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. 2018 Dec 20;97(4):1468–1477. doi: 10.1093/jas/sky480

Enhanced sperm production in bulls following transient induction of hypothyroidism during prepubertal development

Muhammad S Waqas 1,2, Michela Ciccarelli 1,2, Melissa J Oatley 1, Amy V Kaucher 1, Ahmed Tibary 1,2, Jon M Oatley 1,✉
PMCID: PMC6447252  PMID: 30576512

Abstract

Male reproductive capacity is a critical component of cattle production and the majority of genetic gain is made via selective utilization of gametes from desirable sires. Thus, strategies that enhance sperm production increase the availability of elite genetics for use in improving production characteristics of populations on a worldwide scale. In all mammals, the amount of sperm produced is strongly correlated to the number of Sertoli cells in testes. Studies with rodents showed that the size of the Sertoli cell population is set during prepubertal development via signaling from thyroid hormones. Here, we devised a strategy to increase Sertoli cell number in bulls via induction of a transient hypothyroidic state just prior to and extending beyond the period of Sertoli cell proliferation that we found to normally cease between 4.5 and 5 mo of age. In adulthood, these bulls produced a significantly greater number of sperm compared to age-matched controls and their testes contained nearly 2 times more Sertoli cells. Importantly, sperm motility, morphology, fertilizing ability, and viability after cryopreservation were found to be no different for treated bulls compared to untreated control bulls. This strategy of transient induction of hypothyroidism during a defined period of prepubertal development in bulls could prove to be an efficacious approach for enhancing daily sperm production in genetically desirable sires that will, in turn, provide an avenue for improving the efficiency of commercial cattle production.

Keywords: bull, hypothyroidism, Sertoli cells, spermatogenesis

INTRODUCTION

In commercial cattle production, achieving genetic gain for improvement of production characteristics is made through selective breeding. Because millions of sperm are produced per day, the overriding mode for dissemination of genetics has been selective utilization of sperm from desirable bulls (Oatley, 2010). In addition, the advent of technologies for freezing and thawing bull sperm followed by use in artificial insemination has provided a conduit for extended availability of elite genetics on a worldwide scale (Vishwanath, 2003). Furthermore, the application of semen sexing technologies has provided a key tool for precision breeding in cattle industries where a single gender is more desirable such as dairy cattle production (Vishwanath and Moreno, 2018). However, an existing bottleneck has been that the demand for sexed semen from elite sires is greater than the number of sperm than can be collected (Seidel, 2014). Thus, there is need for strategies to enhance the daily sperm production (DSP) level in bulls.

Spermatogenesis is the sum of germ cell maturation steps initiating with the transition of undifferentiated spermatogonia to differentiating spermatogonia. A series of mitotic divisions amplifies the differentiating spermatogonial pool before meiotic prophase is initiated and the cells transition to spermatocytes that undergo 2 meiotic divisions to become haploid round spermatids that undergo spermiogenesis thereby yielding elongated spermatids and eventually spermatozoa (Staub and Johnson, 2018). The entire process occurs in seminiferous tubules, and the sheer number of sperm produced in each round of spermatogenesis is strongly correlated to the size of a somatic support cell population termed Sertoli cells (Berndtson et al., 1987). These “nurse” cells are the only somatic population that is in direct contact with germ cells and their density in adulthood is set by a defined period of proliferation in prepubertal development (Cooke, 1996). Therefore, strategies to increase Sertoli cell number can lead to enhanced DSP in males and would impact genetic gain in cattle populations via increasing the availability of sperm from desirable bulls for use in selective breeding.

In mice and rats, the length of the period of Sertoli cell proliferation during prepubertal development is set by the emergence of thyroid hormone signaling (Holsberger et al., 2003, 2005). Transient induction of hypothyroidism from neonatal development through puberty leads to an extended period of Sertoli cell proliferation resulting in increased population size and DSP compared to the normal physiological state (Cooke et al., 1991, 1994b, 1996; Joyce et al., 1993; de Franca et al., 1995; Maran et al., 1999). In male rodents, transient induction of hypothyroidism leads to testes with ~150% in Sertoli cell number and ~140% in DSP per gram of testis (Cooke et al., 1991, 1994b). Furthermore, the number of Sertoli cells is correlated with the number of spermatogonial stem cell niches in mice which ultimately provides a larger foundation for continuity and robustness of spermatogenesis (Oatley et al., 2011). Intriguingly, a relationship between thyroid hormone levels in the systemic circulation and Sertoli cell proliferation has also been observed in the ram (Oluwole et al., 2013) and the boar (Sun et al., 2015). Furthermore, in bulls the level of the thyroid hormone thyroxine (T4) during prepubertal development is negatively correlated with testicular size at puberty (Cestnik, 1998). In this study, we explored whether transient induction of hypothyroidism in prepubertal bull calves during the time frame of when Sertoli cell proliferation normally occurs would impact spermatogenesis in adulthood.

MATERIALS AND METHODS

Animals and Treatments

All animal procedures were approved by the Institutional Animal Care and Use Committee of Washington State University. Hybrid Angus × Wagyu bulls (n = 6) from the same herd and of similar age were randomly assigned to a treatment or control group. The cohort of experimental bulls was produced from 2 different AI sires. The treatment group received the antithyroid drug Methimazole in tablet form (2 mg/kg body weight) twice daily from 4 to 6 mo of age. Control animals were fed the same diet and managed in identical conditions but did not receive Methimazole tablets. Body weight and scrotal circumference were measured every 2 mo from 4 to 28 mo of age.

Immunohistochemical Staining of Bovine Testis Cross Sections

Pieces of parenchyma (each ~100 mg in size) were collected from testes of bull calves at 1 to 12 mo of age following castration. The samples were fixed by immersion in Bouin’s solution, serially dehydrated in ethanol and paraffin embedded. Cross sections of 5 μm thickness were adhered to glass slides followed by deparaffinization and incubation in boiling Na citrate buffer (pH 6.0) for antigen retrieval. Nonspecific antibody binding was blocked by incubating the sections in 10% normal serum for 1 h at room temperature. The sections were then incubated with primary antibody (anti-Sox9, AB5535 Millipore, 1:100; or anti-Ki67, 550609 BD Biosciences, 1:500. Burlington, MA) diluted in PBS containing 0.5% BSA overnight at 4 °C. On the next day, sections were washed in PBS, incubated with either HRP or fluorophore conjugated secondary antibody at room temperature for 1 h, and then washed in PBS. For the immunofluorescence-stained sections, coverslips were mounted using aqueous solution containing DAPI (Life Technologies, Waltham, MA) for counterstaining of DNA. For colorimetric staining, the sections were incubated with DAB solution (SK-4100; Vector Laboratories, Burlingame, CA) followed by washing in distilled water, counterstaining with hematoxylin, and then coverslips mounted with aqueous medium (H-5501; Vector Laboratories).

Serum Thyroid Hormone Analysis

Blood was collected into red top vacutainer tubes from all bulls every 2 wk from day 0 of treatment until week 16 post-treatment. Serum was separated by centrifugation (3,000 × g for 20 min) and stored at −80 °C prior to assaying for the levels of thyroxine (T4) and triiodothyronine (T3) by ELISA following the manufacturer instructions (BT0046 for T3 and BT0047 for T4, NeoBiolab, Cambridge, MA).

Semen Collection and Evaluation

Semen samples were collected every 2 mo from all bulls by electroejaculation throughout the age range of 18 to 28 mo. Collections from all bulls at each age point were conducted by a single experienced operator in order to provide a layer of standardization to the sampling process. One ejaculate was collected from each bull at each age point. Samples were collected into prewarmed tubes and diluted 1:1 with Triladyl (Minitube, Delavan, WI) extender. The volume of each semen sample was recorded and slides prepared for assessment of sperm morphology using eosin-nigrosin staining and light microscopy at 1,000× magnification. Samples were also analyzed for sperm concentration and motility using a computer-assisted semen analysis (CASA) system (Spermvision, MOFA, Delavan, WI). In addition, semen was cryopreserved in liquid nitrogen at concentration of 1 × 108 cells per mL in 0.5 cc straws. After 4 to 24 mo of cryopreservation, the straws were thawed at 37 °C for 1 min and sperm viability (PI/SYBR staining, MOFA, Delavan, WI) and motility were assessed using CASA.

In Vitro Fertilization of Bovine Oocytes

To assess the fertilizing ability of fresh and cryopreserved sperm from all bulls, in vitro fertilization (IVF) was conducted as described previously (Moreira et al., 2002), with minor modifications. Matured Bos taurus oocytes (from Applied Reproductive Technology Inc.) were washed in HEPES-TALP 3 times in an X-plate at 38 °C followed by dispersion into IVF-TALP media in a 4-well plate (14444, ThermoFisher Scientific, Waltham, MA). Freshly diluted or post-thawed spermatozoa were prepared using percoll sedimentation (10 min, 1,000 × g), washed in SP-TALP media, centrifuged at 200 × g for 5 min and then resuspended in IVF-TALP media. Spermatozoa and oocytes were then combined in a 4-well plate at a concentration of 1,000 spermatozoa per mL of the media followed by incubation at 38.5 °C in an atmosphere of 5% CO2 in air. Approximately, 18 to 20 h post-IVF, putative zygotes were denuded from cumulus cells using hyaluronidase (10,000 IU/mL) and incubated in BSA and Gentamicin-supplemented KSOM+AA (Caisson Labs). Cleavage rate was calculated 24 h post-IVF and blastocyst rate was recorded 8 d post-IVF.

Postmortem Analyses of Testes

At 29 mo of age, all bulls were euthanized and testes and epididymides were recovered and weighed. Fragments of testicular parenchyma at ~100 mg in size were collected from various regions of each testis and fixed in 4% paraformaldehyde. Cross sections of 5 μm thickness were adhered to glass slides and processed by immunostaining for SOX9 expression followed by microscopic assessment. Images of cross sections were captured and the number of SOX9+ Sertoli cell nuclei per seminiferous tubule cross section was quantified as well as measurement of tubule diameter. In total, 10 different cross sections from different regions of the testis, each containing 8 to 10 seminiferous tubules, were analyzed per bull. Thus, for each bull 80 to 100 seminiferous tubules were used for assessment of Sertoli cell number.

Statistical Analysis

All data are presented as the mean ± SEM. For measures of body weight, scrotal circumference, sperm number, sperm motility, and sperm morphology, differences between treated and control groups were determined statistically using a multivariate ANOVA with repeated measures transformation model. For measures of postmortem testis weight, epididymal weights, seminiferous tubule diameter, and Sertoli cell number, a 2-tailed unpaired t-test was used to assess differences. Statistical analyses were carried out using SAS systems software or GraphPad Prism software (GraphPad, Inc.). A P-value of <0.05 was considered significant.

RESULTS

The Postnatal Period of Sertoli Cell Proliferation in B. taurus Bulls

First, in a preliminary experiment, we aimed to determine the age at which Sertoli cell proliferation ceases in bull calves during prepubertal development. Previous studies that examined the testis cell population dynamics in Holstein bulls via cytological approaches suggested that Sertoli cell number plateaus between 6 and 7 mo of age (Curtis and Amann, 1981). To explore this in more depth and pinpoint when Sertoli cells exit the cell cycle, cross sections of testes from hybrid Angus bulls at 1 to 10 mo of age (n = 3 to 5 animals at each age point) were analyzed by co-immunofluorescent staining for expression of the Sertoli cell marker SOX9 (Silva et al., 1996; Barbara et al., 2000; Hemendinger et al., 2002) and the cell proliferation marker Ki67 (Scholzen and Gerdes, 2000). Outcomes revealed Ki67 staining in Sertoli cells at 1 to 4.5 mo age but absence of staining by 5 mo of age which persisted into adulthood (Fig. 1). These observations suggested that Sertoli cell proliferation ceases between 4.5 and 5 mo of age in B. taurus breeds of cattle.

Figure 1.

Figure 1.

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The period of Sertoli cell proliferation in Bos taurus bull calves. Representative images of cross sections from testicular parenchyma of bull calves at 4 to 5 mo and 12 mo of age immunostained for the Sertoli cell marker SOX9 (green staining) and proliferation marker Ki67 (red staining). Arrows denote actively dividing Sertoli cells and arrowheads denote actively dividing germ cells that do not express SOX9. Scale bars are 100 μm.

Transient Induction of Hypothyroidism in Bull Calves During Prepubertal Development

Having determined in our preliminary studies, when Sertoli cell proliferation normally ceases in bull calves, we aimed to devise a strategy for induction of a transient hypothyroidic state just prior and extending beyond the age range (Fig. 2A). To achieve this, we treated hybrid beef bulls (Angus × Wagyu, n = 3) with the antithyroid compound Methimazole (2 mg/kg body weight) twice daily from 4 to 6 mo of age. Control bulls (n = 3) were age matched from the same herd that did not receive Methimazole treatment but housed and managed in identical conditions. Based on the decrease in thyroxine (T4) and triiodothyronine (T3) serum levels at ~3 and 5 wk of treatment, respectively, the induction of a hypothyroidic state occurred by 3 wk of treatment, prior to the bulls reaching 5 mo of age (Fig. 2B and C). Following the treatment period, the serum levels of T3 and T4 returned to normal by ~6.75 and 6.25 mo of age, respectively. Thus, using the treatment strategy, we were able to effectively suppress the production of thyroid hormones just prior to and extending 1 mo beyond the normal age range of when Sertoli cell proliferation ceases.

Figure 2.

Figure 2.

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Transient induction of hypothyroidism in bull calves during prepubertal development. (A) Scheme for transient induction of hypothyroidism just prior to and extending 1 mo beyond normal cessation of Sertoli cell proliferation in bull calves by treatment with the drug Methimazole. (B and C) Serum levels of T3 (B) and T4 (C) in control and Methimazole-treated bull calves during a 5-wk treatment period. (D and E) Body weight (D) and scrotal circumference (E) of control and Methimazole-treated bulls from 4 to 28 mo of age. Data in B–E are mean ± SEM for n = 3 bulls per group and * denotes significantly different at P < 0.05.

To assess the impacts of induced transient hypothyroidism on growth and development, we measured body weight and testis size (assessed by scrotal circumference) every 2 mo for all bulls prior to the treatment period and up to 2 yr of age. Neither overall growth nor testis size was different between the groups prior to or during the treatment period (Fig. 2D and E). In contrast, during the age period when puberty is normally established (10 to 16 mo), mean body weight and scrotal circumference were significantly (P < 0.05) reduced for the treated bulls compared to the control group (Fig. 2D and E). However, by 16 mo of age there was no difference in either parameter between treated and control bulls.

Spermatogenic Output in Bulls Subjected to Transient Induction of Hypothyroidism During Prepubertal Development

Next, we aimed to determine whether transient induction of hypothyroidism during and beyond the period of normal Sertoli cell development alters spermatogenic output in bulls in adulthood. To achieve this, we collected semen samples every 2 mo from 18 mo of age (when body weight was not different between the groups) to 28 mo of age. Overall, the number of total spermatozoa in ejaculates from treated bulls was found to be significantly (P < 0.05) greater compared to control bulls at several of the age points measured (Fig. 3A). Importantly, at 24 to 28 mo of age the samples from treated bulls contained ~30% to 180% (i.e., 1.3- to 2.8-fold) more spermatozoa compared to samples from the control bulls. Although the number of spermatozoa was different between the treated and control groups, the sperm motility and morphology of freshly collected samples were similar (Fig. 3B and C). In addition, the post-thaw survivability and motility following a period of cryopreservation were found to be no different for sperm collected at 24 mo of age from all control and all treated bulls (Supplementary Fig. 2). Furthermore, we tested the IVF capacity of freshly collected and frozen-thawed sperm from 1 control and 1 treated bull at 27 mo of age and determined that there was no difference in cleavage or blastocyst rates (Supplementary Figs. 1 and 3).

Figure 3.

Figure 3.

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Effect of transiently induced hypothyroidism in bull calves during prepubertal development on spermatogenic output in adulthood. (A–C) Total number of sperm (A), percentage of motile sperm (B), and percentage of sperm with normal morphology (C) in ejaculates of control and Methimazole-treated bulls at 18 to 28 mo of age. Data are mean ± SEM for n = 3 bulls per group and * denotes significantly different at P < 0.05.

Testicular Dynamics of Adult Bulls Subjected to Transient Induction of Hypothyroidism During Prepubertal Development

Lastly, at 29 mo of age, we euthanized all bulls and collected testes for examination of seminiferous tubule dynamics. We found that the mean paired testis weight was ~22% higher for treated bulls compared to controls but the difference was not statistically significant (Fig. 4A). In addition, the paired epididymal weight was measured to be significantly (P < 0.05) greater by ~50% in treated bulls compared to controls (Fig. 4B). Morphological assessment of cross sections did not reveal any obvious differences between treated and control bulls and seminiferous tubule diameter was found to be not different (Fig. 4C and D). To assess whether Sertoli cell number was altered in treated bulls, we immunostained cross sections for the marker SOX9 and carried out a quantitative assessment (Fig. 5A and B). Outcomes revealed that testes of treated bulls contained 18.7 ± 2.4 Sertoli cells per cross section which was a significantly (P < 0.05) greater by ~2.3-fold compared to control bulls that contained 8.2 ± 1.4 Sertoli cells per cross section.

Figure 4.

Figure 4.

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Impact of transiently induced hypothyroidism in bull calves on testicular dynamics in adulthood. (A and B) Paired weight of testes (A) and epididymides (B) from control and Methimazole-treated bulls at 29 mo of age. Data are mean ± SEM for n = 3 bulls per group and * denotes significantly different at P < 0.05. (C) Representative hematoxylin and eosin-stained image of a seminiferous tubule cross section from testicular parenchyma of a 29-mo-old bull that was treated with Methimazole during prepubertal development. Scale bar is 100 μm. (D) Diameter of seminiferous tubules in cross sections of testicular parenchyma from 29-mo bulls treated with Methimazole during prepubertal development and their age-matched controls. Data are mean ± SEM for 30 different cross sections from n = 3 bulls per group.

Figure 5.

Figure 5.

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Effect of transiently induced hypothyroidism in bull calves during prepubertal development on Sertoli cell number in adulthood. (A) Representative images of immunostaining for the Sertoli cell marker SOX9 in cross sections of seminiferous tubules from testicular parenchyma of control and Methimazole-treated bulls at 29 mo of age. Scale bars are 100 μm. (B) Quantification of SOX9+ Sertoli cell number in cross sections of seminiferous tubules from testicular parenchyma of control and Methimazole-treated bulls at 29 mo of age. Data are mean ± SEM for 30 different cross sections from n = 3 bulls per group and * denotes significantly different at P < 0.05.

DISCUSSION

The primary approach for achieving genetic gain in cattle production has been through selective utilization of male gametes paired with artificial insemination (Johnson et al., 2011). In the United States, ~80% of dairy cattle are bred with artificial insemination and widespread adoption this technique is a major contributor to the greater than 4-fold increase in average milk production per animal since the 1940s, when artificial insemination was commercialized (Johnson et al., 2011). Thus, strategies that increase sperm production in bulls provides a means to enhance the availability of genetics from elite animals that will, in turn, improve the quality and production efficiency from cattle for the benefit of human consumption (Oatley et al., 2016). The human population is estimated to reach ~10 billion by 2050, and ensuring food security for this projected population growth will require enhanced production from livestock (Béné et al., 2015). Improving the reproductive capacity of male livestock is a key avenue for making these advances.

An approach that has high potential for impacting cattle production is selection of X- vs. Y-bearing sperm (i.e., semen sexing) for use in an artificial insemination setting (Seidel, 2003). Sexed semen can improve the efficiency of herd expansion by shifting the gender ratio of offspring. For dairy cattle production, the efficiency and precision of producing replacement heifers is impacted by artificial insemination with X-bearing sperm from desirable sires. In contrast, the generation of male calves at a higher ratio through artificial insemination with Y-bearing sperm could impact beef cattle production where higher male ratios are of greater value in feedlot operations (Seidel, 2003). An important criterion for a bull to be selected as a sire for semen sexing is generation of an ejaculate containing 6 billion spermatozoa (Butler et al., 2014). At present, many bulls with desirable genetics do not meet the minimum requirement and are therefore excluded from use as breeding stock in a sexed semen setting. In addition, many bulls do not reach peak sperm production until 2 yr of age but attain puberty much earlier; thus, there is lost opportunity for use as a sexed semen sire. For these reasons, the potential impact of many elite bulls on advancing genetic gain in cattle production is not fully realized. Devising strategies that led to increased sperm production in bulls even at a younger adult age could overcome these bottlenecks.

Several previous attempts have been made to enhance sperm production in male livestock but none have proven to be of practical value (Cooke et al., 1994a). Although unilateral castration leads to increased size of the remaining contralateral testis, the total amount of sperm production does not exceed the pre-castration levels from both testes. Also, previous studies have suggested that immunization against the hormones inhibin and estradiol can lead to increased testicular size and DSP in the bull; however, these approaches are transient and require multiple vaccinations thereby leading to limited commercial interest. As an alternative, approaches for increasing the number of Sertoli cells in testes during prepubertal development could produce a long-lasting impact on sperm production in adulthood (Cooke et al., 1994a). The number of Sertoli cells in adult males is fixed, being established during a defined period of proliferation in postnatal development. In mice, Sertoli cells proliferate from birth until 12 to 14 d of age at which point mitotic arrest initiates in response to elevated thyroid hormone (Holsberger et al., 2003, 2005). In cattle, the number of Sertoli cells is highly correlated to DSP (Berndtson et al., 1987) but the timing of population number establishment has been undefined. In the present study, we found that Sertoli cell proliferation ceases between 4.5 and 5 mo of age in B. taurus breeds of cattle. Determining whether the timing is different in other breeds of cattle will be an important assessment with future experimentation should the approach be implemented within the global cattle industry.

Due to a potent influence on inducing cell cycle arrest, suppression of thyroid hormone signaling in rats and mice via transient induction of hypothyroidism spanning and extending beyond the period of Sertoli cell proliferation leads to enhanced population size in adulthood (Cooke et al., 1991; Joyce et al., 1993; de Franca et al., 1995; Maran et al., 1999). In bulls, higher thyroid hormone levels during neonatal life are negatively correlated with testicular size after puberty (Cestnik, 1998), but the impact of transiently inducing hypothyroidism during Sertoli cell development on sperm output as an adult has not been explored. Here, having defined when Sertoli cell proliferation ceases in bulls, we devised a strategy to test this idea. Outcomes of our studies demonstrate that the effects of transient induction of hypothyroidism during prepubertal development on spermatogenic output in adulthood is similar between cattle and rodents, with higher than normal sperm production and a doubling of the Sertoli cell population. These proof-of-concept findings suggest that the mechanisms modulating development of the Sertoli cell population are conserved among mammalian species, at least at the level of hormone signaling.

Beyond the important finding of elevated sperm production in bulls subjected to transient hypothyroidism, a few potentially unexpected impacts on the animal’s physiology should also be considered. First, the induced hypothyroidic state or the increased Sertoli cell content could alter the hypothalamic-pituitary-gonadal (HPG) axis. However, in rodents, this does not seem to be the case as levels of testosterone are similar between males with normal and increased Sertoli cell number (Cooke et al., 1991; Joyce et al., 1993). Another nuance of the induced hypothyroidic state in prepubertal development of both rodents and cattle is a delay in classic signs of puberty. In the current study, based on assessment of body weight and sperm concentration in ejaculates, the age at which puberty was attained in bulls subjected to transient hypothyroidism was delayed by ~4 mo compared to control bulls. However, once puberty was reached there was no difference in body weight between treated and control bulls but sperm production remained elevated in the treated animals. Lastly, the increased epididymal weight that was measured in this study could be a direct or indirect effect of the induced hypothyroidic state which may or may not be a cause for concern. From a direct perspective, the induced hypothyroidism during prepubertal development could have altered morphogenesis of the epididymis. Alternatively, the increased in epididymal weight could be a result of increased sperm production and therefore an elevated sperm reserve. Regardless of any of these nuances, sperm production was found to be elevated in bulls subjected to transient hypothyroidism during prepubertal development and alterations in normal sperm morphology, survival during cryopreservation, or fertilizing ability compared to control bulls were not observed.

Although findings of the current study are encouraging for development of a new approach to enhance sperm production in bulls, there are several nuances that will need to be addressed with future experimentation. First, only 3 bulls of the same breed makeup were examined for an effect of treatment. Thus, the full variation in response among animals both within breed and among different breeds will need to be assessed in a larger number of animals. Second, the possibility that induction of a hypothyroidic state in prepubertal development has a deleterious effect on DNA integrity and the epigenome of sperm produced in adulthood will need to be explored. Third, the mode by which a hypothyroidic state is induced will need to be optimized. In the present study, we attempted to feed the drug Methimazole, which is an antithyroid compound, in pill form to bull calves twice per day for 2 mo by mixing in grain feed. Although effective at times, the animals often refused to ingest the pills thus necessitating the use of a balling gun. This approach is logistically challenging and unlikely to be implementable on a large scale. We propose that an alternative approach should be developed using a slow release implant to deliver the drug. Another potential challenge is the off-label use of propylthiouracil compounds in food animals.

At present, Methimazole administration in livestock is approved in the United States by the FDA for use in treating hypothyroidism only. Thus, administration for improving reproductive capacity leads to adulteration of the meat. However, the intent of treating bulls with the drug is to enhance sperm output rather than to generate an animal that would end up in the food chain. Furthermore, previous studies that fed the drug to feedlot steers demonstrated clearance from tissues within 72 h (Raun et al., 1962). Considering that the withdrawal time between cessation of Methimazole treatment (6 mo of age) and when the breeding bull would enter the food chain is likely years, the off-label use is something that could be overcome. Thus, adulteration of the meat of a retired breeding sire should not be of concern, even if off-label use is not obtained by food animal oversight authorities.

Collectively, the methodology that we have devised in this study provides a novel and relatively simple avenue for enhancing sperm production in bulls. This approach has several possible applications to improve the reproductive capacity of prepubertal bulls that are candidates for use as elite sires. First, an increased level of sperm production in bulls intended for use as sexed semen sires would correspond to fewer animals being eliminated from contention due to not meeting the minimum industry standard. Second, the sheer number of sperm available from bulls in high demand as artificial insemination sires would be less of a limiting factor for expanding utilization of desirable genetics. Both of these potential benefits would lead to enhanced genetic gain in commercial cattle populations and the ability to feed an expanding global population over the coming decades.

Supplementary Material

Supplementary Material
Click here for additional data file. (45.3KB, docx)

ACKNOWLEDGMENTS

The authors thank members of the Oatley lab for helpful discussions. This research was supported by funds from Genus PLC.

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