Production of Offspring from Cryopreserved Chicken Testicular Tissue1
Y. Song and F. G. Silversides2
Agassiz Research Centre, British Columbia, Canada, V0M 1A0
ABSTRACT Cryopreservation of avian germplasm provides a means of genetic banking for future needs in
biological research and animal production. The sperm of
birds can be cryopreserved and used to fertilize eggs.
However, the fertility of frozen-thawed avian semen is
generally much lower than that of mammalian semen
and varies among species or among lines, reducing the
value of semen for the preservation of genetic resources.
In the present study, a simple freezing protocol was used
to cryopreserve testicular tissue of day-old chicks, and
after subsequent transplantation, the frozen-thawed testicular tissue developed functional seminiferous tubules
that produced sufficient sperm to fertilize eggs, resulting
in donor-derived offspring. This study provides an alternative to semen cryopreservation for storage of the male
germline in birds.
Key words: chicken, testicular tissue, transplantation, cryopreservation
2007 Poultry Science 86:1390–1396
INTRODUCTION
Poultry genetic resources are presently maintained as
live animals, which is costly and leaves the populations
vulnerable to disease outbreaks or environmental disasters. Another strategy for conserving genetic diversity
in birds is cryopreservation of germplasm, providing
genetic banking for future needs in biological research
and poultry production. Freezing avian embryos is difficult or impossible because the embryo is attached to a
large yolk. Germline chimeras can be produced by transfer of frozen-thawed blastodermal cells (Kino et al., 1997)
or primordial germ cells (Naito et al., 1994a; Tajima et al.,
1998), allowing cryopreservation of avian germplasm.
Unfortunately, the procedures to produce germline chimeras are complex, the efficiency of germline constitution is low, and depletion of the host germ cells is difficult (Naito et al., 1994b; Song et al., 2005).
Cryopreservation of poultry semen has been investigated extensively since the discovery of the properties
of glycerol as a cryoprotectant (Polge, 1951). However,
the fertility of frozen-thawed poultry semen has been
much lower than that of mammalian semen and the
techniques may not be sufficiently reliable for the cryopreservation of avian genetic resources (Long, 2006). In
addition, there is significant variation in fertility of frozen-thawed poultry semen among species, among lines
(Blanco et al., 2000; Fulton, 2006), and even within lines
(Donoghue et al., 2003). Although cryopreservation of
©2007 Poultry Science Association Inc.
Received January 26, 2007.
Accepted March 9, 2007.
1
Agriculture and Agri-Food Canada Contribution Number 753.
2
Corresponding author: [email protected]
semen is still an important technique for conserving the
male germline, poultry genetic stocks should not been
stored solely as cryopreserved semen because reconstitution of a line may require a redundancy of techniques
to ensure recovery when needed.
Transplantation and cryopreservation of testicular tissue have been studied as means of preserving fertility
in mammals (Woods et al., 2004; Pukazhenthi et al.,
2006), but these techniques have not been explored in
birds. Surgical techniques have recently been developed
to transplant chicken ovarian (Song and Silversides,
2006) and testicular tissue between newly hatched
chicks, with subsequent production of donor-derived
offspring (Song and Silversides, 2007a,b). These transplantation techniques provide an opportunity for cryopreservation of avian genetic material because living
birds can be produced from stored material. The research
described here reports the production of offspring from
cryopreserved testicular tissue.
MATERIALS AND METHODS
Birds
Barred Plymouth Rock (BPR) and White Leghorn
(WL) chicks from pure lines maintained at the Agassiz
Research Centre (Silversides et al., 2007) were used as
donors and recipients of testicular tissue, respectively.
All methods used were approved by the Animal Care
Committee of the Agassiz Research Centre and followed
principles described by the Canadian Council on Animal
Care (1993).
Cryopreservation of Testicular Tissue
Donor testes were isolated from newly hatched BPR
chicks that had been freshly euthanized by cervical dislo1390
1391
CRYOPRESERVATION OF CHICKEN TESTIS
Table 1. Transplantation of frozen-thawed testicular tissue from Barred Plymouth Rock to White Leghorn chicks
Item
Total
Without comb
development
at 3 mo
of age1
With comb
development at
3 mo of age2
9
5
4
8
4
4
5
4
1
Number of chicks receiving transplants
Number of recipients containing
transplanted testicular tissue
Number of recipients with complete
castration
1
Birds were killed at 3 mo of age.
Birds were killed at 11 mo of age.
2
cation. Each testicle was cut into 4 to 5 pieces (from 1.0
to 1.5 mm3 in size) after removal of the tunica albuginea
and tunica vaginalis membranes. Testicular tissue was
transferred into 1.2-mL cryovials (2 birds per vial) and
equilibrated for 25 min at 0°C in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% (vol/vol) dimethylsulfoxide and 10% fetal bovine serum. The cryovials were placed in a Nalgene Cryo1°C freezing container (cat. no. 5100-001, Sigma Chemical Co., St. Louis,
MO) and the container was placed in a freezer at −80°C
for 4 h. The container was designed to achieve a −1°C/
min rate of cooling at −80°C. The cryovials were then
plunged into liquid nitrogen and stored for 4 to 5 mo.
ment were killed at 3 mo of age, and those with normal
male comb development were kept for further study.
Histology
At approximately 11 mo of age, the roosters were euthanized by cervical dislocation. Both donor testicles and
any regenerated host testicles were removed and
weighed. Pieces of tissue of approximately 0.25 cm3 were
removed for histological examination. Tissue samples
from surgically manipulated birds and adult BPR (used
as controls) were fixed in Bouin’s solution overnight,
embedded in paraffin, sectioned at 5 ␮m, and stained
with hematoxylin. Images were captured with a Qimaging Retiga 1300R digital camera (Qimaging Corp., Burnaby, British Columbia, Canada) and an Olympus BX51
microscope (Olympus Corp., Tokyo, Japan).
Transplantation of Frozen-Thawed
Testicular Tissue
The cryovials were removed from the liquid nitrogen
and thawed in a 37°C water bath. The contents were
immediately placed in a petri dish and the testicular
tissue was washed with 3 changes of DMEM containing
10% fetal bovine serum. Eight pieces of frozen-thawed
testicular tissue were transplanted into the abdominal
cavity of the WL chicks using previously described
methods (Song and Silversides, 2007a). Surgically manipulated birds were kept for 2 wk with an initial temperature of 33°C and subsequently reared in a floor pen
with a temperature of 25°C. An oral dose of an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-LaRoche Ltd., Mississauga, Ontario, Canada), was
administered at 100 mg/kg per day for 2 wk after surgery, and then once a week until the birds were 2 mo of
age to prevent immunologic rejection of the transplanted
tissue. Transplanted birds with no male comb develop-
Intramagnal Insemination
Testicular sperm were collected from the donor testicles and used for intramagnal insemination of BPR hens
to test whether the transplants from the frozen-thawed
tissue could produce enough sperm to fertilize eggs.
Testicular sperm were first collected as a fluid suspension that was exuded from the tissue when cut into small
pieces. Subsequently, the tissue was washed with the
same volume of DMEM and collected as a washed suspension. A dose of 0.4 mL of fluid suspension or 0.5 mL
of washed suspension was surgically inseminated into
BPR hens using previously described procedures (Engel
et al., 1991; Song and Silversides, 2007a). The eggs were
collected for 2 wk and incubated to evaluate the fertility
of sperm from cryopreserved testes.
Table 2. Recovery of testes after transplantation of frozen-thawed testicular tissue
Wingband
of recipient
62,508
62,509
62,512
62,517
Weight of
regenerated
host testes (g)
Number
of donor
testes
identified
Weight
of pooled
donor
testes (g)
Fluid
suspension
collected
from donor
testes (mL)
1.0
0
4.4
7.1
1
7
6
1
3.1
10.5
2.0
0.3
0.8
3.0
0
0
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SONG AND SILVERSIDES
Table 3. Production of offspring from sperm obtained from the transplantation of frozen-thawed testicular tissue
Collection
method for
testicular sperm
Fluid suspension1
Eggs collected
Fertile eggs
Chicks hatched
Washed suspension2
Eggs collected
Fertile eggs
Chicks hatched
Day after intramagnal insemination
d 2 to 8
d9
d 10
d 11
d 12
d 13
Total
7
7
7
2
2
2
2
2
1
2
1
0
2
0
0
1
0
0
16
12
10
8
8
8
2
2
1
2
2
2
1
0
0
1
1
1
2
1
1
16
14
13
1
Two hens were surgically inseminated with a dose of 0.4 mL of fluid
suspension from the transplanted testicle.
2
Two hens were surgically inseminated with a dose of 0.5 mL of
washed suspension from the same transplanted testicle.
RESULTS
Growth of Testicular Transplants
from Frozen-Thawed Tissue
Frozen-thawed testicular tissue from day-old BPR
chicks was transplanted into the abdominal cavity of 9
castrated WL chicks of the same age. Four recipient birds
showed the characteristic male comb development by 3
mo of age, but 5 did not (Table 1). Those recipients not
showing comb development by 3 mo of age were killed.
Of these 5 birds, transplanted testicular tissue (Figure
1, panel A) and complete castration were observed in 4
of them. The 4 recipients with male comb development
were kept and killed at 11 mo of age. Of these 4 birds,
transplanted testicular tissue was observed in all of them
(Figure 1, panels B to E) but only 1 was completely
castrated (Table 1 and Figure 1, panel E). Among the 3
hosts with regenerated testes, 1 of the transplanted testis
clearly predominated over the regenerated testis (Table
2 and Figure 1, panel D).
Production of Offspring from Sperm
Collected from Testicular Transplants
Testicular fluid was easily collected from the transplanted testes when the host was completely castrated
or the transplanted testes outgrew the regenerated host
testes (Table 2), but not when the regenerated host testes
predominated over the transplanted testes (Figure 1,
panels B and C). Surgical insemination of sperm collected from 1 transplant (3.1 g in weight; Figure 1, panel
D) produced a total of 23 donor-derived offspring from
the fluid and washed suspensions (Table 3 and Figure
1, panel F). Both fluid and washed suspensions resulted
in fertility for up to 13 d after insemination. Hens surgically inseminated with testicular suspensions from another transplant failed to produce any eggs within 2 wk
of surgery.
Histology of Testicular Transplants
from Frozen-Thawed Tissue
All the seminiferous tubules from the normal adult
testis contained evidence of active spermatogenesis (Figure 2, panel A). In hosts with predominant regenerated
testes, seminiferous tubules in the transplanted tissue
were at various stages of development and only a small
proportion of tubules underwent spermatogenesis (Figure 2, panel B). In the host with a predominant transplanted testis, active spermatogenesis was observed in
seminiferous tubules from the donor testis (Figure 2,
panel C), but spermatogenesis in the regenerated testis
was restricted to a small part of seminiferous tubules
(Figure 2, panel D). In the center of this transplanted
testis, some tubules contained no seminiferous epithelium (Figure 2, panel E) but were full of sperm (Figure
2, panel F).
DISCUSSION
In this study, a simple freezing protocol was used to
preserve chicken testicular tissue, and after thawing and
subsequent transplantation, the frozen-thawed testicular tissue developed functional seminiferous tubules that
produced sufficient sperm to fertilize eggs. This study
extended our previous observations on heterotopic
transplantation of fresh chicken testes between newly
hatched chicks (Song and Silversides, 2007a) and provides an alternative method to semen cryopreservation
for storage of the male germline in birds.
Cryopreservation of avian semen has been studied for
over 50 years, with the results being described in more
than 200 scientific publications. However, a simple universal freezing protocol has not been established for any
species of bird because the susceptibility of avian sperm
to freezing damage varies significantly among lines and
species (Fulton, 2006; Long, 2006). Our successful production of offspring from cryopreserved chicken testicular tissue demonstrated that germ cells in the tissue from
newly hatched chicks can easily be frozen, and that spermatogenesis in the frozen-thawed transplanted tissue is
maintained. Given that the morphology of the testes
and the development of spermatogenesis are essentially
similar in most avian species (Johnson, 1986), we can
expect that newly hatched testicular tissue of most or
all avian species can be preserved in liquid nitrogen
and subsequently used to generate mature sperm when
transplanted into appropriate hosts. Chicken ovaries can
be transplanted between newly hatched chicks with subsequent production of donor-derived offspring (Song
and Silversides, 2007b). Chicken ovaries that were frozen
using the same simple protocol were also transplanted
and underwent development in host chicks (Song and
Silversides, unpublished data). Therefore, cryopreservation and transplantation of testes and ovaries could provide a simple universal protocol for the conservation of
avian germplasm of all species and lines.
CRYOPRESERVATION OF CHICKEN TESTIS
1393
Figure 1. Morphology of testes that developed from frozen-thawed testicular pieces and offspring produced. Donor testicles from hosts in which
the male characteristics were not seen at 3 mo of age (A). The regenerated host testes predominated over the transplanted testes (B, C). The
transplanted testis outgrew the regenerated host testis (D). In a completely castrated host, 5 pieces of donor testes were identified (E) but no eggs
were obtained after surgical insemination of sperm collected. Seven of the 23 Barred Plymouth Rock chicks (F) produced by intramagnal insemination
of sperm collected from the donor testicle shown in D. Bar = 1.27 cm (0.5 in.).
Castration of recipient birds appears to be a critical
factor that affects spermatogenesis in the testicular transplants. In our previous study on transplantation of fresh
chicken testes, sperm could be collected for the production of offspring only in the completely castrated host
(Song and Silversides, 2007a). The present study demon-
1394
SONG AND SILVERSIDES
Figure 2. Spermatogenesis in transplanted donor and regenerated host testes. Seminiferous tubules with active spermatogenesis in a control
Barred Plymouth Rock testis (A). In the host with a predominant regenerated testis, seminiferous tubules and spermatogenesis in the transplanted
donor testis were not fully developed (B). In the host with a predominant transplanted testis, active spermatogenesis was observed in the donor
testis (C) but that in the regenerated testis (D) was restricted, and some tubules in the center of the transplanted testis of this individual lacked
seminiferous epithelium (E) but contained sperm (F). Bar = 100 ␮m.
strated that sperm could be collected from the donor
testis if the host was completely castrated or if the transplanted testis was predominant over the regenerated
host testis. It is generally believed that in avian species,
as in mammals, the development of the testes and the
maintenance of spermatogenesis are dependent on hor-
CRYOPRESERVATION OF CHICKEN TESTIS
mone-controlled interactions in the hypothalamic-pituitary-testis axis (Johnson, 1986). Heterotopic testicular
transplants lack the original vascularization, and transplanted tissue likely needs several days to revascularize
in the host. If the host is completely castrated, serum
levels of luteinizing hormone and follicle-stimulating
hormone rise significantly (Knight et al., 1981), which
provides a positive stimulus for the development of the
transplanted testes (Schlatt et al., 2003; Honaramooz et
al., 2004). After revascularization, the transplanted testis
releases testosterone to establish feedback on gonadotropin release in the recipient chick. The coordinated hormonal interactions between the host and the transplant
induce and maintain active spermatogenesis in the transplanted testis. However, if the host is not completely
castrated, the feedback system in the hypothalamic-pituitary-testis axis is controlled by the regenerated host
testis and the development of the transplanted testis
is restricted.
In the present study, active spermatogenesis was observed in the seminiferous tubules from the frozenthawed testicular tissue and the morphology of most
seminiferous tubules appears similar to that of the control testis, which suggests that most germ cells survived
the cryogenic process. In our previous study on transplantation of fresh testicular tissue (Song and Silversides, 2007a), the seminiferous tubules of the transplants
were enlarged by continued production of sperm with
no release via the efferent duct. In the present study,
the diameter of the seminiferous tubules in transplanted
frozen-thawed tissue was not enlarged, but the amount
of fluid collected from the transplants was similar to
that obtained from the fresh transplants. Histological
analysis suggested that a small number of tubules, in
which the germ cells may have failed to survive the
cryogenic process, became the storage tubules for sperm.
Successful production of 23 offspring from a relatively
small testicle developed from frozen-thawed tissue demonstrated that, although germ cells may have been lost
during the freezing process, the surviving testicular tissue could maintain spermatogenesis and produce
enough sperm for fertilization.
The main purpose of our present research was to demonstrate the feasibility of recuperation of live offspring
from frozen-thawed testes. However, several aspects of
this technique could be refined. First, the efficiency of
castration needs to be improved. In this experiment, 5
out of 9 birds operated on were completely castrated,
and we previously reported (Song and Silversides 2007a)
that only 2 out of 15 chicks were completely castrated,
suggesting that the technique of neonatal castration
could be improved. Second, more than half the chicks
receiving transplants failed to develop male combs by
3 mo of age, indicating a low level of testosterone in the
castrated host. It may be useful to characterize hormonal
changes in recipient birds and administer appropriate
hormones to stimulate development of the transplanted
testes. Finally, the techniques of surgical insemination
sometimes interrupt the egg production cycle, and de-
1395
velopment of a nonsurgical technique for artificial insemination with testicular sperm would be helpful.
ACKNOWLEDGMENTS
The authors would like to thank Beth McCannel, Lee
Struthers, Harold Hanson, Cathy Ingram, Wendy Clark,
and Karli Ryde for care of the experimental birds. Appreciation is also expressed to Tom Forge, in whose
laboratory the microphotography was performed. This
research was funded by the Canadian Poultry Industry
Council, the Canadian Poultry Research Council, and
Agriculture and Agri-Food Canada.
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