Effects of Preservative Conditions of Porcine Ovaries, and the Cultural Terms on the Development of Porcine Preantral follicles and Oocytes In Vitro

• Main Authors: Hsu, Hsi-Fuh, 許勝富
• Other Authors: San-Pao Cheng
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/29597795802199709783

碩士 === 國立中興大學 === 畜牧學系 === 86 === 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).