UK ruminant farmer understanding of copper-related terminology.

Copper provision is not straightforward in ruminants. Other dietary elements such as iron and molybdenum in combination with sulphur are able to interfere with copper availability, absorption and function. These complexities surrounding copper availability in cattle and sheep prevent the simple calculation of copper requirements. Previous research has established that UK farms are failing to provide copper in an effective manner, with some over-supplying and others under-supplying. Copper terminology is not consistently understood by professionals in the agricultural industry; potentially resulting in confusing or misinformation being passed on to farmers. The present work found that most (84 %) farmers felt they lacked understanding of copper related problems and their associated terminology. However, farmers who felt they had the least knowledge appeared more likely to underestimate their knowledge, and those who felt more confident in their knowledge were more likely to be overestimating it. Simple over- and under-supply of copper were only recognised by a small proportion (17 %) as causes of copper related problems while more complex issues were better recognised (27 %) as potential causal factors. However, the more specific terminology relating to the molybdenum-sulphur antagonism was poorly understood by most respondents (96 %).

X-ray absorption spectroscopy of copper and iron in sheep digesta.

BACKGROUND: The bioavailable supply of copper to ruminants has long been problematic. Complexities in supply exist due to interactions with other dietary elements in the rumen, most notably with iron or molybdenum in combination with sulphur, which can result in copper binding preventing its absorption. The molybdenum-sulphur-copper interaction has been extensively studied over the years. However, very little is known about the iron-sulphur-copper interaction, especially its mode of action in the gastrointestinal tract. METHODS: In the present work digesta from the rumen and jejunum of sheep fed a high copper, sulphur and iron diet was analysed using X-ray absorption spectroscopy (XAS). RESULTS: X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) indicated that all of the copper and iron had changed in bonding in the rumen and that the oxidation state of the elements had been reduced into a mix of Fe2+ & Fe3+ and Cu+ with some Cu0. CONCLUSION: The copper compounds were most likely to be thiol co-ordinated in line with Cu+ chemistry. Changes to the copper compounds took place in the jejunum, although thiols were still highly favoured the possible existence of a copper-iron-sulphur complex which also included oxygen and chloride was also observed. This possibly has some resemblance to the crystal structure of bornite.

Comparison of X-ray absorption spectra from copper-loaded bovine and ovine livers.

BACKGROUND: Copper toxicity and hepatic copper accumulation pose a serious risk to ruminant health and production. Differences in the copper-handling mechanisms of cattle and sheep have been noted, not only in comparison to each other, but also in comparison to 'copper-tolerant' monogastric species. Ruminants appear less able to cope with rising liver copper concentration than monogastric counterparts, with sheep in general less able to cope with elevated copper intake than cattle. METHODS: X-ray absorption spectroscopy (XAS) was used to investigate the differences between the livers of these species at high copper status. RESULTS: The X-ray absorption fine structure (XAFS) and X-ray absorption near edge structure (XANES) spectra indicated that the hepatic copper compound is most likely to be bound to metallothionein; consistent with monogastric species. CONCLUSION: Although, most likely stored as copper-metallothionein, there may be a role for glutathione as a short-term, intermediate copper buffer which may have more relevance to sheep than cattle. The potential that thiomolybdate bound copper can be stored in the liver could not be ruled out.

Equine influenza vaccination as reported by horse owners and factors influencing their decision to vaccinate or not.

BACKGROUND: Equine influenza virus is a highly contagious respiratory pathogen that causes pyrexia, anorexia, lethargy and coughing in immunologically naïve horses. Vaccines against equine influenza are available and vaccination is mandatory for horses that participate in affiliated competitions, but this group forms a small proportion of the total horse population. The aims of this study were to: i) identify the equine influenza vaccination rate as reported in 2016 by horse owners in the United Kingdom (UK); ii) examine the demographics of owners and horses which were associated with significantly lower influenza vaccination rates and iii) explore factors that influence horse owners' decisions around influenza vaccine uptake. RESULTS: Responses from 4837 UK horse owners who were responsible for 10,501 horses were analysed. An overall equine influenza vaccination rate of 80% (8385/10501) was reported. Several owner demographic characteristics were associated with significantly lower (p<0.05) reported equine influenza vaccination rates including: some geographical locations, increasing horse owner age, annual household income of less that £15,000 and owning more than one horse. Horse-related features which were associated with significantly lower reported equine influenza vaccination rates included age ranges of <4 years and > 20 years, use as a companion or breeding animal or leaving their home premises either never or at most once a year. The most common reasons cited for failing to vaccinate horses was no competition activity, lack of exposure to influenza and expense of vaccines. In contrast, the most common underlying reasons given by horse owners who vaccinated their horse were protection of the individual horse against disease, veterinary advice and to protect the national herd. Owners of vaccinated horses had less previous experience of an influenza outbreak or adverse reaction to vaccination compared with owners of unvaccinated horses. CONCLUSIONS: This study documented a high rate of equine influenza vaccination as reported by owners in a substantial number of horses in the UK, but this does not reflect the level of protection. Sub-populations of horses which were less likely to be vaccinated and the factors that influence each owner's decision around vaccination of their horses against equine influenza were identified, but may alter following the 2019 European influenza outbreak. This information may nevertheless help veterinary surgeons identify "at-risk" patients and communicate more personalised advice to their horse-owning clients. It may also influence educational campaigns about equine influenza directed to horse owners, which aim to improve uptake of vaccination against this pathogen.

Copper physiology in ruminants: trafficking of systemic copper, adaptations to variation in nutritional supply and thiomolybdate challenge.

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.

Pre-translational regulation of luteinizing hormone receptor in follicular somatic cells of cattle.

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.

Liver copper concentrations in cull cattle in the UK: are cattle being copper loaded?

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.

Effect of monosaccharide sugars on LH-induced differentiation and sugar transport facilitator (SLC2A) expression in sheep theca cells in vitro.

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<0.001). Glucose and galactose were used with equal efficiency so that cell numbers and androstenedione production at the end of the culture were comparable. Pharmacological doses of glucose (16.7 mM) inhibited steroidogenesis (P<0.05). Cell numbers and androstenedione production by cells cultured with fructose were lower than for cells cultured with either glucose or galactose (P<0.001). None of the monosaccharides resulted in the production of lactate. Expression of SLC2A1, SLC2A4 and SLC2A8, but not SLC2A5, mRNA was detected in fresh and cultured theca cells. Large doses (16.7 mM) of glucose and fructose, but not galactose, suppressed (P<0.05) SLC2A expression. The results show that glucose and galactose, but not fructose, are readily metabolised via oxidative pathways to support LH-induced differentiation of sheep theca cells. Further work is required to determine the mechanisms resulting in these differences in relation to the established effects of nutrition on reproductive function.

The effect of short-term nutritional supplementation of ewes with lupin grain (Lupinus luteus) on folliculogenesis, the concentrations of hormones and glucose in plasma and follicular fluid and the follicular levels of P450 aromatase and IRS-1, -2 and -4.

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-17ß (E2), follicular fluid concentrations of glucose, E2, androstenedione and progesterone and the levels of P450 aromatase 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 (P<0.05; χ(2)) in the lupin-fed group. The plasma concentrations of glucose (P<0.05; ANOVA) and insulin (P<0.001; ANOVA) were higher in lupin-fed ewes. The plasma concentrations of FSH were not different but those of E2 were decreased (P<0.001) in the lupin-fed group. Both the follicular fluid concentration of E2 (P<0.05) and the level of P450 aromatase in granulosa cells (P<0.05; ANOVA) were decreased in the lupin-fed group, but only during the follicular phase. The level of P450 aromatase in granulosa cells was positively correlated with the concentration of E2 in follicular fluid (r=0.820; P<0.001; ANOVA). The levels of IRS-1 and -2 in theca and granulosa cell lysates were increased in the lupin-fed group. These data suggest that insulin has a local role in the control of folliculogenesis and is likely to be a mediator of the effects of dietary energy intake on ovulation rate. We suggest that insulin acting through IRS proteins mediates the reproductive actions of insulin in the follicle and that IRS-1 and -2 are nutritionally regulated mediators of the action of insulin in the follicle.

Energy metabolites in pre- and postpartum dairy cattle as predictors of reproductive disorders.

This study aimed to determine values for selected energy metabolites (non-esterified fatty acid [NEFA], ß-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).

Role of the rumen in copper and thiomolybdate absorption.

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 effect of systemic and ovarian infusion of glucose, galactose and fructose on ovarian function in sheep.

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 (P<0.001) insulin responses than glucose (sevenfold increase; 20 min) although the total amount released following fructose and glucose challenge was similar. In experiment 2, low-dose systemic fructose infusion had no acute effect on insulin but did depress FSH (P<0.05), and following the end of fructose infusion, a transient increase in FSH and insulin was observed (P<0.05), which was associated with an increase (P<0.05) in ovarian oestradiol and androstenedione secretion. Systemic infusion of neither glucose nor galactose had a significant effect on ovarian steroidogenesis although glucose acutely suppressed insulin levels. In contrast, ovarian arterial infusion of fructose and glucose had no effect on ovarian function whereas galactose suppressed ovarian follicle number and steroid secretion (P<0.05). In conclusion, this work indicates that fructose and galactose can influence ovarian function in vivo in 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.

The effect of monosaccharide sugars and pyruvate on the differentiation and metabolism of sheep granulosa cells in vitro.

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 (E(2)) production. Dose and type of monosaccharide had a highly significant (P<0.001) effect on FSH-induced differentiation of the granulosa cells, and there was a highly significant interaction (P<0.001). Glucose supported higher levels of E(2) production than fructose, which was in turn higher than galactose (P<0.001). In contrast, pyruvate at low doses supported similar levels of E(2) production as glucose, but higher doses were markedly inhibitory to E(2) production (P<0.001). Cells responded positively to insulin (P<0.001) in the presence of all three monosaccharides. Glucose and the high doses of fructose resulted in the accumulation of lactate (P<0.001), but pyruvate, galactose and the low dose of fructose resulted in low lactate production. SLC2A5 expression was not detected and SLC2A8 expression was not affected, but SLC2A1 and SLC2A4 expression was depressed (P<0.05) by culture in the presence of fructose and glucose. These data show that glucose, metabolised under anoxic conditions to lactate, is the preferred energy substrate to support the gonadotrophin-induced differentiation of ovine granulosa cells in vitro, and that fructose and pyruvate, but not galactose, are alternative energy substrates despite marked differences in the way these substrates are metabolised.

Effect of direct ovarian infusion of bone morphogenetic protein 6 (BMP6) on ovarian function in sheep.

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.

Incidence and treatment of inadequate postovulatory progesterone concentrations in repeat breeder cows.

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 <3ng/mL) in repeat breeder cows was 34% compared with 11.4% in first insemination cows. Treatment with an intravaginal progesterone device for 7 days starting from day 5 or 6 did not improve pregnancy rate. Treatment with 1500 iu human chorionic gonadotrophin (hCG) on day 5 gave an increase in pregnancy rate that was dependent on initial progesterone concentration and significant (P<0.05) in multiparous but not primiparous cows. While the incidence of inadequate day 5 progesterone was high in repeat breeder cows, it was responsive to hCG treatment, although only in multiparous and not primiparous animals.

The effect of short-term nutritional supplementation of ewes with lupin grain (Lupinus luteus), during the luteal phase of the estrous cycle on the number of ovarian follicles and the concentrations of hormones and glucose in plasma and follicular fluid.

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-17beta, and on the follicular fluid concentrations of glucose, inhibin A, estradiol-17beta, 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-17beta were not significantly different. The mean follicular fluid concentration of glucose (small follicles; <3.5 mm) from lupin-fed ewes was elevated (P=0.010) and progesterone lowered (P=0.034) compared to controls. The follicular fluid concentrations of estradiol-17beta, androstenedione and inhibin A were not significantly different. The follicular fluid concentration of estradiol-17beta was positively correlated with androstenedione (r=-0.241; P=0.001) and inhibin A (r=0.734; P< or =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 3.5mm in diameter. These data suggest that the local ovarian actions of nutrients have a role in the mediation of nutritional influences on folliculogenesis.

Does 3D ultrasound offer any advantage in the pretreatment assessment of ovarian reserve and prediction of outcome after assisted reproduction treatment?

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<40 years with basal FSH levels<12 IU/l underwent transvaginal ultrasound in the early follicular phase prior to their first cycle of assisted reproduction treatment. The relationship between the AFC made with each method and outcome was evaluated. RESULTS: The AFC as measured by the 'inversion mode' method (r=0.777) showed a higher, but statistically insignificant, correlation with the number of retrieved oocytes than did comparable measurements made with the '2D equivalent' (r=0.665) and '3D multiplanar' (r=0.687) techniques. Multiple regression analysis showed the AFC measured using any of the three methods was a significantly better predictor of the number of oocytes retrieved (P<0.001) than age, FSH or ovarian volume. AFC was not predictive of non-conception regardless of the measurement technique. CONCLUSIONS: For predicting ovarian respone and outcome, the AFC measured using techniques based on 3D ultrasound offers no statistically significant advantage over a measurement which is limited to information available with conventional 2D imaging.

The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep.

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.

Effect of copper and thiomolybdates on bovine theca cell differentiation in vitro.

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 17alpha-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 microg/ml, copper (chloride) at equimolar concentrations (0-51.6 microg/ml) or equimolar combinations of both media. Lysyl oxidase mRNA expression was investigated with semi-quantitative RT-PCR, whilst expression of cytochrome P450 17alpha-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 microg/ml dose of thiomolybdate and equimolar copper thiomolybdate. Thiomolybdate depressed androstenedione production in a dose-dependent manner at doses greater than 1 microg/ml at 96 h (P<0.05); doses above 20 microg/ml were all greatly reduced at all time points and at 192 h when related to numbers of cells (P<0.001). Copper alone had no effect at physiological doses, but the use of the equimolar copper thiomolybdate reversed the effect of tetrathiomolybdates on androstenedione production at the 20 microg/ml dose. Thiomolybdate supplementation, with and without copper, had no significant effect on the level of lysyl oxidase or cytochrome P450 side-chain cleavage expression. However, cytochrome P450 17alpha-hydroxylase expression was significantly increased (P<0.05) by tetrathiomolybdate, possibly due to a local regulatory system. 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.