38 H. Leibovich et al. Animal Reproduction Science 64 2000 33–47
of variance to detect effects of treatment within day of bleeding. Statistical analyses were conducted using the general linear model GLM procedure in the SAS computer package
Statistical Analysis System; SAS Institute, 1985. Differences of P 0.05 were considered significant and all values are expressed as mean and standard errors of the mean.
3. Results
3.1. Anti-oPL antibody titer Antibodies against oPL were not detected in serum samples obtained from control ewes
or in any of the pre-immune serum samples. In the first experiment, all immunized ewes developed anti-oPL antibodies with titers dilutions giving 50 of specific antibody binding
of 8208 ± 2065 in the first post-immunization period. For the first pregnancy, anti-oPL antibody titers declined on an average to 79, 51 and 24 of first post-immune titers at
60, 80 and 130 days of gestation, respectively. First post-immune titers were correlated r = 0.87; P 0.05 only with titers measured on day 60 of gestation.
All immunized ewes in the second experiment also developed anti-oPL antibodies with first post-immune titers higher than in the first experiment, at 73,051 ± 25,396 Table 1. At
130 days of gestation, anti-oPL antibody titers dropped on an average to 10 and 6 of first post-immune titers in the first and second pregnancies, respectively. Whereas no significant
association was found between post-immune titers and titers at 130 days of gestation in either pregnancy, a significant correlation r = 0.49; P 0.003 was found between
titers measured at 130 days of gestation in the first and second pregnancies. No significant association was observed between anti-oPL titers in the immunized ewes and their lambs’
birth weight, or the ewe milk production.
3.2. Serum concentrations of oPL Distribution of immunoreactive I and bioactive B oPL in serum on days 60, 80
and 130 of gestation for control and immunized ewes in the first experiment are pre-
Table 1 Anti-ovine placental lactogen oPL antibody titers dilutions giving 50 of specific antibody binding in ewes
immunized against recombinant oPL Mean ± S.E.M.
Median Minimum
Maximum First experiment
First post-immune 8208 ± 2065
7465 1643
36080 First gestation, day 60
6481 ± 1613 5945
1222 23870
First gestation, day 80 4230 ± 1147
3510 616
17390 First gestation, day 130
1935 ± 488 1110
616 6166
Second experiment First post-immune
73051 ± 25396 21510
5169 770200
First gestation, day 130 7222 ± 2254
4575 799
53860 Second gestation, day 130
4526 ± 1393 3284
855 24200
H. Leibovich et al. Animal Reproduction Science 64 2000 33–47 39
Fig. 1. Immunoreactive RIA and bioactive bioassay oPL in serum ngnl at 60, 80, and 130 days of gestation in control ewes and in ewes immunized against ovine placental lactogen oPL.
sented in Fig. 1. Concentrations of oPL increased during gestation and I-oPL serum lev- els were always higher than B-oPL levels in the same samples. At 130 days of gesta-
tion, B-oPL serum levels were higher P 0.02 in immunized ewes than in control ewes.
Serum oPL levels at 130 days of the first and second pregnancies in the second exper- iment are presented in Table 2. In control ewes, concentrations of I-oPL in serum were
higher P 0.01 than those of B-oPL, in accordance with results obtained in the first experiment. However, for immunized ewes, the opposite result was obtained: I-oPL con-
centrations were lower P 0.04 than B-oPL concentrations. As in the first experiment, B-oPL levels of immunized ewes were higher P 0.05 than B-oPL levels in control
ewes. As I-oPL levels were relatively low, they were lower P 0.001 than in control ewes.
Table 2 Immunoreactive and bioactive ovine placental lactogen oPL serum levels ngml at 130 days of gestation in
control and oPL-immunized Assaf ewes means ± S.E.M.
a,b
Group Immunoreactive oPL serum levels
Bioactive oPL serum levels First gestation
Control 522 ± 36 a 34–1060
105 ± 12 a 28–266 Immunized
104 ± 42 b 5–438 146 ± 15 b 54–375
Second gestation Control
465 ± 34 a 94–950 149 ± 24 a 38–499
Immunized 53 ± 42 b 0–214
212 ± 27 b 97–825
a
Values in parentheses are range of values.
b
Within pregnancy and type of oPL assay, values with different letters in the same column are different at P
0.05.
40 H. Leibovich et al. Animal Reproduction Science 64 2000 33–47
Fig. 2. Inhibition of ovine placental lactogen oPL-stimulated proliferation of Nb-2 cells a and FDC-P1 cells b by serum obtained from ewe no. 4835 immunized against oPL:
s
pre-immune serum;
j
post-immune serum.
3.3. Serum IGF-I concentrations In the first experiment, concentrations of serum IGF-I on day 130 of gestation did not
differ between immunized 179 ± 32 ngml and control 207 ± 36 ngml ewes. Similar results were obtained in the second experiment where on day 130 of gestation, IGF-I levels
were 190 ± 12 and 194 ± 13 ngml for control and immunized ewes, respectively.
3.4. Neutralization of oPL bioactivity by anti-oPL antisera While neither serum from control, non-immunized, non-pregnant ewes nor pre-immune
serum inhibited cell proliferation following stimulation with oPL, first post-immune serum inhibited proliferation of both Nb
2
-11C and FDC-P1 cells Fig. 2. The IC
50
inhibition values for serum from different ewes were not correlated P 0.05 with antibody titers
of the same samples. 3.5. Free and antibody-bound oPL in serum
IgG-free fractions of serum samples from control and immunized ewes showed oPL bioactivity similar to that measured in those samples before loading them onto the Sephadex
H. Leibovich et al. Animal Reproduction Science 64 2000 33–47 41
Fig. 3. Western blot analysis of bound ovine placental lactogen oPL in fractions containing IgG from control ewes and ewes immunized against oPL. Lanes 1–4: IgG fractions from serum of ewes immunized against oPL;
lanes 5–7: IgG fractions from serum of control ewes; lane 8: 2.5 ng recombinant roPL; lane 9: 5.0 ng roPL; lane 10: 10.0 ng roPL.
column data not shown. No oPL bioactivity was detected in IgG-containing serum frac- tions obtained from control or immunized ewes. However, whereas oPL was not detected
by western blotting in IgG-containing fractions of serum from three randomly selected pregnant control ewes, it was detected in IgG-containing serum samples from four ran-
domly selected immunized pregnant ewes Fig. 3. At 130 days of gestation, an esti- mated 50 of the oPL in the circulation of immunized pregnant ewes was bound to
antibodies.
3.6. Reproductive performance Two immunized ewes in the first experiment and four control and two immunized ewes
in the second experiment, did not lamb until the age of 26 months. Those ewes were culled. Age at first lambing, which in the first and second experiments was 17.0 ± 1.0 and
14.7 ± 0.4 months, respectively, was not affected by treatment P 0.05. The second lambing occurred in the two experiments 7.9 ± 0.4 and 8.4 ± 0.3 months after the first
lambing, respectively, and was also not affected by the treatment P 0.05.
3.7. Lamb birth weights and ewes’ post-partum body weights Prolificacy in the first and second experiments, respectively, averaged 1.85 and 1.32
lambs born per ewe per lambing. The relatively high prolificacy in the first experiment was due to the FecB Booroola gene segregating in the Booroola–Assaf population. Of the
lambs born in the first experiment, 36 and 18 were triplets or more. Treatment, parity and litter size affected P 0.01 birth weight, whereas the effect of lamb sex was significant
P 0.02 only in the second experiment. Lambs born to immunized ewes were heavier P 0.001 than lambs born to control ewes Table 3 and the greater the number of
lambs born in a litter, the greater the advantage in birth weight for lambs born to immunized ewes.
Age at lambing, litter size and treatment did not have significant effects P 0.05 on the ewes’ post-partum body weight. Post-partum body weights of immunized ewes were
numerically higher than those of control ewes averaging, respectively, 67 ± 3 and 63 ± 2 kg.
42 H. Leibovich et al. Animal Reproduction Science 64 2000 33–47
Table 3 Birth weights of lambs born to control ewes and ewes immunized against ovine placental lactogen means±S.E.M.
in their first and second lambings
a
Litter size Lambs born to control ewes
Lambs born to immunized ewes n
Birth weight n
Birth weight First experiment
1 28
4.8 ± 0.2 a 14
5.0 ± 0.2 a 2
50 3.7 ± 0.1 a
26 4.2 ± 0.1 b
3 44
2.8 ± 0.3 a 9
3.9 ± 0.4 b Second experiment
1 60
5.2 ± 0.2 a 35
5.7 ± 0.1 b 2
57 4.5 ± 0.1 a
23 5.3 ± 0.1 b
a
Within rows, values with different letters are different at P 0.05.
3.8. Milk production In the first 108 days of the first lactation, milk production in immunized ewes N = 25;
208 ± 12 l, was greater P 0.01 than that in the control ewes N = 55; 174 ± 8 l. Milk composition was not affected by treatment data not shown.
Similar results regarding the effect of immunization against oPL on milk production were obtained in the second lactation where during the first 110 days of the lactation, milk
production was higher P 0.01 in immunized ewes 271 ± 21 than in control ewes 204 ± 15 l.
We studied lactation curve shape by fitting the Pollot model Pollott and Gootwine, 2000: although milk yield peak occurred for both groups during the first week of lactation, peak
yield was significantly higher in the immunized group than in the control group, being in the first lactation 2.1 ± 0.1 and 1.7 ± 0.1 l, respectively. Other curve parameters, including
a relative decline in milk production rate throughout the lactation, did not differ between the immunized and control groups.
4. Discussion
