PHYSIOLOGY AND REPRODUCTION
Cryopreservation of Chicken Semen of Inbred or Specialized Strains
LARRY D. BACON, DONALD W. SALTER, JOHN V. MOTTA, and LYMAN B. CRITTENDEN
US Department of Agriculture, Agricultural Research Service,
Regional Poultry Research Laboratory, 3606 East Mount Hope Road,
East Lansing, Michigan 48823
FRANK X. OGASAWARA
(Received for publication November 25, 1985)
ABSTRACT Pooled semen from several inbred special chicken strains was diluted with solutions containing
glycerol or dimethylacetamide as a cryoprotectant. One-half milliliter samples in capped glass vials were
frozen at 3 C/min to -35 C in a programmable freezer and stored in a nitrogen vapor tank. One vial of
thawed semen was used to inseminate 4 hens by intravaginal, intrauterine, or intramagnal procedures. The
intramagnal technique required minor surgery but always produced chicks in seven lines in contrast to the
nonsurgical methods. Frozen semen of one strain stored for 29 weeks produced 12 to 14 chicks per vial
when inseminated into 4 hens. This method, therefore, will reliably rescue gene pools from semen after
long-term storage.
(Key words: chicken semen, cryopreservation, inbred strains, artificial insemination)
1986 Poultry Science 65:1965-1971
INTRODUCTION
Fowl semen has been preserved in liquid nitrogen by several methods (Lake and Stewert,
1978; Sexton, 1979). Most studies have employed chicken strains with good semen characteristics. Those studies have indicated many
parameters associated with obtaining good fertilization with cryopreserved semen, including
the collection of good quality uncontaminated
semen, the formulation of the dilutent, the temperature when semen is diluted with solutions
containing cryoprotectants, the type of cryopreservative, methods of storing in vials or straws,
the method and speed of freezing, the method
of nitrogen storage, methods for thawing, and
methods of insemination.
Cryopreservation of semen from inbred chickens with reduced fecundity has had little or no
success (unpublished observation). This study
was undertaken to perfect procedures and attempt intrauterine and intramagnal methods for
insemination of frozen-thawed semen of inbred
'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty
by the US Department of Agriculture and does not imply
its approval to the exclusion of other products that may be
suitable.
lines. A method was developed using the diluents, cryoprotectants, and a programmable nitrogen freezer similar to that described by Lake
etal. (1981).
MATERIALS AND METHODS
Chickens. Initial work on procedures used
semen from 1-year-old males of Line 0, selected
for the absence of endogenous viral genes (AsMmetal., 1979, 1984; Crittenden et al., 1984),
or inbred Line 15I4, selected for susceptibility
to lymphoid tumors (Stone, 1975). Subsequently, semen was frozen from 6-month-old
males of semicongenic strains selected to contain
one endogenous viral (ev) gene, i.e., ev-2, ev-3,
ev-10, ev-U, or ev-12 (Crittenden et al, 1984;
Crittenden, unpublished). Laying hens from
Line 0 and inbred Lines 7, and 6 3 , selected for
susceptibility or resistance to lymphoid tumors,
respectively (Stone, 1975), were used for insemination.
Semen Collection. Males were trained by
semen collection three times a week for 3 weeks
prior to freezing, and a record was kept of volume, appearance, and freedom from foreign material, i.e., urates, feces, or blood. Semen was
collected into a small funnel fitted with a clean,
nontoxic plastic film (parafilm " M " , Marathon
Products, Neenah, WI).1 After males were
1965
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
Department of Avian Sciences, University of California,
Davis, California 95616
1966
BACON ET AL.
Thawing and Insemination. Vials were put in
an ice-water bath and carried to the hen house.
Ice film forming on the tube was removed and
the vial was agitated to resuspend the thawed
semen just prior to insemination. The cryoprotectant was not removed.
Three methods of insemination were used.
1) For normal artificial insemination into the
vagina, semen was drawn into a plastic tube
attached to a 1-ml syringe, and semen (.1 ml/
hen) was expelled into the hen's everted vagina.
2) Intrauterine insemination technique was as
follows (Bobr et al., 1966). A hen without a
palpable hard-shelled egg was everted as for
intravaginal insemination. A special glass cannula containing .1 ml of semen was inserted into
the vaginal orifice and progressively moved
slowly with clockwise turning until passing
through the uterovaginal junction. Semen was
then expelled, with an attached syringe, into the
uterus. The cannula was slowly removed with a
counter clockwise turning. 3) Intramagnal insemination was done by minor surgery (Van
Kreyetal., 1966; Lorenz and Ogasawara, 1968).
A hen with a palpable hard-shelled egg was
placed on its right side, disinfected with 70%
alcohol; feathers were removed from the region
above the ventral feather tract, and the hen was
anesthetized locally by injecting .8 ml of 2%
lidocaine hydrochloride into the skin. Feather
tracts and veins were used to identify an incision
point for a 2 to 3-cm midabdominal incision
through the skin using a scalpel blade. A blunt
long-nosed hemostat was used to penetrate the
peritoneum, and a spreader was used to expose
the oviduct. Gentle pressure posteriorally on the
egg caused the oviduct to emerge as a loop, and
.1 ml of semen was injected using a 1.5-in,
22-gauge needle attached to a 1-ml syringe.
Overlaying skin was clamped with suture
clamps, and the incision was sprayed with disinfectant.
Statistical Analysis. Results were subjected
to the chi-square test.
RESULTS
Insemination Methods Using Different Cryoprotectants. Results of insemination of one vial
of glycerol- or DMA-cryoprotected semen into
4 hens (.1 ml/hen) using intramagnal, intrauterine, or intravaginal procedures are given in
Tables 1 and 2. Intramagnal insemination of
either cryoprotected semen consistently produced chicks, i.e., 8 to 13 chicks from 4 hens
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
trained, a sample of each male's semen was observed microscopically to estimate sperm motility and concentration (Ogasawara, 1981).
Trained males giving adequate amounts of uncontaminated semen with good sperm mobility
were selected. Only semen from the first ejaculate was used, because repeated collection often
resulted in deposits of urates, feces, or materials
that cause immobility or reduced viability of
sperm (Lake, 1957). Clean and macroscopically
acceptable semen was transferred by Pasteur
pipette into a centrifuge tube maintained at 15
C (Lake et al., 1981). This allowed the temperature to equilibrate prior to transfer of the semen
to the cold room. Six lines were handled, with
3 to 10 males per line per freezing trial, in subsequent work.
Preparation for Freezing. All subsequent
manipulations prior to freezing were performed
in a cold room (5 C) with precooled materials
and diluent. Three parts of glycerolized diluent
prepared according to Lake and Stewart (1978),
using glucose instead of fructose, were added
to one part semen in the centrifuge tube. The
semen was quickly mixed by gentle intake and
release with a Pasteur pipette. In some cases
DMA (N-N dimethylacetamide, D-135, Fisher
Chemical) (Lake and Ravie, 1982) was used as
the cryoprotectant. Reagents were kept at 5 C
in the dark and used within 10 days. One-half
milliliter of diluted semen was pipetted into 2
ml screw-capped glass vials (Wheaton), and the
capped vials were placed into coded canes.
Canes were placed into an insulated canister for
transfer to the freezer.
Semen Freezing. Canes were quickly transferred from the canister into the chamber of the
programmable freezing unit previously cooled
to 5 C (Model 900 Controller and Model 972
Freezer, Cryo-Med Programmable Freezing System, Cryo-Med, Mt. Clemens, MI). The sensor
probe was inserted into one vial containing .5
ml of diluted semen. Reference vial and chamber
temperature readings were recorded on a graphic
recorder throughout the freezing cycle. The
freezer was programmed to keep the temperature
rise in the sample minimal at the point of loss
of the heat of fusion. Temperature was lowered
by 3 C/min to -35 C, at which point each cane
was quickly transferred into vapor phase liquid
nitrogen storage unit (Model A4600 or A9000,
Minnesota Valley Engineering, New Prague,
MN). Canes were stored in the lower portion of
the tank where the temperature is estimated by
Cryo-Med to range from -196 to -158 C.
CRYOPRESERVATION OF SEMEN
1967
TABLE 1. Fertility and hatchabilityof eggs from hens inseminated
with Line 0 semen frozen with glycerol1
Hens
IM5
IU
IV
IV (fresh)
(no.
hens)
43
4
5
4
Fertility
Days 1 to 6
Days 7 to 13
Days 14 to 21
Hatchability
of
fertile eggs
(no.
eggs)
(%)
(no.
eggs)
(%)
(no.
eggs)
(%)
(no.
(%
chicks) hatch)
15
10
18
16
53b
0a
21
22
24
17
48b
0a
0a
71b
25
22
29
21
12 a
0*
0a
48b
13
62b
— no eggs —
— no eggs —
30
83b
oa
88b
ab
' Percentages not sharing a common superscript within columns are different (P<.05).
1
Semen was thawed after storage in nitrogen vapor for 2 days and inseminated directly into the hens. Fresh,
undiluted semen was used as control.
2
IM = Intramagnal; IU = intrauterine; IV = intravaginal. Each procedure used .1 ml of thawed semen without
removal of glycerol.
3
The number of hens inseminated.
over 21 days. Only 1 of the 12 hens inseminated
with the intrauterine technique produced fertile
eggs, resulting in 4 chicks. Direct intravaginal
insemination of either cryoprotected semen produced little or no fertility. Fertility was good in
4 hens intravaginally inseminated with fresh unfrozen Line 0 or 15I4 semen. Thus, the intramagnal method was preferable to the intrauterine or
intravaginal insemination procedures.
Maintenance of Fertilizing Potency After
Semen Storage. Line 0 semen, frozen using
glycerol or DMA as cryoprotectants and stored
for 1 day, 7 weeks, or 29 weeks in nitrogen
vapor, was thawed and .1 ml directly inseminated into the hen's magnum. Good fertility was
maintained through 29 weeks of storage (Table
3), and 10 or more chicks were generally produced from one vial of semen regardless of storage time. More chicks were consistently produced from glycerol-cryoprotected than DMAcryoprotected semen. Microscopic examination
of a hanging drop of semen indicated that sperm
protected with glycerol were considerably more
mobile than sperm protected with DMA. Subsequent freezing was, therefore, conducted with
glycerol. However, based on our limited observations, DMA cannot be ruled out, and more
extensive investigations are required (Lake and
Ravie, 1984).
Fertility of Thawed Glycerol-Protected
Semen From Five Lines After Intramagnal Insemination. Pooled semen from five lines
semicogenic for endogenous viral genes (ev) was
frozen on three different occasions using
glycerol. About 6 males were used to obtain
approximately 19 vials of semen on each occasion. After 2 weeks storage, one vial of semen
from each line was thawed and . 1 ml was inseminated into the magnum of each of 3 or 4 hens.
Eight to 20 chicks hatched in 3 of the 5 ev-congenic lines, but ev-3 and ev-10 lines yielded 3
and 2 chicks, respectively (Trial 1, Table 4). Two
more insemination trials were attempted for ev-3
and ev-10 frozen semen. The ev-3 frozen semen
yielded 9 and 1 chicks. Both vials of ev-10 gave
3 chicks each. Thus, these two semen pools
repeatedly gave low fertility but yielded 1 to 9
chicks per vial of frozen semen.
DISCUSSION
This study was done primarily to establish a
procedure to indefinitely store germ plasm of
specialized lines. Lake's freezing method (Lake
etal., 1981) was adapted to fulfill this goal. The
method uses glycerol as the cryoprotectant and
a programmable freezer; adaptations are made
for storage of semen in screw-capped vials in a
vapor-phase nitrogen tank and direct insemination of thawed semen into the hens' magnum
(Ogasawara et al., 1966). The procedure was
satisfactory with inbred lines that have comparatively low fecundity (Stone, 1975). This method
was used to cryopreserve semen from five evcongenic lines. We elected not to reproduce the
lines but are depending on our ability to recover
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
Method
of
insemination
67 b
0a
0a
93 b
54DC
20 a b
0a
88 c
(%)
Days 1 to 6
12
13
20
18
18
18
10
17
(no.
eggs)
100 c
33 b
0a
0*
17^
10 a
71b
3 9 ab
(%)
Days 7 to 13
Fertility
Semen was thawed after storage in nitrogen vapor for 2 days and inseminated directly into the hen. Fresh, undiluted s
' ' Tests of significant differences were made for each line between groups within columns. Percentages not sharing a
9
7
14
14
13
10
7
16
43
4
2
4
4
4
4
4
(no.
(no.
hens)
Hens
3
The number of hens inseminated.
IM = Intramagnal; IU = intrauterine; IV = intravaginal. Each procedure used .1 ml of thawed semen except for 15I 4
later.
2
1
IV (2X)
IV (fresh)
IM
IU
15I4
IV (fresh)
IMJ
IU
IV
Line and
method of
insemination
TABLE 2. Fertility and hatchability of eggs from bens inseminated with semen frozen with d
m http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
CRYOPRESERVATION OF SEMEN
1969
TABLE 3. Maintenance of fertility in frozen-stored Line 0 semen inseminated into the bens' magnum1
Cryoprotectant
and
t i m e stored
Fertility days 1 t h r o u g h 21
(no.
hens)
(no.
eggs)
Hatchability
(%
(no.
fertile
eggs)
fertile
eggs)
(%
(no.
chicks)
hatch)
42
4
3
11
612
26
42
129
21
16
22
59
34
61
52
46°
13
15
14
42
62
94
64
71a
DMA 4
1 day
7 day
2 9 wk
Total
N o n e (fresh) 3
4
3
4
11
4
55
35
48
138
54
14
6
21
41
36
25
17
44
30a
67c
10
5
12
27
30
71
83
57
66*
83a
' ' Tests of significant differences were made between the totals of each cryoprotectant group within
columns. Percentages not sharing a common superscript are different (P<.05).
1
Semen was thawed after storage in nitrogen vapor and .1 ml inseminated into the magnum.
2
The number of hens inseminated and the total eggs produced. Eggs were set on days 7, 14, and 21 postinsemination.
3
One-tenth milliliter of fresh undiluted semen was inseminated into the vagina.
4
Dimethylacetamide.
TABLE 4. Fertility of semen frozen in the presence of glycerol from five ev congenic lines
of chickens after insemination into the hens' magnum1
Trial
and line
inseminated
Fertility days ]1 t h r o u g h 2 1
Hatchabilit>
(%
(no.
hens)
(no.
eggs)
(no.
fertile
eggs)
fertile
eggs)
Trial 1
ev-2
ev-3
<?i>-10
ez>-ll
<?u-12
42
4
3
4
4
612
38
36
38
66
20
7
7
13
33
3 3»b
18a
19a
34ab
50b
Trial 2
ev-i
ev-\0
4
3
46
32
15
6
Trial 3
ev-i
eu-10
4
4
51
48
4
6
(no.
chicks)
(%)
11
3
2
8
20
55a
43a
29a
62a
61a
32a
19a
9
3
60a
50a
8a
12a
1
3
25a
50a
ab
' Tests of significant differences were made for each trial between groups within columns. Percentages not
sharing a common superscript letter are different (P<.05).
1
Semen was thawed after 2 weeks of storage in nitrogen vapor and .1 ml inseminated into the magnum.
2
The number of hens inseminated and the total eggs produced. Eggs were set on days 7, 14, and 21 postinsemination.
3
The number and percent of fertile eggs.
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
Glycerol
1 day
7 wk
29 w k
Total
1970
BACON ETAL.
cedure. Fertility following intrauterine insemination is greatly improved if the hen is inseminated
within .5 hr of oviposition (Ogasawara, unpublished results). In the experiments described,
time of previous egg oviposition was not closely
regulated. Hens that laid an egg in the morning
before 1000 hr were selected for intrauterine and
intravaginal insemination.
The methods for freezing poultry semen are
sufficient to provide chicks from .5 ml of stored
thawed semen, even in inbred lines, provided
insemination is given into the magnum following
minor surgery. Further research is needed to produce chicks successfully without surgery in inbred lines using frozen semen, and those
methods would likely require repeated inseminations using several vials of frozen semen.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance of
Evelyn Young in freezing techniques and operation of the nitrogen freezer, Lenny Provencher
for training roosters and semen collection, and
Kent Helmer for artificial insemination and determination of egg fertility and hatchability.
REFERENCES
Astrin, S. M., E. G. Buss, and W. S. Hayward, 1979.
Endogenous genes are non-essential in the chicken.
Nature 282:339-341.
Bobr, L. W., P. E. Lake, F. W. Lorenz, F. X. Ogasawara,
and H. Krzanowska, 1966. Inhibition of ovulation in
the domestic hen by intrauterine insemination. Poultry
Sci. 44:659-661.
Crittenden, L. B., E. J. Smith, and A. M. Fadly, 1984.
Influence of endogenous viral (ev) gene expression
and strain of exogenous avian leukosis virus (ALV) on
mortality and ALV infection and shedding in chickens.
Avian Dis. 28:1037-1056.
Lake, P. E., 1957. Fowl semen as collected by the massage
method. J. Agric. Sci. 49:120-125.
Lake, P. E., R. B. Buckland, and O. Ravie, 1980. Effects
of glycerol on the viability of fowl spermatozoa-implications for its use when freezing semen. Cryo-Letters
1:301-306.
Lake, P. E., and O. Ravie, 1982. Dimethyl acetamide as a
cryoprotectant for fowl spermatozoa. Poult. Sci.
61(Suppl. l):1497-1498.(Abstr.)
Lake, P. E., and O. Ravie, 1984. An exploration of cryoprotective compounds for fowl spermatozoa. Br. Poult.
Sci. 25:145-150.
Lake, P. E., O. Ravie, and J. McAdam, 1981. Preservation
of fowl semen in liquid nitrogen: Application to breeding programmes. Br. Poult. Sci. 22:71-77.
Lake, P. E., and J. M. Stewart, 1978. Preservation of fowl
semen in liquid nitrogen-an improved method. Br.
Poult. Sci. 19:187-194.
Lorenz, F. W., and F. X. Ogasawara, 1968. Distribution of
spermatozoa in the oviduct and fertility in domestic
birds. VI. The relations of fertility and embryo normal-
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016
the genes of those lines by intramagnal insemination of hens of appropriate lines with thawed
semen if the need arises. Semen of 12 other
lines was also frozen using these techniques;
those thawed semen samples also produced effective fertility following intramagnal insemination (Bacon, Salter, and Motta, unpublished).
The semen cryopreservation, storage, and insemination methods described seem a valid alternative to store genes. Specific genes can be rescued by inseminating thawed semen at a future
time directly into the magnum of a related line,
although there would be some difference in background genes between the selected female line
and the original line. In the case of congenic
lines, this would not be a problem, as long as
the original parent line used for backcrossing in
development of the congenic line exists.
In this experiment, we used normal laying
hens that were available for several inbred lines.
Numbers of hens per line were too limited to
determine if the line of hen influenced the result.
To ensure rapid and successful intramagnal
insemination, hens with excess abdominal fat
should not be used. This may limit the ability
to use this procedure for hens of some lines,
unless they are used at a young reproductive age
under proper feed and environmental conditions
or under conditions that might reduce abdominal
fat, e.g., rearing in large colony cages or floor
pens.
The methods described for male gamete preservation are straight-forward and relatively inexpensive for most research institutions. However,
method of direct insemination of thawed semen
into the magnum requires minor surgery. Because avoidance of surgery would be preferable,
particularly if it required only one vial of semen,
as does the intramagnal injection procedure, several nonsurgical methods for insemination were
attempted. The direct intravaginal insemination
of thawed glycerol-cryoprotected semen (Table
1) or DMA-cryoprotected semen (Table 2) without removal of the cryoprotectant provided little
fertility and no chicks. Lake et al. (1980) have
previously shown similar results with glycerolprotected semen, but if the glycerol is diluted
out of semen after thawing then good fertility
could be established after repeated intravaginal
insemination (Lake et al., 1981). Semen was
inseminated into the uterus by the described
intrauterine device. This device resulted in 1 of
12 hens with fertile eggs that produced 4 chicks
(Tables 1 and 2). Improvement of this method
may provide another alternative nonsurgical pro-
CRYOPRESERVATION OF SEMEN
ity with site of experimental insemination. J. Reprod.
Fertil. 16:445-^55.
Ogasawara, F. X. 1981. Reproductive efficiency in turkeys.
Pages 1-64 in: Western Reg. Res. Publ. W-142. Colorado Expt. Sta. Bull.
Ogasawara, F X., F. W. Lorenz, and L. W. Bobr, 1966.
Distribution of spermatozoa in the oviduct and fertility
in domestic birds. Ill Intra-uterine insemination of
semen from low-fecundity cocks. J. Reprod. Fertil.
11:33^11.
1971
Sexton, T. J., 1979. Studies on the fertility of frozen fowl
semen. 8th Int. Cong. Anim. Reprod. Artif. Insem.,
Krakow 4:1079-1082.
Stone, H. A., 1975. Use of highly inbred chickens in research. US Dept. Agric. Tech. Bull 1514.
Van Krey, H. P., F. X. Ogasawara, and F. W. Lorenz, 1966.
Distribution of spermatozoa in the oviduct and fertility
in domestic birds. IV. Fertility of spermatozoa from
infundibular and uterovaginal glands. J. Reprod. Fertil.
11:257-262.
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 4, 2016