H. Leibovich et al. Animal Reproduction Science 64 2000 33–47 35
the oPL’s role during pregnancy. The aim of the present study was therefore to investi- gate the physiological role of oPL following active immunization of ewe-lambs against
recombinant oPL roPL prepared in our laboratory Sakal et al., 1997.
2. Materials and methods
2.1. Animals and immunization treatments Experimental protocols were approved by the Volcani Center Animal Care Committee.
Two experiments were conducted. The first was carried out at the Central Farm of the Volcani Center at Bet Dagan, where all ewes were kept indoors. Booroola–Assaf crossbred
ewe-lambs, born in the spring of 1996, were assigned to immunized and control groups N = 16 and 36, respectively without knowledge of their genotype at the FecB locus. The
second experiment made use of the Katzenelebugen dairy Assaf flock at Moshav Talmai Elazar: 35 and 68 Assaf ewe-lambs, born between January and April 1998, were assigned
to immunized and control groups, respectively.
In both experiments, immunization of ewe lambs against roPL was started at 6 months of age. At about 8 months of age, all ewe-lambs were introduced into an accelerated breeding
program where mating periods of about 40 days were scheduled every 3–4 months. At the beginning of each mating period, estrus was synchronized in all ewes using progesterone
pessaries Chronogest, Intervet, Boxmeer, The Netherlands and 500 IU eCG Synchroject, Vetimax, Bland, Holland. Ewes were mated following the detection of estrus and if they
returned to estrus in the subsequent cycle, they were mated again. Pregnancy diagnosis was carried out 20–40 days after the end of each breeding period. Ewes that failed to conceive
were resynchronized to estrus and mated again in the next mating period. At lambing, the number of lambs born to each ewe, their sex and birth weights were recorded. Body weight
was recorded for control and immunized ewes 3 days after the first lambing in the second experiment. Lambs were raised by their dams first experiment or separated from their
dams on the day of lambing and moved to an artificial rearing unit second experiment. Ewes were milked only in the second experiment. Milking was performed twice a day at
08.00 and 16.00 h, from the day of lambing. Milk production was recorded weekly until all ewes completed about 110 days of milking.
Ewes in both experiments were synchronized 70–100 days after lambing and mated again in the succeeding breeding period. Ewes remained in the first experiment until they had had
their second lambing. In the second experiment, ewes remained in the experiment until they had completed at least 100 days of milking in the second lactation or up to 12 months after
the first lambing, if they did not lamb again.
2.2. Immunization A 1:1 mixture of the mono and oligo forms of roPL Sakal et al., 1997 was used as the
immunogen. Saline solution 1 ml containing the immunogen 1 mgml was emulsified in an equal volume of Freund’s complete adjuvant Sigma Chemical Co., St. Louis, MO and
injected subcutaneously at several sites over the neck region. Ewes received two booster
36 H. Leibovich et al. Animal Reproduction Science 64 2000 33–47
injections at 3 and 6 weeks after the primary immunization with the same antigen emulsified in incomplete Freund’s adjuvant.
2.3. Antibody detection and measurement Blood samples were collected from the jugular vein and serum was prepared. In the
first experiment, serum was prepared from both control ewes and immunized ewes prior to the immunization procedure pre-immune samples, 3 weeks after the second booster first
post-immune samples and on days 60, 80 and 130 of the first pregnancy. In the second experiment, blood samples were collected and serum was prepared at the pre- and first
post-immune stages, and at 130 days of gestation in both the first and second pregnancies. The ability of anti-oPL antisera to bind roPL was determined using an ELISA. A stock
solution of oPL 2.5 mgml in 0.1 M Na
2
CO
3
buffer; pH 9.6 was prepared and 100 ml was added to each well of a 96-well microtiter plate Nunc-Immuno
TM
, Nunc
TM
, Denmark. Plates were incubated for 3 h at 37
◦
C, then blocked by washing wells three times with PBST PBS with 0.05 Tween 20. Dilutions of antisera ranging from 1:10
4
to 1:3 × 10
6
were prepared with PBST and 100 ml was added to each well. Following overnight incubation at
4
◦
C, the samples were decanted, the plates were washed three times as above and 100 ml of biotinylated rabbit anti-sheep IgG 1:10,000 dilution in PBST; Zemed, San Francisco,
CA was added. Following incubation at 37
◦
C for 3 h, the wells were washed with PBST and 100 ml of alkaline phosphatase Zemed diluted 1:2000 in PBS buffer containing 15
horse serum, 0.5 gelatin and 0.5 BSA was added to each well. After incubation at 37
◦
C for 30 min, the plates were washed and 100 ml alkaline phosphatase substrate 2 mgml
p-nitrophenyl phosphate; Sigma in buffer containing, 0.75 glycine and 1 mM MgCl
2
adjusted to pH 10.5 using 1N NaOH were added. The color intensity was measured at 405 nm after 20 min of incubation by automated plate reader.
2.4. oPL assays In the first experiment, serum levels of oPL were measured at 60, 80 and 130 days of
the first gestation for all immunized ewes and nine control ewes. In the second experiment, serum levels of oPL were determined at 130 days of both the first and second gestations in
24 and 33 immunized and control ewes, respectively. Serum levels of oPL were detected by both the Nb
2
-11C lymphoma cell proliferation bioassay Gertler et al., 1985 and by radioimmunoassay RIA. The oPL antiserum used in the RIA was raised in a rabbit against
recombinant oPL and used at a final dilution of 1:400,000. The antiserum exhibited low cross-reactivity with oPRL and oGH preparations obtained from NIH. The minimum de-
tectable level of oPL was 5 ngml serum. The intra- and inter-assay coefficients of variation were 6.4 and 8.8, respectively.
2.5. Neutralization of oPL activity by anti-oPL auto-antibodies The neutralizing ability of oPL bioactivity by serum from immunized ewes was expressed
in EC
50
values. This value represents the titer required to achieve a 50 reduction in the number of cell doublings following stimulation by roPL, as described previously for
H. Leibovich et al. Animal Reproduction Science 64 2000 33–47 37
human GH Strasburger et al., 1989. Both rat lymphoma Nb
2
-11C cells possessing PRL receptors Gertler et al., 1985 and FDC-P1 cells stably transfected with rabbit GH receptor
Rowlinson et al., 1996 were used in the neutralization assays. 2.6. Serum IGF-I measurements
Serum levels of IGF-I were determined at 130 days of pregnancy for immunized and non-immunized ewes by RIA Breier et al., 1991 and intra- and inter-assay coefficients of
variation were 8.7 and 12.4, respectively. IGF-I concentrations are expressed relative to a reference standard of recombinant human IGF-I Fujisawa Pharmaceutical Co., Osaka,
Japan. Rabbit antiserum to IGF-I UB2-495 was obtained from the Hormone Distribution Program of NIDDK through the NIH National Hormone and Pituitary Program.
2.7. Determination of free and antibody-bound oPL Serum aliquots 250 ml from randomly selected immunized and control ewes were ap-
plied to a Sephadex G-75 column 25 ml bead volume equilibrated with PBS pH 7.0. Then, the column was washed with PBS. The IgG-containing fractions first 12 ml of elu-
ate, as determined in preliminary experiments and the next 10 ml of eluate that contained IgG-free-fractions were collected and concentrated 10-fold using an ultrafiltration system
Amicon Inc., Beverly, MA. Bioactivity of oPL in those samples was determined using the Nb
2
test. Western blotting Towbin et al., 1979 was used to detect oPL in IgG-containing and IgG-free serum fractions.
2.8. Milk composition analysis Milk samples for milk constituent analysis were taken during the morning milking from
immunized and control ewes once, at the milk recording day of week 13 of the lacta- tion. Concentrations of fat, protein, lactose and total solids in milk were measured us-
ing a semi-automated infrared analyzer Milkoscan 134 AB, N. Foss Electric, Hillerod, Denmark.
2.9. Statistical analysis Statistical differences in age at first lambing for each experiment were determined by
the Wilcoxon test. Effect of the experimental treatment on the birth weight of lambs was analyzed by least-squares analysis of variance. The model included effects of treatment of
dam immunized or control, sex of lamb, litter size single, twins, or triplets or more, and parity number first or second. Effect of immunization on post-partum ewes’ body
weight was analyzed by analysis of variance. The model included effects of treatment of dam immunized or control and litter size. Age at first lambing was taken as a covariate.
Milk production records from throughout the period of lactation were subjected to analysis of variance using a model that included experimental group as the main effect, and age at
lambing and days in lactation as covariants. Serum levels of oPL were compared using a variance-weighted Welch F-test. IGF-I concentrations were subjected to one-way analysis
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
