Effect of 2 dosages of prepartum cholecalciferol injection on blood minerals, vitamin D metabolites, and milk production in multiparous dairy cows: A randomized clinical trial.

The objective of the present study was to evaluate the effect of 2 dosages of prepartum cholecalciferol injection on blood minerals, vitamin D metabolites, and milk production. Cows entering their second or greater lactation (n = 158) were randomly assigned to a control group (CON) or one of 2 treatment groups receiving either 6 × 106 IU (6VitD) or 12 × 106 IU (12VitD) cholecalciferol intramuscularly on d 275 ± 1.2 (SD) of gestation. Concentrations of serum total Ca (tCa), phosphate, and Mg were determined on 1, 2, 3, 5, 7, and 10 d in milk (DIM). For a subsample of 30 cows entering the third lactation (n = 10/group), these samples were analyzed for cholecalciferol, 25-hydroxycholecalciferol (25-OHD3), and 24,25-dihydroxycholecalciferol (24,25-[OH]2D3). In these cows, we also determined 1,25-dihydroxycholecalciferol (1,25-[OH]2D3), the biologically most active metabolite, on 1, 2, 3, and 5 DIM. Repeated measures ANOVA was performed to evaluate the effect of different dosages of cholecalciferol on blood minerals, vitamin D metabolites, and milk yield over the first 5 test days after calving. Binary outcomes such as retained placenta and metritis were analyzed using a chi-squared test. Although the 12VitD treatment increased tCa concentrations on 1, 2, and 3 DIM compared with CON, administration of 6VitD increased tCa concentrations only on 1 DIM. Compared with CON cows and 6VitD cows, 12VitD cows had greater serum phosphate concentration during the first 10 DIM. Furthermore, 6VitD cows had greater serum phosphate concentrations compared with CON cows. On the contrary, 12VitD cows had lower serum Mg concentrations during the first 10 DIM compared with CON and 6VitD cows. Cholecalciferol was increased by the treatment and decreased quickly until 10 DIM. In respect to 25-OHD3, the 6VitD treatment resulted in a 4.1-fold increase in comparison to the CON group, while a 6.5-fold increase was observed in 12VitD animals. The vitamin D metabolite 24,25-(OH)2D3 increased linearly with 25-OHD3 serum levels, resulting in the highest concentrations in the 12VitD group. An increase of 1,25-(OH)2D3 until 3 DIM was observed in all cows. However, this rise was most pronounced in the CON group. The incidence of retained placenta was 1.9%, 11.5%, and 29.6%, and that of metritis was 11.5%, 15.4%, and 31.5% for CON, 6VitD, and 12VitD cows, respectively. Although none of the treated cows exerted clinical signs of hypocalcemia, one cow in CON incurred clinical hypocalcemia. Cows of the 12VitD group had a lower milk yield over the first 5 monthly test days compared with the control and 6VitD group (42.2 ± 0.5, 42.0, ± 0.5 and 40.7 ± 0.5 kg for control cows, 6VitD cows and 12VitD cows, respectively). Although no negative side effects were observed in 6VitD cows, we do not recommend the general application of 6 × 106 IU cholecalciferol before calving as positive effects on calcium homeostasis were marginal and restricted to the first DIM. The present findings confirm that the application of 12 × 106 IU cholecalciferol negatively affected milk production on this farm.

Comparison of two different anionic supplements and a low calcium diet fed to transition cows prepartum on DM intake, mineral homoeostasis and performance.

This study evaluates the effects of two rations with a positive dietary cation-anion difference (DCAD) and varying Ca content and two anionic diets on mineral homoeostasis and performance in early lactation cows. For this purpose, 48 pregnant dairy cows stratified for mature equivalent milk production, parity, body condition score (BCS), and BW were randomly assigned to four treatment groups and fed the following rations during the last 3 weeks of gestation: LC: low Ca (0.24% Ca, 1.14% K, DCAD: +86 mEq/kg DM), HC: high Ca (1.23% Ca, 1.17% K, DCAD: +95 mEq/kg DM), AS: Anionic salt (1.21% Ca, 1.21% K, DCAD: -112 mEq/kg DM, and SC: SoyChlor (1.28% Ca, 1.16% K, DCAD: -115 mEq/kg DM). After parturition, all animals were fed a common postpartum diet. Data were collected until 21 days in milk. Urinary pH was significantly decreased with the AS and SC treatment in comparison to the LC and HC groups. The highest prepartum DM intake (DMI) was found in the LC group, while DMI in AS cows was lowest. Postpartum, DMI was significantly greater in LC and SC cows than in animals fed the HC and AS rations. Prepartum serum concentrations of Ca, P, and Mg as well as postpartum serum concentrations of P and Mg did not differ, while postpartum Ca was lower in the HC group, especially 24 and 48 h after parturition. The greatest energy-corrected milk (ECM) yield was observed in cows fed the LC ration prepartum. Interestingly, milk protein production was lower in SC cows compared to LC, HC and AS. This study indicates that a ration containing a restricted Ca content fed prepartum is as suitable as a low DCAD diet to improve Ca balance without compromising DMI and performance.

Does bone mobilization interfere with energy metabolism in transition cows?

The onset of lactation represents a challenge for both mineral homeostasis and energy metabolism in high-performing dairy cows. It has been shown that subclinical and clinical hypocalcemia increases the risk of ketosis and recent studies suggest that bone-derived endocrine factors could play a role in intermediary metabolism. Therefore, we analyzed serum samples from calculated d -7, calculated d -3, d +1, d +3, and d +7 relative to calving from 15 multiparous cows for total Ca, the bone resorption marker CrossLaps, the bone formation marker intact osteocalcin, undercarboxylated osteocalcin (ucOC), insulin, glucose, nonesterified fatty acids, ß-hydroxybutyrate, and insulin-like growth factor 1. Serum concentrations of Ca on d -3 and d +1 were associated with parameters of energy metabolism on d +3 and d +7. As we found large variations for serum concentrations of ucOC already on d -7, we allocated the cows retrospectively to 3 groups: low ucOC, medium ucOC, and high ucOC. These groups differed not only in their ucOC dynamics, but also in insulin sensitivity estimated using the revised quantitative insulin sensitivity index (RQUICKI). High ucOC cows presented with the highest RQUICKI throughout the entire observation period. Our data further support the hypothesis that low serum Ca precedes disturbances of energy metabolism. Furthermore, from our preliminary results it can be assumed that the potential link between mineral homeostasis, bone turnover, and intermediary metabolism should be further investigated.

Randomized clinical trial to evaluate the effects of a prepartum cholecalciferol injection on postpartum serum calcium dynamics and health and performance in early-lactation multiparous dairy cows.

The objectives of the present study were (1) to evaluate the effect of prepartum cholecalciferol treatment on serum Ca concentration during the first 10 d after calving and (2) to evaluate the effect of treatment on subsequent health and performance. Multiparous Holstein cows (n = 377) from one dairy farm were fed a negative dietary cation-anion difference diet (-31 mEq/kg of DM) for the last 21 d of gestation. On d 275, the animals were randomly assigned to a control or a treatment group. Cows in the control group were left untreated, and cows in the treatment group received an injection of 12 × 106 IU of cholecalciferol intramuscularly on the day of enrollment. If treated cows did not deliver the calf within 6 d, they were reinjected with 10 × 106 IU of cholecalciferol. Blood samples were drawn on 1, 2, 3, 5, 7, and 10 days in milk (DIM) and analyzed for serum Ca, P, and Mg concentrations. In a subsample of cows (50 control cows, 35 cows treated once with cholecalciferol, and 15 cows treated twice) serum haptoglobin, nonesterified fatty acids, ß-hydroxybutyrate, and 25-hydroxycholecalciferol concentrations were analyzed on 1, 5, and 10 DIM. Binary data [retained placenta (RP), metritis] were analyzed using logistic regression models. Repeated measures ANOVA with first-order autoregressive covariance was performed to evaluate the treatment effect on milk yield over the first 10 test days after parturition, 25-hydroxycholecalciferol, serum Ca, P, Mg, ß-hydroxybutyrate, nonesterified fatty acids, and haptoglobin concentrations. Cox proportional hazards were used to model the time to event outcomes (time to pregnancy within 200 d, culling until 300 DIM). After enrollment of 31.4% of cows and a preliminary analysis, adverse reactions became apparent, and the study was stopped. Cows treated with cholecalciferol had a greater risk of incurring RP and metritis. The adjusted mean incidences were 2.0%, 7.7%, and 4.0% for RP, and 21.6%, 39.3%, and 33.3% for metritis for control cows, cows treated once, and cows treated twice with cholecalciferol, respectively. Compared with control cows, cows injected once with 12 × 106 IU of cholecalciferol produced less energy-corrected milk on the first (-3.76 kg) and second (-2.75 kg) test days, respectively. Cows injected twice with cholecalciferol (12 × 106 IU of cholecalciferol and 10 × 106 IU 1 wk later) had a reduced milk yield only at first test day (-3.80 kg). Treatment with cholecalciferol led to a significant increase in 25-hydroxycholecalciferol on d 1, 5, and 10 after calving. Serum Ca and P concentrations were significantly increased in cows treated with cholecalciferol, but serum Mg concentrations were significantly reduced. Haptoglobin concentrations were significantly increased on 5 DIM in cows injected once with 12 × 106 IU of cholecalciferol. Although we observed no effect of treatment on culling until 300 DIM, time to pregnancy was delayed by 34 d in cows injected once with 12 × 106 IU of cholecalciferol. In the present study, injection with 12 × 106 IU of cholecalciferol had detrimental effects on health and milk production despite the beneficial effects on Ca homeostasis.

Effects of restricted dietary phosphorus supply to dry cows on periparturient calcium status.

Restricted dietary P supply to transition dairy cows has recently been reported to beneficially affect the Ca balance of periparturient cows. The objective of the present study was to determine whether this effect on the Ca balance can be reproduced when limiting the P-restricted feeding to the last 4 wk of gestation. A total of 30 dairy cows in late pregnancy were randomly assigned to a dry cow diet with either low or adequate P content (0.16 and 0.30% P in DM, respectively) to be fed in the 4 wk before expected calving. After calving, all cows received the same lactating cow ration with adequate P content (0.46% P in DM). Blood was collected daily from 4 d antepartum until calving, at calving (d 0), 6 and 12 h after calving (d +0.25 and d +0.5, respectively) and on days +1, +2, +3, +4 and +7 relative to calving. Blood gas analyses were conducted to determine the concentration of ionized Ca in whole blood ([Ca2+]), and plasma was assayed for concentrations of inorganic phosphorus ([Pi]), total calcium, parathyroid hormone ([PTH]), 1,25-dihydroxyvitamin D ([1,25-(OH)2D3]), and CrossLaps ([CTX]), a biomarker for bone resorption (Immunodiagnostic Systems GmbH). Repeated-measures ANOVA was conducted to study treatment, time, and lactation number effects. The mean [Ca2+] in P-deprived cows remained above the threshold of 1.10 mmol/L throughout the study, and values were higher compared with cows on adequate P supply between d 0 and d +2 and on d +4. The [Ca2+] differed between treatments at the sampling times d 0, d +0.25, d +0.5, d +2, and d +4. Plasma [PTH] and [1,25-(OH)2D3] did not differ between treatments, but P-deprived cows had greater [CTX] than cows with adequate P supply at d +1, d +2, and d +7. These results indicate that restricted dietary P supply to during the last 4 wk of the dry period improves the Ca homeostasis of these cows in the first days of lactation, an effect that seems to be primarily driven by increased bone tissue mobilization.

Effects of diets differing in dietary cation-anion difference and calcium concentration on calcium homeostasis in neutered male sheep.

Feeding low dietary cation-anion difference (DCAD) diets is one strategy to prevent milk fever in cows. The mechanism of action, as well as whether the calcium (Ca) supply of such diets combined with this feeding regimen should meet the requirements, is still unclear. Small ruminants are commonly used as models for cows. The goal of the present study was to demonstrate basic effects of DCAD against a background of different Ca supplies in a sheep model. Twenty-three castrated male East Friesian milk sheep, aged 11 to 12 mo, were randomly assigned to 4 different feeding groups. The ration of each group was either high (highDCAD) or low in DCAD (lowDCAD) combined with adequate (nCa) or restricted Ca supply (lowCa). At baseline, serum and urine were collected from all sheep and a peripheral quantitative computed tomography of the left metatarsus was performed. After a 14-d adaptation period to the different diets, the experiment started (d 0). Urine, feces, and serum were collected on d 0, 4, 7, 14, and 22, and peripheral quantitative computed tomography was performed on d 0 and 22. On d 22, the sheep were killed and sampled for functional studies. LowDCAD was significantly associated with lower urine pH, higher urinary Ca excretion, higher ionized Ca in blood, and higher serum Ca concentrations. Blood pH and bone parameters did not differ significantly between groups. It is unclear from which compartment the high amounts of Ca excreted with urine in the lowDCAD groups originated. Interestingly, lowDCAD resulted in higher renal mRNA abundance of parathyroid hormone receptor but unaffected mRNA abundance of Ca transporters. As neither renal abundance of these transporters nor Ca excretion were influenced by dietary Ca supply, our results support the hypothesis that increased urinary Ca observed with low DCAD diets represents a loss rather than an excretion of surplus Ca.

Review: Regulation of gastrointestinal and renal transport of calcium and phosphorus in ruminants.

In comparison to monogastric animals, ruminants show some peculiarities in respect to the regulation of mineral homeostasis, which can be regarded as a concerted interplay between gastrointestinal absorption, renal excretion and bone mobilisation to maintain physiological Ca and phosphate (Pi) concentrations in serum. Intestinal absorption of Ca or Pi is mediated by two general mechanisms: paracellular, passive transport dominates when luminal Ca or Pi concentrations are high and transcellular. The contribution of active transport becomes more important when dietary Ca or Pi supply is restricted or the demand increased. Both pathways are modulated directly by dietary interventions, influenced by age and regulated by endocrine factors such as 1,25-dihydroxyvitamin D3. Similar transport processes are observed in the kidney. After filtration, Ca and Pi are resorbed along the nephron. However, as urinary Ca and Pi excretion is very low in ruminants, the regulation of these renal pathways differs from that described for monogastric species, too. Furthermore, salivary secretion, as part of endogenous Pi recycling, and bone mobilisation participate in the maintenance of Ca and Pi homeostasis in ruminants. Saliva contains large amounts of Pi for buffering rumen pH and to ensure optimal conditions for the rumen microbiome. The skeleton is a major reservoir of Ca and Pi to compensate for discrepancies between demand and uptake. But alterations of the regulation of mineral homeostasis induced by other dietary factors such as a low protein diet were observed in growing ruminants. In addition, metabolic changes, for example, at the onset of lactation have pronounced effects on gastrointestinal mineral transport processes in some ruminant species. As disturbances of mineral homeostasis do not only increase the risk of the animals to develop other diseases, but are also associated with protein and energy metabolism, further research is needed to improve our knowledge of its complex regulation.

Short communication: Effect of dietary phosphorus deprivation in late gestation and early lactation on the calcium homeostasis of periparturient dairy cows.

Environmental concerns with P of animal origin polluting surface waters are leading to legal incentives aimed at reducing the dietary P content of dairy cow rations to the lowest possible level that does not negatively affect health and productivity. The objective of the present study was to determine the effect of feeding rations with low dietary P content in late gestation on the Ca homeostasis of the periparturient dairy cow. Eighteen multiparous dairy cows were either fed a P-deficient (0.15% P in dry matter antepartum and 0.20% P in dry matter postpartum) but otherwise balanced ration or a control ration with adequate P content (0.28% P in dry matter antepartum and 0.44% P in dry matter postpartum) during the last 4 wk of gestation until d 10 postpartum. Blood was obtained before initiation of P-deprivation (baseline) and -10, -2, +1, +3, and +10 d relative to parturition to be analyzed for plasma concentrations of Ca [Ca] and inorganic phosphate [Pi]. In addition, plasma concentrations of parathyroid hormone [PTH], the bone resorption marker CrossLaps [CTX], 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D were determined in a subset of samples. Feeding a P-deficient diet for 4 wk antepartum positively affected the Ca homeostasis of periparturient cows. Clinical hypocalcemia occurred in 3/9 control and 0/9 P-deprived cows. [Calcium], [PTH], and plasma concentrations of vitamin D metabolites did not differ between groups until parturition, whereas [Pi] was decreased and [CTX] significantly increased in P-deprived animals. At parturition [PTH] was significantly greater in control cows compared with P-depleted cows. The P-deprived cows had significantly higher [Ca] than control cows on d +1 (2.46 ± 0.11 vs. 2.27 ± 0.41 mmol/L) and +2 (2.61 ± 0.13 vs. 2.35 ± 0.25 mmol/L). Plasma [CTX] was significantly higher in P-deprived than in control cows on d +2. Bone resorption and the typical increase in 1,25-dihydroxyvitamin D in periparturient P-deprived cows seemed to occur despite the smaller rise of [PTH], suggesting either greatly increased sensitivity to PTH or bone mobilization independent of PTH. Future studies must explore potentially negative effects of P-deprivation antepartum on health and productivity of the dairy cow in the following lactation.

Modulation of renal calcium and phosphate transporting proteins by dietary nitrogen and/or calcium in young goats.

In young goats, a reduction in dietary nitrogen (N) had an impact on mineral homeostasis although ruminants are able to recycle N effectively due to rumino-hepatic circulation. A solitary calcium (Ca) reduction stimulated calcitriol synthesis and Ca concentrations remained unchanged, whereas a dietary N reduction led to a decrease in calcitriol, which could not be prevented by a simultaneous reduction of N and Ca. In a previous study, it was shown that a reduced dietary N intake caused a decrease in intestinal Ca absorption due to a reduction of intestinal Ca transporting proteins. As no data on the potential role of the kidneys are available, it was the aim of the present study to evaluate whether an N- and/or Ca-reduced diet had an impact on renal Ca and phosphate (Pi) transporting protein expression in young goats. The animals were divided into 4 feeding groups, each receiving an adequate N and Ca supply, a reduced N supply, a reduced Ca supply, or a combined N and Ca reduction for 6 to 9 wk. The protein expression of the renal Ca channel transient receptor potential cation channel subfamily V member 5 (TRPV5) was diminished in N-reduced fed goats (P = 0.03), whereas in Ca restricted animals, the expression remained unaltered. The mRNA and protein expression of the Ca-binding protein calbindin-D28K (CaBPD28K) and the sodium-Ca exchanger 1 (NCX1) were significantly decreased due to the N-reduced feeding (mRNA, P = 0.003; P < 0.0001; protein, P = 0.002; P = 0.02), whereas dietary Ca reduction increased the CaBPD28K and NCX1 mRNA expression (P = 0.05; P = 0.01). The mRNA and protein expression of the parathyroid hormone receptor (PTHR) decreased due to the N-reduced feeding (P = 0.02; P = 0.03). These results confirm that a reduced dietary N intake led to decreased TRPV5, CaBPD28K, PTHR, and NCX1 expression levels, contributing to low levels of calcitriol and plasma Ca. In contrast to this, sodium-phosphate cotransporter type IIa expression and plasma Pi concentration were increased during dietary N reduction, thus indicating that Pi homeostasis is modulated in a calcitriol-independent manner. In conclusion, the modulation of Ca transporting proteins expression in the kidney is not able to prevent changes in mineral homeostasis in young goats receiving an N-reduced diet.

Dietary nitrogen and calcium modulate CYP27B1 expression in young goats.

In livestock, feeding a reduced nitrogen (N) diet is favored for economic and ecological reasons. Ruminants cope more easily with a reduced N diet than monogastric species. However, changes in mineral homeostasis such as a reduction in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) concentrations, calcium (Ca), and IGF1 levels were observed in goats kept on a reduced N diet. The decrease in 1,25-(OH)2D3 occurred even during a simultaneous reduction in dietary N and Ca, whereas a solitary Ca reduction stimulated 1,25-(OH)2D3 synthesis. The aim of the present study was to examine the effects of N- and/or Ca-reduced diets on the expression of 24-hydroxylase (CYP24A1), 1-alpha-hydroxylase (CYP27B1), vitamin D receptor (VDR), retinoid X receptor alpha (RXRα), IGF1 receptor (IGF1R), Klotho, and fibroblast growth factor receptor 1c (FGFR1c) in kidneys of young goats. Four groups were kept on a control diet, an N-reduced diet, a Ca-reduced diet or an N- and a Ca-reduced diet. Renal expression of CYP24A1 was not affected, whereas CYP27B1 expression was significantly diminished in the N-reduced diet fed goats (P < 0.05) and significantly elevated with the Ca reduction (P < 0.001). The VDR expression was not modified, whereas RXRα (P < 0.05) and Klotho expression (P < 0.001) were stimulated during Ca reduction. The IGF1R (P < 0.05) and FGFR1c (P < 0.05) expression were enhanced with the N reduction. From these data, it can be concluded that the downregulation of renal CYP27B1 expression observed with dietary N reduction is probably mediated by a complex interaction between the somatotropic axis and the Klotho/FGF signaling pathway in young goats.

Vitamin D status in growing dairy goats and sheep: Influence of ultraviolet B radiation on bone metabolism and calcium homeostasis.

The aim of this study was to investigate how controlled UVB irradiation in combination with reduced nutritional vitamin D (vitD) supply affects vitD status and Ca metabolism of growing goats and sheep. The hypothesis was that, like dairy cows, goats and sheep are able to compensate for the missing nutritional supply of vitD through endogenous production in the skin, with the consequence of a high vitD status and a balanced Ca homeostasis. Sixteen lambs and 14 goat kids aged 3 and a half months were housed in an UVB free environment and fed hay and a vitD-free concentrate over a period of 13 wk. One group of each species was exposed to UVB lamps daily during individual feeding; the other groups served as controls. Serum, urine, and feces samples were taken at the start and at a monthly interval. Serum was analyzed for vitD metabolites, bone markers, growth hormone, insulin-like growth factor I, Ca, and P. Apparent digestibility and urinary excretion of Ca and P were determined. The left metatarsus was analyzed by peripheral quantitative computer tomography for bone mineral density before starting and at the end of the trial. In wk 13, all animals were slaughtered and samples of skin, rumen, duodenum, kidney, and bone (metatarsus) were collected. Content of sterols of vitD synthesis in the skin, Ca flux rates in rumen and duodenum, expression of vitD receptor in duodenum and kidney, renal and intestinal gene expression of Ca transport proteins, and renal enzymes related to vitD metabolism were determined. The UVB exposure led to lower 7-dehydrocholesterol content in the skin and a better vitD status (higher serum 25-hydroxyvitamin D), but no signs of vitD deficiency were seen in the control groups and no effect of irradiation was detected in the analyzed parameters of Ca homeostasis. Differences between the 2 species were detected: lambs had a higher increase of bone mineral density, lower values of bone markers, growth hormone, and insulin-like growth factor I in serum and higher tachysterol and lower lumisterol content in skin compared with goat kids. The results indicated that growing lambs and goat kids are able to compensate for a vitD-reduced diet by cutaneous vitD synthesis when exposed to UVB irradiation and therefore to keep a high vitD status. In contrast, when a reduced vitD diet is combined with missing UVB exposure, the vitD status drops, but the experimental time was probably too short to induce a vitD deficiency or an effect on Ca homeostasis.

Vitamin D metabolism in growing pigs: influence of UVB irradiation and dietary vitamin D supply on calcium homeostasis, its regulation and bone metabolism.

The aim of this study was to prove whether pigs are able to synthesize vitamin D (vitD) in the skin and to investigate the influence of ultraviolet irradiation (UVB) on vitD status and calcium (Ca) homeostasis of growing pigs. Thirty-two 11-week-old pigs were kept without access to sunlight and divided into four groups receiving the following treatment in a 2 × 2 factorial design: (i) UVB irradiation or not and (ii) vitD in feed or not. Blood, urine and faeces were sampled every third week. In serum, vitD metabolites, Ca, phosphorus (P), magnesium (Mg) and bone markers were analysed. Digestibility of Ca, P and Mg as well as urinary excretion of these minerals was analysed. After 14 weeks, the animals were slaughtered, and samples of skin, intestines, kidneys and bones (metatarsus) were taken for further analyses: sterols of vitD synthesis in the skin, Ca flux rates in the intestines, expression of genes involved in Ca transport in the intestines and kidneys, bone mineral density (BMD) with the aid of peripheral quantitative computer tomography and bone mineral content by ashing the metatarsus. Irradiated animals showed higher levels of 7-dehydrocholesterol and tachysterol in the skin, higher levels of 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol in the serum and higher Ca net flux rates were determined in Ussing chambers. In contrast, the expression of genes involved in Ca transport in the intestines and kidneys was not altered. Similarly, the digestibility of Ca and P as well as the urinary excretion was not affected. With respect to the metatarsus, no differences in mineral contents and BMD were found between groups. At the end of the study, some subclinical signs of beginning vitD 'insufficiency' were observed in the group without access to vitD (represented by higher expression of 1α-hydroxylase in the kidney and increased parathyroid hormone in serum).

Segmental diversity of phosphate transport along the intestinal axis in horses.

For horses, distinct differences in intestinal phosphate transport have been postulated to account for the unique features of hind gut fermentation compared to other monogastric animals and ruminants. So far published data on mechanisms and underlying transport proteins involved in intestinal phosphate transport in the horse are still missing. Therefore we investigated intestinal phosphate transport in horses at both functional and molecular levels. Segmental diversity of intestinal phosphate transport along the intestinal axis was documented using the Ussing chamber technique. A transcellular phosphate secretion in the jejunum was confirmed. Furthermore, 2 sodium-dependent phosphate cotransporters, NaPiIIb and PiT1, were first detected in the equine intestine at mRNA level with PiT1 being expressed in both the small and large intestine, and NaPiIIb being solely expressed in the large intestine. In the colon, unidirectional net flux rates of phosphate were significantly greater compared to flux rates in other segments ( < 0.005) suggesting the colon as a major site for phosphate absorption in horses. Phosphate transport in the colon was mainly transcellular and mediated by a sodium-gradient as documented by Ussing chamber experiments and uptake of phosphate into colonic brush border membrane vesicles. In summary, the present study demonstrated mechanisms and transporters of intestinal phosphate transport in equine intestinal tissues with distinct differences between intestinal segments providing a new basis for a better understanding of intestinal phosphate transport in horses.

Trans- and paracellular calcium transport along the small and large intestine in horses.

Intestinal calcium absorption plays a key role in the maintenance of calcium homeostasis and may either occur by paracellular or transcellular mechanisms. The horse has some unique peculiarities in calcium homeostasis compared to other species including a high absorptive capacity for calcium in the intestine, high plasma calcium concentrations, high renal excretion, and low plasma concentrations of vitamin D metabolites. So far, knowledge about the underlying mechanisms and the regulation of intestinal calcium absorption is still limited concerning this species. Several studies have documented that intestinal calcium transport in horses is not as dependent on vitamin D as in other species. However, published data on other potential regulatory mechanisms are still lacking. In the present study, paracellular and transcellular transport mechanisms for intestinal calcium transport along the intestinal axis were identified in horses using the Ussing chamber technique. Furthermore, the expression of respective transport proteins including transient receptor potential vanilloid member 6, calbindin-D9k and calcium ATPase type 1 in line with the determined calcium flux rates was documented. In respect to regulation of transepithelial calcium transport, novel regulatory proteins for maintaining calcium homeostasis such as B-box and SPRY-domain containing protein and calmodulin were investigated for the first time in equine intestinal tissues in this study. This provides the basis for a new approach for a better understanding of equine calcium homeostasis regulation.

Zeolite A effect on calcium homeostasis in growing goats.

The purpose of this study was to investigate the influence of 2 different concentrations of zeolite A on calcium homeostasis. Seventeen growing goats were divided into 3 groups. Whereas the control group (5 animals) received no supplementation, 2 treatment groups were supplemented with zeolite A at either 1.2 (6 animals) or 1.6 g/kg BW (6 animals), respectively. Blood and urine samples were continually drawn and bone mineral density was measured weekly by peripheral quantitative computed tomography. After 3 wks, the animals were slaughtered and samples were taken from the rumen, duodenum, and kidneys. Plasma concentrations of phosphate ( < 0.001), magnesium ( < 0.001), and 1.25-dihydroxycholecalciferol ( < 0.01) as well as renal excretion of phosphate ( < 0.05) were significantly lower in the treatment groups compared with the control group. Although bone resorption was increased in both treatment groups ( < 0.05), no alterations in bone structure were detected. Determination of gastrointestinal absorption of calcium by Ussing chamber technique and quantification of RNA and protein expression of genes known to be involved in active calcium absorption did not reveal any stimulating effect of zeolite. Plasma calcium concentrations were not altered, probably because of the sufficient dietary calcium supply. However due to the effects of zeolite on 1,25 dihydroxycholecalciferol, bone metabolism and serum concentrations of phosphate and magenesium shown in the present study, potential negative long-termin effects on the animals should be considered whenever rations with zeolite are designed.

Stimulating effects of a diet negative in dietary cation-anion difference on calcium absorption from the rumen in sheep.

The concept of feeding anionic salts in late gestation is widely used to prevent milk fever in dairy cows. While the effects of these diets on renal Ca excretion and tissue responsiveness towards parathyroid hormone have clearly been demonstrated, data on a potential impact on gastrointestinal Ca absorption are conflicting. Therefore, the aim of this study was to investigate the influence of feeding a diet negative in dietary cation-anion difference (DCAD) on ruminal mineral concentrations, fermentation products, electrophysiological properties of rumen epithelia and Ca flux rates. For this purpose, sheep were kept for 3 weeks on diets that were either positive or negative in DCAD. The induction of a compensated hyperchloremic metabolic acidosis could be demonstrated by increased plasma Cl and enhanced concentrations of ionised Ca, while plasma concentrations of HCO3- and base excess were decreased with the low DCAD diet. Neither transmural potential differences nor fermentation products were affected, but ruminal concentrations of Cl and Mg as well as the relation of ionised to total Ca were increased. Ussing chamber experiments revealed alterations of electrophysiological parameters and an increase in the electroneutral component of Ca flux rates from the mucosal to the serosal side of rumen epithelium. As plasma calcitriol concentrations were not affected, it can be concluded that the administration of anionic salts results in a vitamin D-independent stimulation of ruminal Ca transport.

Influence of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on expression of P-glycoprotein and cytochrome P450 3A in sheep.

In order to improve calcium and phosphorus balance, beef cattle and dairy cows can be supplemented with vitamin D. However, different vitamin D metabolites have been shown to increase expression of P-glycoprotein (P-gp, MDR1, ABCB1) and cytochrome P450 3A (CYP3A) in rodents as well as in cell culture systems. As such interferences might have an impact on pharmacokinetics of some drugs widely-used in veterinary medicine, we investigated the expression of P-gp, CYP3A, vitamin D receptor (VDR), pregnane X receptor (PXR) and retinoid X receptor α (RXRα) in sheep either treated orally with 6µg/kg body weight (BW) 25-hydroxyvitamin D3 (OHD3) for ten days before sacrifice or 12h after intravenous injection of 0.5µg/kg BW 1,25-dihydroxyvitamin D3 (1,25- (OH)2D3). Down-regulation of ruminal, jejunal and hepatic, but not renal P-gp could be found with 25-OHD3 supplementation. Interestingly, this effect on P-gp was not observed in tissues from 1,25-(OH)2D3-treated sheep. In contrast, 1,25-(OH)2D3 induced a significant up-regulation of renal and jejunal CYP3A expression, while 25-OHD3 had no impact. Renal expression of VDR and PXR was also increased by treatment with 1,25-(OH)2D3, while jejunal PXR expression was only stimulated in sheep supplemented with 25-OHD3. Either treatments increased renal, but not ruminal, jejunal or hepatic expression of RXRα. These results demonstrate that the impact of large doses of vitamin D metabolites on different target organs and potential interactions with other medications should be further investigated in vitro and in vivo to understand the effects of vitamin D metabolites on metabolism and excretion pathways in livestock.

Renal mechanisms of calcium homeostasis in sheep and goats.

In small ruminants, the renal excretion of calcium (Ca) and phosphate (Pi) is not modulated in response to dietary Ca restriction. Although this lack of adaptation was observed in both sheep and goats, differences in renal function between these species cannot be excluded. Recent studies demonstrated that compared with sheep, goats have a greater ability to compensate for challenges to Ca homeostasis, probably due to a more pronounced increase in calcitriol production. Therefore, the aim of the present study was to examine the effect of 1) dietary Ca restriction, 2) administration of calcitriol, and 3) lactation on Ca and Pi transport mechanisms and receptors as well as enzymes involved in vitamin D metabolism in renal tissues of sheep and goats. Whereas RNA expression of renal transient receptor potential vanilloid channel type 5 was unaffected by changes in dietary Ca content, a significant stimulation was observed with administration of calcitriol in both sheep (P < 0.001) and goats (P < 0.01). Calbindin-D28K was downregulated during dietary Ca restriction in goats (P < 0.05). Expression of the sodium/Ca exchanger type 1 was decreased by low Ca intake in sheep (P < 0.05) and upregulated by calcitriol treatment in goats (P < 0.05). A significant reduction in RNA expression of the cytosolic and the basolateral Ca transporting proteins was also demonstrated for lactating goats in comparison to dried-off animals. Species differences were found for vitamin D receptor expression, which was stimulated by calcitriol treatment in sheep (P < 0.01) but not in goats. As expected, expression of 1α-hydroxylase was upregulated by dietary Ca restriction (P < 0.001; P < 0.05) and inhibited by exogenous calcitriol (P < 001; P < 0.05) in both sheep and goats. However, whereas 24-hydroxylase expression was stimulated to the same extent by calcitriol treatment in sheep, irrespective of the diet (P < 0.001), a modulatory effect of dietary Ca supply on 24-hydroxylase induction was observed in goats (P < 0.05). Taken together, our results confirm observations that modulation of renal Ca excretion does not contribute to maintenance of Ca homeostasis in these ruminants during restricted dietary supply, unlike responses in monogastric animals. The interesting species differences related to vitamin D metabolism might explain the greater capacity of goats to compensate for challenges of Ca homeostasis and should be further investigated.

Influence of UVB exposure on the vitamin D status and calcium homoeostasis of growing sheep and goats.

The purpose of this study was to investigate the influence of exposure to ultraviolet radiation (UVB) on vitamin D status, intestinal calcium absorption and bone metabolism in growing sheep and goats. The hypothesis was that growing sheep and goats are able to synthesise vitamin D within their skin as a result of UVB exposure and that respective consequences for their vitamin D blood levels and the associated parameters can be shown. Fourteen 18-week-old lambs and goat kids were kept in an UVB-free environment and randomly assigned to two groups. One group was daily exposed to UVB (300 watt) for 12 weeks, and the other served as a control group. Except for the exposure to UVB, all animals were kept under the same conditions and fed according to their requirements. Before the start of the experiment and every second week, blood samples were taken. Also the left metatarsus of each animal was analysed by quantitative computer tomography to test for bone mineral status before the start, in week 7 and at the end of the experiment. After 12 weeks, the animals were slaughtered and samples were taken from skin, gastrointestinal tract and kidney for further analyses. In this study, exposure to UVB led to increased serum 1,25-dihydroxyvitamin D (1,25VitD) levels in goat kids, whereas in lambs, serum 25-hydroxyvitamin D (25VitD) levels were increased. In both species UVB-exposed animals showed lower 7-dehydrocholesterol (7DHC) values in skin than their respective control groups. These results indicate that growing goat kids and lambs are able to synthesise vitamin D in the skin when being exposed to UVB.

Epidemiology of cyprinid herpesvirus-3 infection in latently infected carp from aquaculture.

Cyprinid herpesvirus-3 (CyHV-3, koi herpesvirus, KHV) is the causative agent of an economically important disease in carp. The mode of transmission of this virus, especially how the infectious agent is introduced into ponds de novo, is not known in detail. The aim of this study was to investigate the shedding of CyHV-3 from fish with latent infections, under aquaculture conditions. Ponds in Saxony, Germany, with latently infected carp were examined at different times during the production cycle to investigate the influence of fish farming procedures on virus activation and shedding. Carp and water samples were investigated by quantitative real-time PCR. Some of the latently infected carp shed CyHV-3. Virus shedding was induced mainly when the ponds were drained and the carp either harvested or moved to different ponds, and was independent of the water temperature. This indicated that during these times there was a risk that effluent water from the ponds could disseminate the infectious agent. During summer, on-growing carp are infected with low numbers of CyHV-3. These findings are important for disease management strategies in carp aquaculture and for the design of testing protocols for the detection of latent infection in carp populations.