臺灣博碩士論文加值系統

家畜精液之保存主要有液態與冷凍兩種方式，目前豬人工授精產業使用之精液仍以液態保存為主，藉由添加不同類型之稀釋液以延長保存時間。稀釋精液在液態保存期間會因為少數精子死亡，造成精子之凝集現象，使得更多精子死亡。在兔子與綿羊之稀釋精液中添加明膠 (gelatin) 可以使精子均勻分佈，避免精子凝集而沉澱，維持精子之活動力及頭巾膜完整性，並改善生育力，但尚未應用於豬人工授精產業；三仙膠 (Xanthan gum) 是一種廣泛應用於化妝品之增稠劑，也可以使精子均勻懸浮於稀釋精液中，但未有對公豬精液品質影響之研究。本試驗擬開發含有明膠或三仙膠之稀釋液，應用於公豬精液冷藏保存，首先將稀釋液分別保存在 16℃ 3 日、4℃ 14 日或 -20℃ 90 日，檢測稀釋液之 pH 值、滲透壓與總生菌數之變化，評估保存期間稀釋液品質之變化；之後進行精液之稀釋保存，應用流式細胞儀與螢光顯微鏡檢測精液之品質，且以體外受精技術驗證生育力。試驗結果顯示，於 4℃ 保存 14 日與 16℃ 保存 3 日之稀釋液，其滲透壓可以穩定維持，但 pH 值皆顯著較新鮮配製者高 0.2；然而於 -20℃ 保存 90 日之稀釋液相較於 16℃ 保存 3 日者，除了含有 1.5% 明膠之稀釋液外，其他稀釋液之 pH 值顯著較高 (P < 0.05)，但仍在精子可耐受之範圍內。以含有 1.5% 明膠或 0.3% 三仙膠之稀釋液稀釋公豬精液，保存 3 日後，仍可以使精子懸浮於上層，然而未添加明膠或三仙膠之稀釋精液，在保存 6 小時後，精子皆沉澱於下層。添加明膠或三仙膠之稀釋精液，保存至第 3 日時，其精子活力與頭巾完整性均顯著較第 0 日者為低 (P < 0.05)，但仍符合人工授精之合格標準；在保存第 0 日時，添加明膠之稀釋精液之精子膜完整性 (93.8 ± 1.5%) 與粒線體活性 (89.9 ± 0.4%) 均顯著高於其他稀釋精液 (P < 0.05)，但保存至第 3 日時，其精子存活率 (79.3 ± 1.7%) 則顯著低於其他稀釋精液 (P < 0.05)。進一步以體外受精技術驗證精子之生育力，比較有進行搖晃或不搖晃，與添加明膠或三仙膠之稀釋精液，保存 3 日後進行體外受精，其精子穿透率分別為：86.3 ± 20.3%、70.4 ± 7.0%、88.6 ± 7.1% 與 76.6 ± 1.3% 並無顯著差異。綜合上述，添加明膠或三仙膠之稀釋精液，可以維持良好精液品質與良好之生育力。

Livestock semen is preserved in liquid or frozen states. The principal preservation mode of semen used for pig artificial insemination (AI) is liquid storage, which prolongs the storage time of boar semen to different extenders, depending on the extender used. During extended semen storage, the death of some spermatozoa causes agglutination of the cells, causing more of the live sperm to die. The addition of gelatin to semen extenders used for storage of rabbit and sheep semen has resulted in a more uniform distribution of spermatozoa and prevents sedimentation. This has not only maintained sperm motility and membrane integrity but also improved fertility. However, the gelatin-supplemented extenders used for boar are not applicable to pig AI. Xanthan gum (X-gum) is a widely used cosmetic thickener, and it can be used to uniformly suspend semen; however, the effect of X-gum on boar semen quality is unknown. The objective of this study was to develop gelatin- or X-gum-supplemented extenders applicable to refrigerated preservation of boar semen. Initially, the extenders were stored at 16°C for 3 days, 4°C for 14 days, or -20°C for 90 days, and we examined changes in pH value, osmotic pressure, and total bacteria count to evaluate the effect of storage on the quality of the extender. Fresh boar semen was then diluted in the extenders and stored. Semen quality was examined by flow cytometry and fluorescence microscopy, and fertility was verified by in vitro fertilization (IVF). The results indicated that the osmotic pressure of extenders could be maintained during storage at 16°C for 3 days and 4°C for 14 days, but that the pH value was significantly 0.2 higher than that of fresh extenders. Nevertheless, when comparing extenders stored at -20°C for 90 days and at 16°C for 3 days, the pH values and osmotic pressures were significantly higher expected mBTS (+G) (P < 0.05), but were still tolerated by the sperm. Boar semen was extended in 1.5% gelatin- or 0.3% X-gum-supplemented extenders for 3 days. In these extenders, the spermatozoa remained suspended in the upper layer; however, in semen extenders lacking gelatin or X-gum, the semen sedimented after 6 hours. When the semen was extended in gelatin- or X-gum-supplemented extenders, the sperm motility and membrane integrity were significantly lower on the 3rd day than on the 1st day (P < 0.05); however, these factors still met the eligibility criteria for pig AI. The sperm membrane integrity (93.8 ± 1.5%) and mitochondrial function (89.9 ± 0.4%) on the 1st day (P < 0.05) were significantly higher for semen extended in gelatin-supplemented extenders than those of semen extended in other types of extenders. However, the sperm viability (79.3 ± 1.7%) was significantly lower in gelatin-supplemented extenders than in other types on the 3rd day (P < 0.05). Using IVF of pig oocytes, we then compared the fertility of the extended semen between that stored with shaking or non-shaking, and that stored in gelatin- or X-gum-supplemented extenders for 3 days. The sperm penetration rates were 86.3 ± 20.3%, 70.4 ± 7.0%, 88.6 ± 7.1%, and 76.6 ± 1.3%, respectively; there were no significant differences in fertility between semen stored under these conditions. In conclusion, boar semen extended with gelatin- or X-gum-supplemented extenders can effectively maintain semen quality and fertility.

目錄
中文摘要 I
英文摘要 III
誌謝 VI
表次 XI
圖次 XII
附次 XV
壹、前言 1
貳、文獻檢討 3
一、豬人工授精 3
(一) 國外現況 4
(二) 國內現況 4
(三) 授精方式 5
二、公豬精液保存 7
(一) 液態保存 7
(二) 冷凍保存 8
三、稀釋液 9
(一) 稀釋液成分 10
(二) 種類 12
四、精液品質檢測 14
(一) 顯微鏡檢測 14
1. 物理性狀 14
2. 精子濃度 14
3. 精子活力 14
4. 精子形態 15
(二) 流式細胞儀檢測 15
1. 精子存活率 17
2. 精子頭巾完整性 17
3. 精子粒線體功能性 18
4. 精子獲能狀態及膜流動性 18
5. 精子染色質結構分析 19
五、公豬生育力之評估 20
(一) 人工授精 20
(二) 體外受精 20
参、材料與方法 22
一、精液之收集與稀釋 22
二、稀釋液之製備 22
三、稀釋液 pH 值、滲透壓與總生菌數檢測 22
四、精液品質檢測 23
(一) 精子濃度 23
(二) 顯微鏡檢查 23
1. 活力 23
2. 形態 23
(三) 流式細胞儀評估精液品質 26
1. 存活率 26
2. 頭巾膜完整率 26
3. 粒線體功能性 27
五、精子之體外受精 34
(一) 卵母細胞之體外成熟 34
(二) 卵母細胞與精子之體外受精 35
(三) 卵母細胞形態之評估 36
(四) 體外受精之評估指標 36
六、試驗設計 36
(一) 以流式細胞儀檢測公豬精液性狀之有效性 37
(二) 稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 37
(三) 在精液保存期間不同稀釋液對精液品質之影響 38
(四) 應用體外受精技術評估稀釋液對保存精液生育力之影響 38
七、統計分析 39
肆、 結果 40
一、以流式細胞儀檢測公豬精液性狀之有效性 40
二、稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 48
三、在精液保存期間不同稀釋液對精液品質之影響 60
四、應用體外受精技術評估稀釋液對保存精液生育力之影響 68
伍、討論 72
一、以流式細胞儀檢測公豬精液性狀之有效性 72
二、稀釋液保存期間 pH 值、滲透壓與總生菌數之變化 72
三、在精液保存期間不同稀釋液對精液品質之影響 75
四、應用體外受精技術評估稀釋液對保存精液生育力之影響 77
陸、結論 78
柒、參考文獻 79
捌、附錄 86

程伶，2009。歐美養豬新資訊 (二十四) 。現代養豬，31 (1): 19-25。
劉世華，2012。豬隻人工授精站精液生產。現代養豬，33 (10): 69-74。
陳銘正，2003。豬隻人工授精站作業標準。國立宜蘭大學農業推廣季刊，第二十八期。
郭有海、劉世華，2004。豬隻人工授精站標準作業書手冊。財團法人台灣動物科技研究所。
Anil, S. S., A. Larriestra, J. Deen, R. B. Morrison and L. Minion. 2004. A retrospective study on the preserving capacity of a commercial boar semen extender. Theriogenology 62: 425-436.
Bailey, J. L., C. Lessard, J. Jacques, C. Breque, I. Dobrinski, W. Zeng and H. L. Galantino-Homer. 2008. Cryopreservation of boar semen and its future importance to the industry. Theriogenology 70: 1251-1259.
Boonkusol, D., K. Saikhun and P. Ratanaphumma. 2010. Effects of extender and storage time on motility and ultrastructure of cooled-preserved boar spermatozoa. Kasetsart J. (Nat. Sci.) 44: 582-589.
Cheng, W. T. K. 1985. In vitro fertilization of farm animal oocytes. Ph. D. Thesis, University of Cambridge.
Corcini, C. D., F. Moreira, R. Pigozzo, A. S. Varela, N. U. Torres and T. Lucia. 2011. Semen quality and reproductive performance after artificial insemination with boar sperm stored in a gelatin-supplemented extender. Livest. Sci. 138: 289-292.
Didion, B. A., K. M. Kasperson, R. L. Wixon and D. P. Evenson. 2009. Boar fertility and sperm chromatin structure status: a retrospective report. J. Androl. 30: 655-660.
Evenson, D. P., Z. Darzynkiewicz and M. R. Melamed. 1980. Comparison of human and mouse sperm chromatin structure by flow cytometry. Chromosoma 78: 225-238.
Foxcroft, G. R., M. K. Dyck, A. Ruiz-Sanchez, S. Novak and W. T. Dixon. 2008. Identifying useable semen. Theriogenology 70: 1324-1336.
Fraser, L., K. Gorszczaruk and J. Strzezek. 2001. Relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality. Reprod. Domest. Anim. 36 Suppl 2: 325-329.
Funahashi, H., N. H. Kim, T. T. Stumpf, T. C. Cantley and B. N. Day. 1996. Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes. Biol. Reprod. 54: 1412-1419.
Funahashi, H. and T. Sano. 2005. Select antioxidants improve the function of extended boar semen stored at 10 degrees C. Theriogenology 63: 1605-1616.
Gadea, J. 2003. Review: Semen extenders used in the artificial insemination of swine. Span. J. Agric. Res. 1: 17-27.
Garner, D. L., J. R. Dobrinsky, G. R. Welch and L. A. Johnson. 1996. Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SyBr-14. Theriogenology 45: 1103-1113.
Garner, D. L., C. A. Thomas, H. W. Joerg, J. M. DeJarnette and C. E. Marshall. 1997. Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. Biol. Reprod. 57: 1401-1406.
Gil, M. A., C. Alminana, J. Roca, J. M. Vazquez and E. A. Martinez. 2008. Boar semen variability and its effects on IVF efficiency. Theriogenology 70: 1260-1268.
Gillan, L., G. Evans and W. M. Maxwell. 2005. Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63: 445-457.
Graham, J. K. 2001. Assessment of sperm quality: a flow cytometric approach. Anim. Reprod. Sci. 68: 239-247.
Gravance, C. G., D. L. Garner, J. Baumber and B. A. Ball. 2000. Assessment of equine sperm mitochondrial function using JC-1. Theriogenology 53: 1691-1703.
Hong, J. Y., H. Y. Yong, B. C. Lee, W. S. Hwang, J. M. Lim and E. S. Lee. 2004. Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media. Theriogenology 62: 1473-1482.
Huo, L. J., X. H. Ma and Z. M. Yang. 2002. Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage. Theriogenology 58: 1349-1360.
Johnson, L. A., K. F. Weitze, P. Fiser and W. M. Maxwell. 2000. Storage of boar semen. Anim. Reprod. Sci. 62: 143-172.
Kasai, T., K. Ogawa, K. Mizuno, S. Nagai, Y. Uchida, S. Ohta, M. Fujie, K. Suzuki, S. Hirata and K. Hoshi. 2002. Relationship between sperm mitochondrial membrane potential, sperm motility and fertility potential. Asian J. Androl. 4: 97-103.
Kumaresan, A., G. Kadirvel, K. M. Bujarbaruah, R. K. Bardoloi, A. Das, S. Kumar and S. Naskar. 2009. Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa. Anim. Reprod. Sci. 110: 162-171.
Kwon, I. K., K. E. Park and K. Niwa. 2004. Activation, pronuclear formation and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa. Biol. Reprod. 71: 1430-1436.
Latif, A., A. Ijaz, M. Aleem and A. Mahmud. 2005. Effect of osmotic pressure and ph on the short-term storage and fertility of broiler breeder sperm. Pakistan Vet. J. 25(4): 179-182.
Larsson, B. and H. Rodriguez-Martinez. 2000. Can we use in vitro fertilization tests to predict semen fertility? Anim. Reprod. Sci. 60-61: 327-336.
Lòpez-Gatius, F., G. Sances, M. Sancho, J. Yaniz, P. Santolaria, R. Gutierrez, M. Nunez, J. Nunez and C. Soler. 2005. Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen. Theriogenology 64: 252-260.
Lòpez Rodriguez, A., T. Rijsselaere, P. Vyt, A. Van Soom and D. Maes. 2011. Effect of Dilution Temperature on Boar Semen Quality. Reprod. Domest. Anim. Doi: 10.1111/j.1439-0531.2011.01938.x.
Manafi, M. 2011. Artificial Insemination in Farm Animals. InTech. Croatia.
Martinez-Pastor, F., M. Mata-Campuzano, M. Alvarez-Rodriguez, M. Alvarez, L. Anel and P. de Paz. 2010. Probes and techniques for sperm evaluation by flow cytometry. Reprod. Domest. Anim. 45 Suppl 2: 67-78.
Mattioli, M., M. L. Bacci, G. Galeati and E. Seren. 1989. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 31: 1201-1207.
Nagy, S., G. Sinkovics and A. Kovacs. 2002. Viability and acrosome integrity of rabbit spermatozoa processed in a gelatin-supplemented extender. Anim. Reprod. Sci. 70: 283-286.
Newth, M.S and D. G. Levis. 1999. Change in pH boar of semen extenders. Nebraska Swine Report: 3-6.
Purdy, P. H., N. Tharp, T. Stewart, S. F. Spiller and H. D. Blackburn. 2010. Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics. Theriogenology 74: 1304-1310.
Riesenbeck, A. 2011. Review on international trade with boar semen. Reprod. Domest. Anim. 46 Suppl 2: 1-3.
Roca, J., I. Parrilla, H. Rodriguez-Martinez, M. A. Gil, C. Cuello, J. M. Vazquez and E. A. Martinez. 2011. Approaches towards efficient use of boar semen in the pig industry. Reprod. Domest. Anim. 46 Suppl 2: 79-83.
Roca, J., J. M. Vazquez, M. A. Gil, C. Cuello, I. Parrilla and E. A. Martinez. 2006. Challenges in pig artificial insemination. Reprod. Domest. Anim. 41 Suppl 2: 43-53.
Rozeboom, K. J., D. L. Reicks and M. E. Wilson. 2004. The reproductive performance and factors affecting on-farm application of low-dose intrauterine deposit of semen in sows. J. Anim. Sci. 82: 2164-2168.
Ruiz-Sanchez, A. L., R. O'Donoghue, S. Novak, M. K. Dyck, J. R. Cosgrove, W. T. Dixon and G. R. Foxcroft. 2006. The predictive value of routine semen evaluation and IVF technology for determining relative boar fertility. Theriogenology 66: 736-748.
Steverink, D. W. B., N. M. Soede, E. G. Bouwman and B. Kemp. 1998. Semen backflow after insemination and its effect on fertilisation results in sows. Anim. Reprod. Sci. 54: 109-119.
Vyt, P., D. Maes, E. Dejonckheere, F. Castryck and A. Van Soom. 2004. Comparative study on five different commercial extenders for boar semen. Reprod. Domest. Anim. 39: 8-12.
Waberski, D., H. Henning and A. M. Petrunkina. 2011. Assessment of storage effects in liquid preserved boar semen. Reprod. Domest. Anim. 46 Suppl 2: 45-48.
Waterhouse, K. E., P. M. De Angelis, T. Haugan, H. Paulenz, P. O. Hofmo and W. Farstad. 2004. Effects of in vitro storage time and semen-extender on membrane quality of boar sperm assessed by flow cytometry. Theriogenology 62: 1638-1651.
Xu, X., J. Ding, P. C. Seth, D. S. Harbison and G. R. Foxcroft. 1996. In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculate fraction. Theriogenology 45: 745-755.
Xu, X., S. Pommier, T. Arbov, B. Hutchings, W. Sotto and G. R. Foxcroft. 1998. In vitro maturation and fertilization techniques for assessment of semen quality and boar fertility. J. Anim. Sci. 76: 3079-3089.
Yaniz, J., J. I. Marti, M. A. Silvestre, J. Folch, P. Santolaria, J. L. Alabart and F. Lopez-Gatius. 2005. Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity. Theriogenology 64: 1844-1851