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研究生:許勝富
研究生(外文):Hsu, Hsi-Fuh
論文名稱:保存豬卵巢之環境和培養條件對豬腔前濾泡和吝母細胞於離體發育之影響
論文名稱(外文):Effects of Preservative Conditions of Porcine Ovaries, and the Cultural Terms on the Development of Porcine Preantral follicles and Oocytes In Vitro
指導教授:鄭三寶
指導教授(外文):San-Pao Cheng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:畜牧學系
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:122
中文關鍵詞:豬卵巢保存環境培養條件腔前濾泡卵母細胞離體發育
外文關鍵詞:Porcine OvaryPreservative!conditionsCultural termsPreantral follicleOocyteDevelopment in vitso
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哺乳動物卵巢腔前濾泡(preantral follicle)之離體培養,除可用為探
討濾泡發育之機制外,並可培育出大量之卵母細胞供為繁殖相關科技之材
料。為了設定最佳保存豬卵巢組織之環境,以提供為運送卵巢組織時之依
據,以及評估培養模式和培養液中之FSH含量等對不同生長期別之腔前濾
泡於離體生長和卵母細胞之存活率之影響,而進行一系列之試驗。評估保
存豬卵巢於不同溫度[低溫組(5∼10℃)、室溫組(20∼25℃)和高溫組(32
∼37℃)]經歷不同時間(10、30或60分鐘)之影響性發現,僅經歷保存10分
鐘,即將卵巢細切並浸泡於39℃膠原蛋白脢(1.5 mg/ml collagenase)溶
液60分鐘以軟化組織之處理,則收集之腔前濾泡、腔狀濾泡、卵丘-卵母
細胞複合體或裸露卵母細胞等四類來源之卵母細胞之存活率,在不同溫度
組別間之差異不顯著(P>0.05)。當保存卵巢組織之時間延長至30或60分鐘
時,則低溫保存組者之卵母細胞之存活率顯著低於室溫或高溫保存組者(
P<0.05)。若將保存卵巢經歷60分鐘,並將所收集之腔前濾泡,經海藻酸
鈣膠層包埋培養4天,則室溫組之卵母細胞之存活率顯著低於高溫組
者(53.5% vs. 76.5%, P<0.05);惟保存卵巢僅經歷30分鐘者則否(77.9%
vs. 79.7%, P>0.05)。經12天培養後,小型(200∼<300mm )、中型(300
∼<400mm )和大型(400∼<500mm )等不同生長期別之腔前濾泡和卵母細胞
之直徑增加幅度以及卵母細胞之存活率等,在溫度組別間之差異均不顯
著(P>0.05)。為評估導致低溫保存60分鐘之卵巢組織之各級濾泡內卵母細
胞存活率下降之原因,乃直接檢測取自未經39℃膠原蛋白溶液軟化處理
之低溫(5∼10℃)保存10、30或60分鐘之卵巢表面腔狀濾泡之卵母細胞存
活率,結果顯示,卵母細胞之存活率在三種不同時間組別間或不同生長階
段之腔狀濾泡[直徑<3(小型)、3∼<5(中型)和5∼<7 (大型)mm]間之差異
均不顯著(P>0.05)。惟將取自低溫保存 60 分鐘之卵巢之各級腔狀濾泡之
卵母細胞,再置於無(-)或含(+)1.5 mg/ml膠原蛋白脢溶液中,經歷體
溫(39℃)或低溫(5∼10℃)水浴培養60分鐘處理時,就於水浴之溫度而言
,於低溫水浴組(5∼10℃)之三類腔狀濾泡之卵母細胞存活率均顯著較之
置於體溫(39℃)水浴者為高(P<0.05);惟以培養液中添加之膠原蛋白脢,
並不對水浴低溫組或體溫組之卵母細胞之存活率有顯著之影響(P>0.05)。
繼之,為能簡化維持腔前濾泡在培養過程之三度立體結構之包埋處理,乃
比較腔前濾泡培養於已裝填生物薄膜上或經海藻酸鈣膠層包埋或未予包
埋(對照組)等處理之卵母細胞存活率,結果顯示,迄離體培養4天後,應
用海藻酸鈣膠層包埋和直接培養於已裝填生物薄膜等二組之卵母細胞存活
率,均顯著高於未處理之對照組(74.5% 和69.5% vs. 20.5%, P<0.05)。
而經長時程(12天)培養後,海藻酸鈣膠層所包埋之濾泡和卵母細胞之直徑
增加值,雖顯著低於生物薄膜所培養者(P<0.05),然卵母細胞之存活率則
否(47.8% vs. 44.7%, P>0.05),而卵母細胞外觀形態正常率,則反
之(59.1% vs. 29.4%, P<0.05)。在評估培養液中之FSH含量[ 0、1、2和4
mg/ml ]對不同生長期別之腔前濾泡[直徑為200∼<300(小型)、300
∼<400(中型)和400∼<500(大型)mm],於離體發育之影響之試驗結果顯示
,在不含FSH (0 mg/ml)之培養液中,三類不同生長期別之腔前濾泡之濾
泡直徑經培養4天後,均呈負成長,分別較之置於液中含FSH之各組者為
低(P<0.05)。迄培養結束(12天),小型濾泡組之腔前濾泡之直徑和成長之
幅度,培養於含1 mg/ml FSH組者較之培養於含4 mg/ml者為高(P<0.05);
中型濾泡者,則在三種FSH含量組間之差異均不顯著(P>0.05);惟大型濾
泡者,於液中含4 mg/ml FSH組者較之含1 mg/ml組者顯著增加(P<0.05)。
就腔前濾泡之生長期別而言,置於培養液中含1或2 mg/ml FSH時,小型腔
前濾泡之直徑成長幅度均顯著大於大型腔前濾泡者(P<0.05);惟當置於液
中含4 mg/ml FSH時,濾泡成長之幅度在不同生長期別間之差異不顯著(
P>0.05)。資訊也顯示,腔前濾泡之囊腔形成率,隨培養前腔前濾泡直徑
值之增加而增加(P<0.05),也隨液中所含FSH濃度之增加而略提升。惟培
養後之卵母細胞之直徑值或成長幅度並不因腔前濾泡之生長期別,或培養
液中含不同濃度之FSH,而有顯著之差異(P>0.05)。此外,腔前濾泡經離
體培養4天後之卵母細胞之存活率,在液中不含FSH (0 mg/ml)組者,均顯
著低於液中含FSH之各組者(P<0.05)。雖然,於培養過程或迄培養結束(12
天),卵母細胞之存活率在液中含FSH之各組間之差異均不顯著(P>0.05)。
然而,腔前濾泡經離體成長培養後,再將存活之卵母細胞(卵丘-卵母細胞
複合體)供後續成熟培養時,僅源自大型腔前濾泡組並被置於液中含4 mg/
ml FSH培養者,始獲有成熟之卵母細胞(成熟率為5%);同時卵母細胞恢復
減數分裂之比例,隨培養前腔前濾泡之直徑值之增加而增加(P<0.05)。但
並不因液中含有FSH之濃度不同,而有顯著差異(P>0.05)。綜合上述資料
提示,於低溫(5∼10℃)保存時,雖未對卵巢各級濾泡之卵母細胞之存活
率造成立即性之傷害,惟當卵巢組織再經歷高溫(39℃)環境之膠原蛋白
溶液軟化處理60分鐘後,濾泡內卵母細胞之存活率即告下降。而置於室
溫(20∼25℃)保存長達60分鐘之處理,亦不利於腔前濾泡內卵母細胞之後
續存活率。因此,維持在室溫30分鐘或高溫60分鐘之處理,可提供為運送
豬隻之卵巢組織時,設綜合上述資料提示,於低溫(5∼10℃)保存時,雖
未對卵巢各級濾泡之卵母細胞之存活率造成立即性之傷害,惟當卵巢組織
再經歷高溫(39℃)環境之膠原蛋白溶液軟化處理60分鐘後,濾泡內卵母
細胞之存活率即告下降。而置於室溫(20∼25℃)保存長達60分鐘之處理,
亦不利於腔前濾泡內卵母細胞之後續存活率。因此,維持在室溫30分鐘或
高溫60分鐘之處理,可提供為運送豬隻之卵巢組織時,設定環境條件之參
考。若考慮及提高培養後卵母細胞之存活率及增加細胞形態之正常率時,
以海藻酸鈣膠層包埋豬腔前濾泡之方式,不失為目前較理想之選擇。而濾
泡和卵母細胞之生長幅度,確能受不同生長期別(由濾泡直徑所區分)之腔
前濾泡所影響。於離體環境,腔前濾泡和卵母細胞之發育以及卵母細胞之
存活率和後續之成熟率,均與培養前該腔前濾泡之直徑大小或培養液之組
成(如FSH濃度)之關係密切。

The purpose of porcine preantral follicle culture is on
onehand to under-stand the development of ovarian follicles and
toobtain large numbers of oocytes grown and matured in vitro
formanipulatory facilitation of reproductive tech-nology, on the
other hand. In this study, a series of trials were undertaken to
characterize the best conditions for porcine ovary preservation
during trans-portation, and to evaluate the effect of culture
systems and FSH treatments onoocyte growth and survival of
developing preantral follicles in different stages. Thermal
treatments [ low temperature (5-10℃)、room temperature(20-25℃)
andhigh temperature (32-37℃)] with different durationtime
(10、30 or 60 min) wereconducted on porcine ovaries to
evaluatethe effect of preservation temperatureson oocyte
survival rate. The results demonstrated that when preserved for
10min, the survival rates of oocytes collected from preantral
follicles、antralfollicles、cumulus-oocyte-complex and nude
oocytes were not significant differ-ent among all temperature
groups (P>0.05) after the incubation of minced ovarieswith 1.5
mg/ml collagenase at 39℃ for 60 min . When preservation time
was pro-longed to 30 or 60 min, the oocyte survival rates of low
temperature group weresignificantly lower than those of the room
or high temperature group (P<0.05,respectively). After
encapsulated in Ca-alginate gels and cultured in mediumfor 4
days thereafter, the survival rate of oocytes collected from
preantralfollicles of 60 min preservation at room temperature
treatment was significantlylower than that at high temperature
treatment (53.5%vs. 76.5%, P<0.05). However,the difference of
oocyte survival rate was not significant between room and
hightemperature treatments(77.9% vs. 79.7%, P>0.05) when ovaries
were preserved for30 min.After 12-day culture in the embedded
Ca-alginate gels, the diameeters ofpreantral follicles in
different developing stages[ 200-<300(small)、300-<400 (middle)
and 400-<500 (large) mm ] and their oocytes, and the oocyte
survivalrate were also not significant between room and high
temperature groups(P>0.05). To elucidate the low survival
rate of oocytes in developing follicles of porcine ovary
preserved in low temperature for 60 min, ovaries were preserved
at 5-10℃ for 10、30 or 60 min without the softening treatment
by collagenase solution at 39℃, and then the survival rate of
oocytes collected from the superficial antral follicles was
investigated. The results revealed that the oocyte survival
rates were not different among the groups of preservation
timeand among the stages of antral follicles [<3 (small)、3-5
(middle)and 5-<7 (large) mm](P>0.05, respectively). The oocytes
of developingantral follicles preserved at low temperature for
60 min were then cultured for 60 min in the absence (-) or
presence (+) of 1.5 mg/mlcollagenase at 39℃ (body temperature
water bath, BP) or 5-10℃ (lowtemperature water bath, LP),
suggesting that the survival rates of oocytes in different
antral follicles were significantly higher in LP than those in
WP, but the oocyte survival rates either in 39℃ or in 5-10℃
groups was not affected by the presence of collagenase(P>0.05).
Furthermore, to simplify the maintenance of dimensional
integrity of preantral follicles in encapsulation step during
culture, we evaluate the survival rates of oocytes from
preantral follicles cultured inimpregnate membrane or embedded
in Ca-aglinate gels in comparison to control. The results
suggested that the oocyte survival rates of Ca-alginate gels
encapsulation andimpregnate membrane treatments cultured for 4
days were significantly higher than those of control(74.5% and
69.5% vs. 20.5%, P<0.05).The increased diameters of preantral
follicles and oocytes in Ca-alginate gels encapsulation for
long-time culture (12 days) in vitro were significantly lower
than those in impregnate membrane treatment (P<0.05). Although
the oocyte survival rates werenot different between these two
treatments(47.8% vs. 44.7%,P>0.05),surprisingly,the rate of
morphological normality for oocyte further surviving potential
was higher in Ca-alginate gels encapsulated oocytes treatment
than that in impregnate membrane treated oocytes (59.1% vs.
29.4%, P<0.05). Different FSH contents in the media [0、1、2
and 4 mg/ml] were subjected to evaluate FSH effect on growth and
survival of oocytes collected from different stages of preantral
follicles [ 200-<300 (small)、300-<400 (middle) and
400-<500(large) mm]. The results revealed the significantly
decreased diameters of preantral follicles in the three
developing stages under the absence of FSH in comparison to the
group in the presence of FSH after 4-day culture (P<0.05),
suggesting a negative growth without FSH stimuation. After
12-day culture in vitro, the diameters of follicles and
increased diameter percentages in small preantral follicles were
significantly higher in 1 mg/ml FSH treatment than those
cultured in 4 mg/ml FSH (P<0.05). In the middle preantral
follicles, no significant difference was found in the follicle
diameters among groups of different FSH contents (P>0.05), but 4
mg/ml FSH treatment significantly elevated follicle diameters
and increased diameter percentages than 1 mg/ml FSH in the large
preantral follicles (P<0.05). In the culture either with 1 mg/ml
or with 2 mg/ml FSH, the increased diameter percentages of
small preantral follicles were significant higher than those of
large follicles (P<0.05), but there was no significant
difference among developing stages of preantral follicles under
4 mg/ml FSH stimulation (P>0.05). At the end of culture, the
rates of antrum cavity formation in preantral follicles were
increased progressively with preantral follicle diameters and
FSH contents in the media beforeculture (P<0.05). The
diameter and increased diameter percentage of oocyte collected
from different stages of preantral follicles were not affected
by different FSH contents (P>0.05). However, the survival rates
of oocyte collected from preantral follicles of different stages
were lower in the absence of FSH than those in the present FSH
(P<0.05). Although there were no differences of oocyte survival
rates among groups of different FSH contents [1、2 or 4 mg/ml
FSH], the further oocyte maturation (progressing to metaphaseⅡ)
and!oocyte survival after 12-day culture (collected from
cumulus-oocyte-complex) only appeared in large preantral
follicles under the stimulation of 4 mg/ml FSH (maturation
rate5%). The rates of oocytes resuming meiosis increased
progressively with pre-antral follicles diameters (P<0.05). but
were not affected by FSH contents (P>0.05). Taken together,
results of the experiments suggested that low temperature
(5-10℃) of preservation did not cause immediate damage of
porcine oocyte survival rates in different follicles, but when
incubated at 39℃ with collagenase solution for 60 min, the
survival rate of oocyte decreased. Ovaries preserved at room
temperature (20-25℃) for 60 min, also did not favor the oocyte
survival. Consequently, the best preservation conditions of
porcine ovaries during transportation were concluded under
temperatur at 20-25℃and duration time for 30 min or at high
temperature (32-37℃)for 60 min. Treatment of preantral
follicles with encapsulation in Ca-alginate gels provided a good
modle to increase the survival and morphological normality
ratesof oocytes. The growth rates (increased diameter ratio) of
preantral follicles and oocytes cultured in vitro were affected
by different developing stages of follicles under the presence
of FSH suggesting that the survival rate and the meiotic
competence of oocytes maturation grown in vitro were increasing
progressively with preantral follicles diameters and medium
contents (eg: the concentration of FSH).

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