bluuebunny/crossref_metadata_2025 huggingface dataset:
Can foxes be controlled by reducing their fertility?
Authors: Pech R. , Hood G. M. , McIlroy J. , Saunders G.
Published: November 2003

A model based on data from research in New South Wales conducted by the
Cooperative Research Centre for the Biological Control of Vertebrate Pest
Populations suggests that the effectiveness of fertility control in reducing
the abundance of foxes (Vulpes vulpes) can be strongly
influenced by environmental variability. The model includes age-specific
recruitment and survival as functions of resources indexed by rainfall. It is
assumed that fertility control will affect only female foxes and that the use
of a baiting regime to deliver a contraceptive agent will result in fixed
proportional changes in pregnancy rates.
By comparing the variability in the rate of increase of treated and untreated
fox populations, the model predicts that: (i) frequent
baiting, every one or two years, will be more effective than applications of
baits at longer time intervals; (ii) the abundance of
foxes will decline more rapidly with higher levels of fertility control;
(iii) infertility which lasts for only one breeding
season is less effective than permanent sterility which allows for
accumulation of sterile animals in the population; and
(iv) highly variable results are likely to be the
outcome of low-frequency baiting with an agent that produces only temporary
infertility.
Perifusion culture system for bovine embryos: improvement of embryo development by use of bovine oviduct epithelial cells, an antioxidant and polyvinyl alcohol
Authors: Lim J. M. , Reggio B. C. , Godke R. A. , Hansel W.
Published: November 2003

Three experiments were conducted in an attempt to improve a continuous
flow-perifusion system capable of maintaining embryo development for long
periods of time. Bovine embryos (8–16 cells) obtained from static
co-culture with cumulus cells in a serum-free medium were perifused in an
ACUSYST-S cell culture incubator. Culture chambers of the incubator consisted
of a 0·2-mL unit (Chamber 1) connected to a
1·5-mL-1 unit (Chamber 2), with the outflow from
Chamber 1 routed to the inlet to Chamber 2. A bovine embryo culture medium
supplemented with 3 mg mL 1 bovine serum albumin (BSA) and 25 mM HEPES was
used as a perifusion culture medium (PCM). Embryos were perifused in Chamber 2
for 24, 48 and 72 h and further co-cultured in a static system up to 216 h
after insemination. In Experiment 1, conditioning PCM with frozen–thawed
bovine oviduct epithelial cells (BOEC) placed in Chamber 1 enhanced
(P < 0·05) blastocyst formation of embryos in
Chamber 2, after 24, 48 and 72 h of perifusion culture. The proportion of
blastocysts was not further increased by placing BOEC in Chamber 2 along with
the embryos. In Experiment 2, embryos were perifused with PCM conditioned with
BOEC in Chamber 1 for 48 h or 72 h. A higher proportion of perifused embryos
developed to the blastocyst stage after addition of 25 U
mL-1 or 50 U mL-1 of superoxide
dismutase (SOD) to PCM than in its absence. However, blastocyst formation of
embryos perifused for 72 h was not increased after addition of 50 U
mL-1 SOD compared with its absence. In Experiment 3, the
proportions of morulae and blastocysts were not decreased by replacement of 3
mg mL-1 BSA with 1 mg mL-1
polyvinyl alcohol (PVA) in a BOEC-conditioned medium containing 50 U
mL-1 SOD after perifusion for 48 h. In conclusion, PCM
conditioning with BOEC and addition of an antioxidant to the perifusion medium
improved the developmental capacity of perifused embryos. PVA is an adequate
replacement for BSA in the perifusion medium.
Effect of cytochalasin B and cycloheximide on the activation rate, chromosome constituent and in vitro development of porcine oocytes following parthenogenetic stimulation
Authors: Cha S. K. , Kim N.-H. , Lee S. M. , Baik C. S. , Lee H. T. , Chung K. S.
Published: November 2003

Activation rate, chromosome constituent and developmental pattern of porcine
oocytes was examined in the presence and absence of cytochalasin B and
cycloheximide following parthenogenetic stimulation. Treatment with
cycloheximide after ethanol or Ca2+ ionophore treatment
increased the incidence of activation. The percentage of oocytes with two or
more female pronuclei was higher (P < 0· 05)
in oocytes treated with cytochalasin B than in control or
cycloheximide-treated oocytes. Treatment with both electrical stimulation and
cytochalasin B increased the incidence of diploid chromosome spreads, and
accelerated development to the morula and blastocyst stage compared with the
control and cycloheximide-treated groups, suggesting a role of ploidy in the
development of parthenote.
Comparison between in vivo-derived and in vitro-produced pre-elongation embryos from domestic ruminants
Authors: Thompson Jeremy G.
Published: November 2003

In vitro production of ruminant embryos has become
routine and is increasingly available as a commercial service to dairy, meat
and wool producers. However, the efficiency of producing viable embryos and
the development of such embryos after transfer to recipients are perceived to
be inferior to that which occurs in vivo. The present
review outlines the biochemical and morphological similarities and differences
between embryos produced In vitro and those produced in
vivo. Some measures of metabolism are not markedly different between
In vitro- and in vivo-derived
blastocysts. However, at a cellular and subcellular level, differences in
metabolism, morphology and ultrastructure have been described, as has
susceptibility to manipulation and cryopreservation. Most importantly are the
differences in lambing and calving rates and the reports of abnormal fetal
development from embryos produced In vitro. These latter
observations are of major concern, as they suggest that the
In vitro environment may affect subsequent developmental
physiology. At the extreme, these effects may not be expressed until adult
life. Further efforts to improve the efficiency of
In vitro embryo production must be accompanied by a
commitment to assess the long-term consequences of these procedures.
Extra keyword: development.
Evaluation of sperm damage and techniques for sperm clean-up
Authors: Rodriguez-Martinez H. , Larsson B. , Pertoft H.
Published: November 2003

The cryopreservation of bull semen adversely affects the metabolism, motility
and membrane integrity of the spermatozoa, thereby decreasing the fertilizing
ability of the processed sample. The present review covers methods available
for examining the functionality of bull spermatozoa after thawing, including
the use of fluorophores in combination with morphological examination.
Procedures for clean-up, separation and selection of morphologically-normal,
viable spermatozoa in vitro are also reviewed. Among the
reviewed procedures are methods for dilution and washing and the selective
fractionation of sperm subpopulations (especially density-gradient
centrifugation in silica-bead suspensions), as well as methods based on
adherence to glass (differential filtration) and sperm self-migration
(particularly swim-up through hyaluronic acid). Emphasis is placed on
assessing the value of these techniques for diagnostic purposes and for
optimizing the methodology ofin vitro fertilization.
Managing problem wildlife in the ‘Old World’: a veterinary perspective
Authors: Artois M.
Published: November 2003

This paper focuses on mammalian pest species mainly in Europe and Africa and
on implications for animal health, human safety, wildlife management and
animal welfare. Three examples of problem species are presented: the wild boar
(Sus scrofa), the stray dog
(Canis familiaris) and the red fox
(Vulpes vulpes). Several species are reservoirs or
vectors of transmissible diseases of man and of economically valuable domestic
species. The control of these and other infections and the limitation of the
nuisance or damage caused by these pest species involves lethal or non-lethal
methods which are briefly reviewed. Some control measures require veterinary
expertise, and veterinary involvement in managing problem species is likely to
increase. With regard to fertility control, methods are considered which will
allow an appropriate choice of the best technique for the management of
problem animals in various habitats. For desirable native species, traditional
methods of control, especially hunting in the case of game species, is
preferable to contraception. For exotic or feral species, control of fertility
seems to be a worthwhile option.
Nitric oxide synthase regulation during embryonic implantation
Authors: Novaro V. , González E. , Jawerbaum A. , Rettori V. , Canteros G. , Gimeno M. F.
Published: November 2003

It has previously been demonstrated that uterine nitric oxide synthase (NOS)
activity increases before embryonic implantation in rats. The aim of the
present work was to investigate the regulation and the physiological relevance
of the nitric oxide (NO) system in ovoimplantation. The increase in NOS
activity in early pregnancy was found to be independent of the presence of
embryos in the uterus. Whereas the Ca2+-dependent
isoform of NOS increased gradually in the preimplantation days, the
Ca2+-independent isoform increased just at the beginning
of implantation (Day 5, 1800 hours); then the activity of both isoforms
declined. Oestradiol, whose concentration peaks before implantation, might be
regulating NOS activity in the uterus, since treatment of rats with tamoxifen,
a receptor antagonist, reduces the activity of both isoforms to
preimplantation levels. Intraluminal injections of L-NAME (0·5 mg
kg-1), a competitive inhibitor of NOS, reduced by
50% the number of implanted embryos; this suggests that the NO system
plays a role during implantation. The data suggest that oestradiol might be a
modulator of NOS activity during nidation and that NO production is necessary
to achieve a successful embryo implantation.
Regulation of reproductive tract immunoglobulins by oestradiol-17β in the European Red Fox (Vulpes vulpes)
Authors: Jersey James de , Hinds Lyn A. , Bradley Mark P.
Published: November 2003

The effect of the ovarian hormone, oestradiol-17β, on reproductive tract
immunity in the female fox was investigated. Reproductive tract antibody
responses were induced by either Peyer’s patch immunization with a
recombinant fox sperm protein, or by oral immunization with live, attenuated
Salmonella typhimurium. The effect of exogenous
oestradiol-17β or the stage of the oestrous cycle on reproductive tract
immunity was assessed. The secretion of specific vaginal IgA, but not vaginal
IgG, antibodies was reduced by exogenous treatment with oestradiol-17β,
while both specific vaginal IgA and vaginal IgG levels declined during the
period of natural oestrus. It is concluded that oestradiol-17β, and
probably other reproductive hormones, are involved in the regulation of
antibody-secretion in the fox reproductive tract, and that reproductive status
is an important factor to consider in the design and application of vaccines
which aim to induce immunity within the female reproductive tract.
A competitive enzyme immunoassay for follicle-stimulating hormone in ovine plasma using biotin-streptavidin amplification
Authors: Watanabe Hiroko , Miyamoto Akio , Fukui Yutaka
Published: November 2003

To determine follicle-stimulating hormone (FSH) concentration in ovine plasma,
a second-antibody enzyme immunoassay using biotin-streptavidin amplication
(BS-EIA) was developed and was validated by comparison with a routine
radioimmunoassay (RIA). Ovine FSH was labelled with
D-biotinyl-b.epsi;-aminocaproic
acid-N-hydroxysuccinimide ester (biotin-7-NHS). The
minimum and maximum detection limits were 0·40 ng
mL-1 and 25·0 ng mL-1
respectively in the BS-EIA. Plasma samples from three superovulated ewes were
measured for FSH by both BS-EIA and RIA. Correlation between values obtained
by BS-EIA and RIA was 0· 86 (n = 106), and
the linear regression had a slope of 0·88 and an intercept of
-0·07 (R2 =
0·74). The baseline of BS-EIA was slightly lower than that of RIA
(0·69 ng mL-1v. 1·10
ng mL-1). The intra-assay and inter-assay coefcients of
variation in BS-EIA were 13·8% and 16·7%
(n = 106) respectively. The recovery rates were
104· 8 2·7% (mean±s.e.m., n
= 14). Changes in plasma FSH concentration from superovulated ewes
evaluated by BS-EIA corresponded well with the results of routine RIA.
Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa
Authors: Sánchez-Partida L. G. , Setchell B. P. , Maxwell W. M. C.
Published: November 2003

The epididymal compounds taurine, hypotaurine and inositol, and the
antioxidants carnosine and ascorbic acid, were added to Tris-based diluents
containing varying concentrations of glycerol, and their effect on the
post-thaw motility characteristics and fertility of ram spermatozoa was
examined. Overall, the post-thaw motility characteristics of spermatozoa were
better when semen was frozen in the presence rather than in the absence of
glycerol. Only taurine protected spermatozoa during cryopreservation; the
presence of 25 mM or 50 mM taurine significantly improved the post-thaw
percentage of motile spermatozoa but this had no effect on fertility after
cervical or laparoscopic insemination of ewes. Increasing the concentration of
taurine to more than 100 mM significantly reduced the percentage of motile
spermatozoa, compared with the lower concentrations of the amino acid. The
presence of more than 50 mM carnosine or ascorbic acid significantly reduced
all motility characteristics compared with the control diluent. Given that
hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility,
the cryoprotective effect of taurine may be attributable to its osmoregulation
rather than to its antioxidant properties.
Effect of hormone pre-treatment of prepubertal sheep on the production and developmental capacity of oocytes in vitro and in vivo
Authors: O'Brien J. K. , Beck N. F. G. , Maxwell W. M. C. , Evans G.
Published: November 2003

Twenty 36-week-old Merino lambs were given either 3, 1 or 0 treatments of 50
µg oestradiol benzoate and (48 h later) a 1·5 mg Norgestamet
implant left in situ for 9 days (3-, 1- and
0CYCLE+G). On Day 7 after the last implant insertion, and on the same day
for 0CYCLE+G, each lamb received 400 I.U. pregnant mare serum
gonadotrophin and 6 mg follicle-stimulating hormone (FSH). The reproductive
tracts were removed for oocyte collection 24 h after FSH. Reproductive tracts
were also collected from 16-24-week-old lambs (n
= 31) (0CYCLEG). The number of antral follicles per ovary was similar
for the 3-, 1- and 0CYCLE+G treatments. Similar rates of
in vitro maturation and monospermic fertilization were
obtained for all groups. The proportion of blastocysts per cleaved oocyte was
higher for 1CYCLE+G (50·5%) than for 3CYCLE+G
(32·9%), 0CYCLE+G (24·3%), and 0CYCLEG
(11·8%) (P < 0·05). Viable
fetuses were obtained at Day 93 of pregnancy after transfer of embryos from
all treatments. These results indicate that a single treatment with oestrogen
and progesterone, prior to gonadotrophin stimulation, will increase the yield
and developmental capacity of oocytes from prepubertal sheep.
Cloning and characterization of the cDNA encoding the PH20 protein in the European red fox Vulpes vulpes
Authors: Have José ten , Beaton Sandra , Bradley Mark P.
Published: November 2003

The PH20 protein is thought to play a crucial role in mammalian fertilization.
The fox PH20homologue has been cloned from a testis cDNA
library and the deduced protein sequence shows high levels of homology to PH20
proteins isolated from other species. Unlike other PH20 proteins the fox
protein does not appear to be membrane associated through a GPI-linkage nor
does it show the presence of a transmembrane domain at the C-terminus of the
protein. It is in this region that the proteins appear to be least conserved.
Immunolocalization studies on fox sperm show that the PH20 protein is located
on the inner acrosomal membrane. Transcription of PH20
in the fox is seasonally regulated, with the mRNA expressed during those
months when spermatogenesis is at its peak. The PH20 sequence described in
this paper has been submitted to the Genbank database and has the accession
number U41412.
Biochemical parameters regulating forward motility initiation in vitro in goat immature epididymal spermatozoa
Authors: Jaiswal Bijay S. , Majumder Gopal C.
Published: November 2003

An investigation was carried out to analyse the biochemical parameters
influencing forward motility (FM) initiation in vitro in
the goat caput-epididymal immature spermatozoa. Forward motility was induced
in approximately 55% of caput-sperm upon incubation in an alkaline (pH
8.0) modified Ringer’s solution containing theophylline (30 mM) (an
inhibitor of cyclic AMP phosphodiesterase), dialysed epi-didymal plasma (EP)
and bicarbonate. Both EP and bicarbonate induced sperm motility in a
dose-dependent manner, and at saturating doses EP (0.6 mg protein
mL–1) and bicarbonate (25 mM) induced FM in
approx-imately 38% and 44% of the cells, respectively. The
motility-promoting efficacy of EP was attributed to a heat-stable protein
termed ‘forward motility protein’ (FMP). Bicarbonate served as an
initiator as well as a stabilizer of FM and its action was not dependent on
FMP. FMP can induce FM in the caput-sperm, but it is not essential for sperm
motility initiation. Alteration of the medium pH from 6.60 to 8.00 caused a
marked increase in the EP or bicarbonate-dependent sperm FM initiation, as
well as intrasperm pH. At the physio-logical pH, bicarbonate served as a much
more potent motility activator than FMP, although both the motility promoters
showed maximal efficacy at alkaline pH (~7.8). EP as well as bicarbonate
elevated the intrasperm cyclic AMP level. Unlike EP, bicarbonate is capable of
increasing intrasperm pH. The intrasperm pH increased from 6.54 0.02 to 6.77
0.03 during sperm transit from caput to cauda. The data are con-sistent with
the view that FMP activates sperm forward motility by enhancing the intrasperm
cyclic AMP level and that extracellular bicarbonate and pH play a vital role
in the initiation of sperm FM during the epi-didymal transit.
Obesity and reproductive disorders: a review
Authors: Norman R. J. , Clark A. M.
Published: November 2003

Obesity has significant consequences for the reproductive system, depending
upon the amount and distribution of body fat. Epidemiological evidence clearly
shows that being overweight contributes to menstrual disorders, infertility,
miscarriage, poor pregnancy outcome, impaired fetal well-being and diabetes
mellitus. Central adiposity is particularly important in clinical sequelae
associated with an increased body mass index. The advent of assisted
reproduction highlights the problems of being overweight, and the use of
gonadotrophins in ovulation induction and in vitro
fertilization is more difficult when the subject is overweight. Weight loss
has marked effects on improving the menstrual cycle and promoting spontaneous
ovulation and fertility. Results indicate that fertility is improved through
exercise and sensible eating patterns when conducted in a group environment.
The mechanisms for this are unclear but may be associated with changes in
sensitivity to insulin.
Disturbance of the reproductive axis induced by negative energy balance
Authors: Judd Stephen J.
Published: November 2003

Animal reproduction is impaired when intake of energy is so restricted that
activities essential to life are threatened; this is seen as a homeostatic
adjustment that restricts wasteful energy expenditure. Fasting or exercising
to a degree requiring considerable energy expenditure has major effects on the
hypothalamus, including activation of corticotrophin-releasing factor (CRF)
neurons, suppression of thyrotrophin-releasing hormone synthesis, and
increased growth hormone secretion; these are associated with increased
concentrations of hypothalamic neuropeptide Y mRNA and are corrected by
administration of leptin, an adipose-tissue protein with a tertiary structure
similar to the cytokine interleukin-2.
This response to fasting results from a disordered pattern of activity in the
gonadotrophin-releasing hormone (GnRH) pacemaker, characterized by reduced
luteinizing hormone pulsatility, particularly during daytime. Animal studies
have suggested that the response depends on an intact afferent vagal system
from the stomach and the presence of oestrogen. Noradrenergic neurons forming
the A2 group increase the activity of CRF neurons that, in turn, inhibit GnRH
pulsatility. Reproductive impairment due to fasting is reversed by leptin, and
abnormalities of leptin are described in individuals who fast or who develop
exercise-induced amenorrhoea. This paper discusses these changes induced by
negative energy balance and speculates on the involvement of leptin as a
contributor to these abnormalities.
Genetic disorders and spermatogenesis
Authors: McLachlan R. I. , Mallidis C. , Ma K. , Bhasin S. , Kretser D. M. de
Published: November 2003

Male infertility affects one man in twenty and a genetic basis seems likely in
at least 30% of those men. Genetic regulation of fertility involves the
inter-related processes of testicular development, spermatogenesis (involving
germ cell mitosis, meiosis and spermatid maturation), and their endocrine and
paracrine regulation. In regard to spermatogenesis, particular attention has
been given to the Yq11 region, where some spermatogenesis genes
(‘azoospermia factors’) appear to be located. Several candidate
genes have been identified but have not been shown to have a defined or
essential role in spermatogenesis. Microdeletions of Yq11 are found in
~15% of azoospermic or severely oligospermic men. The complexity of the
genetic control of male fertility is demonstrated by the evidence for genes
involved in spermatogenesis and sexual differentiation on the X chromosome and
autosomes. Better understanding of the genetic regulation of normal
spermatogenesis will provide new probes for clinical studies; however, at
present the majority of spermatogenic failure remains without an identified
genetic linkage. The advent of intracytoplasmic sperm injection permits
fertility in many previously sterile men and presents the possibility of their
transmission of infertility; appropriate counselling is required.
Increased chromosome abnormalities in human preimplantation embryos after in-vitro fertilization in patients with recurrent miscarriage
Authors: Simón C. , Rubio C. , Vidal F. , Gimenez C. , Moreno C. , Parrilla J. J. , Pellicer A.
Published: November 2003

Recurrent miscarriage is a pathological condition induced by maternal and
embryonic causes. This paper describes a prospective study to determine the
real incidence of aneuploidy for autosomes 13, 16, 18, 21, 22, and gonosomes
in preimplantation human embryos obtained from patients with recurrent
pregnancy loss after ovarian stimulation in an IVF-ET programme. Our results
indicate that aneuploidy for the chromosomes analysed are abnormally higher in
embryos obtained after IVF from recurrent abortion patients (58%)
compared to non-recurrent abortion patients undergoing IVF. Furthermore,
monosomies are six times more frequent than trisomies (47 : 8) in
preimplantation embryos from recurrent abortion patients. Based on the present
study, preimplantation genetic diagnosis (PGD) of embryos obtained from
patients with recurrent miscarriage could prove advantageous in diagnosing
abnormal embryos and selecting normal embryos for transfer.
The Y-chromosome and reproductive disorders
Authors: McDonough Paul G.
Published: November 2003

Over the past decade the tools of modern molecular biology have provided
unique insights into our fundamental understanding of developmental systems.
These insights have been gleaned from the study of a wide variety of model
organisms including yeast (Saccharomyces cerevisiae),
fly (Drosophila), worm
(Caenorhabditis elegans), and mouse. In man, the first
analysis of developmental systems started with sexual differentiation and
focused on the role of Y-linked genes. The presence of living developmental
mutants in man affecting sexual development and the early technology of
deletion mapping facilitated the isolation and identification of small
segments of putative DNA suspected to contain sex-determining genes. The
isolation of genes such as SRY (Sex Related gene on Y)
has provided the first insights into the molecular biology of human sexual
differentiation. The focus on the Y chromosome has brought further insights
into chromosomal pairing, statural determinants in man, oncogenesis,
spermatogenesis, haploid genomes, and the lineage of man himself. This paper
provides the circumstantial and direct evidence to illustrate the importance
of the Y chromosome in reproductive disorders, and in the analysis of haploid
genomes.
Biological bases of premature ovarian failure
Authors: Gosden Roger G. , Faddy Malcolm J.
Published: November 2003

The ovary is endowed at birth with a fixed number of primordial follicles,
which steadily dwindles throughout life as a result of atresia and recruitment
towards ovulation. In addition to age, the number varies allometrically
between species, larger and longer-lived animals tending to have more
follicles initially and these disappear at a slower rate. A causal
relationship between follicle depletion and menopause clearly exists, and
there is a gradual acceleration of follicle wastage in the human ovary
beginning more than a decade before the end of menstrual life. A mathematical
model has provided confirmatory evidence of this relationship, and indicates
that menopause is triggered by a threshold number of follicles which varies
stochastically with a mean of 1100
Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilized oocytes
Authors: Jung Y. G. , Sakata T. , Lee E. S. , Fukui Y.
Published: November 2003

The uptake and synthesis of 19 amino acids by fresh or frozen–thawed
bovine blastocysts produced by parthenogenesis (PT) or
in vitro fertilization (IVF) were compared in the
present study. Fresh blastocysts, 180 h after IVF or PT activation, and
frozen–thawed blastocysts, 168 h old and cultured for 12 h post-thawing,
were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl
alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA,
respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2:
M2). In Experiment 1, when fresh or frozen–thawed PT blastocysts were
cultured in M1, the uptake of glutamate (in fresh only), aspartate and
arginine, and the synthesis of glutamine and alanine were significantly
enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine,
glycine, arginine and alanine were significantly taken up. It was found that
the glutamine concentrations was significantly higher (P
< 0.001) in the culture medium drops containing embryos than in the drops
without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the
uptake of aspartate and synthesis of alanine were greater
(P < 0.01) than those by IVF blastocysts. When M2 was
used, a significant (P < 0.01) production of serine,
asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT
blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured
in M1, fresh blastocysts depleted more aspartate and glutamate, and produced
more glutamine and alanine than frozen–thawed blastocysts. When cultured
in M2, frozen–thawed blastocysts depleted more threonine
(P < 0.01) than fresh blastocysts. These results
indicate that the uptake and synthesis of amino acids were different in fresh
or frozen–thawed bovine blastocysts derived from PT or IVF. These
differences in amino acid metabolism may be related to the viability of the
blastocysts.
Exogenous protein affects developmental competence and metabolic activity of bovine pre-implantation embryos in vitro
Authors: Eckert J. , Pugh P. A. , Thompson J. G. , Niemann H. , Tervit H. R.
Published: November 2003

The role of exogenous protein during bovine pre-implantation embryo
development in two in vitro production systems was
investigated. Morphological development, survival after vitrification and
metabolic activity before and after vitrification were recorded in blastocysts
generated in vitro in synthetic oviduct fluid (SOF)
medium in the presence of either bovine serum albumin (BSA) or
polyvinyl-alcohol (PVA). Metabolic activity was determined by measuring oxygen
consumption, glucose and pyruvate uptake as well as lactate production.
Development to blastocysts and survival after vitrification were reduced
significantly in medium lacking protein. Of the metabolic parameters measured,
only pyruvate uptake was increased significantly in embryos cultured in medium
supplemented with PVA. Whereas in BSA-supplemented medium pyruvate uptake was
correlated with lactate production, in PVA-supplemented medium glucose uptake
was correlated with lactate production. Lactate production increased
significantly after vitrification as compared with fresh embryos. Thus,
exogenously added protein significantly alters oxidative metabolism. In medium
lacking protein, the additional pyruvate may be used for the maintenance of
intracellular amino acid pools. Vitrification appears to alter glycolytic
metabolic profiles indicating a stress-response. In conclusion, the perturbed
metabolism corresponding to reduced developmental capacity of embryos produced
under protein-free conditions emphasizes the ambiguity between maximum
develop-ment, technical and hygienic requirements and physiological demands of
the early bovine embryo in vitro. The use of
well-defined recombinant proteins might assist in closing this gap.
Influence of maternal bodyweight on adaptation after birth in near-term lambs delivered by Caesarean section
Authors: Symonds Michael E. , Clarke Lynne
Published: November 2003

The present study investigated the influence of maternal bodyweight on
adaptation after birth in near-term lambs delivered by caesarean section. One
twin lamb from a heavy (>60 kg) or light ewe (<55 kg) was randomly taken
and immediately placed into a warm (30˚C) ambient temperature and its
twin then delivered into a cool (15˚C) ambient temperature. Continuous
recordings of colonic temperature were then made followed by measurements of
thermoregulation and brown adipose tissue function up to 6 h of life.
Following caesarean section delivery colonic temperature rapidly declined over
the first 30 min of life, a response that was greater in lambs at 15˚C.
Irrespective of ambient temperature all lambs born to heavy ewes commenced
shivering within 15 min of birth and were able to restore body temperature.
These adap-tations were only observed in warm-delivered lambs born to light
ewes, as cool-delivered lambs failed to shiver, became hypothermic and 4 out
of 6 lambs exhibited respiratory failure. Umbilical vein plasma thyroid
hormone concentrations were lower in lambs born to light compared with heavy
ewes. Plasma T3 concentrations remained lower in
warm-delivered lambs born to light ewes until 2 h after birth when heat
production and colonic temperature were not significantly different from warm
delivered lambs born to heavy ewes. Lambs born to light ewes possessed less
brown adipose tissue than those born to heavy ewes. In conclusion, maternal
bodyweight has a critical influence on survival following caesarean section
birth that is dependent on the ambient temperature into which a lamb is
delivered.
Glycolytic enzyme activity in hypotonically treated boar spermatozoa
Authors: Jones A. R. , Piccolo F.
Published: November 2003

Treatment of washed boar sperm with hypotonic phosphate buffer disrupted the
cytoplasmic membrane and released the soluble contents and
phosphofructokinase, but the other glycolytic enzymes and lactate
dehydrogenase were retained. Addition of the appropriate substrates and
co-factor(s) to preparations of treated cells in phosphate-buffered saline
showed that enzyme activity could be re-instated. This simple preparation
should be of assistance in the investigation of specific sections of the
glycolytic pathway without the use of chemical inhibitors.
Selection of immature pig oocytes for homologous in vitro penetration assays with the brilliant cresyl blue test
Authors: Roca J. , Martinez E. , Vazquez J. M. , Lucas X.
Published: November 2003

The present study was designed to evaluate the brilliant cresyl blue (BCB)
test as a means of selection of immature pig oocytes to improve the
performance of the homologous in vitro penetration
(hIVP) assay carried out with immature oocytes to assess the penetrating
ability of boar spermatozoa. Immature pig oocytes harvested from abattoir
ovaries were incubated in 13 µМ BCB in phosphate-buffered saline,
and selected according to their colour (blue and colourless oocytes) after 90
min incubation at 39°C in 5% CO 2 in air. To
assess the selection criteria of the BCB test (Experiment 1), the diameter and
viability (FDA test) of blue and colourless oocytes were determined.
Differences (P<0.01) were found in both mean diameter
(113.08 ± 0.65 µm v. 100.29 ± 0.96
µm) and the viability rate (100 v. 70.71%)
between blue and colourless oocytes. In Experiment 2, the selection
effectiveness (healthy oocytes relative to inseminated oocytes) and
penetrability (percentage of penetrated oocytes and number of sperm in
penetrated oocytes) of unincubated (selected by morphological criteria) and
BCB incubated oocytes were evaluated. Blue oocytes showed the best
(P<0.001) selection effectiveness and the highest
(P<0.01) penetrability. Finally, the effectiveness of
the BCB test to reduce interassay variability in the results of hIVP assays
was examined (Experiment 3). It was found that more consistent data
(P<0.05) of selection effectiveness and percentage of
penetrated oocytes were obtained when blue oocytes were used. In summary,
these results indicate that the BCB test improves hIVP performance when used
to preselect immature oocytes.
Transgenic modification of cows milk for value-added processing
Authors: Zuelke Kurt A.
Published: November 2003

The application of transgenic technologies in dairy cattle has been restricted
largely to producing potential pharmaceutical or nutriceutical products in the
mammary gland. Broader application of transgenesis in dairy cattle production
will require identifying target traits that are both amenable to transgenic
modification and economically important to the dairy industry. The casein
proteins are the most valuable component of cows milk destined for value-added
processing. The four bovine casein genes lie within a single, multi-gene locus
of approximately 200 kb in length. The working hypothesis is that this
multi-gene locus contains all of the DNA sequences required to regulate the
coordinated expression of all four individual casein genes (i.e. a locus
control region or LCR). The initial research aim is to clone the entire casein
locus into a bacterial artificial chromosome (BAC) vector, thus preserving the
extended 5′and 3′ regions that flank the locus, as well as
maintaining the spatial integrity of the four individual casein genes that
comprise the locus. The author's laboratory has prepared a bacterial
artificial chromosome (BAC) library of genomic DNA from elite dairy cattle.
Partial, non-elite BAC clones of the casein gene locus are being tested in
transgenic mice to establish proof of concept. Advances in nuclear transfer of
transfected somatic cells should improve the efficiency of producing
transgenic calves that possess a BAC casein construct introduced into an elite
genetic background.
Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation
Authors: Lavoir M-C. , Conaghan J. , Pedersen R. A.
Published: November 2003

Several different culture conditions were evaluated for culturing grade 4embryos (containing 2–4 blastomeres and with >50%fragmentation) 68 h after fertilization to the blastocyst stage. Embryos wereco-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium withor without 10% v/v synthetic serum substitute (SSS), co-culturedwith BRL cells in KSOM with or without 10% SSS, or cultured in KSOMwith 100 nM heparin binding epidermal growth factor. The most consistentdevelopment was obtained when embryos were co-cultured with BRL cells in KSOM.Rates of development to the blastocyst stage were between 27% and40%. After reaching the blastocyst stage, continued culture of theseblastocysts was only possible in a medium without serum. In a serum-deprivedmedium cells attached and showed initial outgrowth, but did not survivepassaging. Using another approach, inner cell masses (ICMs), isolated fromblastocysts with high efficiency using immunosurgery, were able to attach to afeeder layer in the presence of serum. Some ICMs differentiated whereas otherscould be succesfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead ofwell-defined colonies, the human colonies were characterized by individualcells and colonies without defined borders.
Chromatin remodelling and nuclear reprogramming at the onset of embryonic development in mammals
Authors: Renard Jean-Paul
Published: November 2003

Two main strategies are used to produce cloned mammals. The first involves the
condensation of donor chromatin into chromosomes directly exposed to the
recipient cytoplasm, whereas the second leaves the donor nucleus in interphase
until the time of the first mitosis. Both strategies, which induce marked
changes in chromatin organization, allow full reprogrammation of
somatic-differentiated fetal and adult cells. This paper reviews some of the
recent data that contribute to our understanding of chromatin remodelling at
the onset of normal development, as well as after the introduction of a
foreign nucleus into a recipient enucleated oocyte. These data indicate that
the coordinated changes in chromatin organization that take place up until the
first cellular differentiations at the blastocyst stage are determinants for
successful cloning. Although some degree of synchronization between the cell
cycle stages of donor and recipient cells is necessary for correct remodelling
of a transferred nucleus, the kinetics of remodelling events occurring during
the one-cell stage appears to be the determining factor for the normal onset
of gene expression.
Age-dependent development and surface ultrastructural changes following electrical activation of bovine oocytes
Authors: Suzuki Hiroyuki , Liu Lin , Yang Xiangzhong
Published: November 2003

Electrical activation of oocytes is a key component of the current mammalian
cloning regimen. The present study examined the surface ultrastructure
alterations of bovine oocytes at different ages fol-lowing electrical pulse
(EP) activation. In Experiment 1, bovine oocytes 22, 30 and 40 h after
in vitro mat-uration were activated by EP. Significantly
higher pronuclear formation (57 or 59%) was obtained for the 30- or
40-h oocytes than for the 22-h oocytes (10%). More oocytes (58%)
in the 40-h group activated spon-taneously than in the 22- or 30-h groups. In
Experiment 2, oocytes were cultured for 1, 2, 4 and 8 h after activation and
then evaluated by scanning electron microscopy (SEM). The zona pelucida (ZP)
meshwork was completely abolished within 1 h after EP in the majority of the
oocytes in the 22- and 30-h groups, but no obvious changes were observed in
the 40-h group. From 2 h onward, the ZP meshwork was gradually restored in
most treated oocytes. It was also noted that the microvilli pattern on the
vitelline surface was altered dramatically after EP treatment, with the
proportion affected increasing with oocyte age. The present study extends the
previous findings that older oocytes are more readily activated and that the
age-dependent surface ultrastructural alterations correlate very well with the
age dependent activation of oocytes.
Novel method for demonstrating nuclear contribution in mouse nuclear transfer
Authors: Munsie Megan , Peura Teija , Michalska Anna , Trounson Alan , Mountford Peter
Published: November 2003

Confirmation of nuclear contribution is essential to all nuclear transfer
experiments. Contribution is easily demonstrated in nuclear transfer progeny
but more difficult to confirm in nuclear transfer embryos. The use of donor
nuclei isolated from lacZ transgenic mice offers a clear
and simple method to demonstrate contribution in nuclear transfer embryos and
offspring. The unique line of transgenic mice (Zin40) used in this study
displays nuclear localised lacZ expression in all cells,
including embryonic blastomeres, and demonstrates distinctive blue nuclei when
treated with X-gal substrate. This characteristic staining pattern provided an
ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos
were generated following serial nuclear transfer of metaphase-arrested nuclei
from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear
transfer blastocysts was confirmed by the generation of live born offspring.
Transgenic blastocysts and all tissue samples from fetuses and pups generated
by nuclear transfer displayed distinctive blue nuclei when stained with X-gal.
This staining pattern was characteristic of the transgenic mice from which the
donor nuclei were isolated and clearly confirmed nuclear origin. The use of
this marker will also allow the opportunity to investigate the developmental
potential of nuclear transfer embryos by examining the contribution of nuclear
transfer embryonic cells in chimaeric embryos.
The relationship between membrane status and fertility of boar spermatozoa after flow cytometric sorting in the presence or absence of seminal plasma
Authors: Maxwell W. M. C. , Long C. R. , Johnson L. A. , Dobrinsky J. R. , Welch G. R.
Published: November 2003

The motility, viability (percent live), capacitation status and
in vitro fertility of boar spermatozoa were examined,
after staining with Hoechst 33342 and flow cytometric sorting in the absence
or presence of seminal plasma. Viability was higher in unstained controls and
when seminal plasma was present in the medium used to collect spermatozoa from
the cell sorter than when seminal plasma was absent or in the staining
extender only, but motility was highest when seminal plasma was included in
the extender only, compared with the controls and other treatments. The
proportions of capacitated spermatozoa were increased by sorting, but were
lower when seminal plasma was present, rather than absent, from the staining
extender and the collection medium. Compared with unstained controls,
extension and staining without sorting only increased the proportion of
capacitated spermatozoa after washing in preparation for
in vitro fertilization. The percentages of polyspermic,
penetrated and cleaved oocytes were lower when inseminated with unsorted
(stained) than control (unstained) spermatozoa, regardless of the presence or
absence of seminal plasma. These parameters were higher for sorted than for
control spermatozoa in the absence of seminal plasma, but in its presence
penetration and cleavage were substantially lower. The proportions of
capacitated spermatozoa were lower when seminal plasma was present in the
collection medium only than in the staining extender or when it was absent
altogether, but the former treatment substantially reduced the proportions of
polyspermic, penetrated and cleaved oocytes, and the proportion of
blastocysts. These findings indicate that sperm capacitation associated with
flow cytometric sorting can be reduced by the inclusion of seminal plasma in
the collection medium, but this treatment reduces the ability of spermatozoa
to fertilize oocytes in vitro under these conditions.
In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids
Authors: Lim J. M. , Lee B. C. , Lee E. S. , Chung H. M. , Ko J. J. , Park S. E. , Cha K. Y. , Hwang W. S.
Published: November 2003

This study was conducted to examine the effects of carbohydrates and amino
acids on the maturation and fertilization of bovine oocytes. To evaluate the
effect of each treatment without any unpredictable interference, oocytes were
cultured in a simply defined medium (modified Tyrode’s medium; mT)
without the addition of hormones and proteins. In Experiment 1, oocyte
maturation to the metaphase-II stage was significantly
(P<0.0001) enhanced after the addition of glucose
(5.6 mМ), lactate (10 mМ) and/or pyruvate (0.5 mМ) to mT
(37–74%) than after no addition (0%). In mT supplemented
with glucose, the addition of 19 essential and non-essential amino acids (aa;
0, 0.01, 0.1, 1, 5 or 10%) did not further improve
in vitro maturation (Experiment 2) or
in vitro fertilization (Experiment 3) of oocytes.
However, more (P<0.05) pronuclear formation after in
vitro-insemination was found in oocytes matured in mT with 1% aa and
glucose than in oocytes matured in mT with glucose alone (56% vs.
35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h
after insemination and pronuclear formation from 8 h (Experiment 4). When
cultured inseminated oocytes were examined up to 192 h post insemination, a
significant (P<0.05) increase in the number of 2-cell
(18 v. 38%) and 8-cell embryos, (7
v. 20%) and morulae (0 v.
8%) was found after the addition of 1% aa to mT with glucose
than after no addition (Experiment 5). A limited number of oocytes matured in
mT with aa and glucose developed to the blastocyst stage (6%). These
results indicate that exogenous carbohydrates and amino acids are
prerequisites for the maturation and fertilization of bovine oocytes
in vitro. Glucose alone promotes the nuclear maturation
of oocytes, whereas amino acids aid the pronuclear formation of fertilized
oocytes.
Apoptosis and cell proliferation in the seminal vesicles and coagulating glands of male Syrian hamsters exposed to diethylstilboestrol (DES)
Authors: Nonclercq Denis , Zanen Jacqueline , Toubeau Gerard , Laurent Guy , Heuson-Stiennon Jeanine-Anne
Published: November 2003

Regression of the accessory sex glands was induced in male Syrian hamsters by
chronic exposure to diethylstilboestrol (DES), an agonist of
17β-oestradiol. Experimental groups (n =
4–5) were killed at increasing time intervals up to 6 months after
initiation of treatment. Organ atrophy was evaluated by morphological
examination. Apoptosis in the seminal vesicles and coagulating glands was
visualized by in situ analysis of DNA fragmentation.
Cell proliferation was monitored by immunostaining nuclei of S-phase cells
after pulse labelling with BrdU. The volume of the seminal vesicles decreased
drastically after 2 weeks of DES administration due to a marked reduction of
secretions in the lumen of the glands. Cell proliferation in the seminal
vesicles was stimulated by chronic administration of DES. Mitotic activity
mostly increased during the first month of treatment, leading to epithelial
hyperplasia associated with progressive hyperplasia of the fibromuscular
stroma. Evidence of epithelial dysplasia and metaplasia, often associated with
an infiltration of polymorphonuclear leukocytes, was observed in animals
exposed to DES for 4 months or more. Regression of the seminal vesicles was
also associated with apoptosis in the gland epithelium. Apoptosis appeared 3
days after starting DES administration and culminated at 1 month. Thereafter
the number of apoptotic cells decreased progressively, but apoptosis remained
present until the end of treatment. In contrast, coagulating glands were less
sensitive to DES. No major morphological changes were observed in these glands
except for a moderate reduction of protein secretion. The levels of the
apoptotic and proliferating cells indices were very low in the coagulating
glands during DES treatment. In conclusion, these data point to different
sensitivities of the accessory sex glands to DES exposure because DES induces
extensive alterations of the normal morphology of the seminal vesicles, but
shows only a moderate impact on the coagulating glands.
Extra keyword: oestrogen.