J. Anim. Sci. 2004. 82:2259-2263
© 2004 American Society of Animal Science
Effect of divergent selection for testosterone production on testicular morphology and daily sperm production in boars
S. Walker,
O. W. Robison,
C. S. Whisnant and
J. P. Cassady1
Department of Animal Science, North Carolina State University, Raleigh 27695-7621
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Abstract
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The objective of this study was to characterize correlated responses in testicular morphology and daily sperm production to divergent selection for testosterone production. Duroc boars from high and low lines (HTL and LTL, respectively) divergently selected over 10 generations for testosterone production in response to a GnRH challenge followed by random selection were used. Testicular tissues were sampled from all available males of generation 20 (HTL, n = 46; and LTL, n = 13). Volume densities for Leydig cells, seminiferous tubules, and Sertoli cells were estimated along with sperm production. The HTL boars had greater volume densities of Leydig cells than did LTL (P < 0.01). Volume density of seminiferous tubules tended to differ between lines (P < 0.07), but Sertoli cell volume densities did not differ (P < 0.27). Sperm production traits, adjusted for age, did not differ significantly between lines. Body, testicular, and epididymal weights were recorded for boars from HTL (n = 82) and LTL (n = 44) from generations 20 and 21. After adjustment for BW, average paired testicular weights for HTL and LTL were 417 and 457 g (P < 0.01), respectively. Epididymal weights, adjusted for BW, were heavier for HTL (P < 0.01) than for LTL. To demonstrate that the selection lines still differed for testosterone production, lines were evaluated in generation 21. Endogenous testosterone production of the HTL (n = 54) and LTL (n = 44) testosterone production line averaged 49.0 ng/mL and 27.8 ng/mL (P < 0.01), respectively. Plasma FSH concentrations did not differ between lines (P < 0.30). Selection for testosterone production in response to a GnRH challenge was an effective method of changing testosterone concentrations, testicular size, epididymal weight, and volume density of Leydig cells. However, daily sperm production per gram of testes was unchanged. Based on the results of this study, selection for testosterone production is not recommended as a method of increasing sperm production in pigs.
Key Words: Pigs • Reproduction • Selection • Testosterone Production
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Introduction
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Female reproductive traits in pigs are low to moderately heritable and sex limited. Greater response to selection would be expected if a trait in males, highly correlated with reproduction in females, could be identified. Testosterone production may be such a trait. Testosterone is a steroid hormone produced in Leydig cells (Plant, 1986
). It is part of a feedback regulation system in which steroidogenesis occurs in Leydig cells in direct response to LH stimulation (Odell et al., 1974). Testosterone then acts on the hypothalamus to regulate GnRH pulse frequency, which in turn releases LH from the anterior pituitary. Both LH and FSH are required for testicular development, spermatogenesis, ovulation, and androgen synthesis (Schinckel et al., 1984). Testosterone can serve as a precursor for estradiol. Thus, it might be expected that selection for increased testosterone (TEST) would result in greater TEST production in males and greater estrogen production in females.
Testosterone production is positively correlated with body growth, and testis size is positively correlated with sperm production in boars (Lubritz et al., 1991). In addition, FSH and LH affect reproduction in both sexes, with hormone production being controlled by the same genes (Robison et al., 1994; Rathje et al., 1995). Robison et al. (1994) hypothesized that divergent selection for TEST would result in an indirect response in various production and reproductive traits. Boars from the high-TEST-production line (HTL) had greater TEST concentrations, testis size, and ADG, and their female offspring had greater litter size and ADG than did boars from the low TEST production line (LTL; Robison et al., 1994).
The effect of divergent selection for testosterone production on male reproduction has not been previously explored. The purpose of this study was to characterize effects of divergent selection for testosterone production on testicular morphology and sperm production traits in boars.
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Materials and Methods
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Duroc boars from lines divergently selected for testosterone production were used. Lines were selected 10 generations for TEST production in response to a GnRH challenge (Robison et al., 1994). Lines were then maintained by random selection within each line. Data were obtained from all available HTL and LTL boars in generations 20 (HTL, n = 46 and LTL, n = 13) and 21 (HTL, n = 54 and LTL, n = 44). Boars were weighed and castrated at an average age of 211 d and weight of 97 kg.
Left and right testes and left epidiymis were weighed. Three randomly selected 1-cm cubes and one 30-g filet were immediately taken from the left testis. Cubes were placed in 15-mL conical tubes filled with 4% paraformaldehyde and immediately placed on ice. Samples were then dehydrated using increasing concentrations of ethanol (concentrations other than 100% are vol/vol) and stored in 70% ethanol at 4°C. The ethanol in each tube was changed weekly until samples were further dehydrated in 85, 95, and 100% ethanol. Tissues were then placed in a 1:1 solution of ethanol and xylene for 45 min, transferred to a clean container with xylene only for 45 min, and transferred again to a clean container of xylene for 45 min. Next, tissues were embedded in paraffin wax blocks. After hardening, the mold was removed and tissues blocks were stored at –20°C. Tissues were cut into 6-mm-thick slices using a microtome, placed on microscope slides, and allowed to dry at room temperature for 8 to 12 h. One slide was made from each tissue block. Two slides were selected and stained for histological evaluation. One set of slides was stained with hematoxylin using eosin as a counter stain for determining Leydig cell and seminiferous tubule volume density. Another set was stained using only hematoxylin to determine Sertoli cell volume density. Slides were allowed to dry completely at room temperature and then permanently mounted. Tissue sections were imaged using a light microscope (Olympus VANOX-S, Olympus, Hamburg) and the Optimet 1.1 video imaging software (BioScan Optimetric). Three randomly selected images were taken at 4x magnification for determination of seminiferous tubule volume density and three randomly selected images were taken at 20x magnification for Leydig cell volume density determination. Seminiferous tubules of similar size and stage of development were selected at 20x magnification from each hematoxylin stained slide for determination of Sertoli cell volume density. Volume densities were determined for each sample. Three samples per animal were counted for Leydig cell and seminiferous tubule volume density, and two samples were counted for Sertoli cell volume density. Each volume density estimate was treated as a repeated measure. Volume density of each sample was estimated by using a coherent test system as described by Weibel (1979). Samples were scored independently by two technicians. Each cell that was at least half filled with the structure of interest was considered a "positive" cell. The ratio of "positive" cells to the total number of cells containing testicular tissue was determined to calculate structure volume density within a sample (SVD). The SVD was multiplied by left testis weight to estimate mass of structure within the left testis (ESM).
The 30-g filet sampled from the left testis was placed in a 50-mL conical tube. Tubes were immediately placed on ice and stored at –20°C. Each filet was divided into three equal sections. Sections were homogenized for 30 s in 200 mL of 0.15% PBS with 0.1% Triton-X-100 (concentrations other than 100% are vol/vol). Three 0.5-mL samples were removed and vortexed with 0.5 mL of trypan blue stain. A drop of solution was then placed on a hemacytometer and number of intact mature spermatids was recorded. Estimates of sperm per gram of testis (SPM/g), daily sperm production per gram of testis (DSP/g), total daily sperm production (TDSP), and total testicular sperm (TTS) were calculated according to procedures outlined by Rathje et al. (1995).
Blood samples were taken from all boars in generation 21 at an average of 190 d of age to determine testosterone and FSH concentrations. Blood was collected in 10-mL tubes containing EDTA using 20-gauge needles. One tube of blood was taken from each boar and immediately placed on ice. A second sample was taken from each boar approximately 90 min later. Samples were placed on ice for transport to the laboratory where samples were centrifuged at 1,600 x g at 4°C for 25 min. Plasma was removed by pipette and split between two tubes. Samples were assayed for TEST and FSH. Plasma from each blood sample was kept separate so that variance in concentration due to time could be estimated. Circulating plasma concentrations of TEST were quantified in duplicate using the RIA procedure described by McKinnie et al. (1988). Intra- and interassay CV for TEST assays were 3.2 and 5.8%, respectively. A RIA to determine FSH concentration was done with samples run in duplicate at the USDA-ARS Meat Animal Research Center in Clay Center, NE, using methods described by McCoard et al. (2003). Samples were run within a single assay, and the intraassay CV for the FSH assay was 12.6%.
Data were analyzed using the following models:
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where Li = line effect, Bj = year effect, u = body weight, Tl = technician effect, Im = effect of animal, a = age, Cn = assay effect, and So = sampling effect. Model I was used for weight of both testes (TOTWT) and left epididymal weight (EPIWT). Model II was used to determine morphological differences between HTL and LTL for both SVD and ESM. Model III was used to analyze sperm production traits. Model IV was used to determine differences in TEST and FSH concentrations between lines. Effect of assay was removed for analysis of FSH data. One issue concerning each model was whether to adjust for BW or age. Obviously, boars may be of a common age or BW and still be physiologically different. It was decided that weight traits would be adjusted for BW and sperm and hormone traits would be adjusted for age. It is important to note that line x weight and line x age interactions were tested and were not found to be significant; therefore, they were dropped from all models.
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Results
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Year and BW (P < 0.01) influenced TOTWT and BW (P < 0.01) affected EPIWT. However, differences between line means were unaffected by the adjustment for weight. Total paired testes weight was greater in LTL (Table 1). However, EPIWT was greater in HTL boars (Table 1). Serum, from Generation 21 boars, was assayed for TEST and FSH concentrations. The HTL had greater TEST (Table 1). Fixed effects of assay (P < 0.01) affected TEST, and age (P < 0.10) affected TEST and FSH; however, neither of these adjustments altered the magnitude of the difference between lines. Concentration of FSH did not differ between lines (Table 1).
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Table 1. Least squares means for body weight, age, testicular weight measurements, and testosterone and follicle stimulating hormone concentrations in boars from lines selected for high and low testosterone production
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Morphological characteristics of testes were measured in boars from Generation 20. Sample volume density and ESM were determined for Leydig cells, seminiferous tubules, and Sertoli cells. Technician and line x technician did not significantly affect SVD or ESM. Leydig cell SVD and ESM (Table 2) were greater in HTL boars. The LTL tended to have greater SVD and ESM for seminiferous tubules than HTL (Table 2). Sertoli cell volume density did not differ significantly between LTL and HTL for SVD or ESM (Table 2).
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Table 2. Least squares means for volume density of Leydig cells, seminiferous tubules, and Sertoli cells in boars from lines selected for high and low testosterone production
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Sperm production traits were determined from Generation 20 animals, and all estimates were adjusted for age. However, including age as a covariate in Model III did not account for a significant amount of error. Least squares means estimates for SPM/g, DSP/g, TDSP, and TTS did not differ between the lines (Table 3).
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Table 3. Least squares means for sperm production measurements in boars from lines selected for high and low testosterone production
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Discussion
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By characterizing the correlated responses to divergent selection for testosterone production, we gain a greater understanding of the biology of the testis. Boars from HTL had significantly larger epididymal weights than LTL boars. Development of the epididymis is under the control of testosterone prenatally and its growth increases around the time of puberty when testosterone concentrations increase (Robaire and Hermo, 1988). A decrease in TEST also has been shown to cause epididymal degeneration (Nair et al., 2002). It is likely that lower TEST levels in LTL boars during development had a negative influence on epididymal growth and ultimately epididymal weight. Increased epididymal weights in HTL are likely due to increased testosterone levels. Sperm storage within the epididymis was not measured in this experiment. However, it may be hypothesized that greater epididymal weight may result in greater capacity for sperm storage, which could improve overall fertility.
Daily sperm production was not different between lines despite higher serum testosterone concentrations in HTL boars. This may be because spermatogenesis can proceed at less than maximal testosterone levels. In the rat, intratesticular testosterone concentrations 4 to 20% of normal were sufficient to maintain spermatogenesis (Rommerts, 1988; Sharpe et al., 1989). This indicates that although spermatogenesis requires testosterone, there may be a threshold effect whereby further increases do not result in more sperm production once a certain level of testosterone is achieved. Peter et al. (1980) reported a low correlation (r = 0.12) between sperm production and the concentration of testosterone in the ejaculate of boars. Neither intratesticular nor ejaculate testosterone were measured in the current study but might be expected to be higher in HTL boars.
Boars from HTL had significantly smaller paired testis weights than did LTL boars. However, testis size is not directly related to testosterone production. Testosterone is synthesized in the Leydig cells of the testes (Tripepi et al., 2000); however, factors that control Leydig cell development are mostly unknown. Leydig cell volume density was significantly higher in HTL, which is directly related to testosterone levels. More Leydig cells would be expected to result in greater testosterone production, as was the case for HTL boars. The joint effect of testis size and Leydig cell SVD may be evaluated by considering the Leydig cell ESM. Because ESM was greater in HTL boars, it was concluded that HTL had more Leydig cells despite having smaller testes. Thus, HTL boars had a greater capacity for synthesizing testosterone. Although it may be true that an increased number of Leydig cells creates greater potential for testosterone production, it may be concluded from hemicastration studies that testosterone production is maintained even after one testicle is removed (Kosco et al., 1989; Ford et al., 2001). It is interesting to compare these results to those of Zanella et al. (1999), who reported boars with low FSH had larger testes than boars with high FSH, but production of testosterone in vitro did not differ. These observations are consistent with the role of testosterone in the endocrine feed back regulation system. Testosterone has negative feedback on LH secretion, whereas FSH is primarily regulated by inhibin, which is produced in Sertoli cells (Bardin et al., 1988). Robison et al. (1994) estimated testis size using calipers and found testis size to be greater in HTL. In the current study, boars were castrated and testes were weighed. Paired testis weight was greater in the LTL. The inconsistency between these results was unexpected. We are unable to offer a biological explanation for these conflicting results.
Sertoli cells play a critical role in maturation of germ cells and sperm production (Krantic and Benahmed, 2000; McCoard et al., 2000; Ford et al., 2001). Also, Sertoli cell proliferation is a primary factor in determining testis size and sperm production capacity (Zanella et al., 1999; França et al., 2000; Ford et al., 2001). Because Sertoli cells do not continue to proliferate after puberty, prepubertal plasma FSH levels are important for Sertoli cell proliferation and determination of sperm producing capacity at maturity (França et al., 2000; Krantic and Benahmed, 2000). Results from this experiment were consistent in that no differences were found between lines for Sertoli cell ESM, FSH concentrations, or measurements of sperm production. Seminiferous tubule volume density tended to differ between lines with LTL having a greater volume density than HTL. It is unknown whether volume density of the tubules differed due to a decrease in tubule diameter or length. However, this slight difference in seminiferous tubule volume density did not result in greater sperm production.
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Implications
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Although divergent selection for testosterone production in boars was successful, traits associated with sperm production were unaffected. At this time, selection for testosterone production is not recommended as a means for increasing sperm production to improve boar fertility.
1 Correspondence: 232B Polk Hall, Box 7621 (phone: 919-513-0262; fax: 919-515-7780; e-mail: joe_uscassady{at}ncsu.edu).
Received for publication August 1, 2003.
Accepted for publication April 12, 2004.
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Abstract
Introduction
