mean number of medium follicles (data not presented). The mean number of large follicles was increased (P < 0.05) from Day 5 until the second day post PG by 1.0 and 1.5 mg pFSH compared with that of the untreated controls. The ovulation rate of heifers injected with pFSH between Day 3 and estrus was higher (P < 0.05) than that of the controls (Table 2). The mean ovulation rate for all pFSH-treated heifers was lower than the mean number of large follicles detected on the second day post PG. Multiple ovulations were detected in all pFSHtreated groups (Table 2). However, there was no significant difference between pFSH treatments in the proportion of heifers which had twin or multiple ovulations. There was also no significant difference between treatments in the interval to estrus after PG (Table 2) or in the estrous response after PG (control: 6/8 (75 %); 1.0 mg pFSH until PG: 7/8 (88 %); 1.0 mg pFSH until estrus: 10/10 (100 %); 1,5 mg pFSH until PG: 9/10 (90 %); and 1.5 mg pFSH until estrus: 9/9
(100%). Experiment 3 Four control heifers did not ovulate (based on the absence o f a CL at ultrasound examination), and their data were excluded from analysis. The mean ovulation rate was higher (P < 0.05) in pFSH-treated heifers than in controls, but there was no difference in ovulation rate between heifers given 1.0 (2.0 + 0.2) or 1.5 mg pFSH (2.2 + 0.4) until estrus. The proportion of heifers with twin ovulations following 1.0 mg or 1.5 mg pFSH was greater (P = 0.011) than in untreated controls (Table 3). However, there was no difference in the percentage of heifers with 2 or more ovulations between the 2 pFSH treatments. Although the percentage of control heifers that exhibited estrus after PG was 64 % (7/11), this was not different (P > 0.05) compared with pFSH-treated heifers (1.0 mg pFSH until estrus: 13/16, 81% vs 1.5 mg pFSH until estrus: 15/16, 94 %). Treatment with pFSH did not affect (P > 0.10) the interval between PG and estrus. Experiment 4 The mean ( + SEM) diameter (mm) of the first dominant follicle at the time of initiation of pFSH for the 3 treatments (pFSH-incline, pFSH-plateau and pFSH-decline, respectively) was 9.0 + 0.3, 13.2 + 0.4 and 13.8 + 0.6. The mean diameter of the dominant follicle was significantly smaller (P < 0.05) for heifers given pFSH at the incline (growth) stage of development of the first dominant follicle than at the plateau (static) and decline phases. There was no difference (P > 0.05) between the pFSH-treated heifers and their respective controls in the mean diameter of the first dominant follicle at the initiation of pFSH treatment. All of the control heifers had a single ovulation after PG. There was no difference (P > 0.05) in the mean ( + SEM) ovulation rate of heifers assigned to the pFSH-incline or pFSH-plateau treatments (Table 4). However, the mean ovulation rate in the pFSH-deeline treatment was higher (P < 0.05) than for either of the other pFSH treatment groups. The proportion of heifers with multiple ovulations (>_ 3 ovulations) in the pFSH-decline treatment group (7/17) was greater (P < 0.05) than in the pFSH-incline
8
1.0 mg FSHp
10
9
1.5 mg FSHp --> PG
1.5 mg FSHp --> Estrus
2.3 + 0.3 b
1.6 +0.3b
2.1 +0.3 b
2.0+0.3 b
1.0 + 0 . 2 a
5
Day of estrous cycle
-+ 0.0 a
3.1 +0.5 ~
22+0.3 b
2.5 + 0.4 b
2.1 +0.4 b
1.0
1
3.3_+0.5 bc
2.2 + 0.3 b
2.2+0.4 b
2.3+0.4 =
1.0 + 0.0"
2
Day after PG
3.1 +0.4 c
1.6+0.3 ab
2.1 +0.3 b
1.3+0.3 ab
1.0 -+ 0 . 0 a
Mean OR
1
6
3
7
8
1
2
2
2
1
3
3
1
-
4
1
5
8a
5 2 + 5a
47_+ 6 a
52-+ 5"
53+
61 -+ 14a
Interval from PG to estrus (hours)
Data are not presented from Days 3 and 4 of the estrous cycle since there were no significant differences between treatments on these days.
2
2
4
2
No. of CL
abc Means in the same column with different superscripts are significantly different (P<0.05).
10
1.0 mg FSHp --> Estrus
--> PG
8
n
Control
Treatment
No. of large (_>10mm) follicles
Table 2. The effect of 1.0 and 1.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until Day 5 or until estrus (induced by a single injection of Cloprostenol [PG] on Day 5) on the mean (_+SEM) number of large (>10mm) follicles on Day 5 of the estrous cycle and on the first and second day after PG; on the mean (_+SEM) ovulation rate (OR); on the number of corpora lutea (CL); and on the interval from PG to onset of estrus (hours _+SEM) in beef heifers. (Experiment 2)
O1 6a
55+
6a
44 + 6"
16
16
38 + 13a
11
Hours from PG to Estrus
2.2+0.4 ~
2.0 +_0.2 b
1.0 + 0.0a
Mean OR
5
5
7
1
ab Means in the same column with different superscripts are significantly different (P
--> Estrus
1.5 mg FSHp
--> Estrus
1.0 mg FSHp
Control
Treatment
slaughter in beef heifers. (Experiment 3)
8
7
2
3
3
2
1
4
No. of CL
Table 3. The effect of 1.0 or 1.5 mg pFSH given twice daily from Day 3 of the estrous cycle (estrus = Day 0) until estrus (induced by a single injection of cloprostenol [PG] on Day 5) on the interval (hours + SEM) from PG to onset to estrus; on the mean (+ SEM) ovulation rate (OR); and on the number of corpora lutea (CL) detected at
¢)
ol o')
17
17
Static Phase
Decline Phase
58 -+ 9=
43_+ 4 a
45_+ 7a
54 +_ 10 a
Hours from PG to Estrus
3.9 -+ 0.8c
1.9+_0.3 b
1.9+0.3 b
1.0 + 0.0 a
Mean OR
5
8
8
12
1
3
5
8
2
2
2
2
3
Means in the same column with different superscripts are sign ficantly different (P<0.05).
19
Growth Phase
abc
12
Control
Treatment 4
5
6
No. of CL 7
8
9
Table 4. The effect of 1.5 mg pFSH given twice daily to coincide with the growth phase (Day 3), static phase (Day 6 - 8) or decline phase (Day 8 +) of the development of the first dominant follicle, followed by a single injection of cloprostenol (PG) 2 days after the initiation of treatment with FSH, on the interval (hours + SEM) from PG to onset of estrus; on the mean (+ SEM) ovulation rate (OR); and on the number of corpora lutea (CL) in beef heifers. (Experiment 4)
10
11
O"1
O1 O~
(b (b
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treatment group (1/19). Although there were numerically more heifers with double ovulations in the pFSH-incline treatment (8/19), this was not significant (P = 0.12) compared with the pFSHdecline treatment (3/17). The estrous response of heifers assigned to the pFSH-incline treatment group was 68% (13/19). However, this was not statistically significant (P > 0.10) compared with that of the pFSH-plateau (15/17; 88 %) or the pFSH-decline (14/17; 82 %) treatment groups. Heifers assigned to the pFSH-decline treatmefit group tended to have a longer interval to estrus after PG (57.8 h) than after the pFSH-incline (44.8 h) and the pFSH-plateau (43.3 h) treatments. However, this effect was also not statistically significant (P > 0.10). DISCUSSION The results of our 4 experiments indicate that low dosages of pFSH administered twice daily from Day 3 of the estrous cycle result in the selection and ovulation of 1 to 2 extra follicles. The administration of pFSH until estrus appears to be essential for the ovulation of these extra selected follicles. Treatment with pFSH did not significantly increase the number of medium follicles, however, the number of large follicles increased following injections of 1.5, 2.0 or 2.5 mg pFSH twice daily in all the experiments. Although variation in the ovulation rate was observed in pFSH-treated heifers, the incidence of twin ovulations was low in Experiments 1 and 2. In Experiments 3 and 4, the incidence of twin ovulations was 47% and 30% respectively. The results from Experiment 4 suggest that the stage of development of the first dominant follicle may influence the ovulation rate and the occurrence of twin and multiple ovulations. In cattle (12), FSH is the most important hormone to stimulate follicular growth to the dominant follicle stage. Recent data have shown that the secretion of FSH during the estrous cycle follows a recurrent pattern of elevation and plateau (1, 26). One day after the elevation in FSH concentration, a new follicular wave emerges. Selection of the dominant follicle occurs as FSH concentrations decline, and dominance is associated with plateau concentrations of FSH. The precise temporal relationship between the decline in FSH concentration and the selection of the dominant follicle remains to be elucidated. Recent data suggest that selection of a single dominant follicle can be delayed by 4 dally injections of pFSH which slows the rate of FSH decline after follicular wave emergence (2). The results of the four experiments do not fully support a previous study (14) which suggested that the fate of subordinate follicles was determined by Day 3. Clearly, the presence of an active dominant follicle when treatment with pFSH was initiated, lowered the ovulatory response compared to other stages of the estrous cycle (9, 10), and in this regard, the lower ovulation rates obtained in our study support this observation. The results from Experiment 4, in which the effects of pFSH at various stages of development of the first dominant follicle were examined, showed significant differences in the ovulation rate between the decline phase and the growth and plateau phases of development. This is most likely due to the loss/lack of physiological dominance of the first dominant follicle from Day 8 forward and supports the hypothesis of higher ovulation rates associated with the absence of a dominant follicle. The administration of pFSH from Day 3 did not increase the number of follicles present during the first wave but appeared to result in more medium follicles attaining large diameters. Since data were not recorded for small (< 5 mm) follicles, it is not possible to say if that
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follicular population was influenced by pFSH. The current data suggest that exogenous FSH from Day 3 onward probably delays the onset of atresia in cohort subordinate follicles observed on Day 6 rather than rescuing atretic follicles, since treatment with FSH was initiated before atresia had begun. Previously, it has been demonstrated that PMSG can "rescue" follicles from atresia (17). It has also been postulated that preselection superovulatory treatments (pFSH) may be acting to delay onset of atresia (2). Data which were generated in this laboratory (22) have shown that a single injection of 7 mg pFSH on Day 1 of the estrous cycle increased the number of medium follicles, decreased the number of small follicles but did not affect the number of large follicles or the total follicle population. Similarly, in prepubertal heifers which received FSH, a shift from small to meditma and large follicles without any effect on the total number of follicles was observed (19). The failure of the FSH-stimulated follicles to ovulate, which was noted in Experiments 1 and 2, may be due to the initiation of atresia as a consequence of reduced FSH concentrations in association with luteal phase LH pulse frequency. In Experiments 3 and 4, data were not recorded on the number of large follicles that were present 2 d after the administration of PG, and on the number of large follicles that subsequently ovulated. Increased FSH concentrations appear to be important in the maintenance of multiple dominant follicles from PG to estrus, suggesting that FSH may play a role in estrogen activity of these follicles, as termination of FSH at the time of initiation of luteolysis resulted in a decreased ovulation rate (1, 26); it may also be that the decline in FSH is responsible for follicle selection. The interaction of insulin-like growth factor-I (IGF-I) and FSH in the induction of the aromatase system is important for estradiol production within the follicle. However, it is possible that if FSH support is withdrawn in the presence of elevated concentrations of progesterone, androgen production declines as a consequence of reduced LH pulse frequencies, and this leads to decreased intrafollicular production of estradiol. In turn, IGF-I concentrations decrease due to effects of IGF binding proteins (IGFBPs; 11). For example, IGFBP 3 inhibits the binding of FSH to its receptor (29), and FSH inhibits the production of IGFBPs within the follicle (11). Therefore, a fall in FSH concentrations may lead to increased IGFBPs, which decrease IGF production (11) and thus follicular atresia may ensue. The development of practical regimens to induce twin ovulations and twin pregnancies in cattle has been an ongoing concern for many years. Regardless of the approaches used, with the exception of embryo transfer, variability in ovulatory response has been a consistent finding (3, 5, 6). The results of our present experiments suggest that a low dose of pFSH (1.0 to 1.5 mg twice daily from Day 3 until estrus) together with the presence of an active dominant follicle resulted in a significant increase in the occurrence of twin ovulations. Although multiple ovulations were detected, some heifers failed to respond to ovarian stimulation by pFSH. However, our data show that a 50% twin ovulation rate can be achieved when treatment is initiated during the first wave of follicular growth in conjunction with low doses ofpFSH. In summary, administration of pFSH during the putative phase of selection of the first dominant follicle can increase the number of follicles detected by 1 or 2. The successful ovulation of most of the selected dominant follicles is dependent on the continued administration
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of pFSH after PG until estrus. Variability in ovulation rate remains a problem even with the low dosages of pFSH used in these studies. REFERENCES 1.
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