Spacing of fetuses and local competition in strains of mice with large, medium and small litters N. W. Bruce and J. R. Wellstead Centre for Human Biology, Department of Anatomy and Human Biology, The Western Australia, Nedlands, Western Australia, 6009 Australia
University of
Summary. The tendency for fetuses to be evenly spaced along the uterine horn and the relationship of this to local competition between fetuses was examined in three strains of mice which characteristically produce large, medium and small litters. Local competition was assessed by correlating, within each uterine horn, the weight of each fetus or placenta with the mean distance to its immediate neighbours. Weights and distances were measured on day 19 of gestation, on the day before expected parturition and distances only on day 7. Average litter sizes (live fetuses) were 16\m=.\3\m=+-\0\m=.\9, 11\m=.\7\m=+-\0\m=.\6and 7\m=.\2\m=+-\0\m=.\5 (mean \m=+-\sem) in the large, medium and small litter strains, respectively (n 7, 7 and 10, respectively). On day 19, the mean distance between fetuses was significantly less (P < 0\m=.\05)in the large strain (10\m=.\1\m=+-\1\m=.\0mm) than in the medium (14\m=.\0\m=+-\1\m=.\2 mm)or small (13\m=.\5\m=+-\1\m=.\0mm) strains. Evenness of spacing, expressed as the standard deviation of distances between fetuses divided by the mean distance, improved from day 7 to day 19 of gestation in all three strains and effectively prevented local competition between fetuses in the medium litter (r 0\m=.\04)and small litter strains (r 0\m=.\17),but not in the large litter strain (r 0\m=.\45,P < 0\m=.\01).Thus, local crowding does not seem to be detrimental to fetal growth in mice, except in strains specifically bred for large litters. =
=
=
=
Keywords: mouse; fetus; local competition; spacing
Introduction In polytocous species it is generally accepted that growth of a particular fetus is affected by compe¬ tition from its littermates. Such competition can be a 'systemic' effect, due to the total number of fetuses in a litter, a 'regional' effect, due to the number of fetuses in a particular uterine horn, or a 'local' effect due to the proximity of immediate neighbours. There is good quantitative evidence supporting the operation of systemic and regional effects in a number of species including rats (Barr et al, 1970; Norman & Bruce, 1979), guinea-pigs (Eckstein & McKeown, 1955) and mice (McLaren, 1965), but the strength of local effects has received less attention. This may be because fetuses tend to be spaced evenly along the uterine horn, thus minimizing crowding or competition at the local level. It is important to note that where evenness of spacing has been properly quantified, as in rabbits, it falls considerably short of the ideal, that is equal spacing between conceptuses (Boving, 1971). To date, however, the situation for mice has not been determined and the effects of local competition have not been fully quantified. In this study, three strains of mice were examined, which characteristically carry small, medium and large litters. It was postulated that local effects would be more prominent in strains carrying large litters if the length of the uterine horn did not increase proportionally. Placental weights were recorded to determine whether any effect on the fetus could be related to changes in placental growth.
Materials and Methods Animals and treatments
Quackenbush, ARC Swiss and CBA/CaH were selected as the large, medium and small litter strains of mice, respectively. All mice were nulliparous, aged 2-4 months and purchased from the Animal Resources Centre, Murdoch, Western Australia. They were kept in an environmentally controlled room (temperature, 17-23°C; relative humidity, 50-70%; lights on from 07:00 to 21:00h). Food and water were freely available. Females were mated during darkness and the next day was day 1 of gestation. Evidence of mating was assessed by the presence of a vaginal plug (day 7 and 19 groups) or by rapid increase in weight following a single exposure to males (day 19 group only); mice in all three strains normally give birth on the morning of day 20. Mice were killed at 10:00 h on the morning of day 7 or day 19 of gestation with an overdose of sodium pentobarbitone. The entire uterus of each mouse, together with oviducts, ovaries, cervix and vagina, was removed and trimmed of fat. Each uterine horn was dissected free from its mesometrium and from its attachment to the vaginal wall and contralateral horn. The number and position of each implantation site was recorded, and for mice at day 19 of gestation, the uterine horn was cut along the antimesometrial border and the conceptuses (fetuses, fetal membranes and placentas) were removed and weighed to the nearest milligram (see Bruce & Norman, 1975; Rahima & Bruce, 1986, for details). The uterine horn was transferred to a water bath, its ovarian end was pinned to a cork board and the cervical end was attached to a thread that ran freely over a pulley to a 2 g weight. This allowed the uterine horn to stretch slightly but evenly since the water bath elevated the horn above the cork board so preventing frictional resistance. After one minute to allow the uterus to respond to the stretch, the cervical end was also pinned to the cork board. The uterine horn was examined through a dissection microscope and pins were carefully positioned in the centre of each implantation (day 7, intact uterine horn) or implantation site (day 19, opened horn) and at the extremities of each horn. The distances between the pins were then measured with dial callipers to the nearest 0-1 mm. Although the technique has been validated for rats (Rahima & Bruce, 1986) it was revalidated for mice since the measurements had to be carried out with greater accuracy. Distances between implantations were therefore measured on six uterine horns at day 19 of gestation with two animals from each strain, as described above. The horns were then
released from the tensioning weight, restretched by an independent observer, pinned and measured again. There were no significant differences between the two sets of measurements; the correlation coefficients between the two sets as carried out on each of the six uterine horns were r 0-938, 0-981, 0-996, 0-922, 0-999 and 0-999. =
Analysis of results.
Evenness of spacing
was
determined
ceptuses and between the uterine extremities and the
the distances between
by measuring
nearest
neighbouring con¬
conceptus for each uterine horn. The coefficient of
variation (standard deviation/mean) of these distances (CVd%) was then used as the estimate of evenness, a low value implying even spacing and a high value, uneven spacing (Rahima & Bruce, 1986). The average of the distances for any one uterine horn (average distance) was used to compare the extent of spacing between strains or between the two stages of gestation examined.
Calculation of the effects of competition between littermates Local effects. Local competition was assessed by correlating the mean of the two distances separating each fetus from its neighbours or uterine extremity with the fetal or placental weight associated with that fetus. Each uterine horn was assessed independently to exclude interaction with systemic and regional effects. An analysis of covariance was then performed on all horns for a particular strain and, since there was no evidence of differences in slopes, the resultant common correlation coefficients were used to express the average relationship for each strain. The appropriate degrees of freedom (d.f.) total number of fetuses examined (the number of horns examined + 3). =
—
interaction with systemic effects was excluded by assessing regional competition: the difference in number of live fetuses between the left and right uterine horns of each mouse was correlated with the number of litters 2). differences in mean fetal or placental weights between the two horns (d.f.
Regional effects. Any
=
—
assessed by correlating the number of live fetuses per litter with either Systemic effects. Systemic competition number of litters 2). the mean fetal or placental weight per litter (d.f. Group means of number of conceptuses and live fetuses per litter and mean fetal and placental weights were derived from a single average value for each mouse (n number of mice examined in each group). For between-strain comparisons of length of uterine horn, average distances between conceptuses and CVd%, each uterine horn was treated independently (n 2 (number of mice examined)). Strain comparisons were based on one-way anovas and the least significant difference statistic (Snedecor & Cochran, 1967). was
=
—
=
=
Results Litter size and maternal
weight
Litter size in the large litter strain was more than twice that of the small litter strain with that of the medium litter strain about halfway between the two (Table 1, Fig. 1). Weight gained during
positively correlated to litter size (r 0-893, < 005; r 0-682, not significant; and r 0-810, < 0-05 for the large, medium and small strains, respectively) and in the large strain resulted in a doubling of total weight over the 19 days. Despite the large range in litter size between strains, there was no evidence that litter size affected the number of fetuses dying before day 19 and there was no difference in sex ratios between strains. Only five pairs of conjoined placentas were found from a total of 292 placentas (two pairs from one mouse in the large litter strain, one pair in the medium litter strain and two pairs from one mouse in the small litter strain). Weights of conjoined placentas, when split, were only 5% lighter than the average of the remaining single placentas in the same horn (pooled result of three strains, paired t test, not significant). Fetuses associated with conjoined placentas were 14% lighter (P < 0-01 ). Because of the small number of conjoined placentas (3-4% of the total), it was decided to include them in all subsequent results. The distance between conjoined placentas was 4-3 mm (observed distance between metrial glands) for the medium litter strain and taken as 01 mm for the small litter and larger litter strains (they appeared to share a single metrial gland). pregnancy
=
=
=
Table 1. General differences among the three strains of mice at Litter size
Large
Number of mice Maternal weight Day 1 (g) Gain (g) Number per mouse
are means
Medium
19 of gestation Small 10
380 ± 1-7 37-2 ± 2-5
16-3 ± 0-9 1-1 ± 0-5 1389 +38 99-4 + 4-4 14-2 ± 1-0 0-473** (99)
Live fetuses Dead fetuses Fetal weight (mg) Placental weight (mg) Fetal weight:placental weight Correlation of fetal weight with placental weight within strains Values
day
+ sem; number in
parentheses
=
32-0 + 0-9" 25-0 ± 1-7" II-7 ± 1-1 ± 1243 + 78-2 ± 160 + 0-378**
0-6" 0-5
10" 2-9" 0-6
(67)
230 + 0-9" 14-6 ± 0-9b 7-2 0-8 936 92-3 10-3
± 0-5" + 0-2 +
17bc
± 3-Ie + 0-4"
0-104(49)
degrees of freedom.
abThe mean values of the medium litter and/or small litter strains are significantly different from that of the large litter strain; 'P < 005, bP < 001, respectively. The mean value of the small litter strain is significantly different from the medium litter strain; < 0-01, respectively. Least significant difference. "Common correlation
significant
Fig. 1. Comparison of gravid uteri (b) medium and (c) large litters.
University of
Summary. The tendency for fetuses to be evenly spaced along the uterine horn and the relationship of this to local competition between fetuses was examined in three strains of mice which characteristically produce large, medium and small litters. Local competition was assessed by correlating, within each uterine horn, the weight of each fetus or placenta with the mean distance to its immediate neighbours. Weights and distances were measured on day 19 of gestation, on the day before expected parturition and distances only on day 7. Average litter sizes (live fetuses) were 16\m=.\3\m=+-\0\m=.\9, 11\m=.\7\m=+-\0\m=.\6and 7\m=.\2\m=+-\0\m=.\5 (mean \m=+-\sem) in the large, medium and small litter strains, respectively (n 7, 7 and 10, respectively). On day 19, the mean distance between fetuses was significantly less (P < 0\m=.\05)in the large strain (10\m=.\1\m=+-\1\m=.\0mm) than in the medium (14\m=.\0\m=+-\1\m=.\2 mm)or small (13\m=.\5\m=+-\1\m=.\0mm) strains. Evenness of spacing, expressed as the standard deviation of distances between fetuses divided by the mean distance, improved from day 7 to day 19 of gestation in all three strains and effectively prevented local competition between fetuses in the medium litter (r 0\m=.\04)and small litter strains (r 0\m=.\17),but not in the large litter strain (r 0\m=.\45,P < 0\m=.\01).Thus, local crowding does not seem to be detrimental to fetal growth in mice, except in strains specifically bred for large litters. =
=
=
=
Keywords: mouse; fetus; local competition; spacing
Introduction In polytocous species it is generally accepted that growth of a particular fetus is affected by compe¬ tition from its littermates. Such competition can be a 'systemic' effect, due to the total number of fetuses in a litter, a 'regional' effect, due to the number of fetuses in a particular uterine horn, or a 'local' effect due to the proximity of immediate neighbours. There is good quantitative evidence supporting the operation of systemic and regional effects in a number of species including rats (Barr et al, 1970; Norman & Bruce, 1979), guinea-pigs (Eckstein & McKeown, 1955) and mice (McLaren, 1965), but the strength of local effects has received less attention. This may be because fetuses tend to be spaced evenly along the uterine horn, thus minimizing crowding or competition at the local level. It is important to note that where evenness of spacing has been properly quantified, as in rabbits, it falls considerably short of the ideal, that is equal spacing between conceptuses (Boving, 1971). To date, however, the situation for mice has not been determined and the effects of local competition have not been fully quantified. In this study, three strains of mice were examined, which characteristically carry small, medium and large litters. It was postulated that local effects would be more prominent in strains carrying large litters if the length of the uterine horn did not increase proportionally. Placental weights were recorded to determine whether any effect on the fetus could be related to changes in placental growth.
Materials and Methods Animals and treatments
Quackenbush, ARC Swiss and CBA/CaH were selected as the large, medium and small litter strains of mice, respectively. All mice were nulliparous, aged 2-4 months and purchased from the Animal Resources Centre, Murdoch, Western Australia. They were kept in an environmentally controlled room (temperature, 17-23°C; relative humidity, 50-70%; lights on from 07:00 to 21:00h). Food and water were freely available. Females were mated during darkness and the next day was day 1 of gestation. Evidence of mating was assessed by the presence of a vaginal plug (day 7 and 19 groups) or by rapid increase in weight following a single exposure to males (day 19 group only); mice in all three strains normally give birth on the morning of day 20. Mice were killed at 10:00 h on the morning of day 7 or day 19 of gestation with an overdose of sodium pentobarbitone. The entire uterus of each mouse, together with oviducts, ovaries, cervix and vagina, was removed and trimmed of fat. Each uterine horn was dissected free from its mesometrium and from its attachment to the vaginal wall and contralateral horn. The number and position of each implantation site was recorded, and for mice at day 19 of gestation, the uterine horn was cut along the antimesometrial border and the conceptuses (fetuses, fetal membranes and placentas) were removed and weighed to the nearest milligram (see Bruce & Norman, 1975; Rahima & Bruce, 1986, for details). The uterine horn was transferred to a water bath, its ovarian end was pinned to a cork board and the cervical end was attached to a thread that ran freely over a pulley to a 2 g weight. This allowed the uterine horn to stretch slightly but evenly since the water bath elevated the horn above the cork board so preventing frictional resistance. After one minute to allow the uterus to respond to the stretch, the cervical end was also pinned to the cork board. The uterine horn was examined through a dissection microscope and pins were carefully positioned in the centre of each implantation (day 7, intact uterine horn) or implantation site (day 19, opened horn) and at the extremities of each horn. The distances between the pins were then measured with dial callipers to the nearest 0-1 mm. Although the technique has been validated for rats (Rahima & Bruce, 1986) it was revalidated for mice since the measurements had to be carried out with greater accuracy. Distances between implantations were therefore measured on six uterine horns at day 19 of gestation with two animals from each strain, as described above. The horns were then
released from the tensioning weight, restretched by an independent observer, pinned and measured again. There were no significant differences between the two sets of measurements; the correlation coefficients between the two sets as carried out on each of the six uterine horns were r 0-938, 0-981, 0-996, 0-922, 0-999 and 0-999. =
Analysis of results.
Evenness of spacing
was
determined
ceptuses and between the uterine extremities and the
the distances between
by measuring
nearest
neighbouring con¬
conceptus for each uterine horn. The coefficient of
variation (standard deviation/mean) of these distances (CVd%) was then used as the estimate of evenness, a low value implying even spacing and a high value, uneven spacing (Rahima & Bruce, 1986). The average of the distances for any one uterine horn (average distance) was used to compare the extent of spacing between strains or between the two stages of gestation examined.
Calculation of the effects of competition between littermates Local effects. Local competition was assessed by correlating the mean of the two distances separating each fetus from its neighbours or uterine extremity with the fetal or placental weight associated with that fetus. Each uterine horn was assessed independently to exclude interaction with systemic and regional effects. An analysis of covariance was then performed on all horns for a particular strain and, since there was no evidence of differences in slopes, the resultant common correlation coefficients were used to express the average relationship for each strain. The appropriate degrees of freedom (d.f.) total number of fetuses examined (the number of horns examined + 3). =
—
interaction with systemic effects was excluded by assessing regional competition: the difference in number of live fetuses between the left and right uterine horns of each mouse was correlated with the number of litters 2). differences in mean fetal or placental weights between the two horns (d.f.
Regional effects. Any
=
—
assessed by correlating the number of live fetuses per litter with either Systemic effects. Systemic competition number of litters 2). the mean fetal or placental weight per litter (d.f. Group means of number of conceptuses and live fetuses per litter and mean fetal and placental weights were derived from a single average value for each mouse (n number of mice examined in each group). For between-strain comparisons of length of uterine horn, average distances between conceptuses and CVd%, each uterine horn was treated independently (n 2 (number of mice examined)). Strain comparisons were based on one-way anovas and the least significant difference statistic (Snedecor & Cochran, 1967). was
=
—
=
=
Results Litter size and maternal
weight
Litter size in the large litter strain was more than twice that of the small litter strain with that of the medium litter strain about halfway between the two (Table 1, Fig. 1). Weight gained during
positively correlated to litter size (r 0-893, < 005; r 0-682, not significant; and r 0-810, < 0-05 for the large, medium and small strains, respectively) and in the large strain resulted in a doubling of total weight over the 19 days. Despite the large range in litter size between strains, there was no evidence that litter size affected the number of fetuses dying before day 19 and there was no difference in sex ratios between strains. Only five pairs of conjoined placentas were found from a total of 292 placentas (two pairs from one mouse in the large litter strain, one pair in the medium litter strain and two pairs from one mouse in the small litter strain). Weights of conjoined placentas, when split, were only 5% lighter than the average of the remaining single placentas in the same horn (pooled result of three strains, paired t test, not significant). Fetuses associated with conjoined placentas were 14% lighter (P < 0-01 ). Because of the small number of conjoined placentas (3-4% of the total), it was decided to include them in all subsequent results. The distance between conjoined placentas was 4-3 mm (observed distance between metrial glands) for the medium litter strain and taken as 01 mm for the small litter and larger litter strains (they appeared to share a single metrial gland). pregnancy
=
=
=
Table 1. General differences among the three strains of mice at Litter size
Large
Number of mice Maternal weight Day 1 (g) Gain (g) Number per mouse
are means
Medium
19 of gestation Small 10
380 ± 1-7 37-2 ± 2-5
16-3 ± 0-9 1-1 ± 0-5 1389 +38 99-4 + 4-4 14-2 ± 1-0 0-473** (99)
Live fetuses Dead fetuses Fetal weight (mg) Placental weight (mg) Fetal weight:placental weight Correlation of fetal weight with placental weight within strains Values
day
+ sem; number in
parentheses
=
32-0 + 0-9" 25-0 ± 1-7" II-7 ± 1-1 ± 1243 + 78-2 ± 160 + 0-378**
0-6" 0-5
10" 2-9" 0-6
(67)
230 + 0-9" 14-6 ± 0-9b 7-2 0-8 936 92-3 10-3
± 0-5" + 0-2 +
17bc
± 3-Ie + 0-4"
0-104(49)
degrees of freedom.
abThe mean values of the medium litter and/or small litter strains are significantly different from that of the large litter strain; 'P < 005, bP < 001, respectively. The mean value of the small litter strain is significantly different from the medium litter strain; < 0-01, respectively. Least significant difference. "Common correlation
significant
Fig. 1. Comparison of gravid uteri (b) medium and (c) large litters.
