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Effect of High-Dose Liothyronine on Semen Quality and Recovery Following Withdrawal in Rabbits

From the Department of Animal Science, Cornell University, Ithaca, New York.

Correspondence to: Dr Robert H. Foote, Department of Animal Science, Cornell University, Ithaca, NY 14853-4801.

Received for publication April 19, 2002; accepted for publication June 6, 2002.

	Abstract

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Endocrine aspects of the thyroid in the pituitary-thyroid-gonadal axis have been studied extensively, but few controlled studies have been conducted on sperm output in males with hyperthyroidism. The rabbit was used as a model to study the effects of hyperthyroidism induced with supraphysiologic doses of tri-iodothyronine (T₃). Semen was collected from 18 sexually mature Dutch rabbits for 9 weeks before the experiment to standardize semen collection procedures, and to provide semen data to equalize treatment groups. Controls were continued on the same regimen for 18 weeks, whereas the treatment group received daily T₃ doses of 10 µ/kg per body weight for 5 weeks, followed by 13 weeks of recovery. Feed consumption was reduced precipitously during T₃ treatment, but it recovered rapidly following termination of T₃ administration. Gradual weight loss and recovery accompanied changes in feed consumption. Loss of libido was not detectable with treatment. Sperm output during 9 weeks following T₃ treatment was 46% of that collected during the standardization period. During the last 4 weeks, sperm output had recovered to 88% of pretreatment values, and subsequently, treated males had normal fertility. Histologic sections showed that the thyroid was inhibited by T₃ treatment and that spermatogenesis was moderately depressed. Both recovered by the termination of the experiment. These studies indicate that effects of induced hyperthyroidism on sperm production are transient.

     Key words: Hyperthyroidism, tri-iodothyronine, rabbit semen, libido, fertility

In adult males, species differences on the effect of hypothyroidism and hyperthyroidism on both spermatogenesis and libido have been reported. Part of these differences are due to the effect of photoperiod on seasonally reproducing animals (Chandrasekhar et al, 1986; Parkinson and Follett, 1994; Anderson and Barrell, 1998). Also, the effect of hypothyroidism in adults after testicular maturation is different from its effect during sexual development. Induction of hypothyroidism in neonatal animals at a critical stage of development results in hypertrophy of spermatogenic cells accompanying an increase in the number of Sertoli cells (Joyce et al, 1993; Cooke et al, 1994; Ariyaratne et al, 2000).

Studies of thyroid dysfunction in men have been directed primarily toward general health or pathology, and not on reproduction (Pacini et al, 1994; Singer et al, 1995; Inzucchi and Burrow, 1999; Hardmann et al, 2001). Effects of thyroid dysfunction on reproduction are not well understood (Veláquez and Arata, 1997). Earlier clinical studies in men often indicated some improvement in semen quality following treatment with T₃ or T₄ (Farris and Colton, 1958; Reed et al, 1958; Arrata et al, 1969), but matched controls and follow-up usually were lacking. Also, if quality of ejaculated semen improved, it could reflect an improvement in libido that was associated with general improvement in health rather than a direct effect on the reproductive system.

Because information on the effect of hyperthyroidism on reproduction in males is minimal, we used rabbits to undertake the study. Rabbits are useful for studying the effects of thyroid alteration on semen quality because the pituitary-thyroid-gonadal relationships are typical of most mammals (Chu, 1944; Maqsood, 1952; Brown-Grant, 1955; Johnson et al, 1958). Furthermore, spermatogenic function in this model has been thoroughly studied, and regular semen collection for the purpose of monitoring chronic spermatogenic function is easily performed (Swierstra and Foote, 1965; Amann, 1981; Blanco-Rodriguez, 2002). Oloufa et al (1951) reported that feeding thyroprotein (iodinated casein) to rabbits at 33°C greatly suppressed semen quality compared with that observed in untreated males housed at 33°C and at 16°C. However, induction of hyperthyroidism was confounded by environmental temperature, and the trial lasted only 5 weeks. This is too short a time to measure possible recovery of spermatogenesis, and no follow-up study was reported.

The present study was designed to test specifically whether or not a supraphysiologic dose of the levo form of T₃ (liothyronine) would decrease semen quality, affect libido, and be transitory in its effects. Furthermore, we wished to determine whether fertility subsequent to treatment would be affected. T₃ was selected because of its desirable experimental property of having a rapid turnover rate and because T₃ is the active form in tissues.

	Materials and Methods

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Animals and Feeding

Twenty-three Dutch-belted males from our colony were housed in individual cages in an animal room maintained on a 12 L:12 D cycle. Their average age was 13 months. They were tested for ease of semen collection. Two ejaculates per day 1 day per week were collected from each male for 9 weeks to obtain pre-experimental semen quality data on each, and to adapt the animals to a standard semen collection routine. Eighteen males that adapted well to this routine were selected for the experiment.

The animals were weighed weekly. They received fresh water ad libitum, and each morning they were fed a complete pelleted ration averaging about 95 g/day. This quantity of feed was designed to maintain healthy males in good body condition. Any feed not consumed by the next feeding was weighed to estimate daily feed consumption.

Experimental Design

The 18 animals assigned to the experiment were ranked on the basis of total motile sperm output per week during the 9-week pre-experimental period. Then they were paired, and members of each pair were randomly assigned to the treatment or control group. The control group continued untreated for 18 more weeks during the experiment. Treated animals received the levo form of T₃ at a daily dose of 10 µ/kg of body weight for 5 weeks, when treatment was discontinued. Semen collections continued during 13 more weeks to allow for recovery. This allowed for 8 to 9 weeks of recovery of spermatogenesis, plus time for quality of ejaculated semen to return to normal. Three males that were not killed for histologic examination at the conclusion of the semen collection study were tested for fertility. The dosage of T₃ was estimated from various reports in the literature with the intent of causing hyperthyroidism, and probably disturbing spermatogenesis without causing serious general stress to the animals. Two additional males from the original group of 23 males that had good libido but from which it was difficult to collect semen were treated with T₃ and were killed at the end of T₃ treatment to examine the thyroids for suppression of function.

Semen Collection and Evaluation

Semen was collected with an artificial vagina using a doe as a stimulus animal. Following brief exposure to the female, the male was allowed to mount the teaser doe, which was held by the semen collector. The time from exposure to ejaculation was recorded. If the male did not ejaculate within 5 minutes, a refusal was recorded. The same procedure was used to obtain a second ejaculate 1 hour after collecting the first ejaculate of semen. Multiple does were kept as stimulus animals to maintain sexual novelty.

Semen was collected in graduated test tubes. Gel was removed immediately following semen collection. The volume of the gelfree ejaculate was recorded. An aliquot of the semen was diluted with phosphate-buffered saline. A 6-µL droplet was placed on a slide, covered, placed on a microscope stage at 37°C, and the percentage of motile sperm was estimated subjectively in several fields. Another aliquot of each ejaculate was processed for estimating sperm concentration in duplicate with two hemacytometer chambers. The total number of sperm and progressively motile sperm in each ejaculate were computed.

Administration of T₃

T₃ was administered subcutaneously daily to ensure that the planned dosage was given. Approximately 0.2 mL of a 100-µg/mL solution of T₃ (10 µg/kg of body weight) was injected per animal daily for 5 weeks. Doses were adjusted according to the weekly weight of each animal. This dose was expected to suppress thyroid-stimulating hormone and thyroid function because T₃ is more effective than T₄ in suppressing thyroid function in rabbits (Brown-Grant, 1955).

Histology of Thyroids and Testes

Tissues were prepared for histologic examination from 5 treated and 3 control animals at autopsy. They were sectioned at 6 µm and stained with hematoxylin and eosin. The other treated males were used to collect semen and inseminate females, and were checked for fertility 1 month after completing the semen collection study. Two additional males with good libido from the original 23 were treated with T₃, and the thyroids and testes were examined at the end of treatment.

Statistical Analysis

Data were summarized on a weekly basis. The 27-week study was a randomized block design, divided into 9 weeks of pre-experimental standardization, 5 weeks of T₃ treatment, and 13 weeks of posttreatment recovery. Controls were maintained on the same semen collection schedule throughout the 27 weeks. Bucks were considered as random and treatments as fixed in the general mixed linear model analysis of variance (Statistical Analysis Systems, 1985). Treatment differences of P .05 were considered to be significantly different. To check that the 9-week pre-experimental period resulted in equalization of the two groups of males, analysis of covariance on total sperm and total motile sperm per week was done. The adjustment was too small to appreciably alter the statistics.

	Results

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Weekly feed consumption and body weight are illustrated in Figure 1 and are summarized by periods in Table 1. Feed consumption and body weight were reduced (P < .05) during the treatment period. Feed consumption returned to normal almost immediately after cessation of treatment. Body weight recovery was slower, but returned to near pretreatment weights by the end of the experiment.

View larger version (28K): [in this window] [in a new window]   Figure 1. Weekly feed consumption and body weight changes in rabbits during 9 weeks of pretreatment, 5 weeks of treatment, and 13 weeks posttreatment. Vertical bars are SE.

Libido was unaffected by treatment (Table 2). Neither the time required to obtain an ejaculate of semen (reaction time) nor the proportion of successful collections was affected. A few samples in both treated and control groups were lost outside of the artificial vagina, or the teaser was inseminated. A few samples were very small, with the semen deposited on the rubber lining of the artificial vagina, so ejaculate characteristics were not measurable. Refusal to mount or ejaculate occurred only 7 times in each group.

A summary of semen characteristics appears in Table 3. The percentage of progressively motile sperm and total sperm per week during the standardization period were similar. Total motile sperm per week in control and treated groups averaged 229 x 10⁶ sperm and 225 x 10⁶, respectively. The percentage of progressively motile sperm was slightly reduced during treatment (P < .05). Likewise, sperm output was beginning to be reduced by the end of the treatment period, and was greatly reduced during the first several weeks after treatment (P < .05). It had returned to near pretreatment values by the last 4 weeks of the experiment.

The two extra males given T₃ for 5 weeks showed complete inhibition of the thyroid gland by the end of treatment, but only moderate interference with the meiotic stages of spermatogenesis. There was a reduction in postmeiotic types of spermatogenic cells but these were not quantified. Animals autopsied at the end of the experiment had normal thyroid glands and testes, as determined by histologic examination. Gross appearance of other organs was normal.

Four treated animals remained, but one became ill from unknown causes. An ejaculate of semen from each of 3 remaining treated animals was collected and used separately to inseminate 3 or 4 does each. All males were fertile, with 8 of 11 does inseminated producing normal litters, ranging from 5 to 7 live young per litter.

	Discussion

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The control and treated groups were equalized by pairing the animals on the basis of 9 weeks of semen collection before treatment. Thus, analysis of covariance did not appreciably improve the statistical equality of the groups.

Based on published reports (Maqsood, 1952; Brown-Grant, 1955; Kar et al, 1958; Hardman et al, 2001) it was expected that the dose of T₃ administered would completely inhibit thyroid secretion. T₃ was chosen over T₄ as the drug to use because of its potency, rapid effects, and rapid turnover (Hardman et al, 2001), and it is the compound prevalent within tissues. It was desired to have the effect of exogenous thyrotropic hormone established rapidly at the beginning of the experiment and be eliminated rapidly at the termination of treatment. The half-life of T₃ is about 7 hours.

The experiment was the first of its kind conducted with sufficient duration to examine both suppression and recovery of spermatogenic function, as measured by sperm output. Oloufa et al (1951) reported a severe depression of sperm production when rabbits received 0.01% of iodinated casein in the diet, and animals were maintained at 33°C. However, this result may have been due in part to the high temperature. Johnson et al (1958) reported that housing rabbits in an environment exceeding 28°C causes stress. The 5-week study by Oloufa et al (1951) was too short to determine whether spermatogenesis would recover because approximately 8 weeks are required to produce sperm from spermatogonia and to transport them through the epididymis. No subsequent data were found that answered the questions that prompted the present experiment.

Feed consumption and body weight (Table 1) decreased, as was expected during the period of hyperthyroidism. Libido was not affected (Table 2), so this was not a factor in obtaining semen and measuring sperm output. This is in contrast to low libido noted in men with thyroid dysfunction (Veláquez and Bellabarba Arata, 1997).

Changes in sperm output were gradual during and following treatment. This is due to the epididymal reserves of the rabbit (Amann, 1981), which were gradually depleted. Spermatogenesis was suppressed by treatment, as indicated by the postmeiotic reduction of spermatogenic cells in the two rabbits that were killed after 5 weeks of treatment. Sperm reserves were then gradually restored as normal spermatogenesis resumed, as indicated by normal testicular histology 13 weeks after cessation of treatment.

The time scale schematically depicting the relationship between changes in spermatogenesis as reflected by output of ejaculated sperm is shown in Figure 2. This relationship is consistent with the known duration of spermatogenesis and epididymal transport in rabbits (Swierstra and Foote, 1965; Amann, 1981). The decrease illustrated in Figure 2 reflects the fact that the number of sperm ejaculated during the first 9 weeks after treatment was only 46% (P < .05) of the number during the pretreatment period of standardization.

View larger version (18K): [in this window] [in a new window]   Figure 2. Estimated time relationships between the effect of T3 on spermatogenic activity and sperm output based on duration of spermatogenesis and sperm transport through the epididymis in rabbits.

More intensive semen collection could have delineated more precisely the changes in sperm output relative to sperm production over time. However, the objective was not to evaluate the effect of T₃ on the kinetics of spermatogenesis but rather to establish whether or not T₃ effects were transient, with a low intensity of semen collection similar to clinical studies conducted with men.

Administration of T₃ or T₄ causes a negative feedback, depressing secretion of thyroid-stimulating hormone and the gonadotropic hormones (Burrow, 1991; Inzucchi and Burrow, 1999; Hardman et al, 2001). Inhibition of thyroid function was apparent in the two rabbits that were killed at the conclusion of T₃ treatment. Whereas the supraphysiologic dose of T₃ reported here decreased sperm output, recovery was rapid following cessation of treatment. Furthermore, treated males were fully fertile after completion of the trial. Thus, there was no permanent effect on reproduction induced by T₃ in these studies.

The extent to which these results apply to men is speculative. Many men are treated with T₄ or a combination of T₃ and T₄ for a variety of conditions associated with thyroid dysfunction (Blackett et al, 1994; Pacini et al, 1994; Singer et al, 1995; Benevìcius et al, 1999; Hardman et al, 2001). However, little attention is given to possible effects on reproduction, except in pregnant women (Burrow, 1991; Singer et al, 1995; Inzucchi and Burrow, 1999; Hardman et al, 2001). Earlier clinical reports on treating subfertile or infertile men with T₃ have suggested that the quality of semen improved (Farris and Colton, 1958; Horrax, 1958; Reed et al, 1958), but thyroid condition was not evaluated. Wortsman et al (1987) obtained no beneficial effects of T₄ with treatment of men with hypogonadism-hypothyroidism.

In conclusion, 10 µg/kg per body weight of T₃ daily for 5 weeks suppressed sperm output, but recovery followed rapidly with a normal output of fertile sperm. This experiment provides the first conclusive evidence that full recovery of spermatogenic function occurs (using the rabbit model) following administration of supraphysiologic doses of T₃. Furthermore, the importance of conducting experiments of sufficient duration to allow for potential recovery following treatment, plus an allowance for the latent effect of testis recovery to be reflected by the output of ejaculated sperm, is evident.

	Acknowledgments

 
The T₃ was supplied by Smith, Kline, and French. Technical help by L. Gray and N. Schales, and manuscript preparation by E. Tumino Van Amburgh is appreciated.

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