الفهرس 
Materials and mithodsOnly 14 pages are availabe for public view
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Abstract
SUMMARY
This work was carried out in IVF laboratory (International Livestock Management
and Training Center, ILMTC) Sakha, Kafr El-Sheikh Governorate, belonging to Animal
Production Research Institute, Agricultural Research Center, Ministry of Agriculture.
This study aimed to investigate the maturation, fertilization and development of
rabbit oocytes in vitro as affected by season of the year, type of culture medium, source of
oocytes and level of osmolarity. The present work comparised two main experiments. Thefirst
one dealt with in vitro maturation (IVM) with checking fetal calf serum (FCS) and
bovine serum albumin (BSA) supplements, in vitro fertilization (IVF) and early embryonic
development in vitro as affected by above mentioned factors. While the second one was
designed to study IVF and early embryonic development for superovulated does slaughtered
at two different periods.
Experiment I:
L A. Some morphological measurements on the ovary:
In this study 14 virgin female New Zealand white rabbits aged 6-7 months old were
used without any treatment (untreated does). Another 14 females were injected with
Pregnant Mare Serum Gonadotrophin (PMSG) hormone (75 IU) and slaughtered after 60
hrs of treatment (PMSG-treated does). Ovaries were excised from both groups to determine
the effect of season of the year on left and right ovaries weights, number of corpora lutea
(CL), number of follicles and number of oocytes obtained/doe. Results obtained for this
experiment could be summarized as follows:
L Season of the year had a significant effect (P<O.OOI) on the left and right ovaries weights
for the untreated group. But this effect was not significant for the PMSG-treated group.
2. Season of the year significantly (P<O.05) affected the number ofCL on the left ovary,
while this effect was not significant for CL presented on the right ovary in the untreated
group. In PMSG-treated group, season had 110 significant effect on the number of Cl,
presented on the both ovaries.
3. Season of the year had a significant effect (P<O.OI) on the number of visible follicles
presented on the left ovary of the untreated group, while this effect was not significant
on the number of the follicles presented on the right ovaries in both groups and those
presented on the left ovary of the PMSG-treated group.
4. Season of the year did not significantly affected the number of oocytes obtained from the
ovaries of the untreated group, while this effect was significant (P<O.05) for the PMSGtreated
group.
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LB. Oocytes in vitro maturation:
Oocytes recovered from untreated and PMSG-treated does at different seasons were
selected, washed and cultured in tissue culture medium (TCM-199) supplemented with FCS
or BSA, then incubated for 20 hrs in 5% C02, 95% air. After 20 hrs of incubation all
oocytes were examined for evidences of maturation. Results obtained could be summarized
as follows:
1. No significant differences among seasons for the maturation rate were obtained.
2. Addition of FCS to TCM-199 medium elevated the percentage of oocytes matured in
vitro than those of BSA (73.1% vs, 54.4%) for untreated does and (81.3 vs. 65.2%) for
PMSG-treated does.
3. Irrespect to the effect of season and protein supplement, PMSG treatment increased
significantly (P<0.05) the percentage of oocytes maturation (73.2%) compared to
(63.7%) for untreated does.
I. C. In vitro fertilization of rabbit oocytes:
The oocytes matured ill vitro pooled and incubated with capacitated spermatozoa ill
vitro for 24 hrs in two media (Brackett’s or TCM-199) with varied osmolality (285, 305
and 325 mOsm/kg) at different seasons. Results obtained could be summarized as follows:
1. No significant differences among seasons were obtained for the fertilization rate.
2. Brackett’s medium elevated the percentage offertilized oocytes in vitro compared with
TCM-199 (31.0 vs. 20.7% for oocytes collected from untreated does and 32.2 vs. 24.3%
for PMSG-treated does).
3. The fertilization rate was significantly (P<0.05) higher (41.3%) for the oocytes cultured
in media of305 mOsm/kg than those cultured in media of285 or 325 mOsm/kg (24.7%
and 10.7%, respectively) for untreated does, while these percentages were 46.8,26.1 and
11.0%, in the respective order for PMSG-treated does.
4. Irrespeet of the season effect, media and osmolarity levels, the fertilization percentage
was non significantly higher for the ooytes collected from PMSG~treated does than those
collected from untreated does (28.3 vs. 25.9%).
I. D. In vitro development of rabbit embryos:
The viability of embryos cultured in the two media and assigned the three levels of
osmolarity were observed carefully at 24 hrs intervals for progress of development to
morula, early blastocyst and blastocyst stages up to six days and the medium was replaced
daily with fresh one. Results obtained could be summarized as follows:
1. In TCM-199 culture medium, 20.8% and 28.9% offertilized oocytes were developed to
morula stage after 72-96 hrs of insemination for untreated and PMSG-treated groups,
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respectively, while in Brackett’s medium 13.9% and 23.1% offertilized oocytes were
developed to the same stage for the respective groups.
2. For untreated and PMSG-treated groups, 16.7% and 18.4%. respectively, offertilized
oocytes cultured in TCM-199 medium were developed to early blastocyst stage after 96-
120 hrs of insemination, while these percentages were 5.6% and 13.5%, respectively, in
Brackett’s medium.
3. Medium of 305 mOsm/kg enhanced the percentage of fertilized oocytes to develop to
morula and blastocyst stages, while fertilized oocytes cultured in media of 285 or 325
mOsm/kg failed to reach these stages of development.
4. Irrespect of the effect of media and osmolarity levels, the percentage offertilized oocytes
reached morula and blastocyst stages were 16.7% and 3.3% for those of untreated
group, while these percentages were 25.8% and 8.9% for those ofPMSG-treated group.
Experiment II:
II. A. Some morphological measurements on the ovary:
In this study, the female rabbits were injected with PMSG hormone (150 IV), 83 hrs
later they were injected with HCG hormone (75 IU) to induce superovulation and these
females were divided into two groups. The female rabbits in the first group were
slaughtered after 10 hrs of HCG injection, while the female rabbits in the second group
were slaughtered after 12 hrs ofHCG injection, at different seasons. Results obtained could
be summarized as follows:
I. Season of the year had a significant (P<O.OOI) effect on the left and right ovaries weights
in the females of group 2, while this effect was not significant on the weight of left and
right ovaries of the group 1.
2. Season of the year did not affect the number ofCL presented on both ovaries in the two
groups.
3. Season of the year significantly (P<0.05) affected the number of follicles presented on left
and right ovaries, also, the total number offollicles on both ovaries ofthe group 1. while
this effect was not significant for group 2.
4. Season of the year did not affected the number of oocytes obtained from the ovaries in
the two groups.
II. B. In vitro fertilization of rabbit oocytes:
Rabbit ovarian follicular oocytes were collected from superovulated does that
slaughtered after 10 hrs ofHCG injection (10 hrs group). Also, rabbit oviductal ova and
follicular oocytes were collected from superovulated does that slaughtered after 12 hrs of
HCG injection (12 hrs group ). These 0\ iductal ova and follicular oocytes were
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inseminated in vitro in a chemically defined media (Brackett’s or TCM -199) with
capacitated sperm in vitroand incubated together for 24 hrs. Results obtained could be
summarized as follows:
1. In 10 hrs group, there were no significant differences in fertilization rate between
Brackett’s and TCM-199 media (49.4 vs. 42.7%).
2. In 12 hrs group, Brackett’s medium improved significantly (P<0.05) fertilization rate than
TCM-199 medium (56 vs. 46.6%).
3. Also, in 12 hrs group, the percentage offertilization of oviductal ova was significantly
(P<O.05) higher than those of ovarian follicular oocytes (61.6 vs, 47%).
IT. C. In vitro development of rabbit embryos:
The fertilized ovarian follicular oocytes that collected from 10 hrs group, also the
fertilized oviductal ova and ovarian follicular oocytes that collected from 12 hrs group were
cultured in Brackett’s and TCM-199 media up to six days after insemination and the
medium was replaced daily with fresh medium. Results obtained could be summarized as
follows:
1. In 10 hrs group, TCM-199 medium enhanced embryo development to morula, blastocyst
and expanding blastocyst stages (44.7, 13.2 and 2.6%, respectively), while these
percentages were 31.8,4.5 and 2.3%, respectively, in Brackett’s medium. However, the
percentage of the embryos cultured in TCM-199 medium and developed to early
blastocyst was significantly (P<0.05) higher than those cultured in Brackett’s medium.
2. In 12 hrs group, the percentage of embryos developed to morula stage in Bracken’ s
medium was slightly higher than those ofTCM-199 medium (45.3 vs. 40.8%), while the
percentages of embryos that developed to early blastocyst, blastocyst and expanding
blastocyst in TCM-199 medium were slightly higher (25.4, ] 1.3 and 5.6%, respectively)
than those in Brackett’s medium (22.1, 9.3 and 3.5%, respectively).
3. Also, in 12 hrs group, the percentages of embryos from oviductal ova fertilized in vitro
that developed to morula. early blastocyst and blastocyst stages were significantly
(P<0.05) higher (61.4, 33.3 and] 7.5%, respectively) than those from fertilized ovarian
follicular oocytes (33, 18 and 6%, respectively). However, the difference between
percentages of embryos that developed to ex-panding blastocyst were not significant (7%
vs.3%).