Panel 5: Animal biotechnology
Lectures
L5.2
L5.1
Recent advances in pig-to-human
organ and cell transplantation
Large animal models for biomedicine
Angelika Schnieke
Technical University of Munich
e-mail: Angelika Schnieke <angelika.schnieke@wzw.tum.
de>
Recent decades have seen a vast increase in our knowledge of the genetic basis of human disease and disease
susceptibility. This has largely been as a consequence of
the abundance of DNA sequence information coupled
with powerful methods of engineering defined genetic
modifications, particularly in mice. It is generally recognised that large animals such as the pig are more relevant
to biomedical research than rodents, being anatomically
and physiologically closer to humans. Livestock species
are already used in biomedical research, for example in
experimental surgery, organ transplantation, and imaging techniques. However very few large animal models
of human disease are currently available. These are restricted to either artificially (e.g. chemically or surgically)
induced disease, or spontaneous disease predispositions
which are often ill-defined. Advanced reproductive and
transgenic techniques are now being extended to large
animal species and it is timely to use these to benefit human health. If biomedical researchers can be provided
with physiologically-relevant models of serious human
diseases, the development of preventive, diagnostic and
therapeutic strategies will be significantly advanced. This
presentation will give an overview of current genetically
engineered large animal models and some we are developing.
Ryszard Słomski1,2, Daniel Lipiński1,2,
Jacek Jura3, Marlena Szalata1,2, Joanna
Zeyland1, Zdzisław Smorąg3
1Department
of Biochemistry and Biotechnology, Poznan
University of Life Sciences, Poznań, Poland; 2Institute of
Human Genetics, Polish Academy of Sciences, Poznań,
Poland; 3Department of Animal Reproduction Biotechnology,
National Research Institute of Animal Production – National
Research Institute, Balice, Poland
e-mail: Ryszard Slomski <slomski@up.poznan.pl>
The use of animals as a source of organs and tissues for
xenotransplantation can overcome the growing shortage of
human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads
to the complement activation and immediate xenograft
rejection as a consequence of a hyperacute immunological
reaction. To prevent a hyperacute rejection it is possible to
change swine genome by human genes modifying the set of
donor’s cell surface proteins. For this purpose in our previous work the gene construct pCMVFut containing the human gene encoding α1,2-fucosyltransferase enzyme (HT, H
transferase) under the human cytomegalovirus (CMV-IE)
immediate early promoter was introduced by microinjection into a male pronucleus of the fertilized porcine oocyte.
As a result of this experiment, the founder male pig was obtained with the transgene mapped to chromosome 14q28.
Approximately 35% of the founder’s progeny demonstrated
presence of the transgene. The RT-PCR analysis revealed expression of a HT gene in different tissues of transgenic pigs.
The objective of present study was to measure the level of
epitope Gal on the cell surface of skin fibroblasts isolated
from transgenic pigs by the use of flow cytometry analysis.
Human complement regulatory proteins (hCRPs) are considered as a way of overcoming hyperacute rejection. CD55
is a decay accelerating factor responsible for inhibiting the C3
convertases of both the classical and alternative pathways.
CD59 as well as CD55 is a glycosyl phosphatidylinositol anchored membrane glycoprotein that is responsible for the
function regulation of a membrane attack complex (MAC).
CD46 has cofactor activity for inactivation of complement
components C3b and C4b by serum factor I, which protects
the host cell from damage by complement. The expression
of human complement regulators in swine cells, especially
CD46, CD55 and CD59 have been proved to be effective in
reducing an impact of complement system on discordant
organs in xenogenic transplantations.
Acknowledgements:
This work was financially supported by Biology of Reproduction
Network, Warsaw, Poland
114
Abstracts L5.3
L5.4
Experimental model for transgenic
pig as a potential bone graft donor in
reconstructions of the facial skeleton
First-in-Poland kidney transplant
from a genetically modified pig. own
model of surgical technique
Jan Zapała1, Grażyna WyszyńskaPawelec1, Zdzisław Smorąg2
Jerzy Skuciński
1Department
of Cranio-Maxillofacial Surgery of the
Jagiellonian University, Kraków, Poland; 2National Research
Institute of Animal Reproduction, Kraków, Poland
e-mail: Grażyna Wyszyńska-Pawelec
<grazynawyszynska@wp.pl>
Bone grafts harvested from transgenic pigs with confirmed integration of human α1,2-fucosylotransferase
gene might be an alternative to autogenous bone grafts
in reconstructive surgery of the facial skeleton. The objective of this study was the assessment of changes in transgenic pig’s scapula after reconstruction of the defect by
autogenous, homogenous and xenogenous bone graft.
Material and methods: 12 transgenic pigs were used in
this study. Autogenous bone graft healing was evaluated
in 3 animals (I group), xenogenous human bone graft in 6
animals(II group) and homogenous bone graft harvested
from normal pig in 3 animals (III group). Radiological
and histological evaluation of specimens was performed
after 1,2 and 4 weeks following surgery.
Results: radiological examination of specimens of the I
and II group revealed features of bone graft healing after 4 weeks. Histological examination of specimens of
the I and II group revealed fibrosis of the granulation tissue and in the II group presence of newly formed bone
trabeculae after 2 weeks.
Conclusion: healing of autogenous and xenogenous human bone grafts inserted into transgenic pig’s scapula was
comparable in radiological and histological examination.
2008
Institute of Public Health, Jagiellonian University, Kraków,
Poland
e-mail: Jerzy Skuciński <jerzy.skucinski@interia.pl>
The own-developed original method of surgical technique
of collecting and transplanting pig kidneys was based on
the collection and transplantation technique that is routinely used in human transplanting. The entire procedure
comprised three stages: en-block kidney collection, perfusion and storage of the organs collected, as well as grafting thereof. The study comprised 5 transgenic gilts with
the α1,3-GT gene blocked and 4 non-transgenic ones. Special attention was paid to appropriate preparation of the
donors for the procedure, maintaining anaesthesia, simple and quick way of accessing and separating the organs
being collected, efficient technique of rinsing and separating the grafts, quick and safe way of transplanting them,
as well as adequate postoperative management. Narcosis
was induced and maintained based on balanced infusion
anaesthesia using sodium thiopental. Both kidneys were
collected en block, and then separated in a container with
ice on the back table. They were rinsed with preserving
fluids at 4oC. Duration of the collection procedure was
70–90 minutes. The kidneys were transplanted sinistrally
into the intraperitoneal space at the level of the 4th-5th
lumbar vertebra. The kidney was transplanted by end-toside type anastomosis of the renal vein and artery to the
posterior caval vein and artery respectively, and ureterovesicostomy without anti-reflux management. Immediately after the procedure, furosemide was administered
and infusion was continued of isotonic fluids, analgesic
agents as well as chinolones. Kidney transplantation time
was 90–120 minutes. Out of the 9 transplantation operations, 8 were successful (one gilt died at 12 days due to
peritonitis). The quantity of urine secreted was 1–2 L/day.
No other complications were found at 2 months post-operation. Adherence to all of the procedures developed
made it possible to perform the first-in-Poland fully successful allotransplantations of kidneys from transgenic
pigs. The results obtained constitute a basis for conducting further studies on application of transgenic pigs for
procuring their organs for xenogenic transplantations in
humans.
Acknowledgements:
The studies presented have been conducted within the framework of the Committee for Scientific Research project.
Vol. 55 EuroBiotech
2008
L5.5
Molecular aspects of elimination of pathogens
and development of xenotransplantation
Ilona Bednarek
Department of Biotechnology and Genetic Engineering,
Faculty of Pharmacy, Medical University of Silesia
e-mail: Ilona Bednarek <dribednarek@sum.edu.pl>
The development of xenotransplantation reveals a solution for the shortage of human donor organs. Pigs are
currently one of the most favored sources of organs and
tissues cause to their low load of microorganisms when
raised under specific pathogen-free conditions, their unlimited availability and the low production costs. Use of
porcine xenografts is currently overshadowed by public
health concern associated with the potential risk for pigderived infection of human cells. A list of organisms to
consider for exclusion from xenograft donors has been
created, it includes viruses: Circovirus, Porcine Adenovirus, Encephalomyocarditis Virus, Influenza Virus, Porcine
Cytomegalovirus, (PCMV), Porcine Endogenous Retrovirus, (PERV), Gamma-Herpes Virus, Porcine Reproductive and respiratory Syndrome Virus, Porcine Parvovirus,
Hendra-like and Menangle Virus, Pseudorabies/Rabies
and Rotavirus.
It is possible to exclude some viruses (like PCMV) from
herds of pigs by early weaning of newborns, but some
viruses remain and serve as a permissive reservoir of
pathogens. Due to the lack of knowledge about the biology of some organisms from donor species in immunosuppressed humans and inability to recognize novel
clinical syndromes resulting from infection with such
pathogens, it is therefore essential for experimental and
clinical xenotransplantation procedures, that specific and
sensitive screening methods are established, as well as
new innovative pathogens evolutionary and functional
analyses are developed.
While infections with known microorganisms can generally be prevented by screening and vaccination, there
is still the risk posed by unknown infectious agents. Additionally pigs harbor several different types of endogenous retroviruses, (like PERVs), in their genomes. Pigs
can be classified into transmitters and non-transmitters
according to whether their peripheral blood mononuclear cells, (PBMC), either do or do not transmit PERV
to human cells in vitro. Pigs harbor three subgroups of
PERV: PERV-A, PERV-B, (both are able to infect human
cells), and PERV-C. There is a possibility to eliminate
from herds pigs harboring PERV-C; PERV-A and B can
not be eliminated. The greatest threat comes from viruses
generated by recombination between members of PERVA and PERV-C, PERV-A/C. These recombinants are able
to infect human cells, and they are characterized by increased infectivity, which correlates with a multimerization of transcription factor binding sites in the viral long
terminal repeats (LTRs). Selection of animals, that do not
harbor PERV-C genomes and animals with low PERV-A
load may significantly reduce the risk of PERV transmission, however it does not eliminate completely PERV
from animals – xenograft donors.
115
Reduction in PERV transmission and expression can be
achieved by RNA interference method, using synthetic
small interfering RNA, (siRNA), or short hairpin RNA,
(shRNA), corresponding to different sequences of PERV
genome. Intracellular transcription of siRNA and stable
siRNA expression can be achieved by incorporation of H1
or U6 RNA Pol III promoters in viral vectors, for example
lentiviral vector. RNA interference is used as an efficient
molecular tool for reduction of chosen gene expression;
however implementation of other methods reveals a solution to overcome the barrier of xenosis. Development of
an antiviral vaccine to protect xenotransplant recipients,
using neutralizing antibodies, intracellular-antibodies,
and elimination of PERV from genome using Cre recombinase systems, all together should lead to effective elimination of PERV from xenograft.
Finally, transgenic, multitransgenic and knock-out approaches, new strategies for cloning pigs and for inactivation of porcine genes by gene targeting, (especially
genes that activate xenograft rejection), give opportunity
to make significant progress in xenotransplantation.
116
Abstracts 2008
L5.6
L5.7
Male specific nuclear remodelling
in sheep somatic cells
Tetraploid and diploid gynogenetic blastomeres
as a carrier cells In mouse chimaeric cloning
Lino Loi, Marta Czernik, Grazyna Ptak
Jacek A. Modliński, Paweł Gręda, Maria
Skrzyszowska, Jolanta Karasiewicz
Department of Comparative Biomedical Sciences, Teramo
University, Italy
e-mail: Loi Lino <ploi@unite.it>
The reversibility of the differentiated status in somatic
cells through Somatic Cell Nuclear Transfer (SCNT) has
been demonstrated in experimental, farm and companion
animals. Besides, the manipulation procedures for SCNT
have been simplified, allowing the reconstruction of large
number of embryos. However, such technical progress
has not been paralleled by an improved knowledge on
basic mechanism controlling nuclear reprogramming.
This gap is witnessed by the low efficiency of offspring
production from SCNT, further complicated by post natal mortality, and the still debated reduced life span of
clones. Despite 10 years having passed since the birth of
the first cloned mammal, little progress has been achieved
in reproductive cloning. On the contrary, the prospect to
use nuclear transfer for the production of patient-tailored
stem cell for cell/tissue therapy is progressing rapidly.
Yet, reproductive cloning has many potential implications in animal breeding. In my Seminar I suggest that
the altered epi/genotype found in cloned embryos arises
from an unbalanced nuclear reprogramming between
parental chromosomes. Probably, the oocyte reprogramming machinery, devised for resident chromosomes,
does not recognize the paternal alleles in a somatic cells. I
shall present in the meeting our approach to balance this
asymmetry through the transient expression in donor
cells of chromatin remodelling proteins physiologically
expressed during spermatogenesis, in order to induce a
male specific chromatin organization to the somatic cells
before nuclear transfer. We demonstrate that the expression of a mouse testis specific protein, Bromo-Domain
Testis specific (BRDT) in sheep fibroblast induce a robust
chromatin remodelling.
Department of Experimental Embryology, Institute of
Genetics and Animal Breeding, Polish Academy of Sciences,
Jastrzębiec, Poland
e-mail: Jacek Modliński <J.A.Modlinski@ighz.pl>
It is known that, at least in the mouse and rabbit, enuclated ½ blastomeres can be used as recipient cells in
mammalian embryo cloning. Also, the technique of supporting the development (within one 2-cell embryo) of
one ½ blastomere (reconstituted with fibroblast nucleus)
with the second normal diploid blastomere resulted in
obtaining rabbits totally derived from the reconstituted
blastomere. Additionally, it was shown that supporting
of single ¼ blastomeres with tetraploid carrier blastomeres (twice enlarged) can yield mice totally derived
from the former, indicating that in such chimaeras whole
ICMs forms from a donor ¼ blastomere. In the mouse,
tetraploid and diploid gynogenetic embryos can develop
to the early postaimplantation stages. It is also known,
that in 2N/4N chimaeras the tetraploid cells are gradually eliminated from embryonic tissues, but can persist
in foetal membranes. The aim of our experiment was to
investigate if tetrapoid and diploid gynogenetic blastomeres can be used as supporting cells in the development
of chimaeric 2-cell embryos in which one 1/2 blastomere
was reconstituted with embryonic/somatic nucleus. Tetrapoid embryos (TE) were produced either by treatment
of very late zygotes with cytochalasin B or by electrofusin
of both blastomeres in 2-cell diploid embryos. For obtaining diploid gynogenetic embryos (DGE), zygotes from
which male pronucleus has been previously removed by
means of selective enucleation (SE) were treated (during
the first cleavage) with cytochalasin B. One blastomere in
the resulting TE and DGE 2-cell embroys was selectively
enucleated and reconstituted with embryonic or somatic
nuclei. The preliminary results indicates that in both
cases some of those chimaeric embryos are able to form
morphologically normal blastocysts.
Acknowledgements:
This study was financed by Scientific Net “Biotechnology of Animal Reproduction” founded by Ministry of Science and Higher
Education.
Vol. 55 EuroBiotech
2008
L5.8
The use of original method of chimeric
somatic cell cloning to create geneticallytransformed embryos/offspring in rabbits
Maria Skrzyszowska1, Marcin Samiec1,
Zdzisław Smorąg1, Ryszard Słomski2,3,
Robert Kalak2, Jacek A. Modliński4
1Department
of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland; 2Department of Biochemistry and Biotechnology,
Agricultural University, Poznań, Poland; 3Institute of
Human Genetics, Polish Academy of Sciences, Poznań,
Poland; 4Department of Experimental Embryology, Institute
of Genetics and Animal Breeding, Polish Academy of Sciences,
Jastrzebiec, Wólka Kosowska, Poland
e-mail: Maria Skrzyszowska <mskrzysz@izoo.krakow.pl>
We wanted to evaluate the capability of a new source
of recipient cytoplasm, different from in vivo- and in
vitro-matured (meiotic metaphase II-arrested) oocytes,
to support the development of reconstructed rabbit
embryos by donor nuclear genome of transgenic somatic cells. Therefore, we decided to use the enucleated intracellular environment (i.e., blastoplast) of 2-cell
embryo-derived blastomeres in the somatic cell nuclear
transfer procedure. In consequence, we developed the
novel technique that led to the production of rabbit chimeric genetically-engineered embryos and offspring
because the donor nuclear transfer took place within
only one blastomere of 2-cell embryos, while the second
one remained intact. Our experimental system, which
was designed for somatic cell cloning, involved the use
of a Tg(Wap-GH1) transgenic female rabbit as a donor
of ear skin-derived fibroblast cell nuclei in the novel
strategy of chimeric cloning. Another method of generation of chimeric transgenic rabbits has been recently
developed by Matsuda et al. (Cloning Stem Cells, 4: 9–19,
2002). In this case, 1-5 blastomere groups isolated from
cloned embryos at morula stages, which had been reconstructed with eGFP gene-transfected fetal fibroblast
donor cell nuclei, were re-aggregated with the cells of
4-8-blastomere-staged embryos originating from fertilized eggs. In conclusion, the cytoplasmic microenvironment of enucleated blastomere (blastoplast) from
rabbit 2-cell embryo was successfully used as a source
of recipient cytoplasm for the somatic cell nuclear genome, enabling the development of the partially reconstructed embryo to be supported by it not only to the
blastocyst stage but also up to term. It indicates that
transgenic adult dermal fibroblast cell nuclei underwent the complete and correct epigenetic reprogramming in the cytoplasm of dividing blastoplast-derived
cells of the 2-cell embryo. The frequency for occurrence
of this process was rather low, because the percentage
of offspring generated by the novel method of chimeric
somatic cell cloning was similar to that obtained commonly using the standard somatic cell cloning procedure. Furthermore, leaving the second embryonic cell
intact improved the structuro-functional quality of the
117
partially reconstructed embryo and allowed the newborn female rabbit to be healthy. The novel technique
of chimeric embryo production can also be considered
as an alternative possibility for the generation of transgenic animals.
118
Abstracts 2008
L5.9
L5.10
Developmental competence of immature
mouse oocytes upon nuclear transfer
Comparison of in vitro developmental
competences and apoptosis occurrence among
the porcine cloned embryos generated using
different methods of oocyte activation
Jacek A. Modliński, Abd-El Nasser
Mohammed, Jolanta Karasiewicz
Department of Experimental Embryology, Institute of
Genetics and Animal Breeding, Polish Academy of Sciences,
Jastrzębiec, Poland
e-mail: Jacek Modliński <zsmorag@izoo.krakow.pl>
In this study selectively enucleated (SE) mouse germinal
vesicle (GV) oocytes were fused to blastomere karyoplast
or foetal fibroblasts (FF). Maturation of reconstituted
oocytes was studied, as well as their pronuclei formation
and preimplantation development after activation or in
vitro fertilization. Earlier studies have shown that activation of completely enucleated (CE) GV oocytes reconstructed with FFs nuclei resulted in formation of pronuclei without visible nucleoli, contrary to pronuclei formed
from 1/8 blastomere nuclei. Since I the course of our experiment a possible role for nucleoli has emerged, therefore enucleolation of GV oocytes was also performed to
find out if nucleoli were indeed indispensable. Timing of
maturation division in SE GV oocytes, but not in CE GV
oocytes, was like in control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomere nuclei developed nucleolated donor pronuclei,
contrary to CE oocytes. SE oocytes reconstructed with
FFs nuclei contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that
the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes
reconstructed with 1/8 blastomere or FFs nuclei failed to
cleave after activation or in vitro fertilization. Control GV
oocytes enucleolated before fertilization seized cleavage
at the 6–8 cell stage, as oppose to intact oocytes, which
in 51% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the
cleavage divisions. Activation of cumulus-enclosed SE
GV oocytes matured in hormone-supplemented medium
and fused to ½ blastomere karyoplasts, yielded morulae
and blastocysts in 45.5% and 23.4% respectively. It cabn
be safely suggested that cumulus cells contributed to succesfull preimplantation development of SE GV oocytes
reconstructed with ½ blastomere nuclei.
Acknowledgements:
This study was financed by the IGAB PAS project (S.III.1.3) and
the Scientific Net “Biotechnology of Animal Reproduction”
founded by the Ministry of Science and Higher Education.
Marcin Samiec, Maria Skrzyszowska
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland
e-mail: Marcin Samiec <msamiec@izoo.krakow.pl>
The aim of the study was to determine the effect of different methods of artificial activation of porcine somatic cell
nuclear-transferred (SCNT) oocytes on the embryo developmental rates and the initiation of apoptosis processes
in the cells of the blastocysts generated. The enucleated
in vitro-matured oocytes were reconstructed with the nuclei of either non-apoptotic adult dermal fibroblast cells
(Group I) or fetal fibroblast cells (Group II). Afterwards,
SCNT oocytes were stimulated using the simultaneous fusion and electrical activation (SF-EA; Groups IA and IIA)
or sequential physicochemical activation (S-PCA; Groups
IB and IIB). Post activation treatments, cloned embryos
were cultured for 6–7 days up to morula and blastocyst
stages. At the end of the in vitro culture, nuclear transfer-derived blastocysts were evaluated intra vitam with
the use of diagnostic conjugate of annexin V and eGFP
protein for the presence of proapoptotic changes in the
cell plasma membrane. The percentages of in vitro cultured cloned embryos that reached the morula and blastocyst stages were 148/294 (50.3%) and 48/294 (16.3%) or
162/271 (59.8%) and 82/271 (30,2%) in Groups IA or IIA,
respectively. In Groups IB and IIB, the rates of embryos
that developed to the morula and blastocyst stages yielded 91/210 (43.3%) and 29/210 (13.8%) or 113/197 (57.4%)
and 45/197 (22.8%), respectively. As measured in relation
to a total number of blastocysts analyzed on apoptosis,
the proportions of embryos, in which annexin V-eGFPpositive cells were detected, were 26.5% (9/34) and 28.3%
(13/46) in Groups IA and IIA, respectively. The frequencies of the embryos with diagnosed apoptotic symptoms
of the cells were 34.5% (10/29) and 37.8% (17/45) in the
populations of blastocysts from Groups IB and IIB, respectively. In conclusion, regardless of the methods used
to activate the SCNT oocytes, the competences of fetal fibroblast cell nuclei to direct the in vitro development of
porcine cloned embryos to morula/blastocyst stages were
higher than those for adult dermal fibroblast cell nuclei.
Furthermore, the SF-EA resulted in higher preimplantation developmental potential of SCNT embryos than the
S-PCA. It has been also shown that lower percentage of
blastocysts, in the cells of which the proapoptotic changes
were detected using the annexin V-eGFP, was obtained
from SCNT oocytes stimulated by the application of the
SF-EA protocol than from these ones stimulated by the
use of the S-PCA protocol.
Vol. 55 EuroBiotech
2008
L5.11
The effect of molecular composition of the
sperm membranes on the cryopreservation
of bull semen: incredible egg yolk
Andre T. Palasz
Ministry of Science and Innovation, Department of Animal
Reproduction, INIA, Madrid, Spain
e-mail: Andre Palasz <Palasz@inia.es>
Cryopreservation of spermatozoa is an integral and essential procedure used in assisted reproduction technologies
of animals and humans. Yet, procedures used to freeze
semen have changed very little in the past 50 years and at
present, even with the best preservation techniques, postthaw sperm survival remains approximately 40 to 50%
of the sperm population in the bull. Two major problems
have always been the formation of damaging ice crystals
and development of high concentrations of salts, which
are damaging, to the sperm cells. This can be partially
overcome by the addition of molar solutions of permeating cryoprotectants such as glycerol (Mazur 1984). However, attempts to cryopreserve sperm in glycerol only
without the addition of egg yolk were shown to be highly
inefficient (Hallak et al., 2000). When bovine serum albumin (BSA) was substituted for egg yolk, post-thaw semen
viability was very low (Grizard et al., 1999). Specifically
chicken egg yolk, rich in phosphatidyl choline (PCH) is
routinely used in extenders for the cryopreservation of
semen from domestic animals (Vishwanath, 2000) and
humans (Jeyendran, 2008). However, despite improvements in collection and processing, including filtration
and gamma radiation, all components of animal origin
are potentially contaminated with viruses and other infectious agents (Rossi et al., 1990). Utilizing egg yolk in
semen extender is of particular concern; according to the
USDA, there were 900000 cases of Salmonella Enteritis
(SE) food poisoning originating from chicken eggs in
U.S.A. in 1999 and only 40000 from all food sources (Stadelman, 1999). Similarly, the potential transmission of the
Newcastle Disease virus provides a strong argument for
the elimination of egg yolk from semen cryopreservation
procedures.
It is noteworthy that the major phospholipid of mitochondria and two other sperm membrane with double
phospholipids bilayer (sperm head and nucleus) is phosphatidyl choline (Parks & Lynch 1992) and as alluded earlier, PCH is present in high concentrations in egg yolk of
chicken but not other avian species (Surai et al., 1999).
Recent results suggest that further progress in improving
the efficacy of semen freezing, requires an understanding of sperm membrane dynamics including specific lipid
composition, distribution in outer and inner sperm membranes, lipid-proteins and lipid-lipid interactions at both
physiological and low temperatures (Cerolini et al., 2001;
He et al., 2001). Cold shock, a phenomenon which is considered to be a main source of cryoinjury, is induced by
sudden cooling, seems to be correlated with membrane
lipid composition. Sperm from different animal species
with similar cold shock resistance have membranes with
a similar chemical nature (Parks & Lynch, 1992). Sper-
119
matozoa metabolize endogenous phospholipids during
in vitro storage (Scoot & Dawson 1968), and a significant
decrease in phosphatidylcholine, in particular, was observed during cooling of turkey spermatozoa (Douard
et al., 2000). At the same time, it was demonstrated that
mammalian membranes can readily modify and take up
exogenous lipids including PCH found in chicken egg
yolk (Spector & Yorek, 1985). Phospholipid-mediated
transfer has been successfully used to incorporate exogenous lipids into human (Arienti et al., 1997), bull (Streiner
& Graham, 1997) and boar (He et al., 2001) spermatozoa.
Although, some of these studies did not show improvement in post-thaw viability of semen but shows that cell
membrane composition can be manipulated and thermo
behavior of such manipulated cells can be changed (Zeron et al., 2000). Those findings were effectively employed
in the preparation of presently available bull semen extenders like Andromed and Biociphos that utilizes plant
origin lipids as an egg yolk substitute. Both extenders
eliminate potential of contamination by pathogens and
viruses but according to recent studies (Thun et al., 2002;
Muiño & Peña, 2007) showed to be less efficacious than
extender supplemented with chicken egg yolk. This may
provide evidence that specific composition of substitute
lipids doesn’t match composition of egg yolk lipids or/
and the physical parameters and homogeneity of produced vesicles are inferior to egg yolk lipids.
Phospholipids in low lipid/water ratio have a very low
critical micelle concentration and naturally produce similar
to biological membranes bilayer structures. However, efficacy of this naturally occurring phenomenon is highly depended on the composition and quality of produced lipids
liposomes. The use of different plant lipids preparations
procedures, surface active components that have abilities
to modify phospholipids vesicles, different antioxidants of
biological and non biological origin and different buffers
use in bull semen extenders on post-thaw semen viability
in vitro and in vivo will be discussed.
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1094.
Vishwanath R, Shannon P (2000) Anim Reprod Sci 62: 23–53.
2008
L5.12
Recent advances in cryopreservation of
mammalian oocytes and embryos
Barbara Gajda, Zdzisław Smorąg
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland
e-mail: Barbara Gajda <bgajda@izoo.krakow.pl>
During the past few decades a significant progress in
cryopreservation of mammalian oocytes and embryos
has been achieved. Live young of at least twenty-five
species have resulted from transfer of cryopreserved
embryos or oocytes. Although cryopreservation of certain mammalian embryos is now a routine procedure,
considerable differences in efficiency exist depending on
the origin of embryos (in vivo or in vitro produced), stage
of development and species. In vitro produced embryos
show a chilling and freezing sensitivity associated with
their lipid content, which can be modified by culture conditions. Ddifferences in cryotolerance between species at
the same stage of development, but in the some species
at all stages of development are observed. For example,
porcine embryos are still among the most difficult objects
to be cryopreserved. So far, two major methods have been
used for oocyte and embryo cryopreservation: conventional slow-rate freezing and vitrification. Conventional
slow freezing represents the first system used for embryo cryopreservation. In this system controlled cooling
rates allow extracellular and intracellular water exchange
without serious osmotic effect and change in cell shape.
This technology has been used successfully for embryo
cryopreservation in various species. Conversely, poor results have been reported for cells more sensitive to chilling such as oocytes of different species and pig oocytes
and embryos. An alternative methods of cryopreservation is vitrification which uses very high concentration
of cryoprotective agents and rapid cooling rates resulting in solidification of solutions. Vitrification is a simple
technology, potentially faster and inexpensive. However,
its use has been more advantageous with chill-sensitive
cells. This review summarizes the progress in cryopreservation of mammalian oocytes and embryos which has
been achieved by using the modification of susceptibility to cryopreservation or vitrification technology such
as: open pulled straw method, electron microscopy grids,
cryoloops, nylon mesh, solid-surface vitrification, microdrop method or centrifugation prior to cryopreservation,
addition of protein, cytoskeleton-stabilizing agents, and
liposomes or application of high hydrostatic pressure.
Vol. 55 EuroBiotech
2008
121
L5.13
L5.14
Molecular characteristics of ancient DNA
samples of aurochs (Bos primigenius)
ART strategies for safeguarding of
endangered breeds and species
Ryszard Słomski1,2,3, Daniel Lipiński1,2, Marlena
Szalata1,2, Joanna Zeyland1, Mirosław S. Ryba3,
Zdzisław Smorąg4, Alexander M. Dzieduszycki3
Jacek A. Modliński1, Paweł Gręda1, Ryszard
Słomski2, Zdzisław Smorąg3, Jolanta Karasiewicz1
1Department
of Biochemistry and Biotechnology, Poznan
University of Life Sciences, Poznań, Poland; 2Institute of
Human Genetics, Polish Academy of Sciences, Poznań,
Poland; 3Polish Foundation for Restoration of the Aurochs,
Warszawa, Poland; 4Department of Animal Reproduction
Biotechnology, National Research Institute of Animal
Production – National Research Institute, Balice, Poland
e-mail: Ryszard Slomski <slomski@up.poznan.pl>
Aurochs (Bos primigenius) was a unique species lasting
through Pleistocene and Holocene for about two million
years. The aurochs was one of the largest animals ever to
inhabit Europe. The range varied over the time it lived
in almost whole Europe except the north most region, in
Asia never crossing Southern Siberia northwards and a
very northern strip of Africa, going little south along the
Nile River. First it disappeared from the Far East, later
from South and Central Asia, than Middle East and Africa. Finally it vanished from southern Europe at the latest
during Roman period. Starting from early Middle Ages
it became extinct from the western and eastern European
lands. The longest the species survived in the very Central Europe (presently Poland), where in spite of being
taken under quite modern protection the last aged cow
died in the year 1627. The most often brought up reason
for dying out of the Aurochs is hunting and pouching.
Another belief is, that growing agriculture pushed them
away further from human sites.
The study on the ancient DNA is quite a challenge. Although more and more research centers undertake the
task, there is still little knowledge on this matter. After
finding and selecting the right material it has been used
to further analysis, i.e. isolation of the confirmed aurochs
DNA. That was the most important step followed by cloning of aurochs DNA in bacterial system for further analysis encompassing DNA sequencing and comparison with
the existing bovine database in search for any similarities
and specific genes. Some of the results have been already
published. Studies of aurochs help also understanding
history of species, its relation to other species and perhaps will help preventing extinction of other animals.
Acknowledgements:
This work was financially supported by Biology of Reproduction
Network, Warsaw, Poland.
1Department
of Experimental Embryology, Institute of
Genetics and Animal Breeding, Polish Academy of Sciences,
Jastrzębiec, Poland; 2Institute of Human Genetics, Polish
Academy of Sciences, Poznań; National Institute of Animal
Production, Balice, Poland
e-mail: Jacek Modliński <J.A.Modlinski@ighz.pl>
The last two centuries have seen a dramatic decline and
extinction of animal species (including several breeds of
domestic animals) and the consequence of these phenomenon is progressive contraction in biodiversity worldwide.
For example, the survival of most wild species in the
Bovidae family and also most species in the Felidae and
Cervidae families is considered by IUCN-WCU as threatened or endangered. The ideal and effective conservation
strategy should involve, of course, the preservation of an
entire environment with all species living in. In certain
cases, however, a specific solutions may be required to
save a particular seriously endangered species. The recent development of ART, such as assisted fertilization,
production of embryos in vitro, freezing gametes, embryos and somatic cells and establishing the genetic banks,
interspecies embryo transfer and somatic cloning of wild
mammals ( gaur, mouflon, banteng, white-tailed deer, African wildcat, wolf), suggests that these techniques could
have clear benefits for conservation biology.
Using the techniques worked out in our laboratory for establishing of several embryo and foetal -derived cell lines
of various livestock species we generated fibroblast cell
lines from Polish endangered sheep breeds and from various rare Cervidae species including milu (Elaphurus davidianus), Thorold’s deer (Cervus albirostris) and also two
extremaly rare mutant strains of red deer (C. elaphus L.),
namely the St. Hubert white deer and the white blazed
deer. Foetal fibroblast from an endangered wrzosówka
sheep breed (Polish Heathehead Sheep) has been successfully used for generation sheep embryonic-somatic chimaeras. The somatic cell lines were also established post
mortem from the skin, ovary and follicular cells of adult
European bison (Bison bonasus bonasus) cow and from the
skin of adult bull. All samples were frozen at passage 1-3
and are stored in liquid nitrogen. The obtained cell lines
are used as a source of donor cells in our experiments
concerning the cloning of European bison. Skin fibroblast nuclei were microsurgically injected into selectively
enucleated bovine zygotes produced in vitro. Selective
enucleation (SE), i.e. removing the chromatin attached
to pronuclear envelope and leaving in the cytoplast nucleoli and liquid contents of pronuclei, has been previously applied in our laboratory to mouse zygotes, leading
to successful embryonic cloning from 1/8 blastomeres, a
task claimed earlier impossible. Bovine zygotes reconstituted with bison somatic nuclei developed in vitro to the
blastocyst stage. The obtained blastocysts were morphologically normal with clearly visible, well developed in-
122
Abstracts ner cell mass. According to our best knowledge, it is the
first attempt to clone European bison using interspecies
somatic cloning technique.
Acknowledgements:
This study was financed by the IGAB PAS project and the Scientific Net “Biotechnology of Animal Reproduction”
2008
L5.15
Application of biotechnological
methods in animal genetic resources
conservation programmes
Jędrzej Krupiński, Elżbieta
Martyniuk, Barbara Gajda
National Research Institute of Animal Production, Balice,
Poland
e-mail: Elzbieta Martyniuk <elzbieta.martyniuk@minrol.
gov.pl>
The ex-situ cryo-conservation of animal genetic resources
provides an important complementary measure to in-situ
and ex-situ in vivo programmes. The Global Plan of Action
for Animal Genetic Resources (FAO, 2007) emphasizes the
need to strengthen ex-situ conservation measures at the
national and regional levels. Enhanced ex-situ conservation requires clear understanding of the current state of
art in the implementation of the various biotechnologies
relevant to the collection and processing of animal genetic
material, which may be used for further reproduction or
reconstruction of individuals. It is also crucial to evaluate the cost effectiveness of current ex-situ conservation
methods and assess the prospects of their refinement over
the next 20-30 years. Understanding current practices,
and assessing the likelihood of significant improvements
in reproductive biotechnology in key livestock species
will enable informed decision-making on the types and
amount of genetic material (semen, oocytes, embryos)
that should be collected and stored from individual donors, to adequately secure their long-term storage and
possible future utilisation. While cryo-conservation is a
proven conservation approach, deep freezing of semen,
oocytes and embryos requires significant financial investments in infrastructure, training of personnel, and substantial operational costs. The first report on the State of
the World’s Animal Genetic Resources (FAO, 2007), made
it clear that most developing countries are lacking the
necessary infrastructure, financial and human resources
to establish national gene banks. To overcome the financial and other barriers, cryo-storage of somatic cells has
been proposed, which are relatively inexpensive to collect and samples can be obtained quickly (Corley-Smith
and Brandhorst, 1999). The pilot study conducted by
Groenevald et al. (2006) demonstrated that collection and
storage of somatic cells could be extremely useful in the
case of rapid genetic erosion and emergency action to
conserve a breed. However, a fundamental question remains. What are the perspectives that breed reconstruction can be cost effectively achieved using somatic cells?
Currently the process of nuclear transfer and cloning is
very costly and its efficiency is relatively low. More precise predictions of rate and speed of enhancement levels
will facilitate selecting conservation approaches that will
best meet both short-term conservation objectives and
long-term use objectives.
Vol. 55 EuroBiotech
2008
L5.16
L5.17
Disease resistant genetically modified
animals – possibilities and perspectives
Transgenic animals as bioreactors
Jacek Jura
National Research Institute of Animal Production,
Department of Animal Biotechnology and Reproduction,
Balice, Poland
e-mail: Jacek Jura <jjura@izoo.krakow.pl>
Infectious disease affects livestock production and animals welfare. Also it has impacts upon both human
health and public perception of livestock production. The
new methods that enables the efficient production of genetically modified animals (GM) to change gene activity
makes the applications of transgenic animals for the benefit of animal and human health increasingly likely. This
technology have specific application where genetic variation does not exist in a given population or species and
where novel genetic changes can be engineered. These
genetically modified animals could be used as a models
to investigate disease progression and evaluate this approach to controlling the disease. In a not distant future
GM animals will be used to complement the traditional
tactics to combat disease, and will provide novel strategies of intervention that are not possible through the traditional approaches. In summary, transgenic animals will
not be the primary tool in the fight against disease but
rather it use will be restricted to specific one.
123
Ryszard Słomski1,2, Daniel Lipiński1,2,
Marlena Szalata1,2, Joanna Zeyland1,
Jacek Jura3, Zdzisław Smorąg3
1Poznan
University of Life Sciences, Department of
Biochemistry and Biotechnology, Poznan, Poland; 2Institute
of Human Genetics, Polish Academy of Sciences, Poznan,
Poland; 3National Research Institute of Animal Production
– National Research Institute, Department of Animal
Reproduction Biotechnology, Balice, Poland
e-mail: Ryszard Slomski <slomski@up.poznan.pl>
Recombinant proteins can be produced in various
prokaryotic or eukaryotic systems and are derived from
bacterial cells, yeast cells, transformed plant and animal
cells, and even from live bioreactors (transgenic animals).
Transgenic farm animals such as cows, pigs, sheep, goats
or rabbits are employed as bioreactors because of their
large scale production potentials, possibilities of obtaining correct glycosylation and post-translation modifications, low maintenance costs, rapid production of transgenic founders as well as high efficiency of expression. A
great number of proteins of pharmaceutical significance
are currently being synthesized in milk and these investigations are at various levels of progress beginning with
the testing of the idea itself and ending with pre-clinical
and clinical tests. Antithrombin III obtained from two
transgenic goats already reached the market as Atryn.
When selecting a transgenic bioreactor, it is necessary to
take into account proteins needed in one year, breeding
and processing potentials, utilization of the recombinant
proteins as well as the time needed to manufacture milk
and its required volume. Transgenic mice provide a good
model needed to determine the usefulness of gene construct as well as in studies of the properties of recombinant proteins but not for their production. Therefore,
they can be used to evaluate the gene construction prior
to the microinjection into farm animals which is more
laborious. Milk is considered as an excellent system for
studying the expression, purification and efficiency of the
applied methods as compared to the expression of proteins in prokaryotic systems. Transgenic rabbits are very
efficient as intermediate in size animals for the production of recombinant proteins at relatively large scale. We
have obtained double transgenic animals expressing human growth hormone (hGH) and human alpha1,3-fucosyltransferase in mammary glands of transgenic female
rabbits. No effect of expression of recombinant proteins
on phenotype and behaviour of females and offspring
were observed. This work was financially supported by
Biology of Reproduction Network, Warsaw, Poland
124
Abstracts L5.18
L5.19
Application of nanotechnology
in animal transgenesis
Biotechnology of the mammary gland expression of milk protein genes and other
genes active in the mammary tissues
Marlena Szalata1,2, Ryszard Słomski1,2
1Poznan
University of Life Sciences, Department of
Biochemistry and Biotechnology, Poznań, Poland; 2Institute
of Human Genetics, Polish Academy of Sciences, Poznań,
Poland
e-mail: Marlena Szalata <slomski@up.poznan.pl>
Nanotechnology is interdisciplinary science, using material and devices built at nanoscale. Nanotechnology cuts
across many disciplines, including physical chemistry,
physics, medicine, veterinary, colloidal science, chemistry, supramolecular chemistry, applied physics, materials
science, and even mechanical and electrical engineering.
It eencompasses biological sciences and basic research
with products applications. Nanotechnology can be applied for cellular biology and medicine, for directed interactions at basic molecular level with high degree of
specificity. In animal biotechnology can be achieved improvement of transgenesis methods by using very precise
delivery of transgene - new genetic information to nucleus. A nanocarrier system incorporated with stimuli-responsive property (e.g., pH, temperature, or redox potential), would be amenable to address some of the systemic
and intracellular delivery barriers. Small molecules and
genes into cells can be delivered using a microfluidic electroporation technique based on constant direct current.
Simple microscale electroporation devices will have the
potential to work in parallel on a microchip platform and
such technology will allow high-throughput functional
screening of drugs and genes. Nanotechnologically prepared transgenic cell synthesizing deficient enzymes and
hormones for regeneration and improvement of natural
physiology processes can be use for animal transgenesis.
Important issue is in vivo monitoring of transgene using
nanotechnologies with aptamers, quantum dots and molecular beacons. It should be also possible to detect any
transgenic transcript and protein, by designing complementary nucleic acids (for mRNA), antibodies or DNA
aptamers (for proteins) along with an appropriate fluorescent reporter molecule. While the investigation and
application of miniaturization technologies to medicine
and biology is progressing rapidly, there has been limited
exploration of microfabricated systems in the area of embryo production. Microfluidics is an emerging technology that allows a fresh examination of the way assisted
reproduction is performed. Microfluidic systems for in
vitro embryo production (IVP) and embryo manipulation
could have a dramatic impact on the development of new
techniques as well as on our basic understanding of gamete and embryo physiology.
2008
Lech Zwierzchowski, Tadeusz Malewski*
Institute of Genetics and Animal Breeding Polish Academy of
Sciences, Jastrzębiec, Poland; *present address: Museum and
Institute of Zoology, Polish Academy of Sciences, Warszawa,
Poland
e-mail: Lech Zwierzchowski <L.Zwierzchowski@ighz.pl>
In the mammary gland, besides of genes encoding caseins
and whey proteins, many other genes are expressed. Cloning of mammary cDNAs from Holstein cattle (ORESTES
program) revealed 6.481 sequences specifically expressed
in the mammary gland and 286.247 ESTs (expression sequence tags). Our search in the Internet resources showed
160 genes expressed specifically in the cow’s mammary
gland. The main milk proteins are caseins. In cattle four
casein genes — αS1, αS2, β and κ — constitute the casein
locus, also containing other genes — stath, his1, fdc-sp. We
identified two bovine BAC clones encompassing the whole
casein locus, and also the stath gene. In the cow’s mammary
gland the expression of the stath gene, together with the
casein genes, occurs exclusively in lactation, suggesting
common regulatory mechanisms.
Recently, gene expression profiling with microarray techniques became very popular. DNA-chips have been used to
analyze gene expression during mammary gland growth
and differentiation. Our study showed that in mice, during
transition from lactation to involution activated is expression of many genes, some with the role in the mammary
cell apoptosis yet unknown. In cattle, transcriptomics is
used to search for genes-candidating for markers of milk
production capacity (QTLs), e.g. by showing their differential expression between cattle of different production types
— dairy or meat. Gene expression profiling in HF and
Hereford cattle showed that previously unexpected genes
may be associated with dairy performance. That may lead
to the development of new molecular indicators of cattle
productivity, so called expression QTLs (eQTLs).
The main goal for the mammary gland biotechnology is
production of bio-pharmaceuticals and nutriceuticals in
transgenic animals. Moreover, other modifications of the
ruminants’ milk are in progress to improve nutritional and
technological properties of the milk. Modifications of the
milk composition may be done either by over-expression
of selected genes (transgenesis) or inactivating of some
genes (KO, iRNA) expressed in the mammary gland.
Production of bio-pharmaceuticals in the mammary gland
of transgenic animals (molecular pharming) becomes now
a reality. However, as so far only one such pharmaceutical – anti-thrombin III (ATryn, GTC Biotherapeutics) produced by cloned transgenic goats, is formally registered
in Europe. Many other pharmaceutical proteins — α-antitrypsin, α-glucosidase, blood clotting factors — are currently subject to clinical trials. The future of other manipulations done on the mammary gland is difficult to predict,
and need many technical improvements before commercialization of genetically modified milk products.
Vol. 55 EuroBiotech
2008
Posters
P5.2
P5.1
Application of domestic duck microsatellite
markers in measuring of genetic
polymorphism in domestic geese
The preliminary results on mitochondrion
activity in small bovine in vitro cultured oocytes
Lucyna Kątska-Książkiewicz1,2,
Hannelore Alm3, Helmut Torner3
125
Krzysztof Andres1, Ewa Kapkowska1,
Urszula Kaczor2
1Department
1Department
of Poultry and Fur Animals Breeding and
Animal Hygiene, 2Department of Sheep and Goat Breeding,
Agricultural University in Kraków, Kraków, Poland
e-mail: Krzysztof Andres <andreskrzysztof@poczta.onet.
pl>
The aim of the investigation is determination of mitochondrial distribution and activity in non-cultured and in
vitro cultured oocytes recovered from early antral ovarian
follicles in cattle. Small, immature bovine oocytes are recovered from slaughtered animals by isolation from small
fragments of ovarian cortex of early antral follicles with
diameter 0.5 to 0.8 mm. Collected follicles were evaluated
on the basis of their morphological appearance and size
and than were opened under stereomicroscope to freed
complexes of cumulus enclosed oocytes surrounded with
mural granulosa cells (COCGs). COCGs were cultured
in vitro (Alm et al., 2006) for 7 days (Day 7) and 14 days
(Day 14). For the control (Day 0) oocytes recovered from
freshly collected COCGs were fixed and stained. Following in vitro culture COCs with normal appearance were
freed from culture, placed in maturation medium for 24
h and then fixed and stained. The mitochondrial-specific
fluorescent and cell-permeant probe MitoTracker Orange
– fluorescent tetramethylrosamine (M-7510) is readily sequestered only by actively respiring organelles, depending upon their oxidative activity. The 4 experiments were
carried out and total number of evaluated oocytes was
108, i.e. 33 Day 0, 29 Day 7 and 36 Day 14. On the basis
of double staining of analysed oocytes (with MitoTracker
Orange & Hoechst 33258, Torner et al., 2004) we concluded that in vitro culture of COCGs prolonged up to 14 days
affected both mitochondrial distribution and activity as
well as chromatin configuration — the main negative effects manifested by degeneration. The investigations will
be continued on a larger scale and in modified culture
systems. It is expected that evaluation of mitochondrial
activity will allow on the more precise determination of
bovine COCGs in vitro culture conditions.
There is still a lack of genetic markers enabling for biodiversity studies in domestic goose, one of the most important domestic bird species. The aim of the study was to
examine the use of domestic duck microsatellite primers
for detecting of polymorphism in goose on the example
of Zatorska geese, a breed belonging to the protected waterfowl genetic resources in Poland.
Ten DNA primer pairs specific for domestic duck microsatellite loci amplification: APH12, APH13, APH15,
APH16, APH20 and APH23 (Maak et al 2003), and
CAUDG7, CAUDG11, CAUDG12, CAUDG13 (Huang et
al 2005), were used in this study. The set of APH markers were previously tested by Maak et al. (2003) in wild
Greylag and Swan geese giving distinct bands in agarose
gel electrophoresis. Also CAUDG markers were initially
evaluated in cross-species amplification in small number
of goose DNA samples from Chinese farm giving PCR
products of two to three alleles by locus (Huang et al.
2005). The current experiment was performed on DNA
extracted from 100 individuals of Zatorska geese. The
PCR was executed in standard conditions with variable
annealing temperature depending on primer used. The
PCR products were separated in 6% polyacrylamide gels
and visualized by silver staining.
The study shows that primers APH15 and CAUDG11
gave no PCR products of expected size in the Zatorska
geese. Locus APH23 was monomorphic in all analyzed
birds. All remaining loci were polymorphic and showed
medium to low level of polymorphism: four alleles were
observed in one locus (APH20), three alleles in two loci
(CAUDG07, CAUDG12), and two alleles in four remaining loci. The mean expected heterozygosity of seven polymorphic alleles averaged of 0.42, with the range from 0.25
(CAUDG07) to 0.53 (CAUDG12). The polymorphic information content (PIC) of those alleles ranged between 0.23
(CAUDG07) and 0.45 (APH20) with the mean being 0.32.
The results revealed that most (70%) of the duck markers
chosen in this paper can be useful in goose biodiversity
studies. It is worth noticed that remaining three markers
were not suitable for Zatorska geese studies, what can
implicate that they could not be useful in microsatellite
studies of every goose breeds with the special emphasis
to those of Polish origin.
of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland; 2Faculty of Biotechnology, University of Rzeszów,
Werynia-Kolbuszowa, Poland; 3Research Institute for the
Biology of Farm Animals, Dummerstorf, Germany
e-mail: Lucyna Kątska-Książkiewicz <lkatska@izoo.
krakow.pl>
References:
Alm H et al (2006) Theriogenology 65: 1422–1434.
Torner H et al (2004) Theriogenology 61: 1675–1689.
References:
Huang Yet al (2005) Genet Sel Evol 37: 455–472.
Maak S et al (2003) Mol Ecol Notes 3: 224–227.
Acknowledgements:
The work was financed by the Ministry of Science and Higher
Education, grant N311 051 32/2819.
126
Abstracts P5.3
P5.4
Analysis of polymorphism of CAST
gene in breeds of Slovak Pinzgau cattle
and Charolais by PCR-RFLP method
The influence of biological preparations on
microbial stabilization in gastrointestinal
tract of broiler chickens
Michal Gábor, Anna Trakovická,
Martina Miluchová
Ivana Nováková1, Martina Fikselová2,
Miroslava Kačániová3, Peter Haśčík4
Department of Genetics and Breeding Biology, Slovak
University of Agriculture, Slovakia
e-mail: Michal Gabor <misogabor@yahoo.com>
1,3Department
Meat tenderness is an important issue in beef cattle production because it has a major impact on consumer satisfaction (Miller et al., 1995). The calpain/calpastatin system
is an endogenous, calcium-dependent proteinase system,
theorized to mediate the proteolysis of key myofibrillar
proteins during postmortem storage of carcass and cuts
of meat at refrigerated temperatures (Koohmaraie et al.,
1995b).
The calpastatin (CAST) gene is the member of the calpains/calpastatin proteolytic system and inhibits μ-calpain — and m-calpain activity and, therefore, plays key
role in the tenderization process. Increased postmortem
CAST activity has been correlated with reduced meat tenderness (Koohmaraie et al., 1995a; Pringle et al., 1997).
The work was oriented to identification of CAST gene
polymorphism and analysis of genotype structure in population of Slovak Pinzgau cattle and Charolais. The polymorphism of CAST gene was studied in a group of 111
heads of cattle (56 steers of slovak pinzgau breed and 55
one-year bulls of charolais breed). Bovine genomic DNA
was isolated by fenol-chlorophorm deproteinization and
ethanol precipitation. The polymorphism of CAST gene
was analyzed by PCR-RFLP method by Schenkel et al.,
(2006).
In the population of cattle of breed Charolais included in
the study was detected homozygote genotype CC with
frequency 38.18%, heterozygote genotype CG with frequency 50.91% and homozygote genotype GG with frequency 10.91%. Results point out that frequency of allele
C was 63.64% and frequency of allele G was 36.36%. In the
population of cattle of breed Slovak Pinzgau cattle included in the study was detected homozygote genotype CC
with frequency 62.50%, heterozygote genotype CG with
frequency 32.14% and homozygote genotype GG with
frequency 5.36%. Results point out that frequency of allele C was 78.57% and frequency of allele G was 21.43%.
Keywords: cattle, PCR-RFLP, calpastatin, CAST gene, frequency
References:
Koohmaraie M et al. (1995a) Expression of Muscle Proteinases and
Regulation of Protein Degradation as Related to Meat Quality. 395–
412, Netherlands
Koohmaraie M et al (1995b) Proc. Meat ´95:Csiro Meat Industry Res.
Con., Session 4A : 1–10, Australia.
Miller MF et al (1995) J Animal Sci 73: 2308–2314.
Pringle TD et al (1997) J Animal Sci 75: 2955–2961.
Schenkel SF et al (2006) J Animal Sci 84: 291–299.
Acknowledgements:
Sources of research financing: project VEGA no 1/4440/07.
2008
of Microbiology, 2Department of Plant
Processing and Storage, 4Department of Animal Product
Evaluation and Processing, Slovak University of Agriculture
in Nitra, Slovakia
e-mail: Ivana Novakova <ivana.novakova@uniag.sk>
Aim of this study was to compare two biological preparations and their efficiency on stabilization of intestinal microorganisms in gastrointestinal tract of broiler chickens.
As a biological material were used broilers Hubbard JV.
The preparations were based on Enterococcus and Lactobacillus strains. They were added daily into the drinking water during the period of feeding. Taking of chyme
samples we have to realize on the end of the feeding. In
the experiments the total enterococci counts on the Slanetz — Bartley agar after 48–72 hours cultivation at 37°C,
number of Lactobacillus on the MRS agar after 72 h after
37°C and counts of Escherichia coli on the McConkey agar
after 24–48 h after 37°C were determined. We have statistically compared counts of Lactobacillus sp., Enterococcus
sp. and Escherichia coli. The positive effect after probiotic
addition was the increase of Lactobacillus sp. and Enterococcus sp. counts and reduction of Escherichia coli count in
the groups where were added the biological preparations
against the groups without addition of the preparations.
Keywords: biological preparation, microbial stabilization, GIT,
chickens.
Vol. 55 EuroBiotech
2008
127
P5.5
P5.6
The influence of selected factors on
the lenght of productive life
Expression of IGF1, IGF2, IGFBP3 and IGFBP5 in
muscles of pigs representing five different breeds
Eva Strapáková1, Peter Strapák2
Barabara Rejduch1, Maria Oczkowicz2,
Małgorzata Witoń2, Agata PiestrzyńskaKajtoch1, Katarzyna Walinowicz2
1Department
of Genetics and Breeding Biology, 2Department
of Animal Husbandry, Slovak University of Agriculture in
Nitra, Slovak Republic
e-mail: Eva Strapáková <Eva.Strapakova@uniag.sk>
The analysis of the some effect to length of productive
life was done in 181001 culled cows of Holstein breed
and in 114020 culled cows of Slovak Simmental breed. In
both breeds the most important factor was the Sire (F =
65.36 +++ in Holstein breed, F = 53.43 +++ Slovak Simmental breed) and the farm effect (F = 49.29 +++ in Holstein
breed, F = 26.32 +++ in Slovak Simmental breed). The factor of milk production at first lactation (F = 30215.0 +++)
and breeding group (F = 3195.74 +++) affirm direction of
selection for intensive increasing of milk production in
Holstein breed. The milk production at first lactation was
important factor in cows of Slovak Simmental breed too
(F = 7736 +++). The lowest effect to length of productive
life was recorded in the age at first calving (F =454.61 +++
respectively F = 2759.81 +++) and the year of culling (F =
4004.61 +++, respectively F = 26.32 +++). The test of effect
of factors affecting length of productive life in Holstein
and Slovak Simmental breeds confirm the most important factors are the Sire, farm, and milk production.
1Department
of Immuno and Cytogenetics, 2 Department of
Animal Breeding and Genetics, National Research Institute of
Animal Production, Poland
e-mail: Oczkowicz Maria <majawrzeska@o2.pl>
IGF1, IGF2, IGFBP3 and IGFBP5 are part of somatotropic
axis and play a crucial role in growth and development.
They are expressed mainly in liver, but also in many other
tissues (muscles, kidney, brain). Insulin like growth factors, their binding proteins and receptors are candidate
genes for meatiness and growth performance of pigs. The
aim of our study was to asses if there are differences in
expression level of IGF1, IGF2, IGFBP3 and IGFBP5 in
muscles of pigs, representing five breeds (Large White,
Landrace, Puławska, Duroc, Pietrain), differing in body
composition and growth performance. Additionally, we
confirmed the effect of A3072G IGF2 mution on expression level, described previously by Van Laere (2003). In
total, we examined 36, two months old gilts. There were
5-6 animals in each breeding group. Total RNA was isolated from longissimus dorsi muscle, using Trizol according to producent protocol. Reverse transcription was
performed with cDNA Archive Kit (Applied Biosystems).
Real-Time PCR was performed with Taq Man probe. Each
target gene was multiplexed with endogenous control
(18SRNA). All Real-Time PCR reactions was performed
in triplicate on 7500 Real-Time PCR System (Applied Biosystems). Data was analysed with 7500 SDS v. 1.3 software
and Statistica v. 5.0. A3072G IGF2 mution was genotyped
using allelic discrimination assay, on 7500 Real-Time PCR
System, according to Carrodeguas (2005). Only Pietrain
animals carried paternally derived IGF2 G3072 allel. As
we expected, IGF2 expression level was approximately 45 fold lower in Pietrain than in other breeds. We did not
find any significant differences in expression level of IGF1,
between analyzed breeds. On the other hand, expression
of IGFBP3 was 2-3 fold higher in Pietrain and Puławska
breed than in other breeds. IGFBP5 expression was the
lowest in Duroc (approximatelly 4-fold lower, comparing
with Landrace). Decreased IGF2 expression in Pietrain
was probably the effect of paternally derived G allel. Further studies are being conducted in order to establish if
observed differences in expression level of IGFBP3 and
IGFBP5 between breeds have biological significance.
References:
Van Leare AS et al (2003) Nature 425: 832–836.
Carrodeguas JA et al (2005) Meat Sci 71: 577–582.
128
Abstracts P5.7
P5.8
Generation of nuclear-transferred rabbit
embryos using handmade cloning method
Polymorphismus ofcasein complex
for human nutrition
Yuriy Kosenyuk, Maria Skrzyszowska
Zuzana Slobodová, Zuzana
Čanakyová, Alojz Kúbek
National Research Institute of Animal Production,
Department of Biotechnology of Animal Reproduction, Balice,
Poland
e-mail: Yuriy Kosenyuk <kosenyuk@izoo.krakow.pl>
Nuclear transfer (NT) is a complex procedure that requires
considerable technical skills. Recently, NT techniques
have been simplified by the use of zona-free oocytes and
developed for somatic cell NT without micromanipulation, i.e. handmade cloning. The aim of the present study
was to determine incorporation modified chemically assisted enucleation in the handmade cloning protocol for
rabbit and to investigate the zona-free embryo development in vitro.
In the somatic cell cloning procedure the in vivo matured
oocytes were used as source of recipient cells. Mature
New Zealand female rabbits were superovulated by injection of 100 IU of PMSG (Serogonadotropin, Biowet)
then after 72 h injected intravenously with 100 IU of hCG
(Biogonadyl, Biomed) to induce ovulation. Ovulated
oocytes were recovered from the oviducts 17-18 h after
hCG injection by flushing with Dulbecco’s PBS. Enucleation of Metaphase II-staged oocytes was accomplished
by demecolcine-induced modified hand made method.
The modified based on zona-free oocyte elongation before cutting chromosomes localization place. The sources
of nuclear donor cells were foetal fibroblasts. Zona-free
cytoplasts were individually washed for a few seconds in
100 μg/ml phytohemagglutinin P in PBS and then quickly
dropped over a single donor cell settled to the bottom of a
microdrop of the diluted donor cell suspension. Formed
cell couples were fused by 3 DC pulses of 2.5 kV/cm for
20 μs each. The reconstructed oocytes were incubated in
B2 medium for 1 h and subsequently treated with 5 μM
calcium ionomycin for 5 min to be artificially activated.
Then nuclear-transferred oocytes were incubated in B2
medium supplemented with 10 μg/mL cycloheximide for
1 h. The zona-free cloned embryos were cultured in the
wells of the well (WOW) system in 20 μl microdrop of B2
medium.
Development abilities of rabbit nuclear-transferred embryos were assessed by cleavage rate. Enucleation rate
was 84.6% (121/143), fusion rate was 95.1% (98/103). Out
of 97 reconstructed oocytes, 55 (56.7%) NT embryos were
cleaved. The frequencies of cloned embryos, that reached
the blastocyst stages, were 1/97 (1.0%). In conclusion, the
presented data indicate the viability of rabbit zona-free
NT embryos, which were derived from oocytes activated
using ionomycin and cycloheximide, were able to develop in vitro to morula and blastocyst stages. The absence of
the zona considerably facilitates the enucleation step and
significantly increases cell fusion rate.
2008
Slovak University of Agriculture, Nitra, Slovakia
e-mail: Zuzana Slobodová <zslobodka@gmail.com>
Main medical opinion are that milk is a nutritious food
of benefit to most people. One particular protein, called
A1 β-casein, which is present in the milk of some but
not all cows, is linked to type 1 diabetes, heart disease
and symptoms of autism and schizophrenia. It may be
implicated in a particular range of auto-immune conditions. This protein is also linked to milk intolerance in
some people. Variants A1 and A2 of β-casein are usually
between many dairy cattle breeds. A1 may play a role in
the a etiology of human illness. A1 may play a role in the
a etiology of human illness. A1 β-casein releases bovine
β-casomorphin7 (BCM7) on digestion. The amount and
stability of BCM7 that is cleaved from A1 beta-casein is
linked to the presence and levels of various enzymes, in
particular dipeptidyl peptidase 4. BCM7 is a strong opioid. Epidemiological records from New Zealand argument
says that consumption of β-casein A1 is associated with
higher national mortality rates from ischaemic heart disease. Therefore, careful attention should be paid to that
protein polymorphism, and deeper research is needed to
verify the range and nature of its interactions with the
human gastrointestinal tract and whole organism. Milk
and milk products are important for prophylaxis and
therapy of various diseases. Fermented dairy products
and yogurts with probiotic bacteria can exhibit protective
function against certain types of cancer. In recent years,
has been much discussion on the use of milk to prevent
cancer.
References:
Kaminski S, Cieslinska A, Kostyra E (2007) J Appl Genet 48:189–
198.
Woodford K (2008) A1 β-casein, Type 1 diabetes, and Links to
other Modern World Illnesses. An invited plenary paper to the
International Diabetes Federation Western Pacific Congress, Wellington, 2 April, 2008. Available at www.lincoln.ac.nz/diabetes.
Zoghbi S, Trompette A, Claustre J, El Homsi M, Garzon J,
Jourdain G, Scoazec J, Plaisancie P (2006) Am J Physiol. Gastrointest Liver Physiol 290: G1105–G1113.
Vol. 55 EuroBiotech
2008
129
P5.9
P5.10
Analysis of polymorphism of beta
lactoglobulin of sheep by PCR-RFLP
Genetically modified soya in rabbit feeding:
detection of the transgene in digestive tract
Martina Miluchová, Anna
Trakovická, Michal Gábor
Weronika Śliwiak1, Dorota Maj1, Piotr
Łapa1, Joanna Pokorska2, Józef Bieniek1
Slovak University of Agriculture, Department of Genetics and
Breeding Biology, Slovakia
e-mail: Martina Miluchova <martina.miluchova@
centrum.sk>
1Department
The work was oriented to identification of LGB gene polymorphism and analysis of genotype structure in population of sheep.
LGB is the major milk whey protein in the ruminants.
This protein, synthesis in the mammary glands during
pregnancy and the lactation stages (Elyasi et al., 2005)
and is important in the evaluation of the milk production
potential and milk fat and protein percentage (Çelik &
Ozdemir, 2006).
AA and AB genotypes are associated with protein and casein content and curd yield. BB LGB genotype sheep were
characterized by a significantly higher protein content of
milk than sheep with the other two LGB genotypes — AA
and AB (Kučinskiene et al., 2005). Bocharev (1998) found
associations between LGB variant AB with higher body
weight, while genotype AA could be linked with sheep
wool density.
The material involved three breeds of sheep: 28 Cigaja, 17
Lacaune and 34 Valaska. Ovine genomic DNA was isolated by salting out method (Miller et al., 1988) and used in
order to estimate LGB genotypes by PCR-RFLP method
(Kučinskiene et al., 2005).
LGB gene in Valaska sheep population the dominance of
heterozygous genotype AB (35.29%) above homozygous
genotype BB (47.06%) was detected. Occurrence of homozygous genotype AA (17.65%) was sporadic, so allele
B (64.71%) dominated above allele A (35.29%). The occurrence of homozygous genotype AA (42.86%) was the most
frequent in Cigaja rams, the homozygous genotype BB
(32.14%) was less frequent. The heterozygous genotype
AB (25%) was detected in lowest numbers in this group of
rams. From these two alleles A (55.36 %) dominated over
allele B (44.64%). In Lacaune breed there were detected
genotypes AA and BB with equal frequency 29.41%. Presence of heterozygous genotype AB (41.18%) was detected
with higher frequency. These results suggest, that alleles
A and B occurred with the same frequency 0.5% in the
population of Lacaune rams.
The aim of this work was to analyze possible influence of
genetically modified (GM) soya addition on the speed of
genes degradation in digestive tract of rabbits and to detect transgene cp4 epsps in contents taken from three significant parts of the tract (stomach, caecum and rectum).
The study was carried out on 24 White Termond rabbits,
divided in 3 groups. Each group was fed in the different
way: first by normal complete pelleted fodder [control
group], second by pellets with 20% soya component (convectional, solvent extract), third by pellets with the same
quantity of GM soybean (solvent extracted). The presence
of DNA fragments in contents of digestive tract of rabbits
was investigated by using the polymerase chain reaction
(PCR) approach. Not only a fragment of transgene (128
bp long) has been investigated, but also a fragment of 118
bp typical for plant’s lectin gene as a control. The long,
detectable fragments of lectin (presented in conventional and GM soya) were found in contents taken from all
places (13 challenges in all experimental groups). On the
contrary, specific primers for genetically modified soybean were detected in four samples taken from caecum
and rectum of 2 animals from the group fed with the GM
soybean meal. Finally, the presence of fragments of lectin
was 3.25 times more frequent then the presence of fragment of transgene. In conclusion, there was no significant
proof that the transgene fragment is less degradable than
lectin — a gene, which is presented in all variety of soya.
Key words: sheep, PCR RFLP, beta-lactoglobulin, LGB
References:
Bocharev V V (1998) PhD thesis: 1–93.
Çelik S et al (2006) Turk J Vet Anim Sci 30: 539–544.
Elyasi G h et al (2005) J Sci & Technol Agric & Natur Resour 9: 134.
Kučinskiene J et al (2005) Veterinarja ir zootechnika 29: 90-92.
Miller S A et al (1987) Nucleic Acids Res 16: 1215.
Acknowledgements:
Sources of research financing: project VEGA no 1/4440/07
of Genetics and Animal Breeding, 2Department
of Cattle Breeding, Agriculture University of Krakow, Kraków,
Poland
e-mail: Weronika Śliwiak <sliwiw@interia.pl>
130
Abstracts P5.11
P5.12
Genetically modified soya in rabbit feeding:
detection of the transgene in tissues
Qualifying of a degree of apoptosis in
stored kidneys harvested from transgenic
and non-transgenic pigs with the method
of luminescence measurement using
lucypherase — preliminary report
Dorota Maj1, Weronika Śliwiak1, Piotr
Łapa1, Joanna Pokorska2, Józef Bieniek1
1Department
of Genetics and Animal Breeding, 2Department
of Cattle Breeding, Agriculture University of Krakow, Kraków,
Poland
e-mail: Dorota Maj <dmaj@ar.krakow.pl>
The use of ingredents and products from genetically
modified plants (GMP) in animal nutrition raises many
questions about the fate of transgenic DNA in the digestive tract and tissues of food-producing animals. The aim
of this study was to detect transgenic and endogenous
plant DNA fragments in muscle, liver and blood of rabbits fed genetically modified soya (Roundup Ready).
From 7 to 12 weeks rabbits (n=24) were fed pellets containing 20% conventional (nonmodified) or 20% genetically modified soya (cp4 epsps gene), and pellets without
soya. Total DNA was extracted from tissues and analyzed
by polymerase chain reaction (PCR) for the presence of
a 118-bp fragment of endogenous soy lectin gene and a
128-bp fragment of cp4 epspsps transgene. Only short
fragments (118-bp) of plant chloroplast DNA (lectin gene)
have been detected from 4 muscle and 4 liver samples but
not in blood of rabbits up to 12 h after the last feeding.
The transgenic DNA sequences (128-bp) were never detected in blood and muscle tissues, howewer 2 liver samples were positive for a 128-bp fragment of the transgenic
cp4 epsps. The presented results are from a pilot study and
further research is needed.
2008
Jerzy Skuciński1, Monika Dąbrowska2, Małgorzata
Starek2, Jarosław Wieczorek3, Grzegorz Kazek4
1Institute
of Public Health, Jagiellonian University, Kraków,
Poland; 2Department of Inorganic and Analytical Chemistry,
Jagiellonian University, Kraków, Poland; 3National Research
Institute of Animal Production, Balice, Poland; 4 Independent
Laboratory of Radioligands, Jagiellonian University, Kraków,
Poland
e-mail: Jerzy Skucinski <jerzy.skucinski@wp.pl>
The research aimed at qualifying of a degree of apoptosis
in kidneys harvested from donor pigs in a unit of time and
in dependence on a kind of preserving fluids and a genetic type of donors (genetically modified v. not modified).
Kidneys were harvested from intravenously anesthetized
donor pigs weighing 30 kg. After harvesting the kidneys
were rinsed with preserving fluids. Directly after this procedure, the kidneys were placed in preserving fluids (temp.
+4°C). Specimens for apoptosis examination were collected
in regular intervals. Apoptosis, also called programmed
or “suicidal” death of a cell, is a very important process of
auto-destruction, generically regulated. It allows highly developed organisms to control a number of cells during puberty and allows for removal of damaged or cancer afflicted
cells. Apoptosis may be divided into 2 stages: in the first one
a process of cellular self-destruction is induced by reception
of extra- and intracellular signals; in the second stage the
genes, whose products participate in the process being now
discussed, become activated. Apoptosis is a physiological
process occurring in a strictly controlled manner, controlled by cellular enzymes, with the participation of energy. In
metabolically active cells ATP levels are constant, however
in dead cells these levels drastically decrease. In the process
of apoptosis the cells indicate the increase of the level of cellular ADP, however, without a distinct decrease of the ATP
level. In the advanced stage of apoptosis also the decrease
of ATP levels may occur. Differently than in apoptosis, in a
process of necrosis a level of ADP increases and hydrolysis
of ATP occurs. In a phase of initial necrosis ATP levels drastically drop as a result of toxic products concentration, what is
linked with relevant increase of cellular ADP. The research is
based on the ATP luminescence present in all metabolically
active cells. The method of luminescence measurement with
the use of lucypherase as the catalyst is based on the following reaction: ATP+lucypherase+O2 oxylucipherine+AM
P+PPi+CO2 + light (562 nm). Intensity of emitted light is linearly dependent on ATP concentration. A selected method
is characterized by high specificity, a wide range of application and easiness of use. These markings were carried out in
temperature of 18-22°C. ADP was measured by its conversion to ATP, and its amount was subsequently marked with
the use of lucypherase. The obtained ADP:ATP ratio was
used to define the mechanism of cellular death (apoptosis
or necrosis).
Vol. 55 EuroBiotech
2008
131
P5.13
P5.14
Effect of in vitro cultured and cryopreservation
on quality of preimplantation porcine embryos
Boar sperm membrane integrity, mitochonrial
activity and motility following storage at 15°C
Magdalena Bryla, Monika Trzcinska,
Barbara Gajda, Zdzislaw Smorag
Monika Trzcinska, Magdalena
Bryla, Zdzislaw Smorag
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland
e-mail: Magdalena Bryła <mbryla@izoo.krakow.pl>
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland
e-mail: Monika Trzcińska <mcala@izoo.krakow.pl>
The aim of this experiment was to investigate the quality
of in vitro cultured and crypreservation porcine embryos
by analysis of DNA fragmentation using the terminal
deoxynucleotidyl transferase (TdT) mediated dUTP nick
— end labeling — TUNEL and DAPI staining. The experiment was done on 2-4 cell embryos produced in vivo and
cultured in vitro for 6 days in NCSU-23 medium until the
expanding blastocyst stage and then vitrified the open
pulled straw (OPS) method. In vitro derived expanded
blastocysts were assigned to one of the following three
groups: control — (1) fixed immediately after cultured,
non vitrified embryos and vitrified embryos — (2) fixed
immediately after vitrification and thawing and — (3)
fixed after vitrification/ thawing and culture for 24h. In
total 180 porcine expanded blastocysts were analysed
for DNA fragmentation. The data were analyzed by Student’s t-test for the number of cell per embryo and Fisher’s test for the percentage TUNEL — positive nuclei per
embryo. The total numbers of nuclei per non vitrified
embryo were not significantly different than in vitrified
embryos from the other groups (1:64.9±22.9, 2:60.2±20.6,
3:55.4±17.3). The TUNEL — positive nuclei in non vitrified embryo were significantly different (P≤0.01) than in
embryos fixed immeditely after vitrification and thawing
(1:12.8±9.2, 2:26.4±15.6), and in fixed embryos after vitrification/thawing and cultured for 24h (3:33.4±16.2). The
percentage of TUNEL index in vitrified/cultured embryos
(3: 60%) was significantly higher (P≤0.01) than in other
groups (1: 20%, 2: 44%). In conclusion, we demonstrated that the total number of nuclei was negatively correlated with the numer of TUNEL -positive nuclei and the
TUNEL index, whereas the number of TUNEL -positive
nuclei was positively correlated with the TUNEL index.
DNA fragmentation and the values of the TUNEL index
increased in vitrification embryos compared to non vitrification embryos. It was also demonstrated, that culture
of vitrificated embryos for 24h significantly caused increasing of fragmentation DNA in these embryos.
Currently, sperm quality is evaluated by conventional
sperm analysis, which determines sperm concentration
and motility. Although the conventional analysis of semen yields considerable information, new methods are
still needed to make the evaluation of sperm more reliable. The purpose of this study was to assess sperm quality
in extended boar semen during storage at 15°C for 6 days
using: Vybrant Apoptosis Assay Kit (Molecular Probes,
Eugene, OR, USA), based on a slight increase in membrane permeability and mitochondrial specific probes
5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimiazolyl carbocyniane iodide JC-1 (Molecular Probes, Eugene, OR,
USA) to measure changes in mitochondrial membrane
potential. Twelve boars were used in this experiment.
Semen (1ejaculate from each male) was collected by the
gloved hand technique. After collection and separation
of gel, the semen was diluted in BTS extender (Beltsville
Thawing Solution, France) and stored for six days at
15°C. The analysis was repeated on the first, third and
sixth days of semen storage. Sperm cell suspensions were
analysed under a fluorescence microscope Nikon Eclipse
E 600 at 40x magnification. Motility was also assessed on
each day of the experiment. Using Vybrant Apoptosis Assay Kit assay we observed 0.5–8% and 0–2% apoptotic
sperm; 7–28% and 29–93% necrotic sperm; and 71–85%
and 8.5–69% live sperm in fresh and stored semen on day
6, respectively. Significant differences in the percentage
of the entire sperm subpopulation among boars were
observed (P<0.01). The percentage of spermatozoa with
dysfunctional mitochondrial was the highest at 6 days of
storage (28–90.5%). The mitochondrial analysis showed
that its function was highly correlated (r=0.936) with the
assessment of viability and with the microscopic estimates of motility. In summery, the detection of changes
in membrane integrity and in mitochondrial membranr
potential in spermatozoa could be a additional criterion
for evaluating sperm quality.
Acknowledgements:
This study was supported by the Polish Comittee for Scientific
Research, grant no. 2P06D 024 30.
Acknowledgements:
This study was supported by the Polish Committee for Scientific
Research, grant no. 2P06D 023 30.
132
Abstracts 2008
P5.15
P5.16
Insect heart as a model for evaluation
of peptide cardiotropic properties
The use of pseudophysiological activation of
oocytes in the somatic cell cloning of goats
Paweł Marciniak1*, Neil Audsley2,
Grzegorz Rosiński1
Marcin Samiec, Maria Skrzyszowska
1Department
of Animal Physiology and Development, Adam
Mickiewicz University, Poznań, Poland; 2Central Science
Laboratory, Sand Hutton, York, England
e-mail: Paweł Marciniak <pmarcin@amu.edu.pl>
Insects has been an excellent model of human diseases
for over a decade. Nowadays they are used as models for
studying metabolic disorders such as obesity and diabetes or neurodegenerative diseases, for example Alzheimer disease. Important researches are made under cardiac
disorders using mainly Drosophila melanogaster as a model
organism.
Insect nervous system produce huge number of bioactive neuropeptides which regulates developmental, behavioural and reproductive processes. Some of them are
known to modulate cardiac action in insects. Several peptide, especially some FMRFamide bioanalogs, are related
to the mammalian ones and cause an effect on higher organisms. Searching for new neuropeptides may lead to
find molecules with new bioactivity.
In this study we use Zophobas atratus beetle semi isolated
heart in a screen for myotropic properties of neuropeptides. We use HPLC and MALDI-TOF MS techniques for
searching peptides in methanol extracts of brain, corpus
cardiacum/corpus allatum complex and ventral nerve cord
of the Zophobas beetle. Continuously perfused with physiological saline, semi isolated beetle heart was exposed to
HPLC fractions and bioactive ones were analyzed using
mass spectrometry.
We found several peptides with cardioactive properties
from among leucomyosuppressin was the most interesting. This decapeptide belongs to the FMRFamide peptide
family related to the some mammals bioanalogs. Native
leucomyosuppressin causes negative reversible chronotropic and inotropic effects on Zophobas myocardium
which is in agreement with the action of the synthetic
one. Primary structure of the peptide was established using MALDI-TOF MS PSD.
Acknowledgements:
*Author was supported by British Council fellowship.
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland
e-mail: Marcin Samiec <msamiec@izoo.krakow.pl>
We have recently developed the novel method of pseudophysiological transcomplementary (transcytosolic)
activation to stimulate the developmental program of
caprine oocytes reconstructed by the somatic cell cloning. The purpose of the study was to examine the in vitro
developmental potential of caprine cloned embryos following the pseudophysiological transcytoplasmic activation of oocytes receiving fetal fibroblast cell nuclei. The
reconstruction of enucleated oocytes was performed by
microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly
into the cytoplasm. The SCNT oocytes were incubated in
B2 medium for 30 min to 1 h before their pseudophysiological transcomplementary activation. The activation
was achieved by electrofusion of clonal cybrids with the
allogeneic cytoplasts isolated from caprine in vitro-fertilized ova (zygotes), which led to the formation of triple
allocytoplasmic hybrids. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The
resulting zygoplast-clonal cybrid couplets were subjected
to plasmolemma electroporation, which was induced by
application of a single DC pulse of 2.4 kV cm-1 for 15 μsec.
The plasma membrane electropermeabilization occurred
in an isotonic dielectric solution deprived of Ca2+ ions.
The transcytoplasmically-activated clonal cybrids were in
vitro cultured for 6–8 days up to morula/blastocyst stages.
A total of 59/74 (79.7%) oocytes reconstructed with fetal
fibroblast cell nuclei were successfully fused with zygoplasts. Out of 59 cultured cloned embryos, 28 (47.5%)
were cleaved. The rates of cloned embryos that reached
the morula and blastocyst stages yielded 23/59 (39.0%)
and 12/59 (20.3%), respectively. In conclusion, the original method of pseudophysiological transcomplementary
(transcytosolic) activation of SCNT doe oocytes, which
has been developed in our laboratory lately and has been
applied to the somatic cell cloning of goats for the first
time, turned out to be reliable and feasible for efficient
stimulation of preimplantation development of clonal cybrids. It has been shown that the relatively high percentages of morulae and blastocysts developed in vitro from
caprine cloned embryos generated by the novel strategy
of pseudophysiological activation of oocytes reconstructed with fetal fibroblast cell nuclei.
Vol. 55 EuroBiotech
2008
P5.17
P5.18
Quality of bovine BCB stained
and non-stained oocytes
Effect of prolactin administered per os
on body gain and health of piglets
Jolanta Opiela
Zdzisław Smorąg1, Florian Ryszka2,
Barbara Dolińska2, Barbara SzczęśniakFabiańczyk1, Lucyna Leszczyńska2,
Mirosław Koska3, Waldemar Kowalski3
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland
e-mail: Jolanta Opiela <jopiela@izoo.krakow.pl>
Brilliant cresyl blue (BCB) staining demonstrates the intracellular activity of glucose-6-phosphate dehydrogenase
(G6PDH) and can be used for the selection of competent
oocytes. The grown oocytes are blue (BCB+) because the
G6PDH activity is too small to reduce the staining. The
growing oocytes stay colorless (BCB-). The results of our
previous experiments seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. In immature oocytes, the Bax transcript level in BCBoocytes was significantly higher (P<0.001) in comparison
to non-stained oocytes. Moreover, there was a tendency of
higher Bax protein expression in immature BCB+ oocytes,
but no relation was found between the activity of G6PDH
and the expression of apoptotic proteins. The presented
results prompted us to check whether BCB staining has
any detrimental impact on chromatin configuration (CC)
and DNA integrity.
Immature oocytes after recovery and morphological selection were subjected to BCB staining (26 μM) for 60 min.
Denuded oocytes were fixed in 4% paraformaldehyde for
60 min. in room temp. and then stored in 1% paraformaldehyde in 4 oC. TUNEL reaction was carried according to
Warzych et al. (2007).
The chromatin configuration was evaluated according to
Liu et al. (2006) classification. Among non-stained oocytes
45% showed diffuse CC pattern, while the rest showed
SN (perinucleolar ring chromatin) configuration with tendency to clumps forming. Among stained oocytes (BCB+
and BCB-) 22.6% showed diffuse patern and almost 40%
presented SN pattern. Interestingly, none of the control
oocytes showed condensed or clumped configuration
comparing to 37.5% of BCB- and 40% of BCB+ oocytes
presenting this pattern. Although there are suggestions
that condensed (C) chromatin represent steps towards
atresia, studies do not confirm that, as the proportions of
C and SN patterns were significantly higher in the healthy
than in atretic bovine oocytes (Liu et al. (2006).
Summing up, it seems that BCB staining has an impact
on the rate of immature oocytes chromatin configuration
changes. BCB staining seems to accelerate the transition
from diffuse CC to GVBD and further progression of
meiosis, which is proceeded by chromatin condensation.
Since only one oocyte (non-stained) out of 73 analyzed
exhibited TUNEL positive labeling, it can be stated that
BCB staining has no impact on DNA integrity.
133
1Department
of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland; 2Pharmaceutical Research and Production Plant
“Biochefa”, Sosnowiec, Poland; 3Experimental Station of Pig
Breeding, Żerniki Wielkie, Poland
e-mail: Zdzislaw Smorag <zsmorag@izoo.krakow.pl>
Prolactin (PRL, mammotropin, lactotropin) is a peptide
hormone secreted by the pituitary gland with a molecular weight of 23 kDa. It has many functions in the body:
it stimulates development of mammary gland, initiates
and sustains lactation in mammals, slows down release
of gonadotropin hormones, is responsible for osmotic
regulation in fish and nesting in birds, stimulates development of crop tissues in some birds, and plays a role
in protein, lipid and carbohydrate metabolism. This hormone has also been proven to modify immune response.
The aim of the study was to evaluate the effect of prolactin administered per os on body weight gain and health
of piglets. Three preliminary experiments on 27 litters of
10 piglets per litter with an equal number of female and
male piglets were carried out. Each experiment consisted
of 6 experimental litters and control. Each piglet from experimental groups received orally 2 ml of Biolactin with
various prolactin contents. The amounts of PRL administered were 0.1 or 1.0 mg in experiment 1, 0.05 or 0.01
mg in experiment 2, and 0.1 or 0.5 mg in experiment 3.
Breeding results of the litters were monitored until 21
days of age. In all 3 experiments, no piglet mortality was
found. In experiment 1, average daily weight gain was
157 g, ranging from 201 g for piglets that received 0.1 mg
of Biolactin to 177 g for piglets that received 1.0 mg of
Biolactin. In experiment 2, no differences in daily weight
gain were observed between control and experimental
groups of piglets that received 0.01 mg of prolactin (181 g
vs. 180 g on average). The experimental group of piglets
that received 0.05 mg of Biolactin was characterized by an
average body weight gain of 164 g (17 g less than in the
control group). In experiment 3, piglets that received 0.1
mg of prolactin (the same dose as in experiment 1) had
an average daily weight gain of 196 g, ranging from 187
g for piglets that received 0.5 mg of prolactin to 157 g in
the control group. It may be concluded from the above
experiments that oral application of 0.1, 0.5 or 1.0 mg of
prolactin has a positive effect on piglet results during the
first weeks of breeding. The research needs to be repeated
on a larger number of litters. In conclusion, the present
study showed a positive effect of prolactin administered
per os on body weight gain and health of piglets during
the first weeks of their lives.
Acknowledgements:
Scientific research supported from the 2006-2008 science funds
as research project No. N 405 002 31/0124.
134
Abstracts 2008
P5.19
P5.20
New polymorphism in second
intron of the CA3 gene in pigs
Polymorphism of H-FABP, LEPR ane
LEP genes in domestic pig breeds
Katarzyna Walinowicz
Katarzyna Ropka-Molik, Mirosław Tyra
Departament of Animal Genetics and Breeding, National
Research Institute of Animal Production, Balice, Poland
e-mail: Katarzyna Walinowicz <kwalinow@izoo.krakow.
pl>
Department of Animal Genetics and Breedeing, National
Research Institute of Animal Production, Balice, Poland
e-mail: Katarzyna Ropka-Molik <kropka@izoo.krakow.
pl>
The carbonic anhydrase III (CA3) gene is a member of the
carbonic anhydrase gene family encoding zinc metalloenzymes, which catalyze reversible hydratation reactions of
carbonic dioxide in order to maintain thermodynamic
equilibrium between CO2 and HCO3-. Carbonic anhydrase III is found in tissues that control energy metabolism, such as muscle, liver and adipose tissue. CA3 genomic DNA consists of seven exons and six introns and
maps to porcine chromosome 4q11->q14. CA3 plays an
important role in muscle diseases (Mokuno et al., 1986).
Expression profile of CA3 showed differences between
two Chinese breeds of Landrace and Tongcheng pigs.
Statistical analysis showed that polymorphism (SNP) at
the position 8607 was significantly correlated with intramuscular fat content of pigs and another SNP site was
associated with percentage of ham (Wang et al., 2007). A
previous study showed that the activity of CA3 in rat adipose tissue decreased with obesity (Takahashi et al., 2001).
This suggests that the CA3 gene might play an important
role not only in muscle development but also in meat flavour and carcass quality. In the present study we tried to
find a new polymorphism which can be associated with
important economic traits in pigs. The pigs used in this
study represented four breeds: Pietrain (n=24), Duroc
(n=24), Polish Large White (n=24) and Landrace (n=24).
Genomic DNA was extracted from frozen blood using
standard methods. Primer pairs for the second intron
were designed using Primer3. Polymorphisms were detected by using the PCR-SSCP method and sequencing
using a Beckman Coulter sequencer. Five polymorphisms
were identified (G/A 2834, G/A 2848, G/A 2867, T/G 2924
and C/T 2894), which had been described by Wang (2007)
in Chinese pigs. New SNPs were confirmed by using the
PCR-RFLP method. Based on NEBcutter analysis, HinfI,
BsrI, Hyp166II and MboI restriction enzymes were applied. The SNP located in the intron might influence the
expression level and rearrange splicing. CA3 is one of the
candidate genes for meat quality. New polymorphisms
found in the second intron of the carbonic anhydrase III
gene could be highly useful for creating a new genetic
marker associated with an important trait of the pig. In
further investigations we will try to find associations for
new SNPs.
The purpose of this study was to detect genetic variation
in porcine H-FABP gene, LEP and LEPR (leptin receptor)
gene, a candidate genes for meat quality traits in pigs. Intramuscular fat (IMF) is the main factor influencing sensory quality of meat. However, pigs are selected for high
lean content by selection for decreased backfat thickness
(BFT) and high growth capacity. It was the main reason
why IMF content of pig meat has decreased below the
suggested level for acceptable meat quality (under optimum 2 to 3%).
Heart fatty acid-binding protein (H-FABP) was postulated to be a candidate that explains part of the variation in
IMF content in pigs. H-FABP is an intracellular proteins
responsible for fatty acid transport from the plasma membrane to the sites of β oxidation and/or triacylglycerol or
phospholipids synthesis. FABPs modulate lipid metabolism and influence intracellular fatty acid concentration. It
has been proved that the mutation in the leptin gene is responsible for the obese phenotype mouse and mutations
in LEPR have been reported to be associated with obesity
in humans and rodents. Thus LEP and LEPR genes are
very interesting as a candidate gene for IMF content.
H-FABP gene, consist of four exons and three introns. To
date, three different point mutation have been identified
in the HFABP gene: HinfI polymorphism in 5’ upstream
region, HaeIII and HpaII in second intron. H-FABP, LEP
and LEPR polymorphism was identified using PCR-RFLP
method in 6 breeds of pigs: Landrace (n=185), White Large
(n=85), Duroc (n=83), Pietrein (n=75), Hampshire (n=17).
H-FABP polymorphism were typed using the primers described by Gerbens (1997) and LEP and LEPR polymorphism using the primers described by Stratil (1998).
The three H-FABP polymorphisms are present in all
breeds tested. Curiously, the Duroc breed has a diffrent
HpaII frequency of genotype than others breeds. It is suprising that the HinfI frequency of genotype is in a state
of disequilibrium in the Landrace breed (hh genotype is
absent). In LEP-HinfI polymorphism TT genotype overcames in all tested breeds. The higest frequency of CT and
CC genotype appeared in Duroc breed. The LEPR-HpaII
polymorphism show high frequency of BB genotype in
all breeds. In Duroc and Pietren breeds, only two LEPR
genotypes are present (BB, AB), due to the low frequency
of allele A.
References:
1. Mokuno K et al (1986) Muscle Nerve 9: 257–260.
2. Wang H.L. et al (2006) Cytogenet Genome Res 115:129–133.
3. Takahashi H et al (2001) Endocrinol J 48: 205–211.
Reference:
1. Gerbens F et al (1997) Mamm Genome 8: 328–333.
2. Stratil A et al (1998) Anim Genet 29: 405–406.
Vol. 55 EuroBiotech
2008
P5.21
P5.22
Post-weaning Multisystemic
Wasting Syndrome in pigs
Insect peptides — new perspective
for cancer therapy?
Zuzana Čanakyová1, Alojz Kúbek2,
Zuzana Slobodová3, Janka Kiśacová4
Małgorzata Słocińska1, Anna
Olejnik2, Mariola Kuczer3
1-4 Department of Genetics and Breeding Biology, Fakulty
of Agrobiology and Food Resources, Slovak University of
Agriculture in Nitra, Slovak Republik
e-mail: Zuzana Čanakyová <zuzaxa@gmail.com>
1Department
Post-weaning Multisystemic Wasting Syndrome (PMWS)
is a serious porcine disease, it tends to be progressive
disease with a high fatality rate in affected pigs. PMWS
belongs to PCVAD (Porcine Circovirus Associated Diseases), it is actually a disease complex, which includes all
diseases associated with PCV-2. PMWS disease causes illness in piglets, with clinical signs including progressive
loss of body condition, visibly enlarged lymph nodes, difficulty in breathing, and sometimes diarrhea, pale skin,
and jaundice. PCV-2 has been shown in excreta and nasal
secretions of infected pigs, this is how it spreads from
pig to pig (Mankertz, 2008) PMWS was first described
in Canada in 1995. Shortly after has been reported from
most pig-producing countries of the world at huge cost
to agriculture. PMWS is caused by porcine circovirus
type 2 (PCV2). PCV-2 is the smallest known porcine virus
and it is ubiquitous in swine herds worldwide. The name
Circovirus was given because it is a member of Circoviridae family and they have their DNA in the form of a
ring. There are two serotypes of Circovirus, that can be
found in pigs. Porcine circovirus-1 is non-pathogenic to
infected pigs, but Porcine circovirus-2 is associated with
PMWS and it is pathogenic. Genetically is possible to detect sequention PCV-2 isolates, they have 1767 to 1768 bp
in length. PCV-2 was first described in Canada in 1995.
(Fenaux et al., 2000). The two main viral genes of PCV-2
are ORF1 and ORF2 which are oriented in opposite directions and which represent 93% of the PCV2 genome.
ORF1 is associated with replication, it is highly conserved
among isolates (Mankertz et al., 1998). The ORF2 gene
encodes capsid protein (Nawagitgul et al., 2002). Host
genetics may also markedly affect the outcome of PCV2
infection and there is increasing evidence of differences
in virulence among PCV2 isolates. We would like to investigate situation of affected pigs with PMWS in Slovak
Republik. We will analyse potencial possitive pigs and
their characteristic differences in genotypes. We would
like to make a review of PMWS situation in our country.Nowdays the results of our research are not indicating PMWS, but in this time we have new methods with
better results. We changed potencial positive samples of
blood into samples of tissue. This is because of higher virus concentration in tissue. We have new High Pure Viral
Nucleic Acid Preparation Kit from tissues. We use PCRRFLP method.
135
of Animal Physiology and Development, Adam
Mickiewicz University, Poznań, Poland; 2Department of
Biotechnology and Food Microbiology, Poznan Unversity
of Life Sciences, Poznań, Poland; 3Faculty of Chemistry,
University of Wrocław, Wrocław, Poland
e-mail: Malgorzata Slocinska <mslocinska@yahoo.com>
The peptides of insect origin with antitumour activity can
be very interesting for scientists because of their potential
use as the therapeutic agents. In our study we have used
two synthetic peptides: DILRG-NH2, pentapeptide with
amidated C-terminus Asp-Ile-Leu-Arg-Gly-NH2, naturally occurring in diapausing pharate first instar larvae
of the wild silkmoth Antheraea yamamai (Yang et al., 2004)
and alloferon, occurring in blood of an experimentally
bacteria-challenged blow fly Calliphora vicina, with amino
acid sequence HGVSGHGQHGVHG (Chernysch et al.,
2002). We have examined effect of these peptides on the
proliferation and the mitochondrial enzymes activity of
human hepatoma cells HepG2 and epithelial adenocarcinoma cells Caco-2. It has been shown, that DILRG-NH2
suppresses proliferation of the hepatoma cells whereas
alloferon decreases activity of the mitochondrial enzymes
in adenocarcinoma cells. Both peptides exhibit the highest activity in concentrations higher than 0.3 mM. The effect of peptides action we observed, may be due rather
to a cell cycle arrest than apoptotic/necrotic activity. The
results obtained indicate that the insect peptides have a
potential to be developed as therapeutic agents for anticancer treatments.
References:
Yang P et al (2004) J Insect Biotechnol Sericol 73: 7–13.
Chernysh S et al (2002) PNAS 99: 12628–12632 .
136
Abstracts P5.23
P5.24
Lipid peroxidation, ATP level and motility
of boar spermatozoa during liquid storage
Quantitative measurements of triglycerides
and phospholipids in the pig oocytes
and in vivo produced embryos
A. Wierzchoś-Hilczer, P. Gogol,
B. Szczęśniak-Fabiańczyk
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland
e-mail: Agnieszka Wierzchoś-Hilczer <awierzch@izoo.
krakow.pl>
Long term efforts to develop a simple and efficient method for cryopreservation of boar semen have been unsuccessful. This means that insemination with frozen semen
generally results in low fertility and fecundity. Although
this line of research has not been abandoned, great emphasis is placed on the improvement of extenders used
for storage of boar semen in about 15oC.Attepmts have
been made to extend the time of semen storage without
reducing fertility indices.
The aim of the present experiment was to check some parameters of boar spermatozoa stored for 7 days. Fresh semen from five boars was used in the experiment. Semen
samples were diluted to a final concentration of 60×106
sperm/ml in BTS extender and stored for 7 days at 15oC.
At 1, 4, and 7 days of storage, each ejaculate was examined
for photon emission intensity. Luminescence was measured at 20oC using an AutoLumat LB953 luminometer
equipped with a cooled photomultiplier with a spectral
response range from 370 to 620 nm. Prior to measurement
of luminescence, spermatozoa were separated from the
seminal plasma and diluents by two-fold centrifugation
(700×g for 15 min) and resuspended in 0.9% NaCl. To the
washed sperm suspension at a concentration of 200×106
cells/ml luminol was added. Emission was induced by
adding 0.3 mM FeSO4 solution. The ATP level in spermatozoa was determined using the bioluminescent method.
For assessment of sperm motility, samples of semen were
incubated at 37oC for 30 min and then the percentage of
motile spermatozoa was evaluated using a contrast phase
microscope equipped with a heated plate at 37°C.
On day 1 the mean value of photon emission intensity was
13.78×105 counts/450s and increased significantly on day
7 to the mean value of 19.31×105 counts/450s (P<0.01). The
increase in photon emission was accompanied by a significant decrease in sperm motility, which averaged 56.46% on
day 1 and 46.04% on day 7 (P<0.01). The ATP concentration
also significantly decreased during 7 days of storage. On
day 7 it reached 63.52% of the level on day 1.
The results of the study showed that reactive oxygen
species and products of lipid peroxidation compromise
motility and ATP production in spermatozoa during boar
semen storage. Induced luminescence assessment in combination with sperm motility and ATP level can give valuable information about the status and function of spermatozoa cells which might be relevant for predicting the
fertilizing potential of the semen.
2008
Marek Romek1, Barbara Gajda2, Ewa
Krzysztofowicz1, Zdzisław Smorąg2
1Department
of Cytology and Histology, Institute of Zoology,
Jagiellonian University, Kraków, Poland; 2Department of
Biotechnology of Animal Reproduction, National Research
Institute of Animal Production, Balice, Poland
e-mail: Marek Romek <bgajda@izoo.krakow.pl>
Changes in the lipid content of pig oocytes and embryos
may be associated with changes in their sensitivity to low
temperature. As was shown previously, differences in the
total lipid content observed in porcine embryos were dependent on the development stage of the embryo. Moreover the qualitative, histochemical evaluation of the lipid
composition in pig embryos shown that zygote contain a
high level of unsaturated hydrophobic lipids and a large
amount of free fatty acids as well as phospholipids. During cleavage the composition of the above mentioned lipids in droplets and cytoplasm changed. However, up to
now, there is no information on the quantity of triglycerides and phospholipids in the single pig oocytes. Thereby
we developed a new method for quantitative estimation
the different types of lipids in pig oocytes and embryos
using scanning confocal microscopy and specific fluorescent dye Nile Red. Porcine oocytes as well as in vivo
produced embryos were collected from ovary follicles or
obtained after flushing oviducts or uterus. Materials were
fixed with and 2% formaldehyde, stained with 100 nM
Nile Red and analyzed in confocal microscope LSM 510
Meta (Zeiss, Germany). To evaluate the level of triglycerides and phospholipids Nile Red was excited using a 514
nm laser lines. Emission spectrums were deconvoluted
into three bands, the first and second ones corresponding
to triglycerides and phospholipids, respectively. Mature
oocytes contained significantly lower content of triglycerides compared with immature eggs while the level of
phospholipids remains almost unchanged during maturation. In embryos produced in vivo, amount of triglycerides decrease significantly with embryo development: remains unchanged from zygote to morula stages but then
decreased at blastocyst to about 82 % of the value founded
in zygotes. The amount of phospholipids in pig embryos
during cleavage decreases too. Their level in blastocyst
is almost twofold lower then that present in blastocyst.
In conclusion, our results showed that pig embryos may
metabolize lipids, especially triglycerides. Additionally,
phospholipids are derived from droplets and used for the
biosynthesis of biological membranes whose surface areas significantly increase during the cleavage process.
Acknowledgements:
This study was supported by Scientific Net of Animal Reproduction Biotechnology.
Vol. 55 EuroBiotech
2008
P5.25
P5.26
Development of caprine cloned embryos
derived from lipofected fetal fibroblast cells
The aspiration efficiency and morphology
of sheep oocytes collected by
laparoscopic ovum pick-up method
Maria Skrzyszowska1, Marcin Samiec1,
Ryszard Słomski2,3, Daniel Lipiński2,3
1Department
of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Balice,
Poland; 2Department of Biochemistry and Biotechnology,
Agricultural University, Poznań, Poland; 3Institute of
Human Genetics, Polish Academy of Sciences, Poznań,
Poland
e-mail: Maria Skrzyszowska <mskrzysz@izoo.krakow.pl>
The purpose of our study was to determine the developmental potential of caprine nuclear transfer-derived embryos reconstituted with fetal fibroblast cells, which had
been genetically modified by lipofection method with
pAlb-GFPBsd gene construct. Positively-selected transgenic fetal fibroblast cells were in vitro cultured up to a
total confluency state and then used for the somatic cell
nuclear transfer (SCNT) as a source of nuclear donor cells.
Enucleated in vitro-matured caprine oocytes were the
source of recipient cells. Single nuclear donor cells were
injected into a perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with a single DC pulse of
2.4 kV/cm for 15 μsec. After a 1-h delay, the reconstructed
oocytes were additionally activated by exposure to 5 μM/
L calcium ionomycin for 5 min followed by treatment
with 2 mM/L 6-dimethylaminopurine for 2 h. The SCNTderived embryos were in vitro cultured in 50-μL droplets
of B2 medium for 24 h. Afterwards, cloned embryos at
2-4-cell stages either were transferred into reproductive
tracts of recipient does or were co-cultured with Vero cells
in B2 medium supplemented with 10% FBS for an additional 120 to 168 h up to morula/blastocyst stages. A total of 76/87 (87.4%) enucleated oocytes were successfully
fused with transgenic nuclear donor cells and intended
to be in vitro cultured. Out of 76 cultured SCNT embryos,
59 (77.6%) were cleaved. From among them, 42 2-4 cellstaged embryos were transferred into 4 recipient females.
Following 6-8 days of in vitro culture, 15/17 (88.2%) and
9/17 (52.9%) cloned embryos reached the morula and
blastocyst stage, respectively. Ultrasonographic diagnostics of gestation, which was performed between Days 30
and 40 after transfer of SCNT embryos into 4 surrogate
recipients, did not confirm the presence of fetuses. In conclusion, it has been shown that the cell nuclei of cultured
fetal fibroblast cells, which had undergone the lipofection
with the pAlb-GFPBsd transgene, were able to support
the preimplantation development of caprine geneticallytransformed cloned embryos to morula and blastocyst
stages. The preimplantation developmental competences
of transgenic nuclear-transferred embryos to reach the
morula and blastocyst stages were relatively high. However, transgenic SCNT embryos exhibiting the expression
of pAlb-GFPBsd fusion protein were unable to implant in
utero and failed to establish pregnancy.
137
Jaroslaw Wieczorek, Yuriy Kosenyuk,
Miroslaw Cegla, Bozenna Rynska
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland
e-mail: Jaroslaw Wieczorek <jarek314@vp.pl>
New techniques for repeated, noninvasive oocytes recovery in living donor including goat and sheep are necessary. The employment of laparoscopy and videosurgery
allowed for the working out the OPU methods. However,
their use is restricted by the relatively low efficacy and
reproducibility of the results. The aim of the study was to
work up the new techniques for repeated recovery of goat
and sheep oocytes useful for culture and fertilization in
vitro and cloning and evaluation of the efficacy of the established method. Oocytes were aspirated with originally
designed catheter for aspiration. The oocytes donors were
45 sheep. Estrus was synchronized with intravaginal
sponges (Chronogest CR, Intervet) for 14 days. Superovulation was obtained by the single injection of eCG (1000
IU IM) 16–24 hours before the removal of the sponges.
Oocytes were collected 24 hours after sponge removal.
The collection oocytes was performed under general anaesthesia lasted about 15–20 min. The endoscope was inserted into the abdominal cavity through umbilicus. Two
trockars for putting the manipulators were inserted 15 cm
below the udder. Oocytes were collected by aspiration of
the follicular fluid from the ovarian follicles. Depending
on the size, the single aspiration of up to 8 follicles was
performed. The collected oocytes were evaluated under
stereoscopic microscope. The following rules of the evaluation and classification of the oocytes were established:
class I – homogenous cytoplasm, at least 3 layers of the
granulosa cells, class II – homogenous cytoplasm, 1-2 layers of granulosa cells, class III – homogenous cytoplasm,
no granular cells, class IV – heterogenous cytoplasm, independent of the granulosa cells. Oocytes class I, II and
III were qualified for the culture. Results: 205 ovarian follicles were aspirated. 137 (66.8%) oocytes were collected,
including 44 in class I, 58 – class II, 16 – class III, 19 – class
IV. 118 (81.6%) of the collected oocytes were qualified
for the culture, including 31.2% – class I, 42.3% – class II,
11.7% – class III. 13.9% of the oocytes were not qualified
for the culture. The obtained results suggest the proposed
technique allows for the collecting oocytes of good quality that can be used for IMV/IVF techniques and cloning.
138
Abstracts P5.27
P5.28
The new extender for bull
sperm sexing technology
Monoclonal antibodies as a tool for
evaluation the gamete quality
Michal Bochenek, Zdzislaw Smorag
Jana Antalíková, Jana Jankovičová,
Katarína Fábryová, Michal Simon
Department of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland
e-mail: Michal Bochenek <mbochen@izoo.krakow.pl>
The aim of the work was to replace the egg-yolk TALP
— standard basic medium used in sperm sexing technology with the new one to get higher sperm viability.
The major components of new medium were: the mixture
of plant-originated proteins and high pressure homogenized plant lipids. Three ejaculates collected from 3 different bulls were used. Immediately after collection the
ejaculates were divided and diluted with standard eggyolk TALP medium (TALP-Y) or the tested new extender
(T-PxL) in 1:2 ratio. Diluted semen was stored for 24 h
in +15°C, then stained, incubated, and sexed according to
the XY Inc. bull semen sexing procedure. The sperm sexing was performed with an SX MoFlo high-speed sorter
at a speed of 3000–4000 cells/s. After collecting about 10
million spermatozoa, both fractions, X and Y, were mixed,
centrifuged at 700×g for 15 min to concentrate the spermatozoa (20 million/mL) and frozen in both commercial Bioxcell (IMV, France) or citrate-based extender containing
homogenized plant lipids (LIPO). For sperm membrane
examination samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20000 spermatozoa were collected for
each sample. The percentage of membrane-intact (“live”)
spermatozoa was taken for statistical analysis. Live/dead
examination was performed after 24 h storage, staining
with Hoechst33342 fluorochrome (preparation for sperm
sexing) and freezing/thawing. Mean percentage of live
spermatozoa after 24h storage/15°C in TALP-Y and T-PxL
was 45.56% vs. 69.64% (P<0.01, test t), after staining with
Hoechst33342: 47.69% vs. 66.34% (P<0.05, test t), after
freezing in Bioxcell: 22.05% vs. 21.23% and after freezing in LIPO: 12.88% vs. 31.29% (P<0.01, test t).It can be
concluded that T-PxL medium should be considered as a
replacement standard TALP-Y medium in bull sperm sexing technology because 1) it results in significantly higher
numbers of live spermatozoa after storage and/or sexing;
2) it eliminates a possible source of transmissible diseases
(such as bovine spongiform encephalopathy); and 3) it
decreases the total cost of the basic media used for the
bull sperm sexing procedure.
2008
Institute of Animal Biochemistry and Genetics Slovak
Academy of Sciences, Department of Immunogenetics, Slovak
Republic
e-mail: Jana Antalíková <jana.antalikova@savba.sk>
Mammalian reproduction is a unique event in which
morphologically disparate gametes recognize each other,
bind and fuse. This event includes highly regulated biochemical interactions between molecules located on the
surface of both gametes as well as substances present in
the natural environment of gametes both in the male and
the female reproductive organs. Only undamaged reproductive organs and gametes can successfully participate
in the process of fertilization. The various methods for
evaluation the quality of human as well as animal gametes have been developed. Application of monoclonal
antibodies (mAb) showed to be suitable means in the understanding of reproductive mechanisms. Antibodies are
valuable tools for identification, isolation and characterization of surface and subsurface components of gametes.
Additionally, antisperm antibodies can be used for monitoring of the integrity as well as acrosomal status of spermatozoa and also as immunological markers that may
elucidate sperm surface changes during capacitation and
acrosome reaction in vitro. In our study, a panel of monoclonal antibodies against bovine sperm antigens has been
produced and further characterized. BALB/c mice were
immunized with whole bull sperm, and their spleen cells
were fused with SP2/0 myeloma cells in the fusion experiment, resulting in the generation of thirteen hybridomas.
In the indirect immunofluorescence assay different reaction patterns of hybridoma supernatants were observed.
From the set of obtained mAbs six of them reacted in the
acrosomal region of sperm head and five mAbs showed
fluorescence signal on the whole spermatozoa. One of
mAb was specific to antigens of the sperm head as whole
and part of tail while the last one recognized molecules
of posterior region of sperm head. To date mAb designed
as IVA-520 against bovine CD46 molecule was fully characterized. Our previous results suggest localization of
CD46 on the outer acrosomal membrane of bovine sperm
and therefore might be used in evaluation of acrosomal
status of frozen-thawed sperm routinely used in the artificial insemination in cattle breeding.
Acknowledgements:
This work was supported by grants VEGA2/6023/6 and APVV
51-024904.
Vol. 55 EuroBiotech
2008
P5.29
P5.30
Effect of exogenous triiodothyronine (T3) on
activity of 3b-HSD in chicken ovarian follicles
The immunogenity of kidney transgenic pig
with human α1,3 fucosylotransferase gene
Andrzej Sechman1, Katarzyna
Pawłowska1, Dorota Wojtysiak2, Danuta
Wrońska-Fortuna1, Janusz Rząsa1
Jarosław Wieczorek1, Jerzy Skuciński2
1Department
of Animal Physiology, 2Department of
Reproduction and Animal Anatomy, University of
Agriculture, Kraków, Poland
e-mail: Katarzyna Pawłowska <k_pawlowska@onet.eu>
Introduction: The enzyme 3β-HSD catalyzes an obligatory step in the conversion of pregnenolone into progesterone as well as precursors of all androgens and estrogens
in the ovary. It is well documented that in the domestic
fowl ovarian steroidogenesis process is strongly affected
by triiodothyronine (T3). However, the mechanism of this
action has not been yet known. The present study was
designed to answer the question whether raise in T3 concentration in blood circulation may influence the activity of 3b‑HSD. Material and methods: Hy-line brown hens
(n=24) were randomly assigned to the 4 treatment groups:
control and three groups of hens injected (i.p.) with 30, 90
or 270 mg T3/day for 7 days. The ovarian stroma, white
follicles, and yellow hierarchical follicles were dissected
from the ovary and snap frozen. The 3b-HSD activity was
measured histochemically according to the procedure
described by Levy et al. [1959]. Results: There were no
differences in 3β‑HSD activity in the stroma and 1–4 mm
white follicles in all experimental groups. However, in
the theca interna layer of 4–6 and 6–8 mm white follicles
the activity of 3β‑HSD following the highest T3 dose treatment was significantly lower (P<0.01) by 46% and 44%,
respectively. In granulosa layer of preovulatory follicles;
particularly in the group treated with 270 mg T3/day a
significant (P<0.01) decline of 3β‑HSD activity by 57% was
noticed in the largest F1 preovulatory follicle. Reduction
in 3β‑HSD activity (by 30%; P<0.01) was also found in the
theca interna layer of F3 follicles following treatment with
dose of 90 mg T3/day. In summary: The results obtained
suggest that in the chicken ovarian follicles T3 plays as a
modulator of ovarian steroidogenesis by influence on the
3β‑HSD activity. Hyperthyroid state in blood circulation
decreases the enzyme activity and caused abnormalities
in ovarian steroids production. The next studies should
be held to elucidated whether 3β‑HSD activity inhibition
starts at transcriptional or/and translational level or there
is other unknown regulatory mechanism.
Acknowledgements
This study was supported by the grant No
2 P06D 031 29 from 2005-2008.
139
1Department
of Biotechnology of Animal Reproduction,
National Research Institute of Animal Production, Poland;
2Institute of Public Health, Faculty of Health Care, Collegium
Medicum, Jagiellonian University, Kraków, Poland
e-mail: Jaroslaw Wieczorek <jwieczor@izoo. krakow. pl>
The possibility of using transgenic pig kidneys for allogenic and heterogenic transplantation was evaluated with
regard to the clinical context. The aim of the study was to
estimate immunological response in donors of the graft
and to assess the influence of transgenesis on immunogenity of the transplanted kidney. There were 3 nontransgenic and 6 transgenic pig donors with a human α1. 3-fucosyltransferase gene. The recipients of the kidneys were
6 nontransgenic and 3 transgenic pigs. Three groups were
selected on the basis of the genetic configuration of donors
and recipients: 1) NN — nontransgenic/nontransgenic, 2)
NT — nontransgenic/transgenic, TT — transgenic/transgenic. The kidneys were transplanted heterotopically to
the intraperitoneal space on the left side at the level of
the 4–5th lumbar vertebrae, with end-to-side anastomosis of the renal vein and artery with vena cava caudale
and aorta, respectively. The immunological response
was evaluated on the basis of the changed levels of IL1, IL-4 IL-6 IL-10 TNF-α and INF-γ in the serum. A very
high increase in the level of each cytokine evaluated was
detected during the first 48–72 hours after engraftment.
The decrease in cytokine level was observed during 11
days after surgery. Weaker immunological response after
the transplantation of transgenic kidneys was observed.
Much lower changes in cytokine level were detected after transplantation of transgenic kidney in comparison to
nontransgenic kidney. The acute rejection of the graft was
found based on the evaluation of cellular and humoral
immunological response. It is concluded that transgenesis had positive effect on the immunological response in
transplant recipients.