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CREATION OF FROZEN ARKS FOR MAMMALIAN SPECIES
CONSERVATION
(Criação de bancos biológicos criopreservados para conservação de mamíferos)
Locatelli Yann 1,2 (PhD)
1
2
MNHN, Réserve de la Haute Touche, 36290 Obterre, France.
INRA, UMR 6175 Physiologie de la Reproduction et des Comportements, équipe Interactions
Cellulaires et Fertilité, 37380 Nouzilly, France.
ABSTRACT
Preservation of endangered species represents a challenging perspective regarding quick
decline of biodiversity. With benefit of human medicine and agronomic research
developments, a wide range of reproductive biotechnologies are now available to
facilitate conservation of mammals threatened with extinction. Cryopreservation of
gametes, embryos and gonadal tissues are important to be performed for many
endangered species on the basis of use of standardized methodologies. Examples of
interspecific gestations have been reported for several mammalian species, indicating
that some species can be restored using frozen embryos transfer even after extinction.
This short review focuses on different illustrations of gametes and tissues
cryopreservation among different mammalian species for creation of biological resource
banks.
INTRODUCTION
Nowadays and whatever the species considered, rapid decline of populations is
observed likely to confirm earths’s sixth mass extinction [9]. According to data from the
International Union for Conservation of Nature (IUCN) more than 20% of mammals are
threatened with extinction in their natural habitat. If conservation effort has shown to be
efficient on Red List Index for mammals (indicator of the changing state of global
biodiversity), it could not prevent its global decrease over the past three decades [33].
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This phenomenon may underline apparition of new threatened species and the
deterioration of status of some already threatened species as well. In some cases,
population loss is fast and cannot be controlled despite protective measures developed
in situ. Precisely designed in these cases, ex situ conservation programs are aimed in
preservation of genetic variability of endangered species during restoration of their
natural habitat. Ex situ programs for mammals consist of captive breeding and are
generally performed by both governmental and non-governmental institutions, reserves
or zoos. The objective of such breeding schemes is to preserve 90% of genetic diversity
within species over 200 years, using non-parented founder animals. The number of
founder animals and population to be kept in captivity depends on generation interval of
the species but is generally estimated to be 50 and 250-500, respectively. The main
difficulty in this aim is thus to prevent inbreeding and sustain heterozygosity to drive
rational genitor exchange between institutions. Achievement of ex situ conservation is
sometimes difficult especially because of genetic loss, inability to conduce genitor
exchange or failure for the species to adapt to captive housing [7].
To prevent genetic loss and facilitate genetic exchange between conservation
centers, assisted reproductive technologies developed for domestic species, such as
semen recovery and cryopreservation coupled to artificial insemination (AI) have been
proposed [18]. With development of second and third generations of animal
biotechnologies (embryo transfer, in vitro fertilization, gametes micromanipulation,
sperm sexing), new possibilities of both genetic management and conservation of
endangered biodiversity is offered. To a greater extent, increasing knowledge in
development and use of biotechnologies such as in vitro fertilization for livestock and
domestic species, allow transposition to close related endangered species. With benefit
of research efforts for human medicine and livestock production, cryopreservation
methods for gametes, embryos or gonadal tissues based on slow freezing and
vitrification turned from thought into possibility. Today, application of cryobiology for
conservation of endangered biodiversity has appeared as an obligatory step for
warranting achievement in conservation programs. Interspecific embryo transfer,
demonstrated to be possible for some ovine [23], bovine [41], equine [68] and deer
species [48], would offer possibilities to gave birth to extinct species using transfer to
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surrogate females. An illustration of possible samples for creation of frozen ark and
their potential use is represented in Figure 1.
Figure 1: possible samples to cryopreserve and their potential use for
constitution of mammalian frozen arks.
Sperm and testicular tissue cryobanking
Since the first success of semen cryopreservation and discovery of the
cryoprotecting properties of glycerol by Polge et al. in 1949 [60], success in sperm
freezing was achieved in many mammalian species that allowed the creation of sperm
banks for human and livestock species. It’s interesting to note that methods of
cryopreservation of sperm based on glycerol supplementation and controlled slow
freezing have not changed much from the principle described by Polge or Lovelock et
Polge [50,60]. Sperm resource banks would allow to both conserve rare genetics for
long periods and to facilitate realization of ex situ programs [36]. Based on ongoing ex
situ program data, Harnal et al. [31] showed in silico that sperm resource banks may
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represent efficient tools for prevention of inbreeding and enhancing genetic diversity for
different mammalian species (tiger, Przewalski’s horse and eld’s deer).
In wild mammals, the first difficulty consists of recovery of good quality
ejaculates. In most cases sperm recovery from non-domesticated animals involves in
electro-ejaculation under general anaesthesia, which can be associated with overstimulation of accessory sex glands and affect seminal plasma composition as
demonstrated in sheep [12]. Interestingly semen may be recovered from valuable males
post mortem via flushing of cauda epididymis, allowing recovery of highly concentrate
of semen. Depending on high variability among mammalian species for the role that
cauda epididymis plays in maturation and storage of sperm [42], this methodology may
allow to recover between 1 to 30 ejaculates equivalent.
This method may be
particularly advantageous in preventing genetic loss of valuable adult males. Seasonal
component is also important to consider and semen recovery must be of course
recovered during the breeding season of the species [47].
In our opinion, successful semen cryopreservation could be obtained with slow
freezing for all mammals using empiric approaches for assessment of extenders,
cryoprotectant (CPA) toxicities, efficiencies and evaluation of the intrinsic permeability
of sperm membrane. However, efficiency of cryopreservation varies highly among
species, individuals within species, and even between ejaculates from individuals,
which is largely attributed to the differences the biophysical characteristics among cell
types or seminal plasma composition.
The primary objective in sperm cryoconservation is preservation of fertility
which may rely on both integrity of DNA, cellular machinery and organites including
the acrosome, and preservation of motility. Good quality semen (ejaculated or
epididymal) from exotic species was successfully cryopreserved using different
extenders and methodologies mainly based on buffered egg yolk or milk media and
using glycerol as cryoprotective agent [34-35]. Table 1 summarizes the different
protocols generally employed for cryopreservation of semen from exotic or endangered
species amongst different families. Interestingly, despite existing differences in
membrane permeability or CPA sensitivity according to species, few variations are
observed in methodology employed, thus resulting sometimes in poor transposition to
target species.
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Table 1. Protocols employed for semen cryopreservation among different mammalian
species.
Family
Extender 1
species
cervidae
Cervus elaphus
Cervus ssp.
Bovidae
Capra Falconeri
Oryx damah
Camelidae
Lama glama
Rhinocerotidae
Rhinoceros unicornis
Extender 2
Citrate- fructose-egg
yolk (20%)
References
Citrate - fructose - egg [44].
yolk (20%) glycerol
(4% final)
Tris – glucose – citrate – /
egg yolk – glycerol 4%
final
[6]
Lactose - egg yolk
(20%)
Hyper-osmotic skim
milk -glycerol (4%
final)
[47]
Necessity of washing of
ejaculate
Skim Milk
Lactose – TEA - glucose
- egg yolk (20%)
Skim milk - glycerol
(7% final)
Locatelli,
unpublished
Lactose – TEA –
glucose - egg yolk
(20%)-glycerol 5%
final
[62]
Tris – egg yolk (10%)
Tris – egg yolk (10%)
– glycerol 7%
[11]
Lactose - egg yolk /
glycerol TEST-egg yolk
extender, 6.25% Me2SO
[32,67]
Acinonyx jubatus
Tes - tris – glucose - egg Tes - tris- egg yolk
yolk (20%)
(20%) - glycerol (4%
final)
[20]
Panthera tigris
Lactose - egg yolk
(20%) -glycerol 4%
Lactose - egg yolk
(20%) - glycerol 4%
[21]
Tes - tris – glucose - egg Tes - tris – glucose yolk (20%)
egg yolk (20%) glycerol (5% final)
[73]
Felidae
Elephantidae
Elephas maximus
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loxodonta africana
Ursidae
Ailuropoda
melanoleuca
Hominidae
Gorilla gorilla
BF-5 : Tes - trisTes - tris- glucoseglucose- fructose -TEA - fructose -TEA -egg
egg yolk (20%)
yolk (20%) - glycerol
4%
[38]
[66]
Tes - tris – glucose - egg /
yolk (20%) -5% glycerol
[45]
Tes - tris – glucose -egg Tes -tris- egg yolk
yolk (20%)
(20%)-glycerol (4%
final)
Long term storage in liquid nitrogen is an objective to consider at time of semen
recovery and processing as seminal plasma or extender proteins may potentially interact
with the sperm plasma membrane and may alter fertility. Thus, when possible,
processing of semen using different cryopreservation methods (extenders/CPA) at time
of recovery may be an important strategy to consider when few data are available on
rare species.
With the progress in human medicine, development of ICSI (intracytoplasmic
sperm injection) methods permitted the use of immature spermatozoa (from caput and
corpus epididymis), motile sperm isolated from testicular biopsies (via Testicular Sperm
Extraction or TESE) or round spermatids thus offering new possibilities for germ cells
cryobanking. Cryopreservation methods for testicular tissue assessed were classical
slow freezing methods using culture media supplementation with DMSO and sucrose.
At thawing sperm that were cryopreserved within the seminiferous tubule can be
extracted for ICSI [2] or whole tissue can be xenografted to immunotolerant mice,
allowing subsequent spermatogenesis and successful sperm differentiation from
spermatogonia as reported in different species. In pig, successful production of viable
piglets [58] was recently reported after fresh tissue xenografting-TESE and ICSI,
indicating suitability of the method. Successful spermatogenesis was also induced after
xenografting cat [65] or bison gonadal tissues [1]. This strategy is particularly
encouraging for the post mortem genetics rescue of prepubertal animals or if death
occurs during the non breeding season.
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Embryo production and banking
In complement to sperm banks, storage of endangered genetic material in the
form of embryos produced by MOET (Multiple Ovulation and Embryo Transfer) or
ovum pick-up associated with in vitro fertilization (OPU-IVF) would allow a reduction
in the number of endangered individuals to be kept in captivity without reducing genetic
diversity. In mammals, IVP may be performed using oocytes deriving from dead
animals (fresh follicle aspiration) to prevent genetic loss. In addition, viable embryos
deriving from non-parented individuals, cryopreserved at initiation of ex situ programs
would allow the restoration of original genetic pattern at any moment of the breeding
scheme.
In this aim, third generation biotechnologies are the best designed tools to
artificially increase female prolificacy by producing numerous embryos using (OPUIVF) [16,72]. IVF also offers a further advantage compared to MOET that optimizes the
utilization of frozen semen from males of high genetic interest, as potentially hundreds
of oocytes can be fertilized using one thawed semen straw. Previous experiments with
deer in our laboratory, indicated that in vitro production of embryos (IVP) was less
invasive than MOET, and IVP was finally retained as the most valuable strategy for
embryo production from “non domestic” species. This is due to reducing handling
procedures and therefore reduced stress for animals. Traditionally for mammals, MOET
utilises estrus/ovarian cycle synchronization, ovarian stimulation, artificial insemination
and embryo recovery. This is associated with at least seven to ten different handlings of
the animal and one or two anesthesia (one for intra-uterine insemination and one for
embryo recovery). The majority of failures from MOET programs encountered
concerned the absence of ovulation or fertilization and an OPU-IVF strategy was
rationally proposed to overcome this problem. However, as integrated technique, OPUIVF may cumulate different limitations and this promising technology was faced with
numerous factors limiting its development in domestic species [56-57,75]. For example,
the materials employed for ovum pick-up, seasonality or stimulation regimen for
follicular growth may affect oocytes yield or their developmental competence as
demonstrated in sheep [10,61]. Similarly, in vitro maturation, fertilization or
development environments may affect overall efficiency of blastocyst production.
Fertilization process may be especially a limiting factor as strong differences can be
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observed in fertility between males, leading to selection of male sires for efficient in
vitro production and precluding the use of sires that may be genetically valuables
[24,70]. Transposition of OPU-IVF to each species consequently requires adjusting
materials, stimulation regimen, and the in vitro production environment.
To date few studies have focused on assessing OPU-IVF wild species. In deer,
attempts were developed on common subspecies for transposition to endangered ones
[8,17,49]. From previous experiment in our laboratory with deer, OPU-IVF has been
demonstrated to be not traumatic and repeatable without affecting the reproductive
function of the donor [49]. In vitro produced embryos can be stored after
cryopreservation with potentially high survival rates after thawing and transfer to
synchronized recipients [46]. In the case of storage of extinct species for which closely
related species or sub-species recipient females are available, cross embryo transfer
seems possible to perform as demonstrated after a successful sika deer embryo transfer
to a red deer recipient [48]. However, to achieve success in embryo production, freezing
or transfer, many adjustments are required before employing the methodology for rare
species conservation. Common methodology developed to support bovine or sheep
embryo development from zygote to the blastocyst stage revealed to be less efficient for
deer embryo. In the case of deer, specific requirements of embryos during early
development may be adjusted by co-culture with oviduct epithelial cells, allowing a
dynamic adaptation of the microenvironment to embryo’s need [46,49]. Similarly, IVF
methods that were successfully developed for human, cat or dog would rationally be
employed for endangered primates, felids, or canids species respectively but may also
require assessment/development
of specific
methodology for
more efficient
transposition. Different successes in embryo production were reported in transposition
of IVP methodologies to endangered species, like Vietnamese sika deer (Locatelli et al.
in press), markhor (Locatelli et al. unpublished), Indian blackbuck [63], rhinoceros
[32,67] and Siberian tiger [19].
Despite sensitivity to chilling injury, embryos from mammals are thought to
support equilibrium and non-equilibrium (vitrification) cryopreservation. Standard
methodology developed for slow freezing consists of equilibrium incubation in
cryoprotectant such as ethylene glycol or DMSO and cooling at a rate of 0.3 to 2 °C/min
preventing ice formation in the cell (vitrification) but not in extracellular solution. Non-
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equilibrium cryopreservation consist of high CPA concentrations exposure driving a
massive dehydratation of cells in association with high cooling rates (1000°C/min),
allowing both intra and extra-cellular vitrification.
As mentioned for semen cryopreservation, success probability in a target
endangered species may rely on ability to define optimal cooling rates with adapted
media and CPA and its concentration. In endangered species, the main difficulty thus
consists in opportunities to perform such observations. A strategy consists of testing the
efficiency of cryopreservation in close related domestic animal model such as sheep for
mouflon, goat for bouquetin, domestic cat for panther or tiger could be used. When
domestic animal models are not available, non endangered related species or subspecies
may also be used in a first time and serve as model (in example European fallow deer
for Mesopotamian fallow deer). To date, modifications made by Wiladsen (1977) to the
original methods from Whittingham et al.1972 for bovine and sheep embryos (imply
slow cooling in 1.5M DMSO at 0.3°C/min to -36°C) still applied as reference method
for cryopreservation of mammalian embryo [76-77].
Vitrification was more recently developed and is consequently less reported in the
literature for endangered species. Generally different CPA are simultaneously combined
allowing to increase viscosity of cryopreservation solution and to minimize intrinsic
toxicity of each CPA. Livebirths were obtained after warming embryos and transferring
into surrogate females in ferret [59,69], llama [3] , camel [64], red deer [46].
Furthermore, viability was observed after warming tiger [19] or macaque [80] vitrified
embryos. In actual fact, few reports demonstrate viability (livebirth) of embryos from
endangered or exotic species after cryopreservation especially if embryos are derived
from in vitro production. Nevertheless, it can be assumed that at least standard slow
freezing methods can be applied for storage of genetics from endangered species.
Oocyte and Ovarian tissue cryopreservation
Oocyte and ovarian cortex cryopreservation are recent technologies that may be
used to store the genetic material of endangered species. During mammalian
folliculogenesis, the oocyte from a growing follicle progressively acquires
developmental competence that can translate at the time of ovulation, to an ability to
resume and achieve meiosis, be normally fertilized by a spermatozoon, develop into an
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embryo and foetus, and then into a healthy individual [55]. The oocyte is a giant and
complex cell that supports the first key reproductive events. To begin with, the size of
the oocyte makes cryopreservation particularly difficult to achieve. For example, at time
of maximum developmental competence, the volume of the oocyte ranges from 1.8x10 -4
µl in mouse to 1x10-3 µl in cattle or humans, making this cell more than others very
sensitive to chilling and maximizes the risk of intracellular ice formation during
freezing. Secondly,
key roles that the oocyte undertakes may be perturbedby
cryoprotecting agents or physical stress after the cryopreservation process: meiotic
spindle disorganization by CPA [40], zona pellucida hardening induced by cortical
granules migration or the early activation of the oocyte [74]. As consequence of
possible intracellular ice formation during slow freezing, vitrification is possibly more
adapted to cryopreserving the oocyte than slow freezing. Depending on species
considered, studies comparing the two methods in terms of survival or damage to
oocyte
at
thawing/warming
are
controversial.
Improvements
were
recently
accomplished in human oocyte cryopreservation using vitrification as an alternative to
standard slow freezing methodologies. Recent studies with human oocytes indicate that
recovery of meiotic spindle integrity was superior when vitrification was
performedwhen compared to slow freezing [14,52]. Similarly in buffalo, developmental
competence was increased after warming when compared to thawing cryopreserved
mature oocytes [25]. In contrast, damage were reduced and developmental competence
was increased after slow freezing when compared to vitrification of rabbit oocytes,
which are particularly difficult to cryopreserve [39]. Mature or immature oocytes can be
succesfully cryopreserved as reported for pig oocytes [29], but some studies indicates
that oocyte maturation may be performed before vitrification as shown for human [22]
or bovine [54]. The use of ICSI or subzonal sperm injection is recommended to
overcome possible zona hardening induced by cryopreservation as mentioned earlier.
Ovarian cortex cryopreservation represents for females, as described for
testicular tissue for males, a challenging and promising approach for gamete storage
from endangered species. In contrast to spermatogenesis, oogenesis is not a continuous
process in mammals. Thus in females, the gamete reserve is defined at birth or a short
time after birth, and consists of oocytes contained in “dormant” primordial follicles and
preantral follicles.
These primordial follicles constitute the ovarian reserve.
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Cryopreservation was successfully achieved for preantral follicles within the ovarian
cortex using standard slow freezing methods performed preferentially with 10%
DMSO-10 % serum, sucrose supplemented media [26,37]. Vitrification methods were
also successfully applied to ovarian tissue despite some difficulties encountered in
reaching real vitrification as a result of the large volume imposed by the size of the
ovarian cortex fragments [4]. The main problem with cryopreserved ovarian tissue is
not the cryopreservation process itself but use of the ovarian reserve at thawing or
warming. As the cryopreserved tissue contains quiescent primordial follicles, support of
folliculogenesis should thus be performed for follicular growth and differentiation into
large antral follicles containing fully competent oocytes. Two strategies are offered after
thawing or warming: in vitro folliculogenesis or xenograft into immunotolerant mice.
To date in vitro folliculogenesis is quite well mastered in rodents, because
characteristics (short growth phase and follicle metabolism) are compatible with
developed in vitro culture methods allowing survival of ovarian explants. What is
generally observed is a rapid activation of primordial follicles differentiation and
growth to the secondary follicle stage during culture probably because of a significant
decrease in AMH secretion. Subsequent growth to the tertiary follicle stage requires an
additional step of culture after isolation of secondary follicles. This two step culture
design (organotypic then isolated follicle culture) is thus required for full support of
folliculogenesis in mammals with long periods of folliculogenesis as demonstrated in
human [71]. The efficiency of ovarian tissue culture remains quite low despite a better
understanding of the specific requirements of cortex tissue or preantral follicles during
culture that allow the growth and differentiation of healthy follicles. Recently,
Margalhaes et al. reported successful embryo production from preantral follicle culture
in goat [51]. Nevhertheless, we can see with sheep [5], goat [51], buffalo [30] or pig
[79] preantral follicle culture from which embryos were successfully produced, that this
methodology is not yet realistic on a large scale but promising.
Xenograft to immunodefficient mice was rationally proposed as an alternative to
in vitro culture to sustain long-lasting folliculogenesis. Despite the probable nonoptimal hormonal environments that were present for xenografts, antral follicles from
different species were successfully recovered from cryopreserved-xenograft ovarian
tissue in immunodefficient rodents, generally two months after grafting and
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gonadotropin stimulation. Such follicular development was reported after orthotopic or
heterotopic xenograft with sheep,cat ([27], marmoset ([13], elephant [28], tammar
wallaby[53], wombat [15,78], pig [43] fresh or cryopreserved tissues.
CONCLUSION
Since the discovery of the cryoprotecting properties of glycerol on sperm cells
by Polge, many achievements have been performed in animal gamete and embryo
cryopreservation. Despite the specific requirements observed between cell types and
according to species, some standardized methodologies were developed and
successfully applied to a wide range of mammals. Often, this knowledge may allow
transposition with reasonable efficiency for preservation of gametes or embryos from
endangered species. More and more, with rapid decline of biodiversity, frozen arks
appear as an obligatory tool for endangered species conservation. Strategies mentioned
did not focus on somatic cells banking for potential use in nuclear transfer cloning, but
it is evident that such resource bank is also important to consider in preservation
programs.
ACKNOWLEDGEMENTS
Frozen ark program performed in Réserve de la Haute Touche is supported by Région
Centre. The author thanks Dr Michael Bertoldo for manuscript proof reading and
language editing.
REFERENCES
1.
2.
3.
Abbasi S, Honaramooz A. Xenografting of testis tissue from bison calf donors
into recipient mice as a strategy for salvaging genetic material. Theriogenology
2011;76: 607-614.
Allan JA, Cotman AS. A new method for freezing testicular biopsy sperm: three
pregnancies with sperm extracted from cryopreserved sections of seminiferous
tubule. Fertil Steril 1997;68: 741-744.
Aller JF, Rebuffi GE, Cancino AK, Alberio RH. Successful transfer of vitrified
Ilama (Lama glama) embryos. Anim Reprod Sci 2002;73: 121-127.
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
117
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Amorim CA, Curaba M, Van Langendonckt A, Dolmans MM, Donnez J.
Vitrification as an alternative means of cryopreserving ovarian tissue. Reprod
Biomed Online 2011;23: 160-186.
Arunakumari G, Shanmugasundaram N, Rao VH. Development of morulae from
the oocytes of cultured sheep preantral follicles. Theriogenology 2010;74: 884894.
Asher GW, Berg DK, Evans G. Storage of semen and artificial insemination in
deer. Anim Reprod Sci 2000;62: 195-211.
Asher GW, Fisher MW, Fennessy PF. Environmental constraints on
reproductive performance of farmed deer. Animal Reproduction Science
1996;42: 35-44.
Bainbridge DR, Catt SL, Evans G, Jabbour HN. Successful in vitro fertilization
of in vivo matured oocytes aspirated laparoscopically from red deer hinds
(Cervus elaphus). Theriogenology 1999;51: 891-898.
Barnosky AD, Matzke N, Tomiya S, Wogan GO, Swartz B, Quental TB,
Marshall C, McGuire JL, Lindsey EL, Maguire KC, Mersey B, Ferrer EA. Has
the Earth's sixth mass extinction already arrived? Nature 2011;471: 51-57.
Berlinguer F, Leoni G, Bogliolo L, Pintus PP, Rosati I, Ledda S, Naitana S. FSH
different regimes affect the developmental capacity and cryotolerance of
embryos derived from oocytes collected by ovum pick-up in donor sheep.
Theriogenology 2004;61: 1477-1486.
Bravo PW, Skidmore JA, Zhao XX. Reproductive aspects and storage of semen
in camelidae. Anim Reprod Sci 2000;62: 173-193.
Cameron RD. Semen collection and evaluation in the ram. The effect of method
of stimulation on response to electroejaculation. Aust Vet J 1977;53: 380-383.
Candy CJ, Wood MJ, Whittingham DG. Follicular development in
cryopreserved marmoset ovarian tissue after transplantation. Hum Reprod
1995;10: 2334-2338.
Ciotti PM, Porcu E, Notarangelo L, Magrini O, Bazzocchi A, Venturoli S.
Meiotic spindle recovery is faster in vitrification of human oocytes compared to
slow freezing. Fertil Steril 2009;91: 2399-2407.
Cleary M, Paris MC, Shaw J, Jenkin G, Trounson A. Effect of ovariectomy and
graft position on cryopreserved common wombat (Vombatus ursinus) ovarian
tissue following xenografting to nude mice. Reprod Fertil Dev 2003;15: 333342.
Cognie Y, Poulin N, Locatelli Y, Mermillod P. State-of-the-art production,
conservation and transfer of in-vitro-produced embryos in small ruminants.
Reprod Fertil Dev 2004;16: 437-445.
Comizzoli P, Mermillod P, Cognie Y, Chai N, Legendre X, Mauge R.
Successful in vitro production of embryos in the red deer (Cervus elaphus) and
the sika deer (Cervus nippon). Theriogenology 2001;55: 649-659.
Comizzoli P, Mermillod P, Mauget R. Reproductive biotechnologies for
endangered mammalian species. Reprod Nutr Dev 2000;40: 493-504.
Crichton EG, Bedows E, Miller-Lindholm AK, Baldwin DM, Armstrong DL,
Graham LH, Ford JJ, Gjorret JO, Hyttel P, Pope CE, Vajta G, Loskutoff NM.
Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity
for oocyte retrieval and in vitro embryo production and cryopreservation in
Siberian tigers (Panthera tigris altaica). Biol Reprod 2003;68: 105-113.
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
118
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Crosier AE, Pukazhenthi BS, Henghali JN, Howard J, Dickman AJ, Marker L,
Wildt DE. Cryopreservation of spermatozoa from wild-born Namibian cheetahs
(Acinonyx jubatus) and influence of glycerol on cryosurvival. Cryobiology
2006;52: 169-181.
Donoghue AM, Johnston LA, Seal US, Armstrong DL, Simmons LG, Gross T,
Tilson RL, Wildt DE. Ability of thawed tiger (Panthera tigris) spermatozoa to
fertilize conspecific eggs and bind and penetrate domestic cat eggs in vitro. J
Reprod Fertil 1992;96: 555-564.
Fasano G, Demeestere I, Englert Y. In-vitro maturation of human oocytes:
before or after vitrification? J Assist Reprod Genet 2012.
Flores-Foxworth G, Coonrod SA, Moreno JF, Byrd SR, Kraemer DC, Westhusin
M. Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini
) embryos to domestic sheep (Ovis aries ). Theriogenology 1995;44: 681-690.
Garcia-Alvarez O, Maroto-Morales A, Martinez-Pastor F, Garde JJ, Ramon M,
Fernandez-Santos MR, Esteso MC, Perez-Guzman MD, Soler AJ. Sperm
characteristics and in vitro fertilization ability of thawed spermatozoa from
Black Manchega ram: electroejaculation and postmortem collection.
Theriogenology 2009;72: 160-168.
Gautam SK, Verma V, Palta P, Chauhan MS, Manik RS. Effect of type of
cryoprotectant on morphology and developmental competence of in vitromatured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or
vitrification. Reprod Fertil Dev 2008;20: 490-496.
Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to
oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum
Reprod 1994;9: 597-603.
Gosden RG, Boulton MI, Grant K, Webb R. Follicular development from
ovarian xenografts in SCID mice. J Reprod Fertil 1994;101: 619-623.
Gunasena KT, Lakey JR, Villines PM, Bush M, Raath C, Critser ES, McGann
LE, Critser JK. Antral follicles develop in xenografted cryopreserved African
elephant (Loxodonta africana) ovarian tissue. Anim Reprod Sci 1998;53: 265275.
Gupta MK, Uhm SJ, Lee HT. Cryopreservation of immature and in vitro
matured porcine oocytes by solid surface vitrification. Theriogenology 2007;67:
238-248.
Gupta PS, Ramesh HS, Manjunatha BM, Nandi S, Ravindra JP. Production of
buffalo embryos using oocytes from in vitro grown preantral follicles. Zygote
2008;16: 57-63.
Harnal VK, Wildt DE, Bird DM, Monfort SL, Ballou JD. Computer simulations
to determine the efficacy of different genome resource banking strategies for
maintaining genetic diversity. Cryobiology 2002;44: 122-131.
Hermes R, Goritz F, Portas TJ, Bryant BR, Kelly JM, Maclellan LJ, Keeley T,
Schwarzenberger F, Walzer C, Schnorrenberg A, Spindler RE, Saragusty J,
Kaandorp S, Hildebrandt TB. Ovarian superstimulation, transrectal ultrasoundguided oocyte recovery, and IVF in rhinoceros. Theriogenology 2009;72: 959968.
Hoffmann M, Hilton-Taylor C, Angulo A, Bohm M, Brooks TM, Butchart SH,
Carpenter KE, Chanson J, Collen B, Cox NA, Darwall WR, Dulvy NK, Harrison
LR, Katariya V, Pollock CM, Quader S, Richman NI, Rodrigues AS, Tognelli
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
119
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
34.
35.
36.
37.
38.
39.
40.
41.
MF, Vie JC, Aguiar JM, Allen DJ, Allen GR, Amori G, Ananjeva NB,
Andreone F, Andrew P, Aquino Ortiz AL, Baillie JE, Baldi R, Bell BD, Biju
SD, Bird JP, Black-Decima P, Blanc JJ, Bolanos F, Bolivar GW, Burfield IJ,
Burton JA, Capper DR, Castro F, Catullo G, Cavanagh RD, Channing A, Chao
NL, Chenery AM, Chiozza F, Clausnitzer V, Collar NJ, Collett LC, Collette BB,
Cortez Fernandez CF, Craig MT, Crosby MJ, Cumberlidge N, Cuttelod A,
Derocher AE, Diesmos AC, Donaldson JS, Duckworth JW, Dutson G, Dutta SK,
Emslie RH, Farjon A, Fowler S, Freyhof J, Garshelis DL, Gerlach J, Gower DJ,
Grant TD, Hammerson GA, Harris RB, Heaney LR, Hedges SB, Hero JM,
Hughes B, Hussain SA, Icochea MJ, Inger RF, Ishii N, Iskandar DT, Jenkins
RK, Kaneko Y, Kottelat M, Kovacs KM, Kuzmin SL, La Marca E, Lamoreux
JF, Lau MW, Lavilla EO, Leus K, Lewison RL, Lichtenstein G, Livingstone SR,
Lukoschek V, Mallon DP, McGowan PJ, McIvor A, Moehlman PD, Molur S,
Munoz Alonso A, Musick JA, Nowell K, Nussbaum RA, Olech W, Orlov NL,
Papenfuss TJ, Parra-Olea G, Perrin WF, Polidoro BA, Pourkazemi M, Racey
PA, Ragle JS, Ram M, Rathbun G, Reynolds RP, Rhodin AG, Richards SJ,
Rodriguez LO, Ron SR, Rondinini C, Rylands AB, Sadovy de Mitcheson Y,
Sanciangco JC, Sanders KL, Santos-Barrera G, Schipper J, Self-Sullivan C, Shi
Y, Shoemaker A, Short FT, Sillero-Zubiri C, Silvano DL, Smith KG, Smith AT,
Snoeks J, Stattersfield AJ, Symes AJ, Taber AB, Talukdar BK, Temple HJ,
Timmins R, Tobias JA, Tsytsulina K, Tweddle D, Ubeda C, Valenti SV, van
Dijk PP, Veiga LM, Veloso A, Wege DC, Wilkinson M, Williamson EA, Xie F,
Young BE, Akcakaya HR, Bennun L, Blackburn TM, Boitani L, Dublin HT, da
Fonseca GA, Gascon C, Lacher TE, Jr., Mace GM, Mainka SA, McNeely JA,
Mittermeier RA, Reid GM, Rodriguez JP, Rosenberg AA, Samways MJ, Smart
J, Stein BA, Stuart SN. The impact of conservation on the status of the world's
vertebrates. Science 2010;330: 1503-1509.
Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000;62:
3-22.
Holt WV. Fundamental aspects of sperm cryobiology: the importance of species
and individual differences. Theriogenology 2000;53: 47-58.
Holt WV, Pickard AR. Role of reproductive technologies and genetic resource
banks in animal conservation. Rev Reprod 1999;4: 143-150.
Hovatta O. Cryopreservation and culture of human ovarian cortical tissue
containing early follicles. Eur J Obstet Gynecol Reprod Biol 2004;113 Suppl 1:
S50-54.
Howard JG, Bush M, de Vos V, Schiewe MC, Pursel VG, Wildt DE. Influence
of cryoprotective diluent on post-thaw viability and acrosomal integrity of
spermatozoa of the African elephant (Loxodonta africana). J Reprod Fertil
1986;78: 295-306.
Jimenez-Trigos E, Naturil-Alfonso C, Vicente J, Marco-Jimenez F. Effects of
cryopreservation on the meiotic spindle, cortical granule distribution and
development of rabbit oocytes. Reprod Domest Anim 2012;47: 472-478.
Johnson MH, Pickering SJ. The effect of dimethylsulphoxide on the
microtubular system of the mouse oocyte. Development 1987;100: 313-324.
Johnston LA, Parrish JJ, Monson R, Leibfried-Rutledge L, Susko-Parrish JL,
Northey DL, Rutledge JJ, Simmons LG. Oocyte maturation, fertilization and
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
120
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
embryo development in vitro and in vivo in the gaur (Bos gaurus). J Reprod
Fertil 1994;100: 131-136.
Jones RC. To store or mature spermatozoa? The primary role of the epididymis.
Int J Androl 1999;22: 57-67.
Kikuchi K, Kaneko H, Nakai M, Noguchi J, Ozawa M, Ohnuma K, Kashiwazaki
N. In vitro and in vivo developmental ability of oocytes derived from porcine
primordial follicles xenografted into nude mice. J Reprod Dev 2006;52: 51-57.
Krzywinski A. Freezing of post mortem collected semen from moose and red
deer. Acta theriol 1981: 424-426.
Lambert H, Citino S, Collazo I, Jeyendran RS. Penetration of zona-free hamster
oocytes by ejaculated cryopreserved gorilla spermatozoa. Fertil Steril 1991;56:
1201-1203.
Locatelli Y, Cognie Y, Vallet JC, Baril G, Verdier M, Poulin N, Legendre X,
Mermillod P. Successful use of oviduct epithelial cell coculture for in vitro
production of viable red deer (Cervus elaphus) embryos. Theriogenology
2005;64: 1729-1739.
Locatelli Y, Mermillod P. Caractéristiques et maîtrise de la fonction de
reproduction chez les cervidés. Productions animales 2005;18 3-25.
Locatelli Y, Vallet JC, Baril G, Touz, L. J, Hendricks A, Legendre X, Verdier
M, Mermillod P. Successful interspecific pregnancy after transfer of in vitroproduced sika deer (cervus nippon nippon) embryo in red deer (cervus elaphus
hippelaphus) surrogate hind. Reproduction, Fertility and Development 2008;20:
160-161.
Locatelli Y, Vallet JC, Huyghe FP, Cognie Y, Legendre X, Mermillod P.
Laparoscopic ovum pick-up and in vitro production of sika deer embryos: effect
of season and culture conditions. Theriogenology 2006;66: 1334-1342.
Lovelock JE, Polge C. The immobilization of spermatozoa by freezing and
thawing and the protective action of glycerol. Biochem J 1954;58: 618-622.
Magalhaes DM, Duarte AB, Araujo VR, Brito IR, Soares TG, Lima IM, Lopes
CA, Campello CC, Rodrigues AP, Figueiredo JR. In vitro production of a
caprine embryo from a preantral follicle cultured in media supplemented with
growth hormone. Theriogenology 2011;75: 182-188.
Martinez-Burgos M, Herrero L, Megias D, Salvanes R, Montoya MC, Cobo AC,
Garcia-Velasco JA. Vitrification versus slow freezing of oocytes: effects on
morphologic appearance, meiotic spindle configuration, and DNA damage.
Fertil Steril 2011;95: 374-377.
Mattiske D, Shaw G, Shaw JM. Influence of donor age on development of
gonadal tissue from pouch young of the tammar wallaby, Macropus eugenii,
after cryopreservation and xenografting into mice. Reproduction 2002;123: 143153.
Men H, Monson RL, Rutledge JJ. Effect of meiotic stages and maturation
protocols on bovine oocyte's resistance to cryopreservation. Theriogenology
2002;57: 1095-1103.
Mermillod P, Oussaid B, Cognie Y. Aspects of follicular and oocyte maturation
that affect the developmental potential of embryos. J Reprod Fertil Suppl
1999;54: 449-460.
Merton JS, Ask B, Onkundi DC, Mullaart E, Colenbrander B, Nielen M. Genetic
parameters for oocyte number and embryo production within a bovine ovum
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
121
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
pick-up-in vitro production embryo-production program. Theriogenology
2009;72: 885-893.
Merton JS, de Roos AP, Mullaart E, de Ruigh L, Kaal L, Vos PL, Dieleman SJ.
Factors affecting oocyte quality and quantity in commercial application of
embryo technologies in the cattle breeding industry. Theriogenology 2003;59:
651-674.
Nakai M, Kaneko H, Somfai T, Maedomari N, Ozawa M, Noguchi J, Ito J,
Kashiwazaki N, Kikuchi K. Production of viable piglets for the first time using
sperm derived from ectopic testicular xenografts. Reproduction 2010;139: 331335.
Piltti K, Lindeberg H, Aalto J, Korhonen H. Live cubs born after transfer of OPS
vitrified-warmed embryos in the farmed European polecat (Mustela putorius).
Theriogenology 2004;61: 811-820.
Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and
dehydration at low temperatures. Nature 1949;164: 666.
Rodriguez C, Anel L, Alvarez M, Anel E, Boixo JC, Chamorro CA, de Paz P.
Ovum pick-up in sheep: a comparison between different aspiration devices for
optimal oocyte retrieval. Reprod Domest Anim 2006;41: 106-113.
Roth TL, Bush LM, Wildt DE, Weiss RB. Scimitar-horned oryx (Oryx dammah)
spermatozoa are functionally competent in a heterologous bovine in vitro
fertilization system after cryopreservation on dry ice, in a dry shipper, or over
liquid nitrogen vapor. Biol Reprod 1999;60: 493-498.
Sambasiva Rao B, Uma Mahesh Y, Lakshmikantan UR, Suman K, Venu Charan
K, Shivaji S. Developmental competence of oocytes recovered from postmortem
ovaries of the endangered Indian blackbuck (Antilope cervicapra). J Reprod Dev
2010;56: 623-629.
Skidmore JA, Billah M, Loskutoff NM. Comparison of two different methods
for the vitrification of hatched blastocysts from the dromedary camel (Camelus
dromedarius). Reprod Fertil Dev 2005;17: 523-527.
Snedaker AK, Honaramooz A, Dobrinski I. A game of cat and mouse:
xenografting of testis tissue from domestic kittens results in complete cat
spermatogenesis in a mouse host. J Androl 2004;25: 926-930.
Spindler RE, Huang Y, Howard JG, Wang P, Zhang H, Zhang G, Wildt DE.
Acrosomal integrity and capacitation are not influenced by sperm
cryopreservation in the giant panda. Reproduction 2004;127: 547-556.
Stoops MA, O'Brien JK, Roth TL. Gamete rescue in the African black
rhinoceros (Diceros bicornis). Theriogenology 2011;76: 1258-1265.
Summers PM, Shephard AM, Hodges JK, Kydd J, Boyle MS, Allen WR.
Successful transfer of the embryos of Przewalski's horses (Equus przewalskii)
and Grant's zebra (E. burchelli) to domestic mares (E. caballus). J Reprod Fertil
1987;80: 13-20.
Sun X, Li Z, Yi Y, Chen J, Leno GH, Engelhardt JF. Efficient term development
of vitrified ferret embryos using a novel pipette chamber technique. Biol Reprod
2008;79: 832-840.
Tanghe S, Van Soom A, Sterckx V, Maes D, de Kruif A. Assessment of
different sperm quality parameters to predict in vitro fertility of bulls. Reprod
Domest Anim 2002;37: 127-132.
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
27 a 29 de junho de 2012.
122
Ciência Animal, 22(1): 106-123, 2012 – Edição Especial
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
Telfer EE, McLaughlin M, Ding C, Thong KJ. A two-step serum-free culture
system supports development of human oocytes from primordial follicles in the
presence of activin. Hum Reprod 2008;23: 1151-1158.
Thibier M. The zootechnical applications of biotechnology in animal
reproduction: current methods and perspectives. Reprod Nutr Dev 2005;45: 235242.
Thongtip N, Saikhun J, Damyang M, Mahasawangkul S, Suthunmapinata P,
Yindee M, Kongsila A, Angkawanish T, Jansittiwate S, Wongkalasin W,
Wajjwalkul W, Kitiyanant Y, Pavasuthipaisit K, Pinyopummin A. Evaluation of
post-thaw Asian elephant (Elephas maximus) spermatozoa using flow
cytometry: the effects of extender and cryoprotectant. Theriogenology 2004;62:
748-760.
Van der Elst J, Van den Abbeel E, Nerinckx S, Van Steirteghem A.
Parthenogenetic activation pattern and microtubular organization of the mouse
oocyte after exposure to 1,2-propanediol. Cryobiology 1992;29: 549-562.
van Wagtendonk-de Leeuw AM. Ovum pick up and in vitro production in the
bovine after use in several generations: a 2005 status. Theriogenology 2006;65:
914-925.
Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to 196 degrees and -269 degrees C. Science 1972;178: 411-414.
Willadsen SM. Factors affecting the survival of sheep embryos during-freezing
and thawing. Ciba Found Symp 1977: 175-201.
Wolvekamp MC, Cleary ML, Cox SL, Shaw JM, Jenkin G, Trounson AO.
Follicular development in cryopreserved Common Wombat ovarian tissue
xenografted to Nude rats. Anim Reprod Sci 2001;65: 135-147.
Wu J, Emery BR, Carrell DT. In vitro growth, maturation, fertilization, and
embryonic development of oocytes from porcine preantral follicles. Biol Reprod
2001;64: 375-381.
Yeoman RR, Gerami-Naini B, Mitalipov S, Nusser KD, Widmann-Browning
AA, Wolf DP. Cryoloop vitrification yields superior survival of Rhesus monkey
blastocysts. Hum Reprod 2001;16: 1965-1969.
Ciência Animal, 22(1), 2012
Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil,
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