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Cobalt and selenium are essential trace elements required for ruminants. There are many different methods of supplementation available to sheep including boluses, in feed, free access minerals, pasture dressing and oral drenches. Recent changes in European legislation have resulted in a reduction in the maximum permitted level (MPL) of cobalt to be included in ruminant diets from 2 mg/kg at 88% DM to 1 mg/kg at 88% DM with a suggested supplementary rate of 0.3 mg Co/kg at 88% DM. This study aimed to determine the efficacy of cobalt plus/minus vitamin B₁₂ and selenium supplementation from oral drenching over a 13 day time period. Seven groups of grass silage fed Suffolk cross mule lambs (n=56) were randomly allocated to one of 7 treatments, a 5 ml oral drench containing 700, 2300, 12000 mg Co/l, with or without 2300 mg vitamin B₁₂/l in a factoral design, with an additional control group which received no drench. All drenches also contained selenium at 625 mg/l. Lambs were weighed on days 0 and 13. The lambs were sampled by jugular venepuncture on days 0, 1, 2, 3, 4, 5, 6, 7, 9, 11 and 13, with samples analysed for plasma selenium and cobalt by ICP-MS, erythrocyte glutathione peroxidase by colourimetric assay and vitamin B₁₂ by immunoassay. The results showed that cobalt via a drench was able to raise plasma cobalt in a dose dependent manner, with each dose level significantly higher than the previous level at all post drenching time points (P

Glucose is a critical metabolic fuel in most mammals although many foodstuffs also contain high levels of the monosaccharides, galactose and fructose. The aims of this work were to determine the insulin response to challenges of these sugars (experiment 1) and to examine the effect of systemic (experiment 2) and direct ovarian (experiment 3) infusion of these monosaccharides on ovarian function in ewes with autotransplanted ovaries. In experiment 1, both fructose (fourfold increase peaking in 2 h) and galactose (twofold increase; 30 min) elicited markedly different (PPPPPin vivoin sheep and that different mechanisms are involved. Thus, fructose exerts stimulatory effects through indirect modulation of peripheral insulin and/or gonadotrophin levels whereas galactose exerts primarily suppressive effects by direct actions on the ovary.

Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 microg/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 +/- 2.8 h in treated vs. 53.3 +/- 2.7 h in controls; P < 0.05) and smaller preovulatory follicles (4.1 +/- 0.2 mm in treated vs. 5.3 +/- 0.1 mm in controls; P < 0.01), which gave rise to smaller corpora lutea (9.5 +/- 0.8 mm in treated vs. 11.7 +/- 0.6 mm in controls; P < 0.05). There was, however, no effect of infusion on ovulation rate. Despite the changes in the size of the ovulatory follicles, when the hormonal data were aligned to the time of the luteinizing hormone surge, there were no differences in preovulatory estradiol, androstenedione, or inhibin A between groups. This study therefore provides strong in vivo evidence to support the hypothesis that BMP6 is an important local regulator of ovarian function and that alterations in BMP6 cellular signaling may explain some of the effects of the FecB mutation in inducing precocious maturation of ovulatory follicles.

BACKGROUND: This study compared the predictive value of the antral follicle count (AFC) measured from stored 3D data by three methods including a '2D equivalent technique' and two 3D techniques, the 'multiplanar view' and 'rendered inversion mode', as a test of ovarian reserve and treatment outcome. METHODS: One hundred consecutive subjects aged

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.

With the release of the Department for the Environment, Food and Rural Affairs/Advisory Committee on Animal Feed Guidance Note for Supplementing Copper to Bovines it was noted that the current copper status of the national herd was not known. Liver samples were recovered from 510 cull cattle at a single abattoir across a period of three days. The samples were wet-ashed and liver copper concentrations determined by inductively coupled plasma mass spectrometry analysis. Breed, age and previous location information were obtained from the British Cattle Movement Service. Dairy breeds had higher liver copper concentrations than beef breeds. Holstein-Friesian and ‘other’ dairy breeds had 38.3 per cent and 40 per cent of cattle above the Animal Health and Veterinary Laboratories Agency (AHVLA) reference range (8000 µmol/kg dry matter), respectively, whereas only 16.9 per cent of animals in the combined beef breeds exceeded this value. It was found that underlying topsoil copper concentration was not related to liver copper content and that age of the animal also had little effect on liver concentration. In conclusion, over 50 per cent of the liver samples tested had greater-than-normal concentrations of copper with almost 40 per cent of the female dairy cattle having liver copper concentrations above the AHVLA reference range, indicating that a significant proportion of the UK herd is at risk of chronic copper toxicity.

Objectives To examine the interobserver reliability of three-dimensional (3D) power Doppler data acquisition from the uterus and ovary. Methods 3D power Doppler angiography was used to acquire endometrial data from 20 patients and ovarian data from a further 20 different patients at various stages of in vitro fertilization. Two different observers each acquired two datasets from all 40 patients resulting in 80 endometrial and 80 ovarian datasets in total. Virtual Organ Computer-aided AnaLysis was used to define the object of interest and semiquantify the power Doppler signal within it. The reliability of measurements of volume and vascularity was assessed by calculating interclass correlation coefficients (ICCs) and limits of agreement between the two observers. Results Whilst volumetric data proved more reliably acquirable than power Doppler data, the interobserver reliability of data acquisition was high overall for both the endometrium and ovary with all measurements obtaining a lower ICC of above 0.9. Limits of agreement revealed minimal disagreement between the two observers for measurements of volume and vascularity within both the ovary and endometrium. The ‘indices of vascularity’ within the endometrium were less than those derived from the ovary and subendometrium, which were comparable. Conclusions 3D ultrasound can be reliably used between observers to acquire power Doppler information from the ovary and endometrium thus supporting the current use and further development of this technique in clinical practice. Copyright © 2004 ISUOG. Published by John Wiley & Sons, Ltd.

We used three-dimensional power Doppler angiography (3D-PDA) to examine the periodic changes in endometrial and subendometrial vascularity during the normal menstrual cycle in 27 women without obvious menstrual dysfunction or subfertility.3D-PDA was performed on alternate days from day 3 of the cycle until ovulation and then every 4 days until menses. Virtual organ computer-aided analysis and shell-imaging were used to define and to quantify the power Doppler signal within the endometrial and subendometrial regions producing indices of their relative vascularity.Both the endometrial and subendometrial vascularization index (VI) and vascularization flow index (VFI) increased during the proliferative phase, peaking approximately 3 days prior to ovulation (P0.001) before decreasing to a nadir 5 days post-ovulation (P0.001). Thereafter, both vascular indices gradually increased during the transition from early to mid-secretory phase. The flow index (FI) showed a similar pattern but with a longer nadir post-ovulation. Smoking was associated with a significantly lower VI and VFI. The FI was significantly lower in women agedor = 31 years and significantly higher in parous patients.Endometrial vascularity, as assessed by 3D-PDA, varies significantly during the menstrual cycle and is characterized by a pre-ovulatory peak and post-ovulatory nadir during the peri-implantation window.

Objectives Three-dimensional power Doppler angiography (3D-PDA) has been largely used for the subjective assessment of vascular patterns but semiquantification of the power Doppler signal is now possible. We examined the intraobserver and interobserver reliability of the semiquantification of ovarian, endometrial and subendometrial blood flow using 3D-PDA, virtual organ computer-aided analysis (VOCAL™) and shell-imaging. Methods 3D-PDA was used to acquire 20 ovarian and 20 endometrial volumes from 40 different patients at various stages of in vitro fertilization treatment. VOCAL was then used to delineate the 3D areas of interest and the ‘histogram facility’ employed to generate three indices of vascularity: the vascular index, the flow index and the vascularization flow index. Intraobserver and interobserver reliability was assessed by two-way, mixed, intraclass correlation coefficients (ICCs) and general linear modeling was used to examine for differences in the mean values between each observer. Results The intraobserver reliability for both observers was extremely high and there were no differences in reliability between the observers for measurements of both volume and vascularity within the ovary or endometrium and its shells. With the exception of the outside subendometrial shell volumes, there were no significant differences between the two observers in the mean values obtained for either endometrial or ovarian volume and vascularity measurements. The interobserver reliability of measurements was equally high throughout with all measurements obtaining a mean ICC of above 0.985. Conclusions 3D-PDA and shell-imaging offer a reliable, practical and non-invasive method for the assessment of ovarian, endometrial and subendometrial blood flow. Future work should concentrate upon confirming the reliability of data acquisition and the validity of the technique before its predictive value can be truly tested in prospective clinical studies. Copyright © 2003 ISUOG. Published by John Wiley & Sons, Ltd.

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the UK dairy herd and, although characterized by reduced or absent oestrus and reduced conception rates, the exact cause remains unknown. The aim of this study was to investigate the expression of mRNA for the copper-dependent enzyme, lysyl oxidase, and the effect of copper and/or copper chelating thiomolybdates on FSH-induced differentiation of bovine granulosa cells cultured in serum-free media. Expression of lysyl oxidase mRNA was investigated using bovine specific primers and RT-PCR on cell lysates obtained from bovine granulosa cells cultured under optimum conditions for 0, 16, 24, 48, 96, 144 and 192 h. The effect of thiomolybdates and copper were investigated by supplementing optimized granulosa cell culture media with ammonium tetrathiomolybdate at 0, 0.1, 1, 10, 100 and 1000 micro g ml(-1), copper chloride at equimolar concentrations (0, 0.0516, 0.516, 5.16, 51.6, 516 micro g ml(-1)) or equimolar combinations of both media. Lysyl oxidase mRNA was expressed by the granulosa cells throughout the 192 h of culture. Thiomolybdate depressed oestradiol production in a dose-dependent manner at doses > 1 micro g ml(-1) and prevented the characteristic clumped appearance of granulosa cells in this serum-free system. Although the supplementation of copper alone had no effect at physiological doses, the use of the equimolar copper and thiomolybdate media ameliorated the effect of tetrathiomolybdates on both oestradiol production and cellular morphology. In conclusion, the results of the present study indicate that lysyl oxidase is expressed by granulosa cells, that thiomolybdates can prevent FSH-induced differentiation of bovine granulosa cells in vitro and that these effects can be reversed by copper supplementation. Overall, these data support the hypothesis that copper-responsive subfertility results from perturbation of the normal pattern of ovulatory follicle growth and development, an effect that may be mediated, at least in part, via lysyl oxidase activity.

Objectives The primary aim of this validation study was to determine the interobserver reliability and validity of measurements of phantom objects of known volume using conventional and rotational techniques of volume calculation according to measurement technique. Methods Two observers each acquired a single three-dimensional ultrasound dataset of three water-filled objects of different size and shape. The same two observers measured all six datasets using both the conventional technique and the newer rotational technique (Virtual Organ Computer-aided AnaLysis, VOCAL™) of volume calculation. Reliability was assessed by calculating intraclass correlation coefficients (ICC) and validity by examining the percentage difference from the ‘true’ volume, as determined by a water displacement technique, by the limits of agreement method. Results All of the techniques were highly reliable (ICC: 0.9962–0.9997) and valid to within 4% of the ‘true’ volumes. There were no significant differences in reliability according to measurement plane or between observers. Measurements made with the 6° rotation step were significantly more reliable than those made by all other techniques with the exception of the 9° rotation step (P < 0.05) and significantly more valid than those made with the 30° rotation step or conventional technique (P < 0.05). Conclusions Volume calculation in the in vitro setting is both reliable and valid but is dependent upon the technique applied, with rotational measurements of volume proving superior to conventional techniques. Copyright © 2003 ISUOG. Published by John Wiley & Sons, Ltd.

Differential regulation of LHR in theca cells (TC) and granulosa cells (GC) is important for normal follicular development. Unlike TC, GC only acquire LH-responsiveness during the later stages of antral follicle development. This study tested the hypothesis that differential LH-responsiveness in these two cell types may be due, in part, to shifts in cellular patterns of alternatively spliced LHR mRNA transcripts which may not be obvious from analysis of total LHR gene expression. It also further explored the role of translation inhibition by an LHR binding protein (LHBP), normally associated with the production of endogenous cholesterol. LHR mRNA variation arises as a result of the alternative splicing of two variable deletion sites (VDS) designated 5′ VDS and 3′ VDS, and it was proposed that differences in cell sensitivity to LH may be due in part to variations in the pattern of the mRNA expression of the receptor variants. The outcomes of the present study support a dynamic multi-facetted regulation of LHR during pre-translation. Not only did the ratio between variants change during antral follicle growth and in vitro cell differentiation but also between TC and GC. Regulation could also be linked to LH concentration feedback mechanisms as the absence of LH caused cultured TC to markedly up-regulate amounts of LHR mRNA. In both TC and GC, LHR mRNA was greatly reduced after treatment to block mevalonate production in the de novo cholesterol pathway, adding further support for a regulatory mechanism linked to enriched cellular amounts of mevalonate kinase.

Signs of clinical copper deficiency in ruminant animals include infertility. Blood copper concentrations alone are not an adequate indicator of copper status, as they take no account of the thiomolybdate challenge to the animal. The use of the ratio of caeruloplasmin activity to plasma copper concentration should provide a better indicator of molybdenum induced copper problems. The objectives of this study were to obtain veterinary feedback on the University of Leeds Trace Element Laboratory's (ULTEL) assessment of copper status and responses to any recommended supplementation. Blood samples from 713 female cattle from 114 herds were assessed for copper status, including plasma copper concentrations, serum caeruloplasmin activities and the ratio between the caeruloplasmin and plasma copper (CP/PlCu ratio). On the basis of only plasma copper analysis 95.0 % of the cattle were classified as being of normal copper status using Veterinary Laboratory Agency reference ranges. However, when the CP/PlCu ratio was also considered only 35.6 % of the cattle would be classed as having normal copper status. Appropriate copper supplementation would be required by 30.7 % of the cattle and a further 27.8 % could also benefit from additional copper. Veterinary responses to a questionnaire (80 % returned) indicated that infertility was a major problem in their clients’ herds. Where copper supplementation was recommended and administered an increase infertility was observed. In herds showing poor or lack ofoestrous behaviour/ later embryo loss (signs of clinical copper deficiency), 91.7 % reported that these signs were reduced by copper supplementation. The relevance of the CP/PlCu ratio as a diagnostic aid in the identification of copper responsive infertility has been supported by the responses of the veterinary surgeons.

Three field trials were carried out to evaluate the performance of a sintered soluble glass copper, cobalt and selenium bolus for maintaining adequate levels of the three trace elements. Trial 1 used 34 growing lambs, trial 2 used 36 growing lambs whilst trial 3 used 50 year-old non productive female sheep. In each trial half the sheep had a bolus administered (bolused) and half remained as controls. Blood samples were taken immediately prior to bolus administration and then at regular intervals throughout each trial. The samples were analysed for copper status (serum caeruloplasmin activity and plasma copper concentration), cobalt status (serum vitamin B12 concentration) and selenium status (erythrocyte glutathione peroxidase activity). For trial 1 the bolused sheep at all post treatment samplings (days 20, 42 and 63) were significantly increased in both cobalt and selenium status (P

Supplemental zinc and selenium were administered to ram lambs grazed on pastures that were not considered to be deficient in either element. The breeding season and polygamy of the ram mean that his requirements for semen production will be relatively large over a short breeding season and this may induce a localised deficiency of zinc and/or selenium, thus resulting in a decrease in semen quality and production.Thirty-three 8-month-old ram lambs were kept at grass and fed a supplement of barley and peas, with ad libitum access to grass silage when grazing became restricted. On day 0, the rams were allocated to two groups by restricted randomisation of live weight. One group each had a zinc, cobalt and selenium soluble glass bolus (Zincosel(R), Telsol) administered with the other group not receiving a bolus to act as a control. Blood samples were taken by jugular venipuncture at day 0 (prior to bolus administration) and at days 23, 44, 65 and 86. Blood samples were analysed for zinc status (plasma zinc concentration) and selenium status (erythrocyte glutathione peroxidase activity). Semen was collected once a week between days 44 and 86, by diversion during a natural mount. Semen quality was assessed by ejaculate volume, spermatocrit, sperm concentration, abnormal morphology, motility, percentage live (negrosin-eosin stain), membrane integrity (hypo-osmotic swelling test (HOS)) and seminal fluid glutathione peroxidase activity and zinc concentration. The bolused lambs had a significantly increased erythrocyte glutathione peroxidase activity (P

An experiment was conducted on 48 ewes during follicular and luteal phases of the oestrous cycle to determine the effect of a 5-day lupin grain supplementation (500 g/day) on folliculogenesis, plasma concentrations of glucose, insulin, FSH and oestradiol-17β (E2), follicular fluid concentrations of glucose, E2, androstenedione and progesterone and the levels of P450aromatase and insulin receptor substrate 1 (IRS-1), -2 and -4 in theca and granulosa cells. Average weight did not differ between lupin-fed and control groups. The numbers of follicles were increased (P2) in the lupin-fed group. The plasma concentrations of glucose (PP2were decreased (P2(P450aromatase in granulosa cells (P450aromatase in granulosa cells was positively correlated with the concentration of E2in follicular fluid (r=0.820;P

The objective of this study was to investigate the effect of three monosaccharides or pyruvate on the ability of gonadotrophins to induce cellular proliferation and differentiation of cultured sheep granulosa cells. Lactate production and levels of mRNA expression for the glucose transporters SLC2A1, SLC2A4, SLC2A5 and SLC2A8 were also determined. No energy source in the culture media reduced cell number (50%) and oestradiol (E2) production. Dose and type of monosaccharide had a highly significant (PP2production than fructose, which was in turn higher than galactose (P2production as glucose, but higher doses were markedly inhibitory to E2production (PPPPin vitro, and that fructose and pyruvate, but not galactose, are alternative energy substrates despite marked differences in the way these substrates are metabolised.

The incidence of low day 5 milk progesterone in dairy cows has been investigated and the efficacy of treating the problem assessed. The incidence of inadequate milk progesterone (empirically defined as

An experiment was conducted using 16 cyclic, Welsh Mountain ewes during the luteal phase of the estrous cycle to determine the effect of a 5-day period of feeding a high-energy high-protein diet (lupin grain; 500 g/day) on folliculogenesis and on the plasma concentrations of glucose, insulin, follicle stimulating hormone (FSH) and estradiol-17β, and on the follicular fluid concentrations of glucose, inhibin A, estradiol-17β, androstenedione and progesterone. Average weight did not differ between lupin-fed and control groups during the experiment. There was a trend for the number of small and large follicles to increase in the lupin-fed group. The plasma concentrations of glucose ( P = 0.012) and insulin ( P = 0.007) were higher during the feeding period in lupin-fed ewes. The plasma concentrations of FSH and estradiol-17β were not significantly different. The mean follicular fluid concentration of glucose (small follicles; P = 0.010) and progesterone lowered ( P = 0.034) compared to controls. The follicular fluid concentrations of estradiol-17β, androstenedione and inhibin A were not significantly different. The follicular fluid concentration of estradiol-17β was positively correlated with androstenedione ( r = −0.241; P = 0.001) and inhibin A ( r = 0.734; P ≤ 0.001) and glucose was negatively correlated with inhibin ( r = −0.241; P = 0.01), but not estradiol ( r = 0.075; P = 0.410) or androstenedione ( r = 0.050; P = 0.564). The lupin grain supplement increased the number of follicles as expected, but this increase was not significant. These changes were reflected in follicular fluid where lupin feeding increased the concentration of glucose and decreased the concentration of progesterone in follicles less than 3.5 mm in diameter. These data suggest that the local ovarian actions of nutrients have a role in the mediation of nutritional influences on folliculogenesis.

This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition stimulates folliculogenesis and ovulation rate. In addition the paper reviews recent findings on the effects of increased levels of nutrition on the blood concentrations of reproductive and metabolic hormones in the ewe and some of the intraovarian changes that take place in response to nutritional stimulation. Finally, in the paper, we propose a model of the physiological mechanism for the nutritional stimulation of folliculogenesis and we review how closely the model fits recent published and unpublished evidence examining the mechanism of flushing. Nutritional stimulation alters the blood concentrations of some metabolic hormones. By using short-term models of nutritional flushing, we have shown that as the blood concentrations of insulin and leptin increase that of growth hormone decreases while that of IGF-I appears unaffected by the nutritional flushing. Nutritional flushing also alters the blood concentrations of some reproductive hormones. Again, using the same model, we have shown that there is a transient increase in FSH and a decrease in oestradiol concentrations in the blood. The changes in oestradiol are particularly evident in the follicular phase of the oestrous cycle. In the ovary, the effect of nutrition is to stimulate folliculogenesis. These changes are associated with intra-follicular alterations in the insulin-glucose, IGF and leptin metabolic systems. The stimulation of these intra-follicular systems leads to a suppression in follicular oestradiol production. The consequence of these direct actions on the follicle is a reduced negative feedback to the hypothalamic-pituitary system and increased FSH secretion that leads to a stimulation of folliculogenesis.

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the national dairy herd. The aims of this study were to determine the effect of copper and/or copper chelating thiomolybdates on LH-induced differentiation by looking at the effects on androgen production, steroidogenic enzymes (cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage) and lysyl oxidase mRNA expression in cultured theca cells maintained under serum-free conditions. The effect of thiomolybdates and copper on LH differentiation was investigated by supplementing (ammonium) tetrathiomolybdate to optimum theca cell culture media at 0–100 μg/ml, copper (chloride) at equimolar concentrations (0–51.6 μg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17α-hydroxylase and cytochrome P450 side-chain cleavage mRNA was examined using real time PCR. Both PCRs used bovine specific primers and cell lysates obtained from bovine theca cells cultured for 6 days and in the presence or absence of the 100 μg/ml dose of thiomolybdate and equimolar copper thiomolybdate. Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 μg/ml at 96 h (PPP In conclusion, these results demonstrate that thiomolybdates can prevent LH-induced differentiation of bovine theca cells in vitro and that these effects can be ameliorated by copper supplementation. Our results also indicate that it is unlikely that the effects of thiomolybdate are mediated at the transcriptional level and further work is required to determine if thiomolybdate exerts its effects through post-translation processing or some other unrelated mechanism. Overall, these data support the hypothesis that copper responsive subfertility results from perturbation of the normal pattern of ovarian steroidogenesis.

An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n= 5; 1 μg/h) or saline (n= 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2α analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 μm using a cryostat at −20 °C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter ≥3.5 mm (1.0 ± 0.36 (s.e.m.) vs 2.4 ± 0.24; control vs leptin;P< 0.02) but had no effect on the number of ≥1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 ± 0.79 vs 7.4 ± 0.81; control vs leptin;P< 0.05), IGFBP-2 (10.0 ± 0.82 vs 5.2 ± 0.87; control vs leptin;P< 0.05) and IGFBP-5 (5.2 ± 1.60 vs 1.2 ± 0.30; control vs leptin;P< 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 ± 0.15 vs 2.2 ± 0.31 mm; control vs leptin;P< 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 ± 0.021 vs 0.39 ± 0.027 arbitrary units; control vs leptin;P< 0.05) and IGFBP-5 (0.46 ± 0.019 vs 0.25 ± 0.053 arbitary units; control vs leptin;P< 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 ± 0.25 vs 1.40 ± 1.17; control vs leptin;P< 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P< 0.05) and increased concentrations of FSH (P< 0.001) and insulin (P< 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles ≥3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production.

Leptin, the metabolic fat hormone, has been shown to have effects on reproduction in mice and to modulate steroid production by cultured ovarian somatic cells in a number of species. However, a direct role of leptin on normal ovarian function in vivo has not been shown. In this paper the effect of passive immunisation against leptin (experiment 1; 20 ml antiserum or non-immune plasma i.v.; n = 6/treatment) and direct ovarian infusion of leptin (experiment 2; 0, 2 or 20 μg recombinant ovine leptin; n = 4/treatment) during the early follicular phase was investigated in sheep with ovarian autotransplants, which allow recovery of ovarian venous blood and regular non-invasive scanning of the ovary. Passive immunisation against leptin resulted in an acute increase (P < 0.05) in ovarian oestradiol secretion but had no effect on gonadotrophin concentrations, ovulation or subsequent luteal function. Conversely, direct ovarian arterial infusion of the low dose of leptin resulted in an acute decline (P < 0.05) in ovarian oestradiol secretion whereas the high dose, which resulted in supra-physiological leptin concentrations, had no effect on oestradiol production compared with the controls. Neither dose of leptin had any effect on gonadotrophin concentrations or ovulation but both doses resulted in an increase (P < 0.05) in progesterone concentrations over the subsequent luteal phase. In conclusion, together these data provide strong in vivo evidence that leptin can modulate ovarian steroidogenesis directly and acutely in ruminants and suggest that leptin is an alternate regulatory system whereby nutritional status can regulate reproductive activity.

Objective To define the changes in endometrial morphometry that occur during the normal menstrual cycle and to examine the discriminatory power of volumetric analysis over conventional two-dimensional measurements in quantifying the effect of compounding factors on endometrial growth. Design The design was that of a longitudinal observational study. Subjects were seen on an alternate-day basis until ovulation and then every four days until the next menstrual period. Setting A University teaching hospital. Population Thirty women with regular cycles and acceptable ‘normal’, menstrual patterns. Methods Three-dimensional ultrasound data were acquired and subsequently analysed by a single observer. Main outcome measures Endometrial thickness and volume. Results Both endometrial volume and thickness increased significantly during the follicular phase (P < 0.001), reaching a plateau around the time of ovulation and remaining relatively stable throughout the luteal phase. These changes in endometrial thickness and volume were highly correlated (R2= 0.767; P < 0.001). Parity was associated with a significantly greater endometrial volume than nulliparity (4.159 vs 2.234 cm3; P < 0.05). Conclusions This study has defined the relative and absolute changes in endometrial growth, both in terms of thickness and volume, throughout the normal menstrual cycle. These data provide a reference for future three-dimensional studies investigating menstrual disorders, pathophysiological change and subfertility.

A serum-free culture system has been developed in ruminants that allows gonadotrophin-responsive induction of oestradiol (E2) production by non-differentiated granulosa cells (GC) from small antral follicles. Critical determinants are dose of FSH and insulin-like growth factor (IGF), and the plating density of the GC. Over the first 16 h of culture when cells remained as a dispersed monolayer, expression declined in FSH receptors (FSHr) (P0.001), IGF type 1 receptor (IGF-1r) (P0.08) and p450 arom (CYP19, P0.001). Characteristic GC clusters formed from 16 h and further enlarged between 24 and 48 h, accompanied by marked increases in FSHr (P0.01), IGF-1r (P0.05), and p450 arom (P0.01) expression, and preceded induction and subsequent peak E2 production, at 96 and 144 h, respectively (P0.01). In conclusion, isolation and dispersion of GC appears to induce reversion to an immature state resulting in loss of receptor expression. Re-establishment of cell-cell communications in the presence of FSH and IGF results in receptor up-regulation and induction of cellular differentiation.

The soluble glass bolus was more effective at 16 weeks after administration than the copper oxide needles. However, the treatments were similar at 20 weeks in supplying extra copper to red deer at pasture. The bolus also provided effective selenium supplementation. The equivocal vitamin B12 results indicate caution in using this parameter to assess cobalt status in this species until further evaluated.

Over the 10-week duration of the trial, the bolus gave the best maintenance of copper status and correction of clinical copper deficiency by preventing uptake of free tetrathiomolybdate into the sheep. The needles and injection also corrected the clinical copper deficiency, with the injection increasing the plasma copper and caeruloplasmin more, whilst the needles were better when the CP/PlCu ratio and TCA insoluble copper were assessed. The chelate was better than the drench for copper status although neither could sustain the correction of the clinical deficiency.

The aim of the present study was to investigate the effects of glucose, galactose and fructose on the LH-induced differentiation and mRNA expression of sugar transport facilitators (SLC2A) by sheep thecal cells derived from small antral follicles cultured under serum-free conditions for 6 days. The dose and type of monosaccharide had a significant effect on LH-induced androstenedione production by theca cells and there was a significant interaction (P

SIR, — We refer to the short communication by [Laven and others (2006)][1]. This reinterprets the data of [Scaletti and others (2003)][2] and questions why copper supplementation did not affect the caeruloplasmin:plasma copper ratios. However, there is an alternative interpretation of this data

This study aimed to determine values for selected energy metabolites (non-esterified fatty acid [NEFA], β-hydroxybutyrate [BHB], urea or urea:BHB ratio), together with a body condition score, associated with an increased risk of cows developing a reproductive disorder and to investigate temporal relationships between predictors and reproductive outcome. A cohort of 98 cows on one farm was monitored weekly from four weeks before to 10 weeks after calving; 89 cows provided sufficient data to calculate commencement of luteal activity (C-LA). Cows with high NEFA × urea (Nu; product of NEFA and urea) values one and three weeks after calving were twice as likely to develop cystic ovarian disease (risk ratio 2). Cows that developed endometritis had high NEFA values one (P=0.02) or four weeks (P=0.04) before calving, or low urea:BHB ratios two weeks before calving, at calving or three weeks after calving (P=0.024, P=0.031 and P=0.001, respectively). Cows that had delayed C-LA had high NEFA values one week after calving (P=0.05) or low urea:BHB ratios three or four weeks after calving (P=0.004 and P=0.003, respectively).

AN investigation into copper deficiency, confirmed by diagnosis of swayback at postmortem examination, and possibly thiomolybdenum toxicity on a pedigree Boer goat unit has led me (MM) and other colleagues to look at whether the presently quoted reference ranges for plasma copper levels in goats are