黴菌毒素種類及培養階段對孤雌激活豬胚後續發育能力之影響

Effects of mycotoxin types and culture stages on the development of parthenogenetic activation porcine embryos

洪國峻(Hung Guo-Jiun)

指導教授 ： 沈朋志 ； 彭劭于

國立屏東科技大學/農學院/動物科學與畜產系所/碩士(2018年)

https://doi.org/10.6346/THE.NPUST.AS.003.2018.D06

摘要

我國製作飼料所需之玉米係絕大多數源自進口，容易因運輸或儲存條件不佳造成黴菌感染而產生黴菌毒素（Mycotoxins），此將降低動物生長及繁殖性能，而造成畜產業之嚴重經損失。目前已知玉米赤黴烯酮（Zearalenone, ZEA）、嘔吐黴素（Deoxynivalenol, DON）及黃麴毒素（Alfatoxin B1, AFB1）對動物生殖之影響較為嚴重。惟迄今針對各種黴菌毒素在卵母細胞成熟階段及胚體外培養危害性之探討則較為闕如。因此，於本研究中擬以豬卵母細胞作為試驗材料，將探討不同濃度之α-ZOL、DON及AFB1在體外成熟 (in vitro maturation, IVM) 階段、體外培養 (in vitro culture, IVC) 階段及不同體外培養階段添加對豬卵母細胞孤雌激活後發育能力之影響。在體外成熟培養（IVM）階段之結果顯示，IVM培養液中添加10 μM α-ZOL組之卵母細胞成熟率（46.2%）顯著低於0 μM（對照組，67.5%）及1.0 μM（62.9%）組；而囊胚發育率則以添加10 μM α-ZOL組（6.4%）顯著低於對照組（38.8%）及0.5 μM（26.1%）組。IVM培養液中添加3 μM DON組之卵母細胞成熟率（38.8%）顯著低於對照組（69.8%）、1 μM（69.6%）及2 μM（63.3%）組；且3 μM DON組之囊胚率（2.7%）亦顯著低於對照組（41.2%）及1 μM（23.0%）。IVM培養液中添加6.25－25 μM AFB1組之卵母細胞成熟率與對照組無顯著差異，且所有AFB1添加組（0-3.6%）之囊胚率均顯著低於對照組（46.4%）。在體外培養（IVC）階段之結果顯示，IVC養液中添加10 μM α-ZOL組之豬胚卵裂率及囊胚率（85.9 and 0 %）顯著低於對照組（99.3 and 40.4%）、0.5 μM（96.1 and 30.0%）及1.0 μM（96.2 and 32.1%）組。IVC培養液中添加3 μM DON組之豬胚卵裂率（83.1%）均顯著低於對照組（97.1%）及2 μM（91.6%）組；而1－3 μM DON組之豬胚皆無發育至囊胚（0%），且均顯著低於對照組（39.7%）。IVC培養液中添加12.5及25 μM AFB1組之豬胚卵裂率（78.7 and 70.8%）均顯著低於對照組（97.9%），而所有AFB1添加組豬胚皆無發育至囊胚。在IVC中不同培養時程之結果顯示，於IVC培養期間，全期（α-ZOL-day 1－7組，90.0%）及前兩天（α-ZOL-day 1－2組，90.0%）培養液中添加10 μM α-ZOL組之卵裂率顯著低於對照組（98.2%）及後五天（α-ZOL-day 3－7組）培養液中添加α-ZOL組（98.3%）；對照組之囊胚率（35.7%）均顯著高於α-ZOL-day 1－7組（2.4%）、α-ZOL-day 1－2組（8.3%）及α-ZOL-day 3－7組（10.4%）。DON-day 1－7組（3.5%）及DON-day 1－2組（15.8%）之囊胚率均顯著低於對照組（43.3%）及DON-day 3－7組（34.9%）。對照組之囊胚率均顯著高於AFB1-day 1－7組（0.0%）、AFB1-day 1－2組（2.1%）及AFB1-day 3－7組（17.8%）；但AFB1-day 3－7組之囊胚率又顯著高於AFB1-day 1－7組及AFB1-day 1－2組。綜合上述結果說明，上述3種黴菌毒素均導致豬卵母細胞於IVM及IVC培養階段之發育能力下降，且發現於豬胚發育阻滯期（指4-cell期）後（day 3－7組）添加黴菌毒素，對豬胚囊胚率之負面影響低於豬胚發育阻滯期前（day 1－2組）添加黴菌毒素。

關鍵字

胚發育率 ； 黴菌毒素 ； 卵母細胞成熟率 ； 豬

並列摘要

Most of corn needed for feed is imported in Taiwan. It is easy to produce mycotoxins by fungal infection due to poor transport or storage conditions. It reduces animal growth performance and reproductive performance, resulting in serious losses in the livestock industry. Zaralenone (ZEA), Deoxynivalenol (DON), and Alfatoxin B1 (AFB1) are known to have serious effects on animal reproduction. The studies so far have focused on assessing the harmful effects of various mycotoxins on the oocyte maturation stage. However, the discussion on the stage of in vitro embryo culture is considerably poor. Therefore, porcine oocytes were used as experimental materials in this study. Different concentrations of α-ZOL, DON and AFB1 will be studied in vitro maturation (IVM) stage, in vitro culture (IVC) stage and different in vitro culture stages on the developmental ability of porcine oocytes after parthenogenetic activation. The results of mycotoxins effect in IVM stage showed that IVM medium with α-ZOL, the maturation rate (46.2%) of oocytes in the 10 μM α-ZOL group was significantly lower than 0 μM (control group, 67.5%) group and 1.0 μM (62.9%) group. The blastocyst development rate was significantly lower in the 10 μM α-ZOL group (6.4%) than in the control (38.8%) and 0.5 μM (26.1%) groups. The maturation rate of oocytes in IVM medium supplemented with 3 μM DON group (38.8%) was significantly lower than in the control group (69.8%), 1 μM (69.6%) group, and 2 μM (63.3%) group. The blastocyst rate was also significantly lower in the 3 μM DON group (2.7%) than in the control group (41.2%) and 1 μM (23.0%) group. The maturation rate of oocytes IVM in the 6.25－25 μM AFB1 groups were not significantly different from the control group, and the blastocyst rate of all AFB1 group (0－3.6%) was significantly lower than the control group (46.4%). The results of mycotoxins effect in IVC stage showed that IVC medium with α-ZOL, the cleavage rate and blastocyst rate (85.9 and 0%) of porcine embryos in the 10 μM α-ZOL group were significantly lower than those in the control (99.3 and 40.4%) group, 0.5 μM (96.1 and 30.0%) group, and 1.0 μM (96.2 and 32.1%) group. The cleavage rate (83.1%) of porcine embryos in IVC culture medium supplemented with 3 μM DON group was significantly lower than in the control (97.1%) group and 2 μM (91.6%) groups. Porcine embryos in the 1 to 3 μM DON group did not able to develop to blastocysts (0%) and were significantly lower than the control group (39.7%). IVC medium with AFB1, the cleavage rate of porcine embryos in the 12.5 and 25 μM AFB1 groups (78.7 and 70.8%) were significantly lower than in the control group (97.9%). None of the porcine embryos in the AFB1 group developed to blastocysts. The results of of mycotoxins effect in different IVC period showed that the 10 μM α-ZOL was added to the whole period (α-ZOL-day 1－7 group), the first two days (α-ZOL-day 1－2 group), and the post-five days (α-ZOL-day 3－7 group). The cleavage rate was significantly lower in the α-ZOL-day 1－7 group (90.0%) and α-ZOL-day 1－2 group (90.0%) than in the control group (98.2%) and α-ZOL-day 3－7 group (98.3%). The blastocyst rate in the control group (35.7%) was significantly higher than in the α-ZOL-day 1－7 group (2.4%), α-ZOL-day 1－2 group (8.3%), and α-ZOL-day 3－7 group (10.4%). The blastocyst rates in DON-day 1－7 (3.5%) and DON-day 1－2 (15.8%) groups were significantly lower than those in the control group (43.3%) and the DON-day 3－7 group (34.9%). The blastocyst rate of the control group (50.6%) was significantly higher than the AFB1-day 1－7 group (0.0%), AFB1-day 1－2 group (2.1%) and AFB1-day 3－7 group (17.8%); but the blastocyst rate in AFB1-day 3－7 group was significantly higher than in the AFB1-day 1－7 group and in the AFB1-day 1－2 group. In conclusion, the above three mycotoxins all resulted in decreasing the developmental competence of porcine oocytes in the IVM and IVC culture stages. It was found that the mycotoxin was added after (day 3-7 group) the porcine embryo developed in the block stage (referring to the 4-cell period), the negative effect on the blastocyst rate of porcine embryos was lower than that before porcine embryo developed in the block stage (day 1-2 group).

並列關鍵字

embryonic development ； mycotoxin ； oocytes maturation rate ； porcine

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