bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-term effects of different thermal conditions during uteroplacental ischaemia on fetal growth of Sprague-Dawley rats
Authors: Asakura Hirobumi , Nakai Akihito , Power Gordon G. , Araki Tsutomu
Published: March 2003

The purpose of the present study was to investigate the short-term effect of various body temperatures on fetal growth during uteroplacental ischaemia. Under mild hyperthermia (n = 6), normothermia (n = 6) and hypothermia (n = 6), a 30-min period of ischaemia was induced in Sprague-Dawley rat dams by clamping the uterine arteries of one uterine horn at 17 days of gestation, leaving the other horn undisturbed. Three days later, the bodyweight of the pups, and the weights of the brains, livers and placentas were compared using the Mann–Whitney U-test. Fetal bodyweight, organ and placental weights were significantly reduced in the uterine horns subjected to ischaemia under the conditions of mild hyperthermia and normothermia (P <0.05), but not with mild hypothermia, compared with the weights in undisturbed uterine horns. It was concluded that both mild hyperthermia and normothermia during ischaemia retard the growth of late-gestation rat pups, in contrast to the sparing effect of mild hypothermia.
Patterns of fetal growth within Large White × Landrace and Chinese Meishan gilt litters at three stages of gestation
Authors: Finch A. M. , Antipatis C. , Pickard A. R. , Ashworth C. J.
Published: March 2003

Low birthweight piglets have an increased incidence of mortality and morbidity. As there are few opportunities to remedy the detrimental consequences of low birthweight after birth, it is important to understand the nature of fetal growth retardation and to identify when low birthweight fetuses deviate from the growth trajectory of their normally grown siblings. The aims of this study were to identify the nature, timing and possible causal factors influencing inadequate fetal growth in Large White × Landrace (LW) and Chinese Meishan (MS) gilts at three stages of pregnancy. Thirty-six per cent of litters contained inadequately grown fetuses. Both intrauterine-growth-restricted (IUGR) and small-for-gestational-age (SGA) fetuses could be identified as early as Day 30 in MS and LW litters and the percentage of litters containing inadequately grown fetuses was similar throughout gestation. MS fetuses, placentas and piglets had less within-litter variation in weight at all stages studied. Inverse relationships were observed between litter size and both minimum and mean weights of MS neonates. No other relationships between fetal size and either uterine position or litter size were observed.
Metalloproteinase 2 activity and modulation in uterus from neonatal streptozotocin-induced diabetic rats during embryo implantation
Authors: Pustovrh Carolina , Jawerbaum Alicia , Sinner Débora , White Verónica , Capobianco Evangelina , González Elida
Published: March 2003

Matrix metalloproteinases (MMPs) are responsible for the remodelling of the uterine extracellular matrix during embryo implantation. Nitric oxide (NO) production is increased at the time when implantation begins. Abnormal tissue levels of MMPs are present in diabetes; elevated NO levels in tissues and an increased oxidative stress are also found. The present work evaluates the uterine MMP2 activity and levels during embryo implantation, as well as the influence of nitridergic compounds and reactive oxygen species (ROS) on the MMP2 enzymatic activity in a model of neonatal streptozotocin-induced diabetic rat. Metalloproteinase 2 activity and levels are increased in diabetic tissues compared with controls (P<0.05 and P<0.002 respectively). The uterine enzymatic activity in diabetic animals decreases in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (P<0.01) and is enhanced (P<0.005) when a generating ROS system (xanthine/xanthine oxidase) is added to the incubating medium. It was also found that uterine superoxide dismutase activity is higher in diabetic rats than in control rats on the day of implantation (P<0.001), suggesting a compensatory antioxidant ability. In conclusion, the results show that the uterine MMP2 activity, which is higher in diabetic animals than in control animals, is modulated positively by NO and ROS during embryo implantation in a model of streptozotocin-induced diabetic rats.
Immunohistochemical distribution of oestrogen and progesterone receptors and tissue concentrations of oestrogens in the cervix of non-pregnant cows
Authors: Breeveld-Dwarkasing V. N. A. , Boer-Brouwer M. de , Möstl E. , Soede N. M. , Weijden G. C. van der , Taverne M. A. M. , Dissel-Emiliani F. M. F. van
Published: March 2003

An immunohistochemical study of the expression of oestrogen (ER) and progesterone receptors (PR) in different regions along the longitudinal and vertical axes of the cervix of non-pregnant cows was performed. Animals were separated into two groups depending on the presence or absence of a functional corpus luteum in their ovaries, as indicated by blood progesterone concentrations. The high progesterone group (HP4) had serum progesterone concentrations >2.0 ng mL–1 (n  = 6) and the low progesterone group (LP4) had serum progesterone concentrations ≤0.5 ng mL–1 (n = 4). Significantly higher concentrations of oestrogen were found in the cervical tissue of animals in the LP4 group than those in the HP4 group (473 ± 53 v.149 ± 46 pg g–1 wet weight; P<0.01). Furthermore, there was a significant effect of tissue layer (epithelium to deep stroma) on the number of ER (P<0.01) and PR (P<0.05) immunoreactive nuclei per 1000 cells. For both ER and PR the proportion of cells expressing the receptor increased from epithelium to subepithelial stroma (P<0.01) and from subepithelium to deep stroma (ER P<0.05; PR P=0.061). When the number of receptor-positive cells were expressed per mm2 tissue, differences between the subepithelial stroma and the deep stroma became even more marked. In addition, the vaginal part of the cervix had significantly more (P<0.01) ER and PR immunoreactive nuclei per 1000 cells than the uterine part, but these differences were no longer apparent when a correction was made for cell density. There was no relationship between progesterone status of the animals, nor local tissue oestrogen concentrations and ER or PR immunoreactivity in the cervix of these non-pregnant cows. Instead, a strong relationship between both longitudinal and vertical positioning of tissue in the cervix and expression of both receptor types was shown. In addition, a strong correlation between ER and PR expression in the subepithelial stroma (R = 0.85, P<0.01) and the deep stroma (R�=�0.83, P<0.01) was evident. In conclusion, these results demonstrate that in studies of steroid hormone receptor expression in the cervix, careful description of sampling site and depth are necessary if the results are to be interpreted meaningfully.
The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro
Authors: Figueiredo F. , Jones G. M. , Thouas G. A. , Trounson A. O.
Published: March 2003

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte–macrophage colony-stimulating factor.
Concentration of calcium, zinc, magnesium, potassium and sodium in the bovine cervical mucus during normal oestrus and oestrus induced by progesterone and/or PGF2α
Authors: Tsiligianni T. , Karagiannidis A. , Roubies N. , Saratsis P. , Brikas P.
Published: March 2003

Ninety-two healthy Friesian cows were used to determine the concentration of calcium (Ca), zinc (Zn), magnesium (Mg), potassium (K) and sodium (Na) in bovine cervical mucus and to compare these concentrations in cervical mucus collected during spontaneous and induced oestrus. Cows were assigned to four groups: (1) no treatment (2) PRID for 12 days plus pregnant mare's serum gonadotrophin (PMSG; 1000 IU, i.m.) at the removal of the PRID (3) PGF2α , two doses (6 mg, i.m.) 11 days apart, and (4) PRID for 7 days plus one dose of PGF2α (6 mg, i.m.) 24 h before removal of the PRID. The cows of the three induced-oestrus groups received two artificial inseminations (AI), whereas those in the spontaneous-oestrus group received only one. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. The results of this study can be summarized as follows: (1) viscosity was significantly lower (P<0.001) and crystallization was significantly (P<0.001) higher in the cervical mucus of cows in the spontaneous-oestrus group compared with cows in the induced-oestrus groups; (2)�Na concentration was similar in all groups; (3) concentration of all the other elements in the spontaneous-oestrus group was significantly lower (P<0.05) than in the induced-oestrus groups; (4) there was significant positive correlation (P<0.001) between viscosity and all elements, and significant negative correlation (P<0.05–P<0.001) between crystallization and all elements.
Susceptibility of reproduction in female pigs to impairment by stress and the role of the hypothalamo - pituitary - adrenal axis
Authors: Turner A. I. , Hemsworth P. H. , Tilbrook A. J.
Published: March 2003

Although it is generally considered that stress can impair reproduction, we suggest that the impact of acute or repeated acute stress or acute or repeated acute elevations of cortisol are of little consequence in female pigs, even if these occur during the series of endocrine events that induce oestrus and ovulation. It is important to understand the impact of acute stress on reproduction because, in the intensive production of livestock, animals are often subjected to short-term challenges. There seems little doubt that reproduction in a proportion of female pigs is susceptible to impairment by severe and prolonged stress or the sustained elevation of cortisol but only when this continues for a substantial period. In female pigs, where reproduction is susceptible to impairment by severe prolonged stress, it is possible that the mediators of this suppression are cortisol, corticotrophin-releasing factor and vasopressin but, in pigs, there is evidence to suggest that adrenocorticotrophic hormone is not involved. Other substances secreted during stress may be involved but these are not considered in this review. It is possible that the mediators of stress act at any level of the hypothalamo–pituitary–ovarian axis. Although a variety of experimental manipulations have provided potential mediators and mechanisms for the stress-induced suppression of reproduction, these experimental manipulations rarely represented physiological circumstances so it is not clear if such mechanisms would be important in a physiological context. The precise mediators and mechanisms by which hormones released during stress may inhibit reproductive processes during severe prolonged stress are yet to be determined.
Semen quality in captive Houbara bustard, Chlamydotis undulata undulata
Authors: Wishart G. J. , Lindsay C. , Staines H. J. , McCormick P.
Published: March 2003

Semen quality in captive-bred Houbara bustards, Chlamydotis undulata undulata, was assessed during three consecutive breeding seasons. In any one season, sperm quality, in terms of the proportion of eosin-permeable spermatozoa and of spermatozoa with abnormally large nuclei, varied among individual males, but not among their ejaculates. Neither the proportion of spermatozoa with large nuclei, nor those permeable to eosin were related to the total sperm output of males. The fertilizing ability of males was related to their mean seasonal proportion of eosin-permeable spermatozoa, but not the proportion of spermatozoa with large nuclei. The ranking of males on the basis of the proportion of spermatozoa with large nuclei in their ejaculates was significantly positively correlated between seasons, although ranking on the basis of sperm eosin-permeability was not. The cause or consequence of producing spermatozoa with large nuclei (and excess DNA) remains to be elucidated, but appears to be a trait that is characteristic of houbara bustard males that is maintained between breeding seasons.
Semen collection from mice: electroejaculation
Authors: Tecirlioglu R. T. , Hayes E. S. , Trounson A. O.
Published: March 2003

The effects of device type (electrostimulator, function generator or computer-generated waveforms), waveform (square, triangle or sine wave), probe type (ring or strip) and anaesthetic compound (ketamine/xylazine combination or pentobarbitone sodium) were investigated on electroejaculation (EEJ) responses of C57Bl × CBA and C57Bl/6J mice. Ejaculates were analysed for total sperm count and motility variables using computer-assisted sperm analyses. Automated computer-generated waveforms delivered through a sound card were more effective and reproducible compared with waveforms generated by function generator and electrostimulator. Sine waves and triangle waves were found to be more effective in producing ejaculate than square waves. As an anaesthetic, pentobarbitone sodium tended to outperform ketamine/xylazine across waveforms and strains. Strip probes failed to produce any ejaculate regardless of the device or waveform employed. Sperm obtained by EEJ exhibited poor motility and C5Bl/6J mice had lower motility variables than C57Bl × CBA mice.
A chronic low dose infusion of insulin-like growth factor I alters placental function but does not affect fetal growth
Authors: Bloomfield Frank H. , Zijl Pierre L. van , Bauer Michael K. , Harding Jane E.
Published: March 2003

Knowledge of the anabolic effects of insulin-like growth factor I (IGF-I) on fetal growth and feto–placental metabolism are derived from studies using large doses of IGF-I. Low doses of enteral IGF-I have trophic effects on the fetal gut, but there are no data on the effects of systemic low doses of IGF-I on fetal growth and feto–placental metabolism. We therefore compared the effects of a chronic infusion of low dose IGF-I (50�μg�day–1, n = 7) with vehicle-infused controls (n = 7) on fetal growth, metabolism and placental transfer capacity in the chronically instrumented late gestation ovine fetus (121–132 days of gestation; term = 145 days). Insulin-like growth factor I infusion did not affect fetal growth or the size of individual organs, including liver, spleen and bone. Placental morphology was altered, and placental clearances of 3-O-[methyl-3H]D-glucose (a non-metabolizable glucose analogue) and [methyl14C]aminoisobutyric acid (a non-metabolizable analogue of amino acids utilizing the system A transporter), were reduced in IGF-I-treated fetuses (P<0.05 v. control). However, fetal and placental metabolite uptake was not significantly different between groups. We conclude that, despite altering placental transfer capacity and morphology, a chronic low dose infusion of IGF-I does not alter fetal growth or metabolism.
Simultaneous detection of viability and sex of bovine spermatozoa
Authors: Révay T. , Kovács A. , Rens W. , Gustavsson I.
Published: March 2003

The viability and sex of bovine spermatozoa were simultaneously evaluated. After viability and acrosome staining with trypan blue/Giemsa, only live spermatozoa became decondensed by a modified papain-dithiothreitol method. Owing to this specific effect, live sperm heads were easily distinguished by their enlarged size and dark violet colour from small, light blue dead sperm heads. In the same sperm sample, X- and Y-chromosome-bearing sperm were distinguished by their fluorescent signal, using fluorescence in situ hybridization (FISH) with an XY paint set and 4,6-diamino-2-phenylindole counterstaining. The combined staining provides a method for morphological and viability evaluation before FISH and permits identification of the proportions of X- and Y-chromosome-containing live spermatozoa in a semen sample. However, only 25% of the undecondensed dead sperm express signals allowing detection of the sex of the chromosome. The method may be an effective tool in evaluating sex-oriented semen samples.
Expression of anti-Müllerian hormone mRNA during gonadal and follicular development in the brushtail possum (Trichosurus vulpecula)
Authors: Juengel Jennifer L. , Whale Lisa J. , Wylde Katherine A. , Greenwood Penny , McNatty Kenneth P. , Eckery Douglas C.
Published: March 2003

The ontogeny of anti-Müllerian hormone (AMH) gene expression in the brushtail possum during formation of the ovary and growth of follicles was examined using in situ hybridization. For comparative purposes, the expression pattern of AMH was also examined in the developing testis. In the female, AMH mRNA was observed in the ovary of 50% (3/6) of pouch young collected around the time of sexual differentiation of the gonad (Days 1–5): the signal was predominately localized to the inner-cortical and outer-medullary region of the ovary. Thereafter, AMH mRNA was not observed in the developing ovary until Days 78–113 of postnatal life when follicles first formed at the cortical–medullary boundary. At this time, AMH mRNA was observed in the cuboidal granulosa cells of some early growing (i.e. transitional) follicles and in the granulosa cells of primary follicles. Thereafter, AMH mRNA was present in granulosa cells at all subsequent stages of follicular growth (i.e. primary through antral), but not in preovulatory follicles. In all cases, once follicles had formed, AMH mRNA was limited to the granulosa cells and was not observed in the surface epithelium, stromal cells, oocytes, theca, corpus luteum, medullary cords, rete or interstitial glands. In the possum testis, Sertoli cells strongly expressed AMH around the time of sexual differentiation of the gonad, but expression decreased to very low levels in adults, suggesting that AMH plays a similar role in brushtail possums to that observed in other mammalian species. In conclusion, localization of mRNA for AMH exclusively to granulosa cells of growing follicles in the brushtail possum is consistent with a central role for this hormone in control of granulosa cell function in marsupials. In addition, expression of AMH in the developing ovary around the time of morphological sexual differentiation raises intriguing questions regarding the possible role of AMH at this time.
Deviant peri-oestrual hormone patterns affect the epithelium of the uterine tube in repeat-breeder heifers
Authors: Båge Renée , Masironi Britt , Sahlin Lena , Rodríguez-Martínez Heriberto
Published: March 2003

In the bovine reproductive tract, the uterine tube is the critical site for a series of events required for fertilization and early embryonic development. In previous studies, a defined category of subfertile heifers, repeat-breeder heifers (RBH), has presented peri-oestrual disturbances (deviating hormone patterns and follicular dynamics) and uterine maternal–embryonic asynchrony. The present study aimed to investigate if tubal function was also affected, by determination of differences in the morphology of the tubal lining epithelium of RBH (n = 4) in comparison to controls (n = 6) during standing oestrus, studied by light and electron microscopy (SEM/TEM), and relate this to steroid hormone concentrations and receptor distribution in the target tissues. Tissue distribution of oestrogen receptor α (ERα) and progesterone receptor B (PRB) was quantified using immunohistochemistry. In particular, secretory cells differed in appearance between RBH and controls. The cells were less lumen protruding, microvilli were fewer and smaller and secretory granules in the apical cytoplasm were more numerous in RBH. Furthermore, the tubal epithelium was conspicuously coated with amorphous material. Morphological differences between categories were not explained hormonally or by steroid receptor distribution, except in two heifers from which uterine tubes were obtained after the luteinizing hormone (LH) surge. The isthmic PRB : ERα ratio was twice as high in the RBH than in the control. The deviating ultrastructure found in RBH, before and after the LH surge, might influence the tubal microenvironment with effects on gamete transport and final maturation and early embryonic development. The present study confirms that previously recorded perturbations in reproductive physiology in RBH are also manifested in the uterine tube, mainly by a deviating ultrastructure of the lining epithelium.
Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen - thawed sorted X- or Y-chromosome-bearing spermatozoa
Authors: Hollinshead F. K. , O'Brien J. K. , Maxwell W. M. C. , Evans G.
Published: March 2003

The fertilizing ability of sex-sorted frozen–thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero–tubal junction (UTJ) with a total of 140 × 106 unsorted (control) or 2–4 × 106 sorted (X or Y) frozen–thawed ram spermatozoa 54�to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon®, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo®). The number of ewes pregnant after insemination with unsorted frozen–thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.
Characterization of viability, mitochondrial activity, acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures
Authors: Huo Li-Jun , Yue Kui-Zhong , Yang Zeng-Ming
Published: March 2003

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen–thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4°C, 15°C, 20°C and 39°C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 ± 1% of the sperm did not take up the Hoechst 33258, whereas 95 ± 2% were stained by SYBR-14 and 96 ± 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15°C and 20°C. There were 62 ± 2% (15°C) and 89�±�2% (20°C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.
Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells
Authors: Ye J. , Flint A. P. F. , Campbell K. H. S. , Luck M. R.
Published: March 2003

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95–100%), but distributed from GVI to GVIV (GVI 56.1 ± 9.1%, GVII 15.3 ± 1.4%, GVIII 21.5 ± 7.1%, GVIV 7.1 ± 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 ± 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1–25 μg mL–1 resulted in 95–100% oocytes being arrested and synchronized at GVII. With >5 μg mL–1 CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 μg mL–1 CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6–12 h, metaphase I (MI) at 10–18�h and anaphase I/telophase I at 16–20 h. After 12 h preincubation with 5 μg mL–1 CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.
Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture
Authors: Rieger D. , McGowan L. T. , Cox S. F. , Pugh P. A. , Thompson J. G.
Published: March 2003

In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 µM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 µM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 µM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 µM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.
Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris
Authors: Fidler Andrew E. , Western Andrea H. , Griffith Nicole , Selwood Lynne , Stent Vicki , McNatty Kenneth P.
Published: March 2003

The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians. To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris. The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCF. The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro. Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF. This source of biologically active marsupial SCF may prove useful in future studies of marsupial development.
Developmental changes in pulmonary and renal angiotensin-converting enzyme concentration in fetal and neonatal horses
Authors: O'Connor S. J. , Fowden A. L. , Holdstock N. , Giussani D. A. , Forhead A. J.
Published: March 2003

Angiotensin-converting enzyme (ACE) has an active role in the control of blood pressure and body fluid homeostasis both before and after birth. This study investigated the ontogeny of pulmonary and renal ACE concentrations in fetal and neonatal horses. Fetal pulmonary ACE concentration increased from 250 days towards term (c. 335 days). Newborn foals showed significantly higher mean concentrations of pulmonary ACE (4.40�±�0.62�nmol min–1 mg protein–1) than both fetuses during late gestation (1.23 ± 0.51 nmol min–1 mg protein–1) and animals aged 1 day to 2 weeks of postnatal age (0.85 ± 0.15 nmol min–1 mg protein–1). Renal ACE was detected in fetal horses from 100 days of gestation but showed no developmental trend during the second half of gestation or in early postnatal life. Overall in the fetus, mean concentrations of renal ACE were also approximately 10 times lower than mean pulmonary values. Renal ACE concentration may be related to the functional immaturity of the equine kidneys. The increase in pulmonary ACE concentration seen towards term in the fetal horse may be induced by the prepartum cortisol surge that occurs very close to delivery in this species. Therefore, premature delivery in this species may interrupt the onset of ACE production in the fetal lungs and circumvent the normal maturation of the renin–angiotensin system.
Development of a porcine follicle-stimulating hormone and porcine luteinizing hormone induced ovulation protocol in the seasonally anoestrus brushtail possum (Trichosurus vulpecula)
Authors: Glazier A. M. , Molinia F. C.
Published: March 2003

Monovulatory brushtail possums (Trichosurus vulpecula) were stimulated with exogenous hormones during seasonal anoestrus to overcome ovarian insensitivity and induce ovulation. Seasonal ovarian insensitivity to pregnant mare serum gonadotrophin (PMSG) was overcome by a new porcine follicle-stimulating hormone/porcine luteinizing hormone (pFSH/pLH) protocol. This protocol was refined because the original treatment produced oocytes with abnormal morphology. Possums (n = 12 per group) received eight injections of pFSH of 1.5, 3.0 or 6.0 mg per injection (at 12-h intervals for 4 consecutive days). Ovulation was induced 12 h after the final pFSH injection with a 4-mg injection of pLH. Control animals were treated with the established protocol of a single injection of 15 IU of PMSG, followed 48 h later with an injection of 4 mg of pLH. All females responded to pFSH/pLH treatment, although optimal stimulation occurred in those receiving 8 × 3 mg pFSH, with 13–14�ovulations and recovery of 11–12 oocytes per female (8 × 1.5 mg pFSH: 13 ovulations, 8–9 oocytes; 8�×�6�mg pFSH: 7–8 ovulations, 4–5 oocytes). In contrast, only seven of 12 females responded to PMSG/pLH and, of those responding, only 2–3 ovulations occurred and only 1–2 oocytes per female were recovered. However, around 80% of oocytes recovered after PMSG/pLH treatment had undergone nuclear maturation (metaphase II/1st polar body) compared with around 60% of oocytes from pFSH/pLH-treated animals. In possums killed from 27 to 39 h after pLH treatment, ovulation onset was first observed from 30 h and by 31.5 h, all animals had completed ovulation. Laparoscopic artificial insemination (LAI) was performed on pFSH/pLH-treated animals to determine whether the oocytes produced were capable of fertilization. Uterine LAI performed 27.5–28.5 h after pLH treatment yielded 11/26 fertilized oocytes (up to 4-cell stage), whereas vaginal LAI performed 13–14 h after pLH treatment yielded 21/53 fertilized oocytes. A proportion of oocytes generated from the refined pFSH/pLH protocol are thus properly mature and capable of fertilization. Further refinement of the protocol is now needed to improve the yield of fully matured oocytes.
Low maternal nutrition during pregnancy reduces the number of Sertoli cells in the newborn lamb
Authors: Bielli Alejandro , Pérez Raquel , Pedrana Graciela , Milton John T. B. , Lopez Álvaro , Blackberry Margaret A. , Duncombe Gregory , Rodriguez-Martinez Heriberto , Martin Graeme B.
Published: March 2003

The nutritional status of females during pregnancy can play a critical role in the postnatal growth and development of the offspring, often leading to permanent changes ('fetal programming'). The Sertoli cells are a strong candidate for fetal programming of future performance because the number of Sertoli cells is highly correlated with adult testicular size and the maximum rate of sperm production. For Merino ewes, we imposed different levels of metabolizable energy (ME) intake (LowME: 70% of requirements for maintenance of ewe body mass and normal growth of conceptus (n = 13); HighME: 110% of those requirements (n = 12)) from Week 10 of pregnancy until parturition and then tested for effects on testicular histology in newborn males. Pregnant ewes were weighed weekly and lambs were weighed at birth and 2 days later. Blood was sampled at the same times. LowME ewes did not gain weight, whereas HighME ewes gained 17% over their pretreatment weight. Birthweights were higher in HighME lambs than in LowME lambs. Paired testes tended to be heavier in the HighME group than in the LowME group (P=0.08). The diameter of the testicular cords did not differ. The absolute volume of testicular cords (0.36 ± 0.02 v. 0.30 ± 0.02 mL for HighME v. LowME, respectively; P=0.03) and the number of Sertoli cells (43.0�±�2.5 v. 34.5 ± 2.0 × 108 for HighME v. LowME, respectively; P=0.018) per testis were both greater in the HighME than in the LowME group. Plasma follicle-stimulating hormone concentrations were not significantly affected at birth or 2 days later. We conclude that undernutrition during pregnancy can reduce testicular development in the newborn. Depending on the ability of the Sertoli cell population to recover between birth and puberty, this may limit the ultimate number of Sertoli cells and, hence, the future capacity for sperm production and fertility.
Effects of electrical stimulation and seminal plasma on the motility of mouse sperm
Authors: Tecirlioglu R. T. , Lacham-Kaplan O. , Trounson A. O.
Published: March 2003

The effects of electric current (in vivo and in vitro) and seminal plasma on epididymal and ejaculated sperm obtained from C57BL × CBA and C57BL/6J mice were investigated by studying motility parameters, fertilization and embryo development. Electroejaculates were obtained by applying a series of computer-generated sinusoidal alternating currents (0.25–3.0 V at 50 Hz) delivered for 1, 2 and 3 s with 1-s rest periods using a four-electrode rectal probe for 4 min. Epididymal sperm obtained from the same mice were either subjected to electric current in vitro in a Plexiglass chamber or incubated in a medium containing seminal plasma for 2 h. In vitro electric current application and incubation in a medium containing seminal plasma significantly (P<0.01) decreased sperm motility. Neither electroejaculates nor epididymal spermatozoa incubated with seminal plasma could fertilize oocytes by conventional IVF (P<0.001), whereas sperm subjected to in vitro electric current had lost little of their ability to fertilize oocytes. Following transfer of embryos generated by intracytoplasmic sperm injection (ICSI), the number of live pups obtained from electroejaculated sperm (10.2%; 6/59) was significantly (P<0.01) lower than from epididymal sperm (50.0%; 22/42). Electroejaculation using a rectal probe had little effect on motility and fertilization capacity of mouse epididymal sperm, whereas the presence of seminal plasma decreased motility and prevented fertilization.
Fertility of male and female emus (Dromaius novaehollandiae) as determined by spermatozoa trapped in eggs
Authors: Malecki Irek A. , Martin Graeme B.
Published: March 2003

Changes in the fertility status of 10 pairs of emus were investigated using egg break-out and numbers of sperm in the perivitelline membrane of the germinal disc (GD) region. After the sexes were separated, sperm in consecutive eggs declined approximately logarithmically at a mean (±SEM, n = 10 females) rate of –0.148 ± 0.021 per log day. Sperm continued to be detected in eggs for 16.5 ± 1.7 days during which 5.6 ± 0.6 fertilized eggs were laid. Fertilized eggs that did not contain detectable sperm were laid by five females for a further 2.2 ± 0.9 days. Based on break-out fertility, the fertile period continued for up to 18.7 ± 2.1 days, for which the mean number of laid eggs was 6.3 ± 0.8. An egg with a 50 : 50 chance of being fertilized would contain 3.5 sperm mm–2 of GD. Based on the sperm decline model, an egg containing that many sperm would be laid 21 days after the last copulation. In emus that were not separated and allowed to incubate their eggs (n = 3 pairs), the number of sperm in eggs laid before and during incubation declined in a manner similar to that after the last copulation and egg-laying stopped after the females had laid 3.3 ± 0.3 eggs. After incubation was terminated, females resumed laying within 8.3�±�1.2�days and the number of sperm in eggs gradually increased but it did not return to pre-incubation levels. In non-incubating emus (three pairs), the number of sperm in eggs declined as laying progressed, although lit was higher during the period when the first seven eggs were laid than during the period when the rest of eggs were laid (214 ± 39 v. 100 ± 16 sperm mm–2 of GD). Sperm numbers varied between successive eggs but a sharp increase followed by a decrease acted as an indicator of recent copulation. There were 8.7 ± 0.3 such increases per laying period (one per 2.8 ± 0.2 eggs), a frequency that suggests that emus copulate once weekly. In conclusion, as long as a female emu is supplied with sperm on a weekly basis, she will be fertile but, when copulations stop, she will stop laying soon after. Male fertility appears to fall towards the end of the laying season and it can be affected by egg incubation at any time of the season.
Development of infertility at young adult age in a mouse model of human Sandhoff disease
Authors: Juneja Subhash C.
Published: March 2003

Sandhoff disease is a human lysosomal storage disease. In a knockout mouse model of Sandhoff disease, which lacks the β-subunit of β-hexosaminidase A (Hex A, αβ subunits) and B (Hex B, ββ subunits), the mutant homozygous mice (Hexb–/–) are healthy until 15 weeks of age when they develop neurodegenerative symptoms. This study was designed to analyse the fertility profile of male and female Hexb–/– mice. Mating behaviour of Hexb–/– mice was assessed at different ages. The ovarian function of Hexb–/– females was determined by superovulation studies. The quality of spermatozoa and ova was assessed by an in vitro fertilization (IVF) procedure. Hexb–/– mice were fertile at a young age. Males were fertile up to the age of 69.3 ± 6.3 days (mean ±�SD) and females were fertile up to the age of 56–63 days. Since both the Hexb–/– sexes showed fertility, the results indicate that Hex A and Hex B (major isozymes of β-hexosaminidase) may not be required for sperm–ovum interactions, in contrast to the widely accepted belief. On the other hand, young adult Hexb–/– males showed a reduction in mating behaviour at the age of 84.8 ± 2.2 days and an absence of mating behaviour at 94.2 ± 2.0 days. Spermatozoa from Hexb–/– mice (aged 109.2 ± 1.8 days) showed a lower IVF rate. Among Hexb–/– females aged 85.6 ± 2.1 days, no mice became pregnant although they were positive for a vaginal plug when caged with fertile males. The number of ova recovered from Hexb–/– females (aged 111.0 ± 3.1 days) and the IVF rate of ova were lower than those of controls. In conclusion, Hex A and Hex B may not be required for sperm–ovum interactions. Mice lacking Hex A and Hex B activities develop infertility at a young adult age in an age-dependent manner.
The phosphatidylinositol signalling system in elongating bovine blastocysts; formation of phosphoinositides, inositol phosphates and stimulation by growth factors
Authors: Hynes A. C. , Sreenan J. M. , Kane M. T.
Published: March 2003

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo elongating cattle blastocysts was investigated using [3H]myo-inositol. Uptake was examined in 13-, 14- and 16-day-old blastocysts and was largely sodium-dependent throughout (P<0.001), indicating the presence of a sodium-dependent inositol transporter. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP and PtdInsP2, and the inositol phosphates of the phosphatidylinositol signal transduction system was examined at Days 14 and 16; incorporation into the three phosphoinositides and into the inositol phosphate species, InsP1, InsP2, InsP3 (including the second messenger, Ins(1,4,5)P3) and InsP4 was detected in both blastocyst stages. The effects of the peptide growth factor, epidermal growth factor (EGF), and the lipid growth factors, lysophosphatidic acid (LPA) and platelet activating factor (PAF), on the activity of the phosphatidylinositol signalling system in 14- and 16-day-old blastocysts were examined. All growth factors significantly stimulated phosphatidylinositol signalling activity. Epidermal growth factor was stimulatory (P<0.001) only in 16-day-old blastocysts, whereas LPA and PAF were active in both 14- (P<0.005 for LPA and P<0.001 for PAF) and 16-day-old blastocysts (P<0.001 for LPA and PAF). These results indicate that the phosphatidylinositol signalling system is present in cattle blastocysts at the elongation stage and is responsive to stimulation by growth factors.
A cytotaxonomic survey of the Pteridophyta of Australia
Authors: Tindale Mary D. , Roy S. K.
Published: March 2003

A cytotaxonomic survey of the ferns and fern allies of Australia (including Lord Howe Island) is presented. Five-hundred-and-twenty-six chromosome counts of 268 Australian species, subspecies, varieties, variants and hybrids are recorded, only a small number having been previously investigated by other botanists on Australian material. Diploids represent c. 62% of the counts on species and c. 38% on polyploids, the latter ranging principally from triploids to a single decaploid and dodecaploid (but no heptaploids). More than one ploidy level has been reported in 19 taxa (almost 8% of taxa). Counts of 10x for Asplenium aethiopicum and 12x for A.�flabellifolium are the highest definite ploidy levels for the Australian pteridophyte flora. Chromosome counts for 29 families and 89 genera are cited. Only diploids were reported for Osmundaceae and Cyatheaceae, but only polyploids for the Psilotaceae, Vittariaceae and Ophioglossaceae. An analysis is given of the levels of ploidy in 248 taxa, excluding the Lycopodiaceae and Hymenophyllaceae. The percentages of diploids and polyploids in Australian species are compared with those of nearby countries. Many species reported on here have never been cytologically investigated before, while others have not been studied previously on Australian material. The following genera have been examined cytologically for the first time: Coveniella Tindale, n = 41; Paraceterach (F.Muell.) Copel., n = 29; 'Oenotrichia Copel.', 2n = 82 (2x); Revwattsia (Watts) D.L.Jones, 2n = c. 328 (8x); and Pteridoblechnum Hennipman (2n = 54). The phylogeny of the genera is discussed in the light of these findings. Certain families such as the Adiantaceae, Cyatheaceae, Hymenophyllaceae, Lindsaeaceae and Marsileaceae were given special attention by collecting as much living material as possible. A number of species-complexes has been found and further chromosome counts added to intercontinental species complexes. The Döpp-Manton and Braithwaite forms of reproductive apomixis have been reported amongst some genera. Endemism, hybridity and apogamy amongst Australian pteridophytes are discussed, as well as homosporous and heterosporous species. The new combination Phymatosorus membranifolius (R.Br.) Tindale is made.
Monograph of Gastrolobium (Fabaceae: Mirbelieae)
Authors: Chandler Gregory T. , Crisp Michael D. , Cayzer Lindy W. , Bayer Randall J.
Published: March 2003

A taxonomic revision with full descriptions and key are presented for the 109 known species of Gastrolobium, including 29 new species described here for the first time. Brachysema, Jansonia and Nemcia are formally placed into Gastrolobium and new combinations have been made where necessary. Included in the revision are full taxonomic descriptions for all species, full synonymies, literature references for original publications, typification, including selection of lectotypes where necessary, distributions complete with maps, and taxonomic and nomenclatural notes. New taxa described herein are G. acrocaroli, G. aculeatum, G. alternifolium, G.�congestum, G. crispatum, G. cruciatum, G. cyanophyllum, G. diabolophyllum, G. discolor, G. elegans, G.�euryphyllum, G. ferrugineum, G. glabratum, G. hians, G. humile, G. involutum, G. melanopetalum, G.�mondurup, G. musaceum, G. nudum, G. nutans, G. reflexum, G. rhombifolium, G. semiteres, G. tenue, G.�tergiversum, G. venulosum, G. whicherensis and G. wonganensis.
New saprobic fungi on fronds of palms from northern Queensland, Australia
Authors: Hyde Yanna , Hyde Kevin D.
Published: March 2003

Collections of decaying palm fronds in tropical rainforests in northern Queensland, Australia, yielded two new genera, Mckenziea and Waihonghopes, and six new fungal species, Kionochaeta filamentosa, Mackenziea livistonae, Monotosporella palmicola, M. clavata, M. sphaerica and Waihonghopes australiensis. The new taxa are described and illustrated and compared with similar taxa.