Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

16 t h International Congress on Animal Reproduction
24 Workshop Abstracts
factors which suppress the rejection of transplanted porcine tissues
and the inactivation or removal of undesirable genes critically
involved in the immunogenic response. This is now possible via
somatic nuclear transfer using donor cells with targeted genetic
modifications. As a first important step towards the production of
suitable porcine donor organs the alpha 1,3 Galactosyltransferase
(α1,3GalT) gene that encodes an important immunogenic epitope has
been knocked out in pigs by a homologous recombination in primary
cell cultures and their use in somatic cell nuclear transfer. The Galepitopes
are the major antigen causing hyperacute rejection of
transplanted pig organs. The transplantation of α1,3GalT-Ko porcine
kidneys and hearts into baboons gave encouraging results, leading to a
significant increase in organ survival, up to 179 days for the heart and
up to 83 days for the kidney without any signs of hyperacute rejection.
However, even with massive immunosuppressive treatment, the acute
vascular rejection and cellular rejection remain major obstacles for
long term survival of a porcine xenograft. It is becoming increasingly
clear that a multiple transgene, multiple knockout approach will be
necessary to produce an optimized xenotransplant donor animal. The
technology for either introducing beneficial genes or removing
undesired genes are available, but genetic modification of pigs and
testing the effectiveness of these modifications in a
xenotransplantation setting in nonhuman primates is a very expensive
and time- and labour consuming, interdisciplinary endeavour.
Prolonged survival of transplanted porcine islets into human patients
for up to 260 days demonstrates the potential of xenotransplantation
as a option for successful treatment of organ failure in the near future.
Workshop 16 - Recent Progress in Andrological
Procedures and Evaluations
Moderator: Peter Chenoweth (Australia)
WS16-1
Andrology procedures and standards: A potential weak
link in animal breeding
Chenoweth, P
School of Animal and Veterinary Sciences, Charles Sturt University, Wagga
Wagga, NSW 2678, Australia
Realisation of the full genetic potential of artificial insemination (AI)
requires the timely insemination of cycling, fertile females with an
effective sperm dose (EFD). In turn, the EFD for each species should
equal or exceed a prescribed number of competent sperm. Assurance
that shipped doses consistently contain an EFD is of great importance
for AI centres and their clients. Such assurance can be provided by
rigorous application of good andrological procedures at the AI centre,
as well as via 3 rd party monitoring by an andrology laboratory (AL).
The assessments conducted by the AL should be both accurate and
precise, reflect relevant “gold standards” for each trait, and be
conducted using good laboratory practices (GLP). Current gold
standards are; sperm motility – CASA; sperm concentration -
haemocytometer and sperm morphology – DIC microscopy (1000X
plus). Determination of the accuracy and precision of each procedure
is also important. Historically, laboratories have used statistical
estimates such as correlation coefficients, coefficients of variation and
regression analyses to provide such information, even though each has
its deficiencies. Data will be provided to illustrate current practices
and results, using the swine and cattle AI industries as examples. The
conclusion is that greater attention can be given to quality assurance
in the AI industries, and this should include improved training,
continuing education and monitoring by a competent 3 rd party
laboratory which has the confidence of the AI industry.
WS16-2
Standardization and product monitoring services in the
animal Andrology laboratory
Althouse, GC
Department of Clinical Studies – New Bolton Center, School of Veterinary
Medicine, University of Pennsylvania, USA
The purpose of a dedicated stud is to manage a continuous flow group
of male animals, and from these animals collect, screen and process
semen. This extended semen product is then distributed to customers
for use in their breeding program. In its simplest form, the stud
contributes 50% of the input into that customer’s reproductive
performance outcome. Consequently, if herd reproductive
performance outcomes come into question, as an external, major
contributor, the stud’s product is frequently one of the first areas to be
examined. This is due, in part, to practicality as the stud’s single
system input (e.g., extended semen doses) is simple to objectively
quantify and compare to contractual expectations. Therefore, given
the influence a stud’s product has on a production system and the ease
in verifying its quality, implementation of a total quality management
program which includes the extended semen product is prudent to
customer satisfaction.
A non-biased, third party is ideal for providing this end product (e.g.,
extended semen dose) monitoring. Although slight variations may
exist between species, in general, basic components in an audit of
extended semen doses includes: dose volume, sample sperm motility,
differential sperm morphology (including acrosomes), sperm
concentration and sperm per dose. From an interpretative standpoint,
information regarding handling and arrival temperature of doses, and
the presence and degree of sample agglutination are of value. In
addition to its monitoring value for dose quality, an appreciation for
stud hygiene and sanitation can be obtained through the screening of
doses for microbial contamination.
Because of the intended use of these data, all analyses performed
should be objective and quantifiable using validated referenced
techniques. Demand is growing amongst the various animal
industries that more uniformity and standardization be applied to
external semen analysis diagnostic services. Veterinary laboratories
which currently provide such services should make efforts to agree
upon protocols and techniques for semen analyses which are
scientifically validated and mutually recognized. Additionally,
consistency in the reporting of results by the laboratories could greatly
aid veterinarians and producers in assessment and interpretation of
results. An example of such a system will be discussed.
WS16-3
Flow cytometry in veterinary andrology: detecting sublethal
damage in spermatozoa
Pena, FJ
Department of Animal Medicine, University of Extremadura, Spain
Thanks to the increasing use of flow cytometry in research in
veterinary spermatology, many new membrane integrity assays have
been developed over the past decade. These assays are important
because of their superior ability to forecast fertility when compared
with other tests, such as sperm motility. This major component of the
sperm quality assessment has generated new investigations with the
aim of developing tests that can detect membrane damage in a very
early state. Using phospholipid transposition tests, early changes in
membrane permeability and fluidity can be assessed in a large number
of spermatozoa using fluorescent probes in combination with flow
cytometry. The kinematics of the apparition of apoptotic markers has
been studied flow cytometrically in equine spermatozoa subjected to
freezing and thawing in our laboratory. Caspase activity, low
mitochondrial membrane potential and increases in sperm membrane
permeability were observed in all the phases of the cryopreservation
procedure. Freezing and thawing caused an increase in membrane
permeability, and changes in the pattern of caspase activity; while
decreases in mitochondrial membrane potential were observed after
centrifugation and cooling to 4ºC and after freezing and thawing. It is
proposed that the sperm mitochondria may be directly involved in the
subtle damage that is present in most of the spermatozoa surviving
freezing and thawing.