Solanum glaucophyllum is a toxic plant with calcinogenic effect that causes enzootic calcinosis (EC) characterized by soft tissue metastatic mineralization mainly in cattle and rarely sheep, buffaloes, pigs, horses, and goats. We describe an outbreak of EC in a herd of 64 goats due to S. glaucophyllum consumption. Thirty-four goats were affected exhibiting hirsutism, stiffening, kyphosis and emaciation. Twelve goats died. Grossly, tissue mineralization was observed in the aorta and carotid arteries, lungs, and heart. Lesions were characterized by multiple rough white plaques, and hardened tissues with loss of elasticity. Microscopically, multisystemic mineralization was observed in aorta and carotid arteries, heart, lung, abomasum, intestine, spleen, lymph nodes, kidney, spleen, and meninges, characterized by extensive granular basophilic deposits of tunica media and/or intima of blood vessels; confirmed as calcium salt deposits with Von Kossa stain. We conclude that ingestion of S. glaucophyllum can cause EC in goats. Though EC is rare in goats under some conditions such as heavy drought and abundant S. glaucophyllum exposure disease can develop.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 μL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Periparturient hypocalcaemia is a widespread metabolic disorder in dairy cows. Clinical and subclinical cases occur primarily in multiparous (Multi) cows, but subclinical cases have also been reported in primiparous (Primi) cows. A preventive strategy was investigated by administering the physiologically active vitamin D3 metabolite, 1,25-dihydroxyvitamin D3 (1,25-dihydroxycholecalciferol, 1,25(OH)2D3) as a rumen bolus. The bolus contained tablets of 1,25(OH)2D3 glycoside extract from Solanum glaucophyllum (SGE), releasing SGE over several days. The aim was to study the effect of a bolus containing 0 (C) or 500 µg (SGE) of 1,25(OH)2D3 on 1,25(OH)2D3 and mineral status in periparturient cows up to three weeks into lactation and on colostrum, milk and calves\' blood mineral contents. The bolus was administered three to four days prior to expected calving to Primi and Multi cows fed a herbage-based diet (dietary cation-anion difference of +522 mEq/kg DM). One C or SGE bolus was applied to 12 Primi and 12 Multi cows. Blood was regularly sampled (and selected a posteriori for antepartum samples) in regard to the actual calving day (d0), immediately prior to bolus application and at day -2, 0.5, 1, 1.5, 2, 4, 8, 11, 15, 18 and 22. Additional samples included urine (at bolus application, d0.5 and d2), colostrum, milk samples (weekly) and calves\' blood (d2). Blood serum 1,25(OH)2D3 increased between d0.5 and d2 in Primi-SGE, but remained unchanged in Primi-C, as did parathyroid hormone (PTH) and Ca in all Primi. Urinary Ca of Primi-SGE was increased on d2, indicating regulation of Ca excess. Three Multi-C cows with confirmed clinical hypocalcaemia needed treatment and thus were excluded from the dataset and replaced. Blood serum 1,25(OH)2D3 and PTH increased while Ca dropped by 40% between d0.5 and d2 in Multi-C, whereas 1,25(OH)2D3, Ca and PTH remained unchanged in Multi-SGE. Blood serum carboxyterminal telopeptide of type I collagen was higher in Primi than in Multi and increased with time, except in Primi-C. Mineral contents in colostrum, milk and blood serum of calves were not influenced to a relevant degree. In conclusion, Primi-C did not, in contrast to Multi-C, develop subclinical hypocalcaemia (<2.0 mmol Ca/l). Prevention of hypocalcaemia with one SGE bolus applied three to four days prior to expected calving was successful in maintaining blood Ca within normal range in Multi over the critical first two days and up to the first three weeks of lactation, without any observed detrimental effects on cows or calves.

1α,25(OH)2VD3 is the most active form of VD3 in animals. It plays an important role in regulating mineral metabolism but also in reproduction. Testes are the main reproductive organs of male mammals. Our research aims to reveal the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets. 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 μg/kg of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides. Sixty days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labelling technique was used to analyse the protein profile of the testes in each group. Western blotting was applied to verify the target of differentially abundant proteins (DAPs). The analysis of morphology and histology of testes showed that a certain concentration of 1α,25(OH)2VD3-glycosides had a positive and significant effect on testicular development. And the results of proteomics analysis showed that of the identified 132,715 peptides, 122,755 were unique peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs focused on proteins closely related to the regulation of testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results indicated that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets\' testes.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 μl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 μl of extract/L containing 0.4 μg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 μg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 μg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ± 3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ± 6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 μl of 1,25(OH)2D3. The ability of 100 μL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 μg/L), 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 μg/L) and 50 nM (20 μg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

Solanum glaucophyllum, a toxic plant known for its calcinogenic effects, causes enzootic calcinosis in ruminant and monogastric animals. We describe an outbreak of enzootic calcinosis that occurred in a herd of 110 horses grazing pastureland heavily contaminated with S. glaucophyllum in Buenos Aires province, Argentina. Ten horses developed clinical signs, and 6 horses died. Clinical signs included abnormal gait (stiff-legged action, short strides), stiffness, thoracolumbar kyphosis, reluctance to move, wide stance, chronic weight loss, weakness, recumbency, and difficulty standing. Autopsy of 2 horses revealed severe mineralization of the aorta, pulmonary arteries, heart, and lungs, consistent with enzootic calcinosis. Although horses usually have very selective grazing behavior, under food restriction conditions, they can ingest the toxic plants and can develop the disease. Enzootic calcinosis should be considered as a differential diagnosis in horses grazing S. glaucophyllum-invaded pasturelands with compatible clinical signs and lesions.

Milk fever (MF) is a metabolic disease in dairy cows around parturition. The clinical lead sign is muscular paresis leading in severe cases to paralysis of the affected animal. Multiparturient animals of high performing dairy breeds are most likely to be affected and have a high probability of recurrence. An acute drop in blood calcium levels causes the disease when the demand for calcium at the onset of lactation exceeds the ability to replete blood calcium levels through mobilization from bone and intestinal uptake. With the understanding of the underlying mechanism, calcium supply management and vitamin D supplementation became prime candidates for MF prevention and therapy. Several strategies have been developed for MF prevention. Application of the active form of Vitamin D, 1,25(OH)2D3, was found to prevent MF effectively. In order to prevent a delayed hypocalcemia, which was occasionally seen after stopping the treatment with 1,25(OH)2D3, a new approach was chosen by applying Solanum glaucophyllum extract (SGE), which contains 1,25(OH)2D3-glycosides, as instant-release (irSGE) in combination with slow-release (srSGE) tablets. In a first study, non-lactating cows were treated with a single bolus of either synthetic 1,25(OH)2D3, irSGE, or srSGE and the results were compared to a control group without treatment. Blood serum levels of 1,25(OH)2D3 (1,25D), calcium (Ca), phosphate (P) and magnesium (Mg) were followed for 11days and the area under the curve (AUC) was calculated. Calcium and phosphate excretion in urine were determined during 15days. While serum concentration of 1,25(OH)2D3 was back to pre-treatment level in the irSGE, srSGE and 1,25(OH)2D3 treated group within 3days, calcium and phosphate levels remained elevated for up to 9days. AUC of serum 1,25(OH)2D3 was 2.89 (1,25D), 3.13 (irSGE) and 4.21 (srSGE) times higher than control. Serum calcium levels were 1.07* (for 1.25D); 1.08* (for irSGE) and 1.12* (for srSGE) times higher than control. Serum phosphate levels were 1.20* (for 1,25D); 1.30* (for irSGE) and 1.41* (for srSGE) times higher than control, with * p<0.05. In a second field study calving cows treated with one bolus containing ir- and sr- tablets of SGE were compared to an untreated control group and to a group treated with 4 boli of commercial calcium salts. As a result, calcium serum levels increased (+19% compared to baseline) around calving after treatment with the single bolus of SGE. The single bolus of SGE lead also to an increase of serum phosphate (+31% compared to baseline). These calcium and phosphate increases were statistically significant (p<0.001) 0-24h after calving compared to the control group and to the group treated with calcium salts. The sample size of the study was too small to draw a conclusion on the effect on MF prevention. In conclusion, application of a single bolus of a SGE extract lead to an increase of serum calcium and phosphate for up to 9days and may thus have the potential to prevent a hypocalcemia and -phosphatemia, an important cause for clinical milk fever.

Solanum glaucophyllum leaves contain high levels of glycosidically bound 1,25 dihydroxyvitamin D3, the most important vitamin D metabolite. The tolerance to this source was evaluated during six weeks with fifty weaned pigs fed increasing levels (0, 2.5, 5, 10 and 20μg 1,25(OH)2D3/kg diet). The diet contained, per kg, 9.7g Ca, 3.5g digestible P and 2000IU cholecalciferol. Ten additional pigs were fed a diet containing 1000IU cholecalciferol/kg, without 1,25(OH)2D3. Weekly plasma and final kidney, bone and urinary mineral contents, bone density and breaking strength served as indicators for possible adverse effects of the supplement. All animals grew well and remained clinically healthy. The measured parameters remained unchanged when 1000 replaced 2000IU cholecalciferol/kg and when 1,25(OH)2D3 was fed up to 10μg/kg. Twenty μg 1,25(OH)2D3 increased plasma Ca and decreased plasma P from the 2nd and the 4th experimental week onwards, respectively. Twenty μg 1,25(OH)2D3 increased final plasma Ca and 1,25(OH)2D3 and reduced final plasma P by respectively 19, 56 and 13%. Twenty μg 1,25(OH)2D3 also increased kidney Ca and urinary Ca by 43 and 69%, respectively, reduced bone breaking strength by 12% and tended to decrease bone ash by 3%. To conclude, 2000IU D3 was not beneficial compared to 1000IU cholecalciferol; up to 10μg 1,25(OH)2D3 per kg diet did not lead to observed adverse effects; 20μg 1,25(OH)2D3 altered the homeostatic regulation of Ca and P thus, may lead to first signs of possible adverse effects, such as soft tissue calcification.