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First published online March 2, 2007
Journal of Experimental Biology 210, 1064-1074 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02724
Characterization of very-low density lipoprotein particle diameter dynamics in relation to egg production in a passerine bird
1 Department of Biological Sciences, Simon Fraser University, 8888
University Drive, Burnaby, British Columbia, V5A 1S6, Canada
2 Poultry Science Department, Texas A&M University, College Station, TX
77843, USA
* Author for correspondence at present address: Department of Biology, Coker Hall, CB 3280, University of North Carolina–Chapel Hill, Chapel Hill, NC 27599-3280, USA (e-mail: ksalvante{at}unc.edu)
Accepted 16 January 2007
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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70 nm in
diameter), which transports lipids to peripheral tissues, to yolk-targeted
VLDL (VLDLy) (30 nm), which supplies the yolk with energy-rich lipid, has
been observed in the plasma of laying domestic fowl. We validated an
established dynamic laser scattering technique for a passerine songbird
Taeniopygia guttata, the zebra finch, to characterize the dynamics of
VLDL particle diameter distribution in relation to egg production. We
predicted that non-gallinaceous avian species that have not been selected for
maximum egg production would exhibit less dramatic shifts in lipid metabolism
during egg production. As predicted, there was considerable overlap between
the VLDL particle diameter distributions of laying and non-laying zebra
finches. But unexpectedly, non-laying zebra finches had VLDL diameter
distributions that peaked at small particles and had relatively few large VLDL
particles. As a result, laying zebra finches, in comparison, had diameter
distributions that were shifted towards larger VLDL particles. Nevertheless,
laying zebra finches, like laying chickens, had larger proportions of
particles within proposed VLDLy particle diameter ranges than non-laying zebra
finches (e.g. sVLDLy: 50% vs 37%). Furthermore, zebra finches and
chickens had similar modal (29.7 nm in both species) and median (32.7 nm
vs 29.6 nm) VLDL particle diameters during egg production. Therefore,
although zebra finches and chickens exhibited opposing directional shifts in
VLDL particle diameter distribution during egg production, the modifications
to VLDL particle structure in both species resulted in the realization of a
common goal, i.e. to produce and maintain a large proportion of small VLDL
particles of specific diameters that are capable of being incorporated into
newly forming egg yolks.)
Key words: VLDL, yolk-targeted VLDL, reproduction, VLDL particle diameter, zebra finch
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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;
Bergink et al., 1974;
Deeley et al., 1975;
Neilson and Simpson, 1973;
Chan et al., 1976;
Wallace, 1985;
Walzem, 1996;
Williams, 1998). As a result
of increased total hepatic lipid production, plasma lipid concentrations
increase from about 3 mg neutral lipid ml–1 plasma in
non-laying turkeys (Meleagris gallopavo) to 21 mg in laying turkeys
(Bacon et al., 1974).
Similarly, plasma triacylglyceride concentration increased from 0.5–1.5
µmol ml–1 plasma in non-laying chickens (Gallus gallus
domesticus) to 20–50 µmol ml–1 plasma in laying
chickens (Griffin and Hermier,
1988). Furthermore, data for egg-laying chickens, turkeys and
quail (Coturnix coturnix) show that there is an oestrogen-dependent
shift in VLDL synthesis from the production of generic VLDL, which ranges in
size from 30 to >200 nm, to smaller, yolk-targeted VLDL, which ranges in
diameter from 15 to 55 nm in domestic fowl
(Chapman, 1980;
Griffin, 1981;
Walzem et al., 1994;
Walzem, 1996;
Speake et al., 1998;
Chen et al., 1999;
Walzem et al., 1999).
Furthermore, whereas generic VLDL has at least six associated apolipoproteins
(including apoA-I, apoB and apoC), VLDLy has only two associated
apolipoproteins, apoB and apoVLDL-II, of which the latter is thought to be
responsible for the decrease in VLDLy diameter
(Chan et al., 1976;
Kudzma et al., 1979;
Griffin, 1981;
Dashti et al., 1983;
Lin et al., 1986;
Schneider et al., 1990;
Speake et al., 1998;
Walzem et al., 1999).
Consequently, the presence of circulating VLDLy in egg-producing females
represents a dramatic shift in lipid metabolism associated with changes in the
composition and structure of VLDL.
The structural changes to circulating VLDL particles directly influence
their in vivo function during egg production
(Walzem, 1996). Whereas the
role of generic VLDL is to transport triacylglycerides throughout the body for
tissue utilization or storage in adipose tissue, the function of VLDLy is to
deliver triacylglycerides to the oocyte, where they will be used as the energy
source for the developing embryo (Walzem,
1996). The smaller diameter of VLDLy is thought to be critical for
enabling the particles to pass through the pores in the granulosa basal lamina
of the ovary, allowing them access to the developing ovarian follicles
(Griffin and Perry, 1985). In
addition, apoVLDL-II also acts as an inhibitor of lipoprotein lipase (LPL),
probably by limiting access to the water needed for triacylglycerol hydrolysis
(Boyle-Roden and Walzem, 2005).
The resistance of VLDLy particles to hydrolysis by extra-ovarian tissues
preserves the triacylglycerol-rich VLDLy for uptake by the developing ovarian
follicles (Walzem, 1996).
Cross-injection studies on turkeys and chickens using labelled generic VLDL
isolated from immature turkeys and labelled VLDLy from laying turkeys and
chickens confirm that immature and laying birds utilize generic VLDL and VLDLy
differently; a greater proportion of generic VLDL was deposited into tissues,
whereas more VLDLy was incorporated into ovarian follicles
(Bacon et al., 1978;
Bacon, 1981). In vivo
studies in laying domestic fowl have detected only low circulating levels of
intermediate-density and low-density lipoproteins, both by-products of the
hydrolysis of VLDL by LPL (Hermier et al.,
1989; Walzem et al.,
1994; Walzem,
1996), providing further evidence for the increased in
vivo resistance of VLDLy to LPL hydrolysis.
Despite the LPL-resistance of VLDLy, chickens and turkeys are able to
incorporate radiolabelled VLDLy from laying females into non-ovarian tissues,
presumably to metabolize them to meet their energetic needs
(Bacon et al., 1978;
Bacon, 1981). Griffin and
Hermier (Griffin and Hermier,
1988) noted that some 10% of VLDLy triacylglycerol can be
hydrolyzed by LPL; given the high plasma concentrations of VLDLy in laying
domestic fowl, this partial hydrolysis may be sufficient to meet the female's
own energetic requirements during laying. Others
(Chen et al., 1999;
Walzem et al., 1999) have
proposed that laying domestic fowl could also potentially meet their energetic
requirements by metabolizing small amounts of generic VLDL that are
synthesized by the avian kidney (Blue et
al., 1980; Tarugi et al.,
1998).
Chickens have been the target of strong artificial selection for prolonged
and consistent egg production, and can maintain high rates of egg production
for over a year (Etches,
1996). It is not known whether non-domesticated, non-gallinaceous
avian species, which have not been selected for maximum egg production,
exhibit such dramatic shifts in lipid metabolism during egg production.
Passerine birds have been shown to experience marked increases in the
concentration of circulating triacylglycerol during egg production
(Christians and Williams,
1999; Challenger et al.,
2001). However, the assay used in these studies measured
triacylglycerides associated with both generic and yolk-targeted VLDL
and was not able to distinguish between the two forms of the lipoprotein. A
dynamic laser scattering technique was used to assess VLDL particle diameter
distribution in domestic fowl (Walzem et
al., 1994; Walzem et al.,
1999). This method provides a frequency distribution of VLDL
particle diameters (in nm), which has been shown to vary in relation to egg
production (Walzem et al.,
1994; Walzem,
1996; Walzem et al.,
1999; Peebles et al.,
2004). At present there is a paucity of data on VLDL particle
diameter distributions for non-domesticated species. In this paper we describe
a modification of the dynamic laser scattering technique for use in a
passerine songbird, the zebra finch. We used this technique to (1)
characterize VLDL particle diameter distributions in non-laying and
egg-producing female zebra finches, (2) estimate the diameter range of VLDL
particles that are available for deposition into the developing eggs of laying
zebra finches, and (3) compare VLDLy dynamics during egg production in zebra
finches and chickens. Non-domesticated birds have not been strongly selected
for egg production and generally experience much more variable environmental
conditions during egg production, including fluctuations in food availability
and low ambient temperature. We, therefore, predicted that laying female
passerines should experience greater selection pressures to be able to
maintain production of larger, potentially generic, VLDL during egg production
in order to meet their unpredictable energetic needs.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Single comb white leghorn chickens of the W-36 strain (HyLine
International, Bryan, Texas, USA) were individually housed in
light-supplemented, fan-ventilated, open-sided houses at the University of
California, Davis, USA. Chickens were given ad libitum access to
water and a corn-soy diet formulated to meet the National Research Council for
Poultry's (NRC) requirements for laying hens (15% protein, <5% lipid, 2900
kcal kg–1) National
Research Council Subcommittee on Poultry Nutrition, 1994), and
were provided with 15 h of light per day. Ambient house temperature varied
from 7–29°C. All animal husbandry and experimental procedures were
conducted in accordance with a protocol approved by the Animal Use and Care
Committee of the University of California, Davis, USA.
Zebra finch breeding and blood sampling
Male (N=36) and female (N=36) zebra finches were weighed
(±0.1 g) at the time of pairing, and tarsus and bill measurements
(±0.1 mm) were taken. Breeding pairs were housed individually in cages
(61x46x41 cm; lengthxwidthxheight) equipped with an
external nest box (15x14.5x20 cm) and were provided with an
egg-food supplement (6 g of a mixture of 62–65 g hard-boiled egg, 13 g
cornmeal, 13 g bread crumbs; 30.2% protein and 13.0% lipid by dry mass) daily
between pairing and clutch completion in addition to the normal seed diet (see
Williams, 1996). Data on
laying interval (days from pairing to initiation of laying) and egg and clutch
size were obtained by checking the nest boxes daily between 09:00 h and 11:00
h. All new eggs were weighed (±0.001 g) and numbered on the day they
were laid. Clutches were considered complete if no new eggs were laid over 2
days. At this time the female was weighed, and the pair was returned to
same-sex, non-breeding cages.
Laying females were weighed and blood samples taken (200 µl from the brachial vein) on the day their first eggs were laid (laying sample). Randomly chosen female zebra finches (N=27) from the same-sex cages were also weighed and blood samples taken (non-laying sample). All blood samples were collected between 09:00 h and 11:30 h into heparinized capillary tubes, and then expelled into EDTA-coated microcentrifuge tubes containing 0.5 mol l–1 disodium-EDTA (3 µl; Sigma-Aldrich Canada, Oakville, ON, Canada) and centrifuged at 2200 g for 10 min in a Baxter Canlab Biofuge 13. A sub-sample (5 µl) of each plasma sample was frozen (–20° C) for triacylglyceride analysis, and the remainder of each plasma sample was placed into an EDTA-coated microcentrifuge tube containing 0.5 mol l–1 disodium-EDTA (5 µl) for VLDL particle diameter distribution analysis. Sodium azide (1% w:v; Sigma-Aldrich Canada) was added to each EDTA-coated tube to prevent mould formation (0.01 µl µl–1 plasma), and the plasma samples were refrigerated (4°C) pending analysis of VLDL particle diameter distribution.
Influence of feeding, fasting and egg-food supplementation on VLDL particle diameter distribution in zebra finches
Two preliminary studies were performed to assess the potentially
confounding effects of feeding vs fasting and diet supplementation
(i.e. the egg-food supplement given to breeding pairs) on various measures of
VLDL particle diameter distribution and circulating triacylglyceride levels.
For the fasting experiment, randomly selected laying female zebra finches were
blood sampled twice; once while in the `fed' state, i.e. ad libitum
access to seed and egg-food supplement and again while in the `fasted' state,
i.e. 15–16 h without access to food. For the `fasted' sample, the seed
and egg-food supplement containers were removed at 19:00 on the night before
blood sample collection. Female zebra finches were weighed and blood sampled
between 10:00 h and 11:00 h on the days that their second and third eggs were
laid (2-egg and 3-egg stages, respectively). The order in which females were
in the fed and fasted states was randomized such that half of the females were
in the fasted state at the 2-egg and in the fed state at the 3-egg stage,
whereas the reverse was true for the other half of the females. Previous
studies on vitellogenin (VTG), the other oestrogen-dependent yolk precursor,
have reported comparable levels of plasma VTG levels at the 2- and 3-egg
stages (Challenger et al.,
2001; Salvante and Williams,
2002). Seed consumption during the 24 h prior to each blood sample
was measured by providing each breeding pair with 30.0 g of seed on the days
the females laid their first and second eggs. Seed was weighed (to the nearest
0.1 g) 24 h later at the 2- and 3-egg stages.
To examine the influence of the high-fat egg-food supplement given to breeding pairs on changes in plasma triacylglyceride levels and VLDL particle diameter distribution during egg production, randomly chosen male zebra finches were weighed and blood samples were taken between 09:00 h and 11:30 h on two separate occasions: as non-breeding individuals on the seed-only diet (seed sample), and during breeding on the egg-food supplemented seed diet (supplemented sample) on the day that their female breeding partners laid their first eggs.
Chicken blood sampling
Blood samples for VLDL particle diameter distribution analysis were
collected from two groups of chickens: immature, non-laying females at 17
weeks of age (N=10) and actively laying females at 29 weeks of age
(N=37). Sampling of the 29-week-old layers coincided with the peak of
laying for the population (i.e. all females were actively laying eggs and the
laying rate for the population was at its peak: 0.9 eggs laid per day). Blood
samples were taken from the brachial vein between 09:00 and 11:00 into
EDTA-coated Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ, USA). Plasma
samples were isolated by centrifugation at 2200 g and were
refrigerated (4°C) pending VLDL particle diameter distribution
analysis.
Triacylglyceride assay
Circulating concentrations of triacylglyceride in zebra finches were
measured enzymatically as an index of total plasma VLDL (i.e. generic VLDL and
VLDLy; Triglyceride E kit; Wako Chemicals, Richmond, BC, Canada; Serum
Triglyceride Determination Kit, Sigma-Aldrich Canada) using the method
developed for domestic fowl (Mitchell and
Carlisle, 1991) and validated for passerines
(Williams and Christians,
1997; Williams and Martyniuk,
2000; Challenger et al.,
2001). Intra- and inter-assay coefficients of variation were 1.85%
(N=6 replicates) and 4.79% (N=13 assay plates),
respectively, using a 19-week hen plasma pool. All assays were run using
96-well microplates and were measured at 540 nm using a Biotek 340i microplate
reader.
VLDL particle diameter distribution assay
Whole plasma contains a variety of different lipoprotein classes, e.g.
VLDL, low density lipoprotein (LDL), high density lipoprotein (HDL).
Therefore, plasma VLDL was isolated as the d<1.020 g
ml–1 fraction of plasma from zebra finches and chickens. The
volume of each zebra finch plasma sample (approximately 100 µl) was
measured and transferred into Beckman Ultra-Clear ultracentrifuge tubes
(13x64 mm, #344088; Beckman Coulter, Fullerton, CA, USA), and NaCl
density solution (d=1.0063; equivalent salt density of undiluted
plasma) was added until a final volume of 1 ml was reached. Alternatively, a
sub-sample (1 ml) from each chicken plasma sample was transferred into
ultracentrifuge tubes. NaCl–NaBr density solution (5 ml;
d=1.0255) was then added to each tube. A blank sample was prepared by
combining NaCl density solution (1 ml; d=1.0063) with NaCl–NaBr
density solution (5 ml; d=1.0255) in an ultracentrifuge tube. The
samples were loaded into a Beckman 50.4 fixed-angle rotor and centrifuged at
148 600 g for 18 h at 14°C in a Beckman L8-70M
ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). Following
centrifugation, the supernatant containing the VLDL portion of the plasma was
isolated from each tube by aspiration with a narrow-bore pipette and
refrigerated (at 4°C) until analysis for VLDL particle diameter
distribution. VLDL particle diameter distribution was measured by dynamic
laser light scattering using a UPA 250 and 7.02 analysis software (Microtrac,
Montgomery, PA, USA) (Walzem et al.,
1994; Véniant et al.,
2000). This technique utilized the Doppler effect as the basis for
diameter distribution determinations by recording light scattering from a
directed laser diode as it passed through the lipoprotein particles. The
magnitude of Doppler-shifting of light scatter that occurs due to the Brownian
motion of the particles was measured as it is proportional to particle
velocity, which is in turn a function of particle diameter, fluid temperature
and fluid viscosity. As both temperature and viscosity were kept constant, the
difference in particle velocity was solely dependent on particle diameter.
Sample measurements were made by placement of the flexible probe-tip into the
sample and activation of the laser diode (
=780 mm laser beam). Light
scattering from the lipoprotein particles was recorded for 3 min for the blank
solution, and 5 min in triplicate for each VLDL sample. The probe was washed
with distilled water and dried between samples.
Data analysis
VLDL particle diameter distribution measurements
To determine the range of VLDL particle diameters involved in egg
production in passerine songbirds, we characterized changes in VLDL particle
diameter distribution in zebra finches in comparison to chickens by
calculating the proportion of VLDL particles that fell within three potential
VLDLy particle diameter ranges: (1) a range based on chicken values (hereafter
referred to as the cVLDLy range), (2) a range based on the proposed sieving
properties of the avian ovary (hereafter referred to as the sVLDLy range), and
(3) a range based on zebra finch values (hereafter referred to as the zVLDLy
range). Walzem calculated VLDLy particle diameter range for laying chickens,
the cVLDLy range, using the regression of the percentage of VLDL particles
within each VLDL particle diameter class against subsequent laying rate, the
most common measure of reproductive effort used for domestic fowl
(Walzem, 1996). Chickens lay
continuously for extended periods. Therefore, laying females were repeatedly
sampled at various times throughout the laying period, and these repeated
measures were incorporated into the VLDLy calculations
(Walzem, 1996). Each of the
resulting correlation coefficients (r) was presented graphically as
y-values for each particle diameter class. The different VLDL
particle diameter classes vary in their ability to support continuous egg
production, and the diameter classes with positive relationships (i.e.
positive correlation coefficients) with laying rate were assumed to have a
better ability to support egg production and were therefore selected to make
up the cVLDLy particle diameter range [21.5–51.1 nm for laying chickens;
Fig. 1A; fig. 6 in Walzem
(Walzem, 1996)]. By contrast,
the sVLDLy range was based on the observation in domestic fowl that the only
VLDL particles observed distal to the granulosa basal lamina of the ovary
during yolk formation, and thus able to reach the plasma membrane of the
enlarging oocyte of the developing ovarian follicles, ranged from 25 to 44 nm
in diameter (Perry and Gilbert,
1979; Griffin and Perry,
1985; Griffin and Hermier,
1988). These studies suggested that pores in the granulosa basal
lamina act as selective sieves, allowing only VLDL particles of certain
diameters to filter into the ovary (Perry
and Gilbert, 1979; Griffin and
Perry, 1985; Griffin and
Hermier, 1988). Finally, the zVLDLy range (10.7–30.4 nm) was
calculated similarly to the cVLDLy range described above with the following
exceptions. Firstly, because zebra finches lay discrete clutches (5–7
eggs), laying females were only blood sampled once, on the day their first
eggs were laid. Therefore, only one set of VLDL particle diameter and
reproductive output data was used per bird (cf. the repeated measures of VLDL
particle diameter and reproductive performance incorporated into the chicken
VLDLy analysis because of their continuous laying). Secondly, because laying
rate is not generally informative in zebra finches (i.e. there is virtually no
variation in laying rate because the majority of females lay one egg per day
without skipping a day until the clutch is complete), we used body
mass-corrected mean egg mass as a measure of reproductive performance in zebra
finches (Fig. 1B). Mean egg
mass varies markedly between individual female zebra finches
(Williams, 1996;
Salvante and Williams, 2002),
but is highly repeatable within individual females between laying bouts
(Williams, 1996), suggesting
that mean egg mass is a distinct phenotypic trait of laying zebra finches.
There is also evidence that egg size reflects a female's `egg laying ability'
or `performance'; females that lay large eggs are more capable of laying
extended clutches in response to egg removal than females that lay small eggs
(Williams and Miller, 2003).
VLDLy particle diameter ranges based on other measures of reproductive
performance in zebra finches (e.g. clutch size, clutch mass) were also
determined but were not used because they encompassed a majority of the VLDL
particle diameter classes (i.e. 30 to >200 nm), making them inconsistent
with other potential VLDLy diameter range estimates. Finally, the modal and
median particle diameter and the range (i.e. width) of each distribution, in
nanometres, and the proportion of very small (<30 nm) and large (>51 nm)
VLDL particles were also determined.
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General statistics
All statistical analyses were performed using SAS
(SAS Institute, 1999). All
percentage data (e.g. percentage of VLDL particles within the various VLDLy
diameter ranges) were arc–sin transformed prior to analysis, however,
non-transformed percentages were used for graphical purposes. Non-normal
variables, as assessed by the Shapiro–Wilk test for normality
(Zar, 1996), were normalized
through log10 transformation (although some non-transformed values
were used for graphical purposes). For intra-specific comparisons of the VLDL
particle diameter distributions of laying and non-laying females,
t-tests were used. When the analyses included variables that were
still not normally distributed after log-transformation (e.g. plasma
triacylglyceride and VLDL particle diameter distribution range of zebra
finches, and modal and median VLDL particle diameter and VLDL particle
diameter distribution range of chickens), non-parametric Wilcoxon rank-sum
tests were performed. The influence of fasting and egg-food supplementation on
VLDL particle diameter distribution was assessed using repeated-measures ANOVA
or ANCOVA (with female body mass as a covariate). If normality of distribution
was achieved following data transformation, then the data were analyzed using
a mixed model, repeated measures ANOVA or ANCOVA with fed–fasted state
for the fasting study or diet for the egg-food supplementation study as the
fixed, repeated factor, and individual bird as a random factor (PROC MIXED)
(SAS Institute, 1999). By
contrast, variables that were still not normally distributed following data
transformation were analyzed using the non-parametric Friedman's test for
treatment differences in a randomized complete block design with individual
birds as blocks that received both treatments (i.e. fed and fasted states or
seed and egg-food supplemented seed diets) in a randomized order (PROC FREQ)
(SAS Institute, 1999). All
values are given as means ± s.e.m., all tests are two-tailed, and the
overall significance level is P<0.05 unless otherwise stated.
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Plasma triacylglyceride and VLDL particle diameter distribution in non-laying and egg-laying zebra finches
Whereas laying and non-laying female zebra finches did not differ in body
mass at the time of blood sampling (P>0.2;
Table 1), laying zebra finches
had higher plasma triacylglyceride levels than non-laying females (Wilcoxon
rank-sum test: Z=–4.008, P<0.0005;
Table 1). By contrast to the
results from laying chickens (see Introduction), non-laying zebra finches had
VLDL particle diameter distributions (Fig.
4A) that were narrow (177 nm wide, cf. 233 nm in laying zebra
finches; Z=–1.980, P<0.05;
Table 1) and peaked at very
small particle diameters (over 55% of particles had diameters smaller than 30
nm, cf. less than 30% in laying zebra finches; t=5.867, d.f.=61.0,
P>0.0001; Table 1)
and contained few large particles (less than 10% of particles had diameters
larger than 51 nm, cf. almost 20% in laying zebra finches; t=3.947,
d.f.=59.2, P<0.0005; Table
1). Furthermore, non-laying zebra finches also had smaller modal
(t=4.405, d.f.=61.0, P<0.0001) and median
(t=5.332, d.f.=61.0, P<0.0001) VLDL particle diameters
than laying females (Table 1).
Therefore, in comparison, laying zebra finches had VLDL particle diameter
distributions that were shifted towards larger VLDL particle diameters
compared to non-laying females (Fig.
4A). Although there was considerable overlap between the diameter
distributions of laying and non-laying zebra finches, laying females still had
greater proportions of VLDL particles within the cVLDLy (t=2.866,
d.f.=30.2, P<0.05) and sVLDLy ranges (t=3.058, d.f.=31.0,
P<0.005; grey shading in Fig.
4A) than non-laying females
(Table 1). However, laying
zebra finches had fewer VLDL particles within the zVLDLy range than non-laying
birds (t=4.581, d.f.=61.0, P<0.0001;
Table 1).
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VLDL particle diameter distribution in non-laying and egg-laying chickens
Non-laying chickens were consistently different from laying chickens in all
measures of VLDL particle diameter distribution. On average, VLDL particle
diameter distributions of laying chickens at 29-weeks of age were narrow, and
peaked at small particle diameters (Fig.
4B), while non-laying chickens possessed wider, less peaked
distributions (Fig. 4B) (range:
Z=4.816, P<0.0001;
Table 1). Laying chickens had a
larger proportion of VLDL particles that fell within the cVLDLy range
(t=9.542, d.f.=10.3, P<0.0001; grey shading in
Fig. 4B) and sVLDLy range
(t=8.909, d.f.=10.3, P<0.0001) than non-laying chickens
(Table 1). Unlike in zebra
finches, non-laying chickens had larger modal (Z=3.818,
P<0.0001) and median (Z=4.797, P<0.0001)
particle diameters, fewer smaller VLDL particles (<30 nm in diameter;
t=25.990, d.f.=43.2, P<0.0001) and more large VLDL
particles (>51 nm in diameter; t=11.314, d.f.=9.1,
P<0.0001) than laying chickens
(Table 1).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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These findings suggest that these factors did not influence the VLDL particle diameter distribution parameters measured.
Estimating VLDLy particle diameter range in zebra finches
Previous studies on egg production in domestic fowl have found that VLDL
particles of different diameters vary in the extent to which they contribute
to yolk formation, and consequently in their ability to support egg production
(Perry and Gilbert, 1979;
Griffin and Perry, 1985;
Griffin and Hermier, 1988;
Walzem et al., 1994;
Walzem, 1996;
Walzem et al., 1999). Basing
VLDLy particle diameter range on the proposed `sieving' properties of the
ovarian granulosa basal laminae of laying chickens and turkeys provided a
comparable estimate of VLDLy particle diameter for zebra finches and chickens,
as the proportion of VLDL particles from non-laying females that fell within
this range was minimal in both species (38% in zebra finches and 19% in
chickens). The majority of VLDL particles of laying chickens (61%) and half of
the particles of laying zebra finches fell within the sVLDLy range. This
suggests that similarities exist in the sieving properties of the ovaries of
different species of birds. Future studies are required to compare the
composition, structure and sieving properties of the ovarian granulosa basal
laminae of domesticated and non-domesticated birds to determine whether
ovarian sieving of VLDL particles influences selection acting on VLDL particle
diameter.
Basing the VLDLy range on Walzem's original correlation method
(Walzem, 1996) also resulted
in comparable estimates of VLDLy particle diameter for zebra finches and
chickens, as the majority of VLDL particles from laying females fell within
the cVLDLy range (63% in zebra finches; 70% in chickens). However, the
majority of VLDL particles of non-laying zebra finches also fell within this
range (51%, compared with only 25% of VLDL particles of non-laying chickens).
Our modified correlation method based on data from laying zebra finches
resulted in a diameter range that encompassed a majority of VLDL particles
from non-laying zebra finches (59%, cf. 25% of VLDL particles from non-laying
chickens within the cVLDLy range). Moreover, the zVLDLy range encompassed only
40% of VLDL particles from laying females (cf. 70% of laying chicken VLDL
particles within the cVLDLy range). The discrepancies between the proportion
of VLDL particles that fell within the cVLDLy in chickens and the zVLDLy in
zebra finches may be due to differences in the way the ranges were calculated.
Many of the VLDL particle diameter classes that were positively associated
with laying rate in chickens, and therefore made up the cVLDLy range, had
statistically significant correlation coefficients (P<0.05 for
r-values greater than 0.444)
(Walzem, 1996). By contrast,
all of the VLDL particle diameter classes that were positively correlated with
residual mean egg mass in zebra finches, and therefore made up the zVLDLy
range, did not have statistically significant correlation coefficients
(P>0.1 in all cases). Consequently, the zVLDLy range appears to be
the least reliable estimate of VLDLy particle diameter range in zebra
finches.
Metabolic shifts in VLDL particle diameter distribution
As predicted, female zebra finches exhibited less dramatic shifts in lipid
metabolism during egg production. However, this was mainly due to the
unexpected finding that the majority of VLDL particles of non-laying zebra
finches were very small in diameter (57% of particles had diameters less than
30 nm). Consequently, the diameter distributions of laying zebra finches
actually shifted towards larger VLDL particles compared to the distributions
of non-laying females. Furthermore, the diameter distributions of both laying
and non-laying zebra finches peaked at small VLDL particles, and therefore
overlapped considerably. Similar results have been reported for comparisons
between growing (i.e. immature) and egg-producing Tsaiya ducks, Anas
platyrhynchos domestica (Lien et al.,
2005). When provided with ad libitum access to food,
domesticated Tsaiya ducks had VLDL particle diameter distributions, as
assessed by transmission electron microscopy, that included more larger
particles (range: 50–75 nm) and had larger mean VLDL particle diameters
during egg production at 30 weeks of age (61.57±1.98 nm) than while
actively growing at 12 weeks of age (range: 35–60 nm; mean diameter:
47.67±2.37 nm) (Lien et al.,
2005). This is in stark contrast to the data from chickens,
wherein less than 10% of the VLDL particles measured at the peak of egg laying
had diameters larger than 51 nm (cf. nearly 60% of particles in non-laying
chickens), resulting in very little overlap between the VLDL distributions of
laying and non-laying chickens.
Laying zebra finches, like laying chickens, had higher circulating triacylglyceride levels and more particles within the sVLDLy and cVLDLy ranges than non-laying females, despite the fact that laying zebra finches had fewer very small VLDL particles, more large VLDL particles, and wider diameter distributions than non-laying females. Furthermore, the VLDL particle diameter distributions of zebra finches and chickens shifted towards similar modal and median VLDL particle diameters during egg production. These results suggest that, regardless of the direction that VLDL particle diameter distributions have to shift, specific changes in lipid metabolism (e.g. increased lipid production and maintenance of a large proportion of small VLDL particles of specific diameters) may be essential for egg production in both domesticated and non-domesticated birds. However, data on reproductive status and VLDL particle diameter distribution from more domesticated and free-living avian species are required to confirm the relationship between changes in lipid metabolism and avian egg production.
The differences in VLDL particle diameter distribution between non-laying
chickens and zebra finches observed in this study may be due to differences in
rates of lipid turnover due to variation in metabolic rate. Based on
allometric scaling of metabolic rate (for reviews, see
Calder, 1981;
Taylor, 1987), smaller
passerine songbirds have higher mass-specific metabolic rates than larger
chickens (Lasiewski and Dawson,
1967; Reynolds and Lee,
1996; McKechnie and Wolf,
2004). Consequently, passerine songbirds also have higher rates of
lipid turnover. When VLDL particles undergo lipoprotein lipase-mediated
metabolism, triacylglycerol is removed by hydrolysis, and surface lipids and
apolipoproteins (e.g. apo-A, apo-C) are transferred to other lipoprotein
particles (e.g. high density lipoproteins) (for reviews, see
Eisenberg, 1986;
Walzem, 1996). This results in
a decrease in VLDL particle size and an increase in particle density, and the
eventual conversion to intermediate density lipoproteins (IDL) and then low
density lipoproteins (for reviews, see
Eisenberg, 1986;
Walzem, 1996). Hermier et al.
(Hermier et al., 1985)
reported that IDL particles from immature chickens had an average diameter of
20.0 nm. Therefore, the abundance of very small VLDL particles observed in
non-laying zebra finches (57% under 30 nm in diameter, cf. <1% in
non-laying chickens) may have actually been IDL particles resulting from the
rapid metabolism of larger VLDL particles.
Differences in the environmental conditions that chickens and zebra finches
are exposed to during egg production may also influence the proportion of
large VLDL particles in circulation. Domestic chickens are generally housed
under conditions that promote optimal egg production, e.g. light-controlled
facilities, a diet regime tailored for egg production, vaccinations against
disease, and husbandry practices that eliminate parasites
(Etches, 1996). Consequently,
they are capable of meeting their own metabolic needs via hydrolysis of the
small VLDLy particles (Bacon and Musser,
1977; Bacon et al.,
1978; Bacon, 1981)
and possibly renal generic VLDL (Walzem et
al., 1999) despite the increased resistance of VLDLy to hydrolysis
by lipoprotein lipase (Bacon et al.,
1978; Bacon, 1981;
Griffin et al., 1982;
Hermier et al., 1989;
Schneider et al., 1990;
Walzem et al., 1994;
Walzem, 1996). Alternatively,
previous studies on laying chickens have suggested that individual hepatocytes
may vary in their functional capacity to initiate apoVLDL-II, and thus VLDLy,
synthesis in response to elevated levels of oestrogen
(Lin and Chan, 1981;
Lin et al., 1986).
Consequently, it is possible that the livers of laying chickens continue to
make small amounts of larger, generic VLDL in sufficient quantities to meet
the laying females' energetic requirements. The rapid and continuous
metabolism of such generic VLDL by highly productive layers would leave
minimal concentrations in circulation relative to VLDLy, making their ready
detection in laying domestic fowl challenging. By contrast, reproduction in
free-living, non-domesticated birds is generally timed to ensure that the
period of chick rearing coincides with the period of peak food abundance
(Perrins, 1970). Therefore,
egg production in these birds often occurs during periods of lower food
availability and unpredictable environmental conditions earlier in the
breeding season (Williams,
1998). Maintenance of large VLDL particles in circulation may act
to buffer the fluctuating, environmentally dependent energetic demands of
these laying birds. The extent to which hepatic VLDLy and renal generic VLDL
contribute to the levels of utilizable VLDL present in laying,
non-gallinaceous birds, and in particular, in free-living birds faced with far
less predictable conditions, remains unknown. Future studies are needed to
assess whether the larger VLDL particles observed in laying zebra finches
contain apoVLDL-II, in order to determine whether these particles are generic
or yolk-targeted VLDL.
In contrast to domesticated birds that have undergone directional selection
for specific traits, such as continuous egg production or rapid growth, the
selective pressures on laying zebra finches, and on non-domesticated birds in
general, are generally focused on maintaining traits that maximize the
trade-off between current reproductive effort and future fecundity and
survival (Williams, 1966;
Stearns, 1992;
Bernardo, 1996). The increased
LPL-resistance of VLDLy may result in selection for the maintenance of larger,
potentially generic, VLDL particles in non-domesticated birds during egg
production, as observed in zebra finches in this study (19%) and Tsaiya ducks
(100%) (Lien et al.,
2005), to ensure that laying females have an ample supply of VLDL
that can be metabolized in case their own energetic demands increase during
egg production due to changes in environmental conditions. Data on VLDL
particle diameter distribution during egg production in many more free-living
avian species, including other gallinaceous and passerine birds, are needed to
determine whether the differences between chickens and zebra finches observed
in this study are, in fact, due to differences in selective pressures on these
birds, or to phylogenetic differences that are unrelated to inter-specific
differences in adaptations to egg production.
In addition to egg production, reproduction in non-domesticated species
generally involves broody behaviour, i.e. incubation and post-hatching
parental care (e.g. provisioning and brooding of young). This is in contrast
to many breeds of domesticated chickens, whose reproductive activity is
limited to egg production as a result of commercial practices (e.g.
photoperiod manipulation, egg removal) and decreases in broodiness,
hatchability and fertility as a consequence of selection for increased egg
production (Emmerson et al.,
1991; Nestor et al.,
1996; Sewalem et al.,
1998) (reviewed by Romanov,
2001). Therefore, given that non-domesticated, laying females must
ensure that they have adequate resources to perform post-laying parental
behaviours, they may limit lipid allocation to current egg production in
exchange for allocating more energy towards self-maintenance (i.e. maintaining
larger VLDL particles) to enhance their chances for survival through the
current reproductive period and beyond, thus maximizing current and
potentially future reproductive effort. Further studies are needed that will
assess the relationships between variation in VLDL particle diameter
distribution during egg production in free-living avian species and both
current and future reproductive success, and maternal survival and
longevity.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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