Table of contents
ABBREVIATIONS AND GLOSSARY
1.1 ORIGINS OF THE PROJECT
1.1.2 Norwegian breeding program of NRF
2.1.1 Spermatozoal structure and function
2.2 SPERM MATURATION
2.3 SPERM TRANSPORT IN THE FEMALE REPRODUCTIVE TRACT
2.3.1 Capacitation and acrosome reaction
2.3.2 ATP production
2.4 EVALUATION OF SPERM QUALITY
2.4.1 The initial semen evaluation
2.4.2 Sperm motility
2.4.3 Sperm viability
2.4.4 Acrosome integrity
2.4.5 ATP Assay
2.5 TECHNIQUES USED FOR SPERM ASSESSMENT
2.5.1 Flow cytometry
2.6 THE AIM OF THE STUDY
3. MATERIALS AND METHODS
3.1 EXPERIMENTAL PLAN
3.1.1 Analysis of selected parameters of fresh and frozen samples from 20 NRF bulls
3.1.2 Analysis of selected quality parameters of frozen samples from eight NRF bulls with known fertility
3.1.3 Analysis of selected quality parameters of fresh and frozen samples from NRF bulls with known proportion percentage live-dead cells
3.3 SEMEN SAMPLES
3.4 PREPARATION OF SPERM SAMPLES
3.4.1 Flow cytometry and luminometer instrumentation
3.5 ANALYSIS OF SPERM QUALITY PARAMETERS BY FLOW CYTOMETRY
3.5.1 Viability and acrosome integrity
3.6 MEASUREMENT OF ATP CONTENT OF BULL SPERMATOZOA USING LUCIFERIN-LUCIFERASE ASS
3.6.1 Optimization of a protocol for measurement of ATP content in bull sperm cells by luminometer
3.6.2 ATP standard curve preparation
3.6.3 Measurements the ATP content of spermatozoa in fresh and frozen semen
3.7 FURTHER OPTIMIZING OF THE PROTOCOL FOR ANALYSIS OF SPERM VIABILITY AND ATP CONTENT WITH KNOWN PROPORTION PERCENTAGE OF LIVE-DEAD SPERM CELLS
3.8 STATISTICAL ANALYSIS
4.1 ANALYSIS OF SPERM QUALITY PARAMETERS
4.1.1 Sperm viability, acrosome integrity and ATP content of fresh and frozen samples
4.1.2 Comparison of decrease of % AIL and ATP content under different incubation times
4.2 ATP CONTENT ADJUSTED FOR % AIL
4.2.1 Categorization of semen samples based on ATP content adjusted for % AIL
4.3 VIABILITY, ACROSOME INTEGRITY AND ATP CONTENT TESTED FOR CORRELATION WITH DAYS NRR
4.4 ANALYSIS OF FRESH AND FROZEN SAMPLES WITH KNOWN PROPORTION PERCENTAGE LIVE- DEAD SPERM CELLS (CONTROL EXPERIMENT)
5.1 FURTHER STUDY
This master work has been carried out at the laboratories of Hedmark university collage. In this work, Professor Elisabeth Kommisrud from Hedmark University College was the main supervisor and Fride Berg Standerholen was a co-superadvisor.
Firstly, I would like to thank my supervisor Professor Elisabeth Kommisrud for her patience, support, ideas, encouragement and guidance. I am very grateful for getting the opportunity to have you as my supervisor. I have learned a lot from being your student.
A further special thanks to Fride Berg Standerholen for the excellent guidance, support, advice and your encouragement in difficult situations
I would like to thank Dr. Arne Linløkken for supporting during statistical analysis. Special thanks to Else Berit Stenseth and Teklu T. Zeremichael for great help in the lab.
Thanks to my wonderful family for supporting throughout studying, my wife Samah Al- Najjar and to my son Mohammed Al-Medhati and my daughters Hana Al-Medhati and Line Al-Medhati
Abbreviations and Glossary
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Acrosome: Membrane enclosed organelle covering the anterior part of nucleus in spermatozoa that contains hydrolytic enzymes for penetration of outer membrane of ovum (zona pellucida).
Acrosome reaction: Fusion of spermatozoal plasma membrane with the outer acrosomal membrane that initiates the release of hydrolytic enzymes from acrosome enabling the sperm to penetrate zona pellucida.
Adenosine triphosphate (ATP): The energy source of sperm cells which is required for the maintenance of sperm cells motility.
Apoptosis: A physiological programmed cell death, which affects cells without an inflammatory reaction in the surrounding tissue.
Capacitation: Membranous and intracellular biochemical transformations on sperm cells that is required to render spermatozoa competent to fertilize an egg.
Estrus: A regularly recurrent state of sexual excitability and during this phase female animal will accept the male and begin ovulate.
Flow cytometry: System for analysis physical and chemical characteristics of particles as they flow in fluid stream through a sensing point. Can be used for measuring relative granularity, size and fluorescence intensity as well as internal complexity of the cells.
Glycolysis: Metabolic process in which glucose is converted to pyruvate and ATP.
Leydig cells: Cells adjacent to the seminiferous tubules in testicle that produce and secrete male sex hormone (testosterone).
Meiosis: The process of cell division in eukaryotes that reduce the number of chromosome in which the change from diploid to haploid takes place, leading to the production of gametes.
Mitosis: A highly ordered process by which chromosomes in a cell nucleus are split into two genetically identical sets in two nuclei and form two individual.
Oxidative phosphorylation: A metabolic pathway in which the mitochondria in sperm cells uses their structure and enzymes to produce energy by the oxidation of nutrients to produce ATP.
Sertoli cells: The epithelial supporting cells of seminiferous tubules that secrete androgen- binding protein and establish the blood-testis barrier by forming tight junction with adjacent Sertoli cells.
Spermatogenesis: Aprocess in which spermatozoa are produced and it is controlled by secretions of the pituitary gland.
Spermiation: The mature spermatids are released from Sertoli cells to the lumen of seminiferous tubule prior to their passage to the epdidymis.
Spermiogenesis: The final stage of spermatogenesis in which spermatids are differentiated into spermatozoa.
Zona Pellucida: A glycoprotein membrane surrounding the plasma membrane of fully grown mammalian oocyte performing an important function during fertilization.
In the present study semen samples from NRF bulls were analysed for sperm viability, acrosome integrity and ATP content. The effect of incubation at 37 °C on sperm viability, acrosome integrity and ATP content was investigated on fresh and frozen semen samples from 20 NRF. Semen was incubated at 37 °C and the sperm quality parameters were analysed at 0 hr and after 6 and 24 hrs of incubation for both fresh and frozen samples. Sperm viability and acrosome integrity was assessed with flow cytometry and ATP content with luminometer. The incubation time at 37 °C had a significant effect on all studied parameters. However, ATP content was significantly more affected than sperm viability and acrosome integrity. After 24 hrs of incubation ATP content of frozen samples decreased approximately to 0 for all tested samples, while percentage of viability and acrosome integrity decreased by 78 ± 11.9 %. Percentage of AIL sperm cells of frozen samples decreased at corresponding rate as in fresh samples. Concerning ATP content there was however, a more marked decline in frozen samples compared to fresh samples during incubation.
Sperm samples from eight NRF bulls with high and low fertility were also analysed for sperm viability, acrosome integrity and ATP content at 0 hr (right after thawing), 3, 6 and 24 hrs incubation at 37 °C. The ATP content adjusted for % AIL at 0 hr was significantly correlated with fertility measured as 56 days NRR, while % AIL or ATP content analysed separately were not correlated to field fertility.
1.1 Origins of the project
The project is a 60 credits constituting Master s Thesis of Hedmark University College Master s Degree program in Applied and Commercial Biotechnology, 2013-2015. This project was carried out in close collaboration with Geno SA, with the aim to find an in vitro method for evaluating bull sperm quality correlated to fertility potential.
Geno SA is a breeding company that is owned by 9800 Norwegian farmers with a major breeding responsibility of Norwegian Red (NRF), the main dairy cattle breed in Norway. The company produces cryopreserved semen from NRF bulls for artificial insemination (AI) in Norwegian farms and for export to countries around the world. Geno s breeding program is based on science and continuous research and development in the areas of dairy cattle breeding e.g. genetics, fertility and artificial insemination. Geno aims high to provide the customers semen doses with good sperm quality, which can indeed help them to increase profits.
1.1.2 Norwegian breeding program of NRF
NRF is the dominant dairy breed in Norway, representing 95% of Norwegian cattle. In recent years, NRF semen has been exported to many countries such as United States, United Kingdom, Canada and Italia. It is reported that NRF probably will continue to be used widely in future (Geno, 2013). NRF was developed in 1939 in Norway of crossbreeding of several breeds, the old Norwegian breeds combined with bulls from Swedish and Finnish breeds. NRF is known with their richness for milk and fertility. The new breed NRF is maintained and developed by Geno. Geno gets all data from Norwegian dairy herd recording system (NDHRS). NDHRS is run by the national dairy cooperative Tine and contains data from a whole range of sources including health card, slaughter houses and laboratory milk analysis. Selected bulls undergo a strict progeny test program and have 200-300 daughters evaluated before considered proven elite bulls. Fertility and health have been included in the net merit index since the 1970s.
In Geno’s breeding program, every year about 230 of the best NRF bull calves from elite sires are selected from herds all over Norway on the basis of pedigree information and the health information of the mother. The bull calves are housed at Øyer testing station, aged approximately three months upon arrival, and being in test until approximately 12 months. About 115 of these are selected and used as test sires for semen production based on their performance at Øyer. Each young bull produces semen (young bull semen) for distribution all over Norway and for later progeny testing. Information from 200-300 daughters is acquired for each sire. Finally, Geno selects 10-12 most valuable dairy bulls as elite sires based on the progeny results. Elite bulls are selected based on their total merit index and average relationship with the population. The eleven features included in the total merit index with their respective relative weights are: milk production (28%), resistance to mastitis (21%), fertility (18%), udder conformation (15%), leg conformation (6%), meat production (6%), temperament (2%), resistance to disease other than mastitis (2%), milkability (1%), calving difficulty (0.5%) and stillbirth (0.5%) (Geno, 2014)
Currently, fertility of AI bulls is routinely measured by the non-return rate (NRR) (Puglisi et al., 2012), the percentage of inseminated heifers and cows that do not return to estrus within specified period of time after AI (56 day) (Fouz et al., 2011). Geno is very interested to identify the most fertile bulls and select ejaculates with good quality for freezing and use in AI. Therefore semen evaluations are very important to differentiate between good and bad semen quality and allow discarding of bulls with poor fertility in an AI program. In this regard the main efforts in this study are directed towards this purpose: the exposure of spermatozoa to stress factors and test the sperm cells’ characteristics in order to develop an objective method for sperm quality test.
NRF constitute an important part of the cattle population in Norway. Male fertility is essential for cattle breeders and breeding companies. To obtain genetic progress for low heritable traits, breeding companies should improve the genetic potential either by selection through progeny testing of bulls (Oltenacu and Broom, 2010) or crossbreeding from different breeds such as NRF by using assisted reproductive technologies such as AI or embryo transfer. (Oltenacu and Broom, 2010). AI is widely used and most the common assisted reproductive technology for livestock improvement (Vishwanath, 2003). AI is a powerful tool to improve fertility potential of bulls.
Spermatozoa are unique mature small male reproductive cell that possess specialized function which is the ability of fertilizing the oocyte and form the zygote (Gadella and Luna, 2014). The mature sperm cell (spermatozoon) is a haploid cell, containing one half of the genetic material necessary for new life. The sperm cells cannot be repaired if severely damaged (Rodriguez-Martinez, 2007).
2.1.1 Spermatozoal structure and function
The bull sperm cell is about 75 -90 µm long (Pesch and Bergmann, 2006) and is comprised of two structural regions, the head and the tail (Eddy, 2006; Senger, 2005) (figure 1). These structures are covered by dynamic plasma membrane (plasmalemma). The spermatozoal head contains the nucleus, acrosome and postnuclear cap. The major part of the head is occupied by the nucleus which is surrounded by a nuclear membrane. The nucleus is composed of oval, flattened and compacted chromatin. Compaction and stabilization of the DNA is a result of a high degree of disulfide cross-linking of the keratinoid proteins. The acrosome is a large vesicle that covers two-thirds of the anterior nucleus. The acrosome reaction includes the release of acrosomal contents (enzymes) to penetrate the zona pellucida (ZP), which is a highly specialized exocytosis during the fertilization. Similar acrosomal morphology is described in bull, boar, ram and stallion (Senger, 2005). The postnuclear cap is a part of the membrane posterior to the acrosome. The sperm tail (flagellum) is essential for sperm motility and is composed of the four parts, capitulum, the midpiece, the principal piece and the terminal piece (Senger, 2005) (figure1). The capitulum fits into the implantation socket, in the posterior of the head. The anterior part of the tail is responsible for neck flexibility during sperm motility. The tail contains axonemal component. This contains two central filaments surrounded by nine pairs of microtubules, which again are surrounded by nine coarse fibers. The outer dense fibers and fibrous sheath are arranged around the axoneme. The sperm midpiece contains several number of mitochondria wrapped helically around the outer coarse fibers of the tail (Senger, 2005). The annulus is a junction of midpiece and principal piece. The principal piece is the longest part of the tail and is characterized by the presence of fibrous sheath (Eddy, 2006) and continues as microtubules that terminates in the terminal piece of the tail (Senger, 2005).
Figure 1. Illustration of the mature sperm cell structure. The sperm consists of two structural regions, a head and a tail. 1. Plasma membrane, 2. Outer acrosomal membrane, 3. Acrosome, 4. Inner acrosomal membrane, 5. Nucleus, 6. Proximal centriole, 7. Rest of distal centriole, 8. Thick outer longitudinal fibers, 9. Mitochondrion, 10. Axoneme, 11. Annulus, 12. Ring fibers A. Head, B. Neck, C. Midpiece,
D. Principal piece and E. End piece. Figure adapted from Human embryology embryogenesis (Embryology)
Spermatogenesis is a continuous process of producing millions of spermatozoa per day (Schlatt and Ehmcke, 2014). This process includes cellular transformation that produce spermatozoa from spermatogonial cells over an extended period of time within seminiferous tubules of testis (Hess and de Franca, 2008). The seminiferous tubules consist of three types of cells: peritubular myoid cells, Sertoli and germ cells (Smith and Walker, 2014) (figure 2). Spermatogenesis is initiated and controlled by endocrine secretions from the pituitary gland that secretes follicle-stimulating hormone (FSH) and luteinizing hormone (LH) under the control of gonadotropin-releasing hormone (GnRH) secreted from hypothalamus. In addition to pituitary and hypothalamus hormone, Leydig cells that are found in the interstitial space play an important role in regulation of spermatogenesis via testosterone secretion, and estradiol-17β hormone which are secreted from Sertoli and germ cells (Cheng and Mruk, 2013). Spermatogenesis can be divided in three major stages: spermatocytogenesis, meiosis and spermiogenesis (Chocu et al., 2012). In mammals, spermatogonial cells are produced from primordial germ cells (McLaren, 2004). During spermatocytogenesis, the spermatogonial stem cells (SSCs) undergo mitotic divisions along the basement membrane of seminiferous tubules. A type A spermatogonia that reside in basal compartment undergo mitotic divisions to form additional germ cells (renewal of the stem cells), however some of which stop proliferating and differentiate into type B spermatogonia and finally develop to primary preleptotene spermatocytes (Cheng et al., 2010). Primary spermatocytes initiate the second stage of spermatogenesis meiosis in which diploid spermatocytes undergo two meiotic divisions to form haploid spermatid. Similar meiotic and spermiogenic phases are described for all mammals (Chocu et al., 2012) Meiosis occurs in the apical (adluminal) compartment after migration of primary preleptotene spermatocytes through blood-testis barrier (junction between Sertoli cells) from basal compartment to adluminal compartment after restructuring blood-testis barrier (Mruk and Cheng, 2011) (figure 2).
Primary preleptotene spermatocytes undergo meiotic division I producing secondary spermatocytes. The meiosis phase includes several changes such as homologous chromosomal pairing, condensation and genetic recombination, cells with completely chromosome known as pachytene spermatocytes and diplotene phase before dividing to form secondary spermatocytes (Schlatt and Ehmcke, 2014). The secondary spermatocytes produced by the first meiotic division undergo meiotic division II to form very small haploid round spermatids (O’Donnell and O’Bryan, 2014).
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Figure 2. Illustration of the seminiferous tubule structure including Sertoli cells, intercellular junction and interstitial tissue and the process of spermatogenesis. Sertoli cells locate on the basement membrane of seminiferous tubule. Adjacent Sertoli cells constitute tight junctions which separate the seminiferous epithelium into basal and adluminal compartments. The figure shows different stages of spermatogenesis. The spermatogonial germ cells proliferate and differentiate into primary preleptotene spermatocytes which in turn undergo meiotic division in the adluminal compartment to form secondary spermatocytes. After completing the second meiotic division haploid spermatid, are formed and further differentiate into sperm cell. Figure taken from Hai et.al. (Hai et al., 2014).
The final phase in production of viable sperm is a highly regulated process termed spermiogenesis where the round, haploid spermatids transform without further division into elongate, highly condensed and mature spermatozoa via a series of complex developmental steps. At least four prolonged steps of spermiogenesis have been categorized including Golgi, capping, acrosomal and maturation (Johnson, 1986) (figure 3, illustrate the first three steps).
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Figure 3. Illustration of the parts of maturation during spermatogenesis: 1. Axonemal structure,
2. Golgi complex, 3. Acrosomal vesicle, 4. Pair of centrioles (proximal and distal), 5.
Mitochondrion, 6. Nucleus, 7. Flagellar primordium, 8. Microtubules, 9. Sperm tail, 10.
Acrosomal cap. Three different stages of spermiogenesis: on the left a fresh spermatid, on the right an immature sperm and in the middle an in-between stage. A rotation of nucleus causes a repositioning of the acrosomal vesicle to occur. This inverts its self like a cap over the nucleus that continues to be condensed (dotted line). The cytoplasm cell components that are no longer needed are discarded and phagocytized by Sertoliʼs cells. The mitochondria are packed thickly (tightly) together around the beginning part of the flagellum (mid-piece). As a sign of its immaturity, the sperm cell on the right that has issued into the lumen still has a bit of cytoplasm around its neck. Figure from Human embryology embryogenesis (Embryology).
The Golgi phase is characterized by the formation of acrosomal vesicles and granules by Golgi apparatus (Hess and de Franca, 2008). These granules are called proacrosomic vesicles which ultimately fuse together forming a larger vesicle, called acrosomic vesicle that settles on one side of the nucleus. Smaller vesicles continue to be added to the larger vesicle increasing its size. In addition, centrioles are migrating from cytoplasm to the base of nucleus, in which a proximal centriole will form attachment point of the flagellum while distal centriole will form axoneme (Senger, 2005). The second phase capping involves the migration of Golgi apparatus towards the caudal part of the cells and flattening the vesicle to form the cap consisting of an outer acrosomal membrane, an inner acrosomal membrane and acrosomal enzymes over the anterior portion of the nucleus. At this phase, the axoneme is formed from distal centriole (Senger, 2005). The acrosomal phase involves the elongation of the nucleus. The acrosome spreads to cover about two-thirds of anterior nucleus. The manchette that consists of microtubules forms in the region of the caudal of the nucleus. Some of these microtubules will form postnuclear cap. The last phase maturation is characterized by assembling mitochondria around flagellum from the base of the nucleus to the anterior one- third of the tail in a spiral formation forming midpiece. The annulus is formed and separates between the midpiece and the principal piece. This phase involves the production of dense outer fibers of the flagellum and the fibrous sheath. The entire spermatozoon is surrounded by plasma membrane and the integrity of this membrane is essential for sperm survival and function (Senger, 2005). The unnecessary sperm cytoplasm is removed and phagocytized by Sertoli cells. Spermatozoa are considered mature when they are released into lumen of the seminiferous tubules at a well regulated process called spermiation (Mruk and Cheng, 2004; Qian et al., 2014), after this process migrate spermatozoa to epididymis for further maturation and storage (Hinton et al., 1996). Spermatogenesis is considered the sum of the total events of spermatocytogenesis, meiosis and spermiogenesis resulting in production of spermatozoa from spermatogonial stem cells (Johnson et al., 2000).
The whole process of spermatogenesis in bull takes about 61 days and time required for each stage is 21, 23 and 17 days respectively (Johnson et al., 2000). The efficiency of spermatogenesis is assessed by measuring the number of the sperm cells produced per gram of testicular parenchyma per day. Adult bull can produce 12˟106 sperm cells/g parenchyma daily (Amann, 1981; Amann et al., 1976; Johnson, 1986; Johnson et al., 2000).
2.2 Sperm maturation
When spermatozoa are released into lumen of the seminiferous tubules in a process termed spermiation, spermatozoa are passively transported into the epididymis. Sperm is still not able to fertilize the oocyte until further modifications take place. The epididymis is comprised of three parts caput, corpus and cauda (Tulsiani and Abou-Haila, 2012) each with specific function. Apart from its role in providing an environment for maturation, it is a site for sperm cells transit, concentration of sperm cells and storage in a quiescent state in cauda until ejaculation (Marengo, 2008). The spermatozoa undergo a process of modification of plasma membrane proteins through its passage in the epididymis. This will determine the sperm fertilizing ability and sperm interaction with the surrounding environment including the ovum in the female reproductive tract. Proteins which are secreted by the epididymis either keep sperm cells in quiescent state in the cauda (by loose binding) or help sperm later to fertilize ovum in the female reproductive tract (by tight binding) (Marengo, 2008). The length of the epididymal duct is about fifty meters long in bull. The duct is surrounded by smooth muscle which helps in transport of sperm cells along the duct. The testicular spermatozoa reach rapidly into epididymis via rete testis and efferent ducts by the pressure of fluid secretion from Sertoli cells and rete testis, pressure from sperm mass produced in the testis and contractions within myoid layer of seminiferous tubule. In addition, spermatozoa transit is facilitated by the movement of cilia on the ciliated cells in the epithelial lining the efferent ducts (Ilio and Hess, 1994). In bull, epididymal transit takes about 7 to 9 days. The cauda storage of sperm cells in males with high ejaculation frequency is 25% to 45% less than sexually rested males. However, sperm cells can have poor viability after relatively long time in the cauda (Senger, 2005). Spermatozoa undergo several morphological, biochemical and physiological alterations in different parts of the epididymis. Spermatozoa in the caput are immotile, infertile and have proximal cytoplasmic droplet. The sperm cells in the corpus show little motility after dilution and some expression of fertility, and they have a distal cytoplasmic droplet, while sperm cells in cauda show normal motility after dilution and fertility potential and have a distal cytoplasmic droplet (Senger, 2005). These changes can be used to ascertain where in the epididymis a fault did occur (Senger, 2005). The final stage of maturation which takes place in epididymis is thought to be dependent on interaction between media in the epididymis released from different regions and sperm cell membrane (Gatti et al., 2004). Epididymal fluid plays an important role in the remodeling of sperm surface (Belleannée et al., 2011). It has been reported that intracellular cyclic adenosine monophosphate (cAMP), calcium and pH regulate sperm functions. These factors are thought to work through protein phosphorylation which is controlled by protein kinases and protein phosphatases. Sperm motility in immature spermatozoa can be stimulated by high protein kinase and low protein phosphatase activities. In addition, protein phosphatase inhibitors play important role in initiation of motility of epididymal spermatozoa in caput and stimulating the motility of spermatozoa in cauda (Huang and Vijayaraghavan, 2004; Smith et al., 1999; Vijayaraghavan et al., 1996). On the other hand, full maturation of spermatozoa to acquire fertilization potential takes place through sperm cells passage in the female reproductive tract.
2.3 Sperm transport in the female reproductive tract
The transport of several millions of spermatozoa deposited in the cow female reproductive tract either by mating or through AI is a complex process. Natural mating of bull and cow takes approximately 1-3 seconds with site of semen deposition in fornix vagina of a volume ranged 0.5 - 12 ml (Senger, 2012). After ejaculation, a big number of spermatozoa are lost from female reproductive tract either by retrograde transport or phagocytosis by leukocyte within female tract (Senger, 2012). The remaining spermatozoa start to travel a long distance through female reproductive tract to the oviduct during three phases: rapid (immediate) transportation, colonization and slow release (Rodriguez-Martinez, 2007). Rapid phase is characterized by the presence of spermatozoa in the oviduct within a few minutes. The contraction of vagina, cervix and uterus in conjunction with copulation is believed the primary force responsible for sperm transport in this phase. Following copulation, different physiological events are involved in sperm transport in female genital tract. (Senger, 2012). Capacitation is initiated as sperm pass through the female reproductive tract and completed in the isthmus of the oviduct (Senger, 2012). In this regard, the spermatozoa with progressive forward motility can successfully penetrate the cervical mucus (Katz et al., 1989; Scott, 2000) and across the uterotubal junction (Gaddum-Rosse, 1981; Scott, 2000). The rapid transport phase is followed by a prolonged phase of sperm migration. However, in cows, colonization of spermatozoa occur in the caudal portion of oviduct isthmus which is considered the functional reservoir for spermatozoa (SR) (Hunter and Wilmut, 1984). Finally, a slow release of spermatozoa from the SR toward the site of fertilization Ampullary isthmic junction (AIJ) in relation to ovulation (Barratt and Cooke, 1991; Rodriguez-Martinez, 2007).
2.3.1 Capacitation and acrosome reaction
Freshly ejaculated mammalian spermatozoa do not possess ability to fertilize an egg until they undergo biochemical and physiological modifications through their passage in the female reproductive tract. These modifications called capacitation which enables spermatozoa to be functionally competent to recognize and fertilize the egg (Abou-haila and Tulsiani, 2009; Tulsiani and Abou-Haila, 2001; Yanagimachi, 1994). The process of capacitation is poorly defined and most understanding of this process is derived from the in vitro studies. Capacitation is mainly dependent upon alterations in sperm membrane cholesterol (Cross, 1998) in which the cholesterol of sperm plasma membrane must be removed (Jones et al., 2010). This alteration is one of the most important events that occurs during sperm capacitation and causes an increase in membrane fluidity as noted in an in vitro capacitation (Rodriguez-Martinez, 2007). Some researchers reported that albumin and high density lipoproteins which is present in the oviductal fluid serve as cholesterol acceptors and aid in removal of cholesterol from sperm plasma membrane in vivo (Killian, 2011; Martínez et al., 2012). In vitro studies on sperm capacitation demonstrated that capacited spermatozoa in the absence of albumin could not undergo a complete acrosome reaction (Hardy, 2002) and in this case, sperm is not able to fertilize an egg. In addition, this physiological transformations in sperm plasma membrane increase the influx of bicarbonate ions (HCO3¯) and calcium ions (Ca2 +) (Breitbart, 2002) which lead to increase adenylyl cyclase activity (Coulter, 2006; Fraser, 1998). This activation results in production of cAMP and most effects of cAMP involve by activation of protein kinase A (PKA) (Buffone et al., 2014) which causes protein tyrosine phosphorylation (Abou-haila and Tulsiani, 2009; Breitbart and Naor, 1999; Naz and Rajesh, 2004).
An intact spermatozoal acrosome is one of the most important functional properties to penetrate ZP during fertilization. Acrosome reaction occurs in acrosome of the sperm (Minervini et al., 2013) and involves the fusion of sperm plasma membrane with outer acrosomal membrane that ensure the release of acrosomal contents (enzymes) at the site of sperm-egg binding (Felipe-Pérez et al., 2008)
2.3.2 ATP production
Mammalian spermatozoa produce ATP by two metabolic pathways, glycolysis and oxidative phosphorylation (OXPHOS) (Rodríguez-Gil, 2013). The mammalian spermatozoon contains many mitochondria (72 mitochondria in bull sperm) (Bahr and Engler, 1970) exclusively located in mid piece and the ATP produced by OXPHOS in inner mitochondrial membrane is transported to microtubules and ensure movement of sperm flagellum. Glycolysis occurs particularly in fibrous sheath of sperm flagellum and glucose which is present in seminal fluid and female reproductive fluid (Mann and Lutwak-Mann, 1981) is the main metabolic substrate for this process that is metabolized to pyruvate and/or lactate. This process occurs in anaerobic condition (Ferramosca and Zara, 2014). Whereas mammalian sperm cells use carbohydrate, lipid and protein as a substrate that can be catabolized by OXPHOS (Ferramosca and Zara, 2014). However, it is unlikely that ATP production in mid piece could be enough to supply energy for active sliding in distal part of long flagellum and support motility (Turner, 2003).
The ATP produced by glycolysis is more likely to be utilized in the distal end of sperm flagellum since glycolytic enzymes such as hexokinase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and other glycolytic enzymes downstream from GAPDH are localized to the fibrous sheath of the flagellum (Garrett et al., 2008a). Some researchers reported that glycolysis is the main source of ATP (Miki et al., 2004; Narisawa et al., 2002; Turner, 2005). It has been reported that ATP produced by glycolysis in freshly ejaculated sperm makes up more than 90% (Silva and Gadella, 2006). On the other hand, other researchers have reported that mitochondria produce an adequate ATP by OXPHOS for sperm motility (Ferramosca et al., 2012; Ruiz-Pesini et al., 1998). Generally, several studies have shown that the sperm cells use different methods to produce energy depending on substrates found in the female reproductive tract (Piomboni et al., 2012; Ruiz-Pesini et al., 2007; Storey, 2008).
2.4 Evaluation of sperm quality
The main goal of livestock breeding industry is to identify genetically superior bull to breed the cows through AI. Therefore, sperm quality and its relationship to fertility potential are a major goal to breeding companies. Evaluation of semen quality is a part of breeding soundness examination which is conducted on bull before use in AI to give optimal fertilization success (Deb et al., 2013).
Semen analysis can be conducted on fresh, cooled or frozen-thawed semen (Mocé and Graham, 2008). In addition several methods have been used today for evaluation of semen quality prior to the use and processing in cryopreservation to ensure efficient breeding (Kasimanickam et al., 2007; Oliveira et al., 2013b). A number of in vitro semen quality parameters have been used such as motility (Farrell et al., 1998), morphology (Saacke et al., 1998) and plasma membrane integrity (Celeghini et al., 2007). However, the results of analysis of such parameters are not always correlated to field fertility (Oliveira et al., 2013b; Sudano et al., 2011; Zhang et al., 1999).
2.4.1 The initial semen evaluation
Immediately, the evaluation of in vitro semen quality is performed after collection (Senger, 2005). These assessments include a visual appraisal of colour, density, presence of dirt or other contaminants, measurements of ejaculate volume and sperm concentration (Al- Makhzoomi et al., 2008). The appearance of the normal ejaculate in bulls is typically a uniform near-white (Mocé and Graham, 2008). In practice the semen sample should be free of blood and flocculent material, as this is indicative of infection in reproductive tract (Mocé and Graham, 2008).
The volume of an ejaculate can be measured by pouring the semen into a graduated cylinder, graduated markings on the collection tube or by weighing the sample (Mocé and Graham, 2008). The sperm concentration means the number of sperm cells/ml of semen sample (Haugan et al., 2007). The sperm concentration can be determined by haemocytometer or a counting apparatus (Kocks and Broekhuijse, 2014). The ejaculate volume and concentration of spermatozoa are important parameters to determine the total number of sperm in the ejaculate by multiplying the volume with sperm concentration/ml. This determines the number of insemination doses to be produced from each ejaculate (Senger, 2005).
2.4.2 Sperm motility
Sperm motility is one of the most widely used tests of sperm quality from the initial stage in raw semen until post-thaw quality control prior to distribution. Sperm motility estimated by light microscopy is highly utilized for assessment of semen quality in all AI laboratories (Vincent et al., 2012). The assay is simple, rapid and inexpensive, can be conducted by placing a drop of semen sample on pre-warmed glass slide at 37 ºC with coverslip and estimating the percent of motile sperm by visual measurement under light microscope (Dhurvey et al.). However, the simple visual estimation of sperm motility using light microscope is very subjective, with different technicians achieving different results on the same series of smears. Currently, Computer Assisted Sperm Analysis is an objective method that gives additional information regarding semen movement attributes.
2.4.3 Sperm viability
The integrity of sperm plasma membrane is often linked to spermatozoal viability. It has been reported that fertility is closely related to integrity of sperm plasma membrane (Correa and Zavos, 1994; Larsson and Rodrıguez-Martınez, 2000; Selvaraju et al., 2008). The principle of most sperm viability assays have been developed to assess whether the sperm plasma membrane is intact or not by using a broad array of membrane-impermeable fluorescent dyes such as DNA dyes, propidium iodide (PI), bis-benzimide (Hoechst 33258), YO-PRO®-1, TOTO®-1 and ethidium homodimer-1 to permeate damaged membranes (McKinnon et al., 2011) and followed by examination with fluorescence microscopy or flow cytometry. In addition, bisbenzmide stain (Hoechst 33258) requires UV light for excitation (Gillan et al., 2005).
PI has been successfully described to identify non-viable sperm cells in boar and bull (Pintado et al., 2000). On the other hand, the membrane-permeant fluorescent probe SYBR-14 is commonly used to assess the integrity of sperm plasma membrane and stains DNA of sperm cells of both membrane intact and membrane compromised (Graham and Mocé, 2005). However, the most commonly assay is using combination of SYBR-14 and PI that differentiate between living cells (green color), dead (red) and dying (combination of green and red color) sperm cells with disrupted membrane (Makarevich et al., 2010).
2.4.4 Acrosome integrity
The assessment of acrosomal integrity of sperm is carried out by different fluorescence techniques (Thomas et al., 1997). The most common method to evaluate acrosomal status is to use fluorescently labeled lectins such as Pisum sativum agglutinin (PSA) and Arachis hypogaea agglutinin (PNA) (Partyka et al., 2012). Acrosome-specific lectins can be conjugated with fluorescence probe as fluorescein isothiocyanate (FITC), phycoerythryn (PE) or Alexa Fluor® (Graham et al., 1990; Kawakami et al., 2002; Nagy et al., 2004b; Partyka et al., 2010; Rijsselaere et al., 2005). The glycoprotein lectins recognize and bind to carbohydrate moieties in different parts of acrosome (Silva and Gadella, 2006). PSA from pea plant is a lectin which recognizes and binds to carbohydrate moieties of acrosomal matrix specially α-mannose and α-galactose (Gillan et al., 2005). The FITC-PSA will only stain acrosome reacted spermatozoa in yellow green (Celeghini et al., 2007; Celeghini et al., 2010; Cross et al., 1986; Graham et al., 1990). PNA from peanut plant conjugated with a fluorochrome such as Alexa 488 will bind and label β-galactose of the outer acrosomal membrane of acrosome reacted and emit green light (Flesch et al., 1998; Graham, 2001; Nagy et al., 2003). This staining technique described above for PSA and PNA has been used by both fluorescence microscopy and flow cytometry (Gillan et al., 2005).
2.4.5 ATP Assay
The quantity of ATP in semen is referable to the given number of motile spermatozoa (Singh et al., 2012; Wood et al., 1986). Quantitative determination of ATP in mammalian spermatozoa using luciferin-luciferase assay has been used in many species as boar (Aalbers et al., 1985), bull (Gumińska et al., 1996), domestic poultry (Wishart, 1982) and human (Mendeluk et al., 1997). The ATP detection reagent contains luciferin, luciferase enzyme, a lytic buffer and ATPase inhibitors. The luciferin serves as substrate and a lytic buffer contains detergent that is necessary to lyse the cells and release cellular ATP. The ATPase inhibitors works to stabilize the released ATP (Sittampalam et al., 2013). The method involves the extraction of ATP from sperm cells and mixing free ATP with luciferin-luciferase reagent. In luciferin-luciferase reaction, a substrate luciferin is oxidized by luciferase enzyme in the presence of ATP-Mg2 + and oxygen to generate photon (light) (Xu et al., 2013).
The light emission can then be recorded and read by luminometer (Rieger, 1997). Luminometer is simple and compose of three parts; a sample chamber which displays luminescence to detector, detector or photomultiplier tube that detects extremely low levels of light and the last part a signal processing method (Roda et al., 2000) (figure 4).
The method measures the total ATP content produced by OXPHOS and glycolysis (Promega, 2015).
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Figure 4. Illustration of luciferin-luciferase reaction. Firstly cellular ATP can be measured by direct lysis of sperm cells by suitable detergent, secondly the released ATP reacts with luciferin to produce oxyluciferin in the presence of Ultra-GloTM luciferase enzyme and leading light emission which is proportional to the number of viable sperm cells. Figure taken from Promega (Promega, 2015).
2.5 Techniques used for sperm assessment
2.5.1 Flow cytometry
Flow cytometer is a system for analysing multiple parameters of the physical and chemical characteristics of single cells up to thousands cells per second as they flow through a stream of fluid (Bergquist et al., 2009). A flow cytometer consists of a light source (laser and arc lamp), the flow cell and hydraulic fluidic system, several optical filters to detect and differentiate specific wavelengths, detectors or photomultiplier tubes (FL1, FL2, FL3) (figure
5) and a data processing unit (Díaz et al., 2010).
The source of light is a laser and arc lamp. Current instruments use a wide variety of lasers which generate a high intense monochromatic light focalized in small volume. Moreover, most common instruments use an air cooled-argon ion laser which produce blue light at 488 nm (Ormerod and Novo, 2008). On the other hand, arc lamp does not give monochromatic light, therefore they require optical filters to detect appropriate wavelength (Ormerod and Novo, 2008). The hydrolauric fluidic system allows the cells to pass detection point in fluid stream one cell at a time.