Bull reproductive development and sperm production enhancement

Abstract

The onset of puberty and daily sperm production of bulls are fundamental aspects that impact natural breeding and semen production centers. Critical molecular and endocrine aspects of testicular development, spermatogenesis and functions are reviewed. Methods to enhance sperm production (gonadotropin supplementation, immunization against inhibin and induction of temporary hypothyroidism) and findings are discussed.

Keywords: Spermatogenesis, Sertoli cell, endocrinology, puberty, hypothyroidism

Introduction

Important aspects of bull testicular development and sperm production have been reviewed.^1-3  Breeding soundness examinations of thousands of bulls are performed every year before they are used for natural breeding or enter artificial insemination centers. However, on their first examination, approximately 20 - 25% of yearling beef bulls fail to meet minimum standards for testicular size and sperm morphology. ^4,5 For high genetic value bulls entering artificial insemination centers for semen collection and cryopreservation, maximum sperm production is required at each collection.

Development of reproductive system and in particular testicular function, starts during fetal life and continues through postnatal period until puberty (Table). These developmental periods are primarily affected by genetics and environmental factors. Testicular function and spermatogenic efficiency are mainly influenced by factors during prenatal period, prepubertal growth phase and post-pubertal maturation phase. Understanding bull gonadal development facilitates optimal management of seedstock dams and young bulls. Objectives are to review bull developmental biology, factors affecting spermatogenesis and potential ways to improve sperm production.

Development of reproductive system in the fetus

Understanding testicular differentiation and development has facilitated substantial improvement in recent years using molecular tools and advancements in genomics. Differences in gene expression can be detected as early as blastocyst stage. ^6,7 Differentiation of inner cell mass into somatic germ layers (ectoderm, endoderm, and mesoderm) follows hatching. Hypothalamus, pituitary gland, and penis derive from ectoderm, whereas gonads, epididymides, ductus deferens and urinary system derive from mesoderm. Somatic germ cells are pluripotent except some yolk sac inner lining cells that differentiate into primordial germ cells (PGCs). Undifferentiated gonad forms after passive or amoeboid migration of PGCs into genital/gonadal ridge. This phenomenon is likely controlled by several molecular signals that are not yet completely understood. High mitotic index is recognized in PGCs and they rapidly populate genital ridge; 1000 - 2000 PGCs are already present in genital ridge by 25 days and gonads can be identified at 28 - 29 days of pregnancy.

Several key player genes (Wilms tumor-1 factor, Lim1 transcription factor, and Steroid factor) are identified in gonadal development. 8,9 Wilms tumor-1 factor and zinc finger like transcription factor (produced by Sertoli cells) have important roles in testicular cord assembly and regulating development of fetal Leydig cells and peritubular myoid cells.¹⁰ 

Differentiation of fetal testes is determined by SRY gene (Y chromosome sex determining region). Expression of SRY begins on day 37 and peaks on day 39. ¹¹ This gene encodes for a protein of high mobility group and regulates several other genes involved in gonadal differentiation into testis. One of these genes, SOX9, is essential in differentiation of fetal Sertoli cells. Furthermore, GATA4 (required for expression of SRY gene and formation of genital ridge) and DMRT are also implicated.

Table. Chronology of major events during fetal and postnatal development of bull reproductive system

Differentiation of bovine testis starts at 41 - 42 days. At this stage, testicular cords lack a lumen and consist of undifferentiated Sertoli cells. By 60 - 70 days, rete testis appears at end of testicular cords and become connected to each other. Concurrently, first generation of fetal Leydig cells (from mesonephros) appear in mesenchyme. After birth, fetal Leydig cells degenerate and are replaced by adult Leydig cells.

External genitalia become masculinized by day 47 under effects of testosterone and androstenedione produced by Leydig cells. By 60 days, scrotum is well developed. Paramesonephric ducts start regressing by day 50 and are completely regressed by day 80. Seminal vesicles and prostate appear by 56 - 58 days. ¹² By end of first trimester, all components of reproductive system are present except for epididymis (begins to form at 110 days.¹³ ) Internal and external genitalia complete development occurs after 100 days. Descent of testes within scrotum occurs at 100 - 120 days of pregnancy. Testicular descent is mediated by insulin-like peptide ³ (from fetal Leydig cells) that initiates transabdominal phase around 80 - 90 days of pregnancy. Inguinoscrotal phase of testicular descent is mediated by androgens. This early maturation of hypothalamo-pituitary gonadal axis in bovine has been confirmed. Concentrations of insulin-like peptide ³ and testosterone in maternal plasma are significantly higher at 4 and 8 months of pregnancy in cows carrying a male versus female fetus.¹⁴ 

Prepubertal development

Onset of puberty in bulls is highly dependent on nutrition and body growth. Slow growth is correlated to slower testicular development and delayed puberty. In some breeds, scrotal circumference is highly correlated to body weight. ¹⁵ At birth, testes are composed of solid (no lumen) chords comprised of primordial germ cells, fetal Leydig cells and undifferentiated Sertoli cells (Figure 1).¹⁶  Accessory sex glands are not functional and penis is adherent to prepuce. Fetal Leydig cells degenerate in first postnatal month and are replaced by adult Leydig cells. GnRH receptors in anterior pituitary increase significantly from 6 - 10 weeks. ¹⁷ Increased frequency of GnRH pulses and corresponding increases in LH concentrations occur during cell differentiation. Number of undifferentiated Sertoli cells increases rapidly. 16,18 During prepubertal period, serum FSH and inhibin concentrations are high, but decline with onset of rapid testicular growth. ¹⁹ FSH has an important role in prepubertal proliferation of Sertoli cells^{, 20} as it increases at 4 - 25 weeks.²¹

Figure 1:

Proliferation of prospermatogonia began at 1 month, and primary spermatocytes appeared ~ 5 months. ²² Age of puberty was negatively correlated with magnitude of LH secretion in first 5 months of life, ²³ a highly heritable trait.²⁴ 

Detachment of the peno-preputial adhesions occurs ~ 6 weeks prior to puberty. As puberty approaches, testicular and epididymal growth accelerate and become almost linear. Low-frequency LH pulses increase serum testosterone concentrations produced by a rapidly increasing number of adult Leydig cells, promoting establishment of spermatogenesis. ¹⁶ Primary and subsequently secondary spermatocytes are detected 5 - 8 months (Figure 1). Mature sperm are present in seminiferous tubules from 8 - 10 months. Puberty (ejaculate with at least 50 million sperm and at least 10% progressive motility) occurs when scrotal circumference reaches 28 - 30 cm in Bos taurus 2 and 27 - 28 cm in Bos indicus.²⁵ 

Following puberty, bulls enter a phase of maturation characterized by increases in accessory sex gland weights, sperm production and proportion of morphologically normal sperm. This phase of maturation varies considerably amongst bulls, lasting 3 - 4 months in Bos taurus.²⁶ 

Effect of nutrition on early development

Nutrition of the dam profoundly affects testicular development. ²⁷ Heifers fed 2.4 x recommended energy and protein requirement in the first 6 months of pregnancy produced calves that had smaller testes and lower serum testosterone concentrations compared to calves from heifers fed 1.9 x or 0.7 x recommended ration. ²⁷ Santa Gertrudis bull calves from dams fed low-protein diet had delays in onset of puberty and sexual maturity than those from dams fed high-protein diet. ²⁸ However mechanism of this action remains unclear.

Bull calves fed diets to achieve 1.4 - 1.5 kg/day average daily gain reach puberty earlier with greater paired testes weight and daily sperm output. ^2,29,30 In general, with an average daily gain of 1 kg/day, puberty occurs at 8 - 12 months. ³¹ Preweaning early development of bulls was largely dependent on maternal milk production. Bull calves born to heifers and aged dams (> 9 years) had smaller scrotal circumference due to lower hypothalamo-pitutary activity. ² However, effects of diet on onset of puberty are confounded by genetics and greater feed intake does not consistently result in earlier puberty.³² 

It is important to note that a high-energy diet, such as often fed in bull testing stations, may have adverse effects on testicular development and spermatogenesis. Young bulls fed high energy diets have better average daily gain and thicker backfat, but their scrotal circumference may not be altered^.2,3,34  However, these bulls have more morphologically abnormal sperm due to poor testicular thermoregulation attributed to increased scrotal fat.^2,33,34 

Enhancement of sperm production

Enhancement of sperm production in valuable bulls is important to satisfy the demand for more doses of semen (particularly sexed semen) for artificial insemination. In addition to care and nutrition of the pregnant dam, several other postnatal strategies were investigated to increase bull sperm production. Unilateral castration increased the contralateral testicular size but failed to increase sperm production. In recent years, our understanding of the role of Sertoli cell as support cell for spermatogenesis, provides new approaches to enhance sperm production.

The role of Sertoli cells in spermatogenesis was studied in a variety of species. ^20,35 Strong positive correlation between Sertoli cell number and sperm production capacity in bulls was established.²⁰ 

Sertoli cell number correlated with number of spermatogonial stem cell niches in mice, critical for spermatogenic activity and sperm production.^36,37 

Postnatal expansion of Sertoli cell population was described in several mammalian species as a 2 wave linear increase in Sertoli cell population. ³⁷ Termination of Sertoli cell proliferation appears to be an extremely important biological point for manipulating spermatogenesis. ³⁷ Increases in testicular estrogen concentrations arrest Sertoli cell expansion in swine but apparently not in bulls. ³⁵ In the latter, based on early cytological studies, Sertoli cell number plateaus at 6 - 7 months. ²² However, based on cell proliferation marker KI67 and expression of Sertoli cell marker SOX9, Sertoli cell proliferation ceases at 4.5 - 5 months (Figure 2).³⁸ 

During prepubertal development, Sertoli cell proliferation is stimulated by FSH but inhibited by thyroid hormone concentrations. This relationship between Sertoli cell proliferation and thyroid hormone concentrations was demonstrated in boars ³⁹ and rams. ⁴⁰ In bulls, thyroxine concentrations were negatively correlated with testicular size at puberty. 41 These observations led to development of hormonal methods during early development to enhance sperm production. These hormonal methods include immunization against inhibin, GnRH injections, FSH injections, and establishment of a transient hypothyroidism.

Immunization against inhibin in early postnatal period increases serum FSH concentrations, testicular size, and daily sperm production in bulls. ⁴² However, it was transient and required multiple vaccinations. There was also a great variability of the response among individuals. Administration of GnRH to prepubertal bulls (120 µg/kg) BID from 4 - 8 weeks hastened puberty by 6 weeks compared to untreated bulls.⁴³

Figure 2.

Bull calves given exogenous FSH every 2 days for 4 - 8 weeks had more Sertoli cells number at 56 weeks and attained puberty 5 weeks earlier than nontreated calves. ⁴⁴ In a more recent study, administration of porcine FSH (30 mg, Folltropin-V in 2% hyaluronic acid) from 35 - 91 days increased endogenous FSH at 70 days and Sertoli cell numbers per seminiferous tubule at 93 days, ⁴⁵ with increase in endogenous FSH due to a positive feedback loop through Activin A produced by Sertoli cells. FSH-treated bulls had on an average 4 more Sertoli cells per seminiferous tubule section than untreated bulls. ⁴⁶ However, there was no difference in spermatogonial cell number.⁴⁶ 

Potential for enhancement of sperm production after induction of transient hypothyroidism in bulls (based on negative relationship between Sertoli cell proliferation and circulating thyroid hormones concentrations) was investigated. ³⁸ To induce hypothyroidism, bull calves received Methimazole (2mg/kg BW, BID) from 4 - 6 months. Age of puberty was delayed with increased sperm production. Bulls subjected to transient hypothyroidism had 30 - 180% more sperm per ejaculate than control bulls, with no effect on sperm motility, morphology, post-thaw survivability or rates of IVF cleavage and blastocyst development. Treated bulls had a 22% higher mean paired testes weight and 50% higher epididymal weight compared to untreated bulls. Finally, treated bulls had a 2.3-fold increase in number of Sertoli cells per cross section of seminiferous tubule than control bulls. These promising results are impetus for further studies.

Conclusion

In the current era of genomic testing, sire selection can be performed very early in life and bull development and sperm production are increasingly important for efficient use of elite sires. In this context, studies are needed to dissect Sertoli cell-specific role in sperm production and methods to enhance it. Current knowledge on prenatal and postnatal bull development suggests that these phases can be modulated through judicious nutritional supplementation of dams and postnatal hormonal treatments of bull.

Conflict of interest :There are no conflicts of interest to declare.

References

1. Brito LFC, Barth AD, Wilde RE, et al: Effect of growth rate from 6 to 16 months of age on sexual development and reproductive function in beef bulls. Theriogenology 2012;77:1398-1405.
2. Brito LFC: Factors affecting bull sexual development. Clinical Theriogenology 2019;11:347-351.
3. McGowan M, Holland MK, Boe-Hansen G: Review: ontology and endocrinology of the reproductive system of bulls from fetus to maturity. Animal 2018;12:s19-s26.
4. Kennedy SP, Spitzer JC, Hopkins FM, et al: Breeding soundness evaluations of 3648 yearling beef bulls using the 1993 Society for Theriogenology guidelines. Theriogenology 2002;58:947-961.
5. Monday JD, Larson RL, Theurer ME, et al: Factors associated with yearling bulls passing subsequent breeding soundness evaluations after failing an initial evaluation. J Am Vet Med Assoc 2018;253:1617-1622.
6. Reijo Pera R, Prezzoto L: Species specific variation among mammals. Curr Top Dev Biol 2016;120:401-420.
7. Heras S, Coninck DIMd, Poucke MV, et al: Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts. BMC Genomics 2016;17:72 DOI 10.1186/s12864-016-2393-z
8. Davies J, Fisher C: Genes and proteins in renal development. Exp Nephrol 2002;10:102-113.
9. Kohler B, Achermann J: Update – steroidogenic factor 1. Minerva Endocrinol 2010;35:73-86.
10. Wen Q, Wang Y, Tang J, et al: Sertoli cell WT-1 regulates peritubular myoid cell and fetal Leydig cell differentiation during fetal testis development. PLoS One 2016; 30:11. DOI:10.1371/journal.pone.0167920
11. Ross D, Bowles J, Hope M, et al: Profiles of gonadal gene expression in the developing bovine embryo. Sex Dev 2009;3:273-283.
12. Vigier B, Prépin J, Jost A: Chronology of development of the genital tract of the calf fetus. Arch Anat Microsc Morphol Exp 1976;65:77-101.
13. Alkafafy M, Sinowatz F: Prenatal development of the bovine epididymis: light microscopical, glycochemical and immunohistochemical studies. Acta Histochemica 2012;114:682-694.
14. Kibushi M, Kawate N, Kaminogo Y, et al: Fetal gender prediction based on maternal plasma testosterone and insulinlike peptide 3 concentrations at midgestation and late gestation in cattle. Theriogenology 2016; 86:1764-1773.
15. McGowan M, Muller T, Lisle A, et al: Recommended minimum scrotal circumference for tropically-adapted b eef bulls in northern Australia. Reprod Domest Anim 2012;47 (suppl. 4):519.
16. Rawlings N, Evans A, Chandolia R, et al: Sexual maturation in the bull. Reprod Domest Anim 2008;43 (suppl. 2):295-301.
17. Amann RP, Wise ME, Glass JD, et al: Prepubertal changes in the hypothalamic-pituitary axis of Holstein bulls. Biol Reprod 1986;34:71-80.
18. Wrobel K: The postnatal development of the bovine Leydig cell population. Reprod Domest Anim 1990;25:51-60.
19. Evans A, Pierson R, Garcia A, et al: Changes in circulating hormone concentrations, testes histology and testes ultrasonography during sexual maturation in beef bulls. Theriogenology 1996;46:345-357.
20. Berndtson W, Igboeli G, Parker W: The numbers of Sertoli cells in mature Holstein bulls and their relationship to quantitative aspects of spermatogenesis. Biol Reprod 1987;37:60-67.
21. Bagu ET, Cook S, Gratton CL, et al: Postnatal changes in testicular gonadotropin receptors, serum gonadotropin, and testosterone concentrations and functional development of the testes in bulls. Reproduction 2006; 132:403-411.
22. Curtis S, Amann R: Testicular development and establishment of spermatogenesis in Holstein bulls. J Anim Sci 1981;53:1645-1647.
23. Evans A, Davies F, Nasser L, et al: Differences in early patterns of gonadotrophin secretion between early and late maturing bulls, and changes in semen characteristics at puberty. Theriogenology 1995;43:569-578.
24. Corbet N, Burns B, Johnston D, et al: Male traits and herd reproductive capability in tropical beef cattle. 2. Genetic parameters of bull traits. Anim Prod Sci 2013;53:101-111.
25. Chase C, Chenoweth P, Larsen R, et al: Growth, puberty and carcass characteristics of Brahman-, Senepol-, and Tulisired F1 Angus bulls. J Anim Sci 2001;79:2006-2015.
26. Lunstra D, Echternkamp S: Puberty in beef bulls: acrosome morphology and semen quality in bulls of different b reeds. J Anim Sci 1982;52:638-648.
27. Sullivan T, Micke G, Greer R, et al: Dietary manipulation of Bos indicus x heifers during gestation affects the prepubertal reproductive development of their bull calves. Anim Reprod Sci 2010;118:163-170.
28. Copping KJ, Ruiz-Diaz MD, Rutland CS, et al: Peri-conception and first trimester diet modifies reproductive development in bulls. Reprod Fert Develop 2018;30:703-720.
29. Brito L, Barth A, Rawlings N, et al: Effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls. Domest Anim Endocrinol 2007;33:1-18.
30. Dance A, Thundathil J, Blondin P, et al: Enhanced early-life nutrition of Holstein bulls increases sperm production potential without decreasing postpubertal semen quality. Theriogenology 2016;86:687-694.
31. Barth AD, Brito LFC, Kastelic JP: The effect of nutrition on sexual development of bulls. Theriogenology 2008;70:485-494.
32. Harstine B, Maquivar M, Helser L, et al: Effects of dietary energy on sexual maturation and sperm production in Holstein bulls. J Anim Sci 2015;93:2759-2766.
33. Bollwein H, Janett F, Kaske M: Effects of nutrition on sexual development of bulls. Anim Reprod (Belo Horizonte) 2017;14:607-613.
34. Bourgon SL, Amorim MDd, Chenier T, et al: Relationships of nutritional plane and feed efficiency with sexual development and fertility related measures in young beef bulls. Anim Reprod Sci 2018;198:99-111.
35. Berger T: Testicular estradiol and the pattern of Sertoli cell proliferation in prepuberal bulls. Theriogenology 2019;136:60-65.
36. Oatley MJ, Racicot KE, Oatley JM: Sertoli cells dictate spermatogonial stem cell niches in the mouse testis. Biol Reprod 2011;84:639-645.
37. Oatley MJ, Kaucher AV, Yang Q, et al: Conditions for long-term culture of cattle undifferentiated s permatogonia. Biol Reprod 2016;95:1-10.
38. Waqas MS, Ciccarelli M, Oatley MJ, et al: Enhanced sperm production in bulls following transient induction of hypothyroidism during prepubertal development. J Anim Sci 2019;97:1468-1477.
39. Sun Y, Yang W, Luo H, et al: Thyroid hormone inhibits the proliferation of piglet Sertoli cell via PI3K signaling pathway. Theriogenology 2015;83:86-94.
40. Oluwole OA, Bartlewski PM, Hahnel A: Relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to Sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs. Reprod Biol 2013;13:150-160.
41. Cestnik V: Higher thyroid hormone levels in neonatal life result in reduced testis volume in postpubertal bulls. Int J Androl 1998; 21:352-357.
42. Bame JH, Dalton JC, Degelos SD, et al: Effect of long-term immunization against inhibin on sperm output in bulls. Biol of Reprod 1999;60:1360-1366.
43. Madgwick S, Bagu ET, Duggavathi R, et al: Effects of treatment with GnRH from 4 to 8 weeks of age on the attainment of sexual maturity in bull calves. Anim Reprod Sci 2008;104:177-188.
44. Bagu ET, Madgwick S, Duggavathi R, et al: Effects of treatments with LH or FSH from 4 to 8 weeks of age on the attainment of puberty in bull calves. Theriogenology 2004;62:861-873.
45. Harstine BR, Cruppe LH, Abreu FM, et al: Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls I: endocrine and testicular development of beef bulls. Theriogenology 2018;105:142-149.
46. Harstine BR, Cruppe LH, Abreu FM, et al: Impact of a timed-release follicle-stimulating hormone treatment from one to three months of age on endocrine and testicular development of prepubertal bulls. J Anim Sci 2017;95:16691679.

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.