A total of 210 pregnant mice was used in this experiment. The mice were mated and judged for age of pregnancy as described in Chapter 2. The growth of the uterus and its gravid components during gestation period were examined, starting just one day after surgery for each group. Examination was performed on five different days of
.. , i
O1cpter3 Effects on length of Gestation and Prenatal Growth 37
pregnancy, ie. day 3, day 7, day 1 1 , day 15, and day 18. Thus, the groups of pregnant
mice operated on, on day 2 of pregnancy were examined five times. the first
examination being day 3 of pregnancy. one day after operation. The other examinations were on day 7, day 1 1 , day 15, and day 18 of pregnancy for the second, the third, the
fourth. and the fifth examination respectively. For the group of pregnant mice operated on, on day 6 of pregnancy, uterine and ovarian examination were performed four times. i.e. on day 7, day 1 1, day 15 and day 18 of pregnancy. In the remaining groups,
pregnant animals operated on, on day 10 and day 14 of pregnancy were examined three
and two times, starting at day 1 1 and day 15 of pregnancy respectively as described in section 2.4 (Chapter 2). Five animals per group were examined (Table 3.2).
Table 3.2 Number of animals autopsied for each designated day of pregnancy per group. Prefix number for each group denotes the day of pregnancy when the operation was performed.
Number of anima1s autopsied on each designated days
of I I 15 2UX 5 5 5 5 5 2UOX 5 5 5 5 5 2S0P 5 5 5 5 5 6UX 5 5 5 5 6UOX 5 5 5 5 6S0P 5 5 5 5 10UX 5 5 5 IOUOX 5 5 5 IOS0P 5 5 5 14UX 5 5 14UOX 5 5 14S0P 5 5 Total 1 5 30 45 60 60
The diameter of the rostral end and middle portion of the uterine loop artery (henceforth these arteries abbreviated as Uo and Ut for the rostral end and middle portion of uterine loop artery respectively) and the diameter of uterine segmental arteries (in this study
these arteries were referred to as placental arteries, abbreviated as PI) at three different
locations (ovarian end, middle point, and vaginal end) were measured using a metal
calliper (see Figure 3.1). Measurement was perfonned at two different times under light
anaesthesia. The first measurement was earned out at surgery and the second one at
autopsy on day 1 8 of pregnancy. At surgery, after the ventral body wall was opened
Olcpter3 Effects on Length ot Gestation and Prenatal Growth "-�:�'�-':'::':::/' . � �
!:
,-,,�-.-,: ,<:.-Kidney
I ' j I : ': " �,,." -""�<,_
..Ovary
Uterine
.;Amniotic
sac
Figure 3,1 Diagram of the uterine vasculature of the mouse near lenn, showing the positions at which diameter of the arteries was measured (arrow heads). Measurements were made on the utero-ovarian artery (Uo) (open arrow, upper left), uterine artery loop (Ut) (open arrow, lower [eft), and on the uterine segmental or placental arteries (PI) at three different locations (ovarian end, middle portion, and vaginal cnd) (solid arrow), For each group, artery diameter was measured two times, at the day of
operation (initial dlam eater) and at the day of autopsy, Le. Day 18 of pregnancy (final
diameter). (Adopted from vom Saa] and Dhar, 1992).
38
-
O1cpter3 Effects on Length of Gestation and Prenatal Growth 39
arteries was determined easily to the nearest 0.02 mm by usmg a metal calliper. A similar procedure was applied when measuring the artery diameter at autopsy on day 1 8 of pregnancy. Measurement on day 1 8 of pregnancy was performed before the blood sample was collected.
After bleeding, mice were killed for the uterine examination by cervical dislocation. The
remaining uterine hom (for both UX and UOX groups) or intact uterus (for the SOP
group) and its ovary was removed, weighed and then placed in a petri dish containing
0.9% (w/v) NaCI solution until examined for gravid components (for horns obtained
from day 18 of pregnancy) or until fixing in Bouin's solution (for horns obtained from
days 3, 7. 1 1 , and I S of pregnancy).
All uterine horns and ovaries collected from the groups of animals at day 18 of pregnancy were examined on collection. After opening the uterus, conceptuses (fetuses, fetal membranes. and placentae) were removed in order from the ovarian end to the vaginal end of the uterine horn. For the SOP group, the left uterine hom was examined first before the right one. Each conceptus was opened and exarnlned before the next conceptus was removed following Norman and Bruce ( l979b). The number of live fetuses was recorded, dissected free from their membranes, separated from their umbilical cords, blotted lightly by transferring them four times on dry petri dish surfaces. sexed visually, and immediately weighed individually to the nearest 0.0001 gram on a Mettler direct reading balance. Visual determination of fetal sex was made by observing the ano-genital papilla distance, where the distance is larger in males than in females (Gruneberg. 1952; Rugh. 1967). Fetal C-R length was determined to the nearest 0.2 rnrn using a metal calliper. Each placenta was carefully separated from its umbilical cord and from feral membranes, blotted lightly as for feruses, and weighed. Care was taken to remove the anached fetal membranes from both the fetus and the placenta. The remaining uterine rissue plus embryonic membranes and umbilical cord were carefully bloued lightly by transferring them onto dry petri dish surfaces four times and weighed totally. The total amount of fetal fluid per horn was obtained by subtraction of total feral weight, total placental weight, and total empty uterine tissue, embryonic membranes and umbilical cord from the total weight of uterine horn (McLaren et ai., 1976). This entire procedure, i.e. preparation. separation of uterine tissue, fetus, and placenta from the embryonic membranes, was conducted on a filter paper moistened with saline solution. This procedure was performed on the live conceptuses only.
Ovaries were examined for weight and number of corpora lutea after all the excessive fat covering the ovary was removed. The number of corpora IUlea was counted visually
Olapter3 Effects on Length of Gestation and Prenatal Growth 40
under dissecting microscope. Corpora lutea were counted in a petri dish three before recording as a unit of data .
Because the uterine horn and its gravid components were more difficult to distinguish and separate by morphology before day 1 8 of pregnancy, they were not dissected for fresh weight measurements. The uterine horns from day 3, day 7, day I I, and day 1 5
of pregnancy, therefore, were fixed i n Bouin's solution until examined. These uterine horns and their gravid components were examined under a dissecting microscope at low magnification.
When examining the fixed uterine horns, the strong smell of Bouin's solution was first removed by washing them in slow running tap water for about 2 minutes. After opening the merine hom, conceptuses were removed in order. Fetuses and placentae (from I I
and 15 days old) were separated from their membranes and/or its umbilical cord, blotted slightly by transferring them to filter paper as many as four times, sexed visually, weighed individually and detennined for C-R length of fetus as described for fresh examination. The remaining uterine tissue plus fetal membranes and umbilical cord were weighed totally as empty uterus. The amount of fetal fluid per horn was calculated as for fresh examination of uterine horn from day 18 of pregnancy.
The uterus obtained from day 3 of pregnancy was examined for uterine weight only when day 7 uterine horn was examined for boch uterine weight and conceptus weight. Fetal weight, placental weight and fetal C-R length were averaged per hom before being used for statistical analysis. Therefore, for the SOP group, which had two uterine horns, the mean values of the right and the left horn for each parameter were averaged.
3.3 Results