在資源貧乏的國家，大多缺乏低成本的精子檢測裝置，以致男性不孕症的精子特性和質量無法得到全面的分析。此外，在現今不孕症的困境中，男性佔了一半的因素，屬相當嚴重的問題。目前人類或畜產動物的精子檢測項目中，泳動的精子數目、精子活力程度和精子的形態皆是透過顯微鏡檢測或電腦輔助精子分析系統(CASA)進行分析。但是，這些儀器都相當昂貴，並且需要特定的操作空間。為了開發低成本和穩定的男性生育檢測裝置，我們研製了一種紙基-MTT的檢測方法，主要以四唑鎓比色法（MTT assay）評估精液中具活動力精子的百分比（即精子活力）。更進一步，我們利用加藥試驗抑制精子中線粒體的代謝活性以及糖解作用中的相關酵素，同時使用攜帶式活力分析系統（iSperm）和紙基-MTT檢測法分析精子活力與酵素的反應機制。我們以紙基-MTT檢測分析法計算擷取面積內的平均訊號強度(AMV)，並進一步評估酵素的作用機制。從結果得知，添加碘乙酰胺(IODO)和3-溴丙酮酸(3BP)這兩種抑制劑皆會使精蟲活力和AMM數值下降，因為這兩種抑制劑皆會針對糖解作用中的甘油醛-3-磷酸脫氫酶(GAPDH)進行抑制。另外，我們發現iSperm與面積平均法(AMM)在分析精蟲活力上具關連性（p<0.05），證實這些被抑制的酵素反應涉及精子泳動。根據我們的實驗，MTT的還原作用與GAPDH的催化具有關連性，並透過NADH促進電子傳遞。基於該添加抑制劑的研究，證實了我們可以利用紙基-MTT分析法評估精子的活力。此種生化檢測法能即時進行數據讀值，並利用檢測試片測定不同物種之精子，依實際使用情況作優化，探討不同代謝機轉在各種生物中扮演的角色，也可檢測藥物對動物生殖能力的影響。最後，我們希望藉由統計不同物種的精子檢測結果，建立精液品質計分評估量表，並透過其中代謝路徑的研究開發相對應的檢測試片達到對症下藥與預防疾病的功效。

In low resources countries, sperm characteristics for male infertility cannot be integral detected. Besides, male infertility is equivalent to half of all infertility cases, is a serious problem nowadays. Mammalian sperm motility has traditionally been analyzed to determine infertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male infertility diagnostics, we developed a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the value of area mean signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-Bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and area mean method (AMM) (p<0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay. The analytical methods for biochemical reactions are conceived for on-site setting via colorimetric readout. Secondly, to optimize our devices with real samples, we anticipate the difference of specimens and locations. This device can be applied to investigate the metabolism in semen and also to evaluate the effects of drugs on animal reproductive capacity. Finally, our goal is to establish a comprehensive assessment of semen quality. The more precisely measurement for semen quality can be achieved by extensive metabolic studies and infected prevention.

Abstract........................................................I
中文摘要........................................................II
誌謝辭.........................................................III
Outline........................................................IV
List of Figures................................................VI
List of Tables................................................VII
Chapter One – Introduction......................................8
Ⅰ、Study Background............................................8
1.1 Infertility.................................................8
1.2 CASA ......................................................11
1.3 iSperm ....................................................13
1.4 MTT .......................................................13
1.5 Glycolysis ................................................14
1.6 Inhibitors ................................................16
Ⅱ、Research Motivation........................................18
Chapter Two – Experimental Section.............................19
2.1 Preparation of paper-based devices........................19
2.2 Porcine sperm preparation.................................19
2.3 Sperm assay and inhibitor treatment experiments...........20
2.4 Sperm motility analysis...................................22
2.5 MTT assay.................................................23
2.6 Statistical analyses......................................26
Chapter Three – Results and Discussion.........................27
3.1 Motility analyses.........................................27
3.2 MTT assay.................................................27
3.3 Inhibitor dose-dependent effects..........................29
3.4 Correlation between sperm motility and AMV in MTT
assay.....................................................31
3.5 Discussion about energy and ATP consumptions for
sperm motility............................................32
3.6 Discussion of MTT reduction in sperm......................34
Chapter Four – Conclusion and Future Outlook...................37
4.1 Future Perspective.........................................37
4.2 Conclusion.................................................39
Supporting Information.........................................41
References.....................................................43

1. United Nations Population Division, United Nations department of Economic and Social Affairs. Available at: http://www.un.org/en/develo pment/desa/population/. (Accessed: 20th April 2017).

2. United Nations, department of Economic and Social Affairs. World Fertility Patterns 2015. Available at: http://www.un.org/en/development/desa/population /theme/fertility/. (Accessed: 20th April 2017).

3. World Health Organization, Department of Reproductive Health and Research. WHO laboratory manual for the examination and processing of human semen Fifth edition. Available at: http://www.who.int/reproductivehealth/publications/ infertility/9789241547789/en/. (Accessed: 21th April 2017).

4. Shady Grove Fertility. Trying to conceive: male factor infertility. Available at: https://www.shadygrovefertility.com/blog/diagnosing-infertility/trying-to-conceive-male-factor-infertility/. (Accessed: 21th April 2017).

5. Esteves, S. C. & Agarwal, A. Novel concepts in male infertility. Int. braz j urol. 37, 1 (2011).

6. Amann, R. P., Waberski, D. Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology. 81, 5-17 (2014).

7. Kime D. E. et al. Use of Computer assisted Sperm Analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals. Aquatic Toxicol. 36, 223–237 (1996).

8. Gadea, J. Sperm factors related to in vitro and in vivo porcine fertility. Theriogenology. 63, 431–444 (2005).

9. Broekhuijse, M. L. W. J., Sostaric, E., Feitsma, H. & Gadella, B. M. Application of computer-assisted semen analysis to explain variations in pig fertility. J. Anim. Sci. 90, 779–789 (2012).
10.Mortimer, S. T., van der Horst, G. & Mortimer, D. The future of computer-aided sperm analysis. Asian J. Androl. 17, 545–553 (2015).

11. Sloter, E. et al. Quantitative effects of male age on sperm motion. Hum. Reprod. 21, 11 2868–2875 (2006).

12. Hirano, Y. et al. Relationships between sperm motility characteristics assessed by the computer-aided sperm analysis (CASA) and fertilization rates in vitro. J. Assist. Reprod. Genet. 18, 213–218 (2001).

13. Hamzelou, J. Are your sperm up to scratch? Phone microscope lets you check. New Scientist. Available at: https://www.newscientist.com/article/2097618-are-your-sperm-up-to-scratch-phone-microscope-lets-you-check/. (Accessed: 15th July 2016).

14. Aidmics Biotechnology Co., iSperm ®. Available at: http://isperm.aidmics.com/. (Accessed: 5th August 2016).

15. Yániz, J. L., Palacín, I., Vicente-Fiel, S., Sánchez-Nadal, J. A. & Santolaria, P. Sperm population structure in high and low field fertility rams. Anim. Reprod. Sci. 156, 128–134 (2015).

16. García-Álvarez, O. et al. Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilization. Reprod. Fertil. Dev. 26 (2013).

17. Holt, C., Holt, W. V., Moore, H. D. M., Reed, H. C. B. & Curnock, R. M. Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials. J. Androl. 18, 312–323 (1997).

18. Kummer, A. B. H. P. et al. Multivariate analyses for determining the association of field porcine fertility with sperm motion traits analyzed by computer-assisted semen analysis and with sperm morphology. Reprod Dom Anim. 48, 747–754 (2013).

19. Hu, J. et al. Portable microfluidic and smartphone-based devices for monitoring of cardiovascular diseases at the point of care. Biotech. Adv. 34, 305–320 (2016).

20. Kobori, Y., Pfanner, P., Prins, G. S. & Niederberger, C. Novel device for male infertility screening with single-ball lens microscope and smartphone. Fertil. Steril. 106, 574–578 (2016).

21. Xu, X. et al. Proc. IEEE. 103, 236–247 (2015).

22. Ford, W. C. Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? Hum. Reprod. Update. 12, 269–274 (2006).

23. Tourmente, M., Villar-Moya, P., Rial, E. & Roldan, E. R. S. Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species. J. Biol. Chem. 290, 20613–20626 (2015).

24. Matsuura, K. et al. Paper-based diagnostic devices for evaluating the quality of human sperm. Microfluid. Nanofluid. 14, 1378–1388 (2014).

25. Matsuura, K., Komiyama, J. & Okitsu, O. Relationship between human spermatozoa motility and enzymatic reactivity. Fertil. Steril. 104, e239 (2015).

26. Nosrati, R. et al. Paper-Based Quantification of Male Fertility Potential. Clinic. Chem. 62, 458–465 (2016).

27. Goodson, S. G. et al. Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation. Biol. Reprod. 87, 75 (2012).

28. Takei, G. L., Miyashiro, D., Mukai, C. & Okuno, M. Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm. J. Exp. Biol. 217, 1876–1886 (2014).

29. Miki, K. et al. Glyceraldehyde-3- phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proc. Natl. Acal. Sci. USA. 101, 16501–16506 (2004).

30. Mukai, C. & Okuno, M. Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement. Biol. Reprod. 71, 540–547 (2004).

31. Stockerta, J. C., Blázquez-Castroa, A., Cañetea, M., Horobinb, R. W. & Villanueva, A. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochemica. 114, 785–796 (2012).

32. Li, X., Xue, X. & Li, P. C. H. Real-time detection of the early event of cytotoxicity of herbal ingredients on single leukemia cells studied in a microfluidic biochip. Integr. Biol. 1, 90–98 (2009).

33. Kazama, M., Asami, K. & Hino, A. Fertilization induced changes in sea urchin sperm: Mitochondrial deformation and phosphatidylserine exposure. Mol. Reprod. Dev. 73, 1303–1311 (2006).

34. Kumar, L. et al. Energy utilization for survival and fertilization - parsimonious quiescent sperm turn extravagant on motility activation in rat. Biol. Reprod. 115, 137752 (2016).

35. Hyne, R. V. & Edwards, K. P., Influence of 2-deoxy-D-glucose and energy substrates on guinea-pig sperm capacitation and acrosome reaction. J. Reprod. Fertil. 73, 59–69 (1985).

36. Huang, L. S. et al. 3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme. J. Biol. Chem. 281, 5965–5972 (2006).

37. Azevedo-Silva, J. et al. The anticancer agent 3-bromopyruvate: a simple but powerful molecule taken from the lab to the bedside. J. Bioenerg. Biomembr. 1–14 (2016).

38. Ehrke, E., Arend, C. & Dringen, R. 3-Bromopyruvate Inhibits Glycolysis, Depletes Cellular Glutathione, and Compromises the Viability of Cultured Primary Rat Astrocytes. J. Neuroscience Res. 93, 1138–1146 (2015).

39. Cardaci, S., Desideri, E. & Ciriolo, R. Targeting aerobic glycolysis: 3-bromopyruvate as a promising anticancer drug. J. Bioenerg. Biomembr. 44, 17–29 (2012).

40. Ganapathy-Kanniappan, S. et al. 3-bromopyruvate: a new targeted antiglycolytic agent and a promise for cancer therapy. Curr. Pharm. Biotechnol. 11, 510–517 (2010).

41. Rodrigues-Ferreira, C., da Silva, A. P. & Galina A. Effect of the antitumoral alkylating agent 3-bromopyruvate on mitochondrial respiration: role of mitochondrially bound hexokinase. J. Bioenerg. Biomembr. 44, 39–49 (2012).

42. Yen, T. H. et al. Evaluating organophosphate poisoning in human serum with paper. Talanta. 144, 189–195 (2015).

43. Kuan, C. M., Lin, S. T., Yen, T. H., Wang, Y. L. & Cheng, C. M. Paper-based diagnostic devices for clinical paraquat poisoning diagnosis. Biomicrofluidics. 10, 034118 (2016).

44. Animal technology laboratories. Agricultural technology research institute. Available at: http://atl.atri.org.tw/ATIT/. (Accessed: 21th April 2017).

45. Tang, Z. et al. Over-expression of GAPDH in human colorectal carcinoma as a preferred target of 3-bromopyruvate propyl ester. J. Bioenerg. Biomembr. 44, 117–125 (2012).

46. Ganapathy-Kanniappan, S. et al. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is pyruvylated during 3-bromopyruvate mediated cancer cell death. Anticancer Res. 29, 4909–4918 (2009).

47. Schmidt, M. M. & Dringen, R. Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes. Frontiers in Neuroenergetics. 1, 1 (2009).

48. Ihrlund, L. S., Hernlund, E., Khan, O. & Shoshan, M. C. 3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs. Mol. Oncol. 2, 94–101 (2008).

49. Gadea, J., Sellés, E. & Marco, M. A. The predictive value of porcine seminal parameters on fertility outcome under commercial conditions. Reprod. Dom. Anim. 39, 303–308 (2004).

50. Mallik, R. & Gross, S. P. Molecular motors: Strategies to get along. Curr. Biol. 14, R971–R982 (2004).

51. Ruiz-Pesini, E. et al. Correlation of sperm motility with mitochondrial enzymatic activities. Clin. Chem. 44, 1616–1620 (1998).

52. Lardy, H. A. & Phillips, P. H. Inhibition of sperm glycolysis and reversibility of the effects of metabolic inhibitors. J. Biol. Chem. 148, 333–341 (1943).

53. Berg, J. M., Tymoczko, J. L. & Stryer, L. Biochemistry 7th revised international Edition, chapter 18 (W. H. Freeman and Company, 2011).

54. Vistica, D. T. et al. Tetrazolium-based Assays for Cellular Viability: A Critical Examination of Selected Parameters Affecting Formazan Production. Cancer Res. 51, 2515–2520 (1991).

55. Berridge, M. V., Tan, A. S., Mccoy, K. D. & Wang, R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica. 4, 14–19 (1996).

56. Berridge, M. V., Herst, P. M. & Tan, A. S. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotech. Annual. Rev. 11, 127–152 (2005).

57. Berg, B. M. Microscopic analysis of MTT stained boar sperm cells. Open Veterinary J. 5, 58–63 (2015).

58. Nasr-Esfahani, M. H., Aboutorabi, R., Esfandiari, E. & Mardani, M. Sperm MTT viability assay: a new method for evaluation of human sperm viability. J. Assist. Reprod. Genet. 19, 477–482 (2002).