Review Article | Open Access

¹Atatürk University Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Erzurum, Turkey
²Atatürk University Faculty of Veterinary Medicine, Department of Biochemistry, Erzurum, Turkey
³Ataturk University, Faculty of Science, Department of Molecular Biology and Genetics, Erzurum, Turkey

*Corresponding author: Ali Dogan Omur, Ataturk University Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Erzurum, Turkey

Abstract

---

Today, it is an important issue to preserve genetic resources and transfer them to future years. The process of freezing semen, which has a wide range of uses such as reproductive biotechnology, protection of the lineage of species and clinical applications, is of great importance in this sense. Freezing the semen in a way that will provide sufficient fertility will make artificial insemination practices more practical and economical, and more accurate and reliable pedigree records will be kept. In addition, it will be possible to use animals with high genetic capacity in large populations. During the freezing-thawing of sperm, membrane lipid phase change, osmotic-mechanical stress and free oxygen radicals occur. As a result, structural deformations in membrane structures and cell organelles and breaks in DNA occur. These negative effects can be reduced by adding antioxidants, various cryoprotectants and chemical substances to semen extenders, and sperm functions are improved after solution. The developments in the light of recent research have been in the direction of increasing the fertility abilities of sperm cells in the cryopreservation process by irradiating them in different spectrum areas. On the other hand, lasers are involved in many areas of our lives due to their linearity, single wavelength, stable radiation and high power. Laser sources are gaining more and more acceptance in various industries thanks to their narrow focusing advantages. Recently, it has been shown that under intense radiation of red LED (Light Emitting Diode) lights, the resistance and capacitation of porcine sperm cells increase in vitro and fertility ability increases in vivo. Consistent results have also been obtained in species such as humans, dogs, bulls, rams and rabbits using devices and systems such as low-energy laser and visible light lamps with wavelengths ranging from 400 nm to 800 nm.
Keywords: Laser; LED; Photostimulation; Semen
Abbreviations: Laser: Light Amplification by Stimulated Emission of Radiation, LED: Light Emitting Diode, CASA: Computer-Assisted Sperm Analysis, VAP: Average Path Velocity (µm/s), VCL: Curvilinear Velocity (µm/s), VSL: Straight-Line (rectilinear) Velocity (µm/s), ROS: Reactive Oxygen Species, CO2: Carbon dioxide, DNA: Deoxyribo Nucleic Acid, RNA: Ribo Nucleic Acid, ATP: Adenosine Triphosphate

Introduction

---

Laser, Working Principle of Laser, Types of Lasers
The equine population of the world is 122.4 million (40 million donkeys, 15 million mules’ 43.3 million Horses and [1]. In the distribution pattern, 98% of all donkeys, 97% of all mules and 384% of all horses are found in the developing countries. The number of equines in Africa is in the range of 17.6 million comprising 11.6 million donkeys, 2.3 million mules3.7 million horses [2].
A laser is a coherent and focused beam of photons. This beam of light is more intense than the light emanating from any source. The laser has a single wavelength. All of the laser waves are of the same frequency and are in phase with each other. Light consists of energy packets called photons. The energy of light is carried by photons. Photons have the property of behaving like both waves and particles. In addition, the wavelength of the photons determines the color of the light.
Light consists of energy packets called photons. The energy of light is carried by photons. Photons have the property of behaving like both waves and particles. In addition, the wavelength of the photons determines the color of the light. The principle of operation of the laser is based on the excitation of photons. Some gases are used to obtain laser light. The most commonly used gas is carbon dioxide. When the carbon dioxide atom is excited by electricity, light or another way, in other words, when energy is given, the electrons of the atom are excited and go from a low energy level to a high energy level. But this excited electron cannot stay in this high energy level forever. Therefore, the excited electron goes from a high energy level to a low energy level. During the transition, it releases energy as photons as much as the difference between the high energy level and the low energy level. The released photon excites the electron of another atom, causing the same photon to be released. The excitation process continues with the energizing of the system. By means of mirrors placed at both ends of the laser, photons are reflected on both sides, thereby stimulating more atoms. The beamed photons create the laser beam [1].
Lasers are classified as high and low power lasers according to their energy densities. High power lasers are also called hot lasers due to the thermal effect they produce and are mostly used in surgical applications. Low-energy lasers act photochemically. They are called low-power laser, low level laser, soft laser.
Argon laser: They are mostly used in eye diseases. It is frequently used in retinal hemorrhages, detachment and glaucoma.
CO2 laser: It is the most suitable type of laser for microsurgical uses.
Neodymium YAG (Ytrium Aluminium Oxide Garnet) laser: It is especially used in tumor treatment and endoscopy.
Helium-Neon (He-Ne) laser: It acts on a wide tissue mass with high dispersion and low absorption. It is the most suitable type of laser for transcutaneous irradiation treatments. It ensures the proliferation of collagen fibers and cells. It reduces pain [2].
Photostimulation: Photostimulation artificially activates biological compounds, cells, tissues and even whole organisms, for this purpose light is used. This method is effective for investigating various relationships between different biological processes [3].

Effect of Photostimulation on Spermatozoa Motility and Oxidative DNA Damage
The ability to successfully fertilize the oocyte relies on the motility ability of the spermatozoa. Sperm motility in both humans and animals has been used as a metric for the viability of sperm samples.
Current methods of assisted reproduction in humans and animals rely on the use of drugs to increase fertility or the direct injection of sperm into the oocyte. These methods are not always successful and additional ways of improving fertilization can be exploited. Many studies have shown that low doses of red light cause an increase in sperm motility and overall fertilization potential in several species [4-7]. One study showed that red light from LEDs can improve sperm quality by increasing the speed of sperm cells and the mitochondrial membrane potential [7]. On the other hand, it has been shown that excitation of 630 nm laser light can lead to the generation of reactive oxygen species (ROS) such as hydrogen peroxide [8]. Low levels of ROS allow the sperm acrosome to react with the oocyte [9], acting as an important messenger in fertilization. However, high levels of ROS can damage cell DNA. Therefore, it is possible that light stimulation causes oxidative DNA damage, which may be more visible in nuclear DNA located near the sperm midpiece, where the cellular mitochondria mass is concentrated. Previous studies have shown that DNA damage can lead to male infertility and that ROS-induced oxidative stress is the main cause of DNA fragmentation [10,11]. Therefore, careful evaluation of DNA damage is important.
For viable fertility to occur, laser irradiation must not cause DNA damage in sperm cells. Spermatozoan DNA damage has been associated with developmental problems and difficulties in embryonic implantation, as well as fertilization dysfunction. The inability of a cell to cope with ROS has been associated with both DNA damage and decreased sperm motility [12].
In a study by Daryl Preece et al (2017), it was shown that exposure to red light can increase sperm motility while producing little or no DNA damage to sperm cells [13]. Since sperm morphology and energy differ significantly between species, it is anticipated that the effect of photostimulation may also vary significantly between species.

Effects of Laser Irradiation on Spermatozoa Functions in Animals
Cryopreservation of spermatozoa has led to wider application of artificial insemination. However, sperm quality decreases after cryopreservation, which reduces their fertility [14]. Exposure to various stresses during cryopreservation physically alters sperm cells and also leads to changes in chemical components necessary for energy metabolism of sperm to support motility [15]. Sperm mitochondrial damage occurs due to destabilization of sperm cell membranes during freeze-thaw procedures, resulting in impaired sperm motility [16]. To date, most research has been done to preserve spermatozoa [17] using suitable extenders [18] to improve freeze-thaw processes and/or various additives [19] during the freeze-thaw process. Sperm mitochondrial function may be adversely affected by the use of some cryoprotectants, namely soy lecithin, as previous findings have revealed [20]. Therefore, various other strategies aimed at improving sperm mitochondrial function need to be evaluated. Application of photobiostimulation with a low-intensity helium-neon (He-Ne) laser has shown to increase the motility of spermatozoa [21]. Photobiostimulation of sperm was first reported in 1969 [22]. This has been proven in several species {human [23], mouse [24], sheep [25] and dog [26] semen}.
On the other hand, there are other animal species where laser Irradiation has positive effects on spermatozoa functions {bull [27], bison [28], stallion [29], turkey [30]}.

Possible Mechanisms of Laser Action in Spermatozoa
Laser irradiation of sperm leads to increased respiration, fructose fermentation, 32 P uptake and Ca2+ absorption, which increases motility and prolongs sperm survival [31]. Photobiostimulation occurs when light is absorbed by porphyrins or cytochromes and the absorbed energy is transferred to oxygen molecules leading to the generation of reactive oxygen species (ROS) [32]. ROS is known as a double edged sword in animal reproduction because higher levels of ROS can cause sperm death due to depletion of ATP and lipid peroxidation causing oxidative stress. Besides, moderate levels of ROS regulate various physiological functions of sperm such as hyperactivation, capacitation, acrosomal reaction, and zona binding [33].
Oxidative phosphorylation in sperm mitochondria leads to the formation of ATP, which is mainly required for sperm motility, and it has been shown that the energetic load of the cell increases after laser irradiation [34]. As a result of long-term storage, sperm mitochondrial senescence occurs, resulting in reduced sperm motility due to reduced ability to produce ATP through mitochondrial respiration [35]. In isolated mitochondria, He-Ne laser irradiation causes an increase in ATP synthesis [36], RNA [37] and DNA synthesis [38]. Sperm mitochondrial ATP is primarily required for sperm motility [39], in addition, mitochondria are also involved in maintaining sperm tail contractility, such as regulation of membrane potential and calcium flux [40].
In conclusion, photostimulation applications are a low-cost method that can be used to improve spermatological parameters. The beneficial effects of photostimulation on spermatozoa include sperm viability, acrosomal integrity, hypo-osmotic swelling response, mitochondrial function, and CASAbased sperm parameters (progressive motility, sperm velocity - VAP, VCL, VSL, distance). The increase in these sperm characteristics may be related to the fact that laser irradiation results in higher cytochrome c oxidase activity, ATP levels and mitochondrial membrane potential, which increases sperm survival. In addition, laser irradiation needs to be standardized, fertility experiments should be performed with laser irradiated spermatozoa and gene expression patterns in laser-treated sperm of various species.

Conflict of interest: The author declared no potential of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author received no financial support for the research, authorship, and/or publication of this article.
ORCID ID: Ali Dogan OMUR https://orcidorg/0000-0002- 2976-4368

References

---

1. Billings CW, Tabak J. (2006) Lasers: The Technology and Uses of Crafted Light (Science and Technology in Focus) Kindle Edition B00D86VGEC, Facts on File (J). [Ref.]
2. Boyraz I, Yildiz A. (2016) Laser Types and using of High Intensity Laser. J Contemp Med. 6: 104-109. [Ref.]
3. Cohen N, Lubart R, Rubinstein S, Breitbart H. (1998) Light irradiation of mouse spermatozoa: Stimulation of in vitro fertilization and calcium signals. J Photochem Photobiol B. 68: 407-413. [PubMed.]
4. Cohen N, Lubart R, Rubinstein S, Breitbart H. (1998) Light irradiation of mouse spermatozoa: Stimulation of in vitro fertilization and calcium signals. J Photochem Photobiol B. 68: 407-413. [PubMed.]
5. Firestone RS. et al. (2012) The Effects of Low-Level Laser Light Exposure on Sperm Motion Characteristics and DNA Damage. J Androl. 33: 469-473. [PubMed.]
6. Karu TI. (2012) Lasers in Infertility Treatment: Irradiation of Oocytes and Spermatozoa. Photomed. Laser Surg. 30: 239-241. [Ref.]
7. Yeste M, et al. (2016) Specific LED-based red-light photo-stimulation procedures improve overall sperm function and reproductive performance of boar ejaculates. Sci. Rep. 6: 22569. [Ref.]
8. Cohen N, Lubart R, Rubinstein S, Breitbart H. (1998) Light irradiation of mouse spermatozoa: Stimulation of in vitro fertilization and calcium signals. J Photochem Photobiol B. 68: 407-413. [PubMed.]
9. de Lamirande E, O’Flaherty C. (2008) Sperm activation: Role of reactive oxygen species and kinases. Biochim. Biophys. Acta - Proteins Proteomics. 1784: 106-115. [PubMed.]
10. Chohan KR, Griffin JT, Carrell DT. (2004) Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation. Andrologia. 36: 321-326. [PubMed.]
11. Kawanishi S, Hiraku Y. (2006) Carcinogenesis with Special Reference to Inflammation. Antioxid. Redox Signal. 8: 1047-1058. [PubMed.]
12. Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin Ea, Aitken RJ. (2008) Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J. Clin. Endocrinol. Metab. 93: 3199-3207. [PubMed.]
13. Preece D, Chow K, Gomez-Godinez V, et al. (2017) Red light improves spermatozoa motility and does not induce oxidative DNA damage. Sci Rep. 7: 46480. [Ref.]
14. Watson PF. (2000) The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci. 60-61: 481-492. [PubMed.]
15. Long JA. (2006) Avian semen cryopreservation: What are the biological challenges? Poult Sci. 85: 232-236. [Ref.]
16. Gillan L, Maxwell WMC. (1999) The functional integrity and fate of cryopreserved ram spermatozoa in the female tract. J Reprod Fertil. 54: 271-283. [PubMed.]
17. Iaffaldano N, Paventi G, Pizzuto R, Dilorio M, Bailey JL, et al. (2016) Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality. Theriogenology. 86: 778-784. [PubMed.]
18. Emamverdi M, Zhandi M, Shahneh AZ, Sharafi M, Akhlaghi A, et al. (2015) Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined homemade or commercial extenders. Anim Prod Sci. 55: 551-558. [Ref.]
19. Mata-Campuzano M, Álvarez-Rodríguez M, Álvarez M, Tamayo Canul J, Anel L, et al. (2015) post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender. Theriogenology. 83: 520-528. [Ref.]
20. Valle ID, Gómez-Durán A, Holt WV, Muiño-Blanco T, Cebrián-Pérez JA. (2012) Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa. J Androl. 33: 717-725. [PubMed.]
21. Karu TI. (2012) Lasers in infertility treatment: Irradiation of oocytes and spermatozoa. Photomed Laser Surg. 30: 239-241. [Ref.]
22. Goldstein SF. (1969) Irradiation of sperm tails by laser microbeam. J Exp Biol. 51: 431-441. [PubMed.]
23. Lenzi A, Claroni F, Gandini L, Lombardo F, Barbieri C, et al. (1989) Laser radiation and motility patterns of human sperm. Syst Biol Reprod Med. 23: 229-234. [PubMed.]
24. Cohen N, Lubart R, Rubinstein S, Breitbart H. (1998) Light irradiation of mouse spermatozoa: Stimulation of in vitro fertilization and calcium signals. J Photochem Photobiol B. 68: 407-413. [PubMed.]
25. Zan-Bar T, Bartoov B, Segal R, Yehuda R, Lavi R, et al. (2005) Influence of visible light and ultraviolet irradiation on motility and fertility of mammalian and fish sperm. Photomed Laser Surg. 23: 549-555. [PubMed.]
26. Corral-Baqués MI, Rivera MM, Rigau T, Rodríguez-Gil JE, Rigau J. (2009) The effect of low-level laser irradiation on dog spermatozoa motility is dependent on laser output power. Lasers Med Sci. 24(5): 703-713. [PubMed.]
27. Fernandes GHC, de Carvalho PDTC, Serra AJ, Crespilho AM, Peron JPS, et al. (2015) The effect of low-level laser irradiation on sperm motility, and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm. PLoS One. 10(3): e0121487. [Ref.]
28. Abdel-Salam Z, Dessouki SHM, Abdel-Salam SAM, Ibrahim MAM, Harith MA. (2011) Green laser irradiation effects on buffalo semen. Theriogenology. 75: 988-994. [PubMed.]
29. Brand˜ao AC, Arruda RP, Andrade AFC, Zaffalon FG, Tarrag´o OFB, et al. (2008) Effect of diode laser on motility, plasma and acrosomal membrane integrity, and mitochondrial membrane potential of cryopreserved stallion spermatozoa. Anim Reprod Sci. 107(3-4): 309-310. [Ref.]
30. Iaffaldano N, Meluzzi A, Manchisi A, Passarella S. (2005) Improvement of stored turkey semen quality as a result of He-Ne laser irradiation. Anim Reprod Sci. 854: 317-325. [PubMed.]
31. Wenbin Y, Wenzhong L, Mengzhao L, Baotian Z, Laizeng AI, et al. (1996). Effects of laser radiation on Saanen buck’s sperm energy metabolism. In Proceedings of the Sixth International Conference on Goats. Beijing, China. [Ref.]
32. Shahar S, Wiser A, Ickowicz D, Lubart R, Shulman A, et al. (2011) Lightmediated activation reveals a key role for protein kinase A and sarcoma protein kinase in the development of sperm hyper-activated motility. Hum Reprod. 26(9): 2274-2282. [PubMed.]
33. Zhang W, Yi K, Chen C, Houa X, Zhou X. (2012) Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. Anim Reprod. Sci 132: 123-128. [PubMed.]
34. Iaffaldano N, Meluzzi A, Manchisi A, Passarella S. (2005) Improvement of stored turkey semen quality as a result of He-Ne laser irradiation. Animal reproduction science. 85(3-4): 317-325. [PubMed.]
35. Vishwanath R, Shannon P. (1997) Do sperm cell age? A review of the physiological changes in sperm during storage at ambient temperature. Reprod Fert Dev. 9: 321-331. [PubMed.]
36. Passarella S, Casamassima E, Molinaro S, Pastore D, Quagliarello E, et al. (1984) Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by heliumneon. FEBS Lett. 175: 95-99. [Ref.]
37. Greco M, Guida G, Perlino E, Marra E, Quagliariello E. (1989) Increase in RNA and protein synthesis by mitochondria irradiated with helium-neon laser. Biochem Biophys Res Commun. 163: 1428-1434. [PubMed.]
38. Vacca RA, Marra E, Quagliariello E, Greco M. (1993) Activation of mitochondrial DNA replication by He-Ne laser irradiation. Biochem Biophys Res Commun. 195: 704-709. [PubMed.]
39. Minelli A, Moroni M, Castellini C, Lattaioli P, Mezzasoma I, et al. (1999) Rabbit spermatozoa: a model system for studying ATP homeostasis and motility. J Androl. 20: 259-266. [PubMed.]
40. Rossato M, Virgilio FD, Rizzuto R, Galeazzi G, Foresta C. (2001) Intracellular calcium store depletion and acrosome reaction in human spermatozoa: Role of calcium and plasma membrane potential. Mol Hum Reprod. 7: 119-128. [PubMed.]