本土黑豬屬於台灣民間品種，為了保留原品種特有之性狀，故冷凍精液之製作方式極為重要。豬隻精液非常敏感，解凍後的冷凍精液活力通常不佳，一般影響精子活力及品質之因素包含：降溫速率、冷凍保護劑、麥管容量及抗氧化劑等。
因此本研究將利用不同製作方式來製作冷凍精液，並解凍精液觀察精子的表現及實施人工授精。沿用前人的冷凍精液製作方式，分為保溫階段（holding time）與平衡階段（equilibration），最後填裝麥管後，放置在液態氮上冷凍，再投入液態氮中直接進行保存；冷凍保護劑分別使用低密度脂蛋白（Low density lipoprotein, LDL）、離心蛋黃與新鮮蛋黃來保護精子減少在冰晶形成時細胞型態被破壞，並添加不同抗氧化劑來減少細胞被氧化。本試驗將對於不同製作方式與不同的冷凍保護劑及抗氧化劑的添加差異進行比較，並進一步對精子之活力及存活率表現做觀察。
研究結果顯示甘油比例，添加5%與6%甘油的精子活力與存活率有顯著差異（p < 0.05），冷卻速率使用階段式降溫能有效提升精子的活力與存活率（p < 0.01）；冷凍保護劑使用LDL或新鮮蛋黃皆優於離心蛋黃；冷凍精液放置於液態氮液面高度3 cm，解凍時利用50℃－12秒解凍冷凍精液其活力優於液態氮液面高度1 cm者（p < 0.05）；冷凍精液放置於液態氮液面高度3 cm及5 cm，解凍時利用50℃－12秒解凍冷凍精液，其活力優於液態氮液面高度1 cm及5 cm，與利用37℃－20秒、70℃－8秒解凍之組合；添加紫錐花萃取物於冷凍精液冷凍解凍精液，活力沒有顯著差異，但是在存活率添加0.1、0.2、及0.3%紫錐花萃取物皆優於無添加組別（p < 0.05）；於冷凍精液解凍進行人工授精之試驗結果中，比較其懷孕率新鮮精液（90%）優於冷凍精液（56%）。為了避免台灣原生品種及種原因生長性能不符效益而逐漸被淘汰，或因重大疫病而撲殺種畜，造成重要豬隻遺傳資源的流失，不斷地改良以獲取更佳的冷凍精液製作方式與條件，對於台灣本土黑豬種原活化運用極其重要，技術的提升不僅是能夠加深產業應用，更是確保珍貴種原永續存在重要的一環。

Domestic black boars in Taiwan are considered a unique species and in order to retain the unique characteristics of the original breed, the methods of freezing its semen is extremely important. Boar semen is very sensitive, and the frozen semen post thawed usually not in a good motility condition. Generally, factors that affect the sperm's motility and quality include: cooling rate, cryoprotectant, straw capacity and antioxidants, etc.
Therefore, this study will use different production methods to observe the performance of sperm, and frozen-thawed semen for artificial insemination. Using the predecessor's method of freezing black boar semen, it is divided into two stages: holding time and equilibration. After the straw is filled, it is placed ontop of liquid nitrogen for freezing, then put into liquid nitrogen for direct preservation. Cryoprotectants that have been previously used by predecessor are low density lipoprotein (LDL), centrifuged egg yolk and fresh egg yolk to protect sperm and reduce cell morphology damage when water crystals are formed, and different antioxidants have been added to reduce cell oxidation. In this experiment, the differences in freezing rate, glycerol ratio, Echinacea extract, liquid level and frozen-thawed temperature will be compared, and further the differences in sperm motility and viability will be discuss.
Additional of 5% and 6% glycerol significantly increases sperm viability (p < 0.05). The results show that stage cooling method can effectively improve sperm motility and viability (p < 0.01).LDL or fresh egg yolk as cryoprotectant is better than centrifuged egg yolk. The frozen semen placed ontop of liquid nitrogen height of 3 cm and 5 cm combine with thawing method 50°C for 12 seconds was better than that of liquid nitrogen at a height of 1 cm and 5 cm. 37℃ for 20 seconds, 70℃ for 8 seconds motility and viability (p < 0.05). The addition of Echinacea extract in frozen-thawed semen was no significant difference in motility. However the viability of adding 0.1%, 0.2%, and 0.3% Echinacea extract better than (p < 0.05) control group and post thawing semen artificial insemination in sows, the pregnancy rate of fresh semen(90%) is better than post thawing semen(56%). In order to avoid the gradual elimination of Domestic Taiwan black boars breeds and breeders due to their inconsistent growth performance, resulting in the loss of important pig genetic resources, continuous improvement has been made to obtain better frozen semen production methods and conditions. Important, the improvement of technology is not only able to deepen industrial applications, but also an important part of ensuring the sustainable existence of precious seeds.

楊逸軒。2014。冷凍保護液成分及冷凍速率對公豬精液冷凍-解凍後精液品質之影響。國立屏東科技大學動物科學與畜產系所碩士論文。
Aisen, E. G., V. H. Medina, and A. Venturino. 2002. Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology 57:1801-1808.
Alkmin, D. V., C. Perez-Patiño, I. Barranco, I. Parrilla, J.M. Vazquez, E. A. Martinez, and H. Rodriguez-Martinez, and J. Roca. 2014. Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate. Cryobiology 69: 203-210.
Amann, R. P., and B. W. Pickett. 1987. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J. Equine Vet. Sci. 7: 145-173.
Anchordoguy, T. J., A. S. Rudolph, J. F. Carpenter, and J. H. Crowe. 1987. Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24: 324-331.
Arriola, J., and R. H. Foote. 1987. Glycerolation and thawing effects on bull spermatozoa frozen in detergent-treated egg yolk and whole egg extenders. J. Dairy Sci. 70: 1664-1670.
Athurupana, R., S. Ioki, and H. Funahashi. 2015. Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender. Theriogenology 84: 940-947.
Axnér, E., U. Hermansson, and C. Linde-Forsberg. 2004. The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa. Anim. Reprod. Sci. 84: 179-191.
Bathgate, R., W. M. Maxwell, and G. Evans. 2006. Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post-thaw sperm quality. Reprod. Domest. Anim. 41: 68-73.
Berndtson, W. E., and R. H. Foote. 1972. The freezability of spermatozoa after minimal pre-freezing exposure to glycerol or lactose. Cryobiology 9: 57-60.
Bianchi, I., K. Calderam, E. F. Maschio, E. M. Madeira, R. da Rosa Ulguim, C. D. Corcini, D. C. Bongalhardo, E. K. Corrêa, T. Lucia, Jr., J. C. Deschamps, and M. N. Corrêa. 2008. Evaluation of amides and centrifugation temperature in boar semen cryopreservation. Theriogenology 69: 632-638.
Bolarín, A., J. Roca, H. Rodríguez-Martínez, M. Hernández, J. M. Vázquez, and E. A. Martínez. 2006. Dissimilarities in sows' ovarian status at the insemination time could explain differences in fertility between farms when frozen-thawed semen is used. Theriogenology 65: 669-680.
Borchers, A. T., C. L. Keen, J. S. Stern, M. E. Gershwin. 2000. Inflammation and Native American medicine: the role of botanicals. Am. J. Clin. Nutr. 72: 339-347.
Breininger, E., and M. Beconi. 2014. Ascorbic acid or pyruvate counteracts peroxidative damage in boar sperm cryopreserved with or without α-tocopherol. Anim. Sci. Pap. Rep. 32: 15-23.
Buhr, M. M., P. Fiser, J. L. Bailey, E. F. Curtis. 2001. Cryopreservation in different concentrations of glycerol alters boar sperm and their membranes. J. Androl. 22: 961-969.
Burley, R. W. 1978. Studies on the apoproteins of the major lipoprotein of the yolk of hen's eggs III. Influence of salt concentration during isolation on the amount and composition of the apoproteins. Aust. J. Biol. Sci. 31: 587-592.
Bwanga, C. O. 1991. Cryopreservation of boar semen. I. A literature-review. Acta. Vet. Scand. 32: 431-453.
Byun, S., E. Lee, and K. W. Lee. 2017. Therapeutic Implications of Autophagy Inducers in Immunological Disorders, Infection, and Cancer. Int. J. Mol. Sci. 18.
Carvajal, G., C. Cuello, M. Ruiz, J. M. Vázquez, and E. A. Martínez, J. Roca. 2004. Effects of centrifugation before freezing on boar sperm cryosurvival. J. Androl. 25: 389-396.
Cejka, C., J. Kossl, B. Hermankova, V. Holan, S. Kubinova, C. Olmiere, and J. Cejkova. 2019. The Healing of Oxidative Injuries with Trehalose in UVB-Irradiated Rabbit Corneas. Oxid. Med. Cell. Longev. 2019: 1857086.
Cerolini, S., A. Maldjian, P. Surai, and R. Noble. 2000. Viability, susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage. Anim. Reprod. Sci. 58: 99-111.
Corcuera, B. D., P. Marigorta, A. Sagüés, F. Saiz-Cidoncha, and J. F. Pérez-Gutiérrez. 2007. Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa. Theriogenology 67: 1150-1157.
Cundell, D. R., M. A. Matrone, P. Ratajczak, and J. D. Pierce, Jr. 2003. The effect of aerial parts of Echinacea on the circulating white cell levels and selected immune functions of the aging male Sprague-Dawley rat. Int. Immunopharmacol 3: 1041-1048.
Cross, N.L. 2007. Effect of pH on the development of acrosomal responsiveness of human sperm. Andrologia 39: 55-59.
de Mercado, E., M. Hernandez, E. Sanz, A. Rodriguez, E. Gomez, J. M. Vazquez, E. A. Martinez, and J. Roca. 2009. Evaluation of l-glutamine for cryopreservation of boar spermatozoa. Anim. Reprod. Sc.i 115: 149-157.
Devireddy, R. V., B. Fahrig, R. A. Godke, and S. P. Leibo. 2004. Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates. Mol. Reprod. Dev. 67: 446-457.
Di Santo, M., N. Tarozzi, M. Nadalini, and A. Borini. 2012. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Adv. Urol. 2012: 854837.
Elbein, A. D., Y. T. Pan, and I. Pastuszak, D. Carroll. 2003. New insights on trehalose: a multifunctional molecule. Glycobiology 13: 17r-27r.
Eriksson, B. M., and H. Rodriguez-Martinez. 2000. Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws. Anim. Reprod. Sci. 63: 205-220.
Esmaeili, V., A. H. Shahverdi, M. H. Moghadasian, and A.R. Alizadeh. 2015. Dietary fatty acids affect semen quality: a review. Andrology 3: 450-461.
Evans, G. 1987. Salamon's artificial insemination of sheep and goats / Gareth Evans, W.M.C. Maxwell. Butterworths, Sydney.
Evans, R. J., D. H. Bauer, S. B. Vaghefi, and C. J. Flegal. 1969. Influence of feeding cottonseed oil to laying hens on the low density lipoproteins of their eggs. J. Nutr. 99: 485-490.
Fang, Y., R. Zhong, X. Zhang, and J. Zhang, D. Zhou. 2018. Boar seminal plasma inhibits cryo-capacitation of frozen-thawed ram sperm and improves fertility following intracervical insemination. Theriogenology 105: 84-89.
Ferrante, A., C. B. Castex, S. Bruno, C. Arraztoa, J. Plaza, D. Neild, and M. Miragaya. 2018. Comparison of Whole and Centrifuged Egg Yolk Added to Kenney's and Lactose-EDTA Extenders for Donkey Semen J. Equine Vet. Sci. 65: 12-18.
Fiser, P. S., and R. W. Fairfull. 1984. The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology 21: 542-551.
Fiser, P. S., R. W. Fairfull, C. Hansen, P. L. Panich, J. N. Shrestha, and L. Underhill, 1993. The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level. Mol. Reprod. Dev. 34: 190-195.
Foulkes, J. A. 1977. The separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine spermatozoa. J. Reprod. Fertil. 49: 277-284.
Fuller, B. J. 2004. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Letters 25: 375-388.
Gadea, J., E. Sellés, M. A. Marco, P. Coy, C. Matás, R. Romar, and S. Ruiz. 2004. Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology 62: 690-701.
Gajalakshmi, S., S. Vijayalakshmi, and D. Rajeswari. 2012. Echinacea purpurea - A potent immunostimulant. Int. J. Pharm. Sci. Rev. Res. 14: 47-52.
Gale, I., L. Gil, C. Malo, N. González, and F. Martínez. 2015. Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen. Andrologia 47: 505-512.
Graham, J. K., and R. H. Foote. 1987. Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing. Cryobiology 24: 42-52.
Gutiérrez-Pérez, O., L. Juárez-Mosqueda Mde, S. U. Carvajal, and M. E. Ortega. 2009. Boar spermatozoa cryopreservation in low glycerol/trehalose enriched freezing media improves cellular integrity. Cryobiology 58: 287-292.
Hernandez, M., J. Roca, J. J. Calvete, L. Sanz, T. Muino-Blanco, J. A. Cebrian-Perez, J. M. Vazquez, and E. A. Martinez. 2007. Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the presence of seminal plasma from good freezer boars. J. Androl. 28: 689-697.
Hezavehei, M., M. Sharafi, H. M. Kouchesfahani, R. Henkel, A. Agarwal, V. Esmaeili, and A. Shahverdi. 2018. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod. Biomed. Online 37: 327-339.
Hirai, M., A. Boersma, A. Hoeflich, E. Wolf, J. Foll, T. R. Aumüller, and J. Braun. 2001. Objectively measured sperm motility and sperm head morphometry in boars (Sus scrofa): relation to fertility and seminal plasma growth factors. J .Androl. 22: 104-110.
Holt, W. V. 2000. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 53: 47-58.
Holt, W. V., and R. D. North. 1994. Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasma membrane damage in cryopreserved ram spermatozoa. Biol. Reprod. 51: 414-424.
Hu, J. H., Q. W. Li, L. Gang, X. Y. Chen, Y. Hai, S. S. Zhang, and L. Q. Wang. 2006. The cryoprotective effect on frozen-thawed boar semen of egg yolk low density lipoproteins. Asian Australas. J. Anim. Sci. 19: 486-494.
Jeong, Y. J., M. K. Kim, H. J. Song, E. J. Kang, S. A. Ock, B. M. Kumar, S. Balasubramanian, and G. J. Rho. 2009. Effect of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes. Cryobiology 58: 181-189.
Johnson, L., and K. Larsson. 1985. Future prospects for deep freezing of boar semen, Proceedings, 1st International Conference on Deep Freezing of Boar Semen, Aug 25-27: 1985: Swedish Univ. of Agricultural Sciences, Dept. of Obstretics and Gynaecology, Uppsala/editors LA Johnson and K. Larsson, Uppsala, Sweden: Swedish Council for Forestry and Agricultural Research, 1985.
Johnson, L. A., J. G. Aalbers, and J. A. M. Arts. 1982. Use of Boar Spermatozoa for Artificial Insemination II. Fertilizing Capacity of Fresh and Frozen Spermatozoa in Gilts Inseminated Either at a Fixed Time or According to Walsmeta Readings. J. Anim. Sci. 54: 126-131.
Johnson, L. A., J. G. Aalbers, C. M. T. Willems, and W. Sybesma. 1981. Use of Boar Spermatozoa for Artificial Insemination. I. Fertilizing Capacity of Fresh and Frozen Spermatozoa in Sows on 36 Farms. J. Anim. Sci. 52: 1130-1136.
Johnson, L. A., K. F. Weitze, P. Fiser, and W. M. C. Maxwell. 2000. Storage of boar semen. Anim. Reprod. Sci. 62: 143-172.
Juarez, J. D., I. Parrilla, J. M. Vazquez, E. A. Martinez, and J. Roca. 2011. Boar semen can tolerate rapid cooling rates prior to freezing. Reprod. Fertil. Dev. 23: 681-690.
Kandror, O., A. DeLeon, and A. L. Goldberg. 2002. Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. Proc. Natl. Acad. Sci. U S A 99: 9727-9732.
King, G. J., and J. W. Macpherson. 1966. Alkaline and acid phosphatase activity, pH and osmotic pressure of boar semen. Can. J. Comp. Med. Vet. Sci. 30: 304-307.
King, G. J., and J. W. Macpherson. 1967. Boar semen studies. II. Laboratory and fertility results of a method for deep freezing. Can. J. Comp. Med. Vet. Sci. 31: 46-47.
Knox, R.V. 2016. Artificial insemination in pigs today. Theriogenology 85: 83-93.
Kunze, H. 1981. Phospholipase A in the semen. Occurrence, properties and possible physiological significance. Fortschr. Med. 99: 1609-1611.
Larsson, K., S. Einarsson, and T. Swensson, 1977. The development of a practicable method for deepfreezing of boar spermatozoa. Nord. Vet. Med. 29: 113-118.
Levitt, J. 1981. Low Temperature Preservation in Medicine and Biology. M. J. Ashwood-Smith , J. Farrant. Q. Rev. Biol. 56: 243-243.
Macías García, B., C. Ortega Ferrusola, I. M. Aparicio, A. Miró-Morán, A. Morillo Rodriguez, J. M. Gallardo Bolaños, L. González Fernández, C. M. Balao da Silva, H. Rodríguez Martínez, J. A. Tapia, and F. J. Peña, 2012. Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77: 1280-1289.
Malo, C., L. Gil, N. Gonzalez, R. Cano, I. de Blas, and E. Espinosa. 2010. Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender. Cryobiology 61: 17-21.
Martins-Bessa, A., A. Rocha, and A. Mayenco-Aguirre. 2007. Incorporation of taurine and hypotaurine did not improve the efficiency of the Uppsala Equex II extender for dog semen freezing. Theriogenology 68: 1088-1096.
Mazur, P. 1984. Freezing of living cells: mechanisms and implications. Am J Physiol. 247: C125-142.
Mazur, P., S. P. Leibo, and G. E. Seidel Jr. 2008. Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions. Biol. Reprod. 78: 2-12.
Merati, Z., and A. Farshad. 2020. Ginger and echinacea extracts improve the quality and fertility potential of frozen-thawed ram epididymal spermatozoa. Cryobiology 92: 138-145.
Meyers, S.A. 2005. Spermatozoal response to osmotic stress. Anim. Reprod. Sci. 89: 57-64.
Mogas, M. T., M. R. d. Álamo, and J. E. Rodríguez-Gil. 2011. Roles of Na+/K+-dependent ATPase, Na+/H+ antiporter and GLUT hexose transporters in the cryosurvival of dog spermatozoa: Effects on viability, acrosome state and motile sperm subpopulation structure. Theriogenology 75: 1669-1681.
Molinia, F.C., G. Evans, P.I. Quintana Casares, and W. M. C. Maxwell. 1994. Effect of monosaccharides and disaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. Anim. Reprod. Sci. 36: 113-122.
Nouri, H., A. Towhidi, M. Zhandi, and R. Sadeghi. 2013. The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen. J. Equine Vet. Sci. 33: 1050-1053.
O'Neill, W., S. McKee, and A. F. Clarke. 2002. Immunological and haematinic consequences of feeding a standardised Echinacea (Echinacea angustifolia) to healthy horses. Equine. Vet. J. 34: 222-227
Pace, M. M., and E. F. Graham. 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39: 1144-1149.
Pagliaro, M., and M. Rossi. 2008. The Future of Glycerol: New Usages for a Versatile Raw Material. Am. J. Ther. 16: 466-467.
Peña, F. J., F. Saravia, I. Núñez-Martínez, A. Johannisson, M. Wallgren, and H. Rodriguez Martinez. 2006. Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation? Anim. Reprod. Sci. 93: 101-113.
Pellati, F., S. Benvenuti, M. Melegari, and T. Lasseigne. 2005. Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC. Phytochem. Anal. 16: 77-85.
Percival, S. S. 2000. Use of echinacea in medicine. Biochem. Pharmacol. 60: 155-158.
Prasad, A. S. 2013. Discovery of human zinc deficiency: its impact on human health and disease. Adv. Nutr. 4: 176-190.
Pursel, V. G., and L. A. Johnson. 1975. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40: 99-102.
Rato, L., M. G. Alves, J. E. Cavaco, and P. F. Oliveira. 2014. High-energy diets: a threat for male fertility? Obes. Rev. 15: 996-1007.
Recuero, S., B. Fernandez-Fuertes, S. Bonet, I. Barranco, and M. Yeste. 2019. Potential of seminal plasma to improve the fertility of frozen-thawed boar spermatozoa. Theriogenology 137: 36-42.
Restrepo, G., C. A. Madrid, L. Prieto, J. E. Duque, and A. Usuga. 2017. Freezing of epididymal canine sperm with centrifuged egg yolk. Rev. de Investig. Vet. del Peru 28: 876-885.
Roca, J., M. Hernandez, G. Carvajal, J. M. Vazquez, and E.A. Martinez. 2006. Factors influencing boar sperm cryosuirvival. J. Anim. Sci. 84: 2692-2699.
Rodriguez-Martinez, H., and M. Wallgren. 2010a. Advances in boar semen cryopreservation. Vet. Med. Int. 2010: 396181.
Roesler, J., C. Steinmüller, A. Kiderlen, A. Emmendörffer, H. Wagner, and M. L. Lohmann-Matthes. 1991. Application of purified polysaccharides from cell cultures of the plant Echinacea purpurea to mice mediates protection against systemic infections with Listeria monocytogenes and Candida albicans. Int. J. Immunopharmacol. 13: 27-37.
Rubinsztein, D. C., C. F. Bento, and V. Deretic. 2015. Therapeutic targeting of autophagy in neurodegenerative and infectious diseases. J. Exp. Med. 212: 979-990.
Rudolph, A. S., J. H. Crowe, and L. M. Crowe. 1986. Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids. Arch. Biochem. Biophys. 245: 134-143.
Safarinejad, M. R., S. Y. Hosseini, F. Dadkhah, and M. A. Asgari. 2010. Relationship of omega-3 and omega-6 fatty acids with semen characteristics, and anti-oxidant status of seminal plasma: a comparison between fertile and infertile men. Clin. Nutr. 29: 100-105.
Salamon, S., I. Wilmut, and C. Polge. 1973. Deep freezing of boar semen. I. Effects of diluent composition, protective agents, and method of thawing on survival of spermatozoa. Aust. J. Biol. Sci. 26: 219-230.
Saravia, F., I. Núñez-Martínez, J. M. Morán, C. Soler, A. Muriel, H. Rodríguez-Martínez, and F. J. Peña. 2007. Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity. Theriogenology 68: 196-203.
Saravia, F., M. Wallgren, S. Nagy, A. Johannisson, and H. Rodríguez-Martínez. 2005. Deep freezing of concentrated boar semen for intra-uterine insemination: effects on sperm viability. Theriogenology 63: 1320-1333.
Sieme, H., H. Oldenhof, and W. F. Wolkers. 2016. Mode of action of cryoprotectants for sperm preservation. Anim. Reprod. Sci. 169: 2-5.
Singleton, W. 2000. Growth and development of AI centers in the USA. Allen Press Inc. Lawrence. KS: 147.
Sugano, H., and I. Watanabel. 1961. Isolations and some properties of native lipoproteins from egg yolk. J. Biochem. 50: 473-480.
Sztein, J. M., K. Noble, J. S. Farley, and L. E. Mobraaten. 2001. Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation. Cryobiology 42: 28-39.
Techakumphu, M., K. Buranaamnuay, W. Tantasuparuk, and N. Am-I. 2013. Improvement of Semen Quality by Feed Supplement and Semen Cryopreservation in Swine, Success in Artificial Insemination - Quality of Semen and Diagnostics Employed. p.17-37.
Thurston, L. M., P. F. Watson, A. J. Mileham, and W. V. Holt. 2001. Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation. J. Androl. 22: 382-394.
Tomas, C., J. Gomez-Fernandez, E. Gomez-Izquierdo, and E. de Mercado. 2014. Effect of the holding time at 15 degrees C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation. Anim. Reprod. Sci. 144: 115-121.
Varo-Ghiuru, F., I. Miclea, A. Hettig, I. Ladoşi, V. Miclea, I. Egerszegi, and M. Zăhan. 2015. Lutein, Trolox, ascorbic acid and combination of Trolox with ascorbic acid can improve boar semen quality during cryopreservation. Cryo Letters 36: 1-7.
Vujkovic, M., J. H. de Vries, G. R. Dohle, G. J. Bonsel, J. Lindemans, N. S. Macklon, P. J. van der Spek, E. A. Steegers, and R. P. Steegers-Theunissen. 2009. Associations between dietary patterns and semen quality in men undergoing IVF/ICSI treatment. Hum. Reprod. 24: 1304-1312.
Vyt, P., D. Maes, S. U. Sys, T. Rijsselaere, and A. Van Soom, 2007. Air contact influences the pH of extended porcine semen. Reprod. Domest. Anim. 42: 218-220.
Wang, P., Y. F. Wang, C. W. Wang, S. H. Bu, J. H. Hu, Q.W. Li, W. J. Pang, and G. S. Yang. 2014. Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing-thawing. Zygote 22: 175-181.
Waterhouse, K. E., P. O. Hofmo, A. Tverdal, and R. R. Miller. 2006. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction 131: 887-894.
Watson, P. F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7: 871-891.
Watson, P. F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60-61: 481-492.
Watson, P. F., and I. C. Martin. 1975. The influence of some fractions of egg yolk on the survival of ram spermatozoa at 5 degrees C. Aust. J. Biol. Sci. 28: 145-152.
Weitze, K. 2000. Update on the worldwide application of swine AI. Boar semen preservation IV: 141-145.
Weitze, K. F., D. Rath, and G. Baron. 1987. New aspects of preservation of boar sperm by deep freezing in plastic tubes. Berl. Munch. Tierarztl. Wochenschr. 94: 485-486, 488.
Westendorf, P., L. Richter, and H. Treu. 1975. Deep freezing of boar sperma. Laboratory and insemination results using the Hülsenberger paillete method. Berl. Munch. Tierarztl. Wochenschr. 82: 261-267.
White, I.G. 1993. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod. Fertil. Dev. 5: 639-658.
Whitworth, K. M., R. R. R. Rowland, C. L. Ewen, B. R. Trible, M. A. Kerrigan, A. G. Cino-Ozuna, M. S. Samuel, J. E. Lightner, D. G. McLaren, A. J. Mileham, K. D. Wells, and R. S. Prather. 2016. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat. Biotechnol. 34: 20-22.
Wu, T. W., F. P. Cheng, I. H. Chen, C. H. Yang, M. Y. Tsai, M. H. Chang, J. H. Wang, and J. T. Wu. 2013. The combinatorial effect of different Equex STM paste concentrations, cryoprotectants and the straw-freezing methods on the post-thaw boar semen quality. Reprod. Domest. Anim. 48: 53-58.
Yeste, M. 2016. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology 85: 47-64.
Yeste, M., E. Estrada, I. Casas, S. Bonet, and J. E. Rodríguez-Gil. 2013. Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels. Theriogenology 79: 929-939.
Yeste, M., E. Estrada, E. Pinart, S. Bonet, J. Miro, and J. E. Rodriguez-Gil. 2014. The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability. Cryobiology 68: 251-261.
Yeste, M., S. Sancho, M. Briz, E. Pinart, E. Bussalleu, and S. Bonet. 2010. A diet supplemented with L-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase. Theriogenology 73: 577-586.
Zak, L. J., A. H. Gaustad, A. Bolarin, M. Broekhuijse, G. A. Walling, and E. F. Knol. 2017. Genetic control of complex traits, with a focus on reproduction in pigs. Mol. Reprod. Dev. 84: 1004-1011.
Zhang, W., K. Yi, C. Chen, X. Hou, and X. Zhou. 2012. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. Anim. Reprod. Sci. 132: 123-128.