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Department of Animal and Range Sciences, New Mexico State University, Las Cruces 88003
2 Correspondence:
Box 30003, Dept. 3I (phone: 505-646-1004; fax: 505-646-5441; E-mail:
dhallfor{at}nmsu.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Anestrus • Propylthiouracil • Reproduction • Sheep • Thyroid Gland
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Introduction |
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Materials and Methods |
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Animals were handled in accordance with accepted guidelines (FASS, 1999) and procedures were approved by the Institutional Animal Care and Use Committee. During both experiments, sheep were maintained in a single pen (4 x 12 m) at ambient temperature and had ad libitum access to water, shade, and salt. Ewes received 1.8 kg of alfalfa hay •animal-1•d-1 (17% CP) for the duration of both experiments. All ewes were cycling at the initiation of each experiment. The antithyroidal compound PTU (Sigma Chemical Co., St. Louis, MO) was used in both trials. The appropriate amount of PTU for each ewe was weighed and placed in gelatin capsules (Torpac, Fairfield, NJ, size 12 EL). Three days’ worth of treatments were weighed and placed in amber vials to prevent photodegradation of PTU. Propylthiouracil was delivered by gavage. Doses of PTU were selected as a result of previous data from our laboratory showing that 5 (Al-Tamimi et al., 1998), 7.5 (Hernandez et al., 1999), and 12 mg (Bollinger et al., 2000) of PTU/kg BW were not sufficient to lower thyroxine concentrations below the 20 ng/mL nadir suggested by Dahl et al. (1995).
Experiment 1. Fourteen cycling Rambouillet ewes (average BW = 78.2 ± 4.5 kg) were stratified by BW and randomly assigned to one of three treatments beginning January 17. Mid-January was selected for initiating treatments because results from a previous experiment (D. M. Hallford, unpublished data) indicated that the majority of mature ewes in this flock became anestrous during the first 2 wk of February. Ewe BW were recorded on d -1 (day before initiation of treatment) and at 2-wk intervals until the end of the experiment. All ewes received a gelatin capsule by gavage for a 35-d treatment period beginning on January 17 (d 0). A control group (n = 4) received blank gelatin capsules, whereas the second group received 20 mg of PTU/kg BW (n = 5) and the third group (n = 5) received 40 mg of PTU/kg BW throughout the treatment period.
Blood samples were collected (jugular venipuncture) from each animal twice weekly for 2 mo before treatments began. Serum progesterone was quantified in these samples to ensure all ewes were cyclic on d 0 (first day of treatment). Beginning on d 0, blood was collected daily for the 35-d treatment period and for 14 d after termination of treatment to measure serum thyroxine concentrations in response to the PTU. Additionally, blood was collected twice weekly to measure serum progesterone until the time when all ewes were anestrous. Blood samples were taken before feeding at each collection. Blood was collected into sterile vacuum tubes (Corvac, Kendall Health Care, St. Louis, MO), allowed to clot at room temperature for 30 min, and then serum was separated by centrifugation at 1,500 x g for 15 min at 4°C. Serum was decanted into plastic vials and stored at -20°C until analyzed.
Experiment 2. Fifteen cycling Rambouillet ewes (average BW = 88.7 ± 3.1 kg) were stratified by BW and randomly assigned to one of three treatments (five ewes/treatment) in a completely random design. Treatments were administered beginning 2 wk earlier (January 2) than the previous experiment because three of four control ewes in Exp. 1 became anestrous during the first 2 wk of the treatment period. Ewe BW were measured on d -1 and twice weekly throughout the experiment. A control group received a blank gelatin capsule for the 35-d treatment period. The second group received 20 mg of PTU/kg BW for 14 d and 10 mg of PTU for the remainder of the treatment period. The third group was dosed with 40 mg of PTU/kg BW for 14 d and 20 mg of PTU/kgBW for the remaining 21 d. The dose of PTU was decreased by approximately 50% after 14 d to reduce effects on BW loss resulting from the highest dose in Exp.1. Blood was collected and handled as described for Exp. 1.
Hormone Analyses
Serum thyroxine was quantified by solid-phase RIA (Richards et al., 1999) with components of a commercial kit from Diagnostic Products Corp. (Los Angeles, CA). The average within-assay CV was 8.2% with a between-assay CV of 6.7% for Exp. 1. Average recovery of added thyroxine for Exp. 1 was 100%. For Exp. 2, the within- and between-assay CV for thyroxine were 5.1 and 10%, respectively, with an average recovery of 98%. Progesterone concentrations were determined using components of a commercial solid-phase RIA kit (Diagnostic Products Corp.) as validated for ruminant serum by Schneider and Hallford (1996). Within- and between-assay CV were 9.9 and 12.4%, respectively, for Exp. 1. Average recovery for Exp. 1 was 86%. Within- and between-assay CV were 7.6 and 9.2%, respectively, and average recovery was 106% for Exp. 2. Anestrus was defined as the day that serum progesterone decreased below and remained less than 1 ng/mL.
Statistical Analyses
In both trials, effects of PTU on thyroxine concentrations and BW were evaluated by split-plot ANOVA for repeated measures (Gill and Hafs, 1971). The models included treatment in the mainplot, which was tested using animal within treatment as the error term. Day and the day x treatment interaction were included in the subplot and were tested using the residual mean square. When a significant day x treatment interaction was detected, treatment effects were examined within day. Date of anestrus was subjected to ANOVA for a completely random design. Means were separated using linear and quadratic contrasts. The percentage of ewes becoming anestrous during and after the treatment period was analyzed by 
2 procedures. All analyses were computed using the GLM or FREQ procedures of SAS (SAS Inst., Inc., Cary, NC).
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Results |
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Serum Thyroxine.
Split-plot ANOVA revealed a treatment x day interaction (P < 0.01) during the 35-d treatment period; therefore, PTU effects on serum thyroxine were examined within day. Serum thyroxine values determined in these samples are presented in Figure 1
. Before administration of PTU on d 0, all ewes had serum thyroxine concentrations of 86.5 ± 8.0 ng/mL. The thyroxine values among the three treatment groups remained similar (P > 0.14) during the first 10 d of PTU treatment. On d 11, serum thyroxine was 90.2 ± 14.0 ng/mL in ewes receiving blank gelatin capsules compared with 75.2 and 44.2 ± 14.0 ng/mL in those receiving 20 and 40 mg PTU/kg BW, respectively (linear effect, P = 0.04). This decrease in serum thyroxine continued throughout the treatment period; and on d 18, values were 83.5, 55.3 and 18.8 (± 13.6) ng/mL in the three respective groups (linear effect, P < 0.01). On d 35 (last day of treatment), serum thyroxine was 65.1, 8.6, and 2.1 (± 4.8) ng/mL in animals receiving 0, 20, and 40 mg PTU, respectively (quadratic effect, P < 0.05). During the recovery period (d 36 through d 49), split-plot analysis of serum thyroxine detected a treatment x day interaction (P < 0.01) necessitating examination of PTU effects within each day (Figure 1). Within 5 d after PTU treatment ended (d 40), both groups receiving PTU had serum thyroxine values above 20 ng/mL. On d 43, the group receiving 20 mg PTU had serum thyroxine values that had rebounded above the controls (quadratic effect, P < 0.05). This trend was observed until d 66. By the end of this 14-d recovery period, serum thyroxine was similar (P = 0.53) among groups with means of 71.4, 73.3, and 57.5 (± 11.8) ng/mL in the three respective groups. This period was designated as the post-recovery period, which began on d 52 and ended on d 84 (Figure 1). On d 70, the group receiving 20 mg of PTU had serum thyroxine concentrations that were similar to the controls, (69.2 and 68.5 ± 6.6 ng/mL, respectively), whereas those receiving 40 mg of PTU had 49.2 ± 6.6 ng/mL. This trend remained consistent among the treatment groups until the end of the experiment on d 84.
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Ovarian Cyclicity. On d 16 ± 12 after initiation of treatment (d 0), controls were anestrus, whereas ewes receiving 20 mg of PTU stopped cycling on d 40 ± 12 and those treated with 40 mg became acyclic on d 81 ± 12 (linear effect, P < 0.01). On d 35 (the last day of the treatment period), 25% of controls were cycling compared with 60 and 100% of ewes receiving 20 and 40 mg of PTU/kg BW (P = 0.06). On d 49 (last day of the recovery period), all controls had become acyclic compared with 40 and 100% of ewes treated with 20 and 40 mg of PTU that continued to cycle (P < 0.01). On d 100, the only group continuing to exhibit cyclic ovarian activity was those receiving 40 mg of PTU (40%, P = 0.12).
Experiment 2
Serum Thyroxine.
A treatment x day interaction (P < 0.01) necessitated examination of PTU effects on serum thyroxine within day. Before administering PTU on d 0, serum thyroxine concentrations were 54.2 ± 3.3 ng/mL in all ewes (Figure 2). Thyroxine concentrations remained similar among the three groups until d 3, when serum thyroxine was 58.8 ± 3.3 ng/mL for controls compared with 38.3 and 43.4 (± 3.3) ng/mL for the groups receiving 20 and 40 mg of PTU, respectively (quadratic effect, P < 0.02). Both PTU-treated groups had serum thyroxine concentrations below 20 ng/mL by d 9 after initiation of the treatment period. On the first day that the PTU dose was decreased by half, serum thyroxine concentrations were 55.2, 6.6, and 1.9 (± 4.0) ng/mL for the three respective treatment groups. Serum thyroxine continued to decrease throughout the treatment period; and on d 35 (last day of the treatment period), values were 57.6 ng/mL for controls and 0.1 and 0.1 (± 2.7) ng/mL for the groups receiving 10 and 20 mg of PTU, respectively (quadratic effect, P = 0.02).
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. On d 36, serum thyroxine values were 54.6, 0.3, and 1.6 (± 3.3) ng/mL for the controls and those receiving 20/10 and 40/20 mg of PTU, respectively. Four days after termination of PTU (d 39), both treated groups had serum thyroxine concentrations above 20 ng/mL. However, thyroxine values continued to differ throughout the 14-d recovery period such that on d 48, serum thyroxine concentrations were 58.1, 34.0, and 32.1 (± 3.6) ng/mL for the three respective treatment groups (quadratic effect, P = 0.03).
Blood samples were also collected twice weekly until all ewes entered anestrus (Figure 2). Split-plot analysis of serum thyroxine during this post-recovery period (d 51 to 107) did not reveal a treatment x day interaction (P = 0.64); therefore, overall effects of treatment on serum thyroxine were examined. The overall serum thyroxine concentrations during the post recovery period were 62.8 ng/mL for controls and 49.1 and 47.1 (± 4.5) ng/mL for the groups receiving 20/10 and 40/20 mg of PTU, respectively (P = 0.06).
Ewe Body Weights. A treatment x day interaction (P < 0.01) was observed for BW. On the day before initiation of treatment (d -1), all ewes weighed 88.7 ± 3.1 kg. Likewise, BW were similar (P > 0.30) among the three treatment groups on all weigh days. By the end of the 90-d observation period, the controls weighed 93.0 kg compared with 87.6 and 86.7 (± 3.3) kg for ewes receiving 20/10 and 40/20 mg of PTU (P = 0.40).
Ovarian Cyclicity. Serum collected twice weekly was used to quantify progesterone and determine day of anestrus, as described in Exp. 1. On d 27 ± 7.7 after initiation of treatment (d 0), controls were anestrus, whereas ewes receiving 20/10 mg of PTU stopped cycling on d 58 ± 7.7, and those treated with 40/20 mg of PTU became acyclic on d 83 ± 7.7 (linear; P < 0.01). On d 35 (the last day of PTU treatment), 40% of controls were cycling compared with 100% of ewes receiving 20/10 and 40/20 mg of PTU/kg BW (P = 0.02). On d 48, 20% of controls continued to cycle compared with 80 and 100% of ewes treated with 20/10 and 40/20 mg of PTU (P = 0.02), respectively. On d 100, the only group with ewes continuing to exhibit cyclic ovarian activity was the one receiving 40/20 mg of PTU (20%, P = 0.34).
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Discussion |
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Serum thyroxine values observed in the two experiments differed from previous studies conducted in our laboratory. Al-Tamimi et al. (1998) used 5 mg of PTU/kg BW in a preliminary experiment to induce hypothyroidism in pregnant ewes. Propylthiouracil at 5 mg/kg BW required 17 d to decrease serum thyroxine to 20.5 ng/mL; however, this dose was not effective in decreasing thyroxine concentrations below 20 ng/mL by the end of the 20-d treatment period. At this dose, thyroxine values among the treatment groups rebounded to control levels within 6 d after PTU treatment ended. A subsequent study used PTU at 10 mg/kg BW for 5 d and 7.5 mg/kg BW for 15 additional days in gestating ewes (Hernandez et al., 1999). Serum thyroxine again responded slowly to treatment with PTU, taking 9 d for thyroxine concentrations of the treated groups to begin to differ from control values, and finally reaching a low value of 18 ng/mL on the last day of treatment. Bollinger et al. (2000) again increased the PTU dose to 12 mg of PTU/kg BW for 5 d and 10 mg PTU/kg BW for the last 15 d of the experiment. The results from this experiment were consistent with the responses in the previous two studies. In all of the studies mentioned, ewes recovered from the hypothyroid state in a fashion similar to that described by Al-Tamimi et al. (1998). A similar decline in serum thyroxine was observed in Brahman cows treated with 4 mg PTU/kg BW (DeMoraes et al., 1998; Bernal et al., 1999).
Sheep appear to be less sensitive than cattle to the goitrogenic effect of PTU feeding, as demonstrated by the comparatively large amount of PTU required to decrease serum thyroxine in ewes. Therefore, the two studies described herein used 20 and 40 mg of PTU/kg BW. At an initial dose of 20 and 40 mg of PTU, serum thyroxine began to decrease within 11 d in Exp. 1 and within 3 d in Exp. 2. Additionally, these levels of PTU were successful in decreasing thyroxine values to nearly undetectable levels by the end of the 35-d treatment period. As seen in previous studies (Al-Tamimi et al., 1998; Hernandez et al., 1999; Bollinger et al., 2000), thyroxine concentrations recovered quickly after the termination of PTU treatment. Decreasing the PTU dose by half after 14 d in Exp. 2 allowed serum thyroxine to return to control values by d 51. These data demonstrate that PTU at 20 mg/kg BW is effective in lowering serum thyroxine below 20 ng/mL, but approximately 30 d of treatment are required to elicit this effect. However, 40 mg of PTU/kg BW resulted in serum thyroxine concentrations less than 20 ng/mL in about 18 d. Thus, the 40-mg treatment produced more desirable effects of thyroxine reduction in terms of the 20-ng/mL level suggested by Dahl et al. (1995) as being necessary to influence LH pulsatility in ovarectomized, estradiol-implanted ewes. In addition, decreasing the dose by half after 14 d of treatment maintained the lower serum thyroxine values. Likewise, both treatments in the present studies induced a much more dramatic lowering of serum thyroxine than that observed by Bollinger et al. (2000), who used 12 mg PTU/kg BW. These data also demonstrate that ewes recover quickly in terms of serum thyroxine even after exposure to high doses of PTU.
Ewe Body Weights
Throughout both experiments, BW were recorded as indicators of animal health and feed intake in response to the chemically induced hypothyroidism. The similarity in BW on d -1 was consistent between the two trials. However, ewes treated with 40 mg of PTU for the entire 35-d treatment period (Exp. 1) lost an average of 6 kg by d 84. This BW loss supports our observation that 40 mg of PTU seemed to affect feed intake. Because the thyroxine concentrations were very low in both PTU-treated groups after 30 d of treatment, we hypothesized that the effect on feed intake may have resulted more from a "toxic" effect of 40 mg than the hypothyroid state. To avoid the apparent "toxic" effect caused by the high dose of PTU, the second experiment was conducted. During the 90-d observation period in the second experiment, ewe BW remained similar on all weigh days. No weight loss was observed in any of the treatment groups. This finding suggests that decreasing the dose of PTU after 14 d allows for the desired decline in serum thyroxine concentrations without adversely affecting ewe health.
Ovarian Cyclicity
The average day that each treatment group entered anestrus was remarkably consistent in the two experiments. A linear increase in the length of cyclicity was observed with increasing amounts of PTU. These data suggest that treating cycling ewes with either 20 or 40 mg of PTU/kg BW, as well as decreasing this amount after 14 d, was effective in extending the period of cyclicity. These data agree with those of Dahl et al. (1995) and Karsch et al. (1995), who suggested that thyroidectomy of ewes during autumn inhibited the onset of anestrus during the subsequent spring. Likewise, O’Callaghan et al. (1993) treated anestrous ewes with thyroxine and extended the anestrous season. However, our data suggest that lowering serum thyroxine with PTU near the end of a normal breeding season can lengthen the period of cyclicity without major adverse effects on the animals. Follet and Potts (1990) showed similar results in Welsh Mountain ewes using the antithyroidal compound methiouracil in combination with controlled day length.
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Implications |
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Footnotes |
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Received for publication June 15, 2002. Accepted for publication September 18, 2002.
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Literature Cited |
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and propylthiouracil during gestation. Pages 226–229 in Proc. Western Sec., Am. Soc. Anim. Sci., Provo, UT.
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-), 20 (-
-), or 40 (-•-) mg of propylthiouracil (PTU)/kg BW. A linear (P < 0.05) decrease was observed beginning on d 11 and continuing through the end of the recovery period with increasing PTU dosage. Serum thyroxine in the recovery period remained different among the treatment groups (P < 0.05) between d 36 and 48. After d 48, controls and ewes receiving 20 mg of PTU/kg BW had generally similar thyroxine values, whereas the group receiving 40 mg of PTU had consistently lower values. By d 84, serum thyroxine was similar (P = 0.19) among the three treatment groups.