The use of oilseeds is an important option for increasing the energy density of ruminant diets in addition to supplying dietary protein to support high rates of production. However, animals may use certain oils or fatty acids with different efficiencies than are currently documented (Su and Jones 1993). Feeding whole soybeans to lactating dairy cows reduces milk protein production (Casper and Schingoethe 1989). Rumsey et al. (1996) recently reported that feeding roasted soybeans (RSB)⁵ reduced weight gain of young beef steers. Although several studies suggest that certain oils or dietary fatty acids affect the thyroid status of ruminants (Kahl et al. 1991, Lough et al. 1993 and 1994, Romo 1995, Norton et al. 1987 and 1988) primarily through changes in peripheral deiodination of the inactive hormone thyroxine (T₄) to the active hormone triiodothyronine (T₃), little is known of the effects oil supplements may have on the hypothalamic release of thyroid-stimulating hormone (TSH). In the research reported by Rumsey et al. (1996), the ability of thyrotropin-releasing hormone (TRH) plus growth hormone-releasing hormone (GHRH) to release growth hormone (GH) was shown to be reduced in young steers fed RSB.

Estrogenic growth promoters are widely used in growing and finishing beef production systems. The estrogenic growth promoter Synovex-S® (SYN, ear implant containing 20 mg estradiol benzoate + 200 mg progesterone) affects the thyroid status in beef steers (Kahl et al. 1978), probably via the T₄ deiodinase mechanism (Rumsey et al. 1985b). Limited reports suggest that estrogens may increase TSH release in sheep (Davis et al. 1978) and rats (DeLean and Labrie 1977). Trenkle (1969) suggested that estrogens increase the sensitivity of the thyroid gland to TSH. Thus an interaction may exist between the feeding of oilseeds and the use of an estrogenic growth promoter with regard to effects on the thyroid axis.

This study was undertaken to determine the influence of feeding a diet supplemented with RSB compared with soybean meal on the ability of TRH and GHRH to stimulate TSH release in young growing beef steers either implanted or not implanted with SYN. We used the combination of hypothalamic releasing hormones (TRH+GHRH) to test, in the same steers, the responsiveness of the pituitary gland to release both GH (Rumsey et al. 1996) and TSH.

MATERIALS AND METHODS

Steers were individually fed either RSB or +RSB diets during the study to gain an estimated 1.3 kg/d. After adaptation to pens and the RSB diet, steers were switched to their assigned diets, implanted as assigned, and fed for 9 wk. The initial 5-wk period was for recording weight gain (Rumsey et al. 1996). During wk 7, 8 and 9, multiple hormone challenges were conducted on each steer to measure TSH, T₄ and T₃ responsiveness. This was followed by an interim feeding period of 3 wk with steers continuing on their respective diets. Steers were then reimplanted as per original implant assignment, continued on their respective diets for an additional 5 wk for recording weight gain and then killed in the Beltsville abattoir as per Rumsey et al. (1996). The liver, kidney and pituitary of each steer were collected and weighed. The pituitary and duplicate 10-g samples of liver and kidney were sealed in plastic bags, frozen in liquid nitrogen and stored at 80°C until analyzed for 5-deiodinase activity (Kahl et al. 1995).

All steers were challenged with three levels of TRH+GHRH (0.1+0.01, 1.0+0.1, 2.5+0.25 µg/kg body wt) over the 3-wk challenge period as described (Rumsey et al. 1996). For each challenge, blood samples were obtained at 10, 0, 5, 10, 15, 20, 30, 45, 60, 120, 240 and 360 min after challenge for T₃ and T₄ analysis and at 10, 0, 15, 30, 45, 60 and 120 min for TSH analysis. Thyroxine-stimulating hormone concentration in plasma was determined by radioimmunoassay (Elsasser et al. 1992), with inter- and intraassay CV of 12.6% and 12.6%, respectively. Thyroxine and T₃ concentrations were determined by radioimmunoassay as reported by Kahl et al. (1992), with inter- and intraassay CV of 4.8% and 6.3% for T₄ and 5.2% and 7.8% for T₃, respectively. The hormone response curves were evaluated for baseline concentration before challenge, area under the curve, peak response (visual evaluation of plasma concentrations), and time from challenge to peak.

Data were analyzed using the GLM procedure of SAS (1988). Response curve data were analyzed as a 2 × 2 × 3 factorial with SYN, RSB, dose level and their interaction as main effects. Differences were considered significant at P  0.05 and to show a trend at 0.05 < P  0.10. Where appropriate, differences between individual means were evaluated using the Duncan's new multiple-range test protected with a significant F test (P < 0.05).

RESULTS AND DISCUSSION

The degree of TSH responsiveness to TRH+GHRH challenge was evaluated among treatments by comparing indicators of response curve characteristics. These indicators were the baseline (zero time) plasma concentration, the number of peaks (one or two), the time lapsed between challenge injection and peaks, the peak concentrations, and the net area under the response curves (total area minus baseline). The evaluation of the T₄ and T₃ response curves was the same as for TSH except only single response peaks were observed. In general, if baseline and peak concentrations vary in a similar manner across treatments and net area across treatments is not different, then the response was considered to suggest a sustained long-term effect of treatment. If baseline concentration is not different across treatments and peak concentrations and net area under the response curve vary in a similar manner across treatments, then an effect of treatment on the immediate sensitivity to hormone challenge is suggested. If baseline and peak concentrations and net area all differ among treatments, then both a sustained treatment effect and an immediate sensitivity are involved. A change in lapsed time between challenge injection and peak is interpreted to be positively related to sensitivity to challenge.

The response curve indicators for TSH for individual challenge dose levels averaged across the SYN and RSB treatments are shown in Table 1. There were no dose level × treatment interactions. Baseline TSH concentration was not different across dose level. This result is consistent with the design, because dose level would not be expected to affect pre-challenge concentrations.

Table 1. Plasma thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) responses to three dose levels of a venous injection challenge of TRH + GHRH in young growing beef steers1 [View Table]

The number of TSH response peaks following hormonal challenge shifted from one peak toward two peaks, which reflects the fact that most steers at the two higher challenge doses displayed the initial increase in TSH, followed by a small reduction and then another increase, presumably a latent stimulation of TSH synthesis. Because of this effect on the number of TSH response peaks, statistical analysis for number of peaks included only data for the middle and high dose levels. There was no significant difference between the middle and high dose levels in the number of TSH response peaks. Similarly, statistical analysis compared only the middle and high dose levels for peak 2 concentration and minutes to peak 2. There was no significant difference between the middle and high dose levels for peak 2 concentration and only a trend (P < 0.10) toward a greater lapse time between challenge injection and peak 2 for the high dose compared with the middle dose.

The overall effects of challenge dose level were to increase plasma concentration of TSH for peak 1 (P < 0.03) and to increase net area under the response curve (P < 0.01). In both cases, the responses to the middle and high doses were similar to each other but different (P < 0.05) from the response to the low dose level. As expected, these response curve changes are consistent with those expected for an immediate sensitivity to hormone challenge.

The effects of SYN and RSB treatments averaged across dose level on the TSH response curve indicators are summarized in Table 2. Across SYN treatments, TSH baseline concentration (P < 0.01), concentrations of TSH for peak 1 (P < 0.03) and peak 2 (P < 0.05), and minutes to peak 2 (P < 0.02) were lower for RSB-supplemented steers than for steers fed soybean meal following hormonal challenge, and area under the TSH response curve tended to be lower (P < 0.08) for the RSB-supplemented steers. These effects of RSB on the TSH response curve are consistent with a sustained long-term effect of RSB and possibly a reduced immediate sensitivity to hormonal challenge.

Table 2. Plasma thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) responses to venous injection challenge of TRH + GHRH in beef steers either implanted (+SYN) or not (SYN) with Synovex-S® and fed soybean meal (RSB) or roasted soybean (+RSB) supplemented diets1,2 [View Table]

The overall effect of SYN was a trend for baseline plasma concentration (P < 0.07), peak 1 concentration (P < 0.06) and peak 2 concentration (P < 0.07) of TSH to be greater for SYN-implanted steers than for steers not implanted. These effects of SYN are consistent with a sustained long-term effect of SYN.

The main effects of SYN on the baseline and peak 2 concentrations of TSH were primarily a result of a greater concentration for the +SYNRSB treatment compared with the other treatments. The SYN × RSB interaction was significant for the baseline concentration of TSH (P < 0.03), and the data showed a trend (P < 0.07) for a similar interaction for peak 2 concentrations. As with baseline and peak 2 concentrations, the highest numerical value for the net area under the response curve was observed for the +SYNRSB-treated steers compared with the other treatment groups, but statistical significance was not obtained for the main effect of SYN or for the SYN × RSB interaction. This may have been a function of the relatively high error variation obtained for this indicator. In general, these interactions would suggest that the sustained long-term effect of RSB on plasma TSH limits the release and/or production of TSH under conditions in which long-term TSH release is enhanced, such as in SYN-implanted steers.

The overall enhancement of TSH status and the sensitivity to challenge by SYN in growing steers is consistent with the overall physiological effect of increased metabolic rate reported earlier (Rumsey and Hammond 1990, Rumsey et al. 1980). Possibly the increased TSH status seen here and the increased GH status reported recently (Rumsey et al. 1996) in SYN-implanted steers are related to the ability of estrogens to enhance the level of cAMP (Dickson 1984), which in turn is associated with release of hypothalamic hormones. Our results are consistent with other reports that indicate estrogens increase the release and sensitivity to TRH in sheep (Davis et al. 1978) and rats (Delean and Labrie 1977). However, SYN did not affect plasma TSH concentrations in beef steers in a study reported by Kahl et al. (1992). In that study, the steers were about 60 kg heavier, suggesting a possible age effect.

The general depression in response by RSB in young growing beef steers may reflect a similar effect on cell membranes and hormone release as suggested in the recent study of GH (Rumsey et al. 1996). Specific fatty acids may affect the fluidity of cell membranes and reduce the affinity to receptor sites (Klausner et al. 1980, Renaud et al. 1985). A specific explanation for the observed interaction between SYN and RSB is not apparent at this time.

Response of thyroxine following thyrotropin-releasing hormone plus growth hormone-releasing hormone challenge. The general pattern of the response curves obtained for T₄ is consistent with that for TSH. The plasma T₄ response curves (Fig. 2) would suggest an attenuated stimulation of T₄ production until after 60 min and a similar initial stimulation to all challenge doses even though the initial TSH response appeared different for the lowest dose. The plasma T₄ concentrations increased with time after the challenge injection, but the response to the lowest challenge dose seemed to be diminishing at 360 min, which is consistent with less TSH response. The response to TRH+GHRH challenge appeared less and slower for T₄ than for TSH, which would be consistent with a secondary or indirect effect of TRH on T₄ release.

The response curve indicators for T₄ for individual challenge dose levels averaged across the SYN and RSB treatments are shown in Table 1. There were no dose level × treatment interactions. Challenge dose increased the lapsed time between challenge injection and peak T₄ concentration (P < 0.01) and the net area under the response curve (P < 0.05). In both cases, the responses to the middle and high doses were similar to each other but different (P < 0.05) from that for the low dose level. Similarly, peak response concentration was numerically increased with dose level, but the differences were not significant. These changes are generally consistent with the effects of dose level on TSH response.

Apparent sensitivity of the thyroid gland to TSH decreased with increasing challenge dose level. Considering the ratio of the net area under the response curve for T₄ and TSH as an indicator of sensitivity, i.e., the units of T₄ produced per unit of TSH, the respective average ratios were 1, 100, 130 and 150 for the increasing challenge dose levels. This suggests that the lowest level of TSH was the most effective in stimulating T₄ production, and this is consistent with a tightly controlled and sensitive metabolic regulating system.

The effects of SYN and RSB treatments averaged across dose level on the T₄ response curve indicators are summarized in Table 2. Across SYN treatments, T₄ baseline concentration and concentration of peak T₄ were lower (P < 0.01) for RSB-supplemented steers than for steers fed soybean meal following hormonal challenge, and area under the T₄ response curve tended to be lower (P < 0.06) for the RSB-supplemented steers. This general effect of RSB on T₄ is consistent with the effect of RSB on TSH. Across RSB treatments, area under the T₄ response curve was greater (P < 0.04) for the SYN-implanted steers compared with those not implanted. As with TSH, the SYN × RSB interaction was significant for baseline T₄ concentration (P < 0.03) and peak T₄ concentration (P < 0.05), primarily as a result of the greater concentrations for the +SYNRSB steers compared with the other treatment groups. As with TSH, the T₄ results indicate that SYN increases the responsiveness of TSH plasma concentrations to TRH+GHRH challenge and that feeding RSB in place of soybean meal reduced this responsiveness, particularly in SYN-treated steers. In general, these results reflect the changes in circulating concentrations of TSH.

Increased plasma concentrations of T₄ have been reported in previous studies with beef steers treated with SYN (Kahl et al. 1978), although this effect of SYN has not been found to be consistent. Decreased 5-deiodinase activity increases circulating concentrations of T₄ (Escobar del Rey et al. 1962, Rumsey et al. 1985a). Although SYN has been shown to decrease deiodinase in beef steers in vivo, this did not occur when beef liver tissue was treated with SYN in vitro (Rumsey et al. 1985b). This suggested an indirect effect of SYN on circulating concentrations of T₄ that could be explained by the stimulatory effect of SYN on TSH observed in the current study.

The effects of SYN and RSB treatments averaged across dose level on the T₃ response curve indicators are summarized in Table 2. Across SYN treatments, both baseline (P < 0.01) and peak (P < 0.03) T₃ concentrations were lower for the RSB-supplemented steers than for the steers supplemented with soybean meal. There were no other significant treatment effects found for T₃ concentration. Unlike the effect of treatments on TSH and T₄, the +SYNRSB treatment did not result in the highest baseline or peak concentration and net area was not affected by treatment. Thus, in general, the results suggest that feeding RSB compared with soybean meal has a sustained long-term effect of reducing plasma T₃ concentration but does not measurably affect the sensitivity of T₃ plasma concentrations to a TRH+GHRH challenge. These results are reasonable because T₃ is the third step in the cascade from the effects of TRH, the result of metabolism of T₄ via deiodinase, and under feedback regulation.

The long-term effects of RSB on circulating T₃ are consistent with the general reduction in concentrations of TSH and T₄ in RSB-fed steers and possibly reflective of a shift in thyroid status because of the TSH and T₄ changes. However, decreased basal plasma concentration of T₃ could also suggest decreased 5-deiodination of T₄ in extrathyroidal tissues. In the current study, we evaluated 5-deiodinase activities in some extrathyroidal tissues. Although different in type, 5-deiodinase activities tended to be decreased both in liver (type I) and pituitary gland (type II) in RSB-supplemented steers compared with soybean meal-supplemented steers at time of killing. Activity of type II 5-deiodinase, which is responsible for the generation of T₃ involved mainly in the local regulation of GH and TSH synthesis (Nunez 1988), tended to decrease in RSB-supplemented steers regardless of SYN implantation (main effect: 0.79 and 0.62 pmol I·h¹·mg protein¹ for RSB and +RSB, respectively; SEM 0.07, P < 0.10). Activity of type I 5-deiodinase in liver, which is responsible for most of the circulating T₃ was affected by RSB × SYN interaction (P < 0.05); RSB supplementation decreased activity in nonimplanted steers (4.35 vs. 3.19 nmol I·h¹·mg protein¹; SEM 0.41; P = 0.07) but was without effect in SYN-implanted animals. This is also consistent with the differences in T₃ concentration seen in this study. Although this reduced deiodinase activity is in contrast to in vitro studies of Norton et al. (1987 and 1988), Kahl et al. (1993 and 1994) and Su and Jones (1993), Romo (1995) recently reported a depression in vitro 5-monodeiodinase activity in the liver of 6-mo-old calves by linoleic acid, which is a predominant fatty acid in whole soybeans. More research is needed in this area. Norton et al. (1987) reported that fatty acid effects on 5-monodeiodinase were concentration dependant, and the influence of saturation by the ruminal environment needs to be considered.

In conclusion, this study evaluated the effect of two current practices used in beef production (feeding RSB and the use of estrogenic ear implants) on the thyroid axis of young growing beef steers. The use of the estrogenic growth-promoter Synovex-S increased the release of TSH and the subsequent release of T₄. Feeding RSB reversed this effect. Thus the substrates necessary for enhancing the thyroid status of beef steers were increased by estrogen and decreased by feeding a RSB supplement. If this enhancement is needed for improved growth, then the results explain why growth was depressed in the RSB-supplemented steers and enhanced in steers given an estrogen ear implant in our recent study (Rumsey et al. 1996).