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* Animal Sciences Department, University of Missouri, Columbia, 65211
Corresponding author:
H. A. Garverick; e-mail:
garverickh{at}missouri.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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, 12 h apart. In experiment 2, 14 lactating cows (7 to 12 d postpartum) received LH (40 µg/h; n = 7) or saline (1 mL/h; n = 7) in hourly pulses for 7 d, beginning 24 h after start of the first follicular wave. Daily samples were used to determine serum concentrations of progesterone (P4), estradiol-17ß(E2), LH, and FSH. Profiles of LH were determined from blood samples collected at 12-min intervals for 8 h on d 3. During infusion of LH, serum P4 and FSH were similar across treatments in both experiments. Serum E2 concentrations were similar in experiment 1, but serum E2 was greater on d 2, 3, and 5 in LH-treated cows in experiment 2. Infusion increased LH pulse frequency and amplitude in both experiments. Formation of cysts did not differ between LH- and saline-treated cows in either experiment (1 of 7 vs. 0 of 5 and 1 of 6 vs. 0 of 7, respectively). Cows that ovulated had similar intervals to ovulation in experiment 1 [6.0 ± 0.1 d (LH) vs. 6.4 ± 0.2 d (saline)], but in experiment 2, ovulation was 14 d earlier in LH-treated cows (5.6 ± 1.8 d vs 19.9 ± 1.5 d). In conclusion, high concentrations of LH are not solely responsible for formation of cysts in lactating dairy cows. Pulsatile infusion of LH stimulated follicular growth and steroidogenesis and decreased time to first ovulation in anestrous postpartum cows.
Key Words: ovarian cyst • luteinizing hormone • ovulation
Abbreviation key: cysts = ovarian follicular cysts, E2 = estradiol-17ß, P4 = progesterone, RIA = radioimmunoassay
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Previous work in our laboratory has demonstrated that elevated serum concentrations of LH are associated with the development and maintenance of cysts (Cook et al., 1991; Hamilton et al., 1995). In these studies, LH profiles of cows during development and maintenance of cysts were characterized by both high frequency and amplitude pulses without a preovulatory-like LH surge. The increase in basal LH concentrations was maintained until cysts spontaneously regressed or were successfully treated. Thus, high peripheral concentrations of LH may be associated with the development and (or) maintenance of cysts. In a preliminary study using cyclic, nonlactating dairy cows, infusion of LH in a high frequency, high amplitude manner for 5 d increased follicular size and delayed ovulation (
10 d versus saline-treated controls) in two of three cows (H. A. Garverick, B. E. Salfen, and R. S. Youngquist, unpublished data). The objective of the current study was to determine whether chronic pulsatile infusion of LH into lactating dairy cows, in a manner that produced high frequency, high amplitude pulses of LH, similar to those observed in cows with cysts, would result in the development of cysts. Alternatively, infusion of exogenous LH may stimulate ovarian follicular development and ovulation.
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MATERIALS AND METHODS |
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(Lutalyse, The Upjohn Company, Kalamazoo, MI) on d 12, and an i.m. injection of 100 µg GnRH on d 10. Ovarian structures were monitored daily by ultrasonography to determine time of ovulation and subsequent emergence of the second follicular wave (d 0). Emergence of the second follicular wave was characterized by recruitment of a new cohort of follicles to grow from 
4 to >5 mm in diameter. One day after the emergence of the second follicular wave (D 1), cows were fitted with indwelling jugular catheters and were randomly assigned to begin receiving hourly pulsatile infusions (each pulse infusion lasting <60 s) of LH (USDA-bLH-B-6; 2.1 S1 Units/mg; <1 % FSH activity; 40 µg/pulse; n = 7) or saline (2 ml/pulse; n = 5), administered via a computer-controlled syringe pump. In preliminary experiments, this level of LH infusion increased circulating LH in a pulsatile manner that was twice that observed in saline-treated cows and similar to cows developing and maintaining cysts (H. A. Garverick, B. E. Salfen, and R. S. Youngquist, unpublished data). On d 2, all cows received two i.m. injections of PGF2 (25 mg/injection) given 12 h apart. Pulsatile infusions of LH or saline were delivered until an ovulation was detected, or until a predetermined termination point (d 5, n = 5; or d 7, n = 7), whichever occurred first. This protocol was similar to a preliminary experiment, in which infusion of LH for 5 d resulted in increased follicle size and delayed ovulation in two of three LH-treated, nonlactating cows (H. A. Garverick, B. E. Salfen, and R. S. Youngquist, unpublished data).
Experiment 2: Early Postpartum Cows
Fourteen early postpartum dairy cows were used in experiment 2. Following parturition, cows were monitored daily by ultrasonography to detect the initiation of the first postpartum follicular wave (d 0; mean = 8 d postpartum; range = 7 to 12 d postpartum). Initiation of the first postpartum follicular wave was defined as emergence of the first developing cohort of follicles, following parturition, to grow 5 mm in diameter. Cows were fitted with indwelling jugular catheters and randomly assigned to receive LH (40 µg/pulse; n = 7) or saline (1 ml/pulse; n = 7) as described in experiment 1, for 7 d, or until ovulation was detected.
Blood Collection and Hormone Analysis
In both experiments, blood samples were collected daily via jugular venipuncture from initiation of study until ovulation (experiment 1) or corpus luteum formation (experiment 2) to assess peripheral concentrations of LH, FSH, progesterone (P4), and estradiol-17ß (E2). Blood samples were also collected at 12-min intervals for 8 h, beginning 48 h after initiation of infusions to verify delivery of LH and to characterize circulating concentrations of LH. Blood samples were allowed to clot overnight at 4°C and centrifuged 24 h later at 1800 x g for 30 min at 4°C. Serum was harvested and stored at -20°C until concentrations of P4, E2, LH, and FSH were determined via radioimmunoassay (RIA).
Serum concentrations of P4 were determined using a direct, solid-phase RIA (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA; Kirby et al., 1997), and daily samples were measured in duplicate 100-µl aliquots of serum in a single assay, with an intraassay CV of 1.8%.
Aliquots of serum used to measure E2 were first subjected to reverse-phase chromatography extraction procedures. Briefly, octadecyl columns (SPEEDISK 96 Silica columns; 20 mg sorbent mass; J. T. Baker, Phillipsburg, NJ) were prewashed with two column volumes of acetone, two volumes of HPLC-grade methanol, and two volumes of distilled water. Steroid-free serum (0.6 ml) was aspirated through columns and was followed by one column volume of 1:4 acetone:water solution. Columns were dried using positive pressure for 3 min, then rinsed with two column volumes of both methanol and distilled water. Following this preparation of each column, 0.6 ml of serum, in duplicate, was aspirated through columns, followed by one column volume of 1:4 acetone:water solution. Columns were dried for 3 min under positive pressure, and the E2-containing fraction was collected by two successive column aspirations with 0.3 ml of methanol. Methanol fractions were evaporated under gentle air stream and by warming to 37°C. Recovery of [125I] estradiol-17ß from chromatography columns were >90%. Fractions were then resolubilized in 0.1 ml of 1% BSA in buffer (0.01 M PO4, 0.15 M NaCl, and 0.01% sodium azide; pH = 7.2), and concentrations of E2 were determined using a previously validated assay (Rozell and Keisler, 1990). Separation of bound and free E2 was performed using a pre-precipitated, sheep antirabbit second antibody, followed by centrifugation at 1800 x g for 30 min. Radioactivity was counted in pellets in a gamma counter for 2 min per tube. The limit of detection was 0.25 pg (90% binding) and intra- and interassay coefficients of variation were 7.3 and 10%, respectively.
Serum concentrations of LH were determined as previously described by Zaied et al. (1980) using anti-oLH TEA # 35 (J. J. Reeves, Washington State Univ., Pullman, WA). Concentrations of LH were measured in duplicate 200-µl aliquots of serum. Intra- and inter-assay CV for three serum pools were 5.2 and 10.8%, respectively, across seven assays.
Serum concentrations of FSH were determined as previously described (Garverick et al., 1988). Concentrations of FSH were measured in duplicate 200-µl aliquots of serum. NIAMDD oFSH RP-1 was used in reference preparations. Intra- and inter-assay CV for three serum pools were 3.2 and 5.5%, respectively, across two assays.
Statistical Analyses
Differences in the proportion of cows forming cysts were determined by Fisher’s exact test (Ott, 1993) using the frequency procedures of SAS (version 8.0; SAS Inst., Inc., Cary, NC). In cows that ovulated, mean time to ovulation from initiation of treatment was determined by analysis of variance (Ott, 1993) using the general linear models procedures of SAS. Differences between treatment means were determined using pairwise t-test comparisons (Ott, 1993). In Experiment 1, time to ovulation was not different between cows infused for 5 or 7 d (P = 0.18); therefore, data were pooled.
Mean serum LH concentrations, pulse frequencies (number of LH pulses/8-h period), pulse amplitudes, and inter-pulse intervals were determined using CLUSTER analysis procedures (Veldhuis and Johnson, 1986) with a minimum LH pulse equaling 1 ng/ml. Effects of treatment on mean LH concentration, pulse frequency, pulse amplitude, and interpulse interval were determined by analysis of variance using the general linear models procedures of SAS. Differences among treatment means were determined using pairwise t-test comparisons (Ott, 1993).
For analysis of daily serum samples, one cow in experiment 1 (LH treatment) and one cow in experiment 2 (LH treatment) were excluded from further analysis due to failure to ovulate. Additionally, one cow from experiment 2 (LH treatment) was excluded from further analysis due to lack of ovarian function. Effects of treatment on daily follicular diameter, P4, E2, LH, and FSH concentrations were determined by analysis of variance using the mixed models procedures of SAS (Littell et al., 1998). Cow within treatment was used as the error term with time serving as the repeated measure. Differences between group means were determined when treatment x time interactions were significant using pairwise t-tests comparisons (Ott, 1993).
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RESULTS |
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0.01), as determined during the frequent blood collection period, in LH- vs. saline-treated cows (Table 1
, Figure 1).
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In cows that ovulated, mean time from detection of the second follicular wave to ovulation did not differ between treatment groups (P = 0.10), and occurred by 6.0 ± 0.1 d in LH-treated cows and 6.4 ± 0.2 d in saline-treated cows. Corpus luteum formation was observed in all cows following ovulation as evidenced via ultrasonography (data not shown).
Mean daily follicular diameter and concentrations of LH and P4 in cows that ovulated are depicted in Figure 2. Follicle diameter increased in LH- and saline-treated cows during the experimental period until ovulation and did not differ between treatment groups (P = 0.49). Mean daily concentrations of LH were numerically greater in LH- vs. saline-treated cows during the infusion period (Figure 2), but treatment, time, and treatment x time interactions were not significant (P 0.23) in the daily samples. While initially greater in saline-treated cows (P 0.05), serum concentrations of progesterone in both groups of cows declined to and remained less than 1 ng/ml following prostaglandin treatment. Mean daily concentrations of FSH and E2 are not shown, as treatment x time interactions were not significant (P = 0.89 and P = 0.55, respectively).
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; P < 0.0001). As shown in Table 1, greater basal concentrations of LH in LH- vs. saline-treated cows were the result of greater LH pulse frequencies and pulse amplitudes (P 0.001) in LH-treated cows.
As in experiment 1, the number of cows forming cysts did not differ between LH- and saline-treated cows (one of six versus zero of seven, respectively; P = 0.46). However, LH treatment reduced the time to first ovulation postpartum (P = 0.0001). In cows that ovulated, mean time to ovulation following initiation of infusion was 5.6 ± 1.8 d in LH-treated cows, whereas mean time to ovulation from initiation of infusion was 19.9 ± 1.5 d in saline-treated control cows. Treatment, time, and treatment x time interactions were not significant for daily concentrations of FSH (P 0.27).
Mean daily follicle diameter and serum concentrations of LH, E2, and P4 are depicted in Figure 3. Mean daily follicle diameter was greater in cows treated with LH vs. saline during the infusion period until ovulation (P = 0.05). In saline-treated cows, the first dominant follicle, postpartum, reached a smaller maximum diameter (13.0 ± 0.9 mm; P = 0.0006) than in LH-treated cows (19.6 ± 1.6 mm) and failed to ovulate. Instead, the first ovulatory follicle in saline-treated cows arose from subsequent waves of follicular growth.
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0.03) in LH- vs. saline-treated cows. Greater mean concentrations of LH were coincident with greater concentrations of E2 on d 2, 3, and 5 (P 0.03) during the infusion period in LH-treated cows. Daily concentrations of P4 were minimal during the infusion period in both LH- and saline-treated cows (P 0.85). However, following ovulation, concentrations of P4 increased to levels greater than 1 ng/mL by d 10 following initiation of LH treatment, but failed to increase in saline-treated cows (P = 0.93) during this period. |
DISCUSSION |
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). Yet, in the current study, experimental alteration of peripheral levels of LH did not result in the formation of cysts in either experiment. Combining both physiological states, 11 of 13 cows treated with LH ovulated by d 7 of treatment. Ovulation was associated with a preovulatory increase in peripheral LH, disappearance of the largest follicular structure, and formation of luteal tissue. During the early postpartum period (experiment 2), pulsatile infusions of LH resulted in ovulation by d 7 (about 14 d postpartum) in five of six cows, vs. zero of seven cows treated with saline. Infusion of LH resulted in ovulation of the first-wave postpartum dominant follicle. In saline-treated cows, none of the first-wave dominant follicles ovulated. Maximum size attained by the first-wave dominant follicle in saline-infused cows was smaller, and mean concentrations of E2 were lower, than in LH-treated cows. Postpartum cows infused with saline ovulated the second or third wave-dominant follicle. Previous studies have shown that while the first dominant follicle can appear as early as 1 wk postpartum, first ovulation does not typically occur until about 20 d postpartum in dairy cows (Savio et al., 1990) and 35 d postpartum in beef cows (Murphy et al., 1990). The ability to ovulate coincides with increases in LH pulse frequency from two to three pulses per 6-h period to five to seven pulses per 6 h (Savio et al., 1990; Stagg et al., 1998). This suggests that low LH pulse frequency is associated with postpartum anestrus. Duffy et al. (2000) have shown that increasing LH pulse frequency alone can decrease interval to first ovulation and (or) increase maximum size of dominant follicles in early postpartum beef cows. These results are consistent with data from the current study in which increases in LH pulse frequency and amplitude were associated with decreased interval to ovulation and increased maximum diameter of the first postpartum dominant follicle.
Our original hypothesis was that the early postpartum period would be more conducive to cyst formation, because development of cysts is greatest during this period of time (Kesler and Garverick, 1982). Concentrations of P4 are typically low early postpartum because corpora lutea are absent before the first postpartum ovulation. A study by Caraty and Skinner (1999) implicated progesterone in the establishment of an LH surge in sheep. However, cows in the current study were capable of producing an LH-surge despite concentrations of P4 remaining below the detection limit of the assay before and during the infusion period. During the postpartum period, infusions of LH were associated with increased follicular growth and steroidogenic output of the first postpartum dominant follicle, because LH-treated cows had greater follicular diameters and serum concentrations of E2 than saline-treated controls. This is consistent with recent work by Duffy et al. (2000), who reported increased peripheral concentrations of E2 following LH treatments to postpartum beef cows. Increases in E2 have been established as potent stimulators of the preovulatory LH-surge (Karsch et al., 1997; Gazal et al., 1998). Therefore, in the current study, treatments of LH increased the estrogenic output of the first postpartum dominant follicle resulting in an LH surge, followed by ovulation and formation of a corpus luteum.
Previous work in our laboratory has shown that both naturally occurring and steroid-induced cysts were associated with the absence of a preovulatory LH surge and functional corpus luteum (Cook et al., 1990, 1991; Hamilton et al, 1995). Findings from other laboratories have also implicated alterations in the LH surge in the etiology of cysts (Yoshioka et al., 1996; Dobson et al., 1997; Christman et al., 2000). A recent study indicated that a large follicle anovulatory condition, similar to cysts, can be induced following an estradiol-induced GnRH surge without subsequent P4 exposure (Gümen et al., 2002). Accordingly, one of the established treatments for inducing cyst regression in dairy cattle is administration of GnRH or a GnRH-like product that, in turn, results in an LH surge (Garverick, 1997). Therefore, while the model used in the current studies was successful in simulating one of the factors associated with cyst development (increased basal concentrations of LH), it failed to simulate other factors associated with the etiology of cysts (absence of an LH surge).
While these data provide evidence that increased basal secretion of LH is not the sole factor responsible for the development of cysts, they do not preclude the involvement of LH in the maintenance of cysts. Previous work from our laboratory has shown that administration of P4 to cows with cysts results in a decrease in both LH pulse frequency and pulse amplitude within 24 h of administration (Calder et al., 1999). Reduction in LH concentrations and increased concentrations of P4 resulted in atresia of cysts and development of normal ovulatory follicles. In addition, some cows that develop cysts resume ovulatory ovarian cycles without exogenous hormonal therapy. In cows with cysts that resume ovulatory ovarian cycles, serum concentrations of LH were lower than in cows that maintained or developed new cysts (Hamilton et al., 1995). Therefore, while high concentrations of LH are not the sole factor responsible for the formation of cysts, high concentrations of LH may be required for the maintenance of cysts.
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CONCLUSIONS |
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Received for publication May 15, 2002. Accepted for publication October 2, 2002.
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