Reproduction in Domestic Animals

234 Pages·2008·12.37 MB·English

by  Biszkup Miklós

Reproduction
Vol. 43 • Supplement 3
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Book of Abstracts
of the
16th International Congress on Animal Reproduction
13–17 July 2008 - Budapest, Hungary
Workshop abstracts
Poster abstracts
Official Organ of
European Society for Domestic Animal Reproduction
ISSN 0936-6768 (Print) European Veterinary Society off Small Animal Reproduction
ISSN 1439-0531 (Online) Spanish Society of Animal Reproduction
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Reproduction in Domestic Animals
Official Organ of European Society for Domestic Animal Reproduction, European Veterinary
Society off Small Animal Reproduction and Spanish Society of Animal Reproduction
Editor-in-Chief Associate Editors Prof Dr. José Luiz Rigo Rodrigues
Universidade Federal do Rio Grande do Sul
Prof. Dr. H. Rodriguez-Mártinez Prof. Dr. W.A. King
Faculdade de Veterinária
Division of Reproduction University of Guelph
Caixa Postal 15004
Department of Clinical Sciences Dept. of Biomedical Sciences
91501-970 Porto Alegre RS, Brazil
Faculty of Veterinary Medicine and Animal Science Guelph, Ont. N1G 2W1, Canada
Swedish University of Agricultural Sciences (SLU)
Prof. Dr. E. Sato
Ullsvägen 14C, Clinical Centre. P.O. Box 7054, Prof. Dr. E. Martinez-Garcia
Laboratory of Animal Production
Ultuna SE-750 07 Uppsala, Veterinary Teaching Hospital
Graduate School of Agricultural Science
Sweden Dept. of Vet. Pathology, University of Murcia
Tohoku University
Telephone: [phone] Campus de Espinardo, 30500 Murcia, Spain
Aoba-Ku, Sendai 981-8555, Japan
Fax: [phone]
E-mail: [email protected] Prof. Dr. M. McGowan
School of Veterinary Science
University of Queensland
St. Lucia QLD 4072l, Australia
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16th International Congress on Animal Reproduction

13-17 July 2008 – Budapest, Hungary

BOOK OF ABSTRACTS

Workshop abstracts

Poster abstracts
SPONSORS, EXHIBITORS & ADVERTISERS
The Organising Committee expresses its gratitude to Minitüb Abfüll- und Labortechnik GmbH & Co. KG,
Current Conceptions Inc. and Alltech Hungary Ltd. for providing travel grants to ICAR congress participants.
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
Table of Contents 3

TABLE OF CONTENTS

TABLE OF CONTENTS..............................................................................................................................................3

WORKSHOP ABSTRACTS........................................................................................................................................5

Workshop 01 - Pathophysiology and Immunology of Postpartum Uterine Disease in Cattle......................................5
Workshop 02 - Factors Affecting Pregnancy Rates in Estrus Synchronization Programs in Beef Heifers..................6
Workshop 03 - Factors Influencing the Embryo Quality..............................................................................................7
Workshop 04 - Imaging techniques in reproduction....................................................................................................8
Workshop 05 - Sustainable development in care of reproduction in small ruminants: efforts and per-spectives.....10
Workshop 06 - Reproduction of Camelidae..............................................................................................................11
Workshop 07 - Physiology, Clinical Relevance and management of postpartum anovulation in dairy cows............12
Workshop 08 - Reproductive Physiology and Management of Reproduction in Farmed Cervidae..........................13
Workshop 09 - Cryopreservation of gametes and embryo.......................................................................................15
Workshop 10 - Artificial Insemination and Related Methods in Pet Carnivores........................................................16
Workshop 11 - International Trade of Semen and Embryos.....................................................................................17
Workshop 12 - New Applications of Technology for Education in Reproductive Science.........................................18
Workshop 13 - Physiological and management factors affecting expression of estrus in cattle...............................19
Workshop 14 - Zoo and Wild Animal Reproduction..................................................................................................20
Workshop 15 - Animals as biomedical models in reproductive (and regenerative) medicine...................................22
Workshop 16 - Recent Progress in Andrological Procedures and Evaluations........................................................24
Workshop 17 - Equine Endometritis.........................................................................................................................25
Workshop 18 - Pregnancy, fetal well-being and the peri-parturient dam..................................................................26

POSTER REVIEWERS.................................................................................................................................................27

POSTER ABSTRACTS.................................................................................................................................................28

Poster 01 - Bovine Reproduction..............................................................................................................................28
Poster 02 - Reproduction of Small Ruminants..........................................................................................................70
Poster 03 - Reproduction of Buffalo and Exotic Bovidae..........................................................................................89
Poster 04 - Reproduction of Camelidae....................................................................................................................93
Poster 05 - Equine Reproduction..............................................................................................................................97
Poster 06 - Porcine Reproduction...........................................................................................................................114
Poster 07 - Reproduction of Pet Carnivores...........................................................................................................125
Poster 08 - Reproduction of Zoo and Wild Mammals.............................................................................................131
Poster 09 - Reproduction of Rabbit and Laboratory Rodents.................................................................................135
Poster 10 - Avian Reproduction..............................................................................................................................139
Poster 11 - Reproduction of Other Vertebrates (Fishes, Amphibians, Reptiles).....................................................140
Poster 12 - Neuroendocrine Control of Reproduction.............................................................................................142
Poster 13 - Molecular Biology of Reproduction.......................................................................................................145
Poster 14 - Ovary and Uterus.................................................................................................................................153
Poster 15 - Pregnancy, Parturition, New-Born Offspring........................................................................................156
Poster 16 - Andrology, Male Genitals.....................................................................................................................160
Poster 17 - Artificial Insemination and Related Techniques...................................................................................172
Poster 18 - Oocyte and Embryo (Including Nuclear Transfer)................................................................................184
Poster 19 - Biomedical Models in Reproductive and Regenerative Medicine.........................................................202
Poster 20 - Gene Modified Animals (Transgenics).................................................................................................203
Poster 21 - New Methods in Care of Reproduction................................................................................................206
Poster 22 - Stress, Diseased State and Reproduction...........................................................................................207
Poster 23 - Conservation of Biodiversity.................................................................................................................209
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
4 Table of Contents

Poster 24 - Toxicology of Reproduction..................................................................................................................211
Poster 25 - Developments in Sustainable Animal Production and Reproduction...................................................214
Poster 26 - Trends in Research, Care and Teaching of Reproduction...................................................................214
Poster 27 - Trace Minerals and Reproduction........................................................................................................215
Poster 28 – Other topics.........................................................................................................................................216

AUTHOR INDEX.........................................................................................................................................................220
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
Workshop Abstracts 5

WORKSHOP ABSTRACTS

Workshop 01 - Pathophysiology and Immunology of implicated. Retention of fetal membranes is associated with impaired
Postpartum Uterine Disease in Cattle prepartum immune function, particularly with reduced activity of
neutrophils. This impairment extends into the postpartum period and
Moderator: Martin Sheldon (UK)
mediates increased susceptibility to subsequent uterine disease and
mastitis.
WS01-1 Puerperal metritis, characterized by malodorous, red-brown uterine
The pathogens and the cause of uterine disease exudate and uterine atony before 14 days postpartum affects up to
40% of dairy cows in some herds. Systemic signs including fever,
Földi, J1,2; Pécsi, A3; Szabó, J4; Kulcsár, M2; Egyed, L5; Huszenicza, G2*
depression or malaise are inconsistent. Reduced dry matter intake in
1Intervet International B.V., Boxmeer, Holland; 2Faculty of Veterinary Science,
late pregnancy, resulting negative energy balance and impaired
Szt. István University, Budapest, Hungary; 3Faculty of Agricultural Science,
immune function contribute to the pathogenesis of this condition as
University of Debrecen, Hungary; 4Faculty of Medical Science, University of
well as clinical and subclinical endometritis. Cows with metritis are at
Debrecen, Hungary; 5Veterinary Medical Research Institute, Budapest,
increased risk for subsequent endometritis. Response to treatment is
Hungary
generally favorable. Most cows with overt uterine disease do not

ovulate early in the postpartum period. Cows ovulating in the face of
A wide variety of bacteria are present in the uterus of all cows at the continued uterine infection may be predisposed to development of
first 10-14 postpartum (pp) days, regardless of disease signs. Mostly pyometra – an accumulation of purulent exudate in the uterine lumen
Streptococcus spp., Staphylococcus spp. and Bacillus spp. were in the presence of a persistent corpus luteum.
isolated from the uterus of cows with physiological involution, while Clinical endometritis is best diagnosed and treated after 4 weeks
Arcanobacterium pyogenes (A. pyo), Escherichia coli (E. coli) and postpartum when purulent material is demonstrable in the uterus or
different Gram negative (GN) anaerobic bacteria namely vagina by ultrasonography, vaginoscopy, manual inspection, or use of
Fusobacterium necrophorum, Prevotella spp. and Bacteroides spp. a specifically designed instrument (Metricheck®) or if the cervix
were predominant in the uterus of clinically diseased animals. Series remains larger than 7.5 cm in diameter. Presence of these signs is
of studies confirmed that clinical and reproductive consequences are associated with reduced likelihood of subsequent pregnancy.
associated with these ‘primary uterine pathogen bacteria’. Although Pregnancy risk is increased by treatment with intrauterine cephapirin
E. coli plays a key role in puerperal metritis (up to 14 days pp) i.e. or systemic prostaglandin F2alpha.
systemic signs of the disease are endotoxin mediated, its influence Many cows without overt signs of infection have persistent uterine
decreases by the time of involution. Clinical endometritis/pyometra inflammation at the beginning of the rebreeding period. This
is mostly the result of a chain of re-infection with A. pyo and GN condition is best diagnosed by endometrial cytology, but may be
anaerobes. A. pyo acts synergistically with F. necrophorum, indicated by presence of a fluid column greater than 3 mm in diameter
Bacteroides spp. and Prevotella spp. Isolation of A. pyogenes at the in the uterine lumen. Subclinical endometritis is associated with
late involution period (28-35 days) is associated with dramatically reduced first service pregnancy risk, increased days open, and
decreased re-conception rate. increased culling risk, making it an extremely costly condition of high
Bovine herpesvirus type 4 (BoHV-4) is a member of Rhadinovirus producing dairy cows.
genus within the Gammaherpesvirinae subfamily. BoHV-4 is unique
among the BoHVs concerning its wide tropism of species as well as
WS01-3
tissue distribution including reproductive organs. BoHV-4 has been
associated with abortion and (puerperal) metritis since 1973, however, The immune system and the mechanisms of uterine
its endometriotropism and the symbiotic relationship between disease
endometrial stromal cells and macrophages persistently infected with Sheldon, IM
the virus has most recently been proven. It supports the concept that Royal Veterinary College, London, UK
BoHV-4, as a secondary pathogen, decreases the local immune
response and as such, promotes bacterial metritis.
After parturition, contamination of the uterine lumen by microbes is
Presence of pathogens in the uterus i.e. (bacterial) contamination does
ubiquitous in dairy cattle. A third of animals develop clinical disease
not always result in inflammation. Even the colonization of the entire
and a third have sub-clinical endometritis. Infections cause infertility
uterine wall i.e. (bacterial) infection as such, does not necessarily
by damaging the endometrium, but they also perturb ovarian function.
mean a clinical disease; it depends on the immune status of the host.
Effects on the ovary include slower follicle growth, reduced secretion
The course of uterine involution may be considered as a ‘see-saw
of oestradiol and fewer ovulations. In animals that ovulate and form a
balance’: in a physiological situation the self-defence mechanisms are
corpus luteum, there are lower plasma concentrations of progesterone
able to counteract the bacterial and/or viral infection.
and uterine disease disrupts luteolysis. The most important pathogens
On a herd level, the most important risk factors of metritis are:
are Escherichia coli, Arcanobacterium pyogenes and Bovine
metabolic disorders i.e. hyperketonaemia and/or ketonuria,
Herepesvirus 4 (BoHV-4). Infection with E. coli precedes infection
hypocalcaemia; dystocia, retained foetal membranes, manual
with A. pyogenes and the development of uterine disease, as well as
intervention at calving (associated with poor hygiene), herd size,
being associated with ovarian dysfunction. The endotoxin of E. coli,
season, parity, high milk production and lack of grazing.
lipopolysaccharide (LPS), is found in high concentrations in the

uterine lumen and ovarian follicular fluid of animals with
WS01-2 endometritis. Detection of microbes and their pathogen associated
The host and nature of postpartum uterine disease in molecules in the genital tract depends on the innate immune system,
dairy cows including pathogen recognition receptors such as the Toll-like
Receptors (TLR). Uterine epithelial and stromal cells, and ovarian
Gilbert, RO
granulosa cells express the TLR4, CD14, MD2 receptor complex for
Cornell University, Ithaca, NY, USA
LPS. In vitro, LPS increases the expression of genes associated with

inflammation in endometrial cells, and LPS switches epithelial cell
Fetal membranes are usually expelled within 6 hours after delivery
secretion from prostaglandin F to E, which may explain how
and are regarded as retained after 12 or 24 hours. The incidence in 2α 2
uterine disease disrupts luteolysis. Granulosa cells treated with LPS
dairy cows is about 5 – 15%. Risk of retention is increased by
have lower aromatase expression and reduced oestradiol production,
abortion, stillbirth, low birthweight, multiple birth, premature
which may explain how uterine disease perturbs ovarian follicle
parturition, dystocia, heat stress and hypocalcemia. Deficiency of
growth and function. Severe uterine pathology is associated with the
antioxidant nutrients such as selenium and vitamin E is also
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
6 Workshop Abstracts

presence of A. pyogenes or viral infection. The exotoxin of A. important production traits in beef cattle (carcass quality, fertility,
pyogenes, pyolysin (PLO), causes epithelial and particularly stromal health & welfare), have increased the need to develop practical and
cell death. Similarly, BoHV-4 is highly tropic for the endometrium, effective programs which efficiently disseminate selected genetics. As
causing a rapid cytopathic effect in stromal and then epithelial cells. the major grazing rangelands for beef cattle are located in tropical and
This tropism may be because viral replication is activated by subtropical regions, development of artificial breeding programs
prostaglandin E, which is constitutively secreted by the stromal cells. which focus on maximizing the reproductive outcome in Bos indicus
2
Further exploration of the molecular mechanism of microbial cattle, the genotypes most suited to these environments, is essential.
infection and immunity will lead to greater understanding of how However, most of the treatment protocols for synchronization of
uterine disease impacts fertility, and provide insights to help develop oestrus have been developed for Bos taurus cattle with the assumption
new therapeutic approaches. being that usage in Bos indicus cattle will result in similar outcomes.
This work was supported by the Wellcome Trust and BBSRC. Although some publications report similar reproductive outcomes,
marked, frequently unexplained variation in response to hormonal
treatments to synchronise oestrus, continue to be important factors
limiting the widespread usage of this technology in extensively
Workshop 02 - Factors Affecting Pregnancy Rates in
managed Bos indicus herds. Progestin and progesterone implants
Estrus Synchronization Programs in Beef Heifers combined with prostaglandin F2α, oestrogen and gondaotrophin
Moderator: Gabriel Bo (Argentina) treatments are most commonly used for fixed-time AI (FTAI) of Bos
indicus females. The reported responses to these treatments in terms
of animals detected in oestrus between 48 to 72 h after implant
WS02-1
removal, and animals diagnosed pregnant to FTAI vary considerably
Reproductive management of cycling and non-cycling (44 to 98% and 37 to 62%, respectively). A series of recently
Bos taurus beef heifers in the USA completed studies conducted in northern Australia have shed some
Lamb, G light on potential causes of the observed variation in response of Bos
North Florida Research and Education Center, University of Florida, Marianna indicus heifers. When 300 cycling disease-free Brahman heifers
weighing 289±30 kg, were randomly assigned to be treated with
oestradiol benzoate (ODB) and intravaginal implants (8 day insertion)
Estrus synchronization and artificial insemination (AI) are
varying in progesterone content (0.78g, 1.56g, 1.9g) followed by
reproductive management tools that have been available to beef
ODB treatment (Day 9) and FTAI 48 to 54 hours after implant
producers for over 30 years. Synchronization of the estrous cycle has
removal, the overall pregnancy rate was 35%. However, the pre- and
the potential to shorten the calving season, increase calf uniformity,
post-AI progesterone profiles of a random samples of 10 heifers from
and enhance the possibilities for utilizing AI. Artificial insemination
each group indicated that 32% (n=30) had plasma progesterone
allows producers the opportunity to infuse superior genetics into their
concentrations of <1ng/ml on day 6 after FTAI, with only 20% of
operations at costs far below the cost of purchasing a herd sire of
these conceiving, compared to 60% in heifers which had progesterone
similar standards. These tools remain the most important and widely
concentrations >1ng/ml. Consideration should be given to
applicable reproductive biotechnologies available for beef cattle
investigating the role of hormonal treatments in inducing suboptimal
operations in the USA. However, beef producers have been slow to
preovulatory follicular and corpus luteum development in Bos indicus
utilize or adopt these technologies into their production systems.
cattle.
Several factors, especially during early stages of development of

estrus synchronization programs, contributed to the poor adoption
rates. Initial programs failed to address the primary obstacle in WS02-3
synchronization of estrus, which was to overcome the late onset of Fixed-time artificial insemination in cycling and non-
puberty in heifers. Additionally, these programs failed to manage the
cycling Bos indicus beef heifers
timing of ovulation precisely, resulting in more days over which
Baruselli, PS1*; Sales, JNS1; Crepaldi, GA1; Sá Filho, MF2; Carvalho, JBP3;
detection of estrus was necessary. This ultimately precluded fixed-
Bo, GA4
time AI (FTAI) with acceptable pregnancy rates. More recent
1Animal Reproduction Department, FMVZ/USP, São Paulo-SP, Brazil;
developments focused on the control of both the corpus luteum and
2FIRMASA-IATF, Campo Grande-MS, Brazil; 3Vale do Paraíba Regional
follicle waves in convenient and economical protocols to synchronize
Station-Apta, Av. Prof. Manoel Cesar Ribeiro, No. 320, Caixa Postal 7,
the timing of ovulation, facilitating FTAI. This new generation of
Pindamonhangaba, SP, Brazil; 4Instituto de Reproducción Animal Córdoba,
estrus synchronization protocols uses two strategies which are key
J.L. de Cabrera 106, X5000GVD Córdoba, Argentina
factors for implementation by producers because they: 1) minimize

the number and frequency of handling cattle; and 2) eliminate the
need for detection of estrus by employing FTAI. However, developing Fixed-time artificial insemination (FTAI) protocols have generally
FTAI protocols in beef heifers has not been straightforward because yielded poor results in B. indicus heifers. It was hypothesized that
of the inability to synchronize follicular waves reliably with follicular growth in Bos indicus heifers might be suppressed by
gonadotrophin-releasing hormone (GnRH). For example, after an circulating progesterone concentrations during the treatment protocol.
injection of GnRH at random stages of the estrous cycle, 60 to 90% of It is speculated that lower steroid hormone clearance rates in Bos
postpartum cows ovulated; whereas, only 48 to 60% of beef and dairy indicus cattle may account for the higher circulating progesterone
heifers ovulated in response to the same treatment. Therefore, most concentrations during progesterone treatment resulting in reduced LH
recent research continues to focus on inducing onset of puberty in pulsatility and follicular growth rates. A series of experiments were
noncycling heifers and enhancing synchrony of ovulation by designed to determine if synchronization of follicle wave emergence
incorporating progestin devices in GnRH-based protocols to facilitate and ovulation with estrogen and subluteal progesterone concentrations
FTAI and improve fertility in heifers. during growth of the ovulatory follicle would improve the synchrony
of ovulation and pregnancy rates following FTAI. Treatment with
PGF at the start of progestin treatment resulted in decreased
WS02-2
circulating progesterone concentrations and increased the follicular
Why are pregnancy rates to fixed-time AI generally lower growth rates, dominant follicle diameters and ovulation rates. In
in Bos indicus than Bos taurus cattle? comparison to CIDR, norgestomet ear implants resulted in increased
McGowan, M*; Butler,S; Phillips, N dominant follicle growth rates leading to ovulatory follicles with
School of Veterinary Science, The University of Queensland, Brisbane, larger diameters. Dose and estradiol ester were important factors in
Australia synchronizing follicle wave emergence. A dose of 2.5 or 5 mg
estradiol valerate resulted in a longer and more variable interval from
treatment to follicular wave emergence than 2 mg of estradiol
The recent developments in quantitative and molecular genetics which
benzoate (EB), which affected preovulatory dominant follicle size
have led to improvements in our ability to select for a range of
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
Workshop Abstracts 7

following progestin removal. Interestingly, satisfactory ovulation rates WS03-2
occurred in both cycling and non-cycling heifers treated with Gene expression in early porcine embryos following
norgestomet ear implants, 2 mg EB at implant insertion and 1 mg EB
serial nuclear transfer
24 h after implant removal. Treatment with eCG at implant removal
also increased dominant follicle growth rate, preovulatory follicle Xing, X; Magnani, L; Lee, K; Wang, C; Cabot, RA; Machaty, Z*
diameter, ovulation rate, and pregnancy rates in Bos indicus heifers Department of Animal Sciences, Purdue University, West Lafayette, IN, USA
that were FTAI. Nevertheless, conception rates in heifers that
ovulated after treatment was lower in non-cycling than in cycling During nuclear transfer (NT), the donor nucleus is reprogrammed so
heifers which may be related to the immaturity of the hypothalamo- that it becomes capable of directing development of the new embryo.
pituitary axis and the reproductive tract. An alternative in non-cycling In most cases reprogramming is incomplete, which leads to abnormal
heifers may be to use a priming treatment with a progesterone expression of genes critical for development. Little is known about the
releasing device 20 to 40 days prior to initiating synchronization underlying mechanisms, but epigenetic alterations induced by factors
treatments. Data suggest that FTAI in Bos indicus heifers can be in the oocyte cytoplasm play fundamental roles. Here we evaluated
successful providing follicle wave emergence is synchronized and the expression of developmentally important genes in pig embryos
methods that result in increased ovulatory follicle growth, such as produced by traditional or serial NT.
reduced progesterone concentrations and treatment with eCG are NT embryos were produced by transferring individual fibroblast cells
employed. into enucleated pig oocytes. These embryos were then either used to
study gene expression, or cultured for 4 days and their cells were
utilized as nuclear donors during serial nuclear transfer (SNT). For
this latter purpose, the blastomeres of 8-16-cell NT embryos were
Workshop 03 - Factors Influencing the Embryo Quality
separated and transferred again into enucleated oocytes. The
Moderator: Li Ning (China) expression of the Oct-4, Nanog and Sox-2 genes were monitored in 2-
Xiuchun Tian (USA) cell and 4-cell-stage NT and SNT embryos. mRNA was isolated from
pools of 20 embryos; gene expression patterns were determined using
quantitative real-time PCR. Transcript levels were quantified using
WS03-1
the comparative threshold cycle method. Embryos produced by in
Modifications in oocyte maturation and/or embryo culture vitro fertilization (IVF) were used as control. Data were analyzed by
to improve quality of pig somatic cell cloned embryos one way ANOVA with Tukey’s test for multiple comparisons.
Zhang, Y1,2, Zhang, K1,3, Wie, H1, Li, J4, Li, Q1, Dai, Y1 & Li, N1* There were dramatic differences in gene expression between embryos
1State Key Lab of Agrobiotech., China Agr. Univ., Beijing 100094, China; at the 2-cell stage. Oct-4 expression in NT embryos was more than
2College of Anim. Sci. Tech., Anhui Agri. Univ., Anhui 230036, China; 600 times lower than in those produced by IVF. In addition, transcript
3Department of Animal Sciences, University of Florida, FL 32611-0910, USA; levels of all three genes were reduced in NT compared to SNT
4Dept. of Genet. Biotech., Facul. of Agr. Sci., Univ. of Aarhus, Tjele, 8830, embryos (p<0.05). At the 4-cell stage, NT embryos had gene
Denmark expression patterns similar to those found in IVF embryos while SNT
embryos exhibited significantly lower Oct-4 and Sox-2 transcript
levels. These results indicate that the recipient ooplasm could
Somatic cell nuclear transfer (SCNT) is regarded as a tool with great
reprogram the expression of Nanog and Sox-2 by the first mitotic
potential in agriculture, medicine and basic research. For pigs, the
division but Oct-4 expression was perturbed compared to that in IVF
efficiency of producing viable offspring from cloned embryos is poor.
embryos. Another round of nuclear transfer led to Oct-4 transcript
Suboptimal oocyte maturation and/or embryo culture are major
levels that were similar to those in IVF embryos. When the NT
bottlenecks limiting commercialization of pig SCNT. Oocytes are
embryos reached the 4-cell stage, their gene expression patterns were
crucial starting material for SCNT therefore we examined effects of
similar to those in the control group. The data imply that in NT pig
oxygen tension, leptin addition during IVM on development of SCNT
embryos, ooplasmic factors can efficiently reset Oct-4, Nanog and
embryos. Blastocyst rates and total cell number per blastocyst of
Sox-2 expression after two mitotic divisions, further reprogramming
SCNT embryos from oocytes matured under low (7%) O2 tension
in the form of another round of NT provide no apparent benefits.
were significantly higher than those of embryos from oocytes matured

under higher (20%) O2 tension (blastocyst rates: 16.3±0.9% vs.
6.5±0.6%, total cell number per blastocyst: 38.4±2.0 vs. 30.4±2.2%, WS03-3
respectively; P<0.05). Addition of 10 ng/mL Leptin during the first Polyploidy in porcine embryos produced in vitro
20±2 hr of IVM was helpful to increase developmental potential and Somfai, T1,4*; Ozawa, M1; Noguchi, J1; Kaneko, H1; Nakai, M1; Maedomari, N2;
quality of SCNT embryos (Zhang et al., 2007, Anim Reprod Sci, Nagai, T3; Kikuchi, K1
101,85-96). To improve the in vitro culture (IVC) we evaluated the 1Division of Animal Sciences, National Institute of Agrobiological Sciences,
effects of NCSU23 and PZM3, and the oxygen tension during IVC on Tsukuba, Japan; 2Graduate School, Azabu University, Japan; 3National
the development of SCNT embryos. IVC with 7% O2 resulted in Institute of Livestock and Grassland Science, Japan; 4Present affiliation:
better blastocyst rates and higher total cell numbers per blastocyst National Livestock Breeding Center, Japan
than IVC with 20% O2. The total cell number per blastocyst was
significantly higher in sequential culture media G3.1/G3.2
In vitro production (IVP) of porcine embryos is stricken by the
(64.3±11.3) compared to NCSU23 (46.3±9.6) and PZM3 (49.4±9.8).
problem of high frequency of polyploidy. The two main reasons for
Addition of leptin or myoinositol into PZM3 was tested and the
polyploidy are 1) polyspermic fertilization and 2) failure of oocytes to
results shown that 1) addition of 50 ng/mL leptin from the 3rd day
reach the metaphase-II (M-II) stage after in vitro maturation (IVM).
after the onset of IVC increased the blastocyst rate and inhibited cell
The objective of the present study is to demonstrate the occurrence
apoptosis of SCNT embryos; 2) addition of 10 μg/mL myoinositol
and consequences of these phenomena. In our IVP system
during the entire IVC slightly increased the total cell numbers per
approximately 60% of fertilized oocytes are polyspermic. We
blastocyst without compromising formation of blastocysts. Employing
separated polyspermic and monospermic zygotes after visualization of
the optimized IVM and IVC systems, we have successfully produced
pronuclei by centrifugation and compared their in vitro developmental
the first SCNT piglet in mainland China, and further transgenic or
ability to the blastocyst stage. Our results show that polyspermy
gene-targeted SCNT piglets. In conclusion, lower O2 tension is
causes reduced cleavage and blastocyst rates; however, cleaved
beneficial to oocyte maturation and development of SCNT embryos,
monospermic and polyspermic embryos have similar ability to
and addition of leptin during IVM or IVC, or myoinositol during IVC
develop to the blastocysts stage. In contrast to monospermic (mostly
at certain concentration and appropriate time could enhance the
diploid) embryos, blastocysts developed from polyspermic zygotes
embryo quality.
showed a higher frequency of polyploidy (21.8%) and mixoploidy
Funded by Major State Basic Research Development Program of
(29.6%), whereas a remarkable proportion (31.3%) of them was found
China
to be diploid. At 36h after IVF, although there was no difference in
16th I n t e r n a t i on al C o ng r es s o n Ani m a l R e pro d u ct i on
8 Workshop Abstracts

the number of nuclei, the number of blastomeres was higher in Workshop 04 - Imaging techniques in reproduction
polyspermic embryos due to early fragmentation. Further studies are Moderator: Gregg P Adams (Canada)
needed to clarify the phenomenon of ploidy correction in polyspermic

zygotes.
During IVM, approximately 30% of oocytes fail to reach the M-II WS04-1
stage and about 60-78% of them are arrested at a certain stage Use of 3-D and 4-D ultrasonography in veterinary
characterized by a metaphase plate without polar body. This stage is research
often referred as “metaphase-I (M-I) arrest”. Our previous studies
Hildebrandt, TB1*, Drews, B1, Kurz, J2, Röllig, K1, Hermes, R1, Yang, S3,
showed, that unlike real M-I oocytes, “M-I arrested” oocytes obtain
Göritz, F1
cytoplasmic maturation and have the ability to develop to the
1Reproduction Management, Leibniz Institute for Zoo & Wildlife Research,
blastocyst stage resulting in polyploid embryos. Chromosome
Germany; 2Institute for Genetik & General Biology, University of Salzburg,
configurations of such oocytes are different from those of M-I oocytes
Austria; 3Yunnan Key Laboratory for Animal Reproduction, Kunming Institute
at 33h IVM and similar to those of M-II oocytes despite of their
of Zoology, CAS, China
tetraploid status. It is suggested that in “M-I arrested” oocytes

segregation of homologue chromosomes may occur during IVM,
however, the extrusion of the first polar body fails and a single Three-dimensional-ultrasonography represents an independent (what
metaphase plate is formed again. is meant by independent?) imaging modality in human medicine with
In conclusion, development to the blastocyst stage is not a perfect a wide range of applications, including prenatal diagnosis,
indicator of embryo quality in porcine IVP systems, since polyploid gynecology, and oncology. Three-dimensional-ultrasonography
embryos can develop to blastocyst stage. Careful selection of M-II allows morphometric measurements in a volume data set and, more
oocytes for IVF, regular monitoring of polyspermy rates and further recently, the analysis of data in tomographic ultrasound imaging mode
improvements of IVF systems are necessary to reduce polyploidy in (TUI). The advantages of three-dimensional-ultrasonography have
porcine IVP systems. also been realized in animal research. Ultrasound equipment used in
animals requires additional components and animal-specific scanners
for transrectal examination. After substantial system modification
WS03-4
transrectal three-dimensional-ultrasonography was applied intensively
Tyrosine kinase receptors (TRK) and bovine early to characterize embryo/fetal development in Asian and African
embryonic development elephants. Three ultrasound systems were used during this study: a
Gómez, E*, Rodríguez, A; Caamaño, JN; Díez. C; Facal, N; Muñoz, M stationary Voluson 530 and a 730 Expert, amd a portable Voluson I
Genética y Reproducción, Serida, Gijón, Spain (General Electric Inc.). Growth curves for elephant embryo/fetal
development were established. In render mode the surface structures
were imaged, and used to depict the transition from the oval-shaped
Neurotrophins (NTs) mediate survival, growth and differentiation by
embryo to the complete fetus with its characteristic trunk. In inverse-
binding to two types of cell surface receptors, the anti-apoptotic Trk
render mode, the fluid-filled compartments were displayed separately.
and the low affinity p75 neurotrophin receptor (p75NTR), also termed
This mode was also applied to imaging of ovarian follicular
as p75NGFR. Subtypes of Trk are NT-specific, such a way NGF binds
development in rhesus macaques during superovulation. In
to TrkA, NT3 to TrkC, and NT4 and BDNF to TrkB; on the contrary,
conjunction with a scientific consultancy for National Geographic,
p75NTR is a common receptor for all NTs. Trk and p75NTR receptors
additional investigations were performed in dogs, primates, hares and
can form complexes, and this association enhances affinity for ligand
dolphins. The main purpose was to identify useful morphological
binding and neurotrophin discrimination.
parameters of the different developmental stages during pregnancy,
In previous work, we suggested a role for NTs during early embryonic
and examine intrauterine fetal behavior. For the latter, four-
development, as we detected TrkA (a truncated isoform), TrkB and
dimensional ultrasound technology (3D in real-time) was applied. We
TrkC proteins in immature oocytes, zygotes, 2-4 cell embryos,
found dramatic differences between species regarding the level of
morulae, expanded and hatched blastocysts, and the inner cell mass of
fetal activity, including some interesting behavioral patterns observed
blastocysts. Using RT-PCR we found mRNA for TrkA, TrkC, p75NTR
from each species throughout their development. According to these
and FGFr2 in all the embryonic stages described above; mRNA levels
preliminary findings, we conclude that intrauterine behavior is an
tended to increase during blastulation, and sharply peaked in hatched
important element during embryogenesis. There is also a strong
blastocysts. However, single (NGF, NT3, NT4 or BDNF, at 20 ng/mL
indication that intrauterine behavior has parallels to known postnatal
each) or pooled NTs in embryo culture with the NT-cooperating
behavior in the different species. Four-dimensional ultrasonography
factor bFGF (2 ng/mL), did not show relevant effects on development
is, a useful tool for identifying species-specific behavioral elements
and cell counts. Comparable results were obtained in a recent
during intrauterine development. The definition of these key
experiment in the absence of bFGF. bFGF was detrimental compared
behaviors and the consequences of their presence or absence for the
to controls without bFGF, while NT3, with or without bFGF,
life history of an individual will improve our understanding of
increased cell numbers in the ICM as opposed to NT4 and pooled
evolution and help in clinical diagnosis of developmental disorders.
NTs. NT4 enhanced cell counts in the trophectoderm as compared to

controls with bFGF and pooled NTs only in the presence of bFGF.
Dose-response studies and new experiments to progress in research WS04-2
with NTs and their receptors are in course. We have provided a novel In vivo imaging of sperm transport and survival in the
description of Trk proteins and TrkA, TrkC, p75NTR and FGFr2 female genital tract using fiber confocal fluorescence
mRNA expression, throughout mammalian embryonic development.
microscopy
This research may help to design future research with NTs in bovine
embryo culture and embryonic stem cells derivation and maintenance. Druart, X1*; Cognié J.1; Baril, G.1; Clément F.2; Dacheux JL.1 and Gatti JL.1
Grant support: AGL2005–04479. Spanish-Hungarian bilateral project 1UMR 6175 INRA, CNRS-Université de Tours-Haras Nationaux, Nouzilly,
HH2005-0015 (TET E-20/2005). M. Muñoz is sponsored by FICYT. France ; 2INRIA, Paris, France

Introduction Fertility of ram sperm is reduced after 24 h of storage
at 15°C in milk diluent despite that the in vitro properties, such as
viability and motility, are well preserved. This might be explained by
a decrease of sperm transport and survival of stored ram sperm in the
female genital tract. We studied the influence of in vitro storage of
ram sperm on in vivo transport in the female genital tract of the ewe
using Fiber Confocal fluorescence Microscopy (FCM, Leica).