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J. Anim. Sci. 2003. 81:249-260
© 2003 American Society of Animal Science

Plasma gonadotropins and ovarian hormones during the estrous cycle in high compared to low ovulation rate gilts1

R. V. Knox2, G. Vatzias3, C. H. Naber and D. R. Zimmerman4

University of Nebraska, Lincoln 68583-0908

2 Current address and correspondence:
Dept. of Animal Sciences, 360 Animal Sciences Laboratory, 1207 W. Gregory Dr., Urbana, IL 61801 (phone: 217-244-5177; fax 217-333-8286; E-mail:
rknox{at}uiuc.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 References
 
Mature gilts classified by low (12 to 16 corpora lutea [CL], n = 6) or high (17 to 26 CL, n = 5) ovulation rate (OR) were compared for plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone, estradiol-17ß, and inhibin during an estrous cycle. Gilts were checked for estrus at 8-h intervals beginning on d 18. Blood samples were collected at 8-h intervals beginning on d 18 of the third estrous cycle and continued for one complete estrous cycle. Analysis for FSH and LH was performed on samples collected at 8-h intervals and for ovarian hormones on samples collected at 24-h intervals. The data were standardized to the peak of LH at fourth (d 0) and fifth estrus for the follicular phase and analyzed in discrete periods during the periovulatory (-1, 0, +1 d relative to LH peak), early- luteal (d 1 to 5), mid-luteal (d 6 to 10), late-luteal (11 to 15), periluteolytic (-1, 0, +1 d relative to progesterone decline), and follicular (5 d prior to fifth estrus) phases of the estrous cycle. The number of CL during the sampling estrous cycle was greater (P < 0.005) for the high vs low OR gilts (18.8 vs 14.3) and again (P < 0.001) in the cycle subsequent to hormone measurement (20.9 vs 14.7). For high-OR gilts, FSH was greater during the ovulatory period (P = 0.002), the mid- (P < 0.05) and late-luteal phases (P = 0.01), and tended to be elevated during the early-luteal (P = 0.06), but not the luteolytic or follicular periods. LH was greater in high-OR gilts during the ovulatory period (P < 0.005), but not at other periods during the cycle. In high-OR gilts, progesterone was greater in the mid, late, and ovulatory phases (P < 0.005), but not in the follicular, ovulatory, and early-luteal phases. Concentrations of estradiol-17ß were not different between OR groups during the cycle. Inhibin was greater for the high OR group (P < 0.005) during the early, mid, late, luteolytic, and follicular phases (P < 0.001). The duration of the follicular phase (from last baseline estrogen value to the LH peak) was 6.5 ± 0.5 d and was not affected by OR group. These results indicate that elevated concentrations of both FSH and LH are associated with increased ovulation rate during the ovulatory phase, but that only elevated FSH during much of the luteal phase is associated with increased ovulation rate. Of the ovarian hormones, both inhibin and progesterone are highly related to greater ovulation rates. These findings could aid in understanding how ovulation rate is controlled in pigs.

Key Words: Follicle-Stimulating Hormone • Inhibin • Luteinizing Hormone • Ovulation Rate • Pigs • Progesterone


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 References
 
Higher ovulation rates may result from increased recruitment of follicles entering the growing pool or from selection of more follicles at luteolysis. Evidence also suggests that increased ovulation rate may arise from selection of greater numbers of follicles over an extended period of the follicular phase in pigs (Zimmerman and Koff, 1988; Zimmerman et al., 1990; Vatzias et al., 1991) and sheep (Cahill et al., 1981; Driancourt et al., 1985).

Investigation into the cause of increased ovulation rate has shown elevated FSH in high- vs low-ovulating females during the pro- and periestrous periods in pigs (Shaw and Foxcroft, 1985; Kelly et al., 1988b; Knox et al., 1991), sheep (Cahill et al., 1981; Mizumachi et al., 1990), and during the late-luteal period in pigs (Knox and Zimmerman, 1993) and sheep (Davis et al., 1981; McNatty et al., 1985). More recently however, Hunter et al. (1996) and Mariscal et al. (1998) failed to detect hormone differences during these periods in pigs. It is not clear whether discrepancies between studies arise due to differences in how follicles are selected for ovulation between genetic lines of pigs. Alternatively, these differences may arise due to how animals are grouped for defining ovulation rate class, or due to which periods are included for analysis of hormone concentrations during the estrous cycle.

Despite the inconsistencies between some of the studies, further characterization of gonadotropin and ovarian hormones during the estrous cycle between pigs differing in ovulation rate could aid in establishing whether the duration of the luteal or follicular phases differ and whether differences in gonadal and gonadotropic hormones exist between high- and low- ovulating pigs. The objective of the present study was to determine whether ovulation rate differences in pigs classified into high and low groups are associated with differences in gonadotropic and gonadal hormones during the estrous cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 References
 
Gilts from the University of Nebraska Gene Pool population, selected for high ovulation rate for nine generations followed by random selection for 13 generations (RS), and those selected randomly over the same period (C) were subjected to laparoscopy using halothane-nitrous oxide anesthesia between d 8 to 14 after expression of their third estrous period. Based on counts of corpora lutea (CL), gilts from both genetic lines were chosen and classified into low- (12 to 16 CL) or high- (17 to 26 CL) ovulation rate (OR) groups. Eleven gilts in all were obtained, six expressing low OR (RS, n = 3 and C, n = 3) and five expressing high OR (RS, n = 3 and C, n = 2). At the fourth estrus, the gilts were 8 to 9 mo of age and between 150 and 180 kg. Blood sampling was initiated on d 18 (d 0 = first d of estrus) of the third cycle and continued for one complete estrous cycle to characterize and compare gonadotropic and gonadal hormone profiles. Ovaries were recovered on d 7 after their fifth estrus and ovulation rate was determined by counting number of CL.

Management

Gilts were maintained in an environmentally regulated confinement facility with concrete slatted floors and under constant light and temperature (22°C). During the previous estrous cycle, and the estrous cycle being evaluated, gilts were tethered in individual stalls. Gilts were fed 1.6 kg of a 14% corn–soybean meal diet once daily prior to d 10 and 1.6 kg of diet twice daily (3.2 kg total) from d 10 through onset of estrus, in order to allow maximal expression of ovulation rate potential. Detection for behavioral estrus was performed at 8-h intervals with fence-line contact with a mature boar beginning on d 18 of each estrous cycle.

Blood Sampling

All gilts were fitted nonsurgically with an indwelling cannula on d 16 of their third estrous cycle. Briefly, gilts had a sterile polyethylene cannula (0.86 mm i.d. x 1.27 mm o.d.) inserted 40 cm into an ear vein through a 14-gauge, 38-mm needle. The cannula was taped in place using elastic tape and hip tag cement. Blood sampling occurred at 4-h intervals from d 18 until 24 h after onset of estrus (d 0), and at 8-h intervals from d 1 to 17 of the estrous cycle and then every 4 h between d 18 and 24 h after onset of the fifth estrus. Blood samples (8 mL) were collected into tubes containing dipotassium EDTA (2 mg/mL) and placed immediately on ice. Samples were stored on ice for <12 h before centrifugation at 800 x g for 15 min. Plasma was aspirated, aliquoted, and stored frozen at -15°C until hormone assays were performed.

Hormone Assays

Plasma concentrations of LH were determined in duplicate 200-µL aliquots by a homologous double-antibody RIA described by Niswender et al. (1970). Intra- and interassay CV averaged 2.6 and 5.1%, respectively. Plasma FSH was quantified in duplicate 300-µL aliquots for the pig based on a modified homologous double-antibody RIA previously described by Kelly et al. (1988b). The FSH antiserum (USDA–98–4P, pFSH ß-antiserum) was characterized previously (Guthrie and Bolt, 1983). Intra- and interassay CV were 2.0 and 11.6%, respectively.

Plasma 17ß-estradiol was measured by RIA validated in our laboratory. Recovery of added mass (1, 2, 4, and 8 pg, 17ß-estradiol) from 400 µL of plasma from each of three independent samples averaged 121 ± 3.9%. Assay determinations of 300, 400, and 500 µL of sample from each of eight independent plasma samples were highly correlated (r > 0.96). Aliquots of plasma samples were extracted in duplicate with 2 mL of diethyl ether. After addition of ether, samples were capped immediately and vortexed for 2 min. The aqueous plasma samples were frozen and the liquid solvent portion was poured off into assay tubes. The diethyl ether extracts were evaporated at 55°C. The extraction was repeated on the plasma samples and the ether extract was added into the assay tube. Again, the ether was evaporated and all samples were resuspended in 200 µL of PBS–0.1% gelatin. Anti-17ß-estradiol antibody (Eli Lilly Co., Indianapolis, IN; lot #022367) raised in rabbits was diluted in PBS–0.1% gelatin and used as first antibody at a concentration of 1:1,000,000. Radioactive tracer (125I estradiol, Amersham Corp., Arlington Heights, IL) was diluted in PBS–0.1% gelatin containing 1:300 normal rabbit serum and added to samples after 24 h. Unbound and antibody-bound radiolabeled estradiol were separated using charcoal (0.5% charcoal with 0.05% dextran) suspended in PBS–0.1% gelatin. Samples were incubated with charcoal for 15 min and then centrifuged at 2,076 x g for 15 min. Concentrations of 17ß-estradiol (pg/mL) in unknown samples were calculated using the four-parameter logistic curve-fitting program (Grotjan and Steinberger, 1977). Recovery rates for 17ß-estradiol averaged 75% and intra- and interassay CV averaged 7.8 and 8.7%, respectively.

Plasma progesterone was determined by RIA and validated in our laboratory. Plasma samples were extracted twice with 2:1 hexane:benzene (vol/vol) and recovery of added mass (6.25, 12.5, and 25 ng progesterone) from 50 µL of plasma from each of five independent plasma samples averaged 88 ± 3.5%. Assay determinations of 20, 40, 60, 80, and 100 µL of sample were all highly correlated (r > 0.95). Aliquots of plasma samples were diluted with PBS–0.1% gelatin to 500 µL. Hexane:benzene (2.5 mL) was added to samples and vortexed for 2 min. Samples were frozen and solvent was poured off into assay tubes and dried under vacuum at 55°C. Extracted samples were rehydrated with 1 mL of PBS–0.1% gelatin. Aliquots of extracted samples (50 µL) were assayed in duplicate. A monoclonal antibody against progesterone-11-BSA (BiosPacific, Emeryville, CA) was used as first antibody at 1:20,000 by dilution in PBS–0.1% gelatin containing 1:400 normal mouse serum. Progesterone-11-{alpha}-hemisuccinate-TME (kindly provided by Dr. A. Belanger, Le Centre Hospitalier de I’Université Laval, Quebec, Canada) was labeled with 125I and used as a tracer; progesterone (Sigma Chemical Co., St. Louis, MO) was used as the standard. Unbound and antibody-bound radiolabeled progesterone was separated by incubation with dextran-coated charcoal (described earlier for estradiol) and centrifuged for 15 min at 2,076 x g. Recovery of progesterone was 61% and intra- and interassay CV averaged 5.6 and 15.5%, respectively.

Plasma inhibin was quantified in duplicate 400-µL aliquots by a homologous double-antibody RIA described by Schanbacher (1988). Porcine inhibin {alpha}-chain [Tyr30](1–0)NH2 (provided by Dr. Nicholas Ling, The Salk Institute, San Diego, CA) was used as a standard and for iodination. Inhibin was labeled with 125I by the chloramine-T method and separated on a Sephadex G–0 column. The inhibin antisera, generated in rabbits, was kindly provided by the USDA Meat Animal Research Center, Clay Center, NE. The antisera was diluted to 1:14,000 and used as first antibody. Donkey anti-rabbit _-globulin was used as second antibody. Intraassay CV averaged 5.2%.

Statistical Analysis

Ovulation rate was classified by final count at slaughter after fifth estrus. Hormone data were analyzed using mixed models procedures of SAS (SAS Inst., Inc., Cary, NC). The model included the fixed effects of ovulation rate class (high vs low), genetic line (RS vs C), sampling period, and their interactions. Genetic line was subsequently dropped from the model because it failed to account for significant variation (P > 0.10). Plasma FSH and LH samples were analyzed from samples collected every 4 h during the ovulatory period, and then every 8 h during the remainder of the estrous cycle. Plasma concentrations of 17ß-estradiol, progesterone, and inhibin were analyzed based on samples obtained at 1300 h throughout the estrous cycle. Analyses of hormone concentrations was performed during discrete periods of the estrous cycle defined by both time of onset of fourth estrus and the peak of LH (ovulatory and luteal periods) or the fifth estrus and LH peak (follicular period), or the initiation of luteolysis (luteolytic period from high progesterone to decline). The analysis by these phases allowed precise determination of the hormone concentrations during specific periods independent of differences in estrous cycle duration. The ovulatory phase included 3 d relative to the LH peak (d -1, 0, and +1), and was also confirmed by onset of estrus. The 5 d of the early luteal phase (d 1 to 5) were defined by the previously characterized second FSH surge and period of rapidly increasing progesterone from the forming CL, the 5 d of the mid-luteal phase (d 6 to 10) by the increasing concentrations of progesterone, and the late luteal phase included 5 d (d 11 to 15) and the period prior to luteolysis. The luteolytic period included 3 d and was defined by the short period of the last observation of elevated progesterone before a rapid decline. The analysis included the day of luteolysis (d 0), 1 d previous (d -1), and 1 d subsequent (d +1). The follicular phase included the 5 d period prior to fifth estrus and included the period from first low baseline progesterone value to the day prior to the peak of LH. Least squares means were then compared using Bonferroni’s test.

The number of CL determined in the third cycle and again after fifth estrus, and measures of duration of time from last baseline estrogen (and first baseline progesterone) value to the LH peak, and the period from the LH peak to last elevated progesterone value before luteolysis, were analyzed using the general linear models procedures of SAS for the fixed effect of OR group. Significant differences between least squares means were determined using Student’s t-test in the predicted difference option of the GLM procedure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 References
 
Ovulation Rate and Estrous Cycle Duration

Gilts previously classified into high- and low-ovulation rate groups by laparoscopic examination during the third estrous cycle by number of CL for high (18.8 ± 0.4, n = 5) and low (14.3 ± 0.6, n = 6) OR, maintained their OR differences (P < 0.001) after their fifth estrus. At fifth estrus, ovulation rate was greater in the high-OR groups (20.9 ± 0.7) compared to the low-OR groups (14.7 ± 0.7 CL, P < 0.001). However, two gilts within the high-OR group expressed normal interestrous intervals (19.0 ± 1.2 d), whereas three gilts exhibited a long interestrous interval (25 ± 1.0 d). Interestrous intervals of gilts in the low-OR class averaged 19.7 ± 0.7 d. The longer duration estrous cycles in the three high-OR gilts was apparently due to a longer luteal period (P < 0.01) characterized by elevated progesterone above baseline lasting 20.4 ± 1.0 d compared to 14.6 ± 1.2 d for gilts with normal estrous cycle durations. In comparison of different phase durations during the estrous cycle in the high- and low-OR groups, the period from the LH peak to the time of peak progesterone was 14.1 ± 0.3 d and was not different between OR groups (P > 0.10). The follicular phase was defined as the period from the last low baseline estrogen value (and first baseline progesterone value) to the LH peak (6.5 ± 0.5 d) and was not influenced by OR group or estrous cycle duration (P > 0.10).

FSH and LH Concentration

In the high-OR group, FSH was higher during the ovulatory period (P = 0.002), the mid- (P < 0.05) and late-luteal phases (P = 0.01), and tended to be elevated during the early-luteal (P = 0.06), but not the luteolytic or the follicular periods (Figure 1Go, Table 1Go). All gilts exhibited an ovulatory FSH surge at estrus and showed a secondary surge of FSH between 1 and 7 d. FSH remained elevated above baseline from d 1 to 15 in gilts expressing normal estrous cycle duration and remained elevated between 1 to 20 d for gilts expressing estrous cycles of greater duration. Patterns of FSH in circulation after the secondary surge were variable; some gilts exhibited multiple surge events, whereas others showed another surge between 8 and 12 d, and others showed no clear surges after the secondary surge (representative patterns in six gilts with varying cycle duration by OR class are shown in Figure 2aGo–f). In almost all cases, FSH appeared to increase just prior to luteolysis.



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  		Figure 1. Least squares means (±SEM) for concentrations of FSH normalized to a 20-d cycle and adjusted to the timing of the LH peaks occurring on d 0 in gilts expressing high (20.9 corpora lutea, CL) or low (14.7 CL) ovulation rate. To adjust for different interestrous intervals, d 15 to 19 includes only the last 5 d prior to the subsequent LH peak.

 

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  		Table 1. Least squares means for concentrations of FSH, LH, progesterone, estradiol-17ß, and inhibin during specific periods of the estrous cycle relative to the peak of LH (d 0) during the third and fourth estrous cycle and in the 5 d prior to fifth estrus (d -5 to -1)
 



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  		Figure 2. (a and b) FSH profiles (note: scales differ for each gilt) in two gilts expressing high ovulation rate with normal 19- to 21-d interestrous intervals; (c and d) FSH profiles in two gilts expressing high ovulation rate with longer duration (24 to 26 d) interestrous intervals; (e and f) FSH profiles in two gilts expressing low ovulation rate with normal 19- to 21-d interestrous intervals.

 
In the high-OR gilts, LH was greater during the ovulatory period (P < 0.005), but did not differ (P > 0.10) during other periods of the cycle (Figure 3Go, Table 1Go). An ovulatory surge of LH coincided with onset of estrus in 10 of 11 gilts and on the day prior to onset of estrus in a single gilt. On average, the LH peak was observed at 6.5 ± 0.7 h after the onset of estrus (mean ± SEM). Based on an 8-h estrous detection interval and a blood sampling interval of every 4 h, the LH peak was estimated to occur at -4 h relative to onset of estrus in one gilt, at onset of estrus in three gilts, at +4 h relative to onset in two gilts, at +8 h in one gilt, at +12 h in two gilts, and at +16 h and +20 h for one gilt each. Estrus was detected at 2100 h in 4 of 11 gilts and at 0500 h in 7 of 11 gilts.



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  		Figure 3. Least squares means (±SEM) for concentrations of LH normalized to a 20-d cycle and adjusted to the timing of the LH peaks occurring on d 0 in gilts expressing high (20.9 corpora lutea, CL) or low (14.7 CL) ovulation rate. To adjust for different interestrous intervals, d 15 to 19 includes only the last 5 d prior to the subsequent LH peak.

 
Ovarian Hormones

Plasma concentrations of progesterone were not different between OR classes during the ovulatory period and early luteal phase, but were greater for high-OR gilts in the mid (P < 0.005), late (P = 0.005), and luteolytic periods (P < 0.005), and not different during the follicular phase (Figure 4Go and Table 1Go). Plasma progesterone was baseline until d 3 after the LH peak, and then increased to reach peak concentration at d 10 where it remained elevated until d 14 in gilts with normal estrous cycle duration and until d 20 in gilts expressing long inter-estrous intervals. At luteolysis, plasma progesterone concentrations declined from high to baseline concentrations over a 2-d period. Plasma estradiol concentrations were not different at any phase during the estrous cycle between high- and low-OR groups (Figure 5Go, Table 1Go). Plasma estradiol remained at baseline from d 2 to the day coinciding with the last day of elevated progesterone. Plasma estradiol increased above baseline for 7 d, including a 5-d period before estrus and both the day of and after the onset of estrus. In the 24-h sampling interval for plasma estradiol, the peak occurred at -1 d before estrus in 76% of gilts and on the day of estrus in 24%. Plasma inhibin concentrations were greater for the high- vs low-OR group during the ovulatory, (P < 0.05), early luteal (P < 0.005), mid luteal (P < 0.005), late luteal (P < 0.001), luteolytic period (P < 0.001), and follicular phases (P < 0.001, Figure 6Go, Table 1Go).



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  		Figure 4. Least squares means (±SEM) for concentrations of progesterone normalized to a 20-d cycle and adjusted to the timing of the LH peaks occurring on d 0 in gilts expressing high (20.9 corpora lutea, CL) or low (14.7 CL) ovulation rate. To adjust for different interestrous intervals, d 15 to 19 includes only the last 5 d prior to the subsequent LH peak.

 


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  		Figure 5. Least squares means (±SEM) for concentrations of estradiol-17ß normalized to a 20-d cycle and adjusted to the timing of the LH peaks occurring on d 0 in gilts expressing high (20.9 corpora lutea, CL) or low (14.7 CL) ovulation rate. To adjust for different interestrous intervals, d 15 to 19 includes only the last 5 d prior to the subsequent LH peak.

 


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  		Figure 6. Least squares means (±SEM) for concentrations of inhibin normalized to a 20-d cycle and adjusted to the timing of the LH peaks occurring on d 0 in gilts expressing high (20.9 corpora lutea, CL) or low (14.7 CL) ovulation rate. To adjust for different interestrous intervals, d 15 to 19 includes only the last 5 d prior to the subsequent LH peak.

 
Correlations Between Hormones

Only significant relationships moderately to highly related (r > 0.45 and P < 0.05) are reported. FSH was positively related to LH concentrations during the ovulatory period (r = 0.57) and to progesterone concentrations during the follicular phase (r = 0.58). FSH was inversely related to estradiol (r = -0.63) during the follicular, ovulatory (r = -0.52), and early-luteal periods (r = -0.46), and inversely related to plasma inhibin during the follicular phase (r = -0.50).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 References
 
The results from this study indicate that when gilts from selected lines are classified into high- and low-ovulation rate groups, high ovulation rate was associated with increased FSH, LH, inhibin, and progesterone concentrations, but not estrogen, when compared to low ovulation rate. It is not clear whether any of the measured hormones actually caused the increased ovulation rate, either directly or indirectly. Yet, there is evidence for involvement of all of these hormones with ovulation rate in pigs. If FSH concentrations in blood are involved in causing high ovulation rate, this may occur due to the role of FSH in recruiting follicles for development and/or decreasing atresia of follicles during the luteal phase and for increased selection of follicles at the end of the luteal or initiation of the follicular phase. The results for FSH from the present study confirm and extend the findings of Kelly et al. (1988b), who reported that gilts from a line selected for higher ovulation rate had greater circulating concentrations of FSH than lower ovulating, nonselected gilts during d 2 to 4 after estrus. One notable difference between the results of the present study and that of Kelly et al. (1988b) is that high- and low-ovulating gilts originated from both the selected and nonselected lines in the present study. The hormone differences observed for high- and low-ovulating pigs, must not be specific to only the line of pigs selected for high ovulation rate, but may be specific to these lines of pigs. In the rat, the large surge of FSH after the ovulatory surge appears to be important for recruitment of follicles (Hoak and Schwartz, 1980), but in the pig, the role of this second surge of FSH has not yet been determined. However, small (<4 mm) follicle populations increase between d 0 to 2 (Guthrie et al., 1995), indicating that the FSH surge may be initiating recruitment of follicles at the beginning of the estrous cycle. During the luteal phase, follicles increase (Kelly et al., 1988a), and increased numbers of follicles have been observed in pigs with greater FSH during the late-luteal and early-follicular phases (Knox and Zimmerman, 1993). Similar results are reported in sheep, with concentrations of FSH increased in the late luteal phase, in ewes with double versus single ovulations (Davis et al., 1981).

Although the reason for the increased ovulation rate is not known, reduced atresia in the medium follicle pool could be the origin since it has been shown that after luteolysis, the number of small and medium follicles declines rapidly as the follicles that will ovulate (>7 mm) first appear (Kelly et al., 1988a; Guthrie et al., 1995). In higher ovulating lines of pigs, a greater number of medium follicles are found on the ovaries prior to estrus (Clark et al., 1982; Kelly et al., 1988a; Knox, 1989). Increased ovulation rate has been related to increased concentrations of FSH and increased numbers of medium follicles in the follicular phase of gilts (Knox, 1992) and to increased concentrations of FSH in sows during the postweaning period (Shaw and Foxcroft, 1985). The recruitment of higher numbers of follicles, independent of the mechanism whereby certain recruited follicles undergo atresia, could indicate the existence of multiple mechanisms to ensure that adequate or increased numbers of follicles can be selected for ovulation.

In the present study, and in the hormone profiles of selected gilts (Mariscal et al., 1998), distinct surges of FSH were observed during the luteal phase. These surges, although of different duration and magnitude, were clearly observed for each animal between d 2 to 5, d 6 to 10, and the 3 to 4 d prior to luteolysis. It remains unknown whether these patterns are related to changes in ovarian follicle populations because follicles were not assessed. However, this may be likely because it has been shown that suppression of FSH during d 10 to 13 of the luteal phase reduced the numbers of 3- to 6-mm follicles by 50% on d 13. This suppression ultimately reduced the numbers of large follicles at estrus (Knox, 1989). Further research is needed to determine the importance of the different periods of FSH release observed in the present study and what role each surge may play in follicle recruitment or maintenance.

Inhibin concentrations were positively related to increased ovulation rate throughout the entire estrous cycle in the present study, although there is limited information on the importance of inhibin concentrations to ovulation rate in pigs. However, Hunter et al. (1993; 1996) reported that higher-ovulating Meishan gilts have greater inhibin during the periestrous period compared to Large White gilts, but this is not translated into differences in FSH concentrations. This could indicate that neither inhibin nor FSH at this period impacts ovulation rate. In contrast, though, Tilton et al. (1994) suggested that the prolific Meishan might be differentially sensitive to different forms of inhibins because ovariectomized Meishan females release greater amounts of FSH following ovariectomy compared to Large White females. The importance of inhibin was demonstrated by King et al. (1993), who when immunizing gilts against the {alpha}-subunit of inhibin, increased FSH 48 to 72 h prior to ovulation and increased ovulation rate by 39%. This would indicate that increases in FSH prior to estrus, but perhaps not at estrus, could rescue medium-sized follicles from early stages of atresia or select additional healthy medium-sized follicles for ovulation. The results of the current study indicate that increased inhibin occurs during the follicular phase in higher-ovulating females. However, with no change in FSH between these OR groups, these results seem confusing. Perhaps these results for inhibin are incomplete because ovarian control of FSH release occurs through inhibins (a and b), activin, and follistatin. Inhibin from porcine follicle fluid has been shown to have suppressive effects on release of FSH from pituitary cells in culture (Miyamoto et al., 1985) and can be detected in circulation (Hasegawa et al., 1988) and from ovarian venous drainage (Sairam and Downey, 1988). Measurement of the inhibin {alpha}-subunit in the present study, and in other studies (Hasegawa et al., 1988; Trout et al., 1992; Hunter et al., 1993), and in studies immunizing against the {alpha}-subunit (King et al., 1993; Wheaton et al., 1998) have not accounted for the effect on free {alpha}-subunit, the impact on the ratio of inhibin forms a and b, or effect on the concentrations of activin and follistatin. Therefore, attempting to relate FSH concentrations to inhibin without knowledge of the other FSH controlling factors may present an incomplete picture.

These results and those of Hasegawa et al. (1988) indicate that inhibin increased during the follicular phase and peaked at estrus. These findings also indicate that during the luteal phase, inhibin declined and was inversely related to concentrations of FSH. Hasegawa et al. (1988) however, observed a substantial increase in inhibin concentration between d 2 and 8 of the luteal phase. The difference between the results of the present study and those of Hasegawa et al. (1988) may be due to differences in the specificity of the inhibin antisera. The antisera used by Hasegawa et al. (1988) was generated in chickens against the {alpha}-subunit of porcine 32-kDa inhibin, whereas the antibody used in this experiment was generated in rabbits against the porcine inhibin {alpha}-subunit. The antibody used in the present study cannot distinguish between monomeric {alpha}-subunit and dimeric inhibin and there is evidence that {alpha}-subunit exists alone in circulation. Guthrie et al. (1997) has characterized numerous forms of inhibins in porcine follicles of different sizes and at different stages of the estrous cycle. Generally, concentrations of all inhibin forms appear to decrease in follicles during the luteal phase and then remain constant during the follicular phase. Li et al. (1997) showed that in the follicular phase, the inhibin ß a-subunit decreased with the advancement of the follicular phase, whereas the {alpha} and ß b-subunits actually increased during the follicular phase, and as follicles underwent final maturation, all subunit expression decreased. This would correlate well with the present finding of lowered inhibin in circulation during the luteal phase and tendency for FSH to be increased during the luteal phase. Further, follistatin is highly expressed during the early follicular phase in pig follicles and may help explain the rapid decline in FSH concentrations during the early follicular phase (Li et al., 1997).

In the present study, LH was observed to be greater in higher ovulating gilts at estrus. This is in contrast to the findings of Kelly et al. (1988b), Hunter et al. (1993), and Mariscal et al. (1998). However, in most of these studies, mean LH was higher during this period in higher- ovulating animals even though it was not statistically significant. The importance of mean LH differences at this time is not known, since in most cases, mean FSH was also higher at the same period.

Progesterone was increased in high-OR gilts that possessed on average five CL more than the average number of CL in the low-OR group. Guthrie et al. (1974) also reported that concentrations of progesterone on d 6 and 12 of pregnancy were increased as ovulation rate increased. Similar increases in progesterone have also been observed during d 4 to 13 in ewes with higher ovulation rates (Cahill et al., 1981). Although not a certainty, this is most likely a result of the increased number of ovulations and not a cause of the increased ovulation rate.

Differences in estradiol concentrations were not observed due to differences in ovulation rate. This is similar to the findings of Guthrie et al. (1974) during the luteal phase and that of Hunter et al. (1993) during the follicular phase. However, higher estrogen has been observed in higher-ovulating pigs by Guthrie et al. (1974) and in sheep (Cahill et al., 1981) just prior to estrus. Perhaps the conflicting information on concentrations and our inability to detect this effect was due to differences between studies in sampling intervals that, in the present study (24-h interval), did not allow the absolute a peak of estrogen to be determined. It is also possible that due to differences in ovulation rate classification and variability in the peak hormone concentrations, estrogen differences may be difficult to detect.

The different durations of estrous cycles were not anticipated at the initiation of the experiment. However, Kelly et al. (1988a) previously reported that gilts selected for high ovulation rate (RS line) had longer interestrous intervals (1.5 d) than lower-ovulating control-line gilts. In the present study, we observed a 4- to 5-d increase in the interestrous interval in three out of five high-ovulating gilts. The prolonged estrous cycles were caused by an extension of the luteal phase because progesterone concentrations did not decline in normal and long estrous cycle females at comparable days after onset of the LH peak. The release of prostaglandin from the uterus appears to be dependent upon the duration and timing of exposure to progesterone (Edgerton et al., 2000). The results of this study indicate that the delay in the timing of luteolysis was not due to a delay in progesterone production because gilts showing extended duration of estrous cycles had similar patterns and concentrations of progesterone after estrus compared to gilts with normal cycle lengths. Additionally, the duration of the follicular phase (interval from the last baseline value for estradiol-17ß to the peak of LH) did not differ between gilts in the hig- and low-OR groups. Long interestrous intervals have been shown to result when gilts are treated with estrogen between d 11 to 16 (Pusateri et al., 1996). However, there was no indication that gilts expressing longer intervals in our study had increased circulating estrogen at this time, nor was progesterone reduced. This might suggest that the reason for the longer interestrous interval may have originated from a failure of the uterus to produce or release prostaglandin (Edgerton et al., 2000), or was due to the failure of the CL to respond to prostaglandin from the uterus due inadequate numbers of receptors (Gasby et al., 1990).


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
References
 
The results of this study indicate that classification of gilts into high- and low-ovulation rate groups allows for the detection of increased plasma concentrations of follicle-stimulating hormone, luteinizing hormone, inhibin, and progesterone during the estrous cycle. Conditions that allow increases in follicle-stimulating hormone throughout much of the estrous cycle may increase the numbers and health of recruited follicles. Understanding how and why increases in follicle-stimulating hormone occur during specific periods of the estrous cycle may allow for the control of follicle populations and their potential for ovulation. A better understanding of hormonal control of follicle development could lead to advancement in methodology for selection of high-ovulating pigs or may lead to improved methods for controlling the estrous cycle and ovulation rate in pigs.


   Footnotes
 
1 Submitted as Journal Series No. 12096, Agricultural Research Division, Univ. of Nebraska at Lincoln. Sincere appreciation is expressed to D. Bolt for supplying purified pFSH and pFSH antibody for the FSH RIA; USDA Meat Animal Research Center, Clay Center, NE, for providing reagents and assistance with the inhibin RIA; S. Rodriguez-Zas, Dept. of Animal Sciences of the Univ. of Illinois for assistance with data analysis, and K. Cull for excellent technical assistance. Back

3 Current address: Dept. of Animal Production, Technological Educational Institute (TEI) of Epirus Kostaki, 47 100 Greece. Back

4 Deceased. Back

Received for publication June 11, 2002. Accepted for publication September 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 References
 


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