Animal measurements and sample collection

Animal measurements and sample collection
Pretreatment blood samples were collected from a subgroup of
vitamin D and control ewes (n = 40) on Day 111 of pregnancy for
subsequent analysis of vitamin D concentrations. At lambing,
between Days 143 and 153 of pregnancy, ewes were intensively
monitored between 0400 hours and 12 midnight each day, for
ewe and lamb sampling to occur before the lamb suckled
following birth. All blood samples were collected via jugular
venipuncture into heparinised vacutainer tubes. Following the
initial collection of blood samples from lambs, only those lambs
that were bled at birth were bled at subsequent time points in the
study. All blood samples collected for vitamin D analysis were
transported on ice, whereas those collected for immunological
analysis were stored at room temperature following collection
and for transport. Following transport, samples collected for
vitamin D analysis were centrifuged for 15 min at 2278g to
isolate plasma, which was then stored at 20
C. For
immunological analysis, whole blood was isolated from blood
samples collected from lambs at birth. The remainder of the
sample, and all other lamb blood samples for immunological
analysis, were centrifuged for 10 min at 952g to isolate plasma,
which was then stored at 80
C.
At birth, all lambs were weighed, and their dam, sex and birth
type were recorded (n = 154). Two 5 mL blood samples were
collected from all single-born lambs and from the first lamb born
only for lambs born in litters, provided they had not already
suckled (n = 91), and the blood glucose concentration of the
sample was measured immediately using a glucometer (AccuChek® Go, Roche Diagnostics, Sydney, NSW, Australia). Rectal
temperatures were taken from most lambs using a digital
thermometer. At least 30 mL of colostrum and a 9 mL blood
sample were collected from all ewes for immunological and
vitamin D analyses, respectively. Colostrum was temporarily
stored at 4
C, before being aliquoted and stored at 80
C for
subsequent immunological analysis.
One-week post-birth (3 days), 5 mL blood samples were
collected from the lambs (n = 91) for immunological analysis.
Lambs were weighed ~2-weeks post-birth. Four weeks post-birth
(1 week), 9 mL blood samples were collected from the
subgroup of control and vitamin D-supplemented ewes
(n = 40) for vitamin D analysis, and two 5 mL blood samples
were collected from the lambs for vitamin D (n = 40) and
immunological (n = 69) analyses. At marking, ~4 weeks postbirth, lambs were weighed and received their primary
vaccinations (Glanvac® 6, Eryvac®, Gudair®, and Scabigard®;
Zoetis). Two weeks following administration of the primary
vaccines (Week 6), lambs were weighed and 5 mL blood
samples were collected from the lambs for immunological
analysis (n = 69). Lambs were then weighed every 2–3 weeks
until weaning at ~14-weeks of age. At weaning, a 5 mL blood
sample was also collected for immunological analysis (n = 65)
and the lambs were administered clostridial booster vaccines
(Glanvac® 6, Zoetis Australia).
Vitamin D concentration assay
Plasma samples collected from ewes at Day 111 of pregnancy,
before treatments commenced (n = 40), and from both ewes and
lambs at lambing (n = 80) and 4 weeks post-birth (n = 80), were
analysed for concentrations of 25-hydroxyvitamin D using liquid
chromatography tandem mass spectrometry (LC–MS/MS) with
electrospray ionisation in positive mode (ESI+). Colostrum
samples (n = 40) collected from ewes at lambing and milk
samples (n = 40) collected from ewes 4 weeks post-lambing
were also analysed for 25-hydroxyvitamin D concentrations.
Briefly, the internal standards d6-25-hydroxyvitamin D2 and
d6-25-hydroxyvitamin D3 were added into samples and, then,
the sample was extracted using acetonitrile to release vitamin D
from vitamin D-binding protein and precipitate proteins. Extracts
were then chromatographed to further isolate vitamin D from
potentially interfering substances and to separate the different
forms of vitamin D (25-hydroxyvitamin D2 (25(OH)D2), 25-
hydroxyvitamin D3 (25(OH)D3) and C-3 epimer of 25-
hydroxyvitamin D3 (C3-epi-25(OH)D3)). The various forms of
vitamin D were quantified in a mass spectrometer (Waters
Micromass Quattro Premier XE) with selected ion mode.
While C3-epi-25(OH)D3 is not currently known to have any
functionally active roles in the body and is not widely included
in the measure of vitamin D status, a significant amount of
C3-epi-25(OH)D3 was detected in the plasma samples in the
present study. Thus, the total 25-hydroxyvitamin D (25(OH)D)
concentrations in the present paper are presented as the total
of 25(OH)D3 and 25(OH)D2, either excluding or including C3-
epi-25(OH)D3.
Whole blood differential cell counts
Whole blood samples collected from lambs at birth and 4 weeks
of age were analysed within 24 h for routine white blood cell
differentials by using the Advia® 120 automated hematology
analyzer (Sequence 284; Siemens, Munich, Germany) in a highoutput pathology laboratory (Clinical Pathology, Murdoch
University Veterinary Hospital, Perth, WA, Australia). Briefly,
the Advia machine adds peroxidase reagents to the samples to
achieve erythrocyte lysis and fixation and staining of white
blood cells. Monocytes and neutrophils are identified on the
basis of light scatter and absorption data generated through
analysis of samples in a peroxidase flow cytometry channel.
Monocyte and neutrophil cell counts are displayed as a
percentage of white blood cells. Manual counts by bloodsmear microscopy were conducted where necessary, such as in
the presence of blood clots or to confirm Advia data.
Whole blood phagocytosis uptake assay
Whole blood samples collected from lambs at birth and 4 weeks
of age were analysed for monocyte and polymorphonuclear
leukocyte phagocytic capacities using flow cytometry.
pHrodo Red Staphylococcus aureus bioparticles® (Life
Technologies, Carlsbad, CA, USA) were resuspended at
1 mg/mL in phosphate-buffered solution (PBS). Resuspended
bioparticles were vortexed and sonicated to remove aggregates,

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