Method and composition for supporting normal blood calcium concentrations in mammals

Abstract

A composition for oral administration to a periparturient mammal at risk of developing hypocalcemia within 0-6 hours after parturition; the composition comprising a form of calcium rapidly absorbable by the periparturient mammal using passive paracellular transport across the intestinal epithelium and a 1-alpha hydroxylated vitamin D compound in an amount sufficient to stimulate active transport of calcium across the intestinal epithelium, the calcium and the 1-alpha hydroxylated vitamin D being administered concurrently to support maintenance of normal blood calcium concentrations in the periparturient mammal.

Claims

1. A composition for oral administration to a periparturient mammal at risk of developing hypocalcemia within 0-6 hours after parturition; the composition comprising a form of calcium rapidly absorbable by the periparturient mammal using passive paracellular transport across the intestinal epithelium and a 1-alpha hydroxylated vitamin D compound in an amount sufficient to stimulate active transport of calcium across the intestinal epithelium , the calcium and the 1-alpha hydroxylated vitamin D to be administered concurrently in the form of a drench or a bolus, tablet or pellet with a density of at least 1.2 kg/L (g/ml) and released from the drench or the bolus, tablet or pellet over a time period less than 2 hours to support maintenance of normal blood calcium concentrations in the periparturient mammal.

2. The composition of claim 1 wherein the form of calcium administered comprises readily water soluble calcium salts comprising calcium chloride, calcium sulfate, calcium propionate, calcium acetate, calcium lactate, or calcium formate, alone or in combination.

3. The composition of claim 1 wherein the form of calcium is calcium chloride and is incorporated in an amount sufficient to induce a compensated metabolic acidosis within 8 hours of administration to support normal sensitivity of tissues to parathyroid hormone.

4. The composition of claim 1 wherein the 1-alpha hydroxylated vitamin D compound comprises 1,25-dihydroxyvitamin D or 1-alpha hydroxyvitamin D or analogs thereof in an amount sufficient to stimulate transcellular rumen or intestinal absorption of calcium.

5. The composition of claim 1 wherein the 1-alpha hydroxylated vitamin D compound is a glycoside of 1,25-dihydroxyvitamin D as found in calcinogenic plants or extracts prepared from the calcinogenic plants.

6. The composition of claim 1 wherein the 1-alpha hydroxylated vitamin D compound is not incorporated in an amount so great that the 1-alpha hydroxylated vitamin D compound causes significant inhibition of the renal 25-hydroxyvitamin D-1-alpha hydroxylase enzyme, thereby inhibiting endogenous production of 1,25-dihydroxyvitamin D, such that delayed hypocalcemia occurs 4-10 days after administration of the composition.

7. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a graphical view showing increase in plasma 1,25-dihydroxyvitamin D by 4 hours after receiving the bolus.

[0028] FIG. 2 is a graphical view showing blood calcium levels post calving comparing treated cows and nontreated cows.

[0029] FIG. 3 is a graphical view showing blood calcium levels post calving comparing treated cows and nontreated cows.

[0030] FIG. 4 is a graphical view showing blood calcium levels post calving comparing treated cows and nontreated cows.

[0031] FIG. 5 is a graphical view showing plasma calcium levels after calving.

[0032] FIG. 6 is a graphical view showing plasma calcium levels after treatment and no treatment.

[0033] FIG. 7 is a graphical view showing plasma calcium levels after treatment and no treatment.

[0034] FIG. 8 is a graphical view showing plasma calcium levels after treatment and no treatment.

DETAILED DESCRIPTION

[0035] This disclosure describes a method of providing a readily soluble source of oral calcium salts to promote rapid paracellular absorption of calcium and which acidify the cow to enhance tissue sensitivity to parathyroid hormone, together with exogenous 1,25-dihydroxyvitamin D or 1-alpha hydroxylated analogs thereof to promote more prolonged and more efficient transcellular absorption of dietary calcium to prevent or mitigate hypocalcemia and milk fever. The method preferably providing the soluble source of calcium salts and exogenous 1,25-dihydroxyvitamin D or analogs thereof shortly after parturition. The soluble source of calcium salts and exogenous 1,25-dihydroxyvitamin D are provided only once. By shortly after parturition is meant that the soluble source of calcium salts and exogenous 1,25-dihydroxyvitamin D or analogs thereof are provided within 6 hours following parturition.

[0036] The terms 1,25-dihydroxyvitamin D and 1,25-OH2D will be used interchangeably herein and should be considered as referring to the same structure.

[0037] The basic structure of 1,25-OH2D comprises a secosteroid. The carbons of this secosteroid are numbered by convention as described in Maestro et al., (2019). Numerous other possible vitamin D compounds that are 1-alpha hydroxylated could be utilized in addition or in lieu of 1,25-OH2D, including but not limited to those listed in Maestro et. al., (2019).

[0038] Unless specifically implied to the contrary, “vitamin D” without a subscript, used alone, as a suffix or prefix, or as a modifier, refers to any of vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D4 (22-dihydroergocalciferol), and vitamin D5 (sitocalciferol).

[0039] Vitamin D secosteroids that are hydroxylated on carbon number 1 in the alpha configuration will be referred to as “1-alpha hydroxylated vitamin D” compounds. The 1-alpha hydroxylated vitamin D compounds include 1,25-dihydroxyvitamin D compounds (i.e., 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D4, and 1,25-dihydroxyvitamin D5); active analogs thereof; or inactive analogs thereof that increase the blood, tissue, or cellular level of a 1,25-dihydroxyvitamin D compound or an active analog thereof.

[0040] “Analogs,” used with reference to 1-alpha hydroxylated vitamin D compounds, refers to biological precursors of 1,25-dihydroxyvitamin D compounds, biological metabolites of 1,25-dihydroxyvitamin D compounds, or any natural or synthetic compound recognized in the art as having a structural similarity to—or being derived from—1,25-dihydroxyvitamin D compounds.

[0041] “Active” used with reference to 1-alpha hydroxylated vitamin D compounds and analogs thereof refers to those analogs that directly produce a vitamin D-dependent effect in a target tissue or target cell without being modified or further metabolized by a non-target tissue, non-target cell, or other site elsewhere in the body. When used to modify “1-alpha hydroxylated vitamin D”, the term “active” refers to 1,25-dihydroxyvitamin D compounds or active analogs thereof. An “inactive” analog of a 1-alpha hydroxylated vitamin D compound also useful to this invention is one that is not directly able to produce a vitamin D-dependent effect in a target tissue or target cell without being modified or further metabolized by a non-target tissue, non-target cell, or other site elsewhere in the body. “Vitamin D-dependent effects” include any of the effects disclosed herein, known in the art, or hereafter discovered that result from administration or treatment of 1,25-dihydroxyvitamin D compounds. Examples of vitamin D-dependent effects include, without limitation, stimulation of transcellular calcium absorption across the rumen and intestinal epithelium and anabolic and catabolic actions on bone.

[0042] Biologically inactive analogs of 1,25-dihydroxyvitamin D that are acted upon within the body to form biologically active 1,25-dihydroxyvitamin D or analogs thereof may be used. They are generally less expensive to utilize than 1,25-dihydroxyvitamin D. One such compound is 1-alpha vitamin D3, which becomes hydroxylated at the carbon 25 position to form active 1,25-OH2D3. Other inactive 1-alpha hydroxylated vitamin D compounds useful in the practice of the invention may contain from one to five pro moieties, which can be at any of positions C-1, C-3, C-24, or C-25 or, indirectly, at position C-26. For the purposes of this disclosure, it is understood that the pro moiety can be appended to any hydroxyl group existing in the cleaved (free) form of 1,25OH2D. For example, in 1,25-dihydroxyvitamin D3, a pro moiety can be appended to the hydroxyl group at positions C-24, C-25, C-3, or any combination thereof.

[0043] In 1-alpha hydroxylated vitamin D compounds, the pro moieties may include sulfate or glycone groups. By “glycone moiety” is meant glycopyranosyl or glycofuranosyl, as well as amino sugar derivatives thereof and other moieties such as glucuronides. The glycone moieties of the vitamin D glycosides can comprise up to 20 glycone units. Preferred are those with a β-glycoside linkage as exist in the calcinogenic plants such as Solanum glaucophyllum, Cestrum diurnum, and Trisetuum flavescens.

[0044] The amount of a 1-alpha hydroxylated vitamin D compound required to be effective for maintenance of normal blood calcium concentration or reducing hypocalcemia will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the veterinary practitioner or animal husbandman. The factors to be considered include the nature of the formulation, the mammal's body weight, surface area, age, general condition, and the particular compound to be administered. In general, a suitable effective dose of 1,25-dihydroxyvitamin D3 equivalent activity is in the range of about 0.1 to about 2 μg/kg body weight, preferably 0.2-1 μg/kg, and most preferred 0.25-0.5 μg/kg.

[0045] The exogenous 1-alpha hydroxylated vitamin D and the calcium salt are preferably administered to the mammal in a preferred form of a bolus. For purposes of this application a bolus comprises a rounded mass typically cylindrical in shape with a density greater than 1.2 g/ml to ensure it does not float on the rumen raft of a ruminant. Any shape is within the scope of this disclosure that is suitable for administration to the particular mammal being treated. Also, for purposes of this application, the term mammal as used herein shall apply to all livestock such as all bovines, buffaloes, hogs, horses, mules, donkeys, sheep, and goats. Of especial interest are dairy cows.

[0046] Numerous documents in the literature, as summarized in Horst et al., (1997), describe the use of 1-alpha hydroxylated compounds administered prior to parturition as a means of preventing or reducing the degree of hypocalcemia an animal might experience at the onset of lactation. These compounds are effective because they stimulate the transcellular or active transport of calcium across the intestinal tract and may also enhance bone calcium release. Use of vitamin D compounds for control of hypocalcemia has not been widely adopted due to three problems. These are timing of administration of the vitamin D compound relative to parturition, possibility of causing toxicity from excessive hypercalcemia, and the possibility of causing inhibition of endogenous synthesis of 1,25-dihydroxyvitamin D, causing hypocalcemia to occur some 4-10 days after the administered vitamin D compound's effects have waned. The timing of administration of prior art required the vitamin D compound be administered within a window of a few days before calving to be effective. The 1-alpha vitamin D compounds of the prior art had to be given at least 12-24 hours prior to calving as the 1-alpha vitamin D requires 12 to 24 hours to initiate transcription and translation of proteins involved in the active transport of calcium across intestinal epithelium. For example, if 1-alpha hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 are administered more than four days prior to parturition they are not effective as the beneficial effects of these compounds on active transport of calcium will have waned before the cow calves. It can be very difficult to accurately predict that a cow is going to calve between 24 and 96 hours after administration of the 1-alpha hydroxylated vitamin D compound. Since this is difficult, prior art generally recommended that a second dose be administered if the cow did not calve within 4-5 days of the first dose being administered (Horst et. al., 1997). Larger doses or use of more potent analogs of 1,25OH2D can extend the window of effectiveness by 1-3 days, but can also cause problems associated with acute hypercalcemia and toxicity. Complicating this is a third problem. As described in Horst et al., (1997), repeated dosing or administration of high doses of vitamin D compounds to prevent hypocalcemia and milk fever can result in substantial inhibition of endogenous synthesis of 1,25-dihydroxyvitamin D. This can result in development of hypocalcemia some 4-10 days after the beneficial effects of the exogenous vitamin D compounds have waned.

[0047] According to the teaching of the document US Pat. No. 9,757,415 a composition comprising Solanum glaucophyllum glycosides for preventing and/or treating hypocalcemia and for stabilizing blood calcium levels in dairy cows can be produced which does not cause excessive hypercalcemia or cause excessive inhibition of the endogenous synthesis of 1,25-dihydroxyvitamin D. The composition utilizes glycosides of 1,25-OH2D3 derived from Solanum glaucophyllum leaves as a source of a 1-alpha hydroxylated vitamin D compound that will be converted to 1,25-OH2D3 by rumen microbial cleavage of the glycoside(s) liberating 1,25-OH2D3. An advantage of this approach is that the leaf material is not expensive and can be considered a natural forage source of a 1-alpha vitamin D compound. Calcium in the form of dolomite is mixed with Solanum glaucophyllum glycoside to form a hard compressed bolus that is only slowly solubilized in the rumen fluids providing a more prolonged release of Solanum glaucophyllum glycoside into the rumen fluids. To be most effective the composition of this patent must be administered between 24 and 72 hours prior to calving. The calcium in the form of dolomitic limestone is not readily soluble in rumen fluid and cannot be absorbed to any great extent by the vitamin D independent paracellular pathway across the rumen and intestinal epithelium. Therefore, the dolomitic calcium will not contribute substantially to maintenance of normal blood calcium concentrations prior to activation of the transcellular calcium absorption pathways initiated by the 1,25-OH2D liberated from the 1,25-OH2D glycosides contained within the S glaucophyllum leaf. The composition could be administered up to 72 hours prior to calving, which is similar to a priori art. Since prediction of the time of parturition can be difficult in the cow, should the cow fail to calve within 5 days of administration of the slow-release bolus a second bolus may be administered. The major obstacle to use of this preparation is, as with most of the a priori art involving use of vitamin D compounds to prevent periparturient hypocalcemia, accurate timing of administration.

[0048] According to the teaching of document US Pat. No. 5,395,622 a bolus comprised of calcium salts, including calcium chloride and calcium sulfate can support improved blood calcium concentrations in periparturient cows for 4-8 hours after administration to a cow. Therefore, as taught by US Pat. No. 5,395,622 the administration of a second bolus of calcium salts is encouraged at 12 or 24 hours after administration of the first bolus to improve blood calcium concentration over a longer period. Goff and Horst (1993) demonstrated that the length of time the blood calcium concentrations will be increased following an oral dose of calcium can be extended 1-4 hours by increasing the dose of calcium administered. However, the dose must be limited as hypercalcemia could develop. More importantly, if large doses of calcium chloride providing more than 3-3.5 equivalents of chloride are utilized the cow risks development of uncompensated metabolic acidosis. As demonstrated in Goff and Horst, (1994), calcium propionate is also a readily soluble source of calcium but it is not acidifying. Calcium propionate may also extend the length of time the dose is able to elevate blood calcium concentrations when compared to calcium chloride (Goff and Horst, 1993).

[0049] The preventive approach of this disclosure has several advantages over the approaches of the prior art. The bolus can be given as soon as the farmer observes that the cow has delivered a calf. The farmer does not have to try to predict the correct time to administer the exogenous 1-alpha hydroxylated vitamin D to the cow prior to calving. In addition, the preferred bolus form of this disclosure is administered as a single dose, obviating the necessity to find and restrain the cow to give multiple doses of an oral calcium supplement to promote normocalcemia as is often recommended when utilizing currently available calcium boluses. These advantages are achieved by provision of a mixture of soluble calcium salts such as calcium chloride and calcium propionate to promote passive absorption of calcium across the rumen and intestinal epithelium to maintain blood calcium concentrations for up to 12 hours after administration of the preparation. This provides the time necessary for the exogenous 1-alpha hydroxylated vitamin D in the preparation to stimulate the active transport of calcium across rumen and intestinal tissues which will support more normal blood calcium concentrations for the next 60-72 hours. The oral calcium salts include calcium chloride and are given in such a way as to promote a small degree of metabolic acidosis, which may promote bone calcium release and endogenous synthesis of 1,25-dihydroxyvitamin D to support normocalcemia. By also including calcium propionate or another non-acidifying readily absorbable calcium source in the preparation a larger total dose of calcium can be safely administered so that more normocalcemic blood calcium concentrations can be maintained for 12 or more hours after administration. The exogenous administration of 1-alpha hydroxylated vitamin D will raise blood 1,25-dihydroxyvitamin D concentrations rapidly, in less than 4 hours, which begins the process of activating active transcellular calcium transport 4-12 hours before the endogenous synthesis of 1,25-dihydroxyvitamin D would normally be expected to occur in response to the onset of hypocalcemia. The exogenous 1-alpha hydroxylated vitamin D allows greater use of diet calcium and any remaining bolus calcium residing in the lumen of the intestinal tract. This synergistic effect of oral soluble calcium salts and exogenous 1-alpha hydroxylated vitamin D compounds allows greater efficacy against periparturient hypocalcemia than either prior art approach alone and with greater ease of use.

[0050] The amount of calcium salt administered should be sufficient to support normal blood calcium concentraAtions for up to 12 hours. This protects the cow from hypocalcemia until the exogeneous 1-alpha hydroxylated vitamin D has had sufficient time to stimulate the more efficient active transport of calcium from the intestinal lumen into the blood of the cow. The amount of 1,25-dihydroxyvitamin D should be sufficient to stimulate intestinal calcium absorption within 12-18 hours of administration. Prior art compositions had to be administered prior to calving to be effective, so they typically included doses of 1-alpha hydroxylated vitamin D compounds in the range of 300-600 ug total to ensure adequate blood levels of 1,25-dihydroxyvitamin D activity would be available throughout the period of time (24-96 hours) they might be administered prior to calving so it could stimulate active calcium transport for the first few days following calving. Because the composition of this invention is administered at a readily identifiable time point, within 6 hours of calving, the amount of 1-alpha hydroxylated vitamin D of this invention found to be effective is less than 300 ug total dose. This reduces the risk of inhibition of the renal 25-hydroxyvitamin D-1-alpha hydroxylase enzyme which can reduce endogenous synthesis of 1,25-dihydroxyvitamin D, as has been observed to cause hypocalcemia to develop 4-10 days after administration of larger doses of 1-alpha hydroxylated vitamin D compounds.

Methods of Production

[0051] The methods described below utilize amounts of calcium and 1-alpha-hydroxylated vitamin D compounds found to be most effective for the dairy cow weighing from 500 to 700 kg. Doses for other species would have to be adjusted to accommodate differing body weight

Preparation of a Solid Dose Oral Preparation (i.e. Bolus)

[0052] In this most preferred preparation for administration to cattle, the calcium salts (preferably 80-150 g CaCl2.Math.xH2O (where x is equal to or greater than 0 and equal to or less than 6) and up to 250 g calcium propionate) and 1-alpha hydroxylated vitamin D compounds (preferably equivalent to 100-280 ug 1,25-dihydroxyvitamin D) are mixed together, along with other materials that may include but are not limited to, compounds that support gluconeogenesis (i.e. propylene glycol, glycerin, propionate), electrolyte balance (i.e. potassium and sodium chloride, magnesium sulfate and chloride), yeast, non-steroidal anti-inflammatory drugs, and rumen fermentable feedstuff (i.e. alfalfa meal, soybean meal). These materials are mixed together with water until a pumpable mixture is formed. It is also possible to add water to a mixing vessel and add the above ingredients to the water and mix until a homogenous pumpable mixture is formed. Regardless of the preparation method utilized to make the pumpable mixture, the pumpable mixture can then be utilized to fill a mold made from paper, polymer, metal or other material that will result in the shape of an item that is able to be swallowed by the animal (i.e. pill or bolus). Upon filling the mold with the homogenous pumpable mixture, the mold will be allowed to rest for a period of 0.5-24 hours (preferably under cooling) until the homogenous pumpable mixture has solidified. Following solidification, the solid product may or may not be coated with a mixture to allow for easier swallowing of the product. The resulting product will be packaged in a way to protect it from breakage and exposure to the elements. Product will be administered to an animal orally. US Pat. No. 5,395,622 and US Published patent Application US20070098810 describe in detail the manufacture of a bolus and both are hereby incorporated by reference in their entirety.

[0053] For ease of administration to the cow the effective doses of the calcium salts and the exogenous 1-alpha hydroxylated vitamin D compounds may be distributed into one large bolus or multiple smaller boluses.

Preparation of a Tablet

[0054] In this version the calcium salts (preferably 80-150 g calcium chloride and up to 250 g calcium propionate) and 1-alpha hydroxylated vitamin D compounds (preferably equivalent to 100-280 ug 1,25-dihydroxyvitamin D) are mixed together, along with materials that are commonly utilized in the manufacture of compression tablets or compression boluses. These materials are termed excipients and may include items such as binders (i.e. dextrose, microcrystalline cellulose), lubricants (i.e. magnesium stearate), flow agents (i.e. dicalcium phosphate, silicone dioxide), disintegrants (i.e. starch), and other excipients. This mixture is then subjected to sufficient force to cause the free-flowing material to be compressed into one solid mass. Once a solid mass has been created any number of these units may be delivered to an animal utilizing an applicator and technique that is known to individual skilled in the art of veterinary medicine or animal husbandry. For ease of administration to the cow the effective doses of the calcium salts and the exogenous 1-alpha hydroxylated vitamin D compounds may be distributed into one large tablet or multiple smaller tablets.

Preparation of a Drench Product

[0055] In this version the calcium salts (preferably 80-150 g calcium chloride and up to 250 g calcium propionate) and 1-alpha hydroxylated vitamin D compounds (preferably equivalent to 100-280 ug 1,25-dihydroxyvitamin D) are mixed together, along with other materials that may include but are not limited to, compounds that support gluconeogenesis (i.e. propylene glycol, glycerin, propionate), electrolyte balance (i.e. potassium and sodium chloride, magnesium sulfate and chloride), yeast, non-steroidal anti-inflammatory drug, and rumen fermentable feedstuff (i.e. alfalfa meal, soybean meal). These materials are mixed together and packaged as dry material with about 85 to 90% dry matter. When utilizing 1,25-dihydroxyvitamin D or similar lipid soluble vitamin D compounds as the form of 1-alpha hydroxylated vitamin D, it will be necessary to incorporate an agent to keep the water insoluble 1,25-dihydroxyvitamin D in solution. Many such materials are known in the arts and include agents such as medium chain triglycerides to form an emulsion with the 1,25-dihydroxyvitamin D vitamin D keeping it suspended in the water of the drench. If the source of 1-alpha hydroxylated vitamin D is a glycoside or glucuronide, as might be contained in material derived from calcinogenic plants such as Solanum glaucophyllum, an emulsifying agent is not necessary as these vitamin D compounds are already water soluble. On farm, the drench mix is added to a suitable amount of water (typically 0.5-20 liters) in a bucket or other suitable vessel and administered orally to the cow shortly after calving via a drench gun, esophageal tube, or tube that extends to the rumen as commonly practiced in large animal veterinary medicine.

Preparation of Gels or Pastes

[0056] In this case the calcium salts (preferably 80-120 g calcium chloride and 100-250 g calcium propionate) and 1-alpha hydroxylated vitamin D compounds (preferably equivalent to 100 to 280 ug of 1,25-dihydroxyvitamin D) are mixed with carriers (i.e. propylene glycol, glycerol, water, or vegetable oils) and thickeners (i.e. xanthan gum, silicon dioxide) to form an emulsion or suspension that is placed into a tube, typically in a volume between 250 and 400 ml. Other compounds may also be included as described above. The tube carrying the mixture is typically designed to fit into a caulking gun for oral administration into the back of the mouth of the cow using techniques familiar to those practiced in the art of animal husbandry and veterinary medicine. For ease of administration to the cow the effective doses of the calcium salts and the exogenous 1-alpha hydroxylated vitamin D compounds may be distributed into one large paste tube or multiple smaller tubes.

[0057] The following examples are intended as illustrations only since numerous modifications and variations within the scope of this disclosure will be apparent to those skilled in the art.

EXAMPLES

Example 1

[0058] Calcium salt boluses were prepared that incorporated either 150 ug 1,25-dihydroxyvitamin D3 or 5, 7.5 or 10 g of leaf from Solanum glaucophyllum, a plant that contains a glycoside form of 1,25-dihydroxyvitamin D3 in its leaves. Within the cow's rumen the 1,25-dihydroxyvitamin D3 glycoside, which is biologically inert, is cleaved by rumen bacterial enzymes to bioactive 1,25-dihydroxyvitamin D3. The batch of leaf material used for this experiment, and all of the following experiments described in this document, was determined to contain the equivalent of 14 ug 1,25-dihydroxyvitamin D3/g leaf utilizing a modification of the method of Gil et al., (2007). The Holstein cows utilized for this study were pregnant and near the end of their lactation and were to be dried off within 2 weeks of the study. These cows were utilized as they should have been in positive calcium balance and therefore would be expected to have relatively low concentrations of endogenous 1,25-dihydroxyvitamin D in their blood allowing better resolution of increases in plasma 1,25-dihydroxyvitamin D that could be attributed to the administration of the boluses. Though our target animal is the cow immediately after calving, previous studies have determined plasma 1,25-dihydroxyvitamin D concentrations can be quite variable (low and very high) depending on the extent of hypocalcemia the cow has experienced prior to calving and time of sampling of the blood. The cows weighed from 600-720 kg and were producing from 16 to 28 kg milk/day. Treatment consisted of administering 2 of the boluses of a single type to a cow so that a cow received boluses comprised of either 300 ug of 1,25-dihydroxyvitamin D, or boluses comprised of 10, 15, or 20 g of S. glaucophyllum leaf in total, supplying the equivalent of 140, 210, and 280 ug 1.25-dihydroxyvitamin D activity. Blood samples were collected into lithium heparin vacutainer tubes from the jugular vein of each cow just before the boluses were administered (time 0) and 1, 4, 24, 48, 72, and 96 hours after the boluses were administered. Plasma was analyzed for concentration of 1,25-dihydroxyvitamin D3 by liquid chromatography-mass spectrometry.

[0059] As shown in FIG. 1, all cows had a significant increase in plasma 1,25-dihydroxyvitamin D by 4 hours after receiving the bolus. Plasma 1,25-dihydroxyvitamin D concentrations remained significantly elevated above pre-treatment 1,25-dihydroxyvitamin D concentration at 48 hours after administration but not at 72 or 96 hours after administration. Plasma 1,25-dihydroxyvitamin D rose more quickly in cows that received synthetic 1,25-dihydroxyvitamin D3 than in cows receiving the 1,25-dihydroxyvitamin D3 glycoside from the Solanum glaucophyllum leaf. This may reflect the time it takes for the rumen bacteria to convert the glycoside to the active 1,25-dihydroxyvitamin D so that it may be absorbed into the blood.

[0060] For examples 2 thru 5, statistical analysis consisted of repeated measures analysis of variance with cow nested within treatment and time after bolus administration as the repeated measure. Treatment plasma calcium means were compared at each time point using Tukey's test of comparison of means and differences are declared to be significant when the probability of the null hypothesis being correct is P<0.075.

Example 2

[0061] Example 2 was performed on a commercial dairy with both Jersey and Holsteins fed a pre-calving diet with a high anion inclusion rate designed to help control hypocalcemia. Only multiparous cows were utilized and cows assigned to a treatment were blocked by lactation number. The effects of treatments on each breed are presented separately and then combined. The treatment protocol was identical for Jersey and Holstein cows and the cows were housed together for the final 21 days prior to calving.

Holsteins

[0062] The average urine pH of Holsteins during the 2 weeks prior to calving was 5.68. The dry matter (DM) intake of the cows was estimated at 23 lbs. (10.5 kg)/day in these cows prior to calving. Seven Holsteins were not administered any boluses at calving. Nine cows received a commercial oral calcium bolus, supplying about 50 g calcium, primarily from calcium chloride. Those Holstein cows received the treatment at calving and again the following morning (12-24 hrs. after the first bolus). Ten Holstein cows were treated with 2 calcium salt boluses which also contained Solanum glaucophyllum leaf a single time within 2 hours of calving. These two boluses supplied 78 g calcium total, the majority from calcium chloride with some from calcium propionate and 14 g Solanum glaucophyllum leaf, supplying 1-alpha hydroxylated vitamin D in the form of glycosides of 1,25-dihydroxyvitamin D determined to be equivalent to 196 ug 1,25-dihydroxyvitamin D3. Plasma samples were obtained from each cow prior to treatment, and 3, 12, 24, 36, 48, and 72 hours after treatment. Plasma calcium concentration was determined using the Arsenazio III reagent method.

[0063] FIG. 2 presents Holstein cow average blood Ca± the standard error of that average. In Holsteins receiving the Ca+SG Bolus, plasma calcium was significantly greater at 36 hours and 48 hours after calving than in cows receiving No Bolus or the Ca Bolus. Both Ca containing boluses significantly increased plasma calcium concentrations 3 hours after calving compared to Holstein cows receiving No Bolus treatment. Only Ca+SG Bolus Holstein cows experienced mean plasma Ca above 8.25 mg/dl (2.06 mM) during the study. No Bolus and Ca Bolus treatment groups average plasma calcium concentration failed to reach even 8.0 mg/dl (2 mM) during the first 3 days after calving.

Jerseys

[0064] The average urine pH of the Jersey cows prior to calving was 5.85 and 25% of the Jerseys had urine pH below 5.5. Dry matter feed intake prior to calving was estimated at 18 lbs. (8.2 kg) DM/day. Ten Jersey cows received a commercial oral Ca Bolus at calving and a second Ca Bolus 12-24 hours later. Fourteen Jersey cows received the 2 Ca+SG Boluses with calcium and Solanum glaucophyllum leaf at calving only. Three Jersey cows received no treatment (No Bolus) after calving. Bolus composition and timing of blood samples was as described above for the Holsteins of this example

[0065] As illustrated in FIG. 3, at the 3-hour time point both boluses, Ca+SG Bolus and Ca Bolus treated cows exhibited significantly increased plasma calcium compared to cows receiving No Bolus. From that point on the Ca Bolus and No Bolus cows had similar plasma calcium concentration. The cows getting the Ca+SG Bolus had significantly higher plasma calcium than cows getting No Bolus at 3,12, 24 and 36 hrs. after calving. Plasma calcium of cows receiving the Ca+SG Bolus was statistically better than in Ca Bolus cows at 24 and 36 hours after calving. Only the Ca+SG Bolus cows had mean plasma calcium above 8.5 mg/dl between 12 and 72 hours of the study.

Combined Jerseys and Holsteins Data

[0066] Since all the cows were similarly housed and fed before and after calving, the results from all cows of Example 2 are combined in FIG. 4. With increased numbers of cows at each time point the statistical power of the study is increased. The Ca+SG Bolus treatment group has greater plasma calcium than the NO bolus treatment group from 3 thru 48 hours after calving and has greater plasma calcium concentration than the Ca Bolus treatment group at 24, 36 and 48 hours after calving. Only the Ca+SG Bolus cows achieved mean plasma calcium concentration above 8.5 mg/dl between 12 and 72 hours.

Example 3

[0067] In Example 3, cows from a herd located on a farm were fed anions in their diet to achieve urine pH between 6 and 6.8. This is generally considered an effective means of preventing clinical hypocalcemia in the dairy cow. The milk production of this herd was 103 lbs. (46.8 kg) / day with 3.9% fat. The average urine pH of these cows the weeks before calving was 6.6. In this study nine multiparous cows got a single commercial calcium bolus (Ca Bolus) at calving (43 g Ca/bolus) and again 12-24 hours after calving, providing a total of 86 g Ca primarily from calcium chloride. Six cows received 2 Ca+10 g SG Boluses at calving only. These 2 boluses were comprised of 78 g calcium primarily from calcium chloride and calcium propionate and 10 g Solanum glaucophyllum leaf total supplying the equivalent of 140 ug of 1,25-dihydroxyvitamin D as the glycoside of 1,25-dihydroxyvitamin D3. Seven cows received 2 Ca+15 g SG Boluses at calving only. These 2 boluses were comprised of 78 g calcium primarily from calcium chloride and calcium propionate and contained 15 g Solanum glaucophyllum leaf supplying the equivalent of 210 ug of 1,25-dihydroxyvitamin D as the glycoside of 1,25-dihydroxyvitamin D3. Plasma samples were obtained from the jugular vein prior to bolus administration (Time=0) and 4, 12, 24, 48, and 72 hours after calving.

[0068] As illustrated in FIG. 5, the Ca Bolus cows had slightly higher plasma calcium concentration prior to any treatment than the cows of either of the Ca+SG Bolus treatments. In Ca+15 g SG Bolus cows the plasma calcium concentration was above 9 mg/dl by day 2. The Ca Bolus cows had plasma calcium concentration below 8.5 mg/dl the first 3 days after calving. Cows in the Ca+10 g SG group had plasma calcium exceed 8.5 mg/dl by day 2. These data suggested a dose of 15 g Solanum glaucophyllum leaf was more effective than a dose of 10 g Solanum glaucophyllum leaf for incorporation into the bolus to prevent hypocalcemia.

Example 4

[0069] This example utilized multiparous Holstein and Holstein X Jersey crossbred cows on a large commercial dairy farm feeding anions to prevent milk fever and hypocalcemia. Urine pH averaged 5.7 in the weeks prior to calving.

[0070] Sixty cows were assigned to one of three treatments based on expected calving date and blocked by lactation number and breed (so there were nearly equal numbers of Holsteins or Holstein X Jersey cows and cows entering their 2nd (N=10) or 3rd and greater lactation (N=10) in each treatment group. The treatments were: A. Two calcium with Solanum glaucophyllum leaf boluses at calving only (supplying 78 g calcium and 1,25-dihydroxyvitamin D3 glycoside equivalent to 196 ug 1,25-dihydroxyvitamin D). B. Gelatin capsule boluses containing calcium, comprised of calcium chloride supplying 40 g calcium/dose. These were administered at calving and again 12-24 hours after calving, for a total of 80 g calcium/cow. C. Cows receiving No Bolus after calving. Plasma samples were obtained shortly after calving and prior to treatment (Time=0) and at 24, 48, 72, 96, and 120 hours after calving.

[0071] The data are presented based on the lactation number the cow was entering. Each treatment group had 10 cows in it. In 2nd lactation cows (FIG. 6), cows in the Ca+SG Bolus treatment group had greater blood Ca concentration than either of the other treatments from 24-96 hours after treatment. FIG. 7 presents plasma calcium concentration in cows entering their 3rd and later lactation. Mean plasma calcium concentrations above 8 mg/dl were attained within 24 hours in the Ca+SG Bolus treatment group. The 3rd or greater lactation cows in the No Bolus group had plasma calcium concentration above 8 mg/dl at 48 hours after treatment. The 3rd or greater lactation cows that received the Ca Bolus attained a plasma calcium concentration by 72 hours after treatment.

Example 5

[0072] In Example 5, multiparous cows were utilized from several small farms that were not utilizing an anionic diet to reduce the incidence of hypocalcemia in the cows. Within a few hours of calving, cows were administered one of four treatments. Six cows were not treated with any bolus after calving (No Bolus). Four cows were treated with intravenous calcium (10.5 g calcium in the form of calcium gluconate) after calving (IV Ca). Twenty-two cows received 2 boluses after calving that contained calcium salts and Solanum glaucophyllum leaf, supplying 78 g calcium and 1,25-dihydroxyvitamin D3 glycoside equivalent to 196 ug 1,25-dihydroxyvitamin D3 (Ca+SG Bolus). Twenty-four cows were treated at calving and again 12-24 hrs. later with commercial oral calcium salt boluses that supplied 40-44 g calcium each for a total calcium dose of 80-88 g, primarily from calcium chloride (Ca Bolus).

All cows had plasma calcium concentration determined prior to treatments being applied and all would be considered to be hypocalcemic (plasma calcium below 8.0 mg/dl). Cows that received No Bolus treatment after calving exhibited a further decline in blood calcium during the first four hours after calving. As illustrated in FIG. 8, in the cows that received No Bolus blood calcium concentration remained below 8.0 mg/dl (commonly considered to be indicative of a cow with subclinical hypocalcemia) until nearly 96 hours after calving. Cows in the Ca Bolus treatment group exhibited a small decline in calcium at the four-hour time point and their blood calcium concentration at the 4-hour time point was significantly better than in cows receiving No Bolus. However, from 12-120 hours after calving their plasma calcium concentrations were similar to those of cows receiving No Bolus after calving. Administration of calcium solution intravenously (Ca IV) resulted in the highest plasma calcium concentration observed at four hours after calving. However, from 24 to 84 hours after calving these cows had the lowest plasma calcium concentration. The intravenous calcium administration inhibited the calcium homeostasis mechanisms of the cow, causing more hypocalcemia to be observed on days 2 and 3 following lactation than if no treatment had been given. Cows in the Ca+SG Bolus treatment exhibited a rise in plasma calcium concentration at each time point after calving and their blood calcium concentration went above the threshold for sub-clinical hypocalcemia of 8.0 mg/dl by 24 hrs. after calving. From 4 hours after treatment thru 84 hours after treatment, cows receiving the Ca+SG Bolus at calving only had significantly higher plasma Ca concentration than cows receiving the commercial oral calcium boluses given two times after calving.