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Serum thyroid hormones and reproductive characteristics of Rambouillet ewe lambs treated with propylthiouracil before puberty
Theriogenology 59 (2003) 1403±1413
Serum thyroid hormones and reproductive characteristics of Rambouillet ewe lambs treated with propylthiouracil before puberty N.H. Wells, D.M. Hallford*, J.A. Hernandez Department of Animal and Range Sciences, New Mexico State University, Box 30003, MSC 3I, Las Cruces, NM 88003, USA Received 13 December 2001; accepted 9 July 2002
Abstract Twenty-four Rambouillet ewe lambs (average weight 43:7 1:2 kg, approximately 6 months of age) were used to examine the effect of thyroid suppression before the onset of puberty on serum thyroid hormones, body weights (BW), and reproductive performance. Beginning in early September, ewe lambs were randomly assigned to three treatments (n 8 lambs/treatment). All animals remained in a single pen (4 12 m) with access to salt, water, shade and alfalfa hay (2.5 kg per animal per day) throughout the experiment. Beginning on Day 0 (®rst day of treatment), all ewe lambs received daily treatments (gavage) for 15 days consisting of 0, 20, or 40 mg 6-N-propyl-2thiouracil (PTU)/kg BW per day. Beginning on Day 15, the 20 and 40 mg treatments were lowered to 10 and 20 mg PTU/kg BW, respectively. All animals were treated for 28 days. Ovarian cyclicity was determined by twice weekly progesterone (P4) analysis. Thyroxine (T4) concentrations were similar on Day 0 (61.6, 54.8 and 56:9 2:5 ng/ml, P 0:17) in ewe lambs receiving 0, 20 and 40 mg PTU/kg BW, respectively. By Day 7, both PTU-treated groups had T4 values less than 20 ng/ml (9.0 and 15:4 2:5 ng/ml) compared with 78.5 ng/ml in controls (P < 0:01). By 7 days after termination of PTU treatment, serum T4 had risen to 29.1 and 26.9 (2.9) ng/ml in the 20/10 and 40/20 PTU groups, respectively. On Day 66, control ewes had 55.0 ng T4/ml compared with 43.1 and 39.0 (2.6 ng/ml) for ewes in the 20/10 and 40/20 groups, respectively (linear, P < 0:01). Serum triiodothyronine (T3) followed a similar pattern to that observed for T4. Ewe lamb BW were similar (P > 0:50) among groups throughout the treatment period. However, following the treatment, PTU-treated ewes tended (P < 0:10) to weigh less than controls. Average Julian day of puberty was also similar (P > 0:50) among treatments (286, 288 and 288 5 days; control, 20/10 and 40/20, respectively). Control ewes had a pregnancy rate of 75%, while both PTU-treated groups had pregnancy rates of 88% (P > 0:20). The administration of PTU resulted in a rapid decline in * Corresponding author. Tel.: 1-505-646-1004; fax: 1-505-646-5441. E-mail address: [email protected] (D.M. Hallford).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 8 4 - 6
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serum T4 and T3 but neither time of puberty nor pregnancy rates were affected by lowered thyroid hormones. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Sheep; Reproduction; Puberty; Thyroxine; Triiodothyronine
1. Introduction The practice of breeding spring born ewe lambs is a management strategy that sheep producers can utilize to increase total production from a ewe ¯ock. Hulet et al. [1] showed that better body condition and heavier weaning weights occurred in ewe lambs exhibiting estrus in the fall of their ®rst year. The exposure to long days of spring and summer, followed by short days in autumn induces photoperiodic cues to stimulate the ewe lamb's ®rst estrus [2±4]. The reproductive cycle in sheep is such that a fall breeding season occurs followed by a season of anestrus. During anestrus, the pulsatile secretion of LH diminishes because of a change in the potency of the estradiol negative feedback on LH [5]. In mature ewes, thyroxine (T4) has been suggested to play a role in seasonal reproduction [6]. Dahl et al. [7] prevented the negative feedback of estradiol by removing the thyroid gland before cessation of the breeding season. This allowed the period of cyclicity to continue longer than normal. Administration of T4 to thyroidectomized ewes caused decreases in LH and the eventual end of cyclicity [7]. Studies by Hernandez et al. [8] and Bollinger et al. [9] examined effects of the thyroid inhibitor propylthiouracil (PTU) on pregnant ewes. In both experiments, oral administration of PTU was effective in lowering circulating T4 concentrations. Recent work [10] demonstrated that large doses of PTU were effective in lowering T4 and preventing the onset of anestrus. Currently no data are available that demonstrate the effect of antithyroidal compounds on puberty. Therefore, the objectives of this study were to examine the effect of thyroid inhibition before the onset of puberty on serum T4, body weight (BW), reproductive performance and offspring performance in Rambouillet ewe lambs. 2. Materials and methods 2.1. Animals Twenty-four spring born Rambouillet ewe lambs (average BW 43:7 1:2 kg, approximately 6 months of age) were used in this experiment. All procedures and facilities described in the following sections were approved by the New Mexico State University Animal Care and Use Committee. Animals were vaccinated at approximately 30 and 60 days of age against enterotoxemia and tetanus. Ewe lambs were weaned at 60 days of age and maintained in a single pen (4 12 m) under ambient conditions (early September to June). All animals were given free access to water, shade and salt throughout the trial. Ewe lambs consumed alfalfa hay (2.5 kg per animal per day; 16% crude protein) and cracked corn (0.22 kg per animal per day). Weights were recorded regularly throughout the trial.
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2.2. Treatments On Day 2 (9 September), ewe lambs were strati®ed by BW and randomly assigned to one of three treatments (n 8 lambs/treatment) in a completely random design. Daily treatments (administered by gavage) beginning on Day 0 (®rst day of treatment) were control (0 mg 6-N-propyl-2-thiouracil (PTU)/kg BW per day), low (20 mg PTU), or high (40 mg PTU) PTU. This dose was administered until Day 14. On Day 15 of the treatment period, the low and high treatments were decreased to 10 and 20 mg PTU/kg BW per day, respectively. The PTU (Sigma Chemical Co., Ltd., St. Louis, MO, USA) was prepared in gelatin capsules (size 12 EL, Torpac Inc., Fair®eld, NJ, USA). Ewe lambs in the control group received blank gelatin capsules. Dahl et al. [7] indicated that administering T4 to mature ovariectomized, thyroidectomized, estradiol-implanted ewes to elevate T4 to nadir levels (approximately 20 ng/ml) was effective in promoting the cessation of LH pulsatility (i.e. initiation of anestrus). Therefore, we attempted to lower serum T4 below 20 ng/ml in ewe lambs to determine PTU effects on puberty. Previous studies conducted in our laboratory using mature pregnant ewes [8,9] demonstrated that doses of 12 mg PTU or less did not decrease serum T4 below 20 ng/ml. However, Hernandez et al. [10] showed that larger doses of PTU (>20 mg) were effective in reducing serum T4 below 20 ng/ml. The larger doses were chosen in an attempt to dramatically reduce serum T4. However, Hernandez et al. [10] also suggested that 40 mg PTU/kg BW may in¯uence feed intake; thus, the PTU doses were decreased by half after 14 days. After the treatment period ended (Day 28), ewe lambs were joined with raddled mature Rambouillet rams for a 34-day breeding season. Breeding dates were recorded twice daily. Weights were recorded on Days 7, 14, 21, 28, 35, 49, 63, 91, 119 and 149. Following a 34day breeding season, all ewes were again maintained in a single pen and managed as described previously until lambing at which time pregnancy percentage, number of lambs born and lamb birth weights were recorded. 2.3. Blood collection Initial blood samples were collected on Day 2 by jugular venipuncture. Daily blood samples were collected from Days 0 through 35 and twice weekly thereafter until the end of the breeding season. Blood samples were collected before feeding into sterile vacuum tubes (Corvac 7, Kendall Health Care, St. Louis, MO, USA). Samples were allowed to clot at room temperature for approximately 30 min before centrifugation at 1500 g for 15 min at 4 8C to separate serum. Following centrifugation, serum was transferred to plastic vials and stored at 20 8C until hormone analysis. 2.4. Hormone analyses Serum T4, triiodothyronine (T3) and progesterone (P4) were quanti®ed by radioimmunoassay using components of commercial kits supplied by Diagnostic Products Corp. (DPC, Los Angeles, CA, USA). Modi®cations to the T4 assay were reported previously [11]. The antibody used in the T4 assay had a 2% crossreactivity with T3 and did not recognize PTU. The P4 assay was modi®ed as described by Schneider and Hallford [12]. The within assay
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coef®cients of variation (CV) were 9.2 and 7.5% for the P4 and T4 assays, respectively, while the between assay CV for P4 and T4 were 8.1 and 13.3%, respectively. Date of puberty was determined by measuring P4 in samples collected twice weekly. Puberty was de®ned as the day that P4 rose above 1 ng/ml and stayed above for two consecutive blood samples. The T3 assay utilized polypropylene tubes coated with an antibody against T3 along with a solution that contained both 125 I-T3 and a substance to interrupt binding between T3 and its transport proteins (tracer solution). A standard solution was prepared by suspending T3 (Sigma T 2877) at 10 ng/ml in 0.01 M phosphate buffered saline containing 1% bovine serum albumin (assay buffer). This stock standard solution was pipetted into the antibody-coated tubes in amounts to provide a standard curve of 0, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 ng T3 per tube. Standard tubes were then normalized to 0.5 ml using assay buffer. Duplicate antibody-coated tubes received 0.4 ml of assay buffer and 0.1 ml of serum from each ewe sample. Each tube then received 1 ml of the tracer solution, after which tubes were vortexed and incubated at room temperature for 3±4 h. Tubes were then decanted and counted for 1 min in a gamma counter. The cross reactivity of the anti-T3 with T4 and PTU was 0.50 and 0.003%, respectively, and the assay sensitivity was 0.07 ng/ml. When 0.1 ng T3 was added to the assay, 28% of the tracer was displaced from the antibody. The within and between assay CV were 9.3 and 12.6%, respectively, and the recovery of an added 5 ng T3/ml was 107%. 3. Statistical analysis Serum T4 and T3 pro®les were analyzed by split-plot analysis of variance for repeated measures [13]. Treatment effects were included in the main plot, and day and treatment by day interactions were included in the subplot. Animal within treatment was used as the error term to examine PTU effects and the residual error was used to test subplot factors. Ewe lamb weights and data for average Julian day of puberty and time from Day 0 to puberty were analyzed by analysis of variance for a completely random design [14]. One ewe lamb in the control group did not achieve puberty during the trial, so she was assigned the maximum Julian date (322) in order to prevent misinterpretation of data. Lamb birth weights, weaning weights, preweaning average daily gains and adjusted weaning weights were also examined by analysis of variance for a completely randomized design. Adjusted weaning weights were calculated as described by Scott [15]. Values for serum T4, ewe BW, lamb birth weights, weaning weights and preweaning average daily gain were separated by linear and quadratic contrasts. Differences among means were considered signi®cant if a probability level less than 0.10 was detected. Treatment effects on pregnancy rates and lamb crop percentages were determined by Chi-square analysis. All analyses were computed using the general linear models and frequency procedures of SAS [16]. 4. Results 4.1. Serum thyroid hormones A PTU by day interaction (P < 0:01) was detected during the 28-day treatment period; therefore, PTU effects on T4 were analyzed within day and results are presented in Fig. 1.
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Fig. 1. Serum thyroxine (T4) in ewe lambs during the treatment period (Days 0 through 28 (top panel) and recovery period (Days 29 through 66). Treatments were 0 (^), 20 (&), and 40 (*) mg of propylthiouracil (PTU) from Days 0 through 14. From Days 15 through 28, PTU dosages were reduced by half.
Serum T4 was similar in ewe lambs receiving PTU compared with values observed in controls (those receiving 0 mg PTU) on the ®rst day of treatment (Day 0; 61.6, 54.8, 56:9 2:5 ng/ml for 0, 20 and 40 mg PTU/kg BW, respectively, P 0:17). However, after 1 day of PTU treatment, serum T4 was reduced (linear, P 0:02) in ewes receiving 20 and 40 mg PTU/kg BW compared with controls. On Day 7, T4 values were much lower in the PTU-treated ewes than in controls (78.5, 9.0 and 15:4 2:5 ng/ml for those receiving 0, 20 and 40 mg PTU/kg BW, respectively; quadratic, P < 0:01). After 2 weeks (Day 14) of PTU administration, ewe lambs receiving PTU had T4 concentrations less than or equal to 1 ng/ml (72.8, 1.0 and 0:4 2:7 ng/ml in 0, 20 and 40 mg PTU/kg BW, respectively; quadratic, P < 0:01).
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On Day 15, the PTU dose for ewe lambs receiving 20 mg PTU was lowered to 10 mg PTU/kg BW, and those receiving 40 mg PTU were lowered to 20 mg PTU/kg BW, while controls remained at the 0 mg PTU/kg BW dose. By Day 22 (7 days after the change in PTU doses), serum T4 was 77.1, 0.2 and 0.3 (2.1) ng/ml in ewes receiving 0, 10 and 20 mg PTU/kg BW, respectively (quadratic, P < 0:01). The lowered doses were suf®cient to hold serum T4 below 20 ng/ml for the remainder of the treatment period (Day 28) as evidenced by serum T4 values less than 1 ng/ml in both PTU-treated groups (80.2, 0.2 and 0:2 2:0 ng/ml in ewes receiving 0, 10 and 20 mg PTU/kg BW, respectively; quadratic, P < 0:01). Daily blood samples were collected for 7 days following termination of PTU treatment (Fig. 1); and although PTU-treated ewe lambs had increasing T4 concentrations, values remained lower than in controls (58.0, 29.1 and 26:9 2:4 ng/ml on Day 35 in ewe lambs treated with 0, 20/10 and 40/20 mg PTU/kg BW, respectively; quadratic, P < 0:01). A twice weekly blood sampling protocol was followed from Days 38 through 66 (Fig. 1). By Day 52 (approximately 3 weeks after PTU treatment ended), the PTU-treated ewe lambs had 41.9 and 36:6 2:6 ng T4/ml (20/10 and 40/20, respectively) compared with 60.9 ng/ml in controls (quadratic, P < 0:05). On Day 66 (last sampling date), the PTUtreated ewe lambs had not completely recovered from the effects of hypothyroidism (55.0, 43.1 and 39:0 2:6 ng/ml for 0, 20/10 and 40/20 mg PTU/kg BW, respectively; linear, P < 0:01). These data indicate that PTU is a very effective means of inducing hypothyroidism in ewe lambs.
Fig. 2. Serum triiodothyronine (T3) in ewe lambs during the treatment period (Days 0 through 28) and recovery period (Days 30 through 34). Treatments were 0 (^), 20 (&), and 40 (*) mg of propylthiouracil (PTU) from Days 0 through 14. From Days 15 through 28, PTU dosages were reduced by half.
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Serum T3 was also quanti®ed and values are presented in Fig. 2. As with T4, a treatment by day interaction was detected (P < 0:01) for T3, necessitating examination of treatment effects within day. Serum T3 followed the same general trend in response to PTU as observed for T4. By 2 days after initiation of PTU treatment, serum T3 was less (P < 0:01) in the two PTU groups than in controls. This difference remained consistent through the treatment period such that on Day 28, serum T3 was 4.11 ng/ml in controls compared with 0.82 and 0.72 ng/ml for those receiving 20/10 and 40/20 mg PTU, respectively (quadratic, P < 0:01). 4.2. Ewe lamb weights Ewe lamb BW are reported in Table 1. Before initiation of treatment (Day 2), all ewe lambs weighed approximately 43 kg (P > 0:20). Weights on Days 14 through 35 were also similar (P > 0:20) among treatments. However, by 3 weeks following the end of the treatment period (Day 49) and again from Days 91 through 146, PTU-treated ewe lambs tended to weigh less (linear, P < 0:10) than did controls. 4.3. Reproductive performance Administration of PTU at doses used in this experiment did not in¯uence reproductive performance. In the present study, reproductive characteristics were similar among treatments (Table 2). Average Julian day of puberty was 286 5:4 in controls and 288 5:4 in both PTU-treated groups (P > 0:05). Time from Day 0 to puberty was Table 1 Weights (kg) of Rambouillet ewe lambs treated daily with propylthiouracil (PTU) for 28 days before beginning a fall breeding season Daya
2d 0d 7d 14d 21d 28d 35d 49e 63d 91e 119e 146e a
PTU (mg/kg BW)b 0
20/10
40/20
S.E.c
43.9 44.2 44.7 45.8 47.4 48.6 49.4 51.2 52.3 57.7 62.8 61.5
43.4 43.7 44.5 46.4 49.3 49.6 48.4 48.8 50.1 53.5 58.1 56.9
43.7 44.6 44.2 46.5 49.1 49.2 48.4 48.0 50.0 54.3 58.8 57.9
1.2 1.2 1.0 1.2 1.2 1.3 1.2 1.0 1.1 1.3 1.4 1.5
Day of trial (Day 0 first day of PTU treatment; 11 September). Ewe lambs received 0, 20, or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10, and 20 mg PTU/kg BW for an additional 14 days. c Standard error based on eight animals per group. d Row values do not differ (P > 0:20). e Row values tend to differ (quadratic, P < 0:10). b
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Table 2 Reproductive responses of Rambouillet ewe lambs treated daily with propylthiouracil (PTU) for 28 days before beginning a fall breeding season PTU (mg/kg BW)a
Reproductive response
Julian day of pubertyc Time from Day 0 to pubertyc,d Pregnancy rate (%)e Lambs born/ewe (n)e
0
20/10
40/20
S.E.b
286 31 75 1.0
288 34 88 0.88
288 35 88 1.12
5.4 5.3
a
Ewe lambs received 0, 20, or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10, and 20 mg PTU/kg BW for an additional 14 days. b Standard error based on eight animals per group. c Date of puberty determined by P4 measured in samples collected twice weekly. Puberty was de®ned as the day that P4 rose above 1 ng/ml and stayed for two consecutive blood samples. Row values do not differ (P > 0:50). d Time from Day 0 (®rst day of treatment) to puberty measured in days. Row values do not differ (P > 0:50). e Row values do not differ (P > 0:20).
31, 34 and 35 5:3 days in ewe lambs receiving 0, 20/10 and 40/20 mg PTU/kg BW, respectively (P > 0:50). Likewise, pregnancy rate and lambs born per ewe were similar (P > 0:20) in both control and PTU-treated ewes. 4.4. Effect of maternal propylthiouracil on offspring Effects of maternal PTU administration on offspring performance (Table 3) were examined in terms of birth weight, actual weaning weight, adjusted weaning weight, and preweaning average daily gain. Birth weights and actual weaning weights were similar (P > 0:90) among groups. Weaning weights adjusted to a 60 day, single ewe lamb, mature ewe basis were also comparable (P > 0:90) among PTU groups (Table 3). Likewise, preweaning gain did not differ (P > 0:90) among treatments. Table 3 Effect of maternal propylthiouracil (PTU) treatment on offspring birth weights, actual weaning weights, adjusted weaning weights, and preweaning average daily gain (ADG). Offspring weights (kg)
Birth weight Actual weaning weight Weaning weight, adjustedc Prewean ADG
Maternal PTU (mg/kg BW)a 0
20/10
40/20
S.E.b
4.7 21.1 28.8 0.30
5.2 19.4 26.0 0.27
4.8 21.3 28.5 0.30
0.4 1.6 1.5 0.02
a Ewe lambs received 0, 20 or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10 and 20 mg PTU/kg BW for an additional 14 days. Row values do not differ (P > 0:90). b Standard error based on 8 animals per group. c Adjusted as described by Scott [15].
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5. Discussion Dahl et al. [7] indicated that administering T4 to mature ovariectomized, thyroidectomized, estradiol-implanted ewes to elevate T4 to nadir levels (approximately 20 ng/ml) was effective in promoting the cessation of LH pulsatility (i.e. initiation of anestrus). In our study, serum T4 and T3 were greatly reduced in ewe lambs receiving PTU compared with controls (P < 0:01) beginning on Day 2 and lasting throughout the sampling period. Levels of serum T3 appeared to recover from the effects of PTU much more quickly than did T4 as evidenced by the values shown in Fig. 2. In fact, 4 and 6 days after PTU administration was terminated, the previously treated ewes had greater serum T3 values (quadratic, P < 0:01) than did controls. The T4 data agree with results reported by Hernandez et al. [10] and suggest that large doses of PTU can lower serum T4 below 20 ng/ml. Ewe lambs appeared to respond more quickly to PTU in terms of T4 reduction than did mature ewes studied by Hernandez et al. [10]. Decreasing the doses to 10 and 20 mg PTU/kg BW on Day 15 was done in an attempt to prevent any adverse health effects because Hernandez et al. [10] noted that 40 mg of PTU may adversely impact animal health in a manner different from hypothyroidism. The administration of PTU may have slightly affected BW after the treatment period as evidenced by decreased weight gain in PTU-treated ewe lambs compared with controls (P < 0:10). Hernandez et al. [10] found that mature ewes treated with 40 mg of PTU tended to lose weight compared with controls. These researchers hypothesized that this effect may have resulted from a `toxicity' caused by the large dosage of PTU resulting in depressed feed intake. Richards et al. [11] observed decreased serum T4, lowered weight gain, and declining feed intake in ram lambs consuming a 20% locoweed diet. The decreased serum T4 concentrations resulting from locoweed toxicosis, along with lowered weights and declining feed intake in the post-locoweed feeding period suggests a relationship between normal thyroid function and appetite. In this study feed intake data were not collected; however, the tendency for lower weights following the PTU treatment period suggests a possible slight toxicity that resulted in depressed appetite. Another aspect for consideration is the observation that along with the trend for decreased weight in hypothyroid ewe lambs, no effect on age at puberty was noted. Because age and weight are considered the major determinants of the attainment of puberty [1], the involvement of the induced hypothyroidism in reaching puberty at a lighter weight warrants further investigation. The drastic reduction in serum T4 and T3 did not hasten puberty or increase conception rates. This ®nding does not support our original hypothesis and does not agree with those reported in mature ewes by other researchers [5±7]. A possible explanation for this different response in ewe lambs and mature ewes may be that mature ewes respond to thyroidectomy in terms of the seasonal response when the surgery is performed during the normal breeding season [6]. In the current study, hypothyroidism was induced during the period immediately before onset of puberty during the ewe lamb's ®rst breeding season, although it could be argued that because our experiment was initiated during September, the onset of puberty may have been confounded with onset of the fall breeding season. In a recent study [17], we examined progesterone pro®les in untreated, nonpregnant ewe lambs for 1 year beginning in September. These females reached puberty on an average of 24 October (S:D: 15 days), became anestrus on an average of 25 December
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(S:D: 26 days), and started estrus activity again by 16 July (S:D: 20 days). These data demonstrate that ®ne wool ewes in southern New Mexico do experience estrus during the summer. However, in an earlier study [18], we monitored progesterone pro®les in ewe lambs beginning at 4 months of age (17 July) and continuing through a fall breeding season. In this study, no cyclic progesterone pro®les were observed until October. Based on these previous studies from our laboratory, we suggest that in the current study, puberty had not been attained before PTU administration began in September. Maternal administration of PTU also did not appear to have an adverse impact on lamb health as suggested by similarities in birth weights, actual and adjusted weaning weights, and preweaning gain (P > 0:90). Although reproduction was not affected by oral PTU administration, these results demonstrate that PTU is an effective means of inducing hypothyroidism in young female sheep. Further studies are needed to examine other possible methods of hastening puberty and improving reproductive ef®ciency in ewe lambs. Acknowledgements Research was supported by the New Mexico Agricultural Experiment Station and is a contribution of Western Regional Project W112. The authors thank R. Rosencrans, M. Jacobsen, B. Jackson, J. Joines, and L. Amezquita for technical and clerical assistance. References [1] Hulet CV, Wiggins EL, Ercanbrack SK. Estrus in range lambs and its relationship to lifetime reproductive performance. J Anim Sci 1969;28:246±52. [2] Foster DL. Mechanism for delay of ®rst ovulation in lambs born in the wrong season (fall). Biol Reprod 1981;25:85±92. [3] Foster DL, Yellon SM, Olster DH. Internal and external determinants of the timing of puberty in the female. J Reprod Fertil 1985;75:327±44. [4] Yeates NTM. The breeding season of the sheep with particular reference to its modi®cation by arti®cial means using light. J Agric Sci 1949;39:1±43. [5] Legan SJ, Karsch FJ. Neuroendocrine regulation of the estrous cycle and seasonal breeding in the ewe. Biol Reprod 1979;20:74±85. [6] Karsch FJ, Dahl GE, Hachigian TM, Thrun LA. Involvement of thyroid hormones in seasonal reproduction. J Reprod Fertil 1995;49(Suppl.):409±27. [7] Dahl GE, Evans NP, Thrun LA, Karsch FJ. Thyroxine is permissive to seasonal transitions in reproductive neuroendocrine activity in the ewe. Biol Reprod 1995;52:690±6. [8] Hernandez JA, Hallford DM, Bollinger JL, Shirley KL, Richards JB. Serum thyroxine, progesterone, and luteinizing hormone and body weight and milk characteristics in ewes treated with PGF2a and propylthiouracil during gestation. Proc West Sec Am Soc Anim Sci 1999;50:226±31. [9] Bollinger JL, Hallford DM, Hernandez JA, Shirley KL. Reproductive, hormonal, and milk characteristics of ewe treated with propylthiouracil and melatonin during late gestation. Proc West Sec Am Soc Anim Sci 2000;51:374±9. [10] Hernandez JA, Hallford DM, Wells NH. Serum thyroxine, body weights, and ovarian cyclicity in ®ne wool ewes subjected to thyroid suppression immediately before onset of anestrus. Proc West Sec Am Soc Anim Sci 2001;52:401±5. [11] Richards JB, Hallford DM, Duff GC. Growth and serum pro®les of ram lambs fed locoweed (Oxytropis sericea) and treated with vitamin E/selenium. Theriogenology 1999;52:1055±66.
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[12] Schneider FA, Hallford DM. Use of a rapid progesterone radioimmunoassay to predict pregnancy and fetal numbers in ewes. Sheep Goat Res J 1996;12:33±8. [13] Gill JL, Hafs HD. Analysis of repeated measurements in animals. J Anim Sci 1971;33:331±6. [14] Steele RG, Torrie JH. Principles and Procedures of Statistics: a biomedical approach. New York: McGrawHill; 1980. [15] Scott GE. The Sheepman's Production Handbook. Denver Co: Sheep Industry Development Program; 1977. [16] SAS. SAS User's Guide: Statistics. SAS Institute Inc, Cary, NC; 1989. [17] Gifford CA, Wells NH, Hernandez JA, Duffey JL, Rosencrans RL, Hallford DM. Serum thyroxine pro®les and environmental conditions during three stages of the reproductive cycle of Rambouillet ewe lambs in southern New Mexico. Proc West Sec Am Soc Anim Sci 2002;53:170±3. [18] Birch MW, Hallford DM, Christensen BL, Schneider FA, Campbell JW. Reproductive performance and serum growth hormone and melatonin in ewe lambs treated with melatonin microspheres and(or) estradiol-17b. Proc West Sec Am Soc Anim Sci 1994;45:279±81.
Serum thyroid hormones and reproductive characteristics of Rambouillet ewe lambs treated with propylthiouracil before puberty N.H. Wells, D.M. Hallford*, J.A. Hernandez Department of Animal and Range Sciences, New Mexico State University, Box 30003, MSC 3I, Las Cruces, NM 88003, USA Received 13 December 2001; accepted 9 July 2002
Abstract Twenty-four Rambouillet ewe lambs (average weight 43:7 1:2 kg, approximately 6 months of age) were used to examine the effect of thyroid suppression before the onset of puberty on serum thyroid hormones, body weights (BW), and reproductive performance. Beginning in early September, ewe lambs were randomly assigned to three treatments (n 8 lambs/treatment). All animals remained in a single pen (4 12 m) with access to salt, water, shade and alfalfa hay (2.5 kg per animal per day) throughout the experiment. Beginning on Day 0 (®rst day of treatment), all ewe lambs received daily treatments (gavage) for 15 days consisting of 0, 20, or 40 mg 6-N-propyl-2thiouracil (PTU)/kg BW per day. Beginning on Day 15, the 20 and 40 mg treatments were lowered to 10 and 20 mg PTU/kg BW, respectively. All animals were treated for 28 days. Ovarian cyclicity was determined by twice weekly progesterone (P4) analysis. Thyroxine (T4) concentrations were similar on Day 0 (61.6, 54.8 and 56:9 2:5 ng/ml, P 0:17) in ewe lambs receiving 0, 20 and 40 mg PTU/kg BW, respectively. By Day 7, both PTU-treated groups had T4 values less than 20 ng/ml (9.0 and 15:4 2:5 ng/ml) compared with 78.5 ng/ml in controls (P < 0:01). By 7 days after termination of PTU treatment, serum T4 had risen to 29.1 and 26.9 (2.9) ng/ml in the 20/10 and 40/20 PTU groups, respectively. On Day 66, control ewes had 55.0 ng T4/ml compared with 43.1 and 39.0 (2.6 ng/ml) for ewes in the 20/10 and 40/20 groups, respectively (linear, P < 0:01). Serum triiodothyronine (T3) followed a similar pattern to that observed for T4. Ewe lamb BW were similar (P > 0:50) among groups throughout the treatment period. However, following the treatment, PTU-treated ewes tended (P < 0:10) to weigh less than controls. Average Julian day of puberty was also similar (P > 0:50) among treatments (286, 288 and 288 5 days; control, 20/10 and 40/20, respectively). Control ewes had a pregnancy rate of 75%, while both PTU-treated groups had pregnancy rates of 88% (P > 0:20). The administration of PTU resulted in a rapid decline in * Corresponding author. Tel.: 1-505-646-1004; fax: 1-505-646-5441. E-mail address: [email protected] (D.M. Hallford).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 8 4 - 6
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serum T4 and T3 but neither time of puberty nor pregnancy rates were affected by lowered thyroid hormones. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Sheep; Reproduction; Puberty; Thyroxine; Triiodothyronine
1. Introduction The practice of breeding spring born ewe lambs is a management strategy that sheep producers can utilize to increase total production from a ewe ¯ock. Hulet et al. [1] showed that better body condition and heavier weaning weights occurred in ewe lambs exhibiting estrus in the fall of their ®rst year. The exposure to long days of spring and summer, followed by short days in autumn induces photoperiodic cues to stimulate the ewe lamb's ®rst estrus [2±4]. The reproductive cycle in sheep is such that a fall breeding season occurs followed by a season of anestrus. During anestrus, the pulsatile secretion of LH diminishes because of a change in the potency of the estradiol negative feedback on LH [5]. In mature ewes, thyroxine (T4) has been suggested to play a role in seasonal reproduction [6]. Dahl et al. [7] prevented the negative feedback of estradiol by removing the thyroid gland before cessation of the breeding season. This allowed the period of cyclicity to continue longer than normal. Administration of T4 to thyroidectomized ewes caused decreases in LH and the eventual end of cyclicity [7]. Studies by Hernandez et al. [8] and Bollinger et al. [9] examined effects of the thyroid inhibitor propylthiouracil (PTU) on pregnant ewes. In both experiments, oral administration of PTU was effective in lowering circulating T4 concentrations. Recent work [10] demonstrated that large doses of PTU were effective in lowering T4 and preventing the onset of anestrus. Currently no data are available that demonstrate the effect of antithyroidal compounds on puberty. Therefore, the objectives of this study were to examine the effect of thyroid inhibition before the onset of puberty on serum T4, body weight (BW), reproductive performance and offspring performance in Rambouillet ewe lambs. 2. Materials and methods 2.1. Animals Twenty-four spring born Rambouillet ewe lambs (average BW 43:7 1:2 kg, approximately 6 months of age) were used in this experiment. All procedures and facilities described in the following sections were approved by the New Mexico State University Animal Care and Use Committee. Animals were vaccinated at approximately 30 and 60 days of age against enterotoxemia and tetanus. Ewe lambs were weaned at 60 days of age and maintained in a single pen (4 12 m) under ambient conditions (early September to June). All animals were given free access to water, shade and salt throughout the trial. Ewe lambs consumed alfalfa hay (2.5 kg per animal per day; 16% crude protein) and cracked corn (0.22 kg per animal per day). Weights were recorded regularly throughout the trial.
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2.2. Treatments On Day 2 (9 September), ewe lambs were strati®ed by BW and randomly assigned to one of three treatments (n 8 lambs/treatment) in a completely random design. Daily treatments (administered by gavage) beginning on Day 0 (®rst day of treatment) were control (0 mg 6-N-propyl-2-thiouracil (PTU)/kg BW per day), low (20 mg PTU), or high (40 mg PTU) PTU. This dose was administered until Day 14. On Day 15 of the treatment period, the low and high treatments were decreased to 10 and 20 mg PTU/kg BW per day, respectively. The PTU (Sigma Chemical Co., Ltd., St. Louis, MO, USA) was prepared in gelatin capsules (size 12 EL, Torpac Inc., Fair®eld, NJ, USA). Ewe lambs in the control group received blank gelatin capsules. Dahl et al. [7] indicated that administering T4 to mature ovariectomized, thyroidectomized, estradiol-implanted ewes to elevate T4 to nadir levels (approximately 20 ng/ml) was effective in promoting the cessation of LH pulsatility (i.e. initiation of anestrus). Therefore, we attempted to lower serum T4 below 20 ng/ml in ewe lambs to determine PTU effects on puberty. Previous studies conducted in our laboratory using mature pregnant ewes [8,9] demonstrated that doses of 12 mg PTU or less did not decrease serum T4 below 20 ng/ml. However, Hernandez et al. [10] showed that larger doses of PTU (>20 mg) were effective in reducing serum T4 below 20 ng/ml. The larger doses were chosen in an attempt to dramatically reduce serum T4. However, Hernandez et al. [10] also suggested that 40 mg PTU/kg BW may in¯uence feed intake; thus, the PTU doses were decreased by half after 14 days. After the treatment period ended (Day 28), ewe lambs were joined with raddled mature Rambouillet rams for a 34-day breeding season. Breeding dates were recorded twice daily. Weights were recorded on Days 7, 14, 21, 28, 35, 49, 63, 91, 119 and 149. Following a 34day breeding season, all ewes were again maintained in a single pen and managed as described previously until lambing at which time pregnancy percentage, number of lambs born and lamb birth weights were recorded. 2.3. Blood collection Initial blood samples were collected on Day 2 by jugular venipuncture. Daily blood samples were collected from Days 0 through 35 and twice weekly thereafter until the end of the breeding season. Blood samples were collected before feeding into sterile vacuum tubes (Corvac 7, Kendall Health Care, St. Louis, MO, USA). Samples were allowed to clot at room temperature for approximately 30 min before centrifugation at 1500 g for 15 min at 4 8C to separate serum. Following centrifugation, serum was transferred to plastic vials and stored at 20 8C until hormone analysis. 2.4. Hormone analyses Serum T4, triiodothyronine (T3) and progesterone (P4) were quanti®ed by radioimmunoassay using components of commercial kits supplied by Diagnostic Products Corp. (DPC, Los Angeles, CA, USA). Modi®cations to the T4 assay were reported previously [11]. The antibody used in the T4 assay had a 2% crossreactivity with T3 and did not recognize PTU. The P4 assay was modi®ed as described by Schneider and Hallford [12]. The within assay
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coef®cients of variation (CV) were 9.2 and 7.5% for the P4 and T4 assays, respectively, while the between assay CV for P4 and T4 were 8.1 and 13.3%, respectively. Date of puberty was determined by measuring P4 in samples collected twice weekly. Puberty was de®ned as the day that P4 rose above 1 ng/ml and stayed above for two consecutive blood samples. The T3 assay utilized polypropylene tubes coated with an antibody against T3 along with a solution that contained both 125 I-T3 and a substance to interrupt binding between T3 and its transport proteins (tracer solution). A standard solution was prepared by suspending T3 (Sigma T 2877) at 10 ng/ml in 0.01 M phosphate buffered saline containing 1% bovine serum albumin (assay buffer). This stock standard solution was pipetted into the antibody-coated tubes in amounts to provide a standard curve of 0, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 ng T3 per tube. Standard tubes were then normalized to 0.5 ml using assay buffer. Duplicate antibody-coated tubes received 0.4 ml of assay buffer and 0.1 ml of serum from each ewe sample. Each tube then received 1 ml of the tracer solution, after which tubes were vortexed and incubated at room temperature for 3±4 h. Tubes were then decanted and counted for 1 min in a gamma counter. The cross reactivity of the anti-T3 with T4 and PTU was 0.50 and 0.003%, respectively, and the assay sensitivity was 0.07 ng/ml. When 0.1 ng T3 was added to the assay, 28% of the tracer was displaced from the antibody. The within and between assay CV were 9.3 and 12.6%, respectively, and the recovery of an added 5 ng T3/ml was 107%. 3. Statistical analysis Serum T4 and T3 pro®les were analyzed by split-plot analysis of variance for repeated measures [13]. Treatment effects were included in the main plot, and day and treatment by day interactions were included in the subplot. Animal within treatment was used as the error term to examine PTU effects and the residual error was used to test subplot factors. Ewe lamb weights and data for average Julian day of puberty and time from Day 0 to puberty were analyzed by analysis of variance for a completely random design [14]. One ewe lamb in the control group did not achieve puberty during the trial, so she was assigned the maximum Julian date (322) in order to prevent misinterpretation of data. Lamb birth weights, weaning weights, preweaning average daily gains and adjusted weaning weights were also examined by analysis of variance for a completely randomized design. Adjusted weaning weights were calculated as described by Scott [15]. Values for serum T4, ewe BW, lamb birth weights, weaning weights and preweaning average daily gain were separated by linear and quadratic contrasts. Differences among means were considered signi®cant if a probability level less than 0.10 was detected. Treatment effects on pregnancy rates and lamb crop percentages were determined by Chi-square analysis. All analyses were computed using the general linear models and frequency procedures of SAS [16]. 4. Results 4.1. Serum thyroid hormones A PTU by day interaction (P < 0:01) was detected during the 28-day treatment period; therefore, PTU effects on T4 were analyzed within day and results are presented in Fig. 1.
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Fig. 1. Serum thyroxine (T4) in ewe lambs during the treatment period (Days 0 through 28 (top panel) and recovery period (Days 29 through 66). Treatments were 0 (^), 20 (&), and 40 (*) mg of propylthiouracil (PTU) from Days 0 through 14. From Days 15 through 28, PTU dosages were reduced by half.
Serum T4 was similar in ewe lambs receiving PTU compared with values observed in controls (those receiving 0 mg PTU) on the ®rst day of treatment (Day 0; 61.6, 54.8, 56:9 2:5 ng/ml for 0, 20 and 40 mg PTU/kg BW, respectively, P 0:17). However, after 1 day of PTU treatment, serum T4 was reduced (linear, P 0:02) in ewes receiving 20 and 40 mg PTU/kg BW compared with controls. On Day 7, T4 values were much lower in the PTU-treated ewes than in controls (78.5, 9.0 and 15:4 2:5 ng/ml for those receiving 0, 20 and 40 mg PTU/kg BW, respectively; quadratic, P < 0:01). After 2 weeks (Day 14) of PTU administration, ewe lambs receiving PTU had T4 concentrations less than or equal to 1 ng/ml (72.8, 1.0 and 0:4 2:7 ng/ml in 0, 20 and 40 mg PTU/kg BW, respectively; quadratic, P < 0:01).
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On Day 15, the PTU dose for ewe lambs receiving 20 mg PTU was lowered to 10 mg PTU/kg BW, and those receiving 40 mg PTU were lowered to 20 mg PTU/kg BW, while controls remained at the 0 mg PTU/kg BW dose. By Day 22 (7 days after the change in PTU doses), serum T4 was 77.1, 0.2 and 0.3 (2.1) ng/ml in ewes receiving 0, 10 and 20 mg PTU/kg BW, respectively (quadratic, P < 0:01). The lowered doses were suf®cient to hold serum T4 below 20 ng/ml for the remainder of the treatment period (Day 28) as evidenced by serum T4 values less than 1 ng/ml in both PTU-treated groups (80.2, 0.2 and 0:2 2:0 ng/ml in ewes receiving 0, 10 and 20 mg PTU/kg BW, respectively; quadratic, P < 0:01). Daily blood samples were collected for 7 days following termination of PTU treatment (Fig. 1); and although PTU-treated ewe lambs had increasing T4 concentrations, values remained lower than in controls (58.0, 29.1 and 26:9 2:4 ng/ml on Day 35 in ewe lambs treated with 0, 20/10 and 40/20 mg PTU/kg BW, respectively; quadratic, P < 0:01). A twice weekly blood sampling protocol was followed from Days 38 through 66 (Fig. 1). By Day 52 (approximately 3 weeks after PTU treatment ended), the PTU-treated ewe lambs had 41.9 and 36:6 2:6 ng T4/ml (20/10 and 40/20, respectively) compared with 60.9 ng/ml in controls (quadratic, P < 0:05). On Day 66 (last sampling date), the PTUtreated ewe lambs had not completely recovered from the effects of hypothyroidism (55.0, 43.1 and 39:0 2:6 ng/ml for 0, 20/10 and 40/20 mg PTU/kg BW, respectively; linear, P < 0:01). These data indicate that PTU is a very effective means of inducing hypothyroidism in ewe lambs.
Fig. 2. Serum triiodothyronine (T3) in ewe lambs during the treatment period (Days 0 through 28) and recovery period (Days 30 through 34). Treatments were 0 (^), 20 (&), and 40 (*) mg of propylthiouracil (PTU) from Days 0 through 14. From Days 15 through 28, PTU dosages were reduced by half.
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Serum T3 was also quanti®ed and values are presented in Fig. 2. As with T4, a treatment by day interaction was detected (P < 0:01) for T3, necessitating examination of treatment effects within day. Serum T3 followed the same general trend in response to PTU as observed for T4. By 2 days after initiation of PTU treatment, serum T3 was less (P < 0:01) in the two PTU groups than in controls. This difference remained consistent through the treatment period such that on Day 28, serum T3 was 4.11 ng/ml in controls compared with 0.82 and 0.72 ng/ml for those receiving 20/10 and 40/20 mg PTU, respectively (quadratic, P < 0:01). 4.2. Ewe lamb weights Ewe lamb BW are reported in Table 1. Before initiation of treatment (Day 2), all ewe lambs weighed approximately 43 kg (P > 0:20). Weights on Days 14 through 35 were also similar (P > 0:20) among treatments. However, by 3 weeks following the end of the treatment period (Day 49) and again from Days 91 through 146, PTU-treated ewe lambs tended to weigh less (linear, P < 0:10) than did controls. 4.3. Reproductive performance Administration of PTU at doses used in this experiment did not in¯uence reproductive performance. In the present study, reproductive characteristics were similar among treatments (Table 2). Average Julian day of puberty was 286 5:4 in controls and 288 5:4 in both PTU-treated groups (P > 0:05). Time from Day 0 to puberty was Table 1 Weights (kg) of Rambouillet ewe lambs treated daily with propylthiouracil (PTU) for 28 days before beginning a fall breeding season Daya
2d 0d 7d 14d 21d 28d 35d 49e 63d 91e 119e 146e a
PTU (mg/kg BW)b 0
20/10
40/20
S.E.c
43.9 44.2 44.7 45.8 47.4 48.6 49.4 51.2 52.3 57.7 62.8 61.5
43.4 43.7 44.5 46.4 49.3 49.6 48.4 48.8 50.1 53.5 58.1 56.9
43.7 44.6 44.2 46.5 49.1 49.2 48.4 48.0 50.0 54.3 58.8 57.9
1.2 1.2 1.0 1.2 1.2 1.3 1.2 1.0 1.1 1.3 1.4 1.5
Day of trial (Day 0 first day of PTU treatment; 11 September). Ewe lambs received 0, 20, or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10, and 20 mg PTU/kg BW for an additional 14 days. c Standard error based on eight animals per group. d Row values do not differ (P > 0:20). e Row values tend to differ (quadratic, P < 0:10). b
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Table 2 Reproductive responses of Rambouillet ewe lambs treated daily with propylthiouracil (PTU) for 28 days before beginning a fall breeding season PTU (mg/kg BW)a
Reproductive response
Julian day of pubertyc Time from Day 0 to pubertyc,d Pregnancy rate (%)e Lambs born/ewe (n)e
0
20/10
40/20
S.E.b
286 31 75 1.0
288 34 88 0.88
288 35 88 1.12
5.4 5.3
a
Ewe lambs received 0, 20, or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10, and 20 mg PTU/kg BW for an additional 14 days. b Standard error based on eight animals per group. c Date of puberty determined by P4 measured in samples collected twice weekly. Puberty was de®ned as the day that P4 rose above 1 ng/ml and stayed for two consecutive blood samples. Row values do not differ (P > 0:50). d Time from Day 0 (®rst day of treatment) to puberty measured in days. Row values do not differ (P > 0:50). e Row values do not differ (P > 0:20).
31, 34 and 35 5:3 days in ewe lambs receiving 0, 20/10 and 40/20 mg PTU/kg BW, respectively (P > 0:50). Likewise, pregnancy rate and lambs born per ewe were similar (P > 0:20) in both control and PTU-treated ewes. 4.4. Effect of maternal propylthiouracil on offspring Effects of maternal PTU administration on offspring performance (Table 3) were examined in terms of birth weight, actual weaning weight, adjusted weaning weight, and preweaning average daily gain. Birth weights and actual weaning weights were similar (P > 0:90) among groups. Weaning weights adjusted to a 60 day, single ewe lamb, mature ewe basis were also comparable (P > 0:90) among PTU groups (Table 3). Likewise, preweaning gain did not differ (P > 0:90) among treatments. Table 3 Effect of maternal propylthiouracil (PTU) treatment on offspring birth weights, actual weaning weights, adjusted weaning weights, and preweaning average daily gain (ADG). Offspring weights (kg)
Birth weight Actual weaning weight Weaning weight, adjustedc Prewean ADG
Maternal PTU (mg/kg BW)a 0
20/10
40/20
S.E.b
4.7 21.1 28.8 0.30
5.2 19.4 26.0 0.27
4.8 21.3 28.5 0.30
0.4 1.6 1.5 0.02
a Ewe lambs received 0, 20 or 40 mg PTU/kg BW daily (gavage in gelatin capsules) for 14 days. After the initial period, doses in the three respective groups were decreased to 0, 10 and 20 mg PTU/kg BW for an additional 14 days. Row values do not differ (P > 0:90). b Standard error based on 8 animals per group. c Adjusted as described by Scott [15].
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5. Discussion Dahl et al. [7] indicated that administering T4 to mature ovariectomized, thyroidectomized, estradiol-implanted ewes to elevate T4 to nadir levels (approximately 20 ng/ml) was effective in promoting the cessation of LH pulsatility (i.e. initiation of anestrus). In our study, serum T4 and T3 were greatly reduced in ewe lambs receiving PTU compared with controls (P < 0:01) beginning on Day 2 and lasting throughout the sampling period. Levels of serum T3 appeared to recover from the effects of PTU much more quickly than did T4 as evidenced by the values shown in Fig. 2. In fact, 4 and 6 days after PTU administration was terminated, the previously treated ewes had greater serum T3 values (quadratic, P < 0:01) than did controls. The T4 data agree with results reported by Hernandez et al. [10] and suggest that large doses of PTU can lower serum T4 below 20 ng/ml. Ewe lambs appeared to respond more quickly to PTU in terms of T4 reduction than did mature ewes studied by Hernandez et al. [10]. Decreasing the doses to 10 and 20 mg PTU/kg BW on Day 15 was done in an attempt to prevent any adverse health effects because Hernandez et al. [10] noted that 40 mg of PTU may adversely impact animal health in a manner different from hypothyroidism. The administration of PTU may have slightly affected BW after the treatment period as evidenced by decreased weight gain in PTU-treated ewe lambs compared with controls (P < 0:10). Hernandez et al. [10] found that mature ewes treated with 40 mg of PTU tended to lose weight compared with controls. These researchers hypothesized that this effect may have resulted from a `toxicity' caused by the large dosage of PTU resulting in depressed feed intake. Richards et al. [11] observed decreased serum T4, lowered weight gain, and declining feed intake in ram lambs consuming a 20% locoweed diet. The decreased serum T4 concentrations resulting from locoweed toxicosis, along with lowered weights and declining feed intake in the post-locoweed feeding period suggests a relationship between normal thyroid function and appetite. In this study feed intake data were not collected; however, the tendency for lower weights following the PTU treatment period suggests a possible slight toxicity that resulted in depressed appetite. Another aspect for consideration is the observation that along with the trend for decreased weight in hypothyroid ewe lambs, no effect on age at puberty was noted. Because age and weight are considered the major determinants of the attainment of puberty [1], the involvement of the induced hypothyroidism in reaching puberty at a lighter weight warrants further investigation. The drastic reduction in serum T4 and T3 did not hasten puberty or increase conception rates. This ®nding does not support our original hypothesis and does not agree with those reported in mature ewes by other researchers [5±7]. A possible explanation for this different response in ewe lambs and mature ewes may be that mature ewes respond to thyroidectomy in terms of the seasonal response when the surgery is performed during the normal breeding season [6]. In the current study, hypothyroidism was induced during the period immediately before onset of puberty during the ewe lamb's ®rst breeding season, although it could be argued that because our experiment was initiated during September, the onset of puberty may have been confounded with onset of the fall breeding season. In a recent study [17], we examined progesterone pro®les in untreated, nonpregnant ewe lambs for 1 year beginning in September. These females reached puberty on an average of 24 October (S:D: 15 days), became anestrus on an average of 25 December
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(S:D: 26 days), and started estrus activity again by 16 July (S:D: 20 days). These data demonstrate that ®ne wool ewes in southern New Mexico do experience estrus during the summer. However, in an earlier study [18], we monitored progesterone pro®les in ewe lambs beginning at 4 months of age (17 July) and continuing through a fall breeding season. In this study, no cyclic progesterone pro®les were observed until October. Based on these previous studies from our laboratory, we suggest that in the current study, puberty had not been attained before PTU administration began in September. Maternal administration of PTU also did not appear to have an adverse impact on lamb health as suggested by similarities in birth weights, actual and adjusted weaning weights, and preweaning gain (P > 0:90). Although reproduction was not affected by oral PTU administration, these results demonstrate that PTU is an effective means of inducing hypothyroidism in young female sheep. Further studies are needed to examine other possible methods of hastening puberty and improving reproductive ef®ciency in ewe lambs. Acknowledgements Research was supported by the New Mexico Agricultural Experiment Station and is a contribution of Western Regional Project W112. The authors thank R. Rosencrans, M. Jacobsen, B. Jackson, J. Joines, and L. Amezquita for technical and clerical assistance. References [1] Hulet CV, Wiggins EL, Ercanbrack SK. Estrus in range lambs and its relationship to lifetime reproductive performance. J Anim Sci 1969;28:246±52. [2] Foster DL. Mechanism for delay of ®rst ovulation in lambs born in the wrong season (fall). Biol Reprod 1981;25:85±92. [3] Foster DL, Yellon SM, Olster DH. Internal and external determinants of the timing of puberty in the female. J Reprod Fertil 1985;75:327±44. [4] Yeates NTM. The breeding season of the sheep with particular reference to its modi®cation by arti®cial means using light. J Agric Sci 1949;39:1±43. [5] Legan SJ, Karsch FJ. Neuroendocrine regulation of the estrous cycle and seasonal breeding in the ewe. Biol Reprod 1979;20:74±85. [6] Karsch FJ, Dahl GE, Hachigian TM, Thrun LA. Involvement of thyroid hormones in seasonal reproduction. J Reprod Fertil 1995;49(Suppl.):409±27. [7] Dahl GE, Evans NP, Thrun LA, Karsch FJ. Thyroxine is permissive to seasonal transitions in reproductive neuroendocrine activity in the ewe. Biol Reprod 1995;52:690±6. [8] Hernandez JA, Hallford DM, Bollinger JL, Shirley KL, Richards JB. Serum thyroxine, progesterone, and luteinizing hormone and body weight and milk characteristics in ewes treated with PGF2a and propylthiouracil during gestation. Proc West Sec Am Soc Anim Sci 1999;50:226±31. [9] Bollinger JL, Hallford DM, Hernandez JA, Shirley KL. Reproductive, hormonal, and milk characteristics of ewe treated with propylthiouracil and melatonin during late gestation. Proc West Sec Am Soc Anim Sci 2000;51:374±9. [10] Hernandez JA, Hallford DM, Wells NH. Serum thyroxine, body weights, and ovarian cyclicity in ®ne wool ewes subjected to thyroid suppression immediately before onset of anestrus. Proc West Sec Am Soc Anim Sci 2001;52:401±5. [11] Richards JB, Hallford DM, Duff GC. Growth and serum pro®les of ram lambs fed locoweed (Oxytropis sericea) and treated with vitamin E/selenium. Theriogenology 1999;52:1055±66.
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[12] Schneider FA, Hallford DM. Use of a rapid progesterone radioimmunoassay to predict pregnancy and fetal numbers in ewes. Sheep Goat Res J 1996;12:33±8. [13] Gill JL, Hafs HD. Analysis of repeated measurements in animals. J Anim Sci 1971;33:331±6. [14] Steele RG, Torrie JH. Principles and Procedures of Statistics: a biomedical approach. New York: McGrawHill; 1980. [15] Scott GE. The Sheepman's Production Handbook. Denver Co: Sheep Industry Development Program; 1977. [16] SAS. SAS User's Guide: Statistics. SAS Institute Inc, Cary, NC; 1989. [17] Gifford CA, Wells NH, Hernandez JA, Duffey JL, Rosencrans RL, Hallford DM. Serum thyroxine pro®les and environmental conditions during three stages of the reproductive cycle of Rambouillet ewe lambs in southern New Mexico. Proc West Sec Am Soc Anim Sci 2002;53:170±3. [18] Birch MW, Hallford DM, Christensen BL, Schneider FA, Campbell JW. Reproductive performance and serum growth hormone and melatonin in ewe lambs treated with melatonin microspheres and(or) estradiol-17b. Proc West Sec Am Soc Anim Sci 1994;45:279±81.