Semen

The semen is a biological fluid containing spermatozoa and seminal plasma. Seminal plasma is the cumulative secretions of accessory sex glands. The mixing of seminal plasma and spermatozoa occurs in the urethra during ejaculation.

Table 20.7 The factors affecting the spermatogenesis

(continued)

Table 20.7 (continued)

Table 20.8 Composition, source, and function of seminal plasma

Table 20.9 Composition of seminal plasma in some domestic animals (in mg/dL unless otherwise stated)

BBU Berger-Broida units; BU Bodansky units; LDH lactate dehydrogenase; SFU Sigma Frankel units; UI international units

Source: ¹ Juyena and Stelletta (2012), ² Wales and White (1965), ³ Talluri et al.

Table 20.10 Production and requirement of spermatozoa for fertilization in domestic animals

^a The number of spermatozoa may be doubled, and possible females to be bred would halve when semen is used in frozen semen technology; however, the potentiality of the animal and characteristics of semen can influence the number of required spermatozoa

Table 20.11 Seminal volume and characteristics of spermatozoa in some free-living wildlife

NA not available

the functions of spermatozoa. Therefore, good quality semen production is one of the main contributors to male fertility and depends upon several factors like breed, age, nutritional status, season, and endocrine factors. Some chemicals and drugs can also modulate semen character.

20.2.1 Formation of Seminal Plasma

Different components of seminal plasma are mainly pro­duced from the accessory sex glands. These components mix at the time of ejaculation. The initial ejaculates comprise the secretions of Cowper (bulbourethral) glands that consti­tute around 5% of total ejaculate. The second fraction of the ejaculate derived from the prostate consists of about 15-30% of the total ejaculates. A small portion of the secretions from the ampulla and epididymis occurs following the second fraction. Finally, the remaining amount of the ejaculate secretes from the seminal vesicle. The composition of semi­nal plasma summarizes in Table 20.8. The biochemical com­position of the semen in different species presents in Table 20.9. Various biochemical tests are generally performed to assess seminal plasma’s biochemical constituents to evaluate the functions of reproductive organs. Fructose is the biomarker for assessing seminal vesicles; citric acid, zinc, and acid phosphatase for the prostate gland; free L-carnitine, glycerophosphocholine, and alfa- glucosidase for the epididymis.

required for fertilization depends on the initial progressive motility and morphological features. In domestic animals, the progressive motility and morphologically normal spermatozoa are generally 70-90%, which is reversed in wild animals. The fresh semen may contain a more morpho­logically abnormal percentage of spermatozoa with less pro­gressive motility in wild species. The clinical terminologies related to spermatozoa concentration and motility summarize in Table 20.12. The morphological abnormalities of spermatozoa are of three types. Primary abnormality or struc­tural deformity is related to hereditary or developmental origin. Secondary abnormalities are associated with func­tional alterations of the reproductive tract. Tertiary abnormalities are due to semen handling and processing. In ruminant and canine species, the sperm motility and viability in the semen are nearly 70-80% and morphological abnormalities are lower than 30%. Morphological abnormalities are evaluated microscopically. In recent years, a computer-assisted integrated sperm analysis system (ISAS) has been used to assess sperm motility, viability, and structural configurations. The DNA integrity of spermatozoa can be evaluated through Comet assay or sperm chromatin structure assay (SCSA). Sperm fluorescence in situ hybridization analysis (FISH) is also performed to assess fertilizing capability of the spermatozoa. The sperm mem­brane integrity can evaluate through the Hypoosmotic sperm swelling test (HOST), and acrosome integrity can analyse through fluorescent staining.

20.2.2 Sperm Concentration in the Semen

The concentration of spermatozoa in the semen mainly varies between species, breed, and ejaculatory fractions (Table 20.10 and 20.11). The effective sperm concentration

20.2.3 Sperm Quality Biomarkers

Protamines (PRM), nuclear proteins, are considered the biomarkers of sperm quality. Mammalian spermatozoa have two types of protamines, PRM1 and PRM2. Their

Table 20.12 Some common terminology used in semen evaluation

proportions vary between species, and altered PRM1 and PRM2 ratios may lead to infertility.

20.2.4 Properties of Semen

20.2.4.1 Volume

The semen volume varies with species (Tables 20.9 and 20.10), age, body size, and between ejaculates. The semen volume is fairly constant with species but may differ between ejaculates. The semen volume increases with age, body size, and vigour. Teasing the bulls is practised to increase semen volume. A decrease in semen volume is seen at a young age, excessive use of males for breeding, incomplete ejaculation, and bilateral seminal vesiculitis. Domestic animals that con­tain lower semen volume have higher sperm concentrations.

20.2.4.2 Colour

The semen of bull and buck is creamy, milky white, or opaque, and buffalo bull is whitish compared to bull semen. The semen colour of stallions, boar, and dogs is pearly white to grey and translucent. The deviation from the normal colour indicates pathological conditions. Orchitis or haemorrhage in the male reproductive system leads to brown, dark red, or pinkish coloured semen. Semen can turn into yellow-green in Pseudomonas aeruginosa infection. An increased number of spermatogenic cells in the semen may cause dull and dirty white semen. Urine contamination leads to yellowish semen colour. Semen appears curdy with the presence of clots in genital tract infections.

20.2.4.3 Odour

The typical odour of the semen is due to the presence of basic amines, like putrescine, spermine, spermidine, and cadaver­ine. Semen may have an unusual smell like strong, fishy due to reproductive tract infections.

20.2.4.4 Viscosity

The consistencies of semen may vary from watery, milky, or creamy, depending on the spermatozoa concentration. The specific gravity of the semen of the bull is 1.0361. There is a positive correlation between spermatozoa’s viscosity and sperm concentration in semen. Various pathological conditions will lead to alteration in the viscosity of semen, viz. less milky (pathology of the epididymis), purulent flocculi (in seminal vesiculitis), and thick viscous (catarrhal conditions of accessory sex glands).

20.2.4.5 pH and Buffering Capacity

The semen has a high buffering capacity (higher than most of the body fluids), which helps maintain its pH near neutrality in an acidic vaginal environment to protect the DNA of spermatozoa. The components contributing to the buffering action of the semen are HCO3/CO2 (contribute 25% of buff­ering activity), proteins (29% of buffering capacity), low- molecular weight components, viz. phosphate, citrate, and pyruvate (47% of buffering capacity). The pH of the rumi­nant semen is slightly acidic, as the bulbourethral gland is smaller in size (Table 20.13). The bulbourethral gland is absent in canines; hence, the pH of semen is also slightly acidic.

20.2.4.6 Osmolarity

The osmolarity of the semen is higher than plasma. The presence of sugars and ionic salts contributes to this high osmolarity. The osmolarity of human semen varies from 250 to 400 mOsm. The osmolality of stallion semen ranges from 331 to 336 mOsm.

Table 20.13 The semen pH varies with species

20.2.5 Ejaculation

Ejaculation is the biological process by which the seminal plasma is forcefully expelled out of the body. The ejaculation occurs through synchronized physiological events and is divided into two phases, emission and expulsion. The spermatozoa moved from the epididymis to the prostatic urethra and mixed with the seminal plasma during emission. During the emission phase, the neck of the bladder is closed to prevent retrograde movement of semen into the bladder. After the bladder neck closure, the spermatozoa coming through the vas deferens mix with prostatic secretions in the prostatic urethra. The spermatozoa containing prostatic secre­tion then mix with seminal vesicle fluid. The secretions from the Cowper ’s glands contribute a little to the seminal fluid. The expulsion phase initiates once the emission phase is over. It mediates through rhythmic contraction of striated pelvic perineal muscles, and the urethral sphincter facilitates the ejection of semen through the penis via the urethral meatus. There are species variations regarding the mixing of spermatozoa with seminal plasma. In bull, ram, and buck, the spermatozoa are mixed instantaneously with the seminal plasma. In contrast, spermatozoa are mixed sequentially with the fluid of the accessory sex glands in the stallion, boar, and dog; hence the ejaculates from these species contain three first ‘sperm free’, followed by ‘sperm rich’ and ‘sperm poor’ fractions of semen.

20.2.5.1 Neural Control of Ejaculation

The process of ejaculation is mediated through complex neurovascular mechanisms. There are several central and peripheral neurological factors involved in ejaculation. The encapsulated nerve endings termed Krause-Finger corpuscles at glance penis and free nerve endings in other portions of the penis are temperature and pressure-sensitive. The tactile receptors in bull and ram are more sensitive to warmth, and the receptors in stallion and boar are more sensitive to slip­periness or pressure within the vagina. During the natural coitus or in an artificial vagina stimulates, these tactile receptors of the penis, and the signals are transmitted through two afferent sensory inputs. The main sensory input comes through the dorsal nerve of the penis. The other sensory afferent input comes through the hypogastric nerve of the paravertebral sympathetic chain. These two afferent inputs terminate at the medial dorsal horn and the dorsal grey commissure of the spinal cord. The efferent nervous system of ejaculation constitutes sympathetic, parasympathetic, and motor nerves. The locations of preganglionic sympathetic and parasympathetic fibres are thoracolumbar segments of the spinal cord and sacral parasympathetic nucleus, respec­tively. The motor neurons are located in the sacral spinal cord. The sympathetic nerves innervate to the smooth muscle of epididymis, vas deferens, seminal vesicles, and prostate

Table 20.14 Role of various neurotransmitters and hormones in the ejaculation process

and facilitate the emission process. The somatic nerves are innervated in ischiocavernosus, bulbocavernosus, and ischiourethralis muscles. The parasympathetic nerves are connected with erectile tissues. Various neurotransmitters and hormones are involved in the ejaculation process. They are summarized in Table 20.14.

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Seminal Characteristics in Wild Animals

Testosterone level is reduced in wild animals due to inbreeding, affecting semen quality. The testosterone concentration of outbred Asiatic lions demonstrated (1.8 ng/mL) is higher than the inbred Indian lions (less than 1 ng/mL), as found in the Gir forest of India, which leads to the production of 60% abnormal with pleomorphic spermatozoa. The high proportions of pleiomorphic spermatozoa have also been reported in wild-born free-living cheetahs (Acinonyx jubatus) in Namibia of Africa, and other sanctuaries or reserves. The captive wild animals may donate more volume of semen, but the concentration of spermatozoa is more in a free-living wild animal.

Learning Outcomes

• Spermatogenesis: Spermatogenesis is a series of complex synchronized processes of mitotic and mei- otic cell divisions, which occur in the seminiferous tubules of the testis. It has two distinct phases, spermatocytogenesis and spermiogenesis. Various hormones like testosterone, FSH and oestrogens have been involved in spermatogenesis. The time required to complete the entire process differs between species, and birds need very little time to complete the spermatogenesis.

• Spermatocytogenesis: Spermatocytogenesis is initiated by mitotic divisions and ends with the early stages of meiotic divisions in a cyclic process called the spermatogenic cycle. The spermatogonia are the germinal epithelium cell layers, considered stem cells. It continuously generates type A spermatogonia and is stored as an intermediate stage. Later, it differentiated into type B spermatogonia followed by primary spermatocytes. The primary spermatocytes generate secondary spermatocytes by first meiotic division with the influence of testosterone after puberty. Secondary spermatocytes undergo second meiotic division and form spermatids through a testosterone-independent process.

• Spermiogenesis: The haploid spermatid transformed into spermatozoa by metamorphic change through spermiogenesis under the influence of FSH. Sertoli cells play a major role in this pro­cess. It has five distinguished steps, Golgi phase, cap phase, acrosomal phase, and maturation stage. The mature spermatozoa contain a distinct head, neck, mid-piece and tail and various cellular organelles. At the end of this phase, the spermatozoa become elongated and leave the Sertoli cells through spermiation.

Nucleus and cellular organelles are typically shaped with the spermatozoa acquiring various proteins and enzymes required for fertilization.

• Spermatogenic efficacy: All the spermatogonia are not converted to spermatozoa; some degenerate dur­ing different stages of spermatogenesis. The effec­tiveness of spermatozoa production depends upon the testicular mass, including the number of stem cells and Sertoli cells and the diameter and length of the seminiferous tubules. Different species have dif­ferent spermatogenic efficacy and can be categorized as high, average, and low. Various stressors, mainly heat stress, reduce the effectiveness.

• Semen: Spermatozoa, along with seminal plasma, constitute the semen. Its characteristics vary in dif­ferent species. The quality of the semen of a partic­ular species also depends upon the breed, age, nutritional status, season, and hormonal status of the animal. Various tests are performed to evaluate semen before using it for breeding purposes.

• Spermatozoa: Morphologically normal

spermatozoa with progressive motility are required for fertilization. Species-specific sperm morpholog­ical features and concentration, along with its via­bility and motility, can be assessed by routine semen evaluation techniques. The integrity of its acrosome, DNA fragment, and some other cellular characteristics can be evaluated by advanced tests.

• Seminal plasma: Different species have different physical and biochemical characteristics. Seminal plasma contains various enzymes, proteins, phospholipids, and other biochemical components to support the spermatozoa in increasing its fertilizing ability and viability.

• Ejaculation: The semen is forcefully expelled out of the body through synchronized physiological events into two phases, emission and expulsion. Several neurohormones and hormones are involved in the complex neurovascular mechanisms of ejaculation.

Exercises

Objective Questions

Q1. Which cells are considered the stem cells of the semi­niferous tubule?

Q2. Which type of cell division occurs in spermatocytogenesis?

Q3. Are spermatids haploid or diploid cells?

Q4. What are intermediate spermatogonia?

Q5. Which stage of spermatocytogenesis is testosterone dependent?

Q6. Which part of the seminiferous tubules holds spermatocytogenesis?

Q7. Why is thermoregulation significant in

spermatogenesis?

Q8. Which phase of spermatogenesis occurs within Sertoli cells?

Q9. Which structure of the spermatozoa contains lysosomal enzymes?

Q10. Significant structural changes occur in which phase of spermiogenesis?

Q11. Which structure is formed in combination with the group of microtubules?

Q12. Citric acid is evaluated to assess the functional activity of which accessory sex gland?

Q13. Which part of the spermatozoa provides energy to the spermatozoa for its movement?

Q14. Spermiation is occurred at which part of the seminifer­ous tubule?

Q15. What is the common duration of the spermatogenic cycle in mammals?

Q16. What is the major cause of the production of pleomor­phic spermatozoa in wild animals?

Q17. In which process the permeability of the sperm mem­brane to bicarbonate is increased?

Q18. Write the name of the following animals serially, according to the concentration of spermatozoa in ejaculate—bull, buck, boar, and tomcat.

Q19. What do you mean by oligozoospermia?

Q20. What is the major source of energy in seminal plasma?

Subjective

Q1. Why is spermatocytogenesis considered a proliferative process?

Q2. Write the different steps of spermiogenesis.

Q3. Describe the spermatogenic cycle.

Q4. What is spermatogenic efficiency?

Q5. How heat stress affects sperm characteristics?

Q6. Write down the unique features of avian spermatogenesis.

Q7. Describe the factors that determine the fertilizing capabilities of spermatozoa.

Answer to Objective Questions

A1. Spermatogonia.

A2. Both the mitotic and meiotic.

A3. Haploid cells.

A4. The resting type A spermatogonia.

A5. First meiotic division.

A6. At the adluminal compartment of the tubule between Sertoli cell and basal layer.

A7. The DNA polymerase and recombinase activities are altered with the fluctuation of testicular temperature.

A8. Spermiogenesis.

A9. Acrosome.

A10. Golgi phase.

A11. Axoneme.

A12. Prostate gland.

A13. Mid-piece.

A14. The lumen of the seminiferous tubules.

A15. 30-75 days.

A16. Inbreeding.

A17. Hyperpolarization.

A18. Buck, tomcat, bull, and boar.

A19. Very less sperm concentration in the fresh ejaculate.

A20. Fructose.

Keywords for the Answer to Subjective Questions

A1. Mitotic divisions, meiotic divisions, number of cells generated from spermatogonia.

A2. Five stages of spermiogenesis, structural changes of the spermatids, spermiation.

A3. Various developmental stages of spermatozoa, longitu­dinal movement, cyclic process.

A4. Degeneration of germ cells, testicular mass, factors alter the spermatogenic efficiency.

A5. Testicular thermoregulation, effect on sperm morphol­ogy, structural deformity.

A6. No stem cell, longitudinal structure, less survivability.

A7. Routine semen evaluation, advance test, morphology and biomarker of sperm quality.

Further Reading

Textbooks

Deviche P, Hurley LL, Fokidis HB (2011) Avian testicular structure, function, and regulation, Ch: 2. In: Hormones and reproduction of vertebrates, Volume 4d (Birds). Elsevier, pp 27-70

Research Articles

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Briz MD, Bonet S, Pinart B, Egozcue J, Camps R (1995) Comparative study of boar sperm coming from the caput, corpus, and cauda regions of the epididymis. J Androl 16(2):175-188

Carluccio A, Tosi U, Battocchio M, Veronesi MC, De Amicis I, Contri A (2006) Citric acid and fructose seminal plasma concentrations and semen characteristics in the stallion. Ippologia Cremona 17(1):29-32

Crosier AE, Pukazhenthi BS, Henghali JN et al (2006) Cryopreservation of spermatozoa from wild-born Namibian cheetahs (Acinonyx jubatus) and influence of glycerol on cryosurvival. Cryobiology 52:169-181. https://doi.org/10.1016/j.cryobiol.2005.10.011

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