Range for
fathers
Range for
unrelated males
Range for
unrelated females
cent advances In human DNA fingerprinting. In: DNA
fingerprinting: approaches and applications (Burke T,
Doll G, Jeffreys AJ, and Wolff R, eds). Basel, Switzerland: Blrkhauser Verlag; 1-19.
Range for
mothers
Jeffreys AJ, Turner M, and Debenham P, 1991b. The effidency of multilocus DNA fingerprint probes for Indlvtdoalization and establishment of family relationships,
determined from extensive casework. Am J Hum Genet
48:824-840.
Lack D, 1968. Ecological adaptations for breeding In
birds. London: Methuen.
Longmire JL, Ambrose RE, Brown NC, Cade TJ, Maechtle TL, Seegar WS, Ward FP, and White CM, 1991. Use
of sex-linked mlnlsatelllte fragments to investigate genetic differentiation and migration of North American
populations of the peregrine falcon (Falco peregrmus).
In: DNA fingerprinting: approaches and applications
(Burke T, Dolf G, Jeffreys AJ, and Wolff R, eds). Basel,
Switzerland: Birkhauser Verlag; 217-229.
0.1
0.2
0.3
0.4
0.5
06
0.7
0.8
0.1
Proportion of bands shared
0.2
0.3
04
0.5
OS
0.7
0.8
Proportion of bands shared
Figure 2. Band-sharing analysis of south polar slcua DNA fingerprints, (a) The frequency distribution of the
proportion of bands shared between each chick and Its putative father (white bars) and presumably unrelated
males (gray bars). Dashed lines represent the range for each distribution. Below is the frequency distribution of
the proportion of bands shared between the three chicks with one or more unattributed bands and the resident
male, (b) The frequency distribution of the proportion of bands shared between each chick and Its putative mother
(white bars) and presumably unrelated females (gray bars). Dashed lines represent the range for each distribution.
Below Is the frequency distribution of the proportion of bands shared between the three chicks with one or more
unattributed bands and the resident female
father. In addition N9 shows a high level
of band sharing with the female (0.607),
being well within the range for a parent.
Therefore the single unattributable band
in the DNA profile of individual N9 is likely
to represent a mutation. This results in a
detected mutation rate of 0.003 mutations
per band per generation.
The maternity of nestling N10 can be assigned to the resident female, while N13
appears to be unrelated to either of the
resident adults. This case of an offspring
(N13) being unrelated to either resident
adult, is most likely explained by adoption. Spellerberg (1971b) found that adult
south polar skua would accept chicks
from other nests that were placed or had
wandered into their territory, while Young
(1963) observed displaced younger chicks
joining neighboring clutches. In addition,
Young (1963) noted that the movement of
a chick into a new territory usually disturbed the original adult-chick relationship, resulting in some cases in the subsequent loss of the resident chlck(s). This
situation could also result from the misidentification of both the resident adults or
intraspecific brood parasitism (egg dumping). Egg dumping has, however, not been
observed in skuas, despite a long series of
intensive behavioral studies, and is unlikely to have gone totally undetected given
the small but very constant clutch size of
this species. Hence, we suggest that our
2 3 8 The Journal of Heredity 1997.88(3)
study is the first reported instance of
adoption of a chick being detected using
DNA fingerprinting.
From Ecology and Evolution, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. D. M. Lambert Is currently at the
Department of Ecology, Massey University, Palmerston
North, New Zealand. We wish to thank the following
people and organizations: Peter Stevens, Cornellla
Bergmann, Dick Bellamy, Iain McDonald, and Patricia
Stapleton, the New Zealand Antarctic Research Program, and the U.S. Antarctic Program. This work was
supported In part by grants from the Auckland University Research Committee. In addition, the work was
supported by the Foundation for Science and Technology grant on DNA tests for sex in birds to D.M.L
Millar CD, Anthony 1, Lambert DM, Stapleton PM, Bergmann CC, Bellamy AR, and Young EC, 1994. Patterns of
reproductive success determined by DNA fingerprintIng in a communally breeding oceanic bird. Blol J Unn
Soc 52:31-18.
Millar CD, Lambert DM, Bellamy AR, Stapleton PM, and
Young EC, 1992. Sex-specific restriction fragments and
sex ratios revealed by DNA fingerprinting in the brown
skua. J Hered 83:350-355.
Mlyaki CY, Hanotte 0, Wa|ntal A, and Burke T, 1992. Sex
typing of Aratlnga parrots using the human mlnlsatellite probe 33.15. Nucleic Acids Res 19-5235-5236.
Rabenold PP, Piper WH, Decker MD, and Mlnchella DJ,
1991. Polymorphic mlnlsatellite amplified on avlan W
chromosome. Genome 34:489-493.
Reid BE, 1966. The growth and development of the
south polar skua (Catharacta maccormicki). Notornis
13.71-89.
Spellerberg IF, 1971a. Aspects of McCormick skua
breeding biology. Ibis 113:357-363.
Spellerberg IF, 1971b. Breeding behaviour of the McCormlck skua Catharacla maccormicki In Antarctica. Ardea 59:189-230.
Watson GE, 1975. Birds of the Antarctic and subantarctlc. Washington, D.C.: American Geophysical Union.
Young EC, 1963. The breeding behaviour of the south
polar skua Catharacla maccormicki. Ibis 105:203-233.
Received October 18, 1995
Accepted July 29, 1996
Corresponding Editor. Robert Wayne
The Journal ol Heredity 1997:88(3)
References
Alnley DG, Morrell SH, and Wood RC, 1986. South polar
skua breeding colonies In the Ross Sea region, Antarctica. Notornis 33:155-163.
Ainley DG, RIWc CA, and Wood RC, 1990. A demographic study of the south polar skua Catharacta maccormicki at Cape Crozler. J Anlm Ecol 59:1-20.
AlnJey DG, Spear LB, and Wood RC, 1985. Sexual color
and size variation in the south polar skua. Condor 87:
427^28.
Bloom SE, 1974. Current knowledge about the avlan W
chromosome. BloSclence 24:340-345.
Court GS, 1992. Ecology oi breeding and non-breeding
south polar skuas (Catharacta maccormicki) on Ross
Island, Antarctica (PhD dissertation). Dunedln, New
Zealand: University of Otago.
Furness RW, 1987. The skuas. Calton: T 4 AD Poyser.
Jeffreys AJ, Royte NJ, Patel I, Armour JAL, MacLeod A,
Colllck A, Gray IC, Neumann R, Gibbs M, Crosier M, Hill
M, Signer E, and Monckton D, 1991a. Principles and re-
Inhibited Feathering: A New
Dominant Sex-Linked
Gene in the Turkey
E. I. Zakrzewska and T. F. Savage
A new mutation has been identified in the
turkey that inhibits the rate of feathering.
The disorder affects the number, size, and
structure of feathers. At hatch, the affected
poults lack flight feathers. As the birds
grow, the expressivity of the disorder may
vary from almost complete absence of
feathers to almost a full feather covering.
Feathers may be twisted and bent, barbs
may be crossing each other, or the calamus may be very fragile. Amino acid com-
position of feathers from the affected birds
is modified, Ala, Asp, Gly, Iso, and Tyr are
significantly lower, and Leu is higher.
Blood chemistry is also altered, with levels
of alkaline phosphatase, glucose, and sodium elevated, and cholesterol decreased
in mutants. The disorder is inherited as a
dominant, sex-linked trait. The gene does
not express itself until after day 16 of incubation, as opposed to a previously described late feathering gene. The symbol
K'lF is proposed for the mutation. A potential industrial application of the trait is
feather sexing of 1 day old turkeys.
Discovery of every new gene in commercial avtan species not only significantly increases the general knowledge of poultry
genetics but can also be very important
from a practical viewpoint. Information
about newly described genes may be utilized to improve the efficiency of production through lower costs of feeding, breeding, and labor. An example of a simple
gene that is already widely used in the
poultry industry is late feathering In chickens. This dominant gene (X) located on
the Z chromosome is responsible for reduced primary feather length in chicks at
the time of hatching. This trait allows efficient and nonintrusive separation of
males from females. This method, called
feather sexing, has been known since the
early 1920s and is commonly used in
chicken production (Warren 1930). Although the K gene may have a slight negative effect on growth rate (Merat 1970)
and egg production (Lowe and Garwood
1976) it still has practical application in
the industry.
With production costs in the turkey industry continually increasing It is very important to minimize production costs. One
area that still has not been Improved is
the separation of males from females at
hatch. Currently sex identification of dayold turkey poults is by cloacal examination. This method requires trained personnel and is very time consuming. A possible
alternative to cloacal examination would
be to utilize a sex-linked gene whose expression could be easily recognized at the
time of hatch and would not have negative
pleiotropic effects.
Current knowledge of the turkey genome and of genes influencing feathering
is very limited (Savage 1990) when compared with that of the chicken (Somes
1990). Of the 35 turkey genes that have
been identified, the majority influence
plumage color (Savage 1990) and feather
appearance (Poole and Marsden 1961;
Smyth 1954). Only one gene, late feathering (K), described by Asmundson and Abbott (1961) is sex linked and dominant.
Unfortunately, in the early 1960s the potential practical significance of that discovery was not quite apparent and the
turkey line carrying the K gene was terminated after only a brief investigation.
This article is devoted to the characterization of a new sex-linked, dominant
gene, with its possible application to the
feather sexing process. The gene, first observed in 1990, was expressed in a single
female within a flock of 5,000 females. That
bird was characterized by its lack of feathers at 8 weeks of age. As the female matured, plumage Improved, however the
feather appearance remained altered. The
growing feathers were silky, while the primary and secondary feathers on the wings
were altered. Some feathers were absent
and existing remlges were narrow, twisted,
and bent. Tail feathers were absent. This
unique female was subsequently reared to
sexual maturity and mated with four normal feathered Medium White males. The
affected offspring demonstrated similar
feather characteristics, suggesting the hereditary nature of the condition. Inhibited
feathering (IF) is the proposed name for
the new genetic feather disorder because
it well characterizes the effects of the
gene. We suggest that according to the
newest nomenclature (Crittenden et al.
1995) the symbol KVFvtM be used in referring to the new mutation, since the possibility of the allellsm with the other sexlinked gene affecting feather growth cannot be excluded (Asmundson and Abbott
1961).
Materials and Methods
Investigation of the Mode of
Inheritance
To determine the mode of inheritance,
several matings were performed involving
birds with inhibited feathering (IF) and
with normal feather development (NF).
The original IF female was artificially inseminated with the semen collected from
four Medium White NF males. Among the
12 offspring from this mating, only the
males exhibited the IF plumage disorder.
They were individually wing-banded at
hatch and raised to maturity, and they
were subsequently mated with NF females.
Of the progeny that hatched, 16 IF poults
displaying the feather anomaly were saved
and subsequently sexed based on the appearance of the secondary sex characteristics. In the following year the matings
consisted of IF females and males that
were mated together. Each year poults selected for further breeding were sexed
when the secondary sex characteristics
were apparent. From each generation a
portion of the offspring were saved and
used for further research. The following
are the types of matings that were performed in the investigation: IF females x
IF males, IF females x NF males, and NF
females x IF males. The data were collected in four consecutive years, 1991-1994.
Sex of the poults was determined either by
examination of gonads of euthanized birds
(McLelland 1990) or by appearance of the
sexual characteristics in older poults.
Individual and pooled chi-square values
for goodness-of-fit for the observed data
were calculated (Snedecor and Cochran
1967) to test the hypothesis that inhibited
feathering condition was the expression of
a dominant, sex-linked gene.
Methods for Describing the Inhibited
Feathering Poults
For the purpose of embryo examination,
eggs were collected from Auburn females,
artificially inseminated with semen from IF
homozygous Bronze males. Thirty eggs
from these matings were incubated and
compared with 15 eggs collected from NF
line Bronze turkeys. Eggs were incubated
in single-stage, horizontal, transferless incubators (Savage et al. 1991). All eggs incubated for the purpose of the embryo examination were removed from the incubator at day 16, and down development of
the embryos macroscopically examined.
The appearance of IF poults was described based on comparisons to the
feather growth pattern of NF poults at the
time of hatching and at 10 weeks of age.
Poults at 10 weeks of age varied in the expressivity of the phenotype and thus were
categorized to groups of birds with similar
plumage appearance. The 10 week old
poults were divided into five categories
and were assigned numerical scores of 15. A score of 1 was reserved for poults
with normal feather development, and the
higher the numeric category score, the
more sparse the plumage. The feather categories were based on a subjective grading of the quality and quantity of the plumage, the area of the skin covered by feathers, the number of flight feathers, the proportion of feathered and naked skin, and
the number of pin feathers.
Biochemical Assays
Blood chemistry analysis. Fourteen IF and
six NF females were randomly selected
Brief Communications 2 3 9
from a flock of 30 week old breeder females prior to photostimulation. Due to financial constraints only females were
studied. Blood was collected by puncture
of the vena humeri profunda in the left
wing (Koch and Rossa 1973). Five milliliters of blood was collected from each female into glass tubes containing sodium
heparin and subjected to analyses (Good
Samaritan Hospital Laboratory, Corvallis,
Oregon). The following chemistry analyses were performed: creatinine phosphoklnase, alkaline phosphatase, lactate dehydrogenase, aspartate amino transferase,
aianine amino transferase, glucose, uric
acid, calcium, phosphorus, total protein,
albumin, cholesterol, triglyceride, sodium,
potassium, and chloride. In this preliminary study, this blood chemistry profile
provides a general assessment of the major physiological functions in the bird.
Feather amino acids. Feathers used for
amino acid determination were collected
from turkeys at 20 and 22 weeks of age. All
IF birds with some development of secondary remiges were selected, then 15 IF
birds of each sex were randomly selected.
In addition, 15 males and 15 females with
normal plumage from the same hatches
were also randomly selected as a control
group. Both groups of birds were fed the
same diet and were maintained in the
same facility.
The third, fourth, and fifth primary remiges on the left wing were cut with scissors about 15 mm above the feather follicle to avoid blood contamination. Each
feather sample was individually washed in
60°C water containing a neutral pH detergent. Feather samples were rinsed in
warm water until the rinse water was free
of debris. A final rinse was performed using distilled water, followed by immersion
of the samples in 95% ethanol to accelerate desiccation of the feather samples. All
samples were then dried in a 37°C oven for
3 h, cooled to room temperature, and subsequently ground using an electric feed
grinder. Pooled samples consisted of
ground feathers obtained from five individuals. Three such pooled samples were
prepared for each sex and feather type. A
total of 12 samples containing feathers
from 60 birds were analyzed for amino
acid composition as previously described
(Savage et al. 1986a).
Scanning Electron Microscopy of
Feather Structure
Primary and secondary remiges were collected from 10 week old IF and NF turkeys
and prepared for scanning electron mi-
2 4 0 The Journal of Heredity 199788(3)
Table 1. Progeny pbenotypes from the matings of normal feathering (K'N/K'N), homozygous dominant
(K'IF/K'IF), heterozygous (K'IF/K'N), and hemlzygoos (K'lF/-) Inhibited feathering turkeys
Progeny phenotypes
Inhibited feathering
Normal feathering
Parental genotypes
Sire
Initial mating
K'N/K'N
Second mating
K'IF/K'N
Other matings
K'IF/K'IF
K'IF/K'IF
K'N/K'N
K'IF/K'N
Dam
K'lF/-
0
K'N/K'lF/K'N/K'lF/K'N/-
Females
Males
5
i
0
0
0
79
12*
0
0
22
74
Observed
Males
Expected
Females
Males
Females
8
0
6.5
0
8"
8"
8
8
43.5
12
20
85
435
12
0
85
46
13
20
86
41
11
0
84
Not sexed.
Only 16 poults were sexed.
croscopy (SEM) as previously described
(Savage et al. 1986b). Feather samples of
approximately 1 cm2 were cut from the upper quarter of the vane.
Results
Mode of Inheritance
The results of the completed matings used
to determine the inheritance are presented in Table 1. In the initial mating of the
original mutant female with normal feathered males, all male progeny exhibited the
feather disorder, whereas all of the females were normal. In the next generation
IF male progeny were mated with NF females. Of 79 poults, 42 were normal and
37 demonstrated the abnormal feathering.
Only 16 IF poults were saved and sexed—
half of them were males. The results from
the other matings are also summarized in
Table 1. Six IF females mated with four homozygous IF males resulted in 46 males
and 41 females and all expressed inhibited
feathering. No NF poults were observed
from these mating types. Three NF females, when mated with one homozygous
IF male, produced 37 IF poults, of which
24 were sexed. The sex ratio observed, 13
males and 11 females, was not significantly different from the expected (1:1). Matings of 12 IF females with 6 NF males resulted In 22 NF females and 20 IF males,
which was in agreement with the expected
segregation ratio. Matings of 5 heterozygous IF males with 15 NF females resulted
in 323 poults. Of these, 153 were NF (79
males and 74 females) and 170 displayed
the IF disorder (86 males and 84 females)
and the chi-square for the four groups was
not significant, P > .75.
Plumage of the Slow Feathering Poults
Intensive feather growth occurs at specific
ages during the life of the bird. The first
such period occurs during embryonic development between days 11 and 16 of incubation. At day 16 down development
has progressed to such a stage that major
developmental defects may be detected
(Asmundson and Abbott 1961). The second intensive phase of feather development occurs between the time of hatch
and 10 weeks of age (Lucas and Stettenheim 1972). We assumed the actual feather
growth could best be observed in these
periods, and therefore we characterized
the feathering of the mutant birds at day
16 of incubation, at the time of hatch, at
10 weeks of age, and at physical maturity
(more than 20 weeks of age).
There are five categories of feathers in
the Gallinaceous birds: (1) remiges and
rectrices, (2) coverts, (3) down feathers,
(4) filoplumes, and (5) bristles. It was observed that down, filoplumes, and bristles
were not affected significantly in IF turkeys. Therefore we emphasize the description of feathers visibly altered by the IF
gene, that is, primary and secondary remiges, wing coverts, and rectrices—the major tall feathers. The classification of the
feather types was based on the description of Chandler (1916).
Sixteen day embryos. Down development
in 15 normal and 30 mutant embryos was
not different. It has been reported that a
sex-linked gene affecting the rate of feathering in the turkey (Asmundson and Abbott 1961) influenced down development
in embryos by inhibiting formation of the
down in the skin and reducing the down
length. This was not observed in our examinations, and therefore our results im-
Figure 1. Comparison of down and feather development in IF and NF turkeys. (A) Wings of 1 day old poults: (left) IF poult, (right) NF poult. (B) IF poult at 16 days of age. ( Q NF
poult at 16 days of age. (D) Eight week old poults: (left and center) IF poults, (right) NF poult. (E) Mature IF females displaying variations in feather development. (F) Mature IF male.
ply that the feather mutation described
herein is different from that described by
Asmundson and Abbott; however, the possibility of allelism cannot be excluded.
Newly hatched poults. At the time of
hatch all IF birds had uniform down, similar to that of NF birds. The IF poults differed from the NF poults only in the absence of flight feathers in the wings (Fig-
ure 1A). As the IF poults grew, differences
between the IF and normal birds became
apparent. At 2 weeks of age IF poults had
only one or two primary feathers present
(Figure IB), whereas NF poults had very
well-developed remiges (Figure 1C). The IF
poults lost their down at an older age, and
the feather development was delayed by
approximately 2 weeks when compared to
normal birds. Between 3 and 6 weeks of
age, all IF poults were devoid of feathers,
with only down remnants present. By 4
weeks of age, small feathers (remiges) began to appear on the wings of some IF
poults. Until this age there were no apparent differences between individual IF
birds.
Ten week old poults. Due to pleiotropic
Brief Communications 2 4 1
variation we arbitrarily established five
classes to which the birds were assigned.
This allowed for a thorough description of
plumage variations.
Class 1. Birds were characterized by
complete and normal feather development
in all feather tracts.
Class 2. These poults had well-developed feathers in the dorsal cervical, interscapular, humeral, the anterior part of the
dorsopelvic and the femoral tracts. The
crural tract, in contrast, exhibited only
"pinlike" feathers in the proximal region
and well-developed feathers in the distal
region. The primary remiges I-K were partially developed (4-9 cm in length), with
the majority of them twisted and bent.
The upper major primary coverts were
also twisted and bent and up to 20 cm in
length. Some of the upper major secondary coverts and secondary remiges were
missing, although not necessarily symmetrical with respect to the body axis. One to
five upper major secondary coverts were
5-7 cm in length. The remaining upper major secondary coverts, if present, were
less than 4 cm long. Secondary remiges
were 1-3 cm long and some of them were
frequently missing. No fully developed coverts were present in the tail. Some IF
birds might have "pinlike" feathers or
very short upper median coverts present
in the tail, while there were no feathers on
the upper part of the prepatagium. The
ventral cervical and pectoral tracts were
covered with very well-developed feathers. Slightly less developed feathers might
be observed in the sternal tracts. Minimal
feathering was observed under the prepatagium and in the ulnar region.
Class 3. Birds in this category had the
dorsal cervical and the edges of the dorsopelvic tracts covered with feathers
about 9-12 cm long. The humeral, crural,
and femoral tracts were covered with very
well-developed feathers and with no differences in these tracts between class 2
and class 3. Some poults might have very
short upper median coverts in the tail
area. The upper major primary coverts
were up to 15 cm long. The primary remiges were about 5 cm long and shorter
than those of normal birds. The upper prepatagium was not feathered. The ventral
cervical and pectoral tracts were covered
with normal, fully developed feathers,
while the other ventral tracts were featherless, except for a slightly visible line of
"pinlike" feathers in the sternal tracts.
Class 4. This category was composed of
birds with one to six primary remiges no
longer than 2 cm and upper major primary
2 4 2 The Journal of Heredity 1997.88(3)
coverts about 4-10 cm long. One to six
secondary remiges and one to six upper
major secondary coverts were also present, although short (2-3 cm). The upper
marginal and under marginal coverts of
the prepatagium were absent. Feathers
about 1 cm long were present on the
boundaries of the ventral and dorsal cervical tracts. The same type of feathers
covered the distal parts of the crural
tracts, whereas on some of the birds the
proximal parts of these tracts were covered with "pinlike" feathers. Slightly longer feathers (about 1.5 cm) were present
on the borders of the anterior part of the
dorsopelvic tract. The longest feathers
(about 10 cm long) were present on the
femoral and pectoral tracts. On the sternal
tract feathers were either missing or very
short (0.5-1 cm).
Class 5. This class consisted of birds
with only one to five primary remiges no
longer than 1 cm and with upper major
primary coverts about 3-7 cm long. Only
one to four secondary remiges and one to
four upper major secondary coverts were
present, though very short (less than 1
cm). Absent were the upper marginal and
under marginal coverts of the prepatagium. Short (about 0.5 cm long) feathers
mixed with "pinlike" feathers existed on
the boundaries of the ventral and dorsal
cervical tracts, constituting four separate
feathered strips along the neck. The same
type of feathers covered the distal parts
of the crural tracts, whereas the proximal
parts of these tracts were naked. The borders of the anterior part of the dorsopelvic tract of some birds were covered with
"pinlike" feathers, while they were naked
on other poults. A few long feathers
(about 5 cm long) were present in the femoral and pectoral tracts. There were no
feathers on the sternal tract.
Mature turkeys. When the IF birds were
24 weeks of age it was no longer possible
to classify them into distinct categories
with respect to feather development. Mature IF birds displayed considerable diversity of feathering, with different tracts affected in different birds (Figure 1E,F). Generally IF males are better feathered than IF
females. Here we provide only a general
qualitative description of how the IF gene
typically affects different feather types in
mature turkeys. It should be kept in mind
that combinations of features described
below could be present on individual turkeys.
The primary remiges are the largest
feathers of the posterior edges of the
wings and were most affected by the IF
gene. Generally remiges are characterized
by their length, size, stiffness and
strength. In NF mature turkey the average
length of the flight feathers is between 2025 cm. The largest feathers on the wings,
the primary remiges, are located in the region of phalanges—digits III and IV (Lucas
and Stettenheim 1972). Considerable variation in the shape and the appearance of
remiges of IF turkeys does not allow a simple and uniform description. About half of
the observed IF birds displayed only fragments of the remiges or are missing all of
them. Some IF turkeys developed remiges,
but their appearance differs greatly from
those formed on the wings of the NF turkeys. Primary remiges present in the
feather tracts of IF birds are those numbered 1-6 (Lucas and Stettenheim 1972),
and the terminal and basal phalanges of
digit III are not covered by feathers. Those
primary remiges that developed were significantly narrower and shorter than those
on NF turkeys. The typical width of the
primary remiges measured at the widest
portion of the vane is about 3.5 cm for NF
females and about 4.7 cm when measured
on NF males. The average width of the primary remiges was about 8 mm and 17 mm
on IF females and males, respectively. The
length of the primary remiges present on
IF turkeys also varied greatly. The length
of some of the IF turkey feathers did not
differ from the length of feathers of NF turkeys, whereas in the most extreme situations feather length did not exceed 0.5 cm.
The shape of the primary remiges also differed greatly from what was considered as
normal (Figure 2A).
Primary remiges present on the wing of
the slow feathering turkey were bent and
twisted. The direction of the bending was
consistently toward the bird's body. The
inner part of the vane was not a flat surface but had an undulating curve. The
vane texture also showed noticeable variation. The proximal region of the pennaceous portion of the vane was closely knit
and appeared as a firm and uniform surface. The veneer (surface) of the distal
part of the vane, however, was wavy due
to bending, and did not appear as flat and
smooth. The barbs located in this area
were not parallel but crossed each other
(Figure 3). In such a situation the proximal
pennaceous barbules were no longer attached to the distal barbules by the set of
cilia and hooklets. In severe situations, a
large portion of the barbules was absent
or it was visible as short immature buds
formed on barbs. The calamus of the altered feather was not different from that
Figure 2. Photographs of the feathers of IF and NF turkeys at 30 weeks of age. (A) Primary remlges of (left) NF turkey, (right) IF turkey. (B) Secondary remiges of (left and
center) IF bird and (right) NF bird, (t) Rectrlces of (left) NF turkey, (center and right) IF turkey.
of normal feather. There were no major differences on the proximal half of the rachis
of the primary remiges when compared
with a normal appearing feather. Often the
distal region of the rachis was twisted 6090 degrees and the ventral groove of the
rachis was present on the side of the
feather rather than on Its ventral side.
Effects of the IF gene on secondary remiges also varied among IF birds. In many
situations several of the secondary remiges were missing. Usually the feather follicle of the absent feather existed in the
skin, however it appeared empty. Similar
to the primary remiges, there were major
differences in the number, quality, and
quantity of the secondary remiges between slow feathering turkeys. Even when
the feathers were present in the feather
follicles, they usually differed significantly
from the corresponding feathers In normal
birds. A significant narrowing of the secondary remiges was noticeable, and this
was not the only feature that distinguished feathers of the IF turkeys from
normal ones. Growing remiges tended to
twist in the proximal region of the shaft,
resulting in reversal of the feather vane by
90-180 degrees (Figure 2B). Similar to the
primary remiges, these feathers might
bend toward the inner side of the feather.
This caused defects in structure of the
vane such as curling of the surface. Some
of the barbs In affected areas were discon-
nected and the hooklets absent (Figure
3B,C,E,F). Severely affected secondary
remiges resembled long and narrow
strings. In some birds several secondary
remiges might appear as normal, whereas
others would be totally missing. In normally appearing feathers, barbules were
arranged in parallel and hooklets were
present in their normal configuration.
These feathers, if separated from the bird,
could not be distinguished from feathers
collected from the turkeys with normal
plumage.
In the majority of the IF turkeys observed, fully developed rectrices were absent. The feather follicles, responsible for
their growth, existed as large empty cavities. Some IF birds, particularly males, developed rectrices, which could be assigned to one of three types based on
their appearance. The first type of retrices
resembled a string, which was not wider
than 1.5 cm, and the length varied between 5-15 cm (Figure 2C). The second
type of feather was extremely small, not
longer than 2.5 cm and not wider than 10
mm (Figure 2C). The third type of rectrices that developed on the IF turkeys might
be described as shorter than 8 cm. For
comparison, the average length of normal
rectrices was about 25-35 cm. The width
of these feathers was not significantly affected. The average width of the widest locus of affected feathers was 41 mm, where-
as the same measurement obtained on the
normal bird was approximately 45 mm
(Figure 2C).
All tail feathers of the IF turkey were
characterized by a fragile and easily
cracked calamus. Unlike normal growing
feathers, it was very difficult to remove
tail feathers from the feather follicle without fracturing the calamus wall. Growing
rectrices were noticeably bent and predisposed to twisting up to 180 degrees, resulting in a disturbed vane texture and disconnection of barbules. Fragments of the
vane were missing in a majority of the
feathers.
Biochemical Assays
Blood chemistry. A comparison of blood
plasma chemistry analyses of IF and NF females at 30 weeks of age is summarized in
Table 2. Only the concentrations of alkaline phosphatase, glucose, sodium, and
cholesterol were significantly different in
the plasma of IF turkey females when compared with NF females. The concentrations of alkaline phosphatase, sodium, and
glucose were higher in IF females, with estimated statistical significance of P < .05,
P < .01, and P < .05, respectively. The cholesterol concentration was lower (P < .01)
in the IF females.
Amino acid content of the feathers. A
comparison of amino acids in the feathers
collected from NF and IF birds Is summa-
Bnef Communications 2 4 3
Figure 3. Electron microscopy of the feathers of IF and NF turkeys at 10 weeks of age (A) Normal feather with well-developed barbules and barblcells (30x). (B,C) Feathers
of IF bird. Arrows point to the site of disconnected barbules (30x). (D) Normal feather with visible structure of barblcells. (E) Feather of IF bird. Arrows Indicate degenerated
barbules (350X). (F) Feather of IF bird. Fusing of barbules and barbicells.
2 4 4 The Journal of Heredity 1997:88<3)
Table 2. Mean plasma biochemical values of Inhibited feathering (IF) and normal feathering (NF)
females
Plasma variable
IF-
Creatlnlne phospholdnase (U/L)
Alkaline phosphatase (U/L)
Lactate dehydrogenase (U/L)
Aspartate amlno transferase (U/L)
Alanlne amino transferase (U/L)
Glucose (g/dl)
Uric Acid (g/dl)
Calcium (g/dl)
Phosphorus (g/dl)
Total protein (g/dl)
Albumin (g/dl)
Cholesterol (mg/dl)
Trigtyceride (mg/dl)
Sodium (mEq/1)
Potassium (mEq/1)
Chloride (mEq/1)
9,113 :
1,032?
643 :
638 :
4.3 :
354':
7.3 :
12.2 :
5.7 :
4.70 :
2.01 :
148*:
159 :
170.5*:
4.96 :
120 :
Table 3. Amlno acid composition* of the feathers
of the inhibited feathering (IF) and normal
feathering (NF) turkeys
NF»
IF
1,063
:52
72
60
0.3
4
0.6
0.2
0.2
0.11
0.06
3
12
0.8
0.13
1
9^50 :
818*:
639 :
506 :
6.5 :
334':
8.2 :
11.9 :
5.4 :
4.48 :
1.92 :
167':
233 :
164.8':
4.93 :
119 :
2369
52
120
35
2.7
8
03
0.2
0.1
0.14
0.11
6
49
13
0.18
2
" Values represent the mean ± SEM lor n - 14.
* Values represent the mean ± SEM for n = 6.
' Values differ significantly (P < .05).
'Values dlfler very significantly (P < 01).
rized in Table 3. There were no significant
differences between males and females in
NF and IF turkeys, and we report pooled
data for NF and IF birds. Concentrations of
alanine, aspartlc acid, glycine, leucine,
and tyrosine were lower (P < .01) in feathers of the IF turkeys, as was the concentration of phenyialanine (P < .05). The
only amino acid whose concentration was
statistically higher in the feathers of IF turkeys was isoleuclne (P < .01).
Discussion
Mode of Inheritance and Phenotype
Description
The data presented in Table 1 are consistent with the hypothesis that Inhibited
feathering is the expression of a dominant
sex-linked gene. Comparison of the IF mutation described herein with other hereditary feather disorders reported in the literature suggests that this is a new mutation. Among several feather mutations
previously described in the turkey, only
"late feathering" was sex-linked and dominant (Asmundson and Abbott 1961). Due
to the same mode of inheritance and similarity of observed gene effects, IF turkeys
were compared with late feathering turkeys. Description of embryo development
in Asmundson and Abbott's late feathering
turkeys suggests that the down formation
is delayed. In our studies there were no
differences in the length of down between
IF and NF turkeys either on day 16 of incubation or at the day of hatch. This observation allows us to conclude that the
down development in IF turkeys during
embryonic development is not affected.
NF
Alanlne
3.25' i= 0.10
Arginlne
5.36 2t 0.11
Aspartlc acid
4.9fr: t0.09
Cystlne
6.27 itO.08
Glutamlc acid 7.89 it 0.17
Gtyclne
5.28'it 0.12
Histldine
0.33 ii 0.01
Isoleuclne
3.83' ir 0.10
Leuclne
6.08*:!= 0.13
Lyslne
0.86 i:0.02
Methlonlne
0.18 itO.Ol
Phenyl alanlne 4.02- 1b 0.10
10.02 i:0.63
Prollne
Serlne
9.90 2:0.27
Threonlne
4.06 i:0.10
Tyroslne
2.28' 1:0.05
Vallne
6.41 ±0.12
4 3 ^ ±0.11
5.45 ±0.12
537'±0.11
6.16 ±0.13
7.98 ±0.15
6.20' i 0.11
0.32 ±0.01
3.48' ± 0 06
6.84' ± 0.13
0.85 ±0.02
0.16 ±0.02
4.43* ± 0.08
10.19 ± 0.70
10.00 ± 0.24
4.02 ±0.08
2.65' ± 0.06
631 ±0.12
• Values represent the mean ± SEM for n = 6.
• Values differ significantly (P < 05).
' Values differ very significantly (P < .01).
The growth of mature feathers on the
body of IF poults is similar to what was
described in late feathering birds. However, in our birds tall feathers (rectrices) do
not develop or are significantly altered. In
late feathering turkeys, rectrices were not
affected and birds developed a full-size
tail. Moreover, in the description of reproductive performances, Asmundson and
Abbott (1961) reported no differences in
the number of eggs produced or in semen
production. Our observations indicate
that egg production and semen production were adversely affected in IF birds.
Several females did not lay eggs, and
those that did produced very few (Zakrzewska 1996). The limited investigation
and description of late feathering turkeys
provided by Asmundson and Abbott
(1961) allowed us to conclude that, despite similar modes of inheritance, the mutations are different from each other. The
possibility of allellsm cannot be eliminated, however, the gene for the late feathering in the turkey is no longer available
and determination of allelism by appropriate matlngs is not possible.
Biochemical Assays
Blood chemistry. There were major differences between the blood chemistry of the
IF and NF females. The level of the alkaline
phosphatase was significantly higher in IF
females when compared to NF females (P
< .05). It has been suggested in literature
that the level of alkaline phosphatase can
be influenced by several factors: genetics
(Singh et al. 1983b; Wilcox 1963: Wilcox et
al. 1963), age (Singh et al. 1983a), onset of
egg production (Savage et al. 1970), as
well as housing system (Singh et al.
1983a,b). The activity of the alkaline phosphatase decreases with age (Banerjee et
al. 1973; Tamaki et al. 1975), and can differ
up to 10 times between immature birds
and adults (Bell 1960). Opposite results,
however, were obtained in a study involving offspring of four lines of White Leghorn females, where essentially linear increase with age was observed (Singh et al.
1983a). Since those studies were performed on different strains of birds, the
contradictory results may suggest that activity and the concentration levels of alkaline phosphatase is genetically determined and varies between strains of birds.
Consequently the differences between
concentration of the alkaline phosphatase
in the blood of IF and NF turkey females
may suggest more complex differences in
physiology other than only rate of feathering. According to Banerjee et al. (1973)
lower activity of alkaline phosphatase is
associated with lower egg production, so
it Is possible to increase egg production
by selection for high levels of alkaline
phosphatase in serum (Gutowska et al.
1943); however, other authors imply the
opposite effect (Rako et al. 1964; Singh et
al. 1983b). In the line of IF turkeys egg production was significantly lower than that
of NF females. The IF females were delayed
in egg production and finished egg production earlier, but eggs were laid less frequently (Zakrzewska 1996).
An increased plasma sodium concentration was observed in the IF females. Hypernatriemia can be associated with elevated serum osmolality. The water loss
may be a result of increased evaporation
Brief Communications 2 4 5
through the skin. Since feathers provide
good insulation, the bare skin with a depleted feather cover may result in increased sensitivity to the higher temperature of the environment (Peguri and Coon
1993). The increased nervousness and activity observed in IF turkeys could result
in panting and could cause evaporative respiratory water loss. The most probable
cause of the difference in the sodium level
would be continued cutaneous and respiratory insensible water loss.
Cholesterol level was significantly lower
In the inhibited feathering line of turkeys
when compared to normal feathered
birds. Cholesterol is considered as one of
the most commonly occurring steroids in
animals and plays an important role in
evaluation of vascular diseases (Bartley
1989). At the same time, it is known that
the cholesterol level decreases steadily as
a reaction to thyroxine replacement therapy (Kaneko 1989b). Therefore it has diagnostic importance in thyroid disorder
evaluation (Lowe et al. 1974). Hypocholesterolemia due to zinc deficiency has been
demonstrated in rats (Koo and Lee 1988;
Lefevre et al. 1985; Schneeman et al. 1986),
pigs (Burch et al. 1975), and humans
(Sandstead et al. 1980). It is suspected
that in IF turkeys the absorption of zinc
from the diet as well as zinc metabolism
may be severely affected (Zakrzewska
1996). Zinc deficiency is known to affect
skin and its derivatives, causing dermatitis
(Garretts and Molokhia 1977; Krieger and
Evans 1980).
The glucose level in the serum of the IF
females was found to be significantly increased when compared with NF females
of the same age. The blood glucose concentration depends on many factors. The
glucose level is a result of the equilibrium
between the quantity of insulin and glucagon in the blood. Also, if the renal reabsorptive capacity for the glucose is exceeded, an additional loss of the glucose
from the system may occur (Kaneko
1989a). Change of the glucose level can
suggest essential differences between the
metabolism of IF and NF turkeys.
Amino acid analysis of the feathers. The
differences in the amino acid composition
of feathers can partly result in abnormal
feather structure. Since the amino acid
contents of barbs and rachis are different,
variations in the mass ratio of barbs to
calamus may result in variation of amino
acid content (Murphy et al. 1990). The IF
turkey feathers contained significantly
less leucine. Nitrogen content in feathers,
which is closely related to content of the
2 4 6 The Journal of Heredity 199788(3)
ma alkaline phosphatase polymorphism and Its association with economic traits In White Leghorns. Ind J
Poult Sci 8:94-102.
dominant protein, keratin, is estimated at
about 16.5% of dry mass of a whole feather (Harap and Woods 1964; Murphy et al.
1990). Low concentrations of the
branched-chain amino acids may result in
feathers that are fragile and more susceptible to deformations.
Bartley JC, 1989. Upld metabolism and Its diseases. In:
Clinical biochemistry of domestic animals (Kaneko JJ,
ed). London: Academic Press; 106-141.
Conclusions
Burch RE, Williams RV, Hahn HKJ, Jetton MM, and Sullivan JF, 1975. Serum and tissue enzyme activity and
trace-element content In response to zinc deficiency in
the pig. Clln Chem 21568-577.
The results of the matings of a mutant turkey confirmed discovery of the new heritable feather disorder In the turkey. Comparisons of the phenotypic attributes of a
previously described late feathering line
of turkeys that was also sex-linked (Asmundson and Abbott 1961) suggest that,
despite some similarities, the disorders
are different. Due to similarity in the localization of the genes in the turkey genome, a test for allellsm should be performed; however, the earlier mutation described in early 1960s is no longer available for research.
Even though the IF line of turkeys can
be potentially useful in commercial turkey
production, its immediate application is
precluded by adverse pleiotropic effects
of the gene. The slow feather development
predisposes the birds to injuries and induces severe stress. Moreover, reproductive performance of IF turkeys is impaired.
However, we observed great variability in
these effects among the affected birds. As
evident from the description of phenotype, some of the IF birds displayed only
slightly inhibited feathering and quickly
developed almost full feather cover. If
such a fast rate of feathering could be
achieved in all IF turkeys, with inhibited
feathering present only at hatch, the new
line of birds would be very useful for commercial use. This should be possible to accomplish through an intensive selection
for faster feathering after hatch.
Intricate results of the blood and feather
analyses suggested more complex pleiotropic effects on the organism of the turkey and require further investigation.
From the Department of Animal Science, Wlthycombe
Hall, Oregon State University, Corvallls OR 97331-6702.
We thank Robert Rltz, a turkey grower of Monmouth,
Oregon, for bringing to our attention the original mutant. This article Is Oregon Agricultural Experiment Station technical paper number 10835. Address correspondence to Dr. Savage at the address above.
The Journal of Heredity 1997*8(3)
Bell DJ, 1960. Tissue components of the domestic fowl.
4. Plasma alkaline phosphatase activity. Biochem J 75:
224-229.
Chandler AC, 1916. A study of the structure of feathers
with reference to their taxonomlc significance. Unlv
Calif Publ Zool 13:243-446.
Crittenden L, Bltgood J, Burt D, 1995. Genetic nomenclature guide. Chick. Trend Genet ll(supplement):3334.
Garretts M and Molokhia M, 1977. Acrodermatltls enterophatlca without hypozlncemla. J Pediatr 91492494.
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RM, 1943. Alkaline phosphatase and egg formation.
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Received October 9, 1995
Accepted July 29, 1996
Corresponding Editor J. James Bltgood
Development of a Chicken
Z-Chromosome-Specific
DNA Library
S. Ambady, S. Ciufo, J. M. Robl,
J. R. Smyth, and F. A. Ponce de Le6n
We have developed a chicken (Gallus domesticus) Z-chromosome-specific DNA library in a phage vector by means of chromosome microisolation and microcloning.
The chromosomal origin, specificity, and
purity was evaluated by fluorescent in situ
hybridization (FISH) on chicken metaphases. Heterologous chromosome painting using this Z-chromosome-specific
probe on turkey (Meleagris gallopavo)
metaphases identified its homologous
Z-chromosome, under the same stringent
conditions as that used in the chicken, indicating a high degree of Z-chromosome
sequence homology among these two
species. This chicken Z-chromosome library will facilitate the development of
Z-chromosome-specific DNA markers that
will be useful for genetic mapping in the
domestic chicken and related avian species. The Z-chromosome-specific DNA
probe will also be useful for studies pertaining to the sex chromosome evolution
in avian species.
Livestock genome maps have progressed
very rapidly in the past few years because
of the availability of highly polymorphic
DNA markers. But in many species the
maps are not dense enough to facilitate a
thorough search for quantitative trait loci
(QTL). This is especially true in the case
of the chicken. The chicken haploid karyotype consists of 39 chromosomes that are
classified into two categories: the macrochromosomes and the microchromosomes. The largest five pairs of macrochromosomes and the Z-chromosome represent about 55% of the total DNA content
of the chicken genome. The Z-chromosome covers about 210 cM of the estimated 2500-3000 cM of the chicken genome
map (Levin et al. 1993). Knowledge on the
genetic composition of the chicken Z-chromosome is limited, in spite of the fact that
this chromosome has the most detailed
linkage map for this species, largely generated by classical linkage test analyses
(Bitgood and Somes 1990). To date, 19
known loci and 14 genetic markers consisting of three chicken middle repetitive
sequence element (CR1) markers, eight
random amplified polymorphic DNA
(RAPD) markers, and three microsatellites
have been assigned to the chicken Z-chromosome (Bitgood and Somes 1990; Cheng
et al. 1995; Saitoh et al. 1993). Moreover,
the avian sex chromosome constitution
differs from that of the mammalian system
since females are heterogametic (ZW) and
males homogametic (ZZ). It has been observed from comparative linkage analyses
that some of the sex-linked genes in mammals are autosomal in chicken, while some
of the sex-linked genes in chicken are autosomal in mammals (Bitgood and Somes
1990).
In order to develop a dense genetic map
for chicken, it is important to generate a
large number of polymorphic markers per
chromosome (Cheng et al. 1995). One way
of achieving this goal is to develop chromosome-specific libraries. Chromosome
flow sorting has been the method of
choice for the generation of chromosomespecific libraries in humans (Fuscoe et al.
1986) and in swine (Langford et al. 1993).
Development of flow-sorted chromosomes
is technically demanding and frequently
yields preparations that have some degree
of contamination with other chromosomes (Hozier and Davis 1992). A more effective and direct way of generating chromosome-specific DNA libraries is by chromosome microisolation and microcloning
of the chromosome of interest. Chromosome-specific libraries generated by chromosome microisolation have been used in
swine (Ambady S et al., unpublished data),
cattle (Ponce de Leon et al. 1996) and
chicken (Li et al. 1992) genetic mapping
studies in order to develop maps for particular chromosomes. Generation of polymorphic markers from chromosome-specific libraries for all of the eight pairs of
the chicken macrochromosomes will help
saturate 55-70% of the chicken genome.
Chromosome-specific DNA can also be
used as heterologous chromosome painting probes in closely and distantly related
species for comparative genome analysis,
study of chromosomal evolution, and for
identifying gross chromosomal abnormalities. This article reports on the generation of a chicken Z-chromosome-specific
DNA library and its use as a painting
probe to identify the Z-chromosome homolog in a related species, the turkey.
Materials and Methods
Microisolation and Microcloning
Chicken metaphases were prepared from
chicken fibroblast cultures following standard procedures, fixed briefly for 5 min
each in 9:1, 5:1, and 3:1 methanol: acetic
Brief Corrrnunications 247