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Ruminants are recognised to suffer from Cu-responsive disorders. Present understanding of Cu transport and metabolism is limited and inconsistent across vets and veterinary professionals. There has been much progress from the studies of the 1980s and early 1990s in cellular Cu transport and liver metabolism which has not been translated into agricultural practice. Cu metabolism operates in regulated pathways of Cu trafficking rather than in pools of Cu lability. Cu in the cell is chaperoned to enzyme production, retention within metallothionein or excretion via the Golgi into the blood. The hepatocyte differs in that Cu-containing caeruloplasmin can be synthesised to provide systemic Cu supply and excess Cu is excreted via bile. The aim of the present review is to improve understanding and highlight the relevant progress in relation to ruminants through the translation of newer findings from medicine and non-ruminant animal models into ruminants.

Background Antagonisms exist in vivo which inhibit copper bioavailability in ruminants. Although the antagonism between iron, sulphur and copper has been well observed in vivo in practice the mechanism by which it acts has not yet been elucidated, nor the compound it creates identified. This results in problems when trying to optimise supplementation to prevent the interaction from occurring or provide a copper source which is able to negate its effects. This work aims to establish if the antagonism between sulphur, iron and copper could be elicited under in vitro rumen replicated conditions and using a range of copper sources to investigate any differences in their participation in the interaction. Methods Rumen simulated conditions were used to test solubility as a proxy for bioavailability of different copper sources. Sources from ionic, hydroxy and organic compounds were tested in de-ionised water and warmed, strained rumen fluid which mimicked duration, agitation, temperature and pH of the rumen. Results All copper sources were less soluble in rumen fluid than in de-ionised water. The addition of sulphide, alone or as part of a sulphur mix with sulphate produced a pronounced reduction in solubility on each of the copper sources. The most soluble were the greatest affected. Conclusion There was no indication that an in insoluble compound containing copper and iron was formed under these conditions. The intricacy of the in vivo rumen is required to elicit the reaction between copper, iron and sulphur.

The concentration of Co in the herbage of a large proportion of grassland farms in Ireland is lower than that required by sheep. The effects of supplementing ewes with Co, and method of supplementation, on ewe and offspring performance, were evaluated over 2 years. The ewes (359 in year 1, 241 in year 2), representing 3 genotypes, were allocated to one of 3 treatments. The treatments were: Control - no supplementation; Drench – Co supplementation via oral drench (31.5 mg Co as CoSO4•7H2O) administered every 14 days; Bolus – Co supplementation via rumen bolus (effective life of ∼6 months) that only contained Co as CoSO4•7H2O with an expected Co release rate of 0.6 mg/day in year 1 and 1 mg/day in year 2. Drench treatment was administered from 7 weeks pre joining, until 6 weeks prior to expected lambing date (early – to mid - March). One bolus was administered to ewes on the bolus treatment at the start of the study (day 0) each year. The ewes were managed as one flock in a rotational-grazing system until housing in early December each year; they were shorn at housing and offered grass silage ad libitum plus a concentrate supplement during late pregnancy. Ewes and lambs were put to pasture within 3 days of lambing. Supplementation with Co, via drench or bolus, increased the concentration of Co (p 0.05) on ewe BW or body condition score at joining (day 49), mid-pregnancy, lambing, 5 weeks post lambing or weaning. Likewise, supplementation had no effect (p > 0.05) on litter size, lamb mortality or number of lambs reared per ewe joined, or on lamb BW at birth or weaning. The only significant animal-performance difference detected between the Bolus and Drench treatments concerned the incidence of lambs classified as ‘born dead or died within 24 h of birth’ (3.1% for Drench, 6.5% for Bolus; p

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

Zinc, cobalt and selenium are essential trace elements for ruminants and all have roles in immune function. The delivery of these elements to grazing livestock can be problematical. The aim of this trial is to determine the effect of the administration of an intraruminal soluble glass bolus to supply zinc, cobalt and selenium to growing lambs at pasture and to determine the effect on humoral immune response via the administration of a novel keyhole limpet haemacyanin (KLH) antigen. On days 0, 17 lambs each had a zinc (15.1% w/w), cobalt (0.52% w/w) and selenium (0.15% w/w) soluble glass bolus (∼33 g) administered (bolused), whilst the 17 lambs received no bolus (control). All of the lambs were grazed together throughout the trial. Trace element status (selenium—erythrocyte glutathione peroxidise activity; cobalt—serum vitamin B12 concentration; zinc—plasma zinc concentration) and live weights were assessed on days 0, 20, 42 and 63, with immunisation with KLH on day 34 and assessment of IgG response by direct ELISA on days 20, 42, 49, 63. Lambs were slaughtered when commercially fit on either day 86 or 121 and livers analysed for copper and zinc concentrations and boluses recovered from the bolus group to assess dissolution rates. The bolused lambs had significantly increased serum vitamin B 12 concentrations ( p p p p p p In conclusion, administration of a zinc, cobalt and selenium soluble glass bolus resulted in an increased antibody response and fulfilled the daily requirements for cobalt, selenium and zinc with an elevated status of these elements compared to unsupplemented controls grazing the same pasture.

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

Balance studies were carried out on four Suffolk-cross lambs which were fed a diet containing only 1.2 mg zinc/kg dry matter; zinc deficiency was induced within three weeks. After a period during which the deficiency was relieved by a pica, the zinc deficient state was re-established. Each sheep was then treated with a soluble glass bolus containing zinc, cobalt and selenium. The plasma zinc concentration of the sheep rapidly increased and was maintained for between six and 10 weeks. The bolus was able to supply the daily requirement of the sheep for zinc, with no detrimental effect on their copper status.

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 rumen is the site of significant interactions between Cu, S and Mo. It also shows reactions between Cu, S and Fe. The interaction between Mo and S results in the formation of thiomolybdates, which in the absence of adequate quantities of rumen Cu are absorbed into the animal and bind to Cu in biological compounds. This is the cause of thiomolybdate toxicity, often misleadingly called Cu deficiency. The effects of thiomolybdates being absorbed into the animal are considered, especially how thiomolybdates bind to Cu-containing compounds such as enzymes, decreasing their activity without removing the active Cu component. The sources of Cu, Mo, Fe and S are examined, including the impacts of water and soil on the animal's intake. Within the present review we have been able to provide evidence that: all classes of thiomolybdates are formed in the rumen; in the absence of available Cu all thiomolybdates can be absorbed into the animal rapidly though the rumen wall or via the small intestine; thiomolybdates bind to Cu in biological compounds and are able to cause problems; effects of thiomolybdate are reversible in vivo and in vitro on cessation of thiomolybdate challenge; the tetra-thiomolybdate form is the most potent Cu binder with decreased potency with decreasing S in the compound. Fe will exacerbate a thiomolybdate problem but will not directly cause it.

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.

Facilitated clinical relevance (FCR) at the University of Nottingham School of Veterinary Medicine and Science is a small-group educational model that promotes student-centered learning. The objectives of this study were: (1) to examine students’ opinions of FCR as a learning approach compared with other teaching methods; and (2) to establish if gender, year of study, or prior exposure to FCR were predictors of attitude. Questionnaires were distributed to undergraduate veterinary students (N = 185) in the first and second year of study. Students’ opinions were assessed by noting their degree of agreement with seven statements using a seven-point Likert-type scale. A total of 162 students (87.6%) completed the survey instrument. The findings indicate that FCR is highly valued among the students. The majority of students regarded FCR as directly relevant for clinical preparation because it helps them to integrate pre-clinical and clinical knowledge and skills in veterinary medicine. However, commonly identified challenges were peer dominance, less cooperative facilitators, and coping with the ambiguity of knowledge and reasoning. No significant differences were detected in attitude between students in the tested variables (p > 0.05). However, being in the first year of study, being male, and having prior exposure to FCR was found to predict a negative attitude (p < 0.006). To increase students’ satisfaction, effective strategies must be designed and implemented to provide support for struggling individuals.

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.

Thirty-three ram lambs at pasture with access to quality grass silage and a barley/pea concentrate were split into two groups by restricted randomisation of liveweight. On day 0, one group (n = 17) was bolused with a zinc, cobalt and selenium soluble glass bolus (Zincosel©, Telsol Ltd) with the other group unbolused as controls (n = 16). The rams were blood sampled on days 0, 23, 44, 65 and 86. Plasma zinc concentrations (PlZn) and erythrocyte glutathione peroxidase activities (eGSHPx) were analysed (Kendall et al., 1999). Collection of semen samples by natural mount and diversion into an artificial vagina were attempted from all rams on days 44, 52, 58, 65, 73, 79 and 86. Semen samples were assessed for ejaculate volume (Vol.), spermatocrit (Sc.), total sperm count (Count), motility (Mot), proportion of live sperm (Live) (negrosin-eosin stain) and abnormalities (total, coiled tail, no tail, two tail, tail droplets and others). The functional integrity of the sperm’s plasma membrane was assessed using the hypoosmotic swelling test (HOS) (Correa et al., 1997). Seminal plasma zinc concentration (spZinc) and glutathione peroxidase activity (spGSHPx) were analysed using the same assays as for the blood. Statistical analysis was carried out by ANOVA using GLM on MINITAB 11 (blood parameters used day 0 as a covariate, proportionality data (Sc., Live, HOS, Mot) used the arcsine transformation and abnormality data used the square root transformation with zero value adjustment. Seven of the control and five of the bolused rams failed to produce a valid semen sample on any of the seven semen collections. The bolused rams had significantly higher erythrocyte glutathione peroxidase activities than the control rams throughout the post bolusing period (p < 0.001). The groups did not have significant differences in plasma zinc concentrations. MOT (d 44,73), HOS (d 73) and Live (d 73,79) were all significantly higher for the bolus group. Count (d 65) and spZinc (d 73) were significantly higher

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

Many sheep in the United Kingdom are grazed on pastures deficient in cobalt and selenium. Proprietary products for selenium and cobalt supplementation often contain copper, which in certain breeds can induce copper toxicity. A soluble glass bolus has been manufactured to supply long term sustained release of cobalt and selenium similar to the commercially available Cosecure, but with the copper content being replaced by zinc. Sub clinical zinc deficiency has been suggested to be involved in disease conditions such as broken mouth periodontitis, footrot and mastitis. Therefore a long term continuous supply of zinc may be beneficial in these situations. This trial was designed to examine the effect of administering a zinc, cobalt and selenium soluble glass bolus on trace element status of over-wintered ewe lambs.

Trace element deficiencies are common in many countries and affect animal health, productivity and welfare. Trace elements that can be limiting in animal nutrition include copper, cobalt, selenium and zinc. Deficiency diseases may be manifested as a consequence of a single or a multiple element deficiency. Deficiency in any of the above trace elements can result in an increased disease susceptibility and a decreased immune function. A soluble glass bolus has been developed for the sustained release of zinc, cobalt and selenium to sheep, using the same technology as the commercially available copper, cobalt and selenium bolus (Cosecure). The aim of this experiment was to observe whether the trace elements released from the bolus would have an effect on the immune function of sheep, as measured by the humoral immune response to a novel antigen.

Trace element deficiencies have been reported throughout the UK and are known to decrease the productivity and welfare of animals. Selenium deficiency is characterised by muscular myopathy, white muscle disease or stiff lamb disease. Subclinical deficiencies can also cause reduced growth rate and impaired immune function. In ruminant animals, cobalt is required for the synthesis of vitamin B12 and deficiency leads to Pine, characterised by emaciation, anaemia and listlessness. Trace element deficiencies can be due to either a single element or multiple elements. Situations occur particularly in sheep where cobalt and/or selenium are deficient but where copper supplementation can be toxic. To correct these deficiencies, a single long-lasting and effective treatment in the form of a soluble glass bolus has been designed to release cobalt and selenium. This new bolus is similar to the commercial Cosecure® bolus, which is a Co/Se/Cu soluble glass, in which zinc replaces the copper.