wildlife management

1. Submitted to : Mam Bushra Allah Rakha
Submitted by : Komal Shakeel
Mishkat Noor

2. Contents
 What are Assisted Reproductive Technologies ?
 Why we use Assisted Reproductive Technologies.
 ARTs used for amphibians
 Cryopreservation
 Artificial Insemination
 In-vitro Fertilization

3. Introduction
 Amphibians play roles as keystone species in their environments;
model systems for molecular, developmental, and evolutionary
biology; and environmental sensors of the manifold habitats
where they reside. The worldwide decline in amphibian numbers
and the increase in threatened species have generated demand for
the development of a suite of reproduction technologies for these
animals (Holt et al. 2003).
 Following are some assisted reproductive technologies that are
used for amphibians
 Sperm Crypopreservation
 Artificial Insemination
 In-vitro Fertilization

4. Assisted Reproductive Technologies
 Assisted Reproductive Technologies (ART) encompass a range of
techniques that manipulate reproductive endocrinology, gametes, and
embryos, for the purpose of enhancing reproductive success. The
emerging field of amphibian ARTs has gained momentum in recent
years due to the alarming rate at which amphibians are declining
worldwide.
 Significant advances have been made in the last decade for amphibian
assisted reproduction including the use of exogenous hormones for
induction of spermiation and ovulation, in vitro fertilisation, short-term
cold storage of gametes and long-term cryopreservation of
spermatozoa.
 Recent advances in reproduction technologies for amphibians include
improved hormonal induction of oocytes and sperm, storage of sperm
and oocytes, artificial fertilization, and high-density rearing of larvae
to metamorphosis.

5. Assisted Reproductive Technologies (ARTs)
 Assisted reproductive technologies (ART) are applied at various levels of the
reproductive cycle and include everything from hormone supplementation
for maturation and release of gametes to artificial insemination or
fertilization, and even embryo management. Currently, ART has been applied
to only a few amphibian species so there is limited knowledge about what the
most efficient and safe methodologies are to induce natural breeding or to
conduct in vitro fertilization (IVF).
 One such technology is genetic resource banking and applied reproductive
technologies for species that are difficult to reproduce reliably in captivity.
 The capacity and application of reproduction technologies to support
amphibian conservation has been increasing since the mid 1990’s ( Browne
and Figiel, 2010).
 Assisted reproductive technologies (ART), such as hormone-stimulated
gamete collection and in vitro fertlisation (IVF), are conservation methods
that can be used to increase reproductive output for breeding and
reintroduction programs when animals fail to breed naturally.

6. Why use Assisted Reproduction
Technologies (ARTs)?
 Some wild amphibian populations have declind to a point in
which captive assurance of colonies have become necessary to
save the species from extinction.
 Critical to the species long tern survival is maintaining and
breeding the funder populations ; however often very little is
known regarding the enviornmental cues that stimulate
reproduction .
 In this scenario,time is working against the
biologist,vaterinanian,or zookeeper and employing ART becomes
necessary to save the species.

7. Assisted Reproduction in Frogs
 Using common species of temperate frogs (e.g., fire-bellied toads, leopard frogs and
bull frogs) the Reproductive Sciences Department is contributing to the
Omaha's Henry Doorly Zoo and Aquarium's commitment to the global efforts for the
amphibian extinction crisis. Non-invasive methods are being tested to induce and
extend the period of sperm release (spermiation) in seasonal frogs in order to
enhance reproductive performance throughout any given year. Methods found to be
effective in these model species will then be applied to more critically endangered
amphibians such as the Mississippi gopher frog.

8. Assisted Reproduction in Hellbenders
 Together with its cousins, the Japanese and Chinese Giant Salamanders, the
hellbender’s number is declining to the point where certain populations are
dying out altogether .so researchers have developed new assisted
reproductive technologies to keep these slimy salamanders around.
 Over the past couple of years ,researchers have been using a new harmone
injection called Amphiplex recently developed for assisted reproduction in
frogs to stimulate egg and milt (seminal fluid) production the the captive
hellbenders.
 Funding from a callaborative SWG grant (State Wildlife Grant) through the
Tennessee Wildlife Resouses Agency supported statewide surveys ,disease
testing ,gene banking,and genetic analysis for Tennessee hellbenders.
 The grant also funded the development of a new technique called eDNA
(enviornmental DNA) for determining if hellbenders are still present in
stream from minute amount of DNA taken from a 1 litre sample of stream
water.

10.  Sperm after cryopreservation from the hellbender
(Cryptobranchus alleganiensis) recovered motility

11. Cryopreservation in Amphibians
 Cryopreservation and other assisted reproductive technologies
(ARTs) could play a role in slowing the loss of amphibian and
reptile biodiversity and managing threatened populations through
genome storage and the production of live animals from stored
material.
 For amphibians, the generation of live offspring from
cryopreserved spermatozoa has been achieved, but the
cryopreservation of oocytes and embryos remains difficult to
achieve.
 Cryopreservation and ARTs are more developed and advanced
for amphibians than reptiles.

12. Cryopreservation in Amphibians
 Cryopreservation, the preservation of cells and tissues by freezing .
 The development of reproduction technologies in Anurans has enabled successful
fertilization and the pro-duction of offspring with the cryopreserved testicular sperm
from a range of amphibian families (Browne et al.,1998; Browne and Figiel, 2010;
Mansour et al., 2009, 2010).
 Testicular sperm was first cryopreserved with the recovery of motility and fertility in
1996 with the European common frog (Rana temporaria) at the Institute of Cell
Biophysics, Pushchino, Russia (Kaurova et al.,1996, 1997), and subsequently with
the cane toad (Bufo marinus) at the University of Newcastle, Australia (Browne et
al., 1998).
 The rapid development of reproduction technologies for amphibians was shown by
the production of the first juvenile frog from hormonally induced then cryopreserved
sperm in 2010.

14. Artificial Insemination
 The collection of sperm is the first step in artificial insemination
protocols. With externally fertilizing amphibians, the collected sperm
can be used for direct artificial fertilization by placing over oocytes or
through injection into oocytes with intra-cytoplasmic sperm injection
(ICSI) ( Browne and Figiel, 2011).
 For internally fertilizing amphibians and reptiles sampled sperm can
be used for artificial insemination (Larsen and Cardeilhac et al., 1996).

16. In-vitro Fertilization (IVF)
 The terms artificial fertilisation and in vitro fertilisation (IVF) are
often used interchangeably within the literature for amphibian
studies.
 . In vitro fertilisation for amphibians has been performed for
more than 50 years, predominantly for studies on early
embryonic development or for commercial production of
laboratory species such as Xenopus laevis.
 The development ofART for amphibians, especially IVF, is
simplistic when compared with mammals because of the many
advantages of external fertilisation in water.
 Once gametes are obtained from both sexes, IVF is a rather
simple process for anurans

17. In-vitro Fertilization
 Recently, Bufo baxteri tadpoles were produced by IVF in
our laboratory, representing the first time an endangered
amphibian has ever been produced by ART (Browne et al.
2006b).

18. CONCLUSION
 Techniques established for the sampling and cryopreservation
of fish sperm have been applied to Anuran sperm since the late
20th century. With improved techniques for the harvesting of
oocytes, and the successful cryopreservation of hormonally
induced sperm from spermic urine, the resulting technologies
now support the general use of gene banking of many Anurans.
Institutions in Russia, Europe, Australia, USA and China are
involved in developing reproduction technologies for many
other amphibians.
 Further research with internally fertilizing amphibians, and all
reptiles on the sampling, cryopreservation, and insemination
with semen, is needed to assure perpetuation of their genetic
variation

19. ThankYou