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A
Method for Determining the Activity
State of Hai r Follicles
Al lan
J
N i xon
New Zealand Pastoral Agriculture Research Institute Ruakura
Agricultural Centre Private Bag 3
123
Hamilton New Zealand
ABSTRACT
A
histological method is described
for determining the proportion of
growing
h i r
follicles
h kin
samples.
A
variation of the Sacpic
s ta in ing method, modified for bulk processing.
produces high contrast
staining
of the principal
tissue
types
present in skin
In
particular,
the
inner root sheath is accentuated, facilitating de-
tection of active follicles. Skin preparations from
a range of species are used to illustrate structural
characteristics of follicles
viewed
in cross section
at variousstages of the hair cycle and to establish
criteria for classification of the sta te of activity
of follicles. The hair cycle may be divided into
quiescent
and
active states a t the points of rapid
transition (early pronanagen and mid catagen).
Data from repeated skin biopsies from ferretsand
goats are also used to demonstrate quantitative
estim ation of follicle activity, change
in
com-
pound follicle size, and the relationship between
follicle
type
and fiber medullation.
Key words: ferret, fiber, goat, hair cycle, hair
follicle, pelage. Sacpic,
skin
he sta inin g of sections of skin with
T ndigo carmine, picric acid, and
saf-
ranin or basic fuchsin wa s
first
described
by Auber (1952). Variations on this
method, known as the Sacpic method,
have been applied for discriminating
quiescent hair follicles from those growing
a fiber. Counts of quiescent and active
follicles have been used to describe pelage
changes, and more recently, experimental
effects on fiber growth (Ryder 1960. 1976,
Nixon et al.
1991
b). Quantitative studies
involving large numb ers of sect ions an d
unique samples require a reliable an d sim-
ple staining procedure: however, pub-
1052-0295/93/6806-3 16/ 3.00/0
BlOTECHNlC & HISTOCHEMISTRY
Copyright
Q 1993 by
Williams & Wilkins
olume 68
Number
6
lished variations of t he Sacpic method
have critical regressive staining and dif-
ferentiation steps, and recommended
staining times vary widely (Ryder and Ste-
phenson
1968,
Maddocks and Jackson
1988).
Furthermore, there
is
no general
explanation of th e criteria used for assess-
ment of follicle activity. Although the most
suitable criteria differ among species and
breeds, they have
in
common changes in
follicle an d f iber morphology which occur
in the course of the hair cycle (Chase
1954). A guide to identification of these
features in transverse sections is needed.
The purpose of t hi s report is to describe
a modified Sacpic method for trea tment of
slides en
mass ,
an d to outline the general
procedure for objective determination of
hair follicle activity an d follicle numbers
from skin sections stained by this method.
Applications of the method are demon-
strated in two dissimilar species.
MATERIALS AN D METHODS
Tissue Collection and Processing
Samples of sk in were collected to compare
staining in
a
range of mammal ian species
an d breeds. Mid-side biopsy samples were
taken from Cashmere goats
Capra hir-
cus) ,Merino an d Romney sheep (Ou i s
a r
ies).and brushtail possums Trichosurus
uulpecula).Skin sa mples from rabbit Or-
yctolagus cuniculus), mouse Musmus-
culus)
an d chimpanzee
Pan
troglodytes)
were collected post mortem.
To
demonstrate some applications of fol-
licle counts, repeated skin biopsies were
316
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Hair Follicle
Act iv i t y
31
7
taken from two widely differing commer-
cially farmed spec ies over times of sea-
sonal pelage change. Six 16-week-old fer -
rets were sampled at 28-day intervals
starting
3
March 198 9, before, during and
after their moult from juvenile to adult
winter pelage. Skin samples were collected
from the mid-lateral trun k of anesthe tized
ferrets using
6
mm diameter disposable
biopsy punches (Stiefel Laboratories,
Wooburn Green, England). Ski n sam ples
8-10 mm diameter were obtained on 3
August and 1 9October 1989 from six non-
breeding female Angora goats by t he sni p
biopsy method using dog-tooth forceps
and curved scissors. These dates corre-
spond to times of w inter minim um an d
spring peak in follicle activity (Nixon et al.
1991
a).
Samples were fixed in phosphate buf-
fered formalin for 48 hr or longer, or in
Bouin’s fluid for 6-18 hr , then processed
overnight through graded concentrations
of ethanol and toluene to paraffin wax.
Thinne r skin samples (ferret, opossum,
rabbit and mouse) were laid fla t on
a
ma-
nila card to maintain their shape during
fixation and processing. Transverse and
longitudinal 7-8 pm sections of hair folli-
cles were cut on
a
rotary microtome.
Stain Preparat ion
The sta ins were prepared
as
follows.
Celestine blue
celestine blue
B
2.5 g
(C.I. 51050)
ferric ammonium sulfa te, 500 ml
5
aqueous solution
boil for 3 min ; cool and 70 ml
filter, add glycerol
Safranin
safranin
(C.I.
50240)-
6 g
50
ethanol 300 ml
mix thoroughly an d
filter before use
Picric acidlethano1
absolute ethanol 300 ml
picric acid, saturated 5 ml
alcoholic solution
Picro-indigo
carmine
indigo carmine (C.I. 73 0 15 )
1 g
picric acid, satur ated 300 ml
Formulations of other stan dard reagents
aqueous solution
were
as
described by Humason (1979).
Stain ing Procedure
The staining was carried out as follows.
1) dewax and rehydrate
2) mordant in celestine blue for 5 min
3)
rinse in tap water
4) stain
in Gill’s hematoxylin for 5 min
5)
rinse in tap water
6)
blue in Scott’s ta p water for 2-5 min
7)
rinse in tap water
8) stain in 2 safranin for 5 min
9)
rinse in
70
ethanol followed by
a
rinse in 95 ethanol
10) differentiate in absolute picric acid/
ethanol for
3
min
11) rinse in 95 ethanol, 70 ethanol,
then ta p water
12) stain in picro indigo carmine for 1 min
13) rinse in tap water
14) dehydrate, clear and moun t.
Assessment of f o i c eAct iv i ty
Serial longitudinal and transverse sec-
tions of hair follicles were prepared to elu-
cidate follicle structure
in
active and
quiescent state s. Th e structu re of follicles
in transverse section was compared
among various depths within t he skin an d
at different stages of th e ha ir cycle. Fea-
tur es of t he follicle and fiber examined
were: melan in pigmentation , medullation.
th e b rush (or “club”)end of a fully grown
fiber, bru sh fiber shedding, production of
an inner root sheath
(IRS),
flatt ening of
outer root sheath (ORS) cells, an d forma-
tion of secondary hair germ, epithelial
st rand or follicle bulb. Descriptions of fol-
licle morphology are given by Chase
(1
954),
Straile et al.
(196
1)an d Montagna
and Parakkal (1 974). Specific characte r-
istics of a nim al fibers ar e described by
Brunner an d Coman (1
974).
Presence or absence of these structur al
feat ures was noted. T he most suitable of
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318
Biotechnic
&
Histochemistry
these criteria for determining follicle ac-
tivity were es tablished for each species by
referring to longitudinal sections and to
the struc ture of the full-length fiber.
Fol l i c le Count ing
Active and quiescent follicles were
counted in the sam ples of ferret an d An-
gora goat skin . Approximately 50 sections,
7 km th ick, were cut per sample , but only
every alt ern ate serie s of five sec tions were
stained . Follicles viewed in tra nsv ers e sec-
tion were drawn free-hand on paper. For
both species, five to ten complete follicle
groups were recorded. The me an number
of follicles SEM counted per sample was
237
31 for ferrets and 233 26 for
goats. Additional primary follicles were
counted in goa ts where possible to improve
the accuracy of estimate s of primary fol-
licle activity. Original primary follicles
were identified by the ir position within t he
group, accessory glands a nd arrector pili
muscles. Each follicle was classified ac-
cording to th e pha se of the hai r cycle.
Numbers of active and quiescent follicles
of each follicle type were then counted
from th e drawings.
Raw counts from ferret skin were used
to calculate percent activity and number
of follicles per compound follicle (follicle
bundle). In Angora goats, percent activity
was determined for primary and second-
ary follicles separately. In addition, the
presence of medullated fibe rs was noted
to estimate the percent medullation in
each follicle type.
RESULTS
AND DISCUSSION
Stain ing
Similar staining resu lts a re obtained with
skin from members of phylogenetically di-
verse species. Tissue types a re clearly de-
fined in a trichrome-like man ner, and con-
sistent sta in intensities a re produced with
the modified Sacpic method using either
fixative. Results are: nuclei, blue-black:
kera tin, yellow; collagen, blue: in ner root
she ath , bright red; outer border of brush-
ends, orange: outer root sheath, pale
green; smooth muscle and erythrocytes,
green . [Figs 1-3.)
The Sacpic stain is well suited for visual
as sessmen t of follicle activity because it
accentuates the inner root sheath which
accompanies a growing fiber. With hema-
toxylin and eosin staining, the inner root
sheath is eosinophilic in some cases and
basophilic in others. Safranin used with
picric acid in the procedure presented here
stains keratinized tissues. including cor-
nifi'ed epidermis, fiber medulla, but not
fully hardened fiber. Gurr (1962) sug-
gested th at thi s effect is due to formation
of a n insoluble safran in-p icra te complex.
The Sacpic method presented here dif-
fers from other variations in that Weig-
er t's iron hematoxylin is replaced by Gil l s
hematoxylin. An iron mordant, provided
by the iron alum in the celestine blue mix-
ture. enables nuclear staining to with-
stand the subsequent acid treatment, and
no acid alcohol destaining step is required.
It ha s been noted (Auber 1952. Ryder and
Stephenson 1968) that safranin may be
replaced as an inner root sheath stain
by 1 basic fuchsin in
50
ethanol:
however, inconsisten t results have been
obtained with different st ocks of ba-
sic fuchsin, which is a variable mixture
of triaminotriphenylmethane homologs
(Sehlinger and Nettleton 1987).Both
1
basic fuchs in (Gurr, London) and 1 par-
arosanilin (Sigma, St. Louis,
M O )
give a
more crimson color, but similar distribu-
tion of sta ining to th at of sa franin . Stain-
ing time for any of th ese three red sta ins
may be reduced to
as
short
as
2 min with-
out markedly affec ting the result, provided
adequate picric ethano l treatment is main-
tained. Stain ing times throughout the pro-
cedure are optimized to give uniform
differentiation and are convenient for
processing successive racks of slides at
5
min intervals. All sta in solutions keep well
an d can be used repeatedly. Picric ethanol
should be changed when it becomes dark
with safranin.
Assessment of Fol l ic le Act iv i ty
Sacpic stained transverse sections are
useful for estimating follicle activity be-
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Hair Foll icle Activity
319
Fig. 1. Quiescent (telogen) follicles i n rabbit skin. Sacpic
staining of transverse sections shows orange brush end in a
large follicle and dermal pap illa cells (arrows) below
secondary hair germ in smaller follicles. Bar 100 pm.
Fig. 2. Active (anagen) follicles in Angora goat skin. Cross
sections through the distal to medial regions of the follicles,
stained by the Sacpic method. The bulb, or proliferative zone
(p), consists of germinal epithelium enclosing the dermal
papilla. The elongation zone (e) contains undifferen tiated
keratinocytes. In the keratogenous zone (k) outer root sheath
cells have a large weakly staining cytoplasm, Henle’s layer
stains red, Huxley’s layer is green, and cortical cells are red
or brown. I n the consolidation zone (c), the outer root sheath
is pale green, the inner root sheath red, and the cortex
yellow. Bar
= 100
pm.
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Fig. 3. Transverse sections of Angora goat hair follicles
stained with Sacpic. A ) Section at the level of the sebaceous
gland duct,
180
pm from the epidermis.
6)
Same follicles sectioned at 740 pm from the epidermis. Follicle
1 is a primary
follicle associated with arrector pili muscle ap), sebaceous
gland sb) and sweat gland sw). All other follicles are
secondary follicles. Follicles
2 ,
3 and 7 possess a hair germ and are quiescent in telogen).
Follicles 1 and 6 have a brush
end which is usually present in quiescent follicles. Follicles 8
and 10 contain bo th the brush end of an old fiber and the
tip of a new growing fiber indicated by arrows); therefore,
these are active in proanagen). Follicles 4, 5 and 9 have a
yellow staining fiber surrounded by a red inner root sheath.
These are active in anagen). Note that follicle activity state
cannot be determined for most follicles from section
A,
since they are observed at the follicle neck. However,
follicle
2 is seen only as a hair germ in section A and is absent from
section B. Bars = 100 pm.
320
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Hair
Fol l ic le Act iv i ty
32 1
cause a suitable number of follicles ca n be
observed simultaneously and the group
struc ture and follicle type information ca n
be obtained readily. An underlying as-
sumption of this method
of
skin analysis
is that the continuous hair cycle c an be
readily divided at two points separating
“active”and “resting” states . If the funda-
mental cha rac ter isti c of follicle activity is
fiber growth, then this class comprises
mostly those follicles in the anagen phas e
of t he hair cycle, together with some in
the brief proanagen and early catagen
phases. Conversely, quiescent follicles are
generally in telogen, although some late
catagen and early proanagen follicles will
also be classified
as
resting because they
are indistinguishable from those in telo-
gen in Sacpic stained cross sections.
Structural characteristics observed in
Sacpic stained follicles ar e summarized in
Table 1. The occurrence of each feature
is
given in hair cycle phases grouped into
active and quiescent cla sses as described
above.
All
of the feat ures examined are
potentially useful as activity criteria , al-
though their applicability varies among
species. In genera l, the key to activity as-
sessment is the ability to recognize these
characte ristics of hair cycle phases in fol-
licle cross sections at the appropriate ski n
depth. Figure 4 furthe r illustrates the re-
lation between hair cycle phase and view
of the follicle in transver se section. The
appearance of the upper follicle neck (Fig.
3A, Fig. 4. level A is similar in active and
quiescent follicles. Most useful informa-
tion c an be obtained from sections a t the
level conta ining th e lower lobe of t he se -
baceous gland [Fig. 4, level B).
In follicles with a growing fiber sec-
tioned
at
this level,
a
distinct red stained
inner root sheath surrounds the fiber.
This feature provides a common and reli-
able indicator of ac tivity (Figs. 3 and
5).
Distally at t he keratogenous zone, the n u-
cleated cortical cells of active follicles
stain with safranin and/or hematoxylin
(Fig.
2).
The d iameter of the follicle in-
creases in the region of t he bulb and the
dermal papilla is surrounded by undiffer-
entiated germinal epithelial cells which
sta in only with hematoxylin.
Features of th e fiber can also provide
useful activity criteria depending on spe-
cies , breed or color form (Table 1). In pig-
mented animals, a high concentration of
melanin granules
is
present in growing
fibers. In rodents, rabbits, possums and
ferrets, all fiber s are medullated over most
of their length and the medulla is visible
in th e follicle while the fiber
is
growing. In
such fur-bearing species, pigmentation or
medullation a re therefore convenient in-
dicators of follicle activity (Fig. 5); ow-
ever, in nonmedullated white colored ani-
mals, such
as
sheep and Angora goats, the
inne r root sheath remai ns the main crite-
rion of fiber growth in sections a t the se-
baceous gland level.
Table
1.
Structural Features of Hair Follicles through the Hair Cycle
used as Activ ity Crit eria
Active Quiescent
Species in which Feature s
Applicable
l te
elogen
eature Early
Proanagen Anagen
Catagen Catagen
Fiber attributes
A A A All colored mammals
A A A
Ferret, mouse, possum, rabbit
P A’ A Ferret, mouse, possum, rabbit
A * P’
P
A l l
species except mouse
A A P/A
Goat, sheep
Fiber pigment
P/A’ P/A
Medulla
A P/A
Cuticle scale
P/A
Brush end
P
P/A
Shed fiber
P/A A
P
Follicle attributes
Distinct I R S
P/A‘ P P/A*
A* A Al l
species
Follicle bulb
P/A’ P P/A‘
A A A l l species
Flatened
ORS
cells
P/A*
A A‘
P/A‘
P
Al l species
Dormant hair germ
A’
A A*
P/A’
P
A l l species
Epithelial strand
P/A* A A’ P* P Al l species
’ Let ters deno te whether a fea ture was a lways present P ) o
r a lways absent A ) , o r som et imes present P /A ) .
*
Structure undergoes transi t ion at or c lose to this phase.
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322 Biotechnic & Histochemistry
V i e w
i n
T S
A
B
C
D
UJ
T e l o g e n P r o a n a g e n A n a g e n C a t a g e n
fibre
outer
root
A A
A
sheath
brush
end
hair
germ
B
fibre
tip
D
inner B
root
sheath
C
dermal
~
apilla
germinal
matrix
Fig.
4.
Relationship between hair cycle stage and view of
follicles
in transverse section. Generalized structure of tollicles
at the four main stages of the hair cycle are shown as they
appear
in
longitudinal section. Diagrams of the structures
seen in cross section through each fol licle at planes A to D
are shown below (cf. Figs. 1-3 . Telogen follicles are
considered quiescent. Anagen, proanagen, and early catagen
phases are active.
Quiescent follicles can be identified by
their compact hair germ and dermal pa-
pilla cells
(Fig.
l) . They usually contain
the brush (or club) end of a fully grown
fiber; however, this
is
occasionally shed
leaving
a
root .sheath with
a
collapsed
empty lum en. In some cases, there may be
a n appreciable num ber of follicles in
which the old brush end fiber has shed
before growth of the new one begins, e.g..
in feral goats (Nixon et al. 1991a). The
outer root s he ath cells of quiescent folli-
cles are often columnar and radially or
spirally arranged (Fig. 1) n contra st to the
large cytoplasmic volume and rounded
shape of this cell type during anagen. Care
must be taken to count follicles from sec-
tions above the hair germ level to ensure
inclusion of all shor ter quiescent follicles
(Fig.
3).
Follicles in transition (proanagen and
catagen) are more difficult to classify from
cross sections. Mid to late proanagen fol-
licles (proanagen
111
to V can be recognized
by the presence of
a
fine fiber tip (Nixon
et al. 1993).The new fiber is usually lo-
cated adjacent or distal to an existing
brush end (Figs. 4 and 3). Such follicles
can usually be classified
as
active, al-
though placing them in a separate cate-
gory can provide meaningful information
on initiation of fiber growth (Nixon et al.
1991a). Near the end of the fiber growth
period, medullation and melanogenesis
cease. In species where the se features are
used as the main activity criteria, the pres-
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Hair
Fol l icle Activi ty
3 2 3
Fig.
5
Ferret hair follicle groups A ) before and B) during growth of
winter pelage. Both groups comprise a trio of
compound follicles (bundles). Prior
to
moult, follicles contain brush ends indicating that they are
quiescent. Growing
follicles possess red inner root sheaths and medullated fibers.
Note the increased number of follicles formed by
branching at the ental side of each bundle as fiber growth is
initiated. Bars = 5 0 pm.
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324
Biotechnic & Histochemistry
ence of a n inner root she ath and /or cuticle
scale pattern c an also be used. In this way,
early to mid catagen (catagen I to V) may
be regarded
as
active. The remnants of
inner root sheath may still be observable
in late catagen (catagen VI to VIII), but
fiber elongation has virtually ceased and
a brush end h as formed (Straile et al.
1961).Such follicles can be classified as
quiescent when they a re identified.
In practice, transitiona l follicles ar e en-
countered infrequently due to the rela-
tively short duration of these phase s in
most species. For example, morphologi-
cally distinct proanagen has been de-
scribed as lasting 10-16 days in goats
(Nixon et al. 1993) and approximately 8
days in ferre ts (Saywell an d Nixon 1992),
compared with
a
total growth period of
about
5
months and 60 days in each spe-
cies, respectively. In shorter term studies
involving proanagen and catagen phases,
alternative staining and assessment
methods using longitudinal sections may
be required.
Appl ica t ions of Fol l i c le Count ing
Data from a series of repeated ski n sam-
ples scored using t he crite ria above reflect
pelage growth more directly than obser-
vation of external fibers alone. It
s
possi-
ble to determine the time a nd duration of
fiber growth. For example, autumn fur
growth of juvenile ferrets commenced in
virtually all follicles by 29 March and wa s
completed in
30
of follicles by
26
April
(Table 2). This indicates that the entire
winter coat was grown over a period of
approximately 8 weeks. Furthermore, the
growth of th e new coat involved the for-
mation of new follicles. A 216 ncrease
in the number of follicles per compound
follicle (Fig.
5)
reflected an observed in-
crease in pelage density. Other mammals
Table 2. Changes in Ferret Hair Follicle Activ ity and
Compound Follicle bundle) Size during Autumn Moult
~~~ ~
Follicles perample Follicle
Date Activity ( ) Bundle
March 0.0
0.0 6.50 0.37
29 March
99.8 f 0.2 9.40 f 0.95
26 April
70.4 f
12.7
14.05 0.51
exhibit similar variation in the number of
derived follicles (Rougeot and Thebault
1983, Kondo et
al.
1988, Nixon 1990).
These changes in the follicle population
ar e most readily identified and quantified
from s kin sections.
Follicle counts from Sacpic stained sk in
sections can reveal differences in behav-
ior between developmental follicle types
(Lyne
1966).
In goats and sheep the ratio
of secondary to primary follicle does not
vary in matu re anim als; however, activity
cycles often differ between follicle types.
For example, in Angora goats (Table 31,
primary follicles showed discontinuous
growth; the majority of th is follicle type
were quiescent in late winter. The coarser
fibers produced by this follicle type are
shed and replaced in spring. Activity in
secondary follicles remained high in
spring, suggesting tha t growth of finer f i -
ber in these follicles was continuous or
asynchronous.
The description of medullation (Table
3)
is an example of t he use of skin histology
to relate follicle type to fiber characteris-
tics. These data show that most fibers
growing in primary follicles possess a me-
dulla, but that fiber from secondary folli-
cles is generally nonmedullated. When ac-
tivity is low (i.e., fibe rs ar e not growing),
medullae cannot be observed in the folli-
cles. Therefore, one m ust consider the hair
growth cycle when examining fiber attri-
butes from skin sections.
Table
3.
Angora Goat Hair Follicle Activity and Fiber Medullation in
Winter and Spring
Primary Secondary Primary Secondary
Sample Date Activity Activity Medullation Medullation
( I ( I ( I
W
3
August
34.3 11.6 94.7 3.3 2.8 - 2.8 0.0 f 0.0
19 October 98.4 f 1.0 94.4 3.0 87.9 .1
0.8
f 0.5
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Hair Fol l ic le Act iv i ty
325
The data presented here illustrate the
potential us es of observing fiber growth
within the follicle, but for descriptive stu d-
ies of full activity cycles, more frequent
sampling
is
generally required. Consider-
ation must also be given to source s of var -
iation when establishing the number of
animals a nd follicles to
be
sampled. Skin
histology methods are labor-intensive but
are suited to measuring experimental ef-
fects in small groups of anima ls, particu-
larly in relation to tim ing of onset of f iber
growth.
A C K N O W L E D G M E N T S
Some tissues were provided by Dr.
A.
J .
Pearson and Dr. M. D. Bown. Technical
assi stance wa s provided by Mr. D.
P .
Say-
well an d Mr. M. G. Ashby. T he c ritique of
M r .
B.
J .
Young on
staining
technique was
appreciated.
REFERENCES
Auber.
L.
1952. Observations on the follicles pro-
ducing wool fibres with special referenc e to ker-
atinisation. Tran s. R. SOC.Edinb.
62:
191-254.
Brunner. H . and Coman, B. J. 1974.
IdentiJication
o Mammalian
Ha i r . Inkata Press, Melbourne.
Chase, H. B. 195 4. Growth of hai r. Physiol Rev. 34 :
Gurr, E. 1962.
Staining Animal Tis sue s. Practical
and Theoretical.
Leonard Hill, London. p. 12
Humason, G.
L.
1979.
Animal Tiss ue Techniques.
4th Ed. Freeman, Sa n Francisco. pp. 113, 545.
Kondo.
K. .
Kohno,
K. .
Nishiumi, T., Son-yan, J.,
Shimizu. Y an d Ohsugi. T. 1988. Determina-
tion of hair density In mink
Mustela uison).
Scientifur 13:
15-18.
113-125.
Lyne, A. G. 1 966. The deve lopmen t of hai r follicles.
Aust. J . Sci. 28: 370-377.
Maddocks, L. G. an d Jac kso n, N. 1988.
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