| Summary: | 碩士 === 國立中興大學 === 畜牧學系 === 84 === To establish the technology of isolation and culture
of preantral follicles from porcine ovaries, it would have
two purposes, one is to understand the development of ovarian
follicle and the other is to obtain large numbers of oocytes
grown and matured in vitro for manipulation in study of
reproductive technology. In this study, there was a series
of trials to understand the effect of the concentrations
of collagenase in media and incubation time on the isolation
of preantral follicle which has normal morphology and function,
then to test the effect of the coating matrix and porcine
follicle at various stages of development on the growth and
sequence maturation of porcine oocytes in vitro.
When minced ovaries were incubated in media contained various
collagenase concentrations (0, 0.75, 1.5, and 3 mg/
ml, respectively) for 60 min, the results revealed that the
media contained 1.5 and 3 mg/ml collagenas, which
were both significantly more than that in the control (
728, 683, and 332 for 1.5, 3 mg/ml and control groups
respectively, p<0.05). The total number of the preantral
follicles and the number of the smaller preantral follicle
(diameter <300 mm) recovered in the enzymatic treatments
were significantly more than that in the control (p<0.05).
When minced ovaries were incubated in the media contained 1.5
mg/ml collagenase for various periods (30, 45, 60 and 75 min,
respectively), the number of the recoverable preantral follicles
significantly increased (p<0.05) progressively with the
incubation time. The recoverable number in the 75 min-
incubated treatment (913) was significantly more than
in the 45 min-incubated treatment (497) (p<0.05) and was
also more, though not significantly, than the other two
treatments (700 and 778 for 30 and 60 min- incubated groups
respectively). The survival rate [assayed by FDA (3', 6'-
fluorescein diacetyl)] of all kinds of porcine oocytes would
not be different when incubation time progressed from 30
min to 75 min (p>0.05). If the isolated follicles from
different incubation time (60 or 75 min) were embedded in
sodium alginate gel and then were cultured in growth media [MB
752/1 (95 ％ ) + FCS (5 ％ ) + FSH (2 mg/ml) + E2 (1 mg/ml)]
for 4 days. The survival rate of oocytes in preantral
follicles in the 75 min-incubated treatment was significantly
low than that in 60 min-incubated treatment (30 ％ vs. 60 ％,
p<0.05). It's important that the coating matrix will
allow the three dimensional integrity of the follicle unit to
remain intact and maintain oocyte-granulosa connections.
Preantral follicles were grown within a three dimensional
matrix of oocytes in sodium alginate gel was significant
high than in collagen gel or in control of uncoating (52
％ vs. 30 ％ or 18 ％, P<0.05). Furthermore, we
investigated the effect of the growth stages of preantral
follicle before cultured on the development of follicle (the
diameter of follicles and oocytes) and the survival rate of
oocytes after long terms cultured in vitro. Freshly
isolated preantral follicles were separated into three classes
according to oocyte diameter, <70 mm (small)、 70~80 mm
(middle) and 80~90 mm (large), and were embedded in sodium
alginate gel and then were cultured for 16 days. At the end
of culture, the diameter of the follicles increased from 260
mm, 320 and 390 mm to 520 mm, 600 mm and 610 mm, the diameter
of the oocytes increased from 58 mm, 71 mm and 84 mm to 86 mm,
106 mm and 114 mm, forming rate of antrum cavity was 0 ％, 4
％ and 14 ％, and survival rate of oocyte was 10 ％, 28 ％ and
25 ％, respectively for the small, middle and large groups.
The results revealed that there was a close relationship
among the development of preantral follicles, survival
rate of the oocytes after cultured and the diameter of the
oocytes in preantral follicles before cultured. The
development of the follicles and the oocytes,and the survival
rate of the oocytes in large group was better than that
in small groups. The mean diameter of the oocytes in large
group had the largest size (115 mm) when were cultured for 12
days. The meiotic competence of oocytes grown in vitro
(from large group of preantral follicles were cultured for
12 days) was assessed and compared with that of oocytes
grown in vivo (from ovarian follicles with diameters less
than 1 mm). After maturation culture, all of the oocytes
with diameters of >90mm grown in vivo, progressed to metaphase
II, were significant high than that oocytes grown in vitro
[61 ％ (169/274) vs. 2 ％ (2/103), p<0.05]. Progression to
metaphase II was observed in 16.7 ％ of oocytes with
diameters greater than 110mm. All of the oocytes grown in
vitro and in vivo, with diameters less than 90mm, remained
at germinal vesicle stage.
Results of the experiments revealed that the isolation of
preantral follicles with biological activity was promoted by
the incubation of the minced porcine ovarian tissue in the
medium containing 1.5 mg/ml collagenase for 60 min. When the
preantral follicle embedded in the sodium alginate gel, it's
advantage for the survival rate of oocytes after
cultured. The preantral follicles at various stages of
development embedded in the sodium alginate after cultured for
16 days, the diameter of follicles and oocytes in all groups
increased progressively and were reached the maximum at a 12
days' culture. The meiotic competence of the oocytes
grown both in vitro and in vivo were increasing
progressively with oocytes diameter. Only a few of oocytes
with diameters greater than 110mm progressed to metaphase
II after maturation culture.
|
|---|
