Temporary suppression of pulsatile LH release following a single injection of a GnRH agonist (deslorelin) in ovariectomised Holstein dairy cows

Animal Reproduction Science 70 (2002) 37–47

Temporary suppression of pulsatile LH release following a single injection of a GnRH agonist (deslorelin) in ovariectomised Holstein dairy cows A.M. Padula, J.M. Borman, P.J. Wright, K.L. Macmillan∗ Veterinary Clinical Centre, University of Melbourne, 250 Princes Hwy, Werribee, Vic. 3030, Australia Received 8 July 2000; received in revised form 16 November 2001; accepted 19 November 2001

Abstract The objective of the experiment was to investigate the potential for using a single injection of the GnRH agonist [D-Trp6 , Pro9 −des-Gly10 -NH2 ] GnRH-ethylamide (deslorelin) to suppress LH secretion in ovariectomised Holstein cows. Each dose of 10, 100 and 1000 ␮g deslorelin was injected intravenously into each of four ovariectomised cows on day 0. Blood samples were collected hourly on day 0 to profile the induced LH release. Frequent serial blood samples were collected at 10 min intervals over 4 h on days −3, −1, +2, +4 and +6. The injection of deslorelin induced a surge-like release of LH that begun within 1 h in all cows. There was no difference between deslorelin doses in terms of maximum LH concentration, area under the LH curve (AUC) or log10 (AUC). The average interval from injection to maximum LH concentration was longer for cows receiving 1000 ␮g than in those receiving 10 ␮g (3.5 versus 1.5 h; P < 0.01), though no different to 100 ␮g (2.8 h; P > 0.1). This relationship was described by a logarithmic function of deslorelin dose in micrograms (R 2 = 73.3%, P < 0.01). Pre-treatment smoothed mean LH concentration was significantly correlated with peak LH concentration of the induced surge: max LH = 5.37 + 9.57 × pre-amplitude (R 2 = 33.2%, P = 0.05). Similarly, LH pulse amplitude pre-deslorelin was also correlated with peak LH of the induced surge max LH = 0.07 + 12.9 × pre-amplitude (R 2 = 53.7%, P = 0.07). Pulsatile release of LH was suppressed only with the 1000 ␮g dose on day +2. Suppression was characterised by a reduction in mean LH, smoothed mean LH and LH pulse amplitude. By day +4, LH parameters were no different to pre-treatment ones. Pulse frequency was not affected by the treatment, although a small non-significant reduction at day +2 for 1000 ␮g dose was observed (3.9 versus 2.8, P = 0.14). In conclusion, temporary suppression of LH output for at least 48 h occurred following a single intravenous injection of 1000 ␮g of deslorelin, even though there were similar peak LH concentrations were for the three doses. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Dairy cattle; Postpartum; Anoestrus; GnRH agonist; Deslorelin; Oestradiol

∗ Corresponding author. Tel.: +61-3-9731-2234. E-mail address: [email protected] (K.L. Macmillan).

0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 9 1 - 9

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1. Introduction Since the initial discovery of GnRH as the hypothalamic factor which controls pituitary gonadotrophin release (Schally et al., 1971), numerous GnRH analogs have been developed and explored for their pro-fertility potential (Karten and Rivier, 1986). Paradoxical anti-fertility effects were soon revealed once more potent analogs were developed. Desensitisation of the female bovine pituitary to GnRH has been achieved with continuous treatment using the GnRH agonists deslorelin (Padula et al., 1999; Bergfeld et al., 1996b; D’Occhio et al., 1996) and buserelin (Gong et al., 1995, 1996). Secretion of LH pulses is suppressed during these treatments (Gong et al., 1996). The capacity of a single injection of a GnRH agonist to induce pituitary suppression and induce anoestrus in cows has not been described. The sensitivity of cattle to deslorelin was demonstrated when treatment for 3 days resulted in a prolonged period of anoestrus in postpartum dairy cows implanted within 3 days of calving (Padula et al., 2000). Injection of buserelin every 3 days was able to prevent ovulation in Holstein cows undergoing regular oestrous cycles (Thatcher et al., 1993). Buserelin injected four times daily between day 9 and 12 post-oestrus significantly prolonged oestrous cycle lengths by around 6 days (Milvae et al., 1984). Successive injections of 50 ␮g GnRH into anoestrous or ovariectomised ewes resulted in diminished LH responses unless the interval between injections was extended to beyond 72 h (Rippel et al., 1974). Similarly, in a study involving serial injections of buserelin, fertirelin and native GnRH, a significant interaction between GnRH form and sequence was found; doses of, or >500 ␮g GnRH, 50 ␮g fertirelin and 10 ␮g buserelin resulted in reduced LH output when given every 48 h, suggesting that some degree of desensitisation had occurred (Chenault et al., 1990). Rodent models have been widely used for potency evaluation of GnRH agonists (Karten and Rivier, 1986; Hahn et al., 1984; Schally et al., 1980). Using these in vivo models, deslorelin has been shown to be 7 (Coy et al., 1976), 15 (Dutta et al., 1978) and 55 (Hahn et al., 1984) times more potent than native GnRH. When in vitro assay systems such as LH release from cultured pituitary cell layers and GnRH radioligand binding affinity were used, deslorelin was 8–198 (144 mean) (Fujino et al., 1974), 10–26 (mean 15) (Perrin et al., 1980) or 76 (Barron et al., 1984) times more potent than GnRH. Equivalent comparative studies with deslorelin have not been completed in cattle, although buserelin and fertirelin have been evaluated (Chenault et al., 1990). One application for suppression of LH output could be prevention of early ovulation in the postpartum dairy cow (Padula et al., 1999). The use of a short deslorelin treatment (∼3 days) has demonstrated the sensitivity of the bovine pituitary to downregulation by suppressing resumption of ovulatory activity for at least 20 days longer in postpartum cows than in untreated herdmates (Padula et al., 2000). Much shorter treatments may be possible and achieve similar results. It is presumed that the mechanism mediating this prolonged anovulation involves suppression of LH pulse secretion, with full recovery requiring restoration of pituitary content of LH. Total pituitary content of LH and FSH was decreased >20-fold in bulls killed after treatment with naferelin for 15 days (Melson et al., 1986). No studies have been published in cattle on the time required for restoration of pituitary gonadotrophin content following a downregulation treatment.

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The objectives of this experiment were to investigate the potential of a single injection of deslorelin to suppress LH output in long term ovariectomised Holstein cows. A log10 dose approach was used to compare LH pulse profiles every 48 h for 6 days. 2. Materials and methods 2.1. Treatments Long-term ovariectomised, mature Holstein–Friesian cows were randomly allocated to receive either 10 ␮g (n = 4), 100 ␮g (n = 4) or 1000 ␮g (n = 4) of the agonist [D-Trp6 , Pro9 −des-Gly10 -NH2 ]GnRH-ethylamide (deslorelin) dissolved in 0.9% physiological saline administered as a single intravenous bolus via an indwelling jugular catheter. Cows were housed indoors in an open sided cattle shed at the Victorian Institute of Animal Science (VIAS) in Werribee and fed grass hay in individual stalls for the duration of the experiment. Prior ethics approval was obtained through the VIAS Ethics Committee. 2.2. Blood sampling Jugular catheters were inserted 24 h before the first sampling period and cows accustomed to the shed overnight before the first day of sampling. Blood samples were collected at hourly intervals for 8 h on the day of deslorelin injection (day 0). On days −3, −1, +2, +4 and +6 relative to deslorelin injection, samples were collected at 10 min intervals over 4 h to characterise patterns of LH secretion. Sampling commenced at 9 a.m. each day. Blood samples (10 ml) were collected into syringes at 10 min intervals during the 4 h sampling session. Each sample was placed into a heparinised centrifuge tube and plasma separated by centrifugation. Catheters were flushed with heparinised saline (50 iu/ml) after every sample collection. Plasma was stored frozen at −20 ◦ C until subsequently assayed for LH. 2.3. Luteinising hormone assay Every sample from days −3, −1, +2, +4 and +6 from an individual cow was run in a single assay to avoid adding any effect of inter-assay variation. Samples collected at hourly intervals from all cows on day 0 were quantified in one assay. LH was assayed using a modified double antibody precipitation radioimmunoassay (Wright et al., 1980). Phosphate buffered saline (PBS) was used throughout the assay. Ovine LH standards (80, 40, 20, 10, 5, 2.5, 1.25, 0.6, 0.4, 0.15 ng/ml) were prepared in plasma from a hypophysectomised–ovariectomised (HYPOX) ewe. Internal quality control samples were made by spiking HYPOX plasma with the stock ovine standard to give approximate final concentrations of 10.0, 5.0 and 0.5 ng/ml. The primary antibody was equine anti-bovine LH (Snook, 1968) diluted 1:300,000. Bovine 125 I-LH was prepared using the chloramine T method, purified by high-pressure liquid chromatography and diluted to produce 12,000 counts per minute in a 100 ␮l aliquot. A 100 ␮l sample volume was used for standards, quality controls and unknown samples. These were incubated along with 100 ␮l

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of the first antibody, 100 ␮l of bovine 125 I-LH and 100 ␮l of equine gamma globulin at 37 ◦ C for 24 h. Separation was performed by addition of the second antibody (goat anti-horse) using 100 ␮l of a 1:160 dilution. This mixture was incubated at 4 ◦ C overnight. A 1.0 ml volume of a 10% polyethylene glycol solution was then added and the mixture incubated at 4 ◦ C for 30 min before being centrifuged at 3000 × g for 15 min so that the supernatant could be aspirated and the remaining pellet counted for 1 min in a gamma counter. The minimum detectable concentration was 0.15 ng/ml. Within assay variation averaged 9.3, 8.5 and 13.3% for high, medium and low standards. Between assay variation averaged 11.8, 10.2 and 18.0% for high, medium and low standards. 2.4. Statistical analysis All pre-treatment (days –3 and –1) LH measurements for each cow were compared by t-test. Since there were no differences, they were pooled to provide pre-treatment mean pulse amplitude, pulse frequency and smoothed/arithmetic mean LH for each cow. The software package PULSTER (Merriam and Wachter, 1982) was used to calculate the number of LH pulses and the amplitude of each pulse (for discussion see (Turek et al., 1994)). A linear CV function was used to define the assay variation: CV(%) = 9.53 + 0.168X. The mean pulse amplitude per cow per 4 h period was used whenever multiple pulses were detected. Arithmetic mean and Pulster® smoothed mean LH concentrations for each group across time were examined using a repeated measures analysis of variance (ANOVA) in a mixed model using the computer software package SAS Analyst 8.0 (SAS Institute, 1999). The PROC MIXED command with REML estimation was used to create a fixed-and-random effects model suitable for repeated measurements (Littell et al., 1998). Cow was classified as a random effect. The model was of the form: mean LH = day, treatment and the interaction of day across treatment. Pre-planned orthogonal comparisons were compared by t-test with mean pre-treatment concentrations (per cow) at day +2, +4 and +6. The SLICE option of the LSMEANS subcommand was used to partition the interaction term whenever the interaction was significant (see SAS online docs at www.sas.com). Smoothed LH means were analysed exactly as for arithmetic means. Pulse frequency data were analysed using a similar mixed model approach and pre-planned contrasts by t-test with pre-treatment means were used. The area under the curve (AUC) for day 0 was calculated using the trapezoidal rule and log10 transformed prior to analysis. One-way ANOVA was used to compare the LH response among dose groups to the deslorelin injection on day 0. 3. Results 3.1. Smoothed mean LH Smoothed mean LH concentrations were lower than arithmetic mean LH concentrations, but were less variable. Statistical conclusions were the same for both. There was no effect of dose (P > 0.25) and no dose × day interaction (P > 0.25, Fig. 1). There was an effect

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Fig. 1. PULSTER derived smoothed mean LH concentrations by day for each dose group. Shown as mean ± S.E. of the mean.

of day (P < 0.01). Pre-planned contrasts of pre-treatment means with days +2, +4 and +6 showed no changes for 10 or 100 ␮g dose groups. The 1000 ␮g dose reduced smoothed mean LH to 52% of pre-treatment (1.69 versus 0.88 ng/ml, pre versus day +2, P < 0.01), but it had recovered by day +4 and was similar to pre-treatment by day +6 (P > 0.1). 3.2. LH pulse amplitude The interaction of dose × day was considered to be significant (P = 0.053). The SLICE command was used to partition the interaction (Table 1) showing that only the 1000 ␮g dose was involved (P < 0.01) and that the interaction was occurring on day +2 (P = 0.051). Mean LH pulse amplitude was reduced to 35% of pre-treatment in the 1000 ␮g dose (1.70 versus 0.60 ng/ml, pre versus day +2, P < 0.01). Pulse amplitude had returned to similar pre-treatment levels by day +4 (1.70 versus 1.41 ng/ml, pre versus day +4; P > 0.05) (Fig. 2). Table 1 Summary of LH pulse amplitude data as determined by PULSTER software (n = 4 per group)a Dose (␮g)

Pre

Day +2

Day +4

Day +6

10 100 1000

1.67 ± 0.5 1.44 ± 0.3 1.70 ± 0.6∗

1.67 ± 0.5 1.44 ± 0.7 0.60 ± 0.3∗∗

1.71 ± 0.6 1.60 ± 0.9b 1.41 ± 0.4∗

1.62 ± 0.6 1.61 ± 0.3 1.57 ± 0.8∗

Means ± S.D. (ng/ml). One cow with no pulses in this group. ∗ P < 0.5. ∗∗ P < 0.01. a

b

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Fig. 2. Plot of mean LH pulse frequency (top) and amplitude (bottom) for each dose group across time. Error bars represent S.E. of the mean.

Individual cow LH secretion profiles over time for the 1000 ␮g dose group were similar. A marked suppression of LH pulse amplitude was observed following deslorelin injection (Fig. 3). 3.3. LH pulse frequency There was no change (P > 0.1) in LH pulse frequency with all cows producing around 1 pulse per hour across all days (Fig. 2). There was a small, but non-significant drop in

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Fig. 3. LH response to an intravenous injection of 10, 100 or 1000 ␮g of deslorelin. Error bars represent S.E. of the mean.

pulse frequency between pre and day +2 in to 1000 ␮g dose (3.86 versus 2.81 pulses/h, P = 0.14). Two cows in the 1000 ␮g group produced only 1 pulse over the 4 h bleeding period on day +2 (Table 2). 3.4. LH release immediately post-deslorelin injection There was a large amount of variation among individual cows in the magnitude of the initial LH release following deslorelin injection. Injection of deslorelin caused a marked increase in plasma LH in all cows by 1 h post-injection. Similar peak concentrations were reached across all doses (P > 0.1, Table 2). Areas under the LH curves were similar for all doses (P > 0.1). The time interval from injection to peak LH level progressively increased with dose such that: hours to max LH = log10 (dose) + 0.583, (R 2 = 73.3%, P < 0.0001) (Fig. 4). Table 2 Summary of immediate LH response following 10, 100 or 1000 ␮g of deslorelin injected intravenouslya Dose (␮g)

LH AUC

Log10 (AUC)

LH max (ng/ml)

Hours max LH

10 100 1000

65.9 ± 40.7 89.3 ± 37.9 116.7 ± 58.8

1.82 1.95 2.07

19.8 ± 11.3 23.3 ± 8.9 19.8 ± 7.8

1.5 ± 0.6∗ 2.8 ± 0.5∗∗ 3.5 ± 0.6∗∗

Blood samples collected at hourly intervals for 8 h. Data presented as means ± S.D. P < 0.5. ∗∗ P < 0.01. a

∗

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Fig. 4. Sequential LH pulse profiles for a typical cow injected with 1000 ␮g deslorelin on day 0. Samples collected at 10 min intervals over 4 h.

Pooling of results from all cows (because no dose response) revealed a significant correlation between smoothed mean LH pre and maximum LH concentrations after deslorelin and the relationship was described by the equation: max LH = 5.37+9.57×pre-LH (R 2 = 33.2%, P = 0.05). Analysis of pooled results for LH pulse amplitude indicated a similar relationship: max LH = 0.07 + 12.9 × pre “pulse amplitude” (R 2 = 53.7%, P = 0.007). 4. Discussion Injection of deslorelin induced a marked increase in circulating plasma concentrations of LH within 1 h (Fig. 4). This response is typical for the initial stimulation induced by GnRH

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and GnRH agonists (D’Occhio et al., 2000). Similar responses were observed between all doses (10, 100 and 1000 ␮g), although there was wide variation amongst individual cows. The minimum dose required to induce a physiological LH surge in some cows would appear to be <10 ␮g. Increasing the dose only resulted in a slight prolongation of the interval to peak LH concentration, and this tended to increase the area under the LH curve. The delay in reaching maximum levels probably reflected the continued stimulation of gonadotropes by deslorelin that had not been cleared from the body. The half-life of deslorelin in cattle is unknown. One study using a labelled [D-Trp6 ]GnRH in humans indicated a relatively short half-life of 60 min for this agonist compared to 6 min for native GnRH (Barron et al., 1982). LH pulse amplitudes were reduced by a 1000 ␮g dose only at day +2, with recovery similar to pre-levels by day +4. Some degree of gonadotrope GnRH receptor downregulation had presumably occurred following the 1000 ␮g dose and was restored by day +4, since pulses of LH could be clearly distinguished. Injection of 8 or 10 ␮g buserelin every 3 days (Thatcher et al., 1989) and 100 ␮g deslorelin twice-weekly (Padula AM, unpublished data) induced anoestrus in previously cycling cows. The transient suppression of LH following 1000 ␮g deslorelin does not adequately explain the persistent state of anoestrus induced in cows given a much lower dose. It is possible that restoration of pituitary content limits complete recovery from anoestrus induced with a GnRH agonist by inhibiting the production of an adequate LH surge. Examination of pituitary gonadotrophin content after agonist treatment may reveal more about the recovery process following receptor downregulation. Treatment of early postpartum cows with an absorbable deslorelin implant designed to release over 2–3 days resulted in a highly variable interval between calving to first ovulation that was prolonged for at least 20 days compared to control cows (Padula et al., 2000). Factors other than suppression of pulsatile LH output may be involved in the continued maintenance of anoestrus in cows intermittently treated with a potent GnRH agonist. The use of smoothed mean LH concentrations reduced the variability commonly detected when arithmetic means are used and probably better represents the situation when attempting to compare pulsatile to non-pulsatile LH profiles. The smoothed mean LH concentrations in the 1000 ␮g dose group were only slightly reduced (1.70 ± 0.25 versus 0.89 ± 0.24 ng/ml; pre versus day +2, P < 0.01). This was consistent with other studies with cows during chronic GnRH agonist treatments that have reported either no change (Bergfeld et al., 1996a) or a slight increase in basal LH (Gong et al., 1995, 1996). The significant correlation between smoothed mean LH concentration and LH pulse amplitude pre-deslorelin with acute LH response to deslorelin may indicate a relationship between the magnitude of the surge and releaseable stores of LH. The obvious difficulty in directly examining the relationship between LH surge magnitude and pituitary content in normal cows was recognised. In GnRH pulsed hypothalamo-pituitary disconnected ovariectomised ewes, LH pulse amplitude decreased as the pulse frequency of GnRH was increased. The authors interpreted LH pulse amplitude to reflect releasable stores of LH (Clarke and Cummins, 1985). 5. Conclusions Doses of 10,100 and 1000 ␮g of deslorelin produced similar surges in LH output from the pituitary with the 1000 ␮g dose slightly prolonging the time interval until maximum

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LH concentrations were reached. LH pulsatility was depressed only in the 1000 ␮g group at 48 h post-injection. It had recovered by day +4 to pre-treatment levels. Depression of LH involved a marked reduction in pulse amplitude as well as a small, but non-significant reduction, of mean pulse frequency over 4 h. A single 1000 ␮g dose of deslorelin given intravenously could be used to temporarily suppress LH output primarily by restricting pulse amplitude for at least 48 h.

Acknowledgements The authors wish to thank Dr. Tim Trigg of Peptech Animal Health for generous supply of the deslorelin used in this study. Thank you to Terry Squires of VIAS for technical support.

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