TECHNICAL FIELD

The present invention relates to an injection formulation. More specifically, the invention relates to a formulation for the administration of a combination of the elements selenium and iodine to mammals to prevent nutritional deficiencies of these elements. The invention also relates to a method of determining whether or not to administer the formulation of the present invention.

BACKGROUND ART

In agriculture, the prevention of element deficiencies is important to ensuring livestock health. This is of particular importance during times of nutritional challenge such as during the pre-mating season, during pregnancy, or where feeds and forages are deficient in nutrients.

Two key elements are selenium and iodine. For both elements, deficiency pre-mating can reduce fertility and fecundity, while deficiency during gestation causes deficiency in utero so that progeny are at risk of being born dead or dying shortly after birth (perinatal mortality). Inadequate selenium can also lead to white muscle disease and poor growth in young mammals. Inadequate iodine can cause enlargement of the metabolism-regulating thyroid endocrine gland. It should be appreciated that meeting element nutritional requirements of the progeny is essential during foetal development and becomes critical during the latter half of gestation.

Existing products on the market to prevent iodine and selenium deficiencies are Flexidine™ and Deposel™. These are used to treat and prevent deficiencies of these elements. They exhibit a slow release effect over an extended time period which is advantageous to avoid extra labour

requirements.

Flexidine™ is a sustained release iodine intramuscular injection to treat and control clinical and sub-clinical iodine deficiencies in mammals. It contains approximately 260 g iodine/litre and a vehicle carrier of 740 g/litre peanut oil (from NZFSA website ACVM register).

Deposel™ uses a different carrier to Flexidine™ with barium selenate particles mixed through providing a selenium source. Like Flexidine™, it forms a long acting depot on administration releasing selenium over time. Deposel™ is injected subcutaneously.

Simply mixing and administering the above two formulations is anticipated as resulting in technical difficulties. In the inventor's experience, the adjuvants and vehicles used in Flexidine™ and Deposel™ are sufficiently different that the two formulations would form a coarse emulsion and therefore might not release iodine and/or selenium at sustained and predictable rates over the desired period of time. In particular, it is anticipated that barium selenate particles used in Deposel™ would not stay suspended in the oil-based Flexidine™ product.

A further problem with the above products is that they are administered via different routes i.e. intramuscular administration for Flexidine™ and subcutaneous administration for Deposel™. This in itself suggests that each formulation exhibits different modes of action and would not obviously combine.

It should be appreciated that it would be advantageous to have a formulation that can simultaneously supply both a selenium source and an iodine source to mammal in one injection. It would be expected that a combination formulation would at least have lower labour costs and more convenience due to less handling than administering two separate injections.

It would also be advantageous to have a product that uses simple and inexpensive raw materials, and that the process to manufacture the product would be simple to complete whilst still providing adequate suspension

characteristics to enable therapeutic release characteristics for an extended period of time.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non- specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

Formulation

According to one aspect of the present invention there is provided a formulation for parenteral administration which includes:

iodised oil; and,

a selenium source; and,

characterised in that the formulation also includes sufficient thickening agent such that, on administration, the formulation forms a depot which releases a sustained therapeutic amount of selenium and iodine over a time period of at least 90 days.

For the purposes of the specification, the term 'parenteral' refers to the form being taken into the body or administered in a manner other than through the digestive tract such as by intramuscular or subcutaneous injection.

The term 'iodised' refers to the endogenous unsaturated fatty acids from the oil covalently-bound to iodine therefore resulting is no free iodine being present.

The term 'depot' refers to a formulation that tends to stay at the administration site so that absorption occurs over a prolonged time period.

The term 'sustained' refers to the depot releasing agent or agents at a semi continuous to continuous rate and not delivering a bolus 'hit' of agent or agents.

Preferably, the oil is characterised by being non-allergenic; thin and flowing at room temperature with a viscosity of approximately 50-100 mPa-sec @ 23°C; containing a high proportion of polyunsaturated fatty acids; and slow to deteriorate by oxidation. In preferred embodiments, the oil is vegetable oil. More preferably, the oil is selected from: arachis oil, poppyseed oil, walnut oil and combinations thereof. Preferably, the iodine is bound to the oil at a concentration of 20-40% (w/w).

Preferably, the selenium source is barium selenate. It is the inventor's experience that barium selenate is preferable due to the increased length of time for release and rate of release. Other selenium sources envisaged by the inventor may include sodium selenate although it is the inventors' experience that these have been found less effective for this agricultural

application. More preferably the selenium source is in particulate form. Most preferably, the selenium source is of a well-defined micronised particle size. It is the inventors' experience that, unexpectedly, the particle size may be a critical determinant of the rate and duration of absorption of the injected selenium depot. In preferred embodiments, the particle size is less than 10 μm. More preferably, the particle size is a controlled distribution within 0.1 to 10 μm.

In preferred embodiments, the formulation is administered by injection. Most preferably, the injection is administered intramuscular.

Preferably, the thickening agent is beeswax. It is the inventors' understanding that beeswax provides optimum suspension and flow characteristics for the formulation that thickens the mixture and slows settling of the selenium source out of the oil solution or the depot once administered. In preferred embodiments, the thickening agent is added at a concentration of 0.5% to 5% (w/w). It is the inventor's experience that this concentration range avoids the formulation becoming too thick at low temperature whilst still minimizing settling of the suspension. More preferably, the thickening agent is of a suitable pharmaceutical grade.

Preferably, sufficient thickening agent is added to result in a formulation with a viscosity of approximately 200-1000 mPa-sec @ 23°C. More preferably, sufficient thickening agent is added to result in a formulation with a density of approximately 1.03 g/ml when measured at 23°C.

Preferably, the prolonged time period for release is at least a time period of 90 days. More preferably, the time period for release is at least approximately 160 to 200 days.

Method of Administration

According to a further aspect of the present invention there is provided a method of elevating the levels of selenium and iodine in a mammal by

administration of a formulation to a mammal, the formulation including:

iodised oil; and,

a selenium source; and,

characterised in that the formulation also includes sufficient thickening agent such that, on administration, the formulation forms a depot which releases a sustained therapeutic amount of selenium and iodine over a time period of at least 90 days.

Preferably, the formulation is administered to a pregnant mammal. More preferably, the formulation is administered during the first half of gestation. This should not be seen as limiting, as based on results found for Flexidine™ and Deposel™ it would be expected by a person skilled in the art that similar results would be found if the formulation were administered pre-mating. It should be appreciated that the formulation may also be used in male mammals and non-pregnant mammals for example, to prevent a nutritional deficiency where mammals are fed nutrient deficient feeds.

Preferably the mammal is a ruminant. More preferably, the mammal is a sheep, cow or deer. Most preferably, the mammal is a ewe.

In preferred embodiments, approximately 4.3 to 8.0 mg iodine/kg live weight and approximately 0.64 to 1.2 mg selenium/kg live weight are administered to a mammal.

Preferably, the depot releases a sustained supplement of iodine and selenium over a time period of at least 90 days. More preferably, the time period for release is at least between approximately 160 to approximately 200 days.

Preferably, the iodine release rate is approximately 2.5 mg per day.

Preferably, the selenium release rate is less than approximately 0.30 mg per day.

Preferably, iodine and selenium levels are elevated in the mammal, and iodine and selenium transfer to progeny during gestation.

Preferably, the mammal to which the formulation is administered produces milk with elevated levels of selenium and iodine, which are transferred to progeny until weaning.

Preferably, administration of the formulation prevents enlargement of the thyroid gland (goitre) in the progeny. In the preferred case where the formulation is administered to a pregnant ewe, the ratio of progeny lamb thyroid weight to lamb body weight at birth is maintained at less than 0.40 g/kg.

It should be appreciated by those skilled in the art that a ratio of lamb thyroid weight to lamb body weight of less than 0.40 g/kg is a healthy level indicative of normal thyroid function. Where this ratio increases, the increased ratio is indicative of a lack of iodine nutrient in the lamb.

Method of Manufacture

According to a further aspect of the present invention there is provided use of a formulation in the manufacture of a medicament to elevate levels of selenium and iodine in a mammal wherein the formulation includes:

iodised oil; and,

a selenium source; and,

characterised in that the formulation also includes sufficient thickening agent such that on administration the formulation forms a depot which releases a sustained therapeutic amount of selenium and iodine over a time period of at least 90 days.

According to a further aspect of the present invention there is provided a method of manufacture of a formulation to provide selenium and iodine at a

sustained rate for a time period of 90 days including the steps of:

(a) mixing oil with a thickening agent;

(b) stirring and heating the mixture of step (a);

(c) sterilising the mixture of step (b)

(d) cooling the mixture of step (c);

(e) adding an appropriately micronised selenium source to the cooled mixture of step (d) and homogenising the resulting suspension.

Preferably, the thickening agent is added at a concentration of 0.5 to 5% (w/w).

Preferably, step (c) sterilisation is completed by filtration through a sterilising filter.

Preferably, the selenium source is sterilized prior to being added in step (e). Most preferably, sterilization of the selenium source is completed by gamma irradiation. Preferably, the selenium source is added during step (e) aseptically to the cooled mixture of step (d).

Method for Determining Administration

According to a further aspect of the present invention, there is provided a method of determining whether to administer an iodine-containing supplement to a ewe by measuring the serum iodine concentrations in the ewe during early gestation and wherein, if the ewe serum iodine concentration is less than approximately 50 μg/L, an iodine deficiency is indicated and an iodine containing supplement is administered.

According to a further aspect of the present invention, there is provided a method of determining whether to administer an iodine-containing supplement to an animal by testing the thyroid-to-birthweight ratio of dead

new-born lambs and wherein, if the lamb thyroid to birthweight ratio is greater than 0.5 g/kg or, in more severe cases, greater than 0.8 g/kg, an iodine deficiency is indicated and an iodine containing supplement is administered to the flock before the following year of pregnancy. For the purposes of this specification, the term flock refers to sheep grazed on a related site area and having similar feed characteristics.

The above methods have been found by the inventor as being more reliable than existing biochemical criteria and benchmark values published and used to assess the iodine status of grazing animals.

Preferably, the above methods are used to determine whether or not to administer the formulation of the present invention as a parenteral supplement to prevent or treat nutritional deficiencies. It should be appreciated also, that the above methods assist in determining the timing of administration i.e. pre-mating if deficiencies are very poor or later if deficiencies are of a less acute nature.

It should be appreciated from the above description that there is provided a formulation that delivers both iodine and selenium in one vehicle and which, when administered pre-mating or during the first half of gestation, maintains mammal iodine and selenium levels well above unsupplemented levels for at least between approximately 160 to approximately 200 days. The supplementation effect transfers from the treated mammal to progeny as indicated by elevated element concentrations in the mammal milk and progeny blood and serum. The formulation can be used to prevent a number of diseases in mammals attributable to dietary insufficiency of selenium and/or iodine. This may have useful applications in farming where livestock feeds and forages lack these elements or where livestock are in nutritionally challenging lifecycle stages such as during pregnancy and lambing. The above method of determining iodine levels also assists in determining the need to administer the above formulation and administration timing.

The above formulation overcomes difficulties of the prior art by providing a convenient one step formulation, is simple to manufacture, uses simple ingredients, and is stable and efficacious.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 shows a graph indicating the selenium response to treatment with a 4 ml dose of the invention formulation and feeding regimens as measured from mean blood selenium levels in ewes fed pasture or kale;

Figure 2 shows a graph indicating the selenium response to treatment with a reduced (3 ml) dose of the invention formulation and feeding regimens as measured from mean blood selenium levels in ewes fed pasture or kale;

Figure 3 shows a graph of the selenium response to treatment with a 4 ml dose of the invention formulation and feeding regimens as measured from mean milk selenium levels on the first day of lactation and through the remainder of the study;

Figure 4 shows a graph of the selenium response to treatment with a reduced (3 ml) dose of the invention formulation and feeding regimens as measured from mean milk selenium levels on the first day of lactation and through the remainder of the study;

Figure 5 shows a graph of the mean blood selenium concentration of lambs born to ewes treated with a 4 ml dose of the invention formulation and feeding regimens;

Figure 6 shows a graph of the mean blood selenium concentration of

lambs born to ewes treated with a reduced (3 ml) dose of the invention formulation and feeding regimens;

Figure 7 shows a graph indicating the iodine response to treatment with a 4 ml dose of the invention formulation and feeding regimens as measured from mean serum iodine levels in ewes fed pasture or kale;

Figure 8 shows a graph indicating the iodine response to treatment with a reduced (3 ml) dose of the invention formulation and feeding regimens as measured from mean serum iodine levels in ewes fed pasture or kale;

Figure 9 shows a graph of the iodine response to treatment with a 4 ml dose of the invention formulation and feeding regimens as measured from mean milk iodine levels on the first day of lactation and through the remainder of the study.

Figure 10 shows a graph of the iodine response to treatment with a reduced (3 ml) dose of the invention formulation and feeding regimens as measured from mean milk iodine levels on the first day of lactation and through the remainder of the study;

Figure 11 shows a graph of the mean serum iodine concentration of lambs born to ewes treated with a 4 ml dose of the invention formulation and feeding regimens;

Figure 12 shows a graph of the mean serum iodine concentration of lambs born to ewes treated with a reduce (3 ml) dose of the invention formulation and feeding regimens;

Figure 13 shows two graphs indicating the distribution of thyroid to birthweight ratios for necropsied lambs born to six groups of ewes (lower panel). The ewes were fed exclusively pasture (<>,♦) or pasture followed by a goitrogenic brassica forage during the latter half of gestation (D ₁ B). Each symbol

represents a lamb that came from a ewe which had received supplemental injectable iodine (♦,■) or was unsupplemented (O ₁ D). The probability of a flock response to iodine treatment is predicted by a cumulative distribution curve flanked by 95% confidence intervals (upper panel).

BEST MODES FOR CARRYING OUT THE INVENTION

A series of experiments are now described showing a preferred method of manufacturing the formulation and the effect of administration to mammals of the formulation of the present invention. These examples should not be seen as limiting as it should be appreciated that a variety of variations may be incorporated without departing from the scope of the invention as described.

Experiment 1 : Manufacture of Formulation

The steps below outline a preferred method of producing a 500 litre batch of formulation:

(a) 450 kg of iodised oil is weighed into a clean dry process vessel.

(b) Thickening agent is weighed out and added to the oil at a concentration of 0.5-5% (w/w).

(c) The contents of the tank are stirred and heated.

(d) The heated material is sterilised by filtration through a sterilising filter into a clean, sterile receiving vessel and allowed to cool.

(e) Sterile selenium in the form of a pure powder barium selenate compound, micronised to an appropriate particle size distribution, is added aseptically to the material and the resulting suspension is homogenised.

(f) The finished product is filled into sterile plastic flexi packs for retail sale. Flexi packs of 100-500 ml volume may be used.

The above formulation produces an injectable formulation with each ml of formulation containing 12-15 mg of selenium as barium selenate and approximately 100 mg of iodine as iodised arachis oil. A 4 ml injection (preferably administered intramuscularly by automatic injector syringe with 16G x 9 mm needle) therefore provides a slow-release depot of 48-60 mg selenium and approximately 400 mg iodine.

The above concentrations of active components have been found by the inventor to provide biologically appropriate, safe and efficacious doses in a conveniently dispensed volume.

Experiment 2: Animal Field Trials

The aim of this experiment was to evaluate the efficacy and welfare aspects of the formulation manufactured according to the method described in Experiment 1. This experiment was conducted and documented in accordance with the principles of Good Clinical Practice-Veterinary (CGP-V). Ethical and scientific quality standards were audited.

A key outcome desired from administering the formulation is to increase selenium and iodine status of the animal in order to avoid problems associated with deficiencies in these elements, including low or no fertility of the untreated animals and excess perinatal mortality of their progeny (related to selenium and iodine deficiencies), and enlarged thyroid glands in the progeny (goitre; a symptom of iodine deficiency).

Experiment 2 Method - Animal Selection and Identification

In order add additional challenge to this test of the formulation effectiveness, the experimental animals grazed a crop of winter kale. This brassica crop is

low in iodine content and contain iodine-sequestering goitrogens. Pregnant ruminants fed such crops during gestation are at increased risk of giving birth to progeny with goitre (cf. Sinclair and Andrews 1954).

Animals - An objective of the study was to determine the effect of treatment on measures of reproductive performance, including lambing percentage, perinatal mortality and incidence of goitre in lambs, therefore a large flock of pregnant ewes was required. The test animals were mixed-age Romney ewes, weighing 55-65 kg, and identified with a numbered eartag. The sheep were sourced from sheep farms in the North Island of New Zealand.

Animal Management - All sheep were managed according to established husbandry and animal health protocols.

The grass pastures used for the trial were determined to have low selenium concentration (<0.03 mg selenium/kg dry material) and low to moderate concentration of iodine (0.15-0.24 mg iodine/kg dry material). These concentrations are so low as to not meet sheep nutritional requirements for dietary intake, therefore setting up a significant challenge to the effectiveness of the invention formulation to adequately provide selenium and iodine supplementation for the ewe and lamb. Approximately four hectares of an alternative grazing crop, brassica kale, were sown and established on site in late spring (November) prior to the trial commencing. This crop also has low iodine content and contains iodine-sequestering goitrogens typical of brassica species (Stoewsand 1995), therefore adding additional challenge to the test of formulation effectiveness.

Inclusion and Exclusion Criteria - In late summer (February) an available flock of 507 Romney ewes was identified from which 5-6 ewes were selected for blood sampling to confirm initial low selenium and iodine status. This flock met the inclusion criteria of <500 nmol selenium/L blood and <40 μg iodine/L serum. Therefore the flock, and by extension all ewes in it, was eligible for use in the study.

Animal Health & Concomitant Medication - In order to have a short and

predictable lambing period, in early April (mid autumn) (Day -63) all ewes were oestrus-synchronized by controlled internal drug-releasing devices (CIDRs) containing progesterone. The CIDRs were removed in April (Day - 53) and the ewes then run with 25-30 harnessed Suffolk rams for 32 days (Day -20). The ewes mated in the first cycle were scanned in early June (Day -4) to assess their pregnancy status as single, twin, triplet, or not pregnant. Pregnancy status was noted for sorting purposes by putting unambiguous paint marks on each ewe.

Allocation - Four days later, 386 of the pregnant ewes were selected (about 70% carried twins or triplets), marked with eartags specifically colour coded for this study, then randomly assigned to groups for treatment as outlined in Table 1 below. This was 08 June (early winter), designated as Day 0 of the study.

Mortality - Ewe deaths were not related to treatment. The overall ewe mortality rate was 9% based on the difference between the number of ewes treated at Day 0 (386) and number of ewes accounted for at the time of docking on Day 126 (350).

Experiment 2 Method - Experimental Design and Treatments Scheme

The basic design of the study was a 2 x 2 factorial (n=89 ewes per group). The classification factors were: +/- treatment with 4 ml of formulation and +/- feeding with kale. Note that the ewes on kale were fed this crop only for a 62 day winter period starting early July and finishing 1 ^st September (early spring). It should be appreciated that these months comprise the latter half of pregnancy which is a critical period for foetal development. Pasture grass was fed at all other times.

An additional portion of the field study was carried out simultaneously at the same site. For this, three extra groups (n=10 ewes per group) were randomly assigned to determine dose-response aspects of the formulation. Group PV was treated with 4 ml of only the formulation vehicle (containing peanut oil and beeswax with no active ingredients) to determine if the vehicle

had any unexpected effects. Groups P3 and K3 were treated with 3 ml of the complete formulation. Group P3 grazed exclusively on pasture while Group K3 grazed kale in winter as described above. Some of the results from these extra groups are included in this report.

Groups treated with the invention formulation (namely Groups P4, K4, P3 and K3) received a single intramuscular injection of either 4 ml or 3 ml of formulation in the anterior neck region using an automatic syringe fitted with a 16G x 9 mm needle, on Day 0 according the scheme in Table 1 below.

Based on the composition of the formulation, a 4 ml injection supplied 60 mg selenium and 400 mg iodine, while a 3 ml injection supplied 45 mg selenium and 300 mg iodine.

As summarized in Table 1 , a total of seven treatment groups were used, arranged to evaluate three dose levels of the formulation (0, 3 and 4 ml) as well as two types of forage feeding regimen (pasture and kale).

Table 1 - Dosage Regimes

Group n Injection Feeding regimen

"PO" pasture control 89 untreated pasture «P4» pasture treated 89 4 ml formulation pasture

"KO" kale control 89 untreated pasture + winter kale "K4" kale treated 89 4 ml formulation pasture + winter kale

"P3" pasture 3 ml treated 10 3 ml formulation pasture "K3" kale 3 ml treated 10 3 ml formulation pasture + winter kale

"PV" pasture vehicle only 10 4 ml vehicle pasture

At the start of the experiment, subsets of ewes were randomly selected and designated as "monitor ewes". These were 15 ewes from each of Groups PO, P4, KO and K4, and all 10 ewes from Groups PV, P3 and K3.

At Day 7, all monitor ewes were examined for presence of lesions or any untoward reaction at the site of injection. Only one small site reaction (a 5

mm x 5 mm bump) was found. Although a second check of the same animals was planned for Day 14, this was deemed unnecessary.

All ewes were grazed on grass pasture as a single mob from mating (Day - 53) until Day 23 when the ewes in Groups KO, K4 and K3 were grazed on kale for 62 days starting early July and finishing September 1 ^st (mid winter to early spring). The ewes in Groups PO, P4, P3 and PV remained on pasture. On Days 85-86 all the kale-fed ewes were returned to pasture, and all groups were placed in separate pastures for lambing. Mean date of lambing was September 12th (early spring) (Day 96). During lambing the ewes and their living lambs were matched and recorded.

All lambs born dead in Groups PO, P4, KO and K4 were retrieved and marked with eartags colour-coded to their treatment group. Lambs were delivered to a pathology laboratory for necropsy. Lamb thyroid glands were dissected out to determine the incidence of goitre as indicated by a ratio of thyroid weight (measured to nearest 0.1 g) to birthweight (nearest 0.1 kg) greater than about 0.4 g/kg.

Once lambing was complete all ewes and their lambs were grazed on pasture as a single mob until weaning in late December (summer).

Perinatal mortality was calculated as the number of dead new-born lambs divided by the number of ewes at Day 0. Flock lambing percentage was calculated at docking time as the number of live lambs docked at Day 126 divided by the number of ewes at Day 0.

Experiment 2 Method - Fate of Animals

The field trial finished in mid December (summer). At that time all ewes (treated and untreated) were returned to the breed flock. All lambs (now weaned) continued to graze pasture until March-April (autumn) when they were sent to a meat processing facility.

Experiment 2 Method - Sampling

In order to determine effects of the formulation on animal selenium and

iodine status, blood samples were collected from the monitor ewes:

• at treatment time at Day 0;

• throughout pregnancy at Days 23 and 63;

• from ewes and their lambs from lambing time until weaning at Days 99, 127, 161 and 192.

At each time of sampling, two 9 ml vacutainers (one Red top with no anticoagulant suitable for serum and one Purple top with K3EDTA suitable for whole blood) were used to collect blood from the jugular vein. At each bleeding, at least 7 and up to 15 of the samples per group were analysed for selenium and iodine concentrations. Any remaining samples were stored and analysed only if a monitor ewe died or more analytical information was required.

Post-lambing, blood from lambs of the monitor ewes was also collected (using two 9 ml vacutainers by the above method and timetable).

During lactation, milk samples (5-120 ml) were collected from lambed ewes at the same time that the ewes and lambs were bled according to the above timetable, with samples taken from 7 to 11 of the available monitor ewes in each group. The initial milk samples were collected 24-36 hours post- lambing and were true milk, not colostrum.

At about monthly intervals, representative samples of herbage from the pastures and fields grazed by the sheep were collected according to standard operating procedure. Samples from separate pastures were combined ("bulked") together.

Experiment 2 Method - Preparation of Samples

On each day of blood collection, the Red top vacutainers were centrifuged at 2000 x g for 20 minutes, then the serum harvested. This serum and the whole blood in the Purple top vacutainers were stored at 4°C until couriered for analysis of iodine and selenium, respectively.

Milk samples were stored at 4 ⁰ C until couriered for analysis of selenium and

iodine.

Pasture samples were stored at 7-9 ⁰ C until dried at 60 ⁰ C for 2 days, then ground and stored in airtight plastic containers until analysis of selenium and iodine.

Experiment 2 Method - Analytical Methods

Blood and milk selenium determinations and pasture selenium determinations were carried out using a semi-automated fluorimetric method (Watkinson 1979). Limits of quantitation were 70 nmol/L and 0.01 mg/kg Dry Matter (DM).

Determinations of iodine in serum, milk and pasture were carried out using an organic alkali digestion procedure followed by inductively coupled plasma mass spectroscopy (ICP-MS) according to the method of Fecher et al (1998). Limits of detection were less than or equal to 1.0 μg/L and 0.05 mg/kg DM.

Experiment 2 Method - Statistical Methods

Significant differences between treatments for blood and milk selenium, serum and milk iodine, as well as thyroid:birthweight ratios in new-born lambs were determined by analysis of variance of repeated measures using Minitab version 14.1 (Minitab Inc, State College, PA, USA). Area-under-the-curve (AUC) summary statistics are a type of weighted average suited to curvilinear responses over irregular-interval sampling, which were calculated via the trapezoidal rule to the last measurement.

Experiment 2 Results

Based on external checks of the site of injection, negligible lesions or adverse reactions to the formulation were visible or palpable.

Experiment 2 Results - Pasture Composition

Mean pasture selenium concentration was 0.03 mg/kg DM (range 0.02 -

0.05). Mean pasture iodine was 0.15 mg/kg DM (0.08 - 0.24). Kale stems and leaves were very low in selenium and iodine, with plant means of 0.02 mg selenium/kg DM and <0.05 mg iodine/kg DM, respectively.

Table 2 - Seasonal changes in selenium and iodine concentrations of pasture and kale (mg/kg DM).

Pasture Kale leaf Kale stem Pasture Kale leaf Kale stem

Month selenium selenium selenium iodine iodine iodine

June 0.04 0.04 0.02 0.24 <0.05 <0.05

July 0.05 0.03 0.03 0.13 <0.05 <0.05

August 0.03 <0.01 0.01 0.09 <0.05 0.05

September 0.02 0.03 0.03 0.08 <0.05 <0.05

October 0.03 0.23

November 0.02 0.11

December 0.03 0.19

Mean 0.03 0.03 0.02 0.15 <0.05 <0.05

Experiment 2 Results - Lambing Statistics

Perinatal mortality was calculated as the number of dead new-bom lambs divided by the number of ewes at Day 0. Flock lambing percentage was calculated at docking time as the number of live lambs docked at Day 126 divided by the number of ewes at Day 0.

Treating ewes with the invention formulation resulted in fewer dead lambs and concomitantly higher lambing percentage, regardless of pasture or kale feeding regimen.

Table 3 - Lambing statistics, including lambing percentage and lamb perinatal mortality.

Ewes Lambs Lambs Lambing Perinatal at Day 0 born alive born dead percentage mortality

Pasture untreated 99 130 27 131% 27%

Pasture 4ml 99 132 16 133% 16%

Kale untreated 89 120 24 135% 27%

Kale 4 ml 99 142 18 143% 18%

Experiment 2 Results - Lamb Thyroid: Birthweight Ratios and Incidence of Goitre

All dead lambs (16-27 per group) were retrieved, necropsied and the thyroid:birthweight ratio measured as g/kg. In Group KO, mean birthweight was significantly lower (P<0.01) and mean thyroid weight was significantly higher (P<0.01) than in other groups.

The results in Table 4 show that supplementation by the formulation of the present invention successfully prevents selenium and iodine deficiency as measured by lamb thyroid ratio.

Table 4 - Thyroid weight and birthweight of dead new-born lambs

(mean ± std err).

Birthweight ¹ Thyroid weight Thyroid:birthweight n (kg) (g) ratio (g/kg)

Pasture untreated 27 4.6 ± 0.23 3.2 ± 0.59 ^a 0.70 ± 0.14 ^a

Pasture 4ml 16 4.6 ± 0.35 1.3 ± 0.16 ^b 0.27 ± 0.03 ^b

Kale untreated 24 3.2 ± 0.15 ^B 6.4 ± 1.13 ^A 2.13 ± 0.43 ^A

Kale 4 ml 18 4.4 ± 0.30 ^A 1.8 ± 0.18 ^B 0.42 ± 0.03 ^B

¹ Within a diet regimen, means with different superscripts are significantly different: pasture groups (a, b) P<0.05; kale groups (A ₁ B) P< 0.01.

Experiment 2 Results - Description and Calculation of Daily Release Rates of the Active Ingredients

The daily release rates of iodine and selenium from the injected depot can be estimated from changes in blood concentrations of these active ingredients.

Figure 1 shows the selenium response to treatment with a 4 ml dose of the invention formulation and feeding regimens as measured from mean blood selenium levels in ewes fed pasture or kale.

The initial mean blood selenium concentration of all these ewes was 304 nmol/L, decreasing over the course of the study to 77 nmol/l for untreated ewes. Treatment with 4 ml of the invention formulation (containing 60 mg of selenium) markedly increased and maintained ewe blood selenium levels for the entire duration of the study i.e. at least 192 days. Blood selenium response peaked near lambing time, at about 22-fold greater levels than equivalent controls. Regardless of feeding regimen the patterns of blood selenium responses were similar and not significantly different.

Figure 2 shows the selenium response to treatment with a reduced (3 ml) dose of the invention formulation and feeding regimens as measured from mean blood selenium levels in ewes fed pasture or kale.

Figure 3 shows that the mean milk selenium concentration of untreated ewes was approximately 70-75 nmol/L on the first day of lactation and throughout the course of the study. Milk from ewes treated with a 4 ml dose of the invention composition was substantially enriched in selenium, at about 3.5-fold greater than controls on the first day of lactation. This milk selenium level was still significantly greater than controls at weaning time, being 191 nmol/L. Only on the first day of lactation did the feeding regimen affect milk selenium responses, with Group K4 greater than Group P4 (313 vs. 235 nmol/L, P<0.05).

Figure 4 shows results when a 3 ml dose of the invention composition was used.

As shown in Figure 5, the initial mean blood selenium concentration of lambs born to untreated ewes was 84 nmol/L, decreasing from birth to weaning to 70 nmol/L. Blood of lambs from ewes treated with the invention composition was substantially higher in selenium, at about 14-fold greater than controls at birth. Mean blood selenium was still significantly greater than controls at weaning time, at a level of 527 nmol/L. Regardless of feeding regimen the patterns of blood selenium responses were similar and not significantly different.

Figure 6 shows results when a 3 ml dose of the invention composition was used.

As shown in Figure 7, the initial mean serum iodine concentration of all ewes was 43 μg/L, increasing slightly over the course of the study to 49 μg/L for untreated ewes. Treatment with 4 ml of the invention formulation (containing 400 mg of iodine) increased and maintained ewe serum iodine for at least 161 days. Effects ranged from 6-fold increase at Day 63 (P<0.001) to a 1.4- fold increase at Day 161 (66 versus 48 μg/L, P<0.001). Serum iodine showed a marked decrease near lambing time, when demands of the rapidly growing foetus and the start of lactation drew heavily on ewe iodine stores to provide sufficient iodine. Ewe serum iodine rose again at Day 127, reflecting the lower iodine demand of later-stage milk. Regardless of feeding regimen the patterns of serum iodine responses were similar and not significantly different.

Figure 8 shows results when a 3 ml dose of the invention composition was used. Figure 9 shows that the mean milk iodine concentration of untreated ewes was 26 μg/L on the first day of lactation and rose slightly over the course of the study to 46 μg/L. Milk from ewes treated with the invention formulation was substantially enriched in iodine, at 15 to 21 -fold greater than controls on the first day of lactation, and this milk iodine level was still significantly greater than controls at weaning time, at 139 μg /L. Regardless of feeding regimen the patterns of milk iodine responses were similar and not significantly different (even at the start of lactation when Group K4 was greater than Group P4: 542 vs. 383 μg /L, P=0.17).

Figure 10 shows results when a 3 ml dose of the invention composition was used. As shown in Figure 11 , among lambs born to untreated ewes, initial serum iodine concentrations were affected by feeding regimen, with the serum in Group KO lambs from ewes fed kale at 132 μg/L versus 78 μg/L in Group PO lambs (P<0.001 ). By weaning time the mean serum iodine in all lambs was 63 μg/L.

Serum of lambs from ewes treated with the invention formulation was not affected by feeding regimen but was significantly higher in iodine than controls from birth to Day 161. At birth this difference was 2.4 to 3.8-fold greater and at Day 161 it was 1.4-fold greater (99 vs. 70 μg/L, P<0.01 ).

Figure 12 shows results when a 3 ml dose of the invention composition was used.

Experiment 2 Results - Selenium and Iodine Responses to Vehicle Only

Group PV was injected with 4 ml of the invention vehicle (containing peanut oil and beeswax with no active ingredients) to determine if the vehicle had any unexpected effects. That group of ewes was fed exclusively pasture and so is directly comparable to Group PO.

In all but one instance the concentrations of blood and milk selenium and serum and milk iodine in vehicle-treated ewes and their lambs were not significantly different from the untreated ewes and lambs. However the serum iodine of Group PV lambs on Day 99 was slightly greater than the serum iodine of Group PO lambs (106 vs. 78 μg/L, P<0.05). Although this might be a real effect, a "rogue" difference among the dozens of separate PV vs. PO comparisons is not unexpected when the overall alpha-level is not controlled (known in statistics as the Multiple Comparisons Problem).

Experiment 2 Results - Selenium and Iodine Responses to Lower Dose Treatments

As described above, two extra groups of 10 ewes per group were assigned to determine selenium and iodine responses to a lower dose of the invention

formulation. Groups P3 and K3 were treated with 3 ml of formulation and the results found are shown in Figures 2, 4, 6, 8, 10 and 12.

Based on composition of the formulation, the 3 ml injection supplied 45 mg selenium and 300 mg of iodine. Group P3 grazed exclusively on pasture while Group K3 was fed kale during winter as described previously.

Appraisal of results showed that lower dose treatment with the invention formulation produced responses roughly proportional to dose size. As an indication of the responses relative to a "standard" 4 ml dose, areas-under- the-curve (AUC) have been calculated:

• calculations compare AUC from Day 0 through Day 192 for blood selenium and serum iodine responses in ewes treated with 3 ml or 4 ml dose;

• calculations compare AUC from Day 99 through Day 192 for milk selenium and iodine responses, and for lamb blood selenium and lamb serum iodine responses.

, . .. . . . . (AUC of P3 - AUC of PO)

An example of the generic formula is: -) (

(AUC of P4- AUC of PO)

Table 5 below summarises the animal responses that are apparent from the 3 ml dose results shown in Figures 2, 4, 6, 8, 10 and 12.

The measures of iodine response in ewes and lambs scaled more-or-less with dose size. However, selenium response in the pasture-fed ewes (Group P3) unexpectedly did not scale with smaller dose in that a 3 ml dose was just as potent as a 4 ml dose, as shown by measurements of ewe blood selenium concentrations for Group P3 versus Group P4.

A follow-on effect of the unexpectedly high ewe blood selenium level was that ewe milk selenium levels and lamb blood selenium levels were also unexpectedly higher than that anticipated.

These results show that a user can expect comparable results for smaller

dose sizes as well as a preferred 4 ml dose. This is particularly the case if ewes treated are less deficient in selenium and iodine at treatment time.

Table 5 - Comparisons of the AUC of Groups P3 vs. P4, and Groups K3 vs. K4 over the entire experiment duration (i.e. to Day 192)

Relative response

Group Analysis (AUC of 3 ml / AUC of 4 ml)

P3 vs. P4 Ewe blood selenium (nmol/L) 101 %

K3 vs. K4 Ewe blood selenium (nmol/L) 52%

P3 vs. P4 Ewe serum iodine (μg/L) 61%

K3 vs. K4 Ewe serum iodine (μg/L) 60%

P3 vs. P4 Milk selenium (nmol/L) 109%

K3 vs. K4 Milk selenium (nmol/L) 91%

P3 vs. P4 Milk iodine (μg/L) 50%

K3 vs. K4 Milk iodine (μg/L) 87%

P3 vs. P4 Lamb blood selenium (nmol/L) 96%

K3 vs. K4 Lamb blood selenium (nmol/L) 65%

P3 vs. P4 Lamb serum iodine (μg/L) 53%

K3 vs. K4 Lamb serum iodine (μg/L) 65%

Experiment 2 Results - Treatment Timing and Carry-over to Lambs

Treating ewes during pregnancy (near scanning time about the end of the first trimester) with 60 mg selenium as barium selenate in a 4 ml dose of the invention formulation markedly increased selenium concentrations in the blood of ewes to >250 nmol/L for at least 192 days, and in lambs from birth to at least weaning.

Selenium from the invention formulation readily crosses the placenta, as demonstrated by the selenium status of new-bom lambs from treated ewes being much higher than lambs from control ewes. Similarly, selenium from

the invention formulation is readily secreted into milk and therefore the selenium status of weaned lambs from treated ewes was higher than lambs from control ewes.

In terms of iodine levels, treating ewes during pregnancy (near scanning time about the end of the first trimester) with 400 mg iodine as an iodised oil in a 4 ml dose of the invention formulation significantly increased serum iodine concentrations of the treated ewes 1.4 to 6-fold over the untreated ewes for at least 161 days, regardless of whether they were fed pasture or kale pre- lambing.

Like selenium, iodine from the invention formulation also readily crosses the placenta as serum iodine concentrations of the new-born lambs from treated ewes were 2.4 to 3.8 fold greater than those from untreated ewes for at least 60 days. Iodine was also secreted into milk and, regardless of prelambing diet, the miik from treated ewes at 24-36 hour post-partum contained 15-21 times as much iodine as that from untreated ewes.

Among treated ewes there was significantly less perinatal mortality (Table 3) and significantly lower incidence of goitre in new-born lambs (Table 4).

The invention formulation can therefore be administered with confidence during pregnancy to ewes to increase the selenium and iodine status of ewes and lambs from selenium deficient flocks, thus ensuring good lamb growth and reduced incidence of goitre in new-born lambs.

Although not tested during this study, it should be appreciated by those skilled in the art that it would be reasonable from these results that treatment pre-mating would also increase ewe selenium status, which is an important management strategy to ameliorate ewe infertility related to trace element nutritional insufficiency.

Experiment 2 - Conclusions

This dose response study evaluated the invention formulation containing selenium and iodine administered to pregnant ewes grazing selenium and

iodine deficient pastures and/or fed brassica kale during the latter half of gestation.

The results show that:

> treatment with 3 ml or 4 ml of invention formulation significantly and proportionally increased selenium and iodine status of ewes and their lambs.

> Treatment during early gestation with 4 ml to provide 60 mg selenium and 400 mg iodine reduced deficiency-associated perinatal mortality and reduced the incidence of goitre in lambs.

> There was no evidence of interaction between the release rates of selenium and iodine.

> Although not tested during this study, it is reasonable to assume that treatment of ewes pre-mating would also increase ewe iodine and selenium status regardless of the pre-lambing diet of pasture or brassica.

Experiment 3 - Assessing and Predicting Iodine Deficiency

In contrast to selenium, marginal iodine deficiency is not readily diagnosed, measurements to assess iodine status of sheep are still poorly defined, and robust biochemical criteria are needed to identify flocks that will respond to iodine supplementation. Although reference ranges for one such biomarker, concentrations of thyroid iodine hormones T ₄ and T ₃ , have been published these are not satisfactory as they have not been directly associated with improved reproductive performance or with the presence or absence of goitre in new-born animals (Clark et al l 998). Indeed, a recent iodine supplementation study with ewes (Grace et al 2001) showed that there was no change in serum T ₄ and T ₃ concentrations due to treatment with iodine.

It would therefore be desirable to have a better method of determining

whether or not iodine supplementation is required. Measurements might be made in ewes or in lambs. In the latter case, advise for supplementation would be relevant to the following year's season of pre-mating, gestation and lambing.

In this experiment, methods of determining the need to supplement with iodine are shown based on:

• evaluating flock iodine status pre-lambing, as measured by iodine concentrations in ewe serum during gestation;

• quantifying the risk of goitre in next year's lambs, as measured by thyroid : birthweight ratios of dead new-born lambs.

Experiment 3 Methods

Data was collected from two separate farm trials of sheep treated or not treated with injectable iodine supplements then fed exclusively pasture or pasture plus goitrogenic brassica forages (n=89 or 350 per treatment group).

Serum iodine concentrations in pregnant ewes were measured during early and late gestation. The incidence of enlarged thyroid glands (goitre) and thyroid to birthweight ratios (as g/kg) in dead new-born lambs was determined by necropsy.

The relationship of goitre and ewe iodine supplementation was calculated by probit analysis of the ratios versus presence or absence of supplementation.

Experiment 3 Results

Ewes from supplemented flocks had higher serum iodine concentrations at early gestation than did ewes not receiving supplements (155 vs 44 μg/L, p<0.001 ), and lamb thyroid-.birthweight ratios were smaller (30 vs 1.6 g/kg; p<0.001).

For all iodine-supplemented groups, ewe serum iodine concentrations at early gestation were uniformly high at 155 + 13 μg/L (n=40) and not different

by farm or forage (data not shown). The thyroid:birthweight ratios in lambs from those groups were consistently low at 0.30 ± 0.01 g/kg (n=110).

For the unsupplemented groups of ewes fed exclusively pasture or pasture followed by a goitrogenic brassica forage during the latter half of gestation, the mean (± SEM) serum iodine concentrations and lamb thyroid:birthweight ratios are shown in Table 6. The group with serum iodine >60 μg/L had thyroid :birthweight ratios of 0.21 ± 0.01 g/kg. In contrast, the three groups with ewe serum iodine <50 μg/L during gestation had lambs with thyroid:birthweight ratios >0.70 g/kg. (Note that the number of lambs assessed is not a comparison of rates of perinatal mortality between treatment groups.)

Table 6 - Thyroid.birthweight ratios of necropsied new-born lambs and serum iodine concentrations of ewes from unsupplemented groups

Farm location Farm A Farm B

Forage ^a Brassica Pasture Brassica Pasture

Lambs n 24 27 34 34 thyroid:birthweight ratio g/kg 2.1 ± 0.4 0.70 ± 0.14 2.0 ± 0.4 0.21 ± 0.01

Ewe serum iodine ^b at early gestation ⁰ μg/L 41 ± 4 40 ± 2 37 + 3 61 ± 4 at late gestation ^d μg/L 49 + 7 39 ± 2 47 ± 2 93 ± 7 ^a The brassica forage crop was kale (Brassica oleracea spp. acephala) at Rangitikei and swedes

(Brassica napus spp. napobrassica or rapifera) at Southland. ^b Serum was measured in ten monitor ewes per treatment group. ^c Serum was collected at ultrasound scanning time, 52-80 days after joining with the ram. ^d Serum was collected at 21-28 pre-lambing or at lambing time.

The thyroid:birthweight results are summarized in Figure 13 which shows the distribution of thyroid to birthweight ratios for necropsied lambs born to six groups of ewes (lower panel) . The ewes were fed exclusively pasture (<>,♦) or pasture followed by a goitrogenic brassica forage during the latter half of gestation (D ₁ B). Each symbol represents a lamb that came from a ewe

which had received supplemental injectable iodine (♦,■) or was unsupplemented (O, D). The probability of a flock response to iodine treatment is predicted by a cumulative distribution curve flanked by 95% confidence intervals (upper panel).

Ratios greater than 0.5 g/kg were statistically associated with goitre in lambs, while ratios greater than 0.8 g/kg indicated with 90% probability that the ewes had inadequate iodine status and would have responded to iodine supplementation in terms of preventing lamb goitre.

The above results therefore show that ewe serum iodine concentrations measured during early gestation can identify low iodine status in time to treat ewes prior to mid-gestation and the thyroid:birthweight ratio of dead newborn lambs can be used to predict a flock's response to iodine supplementation for the following year. More specifically, ewe serum iodine concentration of <50 μg/L or lamb thyroid to birthweight ratio levels of >0.8 g/kg indicates an iodine deficiency in a flock and need for supplementation.

It should be appreciated from the above experiments that there is provided a formulation that delivers both iodine and selenium in one vehicle and which, when administered pre-mating or during the first half of pregnancy, maintains animal iodine and selenium levels well above unsupplemented levels for at least between approximately 160 to approximately 200 days. The supplementation effect transfers from the treated animal to progeny as indicated by element concentrations in the animal milk and progeny blood and serum.

The above formulation overcomes difficulties of the prior art by providing a convenient one step formulation, is simple to manufacture, uses simple ingredients, and is stable and efficacious.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

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Grace ND, Knowles SO, Sinclair, GR. 2001. Effect of pre-mating iodine supplementation of ewes fed pasture or a brassica crop prelambing on the incidence of goitre in newborn lambs. Proc. NZ Society of Animal Production. 61 :164-167.

Sinclair DP, Andrews ED. 1954. Goitre in new-borne lambs. New Zealand Veterinary Journal 2:72-79.

Stoewsand GS. 1995. Bioactive organosulfur phytochemicals in Brassica oleracea vegetables— a review. Food and Chemical Toxicology 33:537-543.

Watkinson JH. 1979. Semi-automated fluorometric determination of nanogram quantities of selenium in biological materials. Analytica Chemica Acta 105:319-325.