阅读说明：本技术 妊娠非人哺乳动物中的贫血的治疗或预防 (Treatment or prevention of anemia in pregnant non-human mammals ) 是由 克里斯蒂安·冯德尔·雷克 托比亚斯·S·克里斯滕森 汉斯·安德烈亚森 拉尔斯·吕克·汤姆森 于 2015-10-27 设计创作，主要内容包括：本发明涉及一种在增加妊娠非人哺乳动物中的血液血红蛋白浓度的方法中使用的铁碳水化合物复合物,其中对具有105g/L以下的血液血红蛋白水平的所述妊娠非人哺乳动物施用一个或多个剂量的包含1800mg以上/剂量的元素铁的量的铁碳水化合物复合物。所述方法涉及以下另外的效果：降低来自具有105g/L以下的血液血红蛋白水平的妊娠非人哺乳动物的死产后代率,增大出生3天内和/或断奶时后代幼崽的血液血红蛋白浓度,或增大具有105g/L以下的血液血红蛋白水平的妊娠非人哺乳动物的后续产次中的窝产仔数。(The present invention relates to an iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration in a pregnant non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to said pregnant non-human mammal having a blood hemoglobin level of 105g/L or less. The method involves the following additional effects: reducing the rate of stillbirth offspring from the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less, increasing the blood hemoglobin concentration of offspring pups within 3 days of birth and/or at weaning, or increasing litter size in subsequent parity of the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.)

1. An iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration in a pregnant non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

2. An iron carbohydrate complex for use in a method of reducing the rate of stillbirth offspring from a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less is administered one or more doses of an iron carbohydrate complex in an amount comprising 1800mg or more per dose of elemental iron.

3. An iron carbohydrate complex for use in a method of increasing the blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

4. An iron carbohydrate complex for use in a method of increasing litter size in a subsequent parity of a non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

5. An iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 200mg per dose of elemental iron is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

6. An iron carbohydrate complex for use in a method of increasing litter size in a subsequent parity of a non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising 200mg or more per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

7. An iron carbohydrate complex for use in a method of increasing the rate of live offspring from a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less is administered one or more doses of an iron carbohydrate complex at a dose of 6mg/kg BW (body weight) or more, wherein the non-human mammal is a pig.

8. An iron carbohydrate complex for use in the method of increasing blood hemoglobin concentration of offspring pups according to claim 5 or in the method of increasing litter size in subsequent parity according to claim 6, wherein the dose of iron carbohydrate complex comprises an amount of elemental iron of 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg or more.

9. The iron carbohydrate complex for use in a method according to any of claims 1 to 8, wherein said non-human mammal is a pig.

10. The iron carbohydrate complex for use in a method according to any of claims 1 to 9, wherein the dose of iron carbohydrate complex is administered parenterally.

Technical Field

The present invention relates to the treatment of anemia (anaemia) in pregnant non-human mammals using iron carbohydrate complexes (iron carbohydrate complexes). The present invention generally improves hemoglobin levels and/or parameters measured on offspring, such as mortality, mortality during lactation, increase in hemoglobin levels, and growth rate during lactation in non-human mammals.

Prior Art

Several studies have shown that anemia is very common in sows, although it is rarely diagnosed in veterinary practice. Recent studies in denmark show that 59% of sows in a large commercial denmark herd have blood hemoglobin (Hb) concentrations below 110g/l (Jensen et al, 2014), which is considered the lower reference value for pregnant sows (Thorn, 2010).

The most common cause of anemia is iron deficiency. Because iron is involved in oxygen transport, the low iron state will have a variety of visible and invisible effects on the health and production of sows and their offspring litters, both in the synthesis of DNA and in many other processes that maintain the normal structure and function of the cells (Morris et al, 1995).

It has recently been shown that high maternal hemoglobin levels during pregnancy are associated with a reduced stillbirth piglet rate (Jensen et al, 2014). The relationship between hemoglobin levels and dead yields in sows has been studied earlier in sows that are less fertile than the current breed (Archibald and Hancock, 1939, Moore et al, 1965, Zaleski and Hacker, 1993).

It has also been shown that the stillborn piglets have lower hemoglobin values than the live piglets (Svetina et al, 2006; Zaleski and Hacker, 1993), and that 75% of the stillborn piglets die during labour (Glastonbury, 1977; Leenhouwers et al, 1999). In addition, hemoglobin levels in sows were correlated with those of offspring piglets (Jensen and Nielsen, 2013). The most obvious explanation for the observed effect of high hemoglobin levels on mortality rates is the reduced risk of hypoxia in sows and piglets and the increased vitality of piglets during delivery. Thus, improvement of hemoglobin levels in sows may open an effective means of reducing stillbirth.

In denmark, the stillbirth losses averaged 1.7 piglets/litter (Vinther, 2013) and thus presented serious economic and welfare problems in pig production. Herd intervention to reduce the number of stillbirths is often difficult to apply and may only contribute rarely to infectious or regulatory factors.

The standard procedure is to supplement piglets with iron during lactation and the industry standard is to inject 200mg of elemental iron during the early part of life. Until the resulting hemoglobin production was achieved, piglets relied solely on iron obtained from the placenta in the uterus of sows during pregnancy.

Attempts to increase hemoglobin levels in sows by injecting a 2g amount of Gleptosil 7 and 4 weeks before the expected farrowing were unsuccessful (Auvigne, 2009). For sows having 107.7 to 117.3g/L hemoglobin in the blood, parenteral administration of iron carbohydrate does not result in an increase in blood hemoglobin concentration.

Piglets born at high hemoglobin levels have an increased chance of survival until weaning and it is an object of the present invention to provide pregnant sows with an iron supplement which can increase the hemoglobin level of the sow and/or improve the parameters measured on offspring litters.

Disclosure of Invention

The present invention relates to an iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration in a pregnant non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to said pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

In another aspect, the invention relates to an iron carbohydrate complex for use in a method of reducing the rate of stillbirth and offspring from a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less is administered one or more doses of an iron carbohydrate complex in an amount comprising elemental iron of 1800mg or more per dose.

In a further aspect, the invention relates to an iron carbohydrate complex for use in a method of increasing the blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

In a further aspect, the invention relates to an iron carbohydrate complex for use in a method of increasing litter size in a subsequent parity of a non-human mammal, wherein one or more doses of the iron carbohydrate complex comprising an amount of elemental iron above 1800mg per dose are administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

Iron deficiency anemia during pregnancy is considered a common problem in most mammals due to increased loss of blood volume and altered metabolism associated with fetal growth. It has been studied in detail and is commonly treated in humans. Iron deficiency anemia is most well understood in pigs in non-human mammals, and most work has focused on iron deficiency anemia in piglets on a full-milk diet, where this condition is endemic. Unlike most other non-human mammals, iron deficiency anemia and its treatment have also been studied in pregnant sows, and the present invention is therefore based on sows as an example of pregnant non-human mammals. Although the invention is described herein primarily using pigs as an exemplary non-human mammal, it will be apparent to those skilled in the art that the invention may be carried out on any non-human mammal. In a certain aspect of the invention, the non-human mammal is a pig, horse, camel, sheep, goat or cow.

An early warning that blood hemoglobin levels fall below 105g/L is a reduced ferritin concentration level, since ferritin levels represent the iron stores of the organism. The present invention therefore also encompasses prophylactic treatment of a pregnant non-human mammal with a low ferritin level predicting a blood hemoglobin level to be reduced below 105g/L if the pregnant non-human mammal is not treated according to the methods of the present invention.

The dose in the context of the present invention may be a single administration or more than two administrations on the same day. Alternatively, two or more administrations may be administered to the non-human mammal over a period of 5 days or less, such as 4, 3, or 2 days. Each administration may be provided by enteral or parenteral routes. In a preferred aspect of the invention, the dose is administered parenterally. Preferred parenteral administration is by injection or infusion. In a certain embodiment, two injections or infusions are administered on the same day to each side of the neck of the non-human mammal.

According to the invention, one or more doses, such as two, three, four, five, six or more doses, are administered to the non-human mammal during pregnancy. Furthermore, when more than two doses are administered, the time interval between each dose is more than 1 week, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more. When two doses are administered, the first dose is administered 15 to 3 weeks before the expected parity and the second dose is administered 8 to 1 week before the expected parity.

It is believed that the iron carbohydrate complexes of the present invention increase blood hemoglobin in anemic non-human mammals, such as sows. This is important in itself as it improves the health and overall well-being of the sow. Furthermore, in certain embodiments, the inventors of the present invention have now found that administration of the iron carbohydrate complexes of the present invention to anemic pregnant sows also has an effect on the offspring of sows. Thus, when the iron carbohydrate complex is administered to a pregnant poor sow one or more times prior to farrowing, administration will also increase the blood hemoglobin level of the offspring piglets.

As a first effect, administration to sows will reduce the stillbirth piglet rate. Stillbirth means lung inflation (inflate). In another embodiment, the ratio of mummified piglets is decreased. Mummified piglets are defined as piglets in which the lungs are not inflated. Thus, a higher percentage of pups will be live. When the term litter size is used, it is to be understood as the total size or number of stillborn and live piglets. In addition, administration to sows may increase the blood hemoglobin concentration, survival, health, and/or growth of offspring litters until weaning. In a preferred aspect of the invention, the average stillbirth progeny rate is less than 12%, such as 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less. In another aspect of the invention, the average survival rate up to weaning is 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The average survival rate was calculated based on live piglets (i.e. the total number of piglets minus dead or mummified piglets) and piglets surviving at weaning.

Thus, more offspring from a litter will survive from farrowing until weaning compared to the case where no iron carbohydrate complex is administered to a pregnant anemia sow. Furthermore, at weaning the offspring will be healthier, e.g. expressed in health parameters such as increased weight gain or higher blood hemoglobin concentration. When the iron carbohydrate complex is administered to a pregnancy-anemic sow, the offspring will gain more body weight and/or higher blood hemoglobin concentration by weaning than if the iron carbohydrate complex was not administered to a pregnancy-anemic sow. In a certain embodiment, the inventors of the present invention have observed that when an iron carbohydrate complex is administered to pregnant anaemic sows according to the present invention, the need for enteral or parenteral administration of additional iron to piglets during lactation is reduced. Thus, the present invention simplifies the work of pig breeders and improves the overall well-being of both sows and offspring.

With the treatment of the invention, the average litter size can be increased to a level in which the average litter size is 13 or more, such as 14, 15, 16, 17 or more, in subsequent parity. Furthermore, the mean hemoglobin concentration in pregnant sows is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more. Furthermore, in a preferred aspect of the invention, the average stillbirth generation rate is reduced by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more. In a certain embodiment of the invention, the mean blood hemoglobin concentration of the offspring pups increases by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more, up to weaning. In one aspect of the invention, the average litter size of the pregnant sow in subsequent parity increases by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

Sows treated according to the present invention may be in various health conditions as reflected by back fat thickness (back fat thickness). In a preferred aspect, the sow has a backfat thickness of 17mm, 16mm, 15mm, 14mm, 13mm, 12mm, 11mm, 10mm or less. Likewise, a sow may already have one or more parity; preferably, the parity of the sows is 2, 3, 4,5, 6, 7, 8, 9 or higher for the treated sows.

In the context of the present invention, the iron carbohydrate complex is iron ions or comprises Fe suitable for administration to pigs³⁺And/or Fe²⁺Any complex of iron particles with a carbohydrate. In a certain aspect of the invention, it is preferred that the iron carbohydrate complex may penetrate the placenta of the sow and thus enter the fetus. Iron carbohydrate complexes are well known to the skilled person and the specific choice of suitable iron carbohydrate complexes is within the knowledge of the skilled person. In a preferred embodiment, the iron carbohydrate complex is formulated as an injectable veterinary composition having a content of elemental iron complexed with a carbohydrate in the interval 5-25% (w/v), e.g. 50-250 g/L. The veterinary composition may comprise adjuvants conventionally used in the art. In addition, veterinary compositions comprising iron carbohydrate complexes may or may not contain a preservative (preservative), such as phenol in an amount of 1-10 g/L. In a certain embodiment, the amount of phenol is less than 1 g/L. Disinfection is obtained in an alternative manner if required.

The iron carbohydrate complex may be selected from a wide variety of different substances. Preferably, the iron carbohydrate complex is selected from the group comprising: iron carboxymaltose, iron polyglucose sorbitol carboxymethyl ether complex, iron mannitol complex, iron dextran, iron hydrogenated dextran (iron hydrogenated dextran), carboxyalkylated reduced oligo-and polysaccharides, iron sucrose, iron gluconate (iron gluconate), iron dextrin, iron hydrogenated dextrin, iron polymaltose, iron hydrogenated polymaltose, iron polyisomaltose, iron hydrogenated polyisomaltose, iron sugar complex (iron saccharoide complex), ferric pyrophosphate, iron sorbitol, glucoheptanoic acid, oxidized dextrin, oxidized dextran, oxidized oligo-and polysaccharides or mixtures thereof. In a certain embodiment, the iron is complexed with dextran glucoheptonic acid. This iron carbohydrate complex is also known as dextran iron oxide (Gleptoferron) and is available as Gleptosil (alsote Limited Animal Health, York, GB) and ursoflerran (Serumwerk Bernburg AG, Bernburg, D). In another embodiment, the iron is complexed with hydrogenated dextran. Commercial iron-hydrogenated glucan complexes are available from Pharmacosmos A/S, Holbaek, Denmark as Uniferon, CosmoFer, MonoFer, and DiaFer.

The carbohydrate component of the iron carbohydrate complexes of the present invention may have any suitable molecular weight. It is generally preferred to use a carbohydrate component having a molecular weight wherein the carbohydrate component of the iron carbohydrate complex has a weight average Molecular Weight (MW) of 800 to 80,000 daltons, preferably 800 to 10,000 daltons. A weight average molecular weight within this range can be more easily delivered from a pregnant sow to a fetus. In a preferred aspect, the apparent molecular weight of the iron carbohydrate complex is an apparent molecular weight of 500.000 daltons or less, such as 400.000 daltons or less.

The dosage of iron carbohydrate complexes is generally calculated on the basis of elemental iron and is generally in the range 1800mg to 10.000 mg. In a certain embodiment, the dose is 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg 4100mg, 4200mg, 4300mg, 4400mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 53000 mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7300mg, 7400mg, 7900mg, 9500mg, 8400mg, 9600mg, 9700mg, 9200mg, 9600mg, 9700mg, 9600mg, 9700mg, 9600mg, 9700mg, 9600mg, 96, 10000mg or more. In certain cases, it may be suitable to administer a dose of 1650mg, such as 1700mg, such as more than 1750mg of elemental iron.

Dosages within this range will be applicable to sows of typical size (size), for example having a Body Weight (BW) of about 250 kg. Because sow weight varies with breed and nutrition, in some embodiments the dose may be better expressed based on sow weight. Thus, the dosage of iron (i.e., elemental iron) can range from 6mg/kg BW to 30mg/kg BW, e.g., 7mg/kg BW, 8mg/kg BW, 9mg/kg BW, 10mg/kg BW, 11mg/kg BW, 12mg/kg BW, 13mg/kg BW, 14mg/kg BW, 15mg/kg BW, 16mg/kg BW, 17mg/kg BW, 18mg/kg BW, 19mg/kg BW, 20mg/kg BW, 21mg/kg BW, 22mg/kg BW, 23mg/kg BW, 24mg/kg BW, 25mg/kg BW, 26mg/kg BW, 27mg/kg BW, 28mg/kg BW, 29mg/kg BW, 30mg/kg BW, or more.

The iron carbohydrate complexes of the present invention are suitable for use in a method wherein the iron carbohydrate complexes are injected into sows having a blood hemoglobin level of 105g/L or less. However, it is expected that the iron carbohydrate complex will have the desired effect on all sows, regardless of blood hemoglobin levels. Thus, it is expected that the iron carbohydrate complex may also be effective if the sow has a blood hemoglobin level below about 110g/L, such as 109g/L, 108g/L, 107g/L, 106g/L or lower. Typically, the treated sow has a blood hemoglobin level of 105g/L or less, such as 104g/L, 103g/L, 102g/L, 101g/L, 100g/L, 95g/L, 90g/L, 85g/L, 80g/L, 75g/L, 70g/L, 65g/L, 60g/L, 55g/L, 50g/L, 45g/L, 40g/L or less. Sows with blood hemoglobin at these levels may be generally referred to as "anemic".

Sows to be treated with the iron carbohydrate complex are usually of the second parity or higher. It is generally believed that the blood hemoglobin level of sows will decrease with increasing parity (Normand et al, 2012) and the inventors of the present invention have surprisingly found that the iron carbohydrate complexes of the present invention are particularly effective when administered to sows of a second or higher parity, such as a third parity, a fourth parity, a fifth parity, a sixth parity, a seventh parity, an eighth parity, a ninth parity or higher. However, it is expected that the iron carbohydrate complex will also be effective if administered to anemic sows in the first parity.

In a preferred aspect of the invention, the iron carbohydrate complexes of the invention are used for parenteral administration by injection, for example Intramuscular (IM), Subcutaneous (SC) or Intravenous (IV) injection. IM injections are preferred, optionally as bolus injections (bolus injections). The expected time of farrowing is well known to the skilled person and may be calculated from the date of conception or pregnancy. The determination of the timing of administration is therefore calculated from the date of conception or pregnancy.

In order to obtain optimal effect on piglets it is generally believed that the final dose should be administered at the latest 1 week, preferably 2-4 weeks before farrowing. Within the last 20 days of pregnancy, the weight of the fetus is expected to double. The first dose should typically be delivered 15 weeks prior to farrowing. However, the above indications are only instructive and an iron carbohydrate complex administered outside the preferred period of 15 to 2 weeks prior to farrowing may provide a valuable effect.

The iron carbohydrate complex dose, such as two, three, four, five, six or more doses, is administered to the pregnant anaemic sow at one or more time points during the pregnancy period, and preferably over a period of 15 weeks to 2 weeks prior to farrowing in the sow. Thus, at each administration, 1,800mg to 10,000mg of elemental iron formulated as an iron carbohydrate complex is administered (e.g., IM, SC, or IV) to a pregnant anemia sow. The dosage at each administration may be the same or the dosage may be different. For example, the first injection may be greater than the second injection, such as greater than 500mg, 1000mg, 1500mg, 2000mg, or vice versa. Usually, two doses administered within a period of 9 to 3 weeks prior to farrowing are sufficient. The time interval between each dose may be 1 week or more, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more.

The iron carbohydrate complex may be administered in conjunction with other veterinary drugs. Another example of a veterinary drug type includes vaccines, such as PRRS vaccines. Antibiotics may also be administered in conjunction with the iron carbohydrate complex. Examples of antibiotics include amikacin (amikacin), aminopenicillin (aminopenicillin), amoxicillin (amoxicillin), azithromycin (azithromycin), cephalosporin (ceohalospirin), ciprofloxacin (ciprofloxacin), clindamycin (clindamycin), doxycycline (doxycline), enrofloxacin (enrofloxacin), erythromycin (erythromycin), penicillin (penicilin), gentamicin (gentamicin), kanamycin (kanamycine), lincomycin (comilinycin), marbofloxacin (marbofloxacin), metronidazole (metronidazole), novobiocin (novobiocin), orbifloxacin (orbifloxacin), penicillin g (penicilin g), penicillin (penicillinase-resistance), penicillinase (penicillinase-resistance), methicillin (methicillin), and tetramethosin (trimethoprim). In a certain aspect, the iron carbohydrate complex is administered in conjunction with erythropoietin (erythropoetine) to stimulate the production of blood cells.

The total amount of iron carbohydrate complexes calculated as elemental iron is at least 1,800mg of elemental iron or more. In a preferred embodiment, the total amount of elemental iron is 2,000mg, 2,500mg, 3,000mg, 3,500mg, 4,000mg, 4,500mg, 5,000mg, 6,000mg, 7,000mg, 8,000mg, 9,000mg, 10,000mg of elemental iron or higher. It is also contemplated that the total amount of iron may be divided into multiple administrations, some of which may contain less than 1800 mg/L.

Exemplary embodiments

1. An iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration in a pregnant non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

2. An iron carbohydrate complex for use in a method of reducing the rate of stillbirth offspring from a pregnant non-human mammal, wherein the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less is administered one or more doses of an iron carbohydrate complex in an amount comprising 1800mg or more per dose of elemental iron.

3. An iron carbohydrate complex for use in a method of increasing the blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

4. An iron carbohydrate complex for use in a method of increasing litter size in a subsequent parity of a non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 1800mg per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

5. An iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising more than 200mg per dose of elemental iron is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

6. An iron carbohydrate complex for use in a method of increasing litter size in a subsequent parity of a non-human mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising 200mg or more per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

7. An iron carbohydrate complex for use in the method of increasing blood hemoglobin concentration of a offspring pup of item 5 or in the method of increasing litter size in a subsequent parity of item 6, wherein the dose of iron carbohydrate complex comprises an amount of elemental iron of 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg or more.

8. The iron carbohydrate complex for use in a method according to any of claims 1 to 7, wherein the non-human mammal is a pig, horse, camel, sheep, goat or cow.

9. The iron carbohydrate complex for use in a method according to any one of claims 1 to 8, wherein the non-human mammal is a pig.

10. The iron carbohydrate complex for use in a method according to any of claims 1 to 9, wherein the dose of iron carbohydrate complex is administered parenterally.

11. The iron carbohydrate complex for use in the method according to any one of claims 1 to 10, wherein the blood hemoglobin level of the non-human mammal is 104g/L, 103g/L, 102g/L, 101g/L, 100g/L, 95g/L, 90g/L, 85g/L, 80g/L, 75g/L, 70g/L, 65g/L, 60g/L, 55g/L, 50g/L, 45g/L, 40g/L or lower.

12. The iron carbohydrate complex for use in a method according to any one of claims 1 to 11, wherein the sow has a backfat thickness of 17mm, 16mm, 15mm, 14mm, 13mm, 12mm, 11mm, 10mm or less.

13. The iron carbohydrate complex for use in the method according to any one of claims 1 to 12, wherein the non-human mammal has a parity of 2, 3, 4,5, 6, 7, 8, 9 or higher.

14. The iron carbohydrate complex for use in a method according to any one of claims 1 to 13, wherein the dose is 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4200mg, 4300mg, 4400mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7900mg, 7400mg, 7900mg, 8400mg, 7900mg, 8400mg, 7900mg, 8400mg, 7900mg, 4700mg, 6400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8200mg, 4700mg, 8400mg, 4700mg, 6400mg, 4700mg, 8200mg, 6400mg, 4700mg, 6400mg, 8200mg, 4700mg, 8200mg, 4700mg, 8200mg, 4700mg, 8200mg, 4700mg, 8400mg, 4700mg, 6400mg, 4700mg, 8400mg, 4700mg, 6400mg, 4700mg, 6400mg, 47, 9200mg, 9300mg, 9400mg, 9500mg, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg or more.

15. The iron carbohydrate complex for use in the method according to any one of claims 1 to 14, wherein one or more doses, such as two, three, four, five, six or more doses, are administered to the non-human mammal during pregnancy.

16. The iron carbohydrate complex for use in a method according to any one of claims 1 to 15, wherein when more than two doses are administered, the time interval between each dose is more than 1 week, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more.

17. The iron carbohydrate complex for use in a method according to any one of claims 1 to 16, wherein two doses are administered, the first dose being administered 15 to 3 weeks before the expected parity and the second dose being administered 8 to 1 week before the expected parity.

18. The iron carbohydrate complex for use in the method according to any one of items 1 to 17, wherein the dose is 6mg/kg BW (body weight) or more, such as 7mg/kg BW, 8mg/kg BW, 9mg/kg BW, 10mg/kg BW, 11mg/kg BW, 12mg/kg BW, 13mg/kg BW, 14mg/kg BW, 15mg/kg BW, 16mg/kg BW, 17mg/kg BW, 18mg/kg BW, 19mg/kg BW, 20mg/kg BW, 21mg/kg BW, 22mg/kg BW, 23mg/kg BW, 24mg/kg BW, 25mg/kg BW, 26mg/kg BW, 27mg/kg BW, 28mg/kg BW, 29mg/kg BW, 30mg/kg BW or higher.

19. The iron carbohydrate complex for use in the method according to any one of claims 1 to 18, wherein the average stillbirth generation rate is 12% or less, such as 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

20. The iron carbohydrate complex for use in the method according to any one of claims 1 to 19, wherein the average survival rate up to weaning is above 80%, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

21. The iron carbohydrate complex for use in the method according to any one of claims 1 to 20, wherein the average litter size is 13 or more, such as 14, 15, 16, 17 or more.

22. The iron carbohydrate complex for use in the method according to any one of claims 1 to 21, wherein the mean hemoglobin concentration in pregnant sows is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

23. The iron carbohydrate complex for use in the method according to any one of claims 1 to 22, wherein the average stillbirth generation rate is reduced by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

24. The iron carbohydrate complex for use in the method of any one of claims 1 to 23, wherein the mean blood hemoglobin concentration of the offspring pups up to weaning is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

25. The iron carbohydrate complex for use in the method according to any one of claims 1 to 24, wherein the average litter size of the pregnant sow in subsequent parity is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

26. The iron carbohydrate complex for use in a method according to any of claims 1 to 25, wherein said iron carbohydrate complex is selected from the group comprising: iron carboxymaltose, iron polyglucose sorbitol carboxymethyl ether complex, iron mannitol complex, iron dextran, iron hydrogenated dextran, carboxyalkylated reduced oligosaccharides and polysaccharides, iron sucrose, iron gluconate, iron dextrin, iron hydrogenated dextrin, iron polymaltose, iron hydrogenated polymaltose, iron polyisomaltose, iron hydrogenated polyisomaltose, iron saccharine complex, iron pyrophosphate, iron sorbitol, glucoheptanoic acid, dextrin oxide, dextran oxide, or mixtures thereof.

27. The iron carbohydrate complex for use in a method according to any one of claims 1 to 26, wherein said iron carbohydrate complex is an iron hydrogenated dextran.

28. The iron carbohydrate complex for use in a method according to any of claims 1 to 27, wherein the carbohydrate component of the iron carbohydrate complex has a weight average Molecular Weight (MW) of 800 to 80,000 dalton, preferably 800 to 10,000 dalton.

29. The iron carbohydrate complex for use in a method according to any of claims 1-28, wherein the apparent molecular weight of the iron carbohydrate complex is an apparent molecular weight of 500.000 dalton or less, such as 400.000 dalton or less.

30. The iron carbohydrate complex for use in a method according to any one of claims 1 to 29, wherein the parenteral administration is selected from the group consisting of: intramuscular (IM), Subcutaneous (SC), bolus injection, and Intravenous (IV) injection or infusion.

31. The iron carbohydrate complex for use in the method according to any one of items 1 to 30, wherein the total amount of elemental iron is 2,000mg, 2,500mg, 3,000mg, 3,500mg, 4,000mg, 4,500mg, 5,000mg, 6,000mg, 7,000mg, 8,000mg, 9,000mg, 10,000mg of elemental iron or higher.

32. The iron carbohydrate complex for use in a method according to any of claims 1 to 31, wherein the amount of reproductive toxic preservative in a formulation comprising the iron carbohydrate complex is 1g/L or less.

33. The iron carbohydrate complex for use in a method according to item 32, wherein the reproductive toxic preservative is phenol.

34. The iron carbohydrate complex for use in a method according to any one of claims 1 to 33, wherein the dose is a unit dose.

35. The iron carbohydrate complex for use in a method according to any one of claims 1 to 34, wherein the dose is administered within 2 minutes or less.

36. The iron carbohydrate complex for use in the method according to any one of claims 1 to 35, wherein the iron carbohydrate complex is dissolved or suspended in an aqueous liquid optionally comprising one or more veterinarily acceptable adjuvants, forming a liquid veterinary composition.

37. The iron carbohydrate complex for use in a method according to any one of claims 1 to 36 wherein the concentration of the elemental iron of the iron carbohydrate complex in the liquid veterinary composition is in the range of 5g/100mL to 25g/100 mL.

38. A method of increasing blood hemoglobin concentration in a pregnant non-human mammal, wherein one or more doses of elemental iron in an amount of 1800mg or more per dose of an iron carbohydrate complex is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

39. A method of reducing the rate of stillbirth offspring from a pregnant non-human mammal, wherein one or more doses of elemental iron in an amount of 1800mg or more per dose of an iron carbohydrate complex are administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

40. A method for increasing the blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of elemental iron in an amount of 1800mg or more per dose of iron carbohydrate complex is administered to a pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

41. A method for increasing litter size in a pregnant non-human mammal during a subsequent parity, wherein one or more doses of elemental iron in an amount of 1800mg or more per dose of iron carbohydrate complex are administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

42. A method for increasing blood hemoglobin concentration of offspring pups within 3 days after birth and/or at weaning, wherein one or more doses of an iron carbohydrate complex in an amount comprising elemental iron above 200mg per dose is administered to said pregnant non-human mammal having a blood hemoglobin level below 105 g/L.

43. A method for increasing litter size in a subsequent parity of a mammal, wherein one or more doses of an iron carbohydrate complex in an amount comprising 200mg or more per dose of elemental iron is administered to the pregnant non-human mammal having a blood hemoglobin level of 105g/L or less.

44. The method for increasing blood hemoglobin concentration of offspring pups according to item 42 or the method for increasing litter size in a subsequent parity according to item 42, wherein the dose of iron carbohydrate complex comprises an amount of elemental iron of 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg or more.

45. The method according to any one of claims 38 to 44, wherein the non-human mammal is a pig, horse, camel, sheep, goat or cow.

46. The method according to any one of claims 38 to 45, wherein the mammal is a pig.

47. The method according to any one of claims 38 to 46, wherein the dose of iron carbohydrate complex is administered parenterally.

48. The method of any one of claims 38-47, wherein the blood hemoglobin level is 104g/L, 103g/L, 102g/L, 101g/L, 100g/L, 95g/L, 90g/L, 85g/L, 80g/L, 75g/L, 70g/L, 65g/L, 60g/L, 55g/L, 50g/L, 45g/L, 40g/L or lower.

49. The method according to any one of claims 38-48, wherein the sow has a backfat thickness of 17mm, 16mm, 15mm, 14mm, 13mm, 12mm, 11mm, 10mm or less.

50. The method of any one of claims 38-49, wherein the non-human mammal has a parity of 2, 3, 4,5, 6, 7, 8, 9 or higher.

51. The method of any one of claims 38 to 50, wherein the dose is 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg 4100mg, 4200mg, 4300mg, 4400mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7400mg, 7900mg, 7600mg, 9500mg, 8100mg, 8400mg, 9200mg, 7900mg, 8400mg, 7900mg, 8400mg, 7900mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700mg, 8400mg, 4700, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg or more.

52. The method according to any one of claims 38 to 51, wherein one or more doses, such as two, three or more doses, such as four or more doses, are administered to the non-human mammal during pregnancy.

53. The method according to any one of claims 38 to 52, wherein when two or more doses are administered, the time interval between each dose is 1 week or more, such as 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more.

54. The method of any one of claims 38-53, wherein two doses are administered, the first dose being administered 15 to 3 weeks before the expected parity and the second dose being administered 8 to 1 week before the expected parity.

55. The method according to any one of claims 38 to 54, wherein the dose is 6mg/kg BW (body weight) or more, e.g., 7mg/kg BW, 8mg/kg BW, 9mg/kg BW, 10mg/kg BW, 11mg/kg BW, 12mg/kg BW, 13mg/kg BW, 14mg/kg BW, 15mg/kg BW, 16mg/kg BW, 17mg/kg BW, 18mg/kg BW, 19mg/kg BW, 20mg/kg BW, 21mg/kg BW, 22mg/kg BW, 23mg/kg BW, 24mg/kg BW, 25mg/kg BW, 26mg/kg BW, 27mg/kg BW, 28mg/kg BW, 29mg/kg BW, 30mg/kg BW, or more.

56. The method according to any one of claims 38 to 55, wherein the average stillborn generation rate is 12% or less, such as 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

57. The method according to any one of claims 38 to 56, wherein the average survival rate up to weaning is 80% or more, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

58. The method according to any one of claims 38 to 57, wherein the average litter size is 13 or more, such as 14, 15, 16, 17 or more.

59. The method of any one of claims 38-58, wherein the average hemoglobin concentration in a pregnant sow is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

60. The method of any one of claims 38-59, wherein the average stillborn generation rate is reduced by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

61. The method of any one of claims 38-60, wherein the mean blood hemoglobin concentration of the offspring pups increases by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more, up to weaning.

62. The method according to any one of claims 38 to 61, wherein the average litter size of the pregnant sow in subsequent parity is increased by more than 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50% or more.

63. The method according to any one of claims 38 to 62, wherein the iron carbohydrate complex is selected from the group comprising: iron carboxymaltose, iron polyglucose sorbitol carboxymethyl ether complex, iron mannitol complex, iron dextran, iron hydrogenated dextran, carboxyalkylated reduced oligo-and polysaccharides, iron sucrose, iron gluconate, iron dextrin, iron hydrogenated dextrin, iron polymaltose, iron hydrogenated polymaltose, iron polyisomaltose, iron hydrogenated polyisomaltose, iron saccharine complex, iron pyrophosphate, iron sorbitol, glucoheptanoic acid, oxidized dextrin, oxidized dextran, oxidized oligo-and polysaccharides or mixtures thereof.

64. The method of any one of claims 38-63, wherein the iron carbohydrate complex is an iron hydrodextran.

65. The method according to any one of claims 38 to 64, wherein the carbohydrate component of the iron carbohydrate complex has a weight average molecular weight of from 800 to 50,000 daltons, preferably from 800 to 10,000 daltons.

66. The method according to any one of claims 38 to 65, wherein the apparent molecular weight of the iron carbohydrate complex is an apparent molecular weight of 500.000 daltons or less, such as 400.000 daltons or less.

67. The method according to any one of claims 38 to 66, wherein the parenteral administration is selected from the group consisting of: intramuscular (IM), Subcutaneous (SC) and Intravenous (IV) administration.

68. The method of any one of claims 38-67, wherein the total amount of elemental iron is 2,000mg, 2,500mg, 3,000mg, 3,500mg, 4,000mg, 4,500mg, 5,000mg, 6,000mg, 7,000mg, 8,000mg, 9,000mg, 10,000mg of elemental iron or higher.

69. The method according to any one of claims 38 to 68, wherein the amount of reproductive toxic preservative in the formulation comprising the iron carbohydrate complex is 1g/L or less.

70. The method of 69, wherein the reproductive toxic preservative is phenol.

71. The method of any one of claims 3887-70, wherein the dose is a unit dose.

72. The method according to any one of claims 38-71, wherein the dose is administered within 2 minutes or less.

73. The method of any one of claims 38 to 72, wherein the iron carbohydrate complex is dissolved or suspended in an aqueous liquid optionally comprising one or more veterinarily acceptable adjuvants to form a liquid veterinary composition.

74. The method according to any one of claims 38 to 73, wherein the concentration of the iron carbohydrate complex in the liquid veterinary composition is in the range of 5g/100mL to 25g/100 mL.

75. A veterinary composition comprising an iron carbohydrate complex and optionally a veterinarily acceptable adjuvant, wherein the concentration of phenol is below 1 g/L.

76. The veterinary composition according to claim 75 comprising iron carbohydrate complex and optionally veterinarily acceptable auxiliaries, which is a unit dose comprising iron carbohydrate complex in an amount of 1800mg or more of elemental iron.

77. The veterinary composition according to item 75 or 76, wherein the unit dose is 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg, 3100mg, 3200mg, 3300mg, 3400mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg 4100mg, 4200mg, 4300mg, 4400mg, 4500mg, 4600mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 5800mg, 5900mg, 6000mg, 6100mg, 6200mg, 6300mg, 6400mg, 6500mg, 6600mg, 6700mg, 6800mg, 6900mg, 7000mg, 7100mg, 7200mg, 7400mg, 7900mg, 9500mg, 8100mg, 8400mg, 7900mg, 8400mg, 9400mg, 7900mg, 8400mg, 7900mg, 8400mg, 9400mg, 4700mg, 8400mg, 4700mg, 9400mg, 4700mg, 94 mg, 4700mg, 8400mg, 4700mg, 9400mg, 4700mg, 8400mg, 9400mg, 4700mg, 8400mg, 4700mg, a including, 9600mg, 9700mg, 9800mg, 9900mg, 10000mg or more.

78. The veterinary composition according to any one of claims 75 to 77, further comprising erythropoietin.

Detailed Description

The present invention relates to an iron carbohydrate complex for use in a method of increasing blood hemoglobin concentration, for use in a method of reducing the rate of stillbirth piglets, for use in a method of increasing the survival, health and/or growth of offspring litters until weaning, or for use in a method of increasing litter size.

The experimental protocol supporting the present invention is described below.

1 materials and methods

1.1 target and study population

The study was conducted in a commercial sow herd, which is representative of a dense sow herd in denmark. The study population consisted of pregnant sows with low hemoglobin values and their offspring litters up to weaning. Pregnant sows between 60-80 days of gestation were selected for the study.

1.2 study design

The study was a randomized clinical trial. Approval from Danish Animal Experiments institute (Danish Animal Experiments) and Danish Medical administration (Danish Medical Agency) was obtained prior to the start of the study. The study was conducted according to the protocol.

1.3 research cell

The study units were sows and their offspring litters.

1.4 herd selection

The incidence (prevalence) study of anemia in danish sows was performed in 5 herds selected under professional veterinary pig practice. Herds with high incidence of anemia were selected for study.

1.4.1 herd selection criteria

1. Herd size of at least 1000 sows

2. Good record keeping of herd data

3. Positive herd owners

4. Iron supplementation of piglets by injection of 200mg of iron dextran on day 4 only

1.4.2 herd elimination criteria

1. Providing additional iron (other than in the feed formulation) to the sow orally or by injection

2. Breeding or breeding herds

3. Parturition induction, e.g. using prostaglandins

4. Apparent infection or management problems that may affect stillbirth

1.5 sample size

The sample size required for estimating morbidity is calculated by the following equation:

N＝(Z_(1-α/2) ²σ²)/L²(J.P.T.M et al 2001)

N is the number of samples selected

α -0.05 (95% confidence level) ═>Z_(1-0.05/2)＝Z_0.975＝1.96

Standard deviation of sigma ═

L is the expected absolute error

In herds where 50% of sows had anemia (<100g Hb/liter), a sample size of 50 animals was sufficient to determine the incidence with +/-13% tolerance.

1.6 identification of sows for clinical trials

All multiparous (multipartous) sows from a sufficient number of weekly batches corresponding to 200 animals will be identified on days 60-80 of gestation. Sows in the first parity will be excluded.

An initial measurement of hemoglobin in 200 sows would be made by haemocure measurement. Healthy sows with less than 100g/l hemoglobin will be included in the study.

1.7 blood sampling

At the beginning of the trial (eleventh week of pregnancy) and one week before the expected farrowing, the sows were restrained and 5mL of blood was sampled from the jugular vein in EDTA and common vacutainer tubes. The samples will be analyzed by complete hematology examination (complete haematology), serum iron, TIBC and serum ferritin as described in sections 4.6.3 and 4.8. Transferrin saturation will also be calculated as described in section 4.9.

1.8 hematology of sows

All 200 EDTA-stabilized blood samples will be first tested for hemoglobin concentration by HaemoCue. The equipment required for this test will be supplied by Pharmacosmos. The selection of 100 anemic sows would be based on this test.

Samples from 100 selected sows will be subjected to a complete hematological examination (baseline measurement) at the University of Copenhagen (University of Copenhagen).

Blood samples were analyzed for red blood cell count (RBC), white blood cell count (total white blood cell count and differential white blood cell count), platelets, Mean Platelet Volume (MPV), red blood cell distribution width (RDW), hemoglobin concentration (Hb), Hemoglobin Distribution Width (HDW), Hematocrit (HCT), Mean Cell Volume (MCV), mean red blood cell hemoglobin (MCH), and Mean Cell Hemoglobin Concentration (MCHC). Reticulocyte markers, including reticulocyte count (absolute and relative), reticulocyte hemoglobin content (Chr), mean reticulocyte hemoglobin concentration (CHCMr), reticulocyte volume (MCVr), reticulocyte red blood cell distribution width (RDWr), and reticulocyte hemoglobin distribution width (HDWr) will also be analyzed.

Serum samples were stored until the study sows were identified for clinical trials.

1.9 recording of sow data

For each sow, the following recordings were made: the age of the sow, the age of the first insemination, the number of stillborn and live piglets in the previous parity, the date of insemination, the date of farrowing, and the parity of the sow.

2 sample Scale calculation for clinical trials

The sample size in each group required to calculate the difference between the animal groups was calculated by the following equation (Graat et al, 2001)

N＝2×(Z_α+Z_β)²*SD²/Δ²

Wherein

N is the number of sows required in each group

Z_α、Z_βNormalized normal distribution at a given confidence and test force (power) level

SD-standard deviation

Delta is the difference of the estimation

The constant 2 means that SD is equal in both groups.

We assume a at 0.05, Z0.05 ═ 1.96 and 80% proof force, Z_0.20Two-way test at 0.84.

Based on previous studies, it was assumed that the difference in hemoglobin (Δ) between the two groups was 10g/L and SD was set to 15 g/L.

When the difference in hemoglobin concentration between the treated and control groups was assumed to be 9g/L, 50 sows per group were required.

2.1 laboratory analysis of frozen samples

Serum samples obtained from selected 100 sows (50 in the treatment group and 50 in the control group) were analyzed for serum iron, Total Iron Binding Capacity (TIBC) and serum ferritin, if possible. Serum iron and total iron binding capacity were analyzed at the Central Laboratory of the University of Copenhagen (Central Laboratory, University of Copenhagen), whereas at the ritzmann Laboratory,Ludwig-Maximilians-serum ferritin measurements were performed by Munich.

2.2 calculation of transferrin saturation (TfS)

Transferrin saturation of all blood samples was calculated using the following formula: TfS (%) ═ serum iron/TIBC × 100.

2.3 measurement of backfat thickness

Baseline backfat measurements of sows were done using an ultrasound device. Measurements were made at the last floating rib of 7cm on each side of the midline of the back, according to the manufacturer's instructions. Backfat measurements will be taken at the beginning of the trial, at the time of farrowing and just before weaning.

2.4 randomization of sows

Selected sows were randomly assigned to two groups:

1. control group

2. Treatment group

Randomization was performed using a polar numbering with the ear tag of the sow.

2.5 treatment of sows

Preparation of test and control substances and equipment for injection will be provided by Pharmacosmos.

A first dose

Sows in the treatment group will receive a 12.5mL (2,500mg) iron injection (Uniferon) intramuscularly in the neck area on day 70 of pregnancy (6 weeks prior to farrowing). The control group will receive 12.5mL of isotonic saline intramuscularly in the neck region on the same day. Any reactions due to iron injection were commented on and treated accordingly.

A second dose

On day 84, two weeks after the first dose of iron (4 weeks before farrowing), the sow will receive a second dose of 12.5mL (2,500mg) of iron injection (Uniferon) intramuscularly in the neck area. In addition, control sows will receive a second dose of isotonic saline.

2.6 management of sows of piglets

All sows were managed according to standard operations on the farm during the whole study period.

2.7 recording and Classification of stillbirth piglets

All fully developed, dead piglets were collected in litters at farrowing. Dead piglets were necropsied and lungs were tested. If the lungs sink in water, the piglets are considered to be stillborn. The stillbirth piglets were classified as follows:

1. non-fresh dead birth: showing signs of deterioration, brown skin colour-these piglets are likely to die more than one week before farrowing begins (Randall and Penny 1967).

2. And (3) stillbirth before delivery: no external signs of decay were shown, but all abdominal organs had the same brick-red color due to hemolysis and autolysis-these pigs died in utero within a few days before farrowing (Bille et al, 1974).

3. And (3) stillbirth in delivery: normal abdominal organs were coloured, but mucus and/or meconium were present in the trachea, indicating that piglets died during farrowing.

Mummified fetuses were recorded but were not included in the trial. If practical, the gender was recorded and individual body weights were obtained for each of the stillborn and live piglets.

2.8 hematology of piglets

Blood was collected from the anterior vena cava from a subset of two live piglets per litter. The piglets were randomly selected among all piglets in that particular pup. Blood was collected from two dead pups per litter according to the Rootwelt et al, 2012 guidelines.

Complete hematological examination of blood, including serum iron, TIBC and serum ferritin, was performed as described in sections 1.8 and 2.1. Lactate will also be measured in each piglet.

2.9 adverse reactions in sows

During the study, 20% of sows were expected to be culled at weaning. Among these sows, 10 animals were selected for the purpose of facilitating the study of the injection site response. The injection site was visually assessed to determine the extent of tissue damage due to the injection. Samples for histological examination will be fixed in formalin.

The number of total born piglets in the treated animals will be compared to those of the control group. The backfat thickness of near weaned sows will be recorded.

3 statistical analysis

SAS 9.3 was used for data analysis. Using a general linear model, the PROC GLM program in SAS was used, compared to the hematology of sows before and after iron injection. Differences in hematology between control and treatment sows were calculated using a general linear model using the PROC GLM program. Explanatory factors will include baseline Hb values, total piglet birth number and sow parity. The probability of stillbirth of piglets in each control and treatment group of sows was calculated using the generalized linear model using the PROC LOGISTIC program. In this procedure, Hb measured at baseline, the number of total born piglets, the parity of the sows, the gender of the piglets are explanatory variables considered for the analysis.

Hematological differences between live and dead piglets were performed using a linear MIXED model using PROC MIXED program with sows as the randomising factor. Other explanatory variables would include litter size, sow parity and sow hemoglobin.

Example 1

100 sows were selected from the herd showing a high incidence of anemia. For practical reasons, the study was carried out in two batches. All data are summarized.

At the beginning of the test (i.e. week 8 of pregnancy), approximately one week before the expected calving and 4 weeks after calving, 5ml of blood was sampled from the jugular vein in EDTA. Blood samples were labeled Hb _ S1, Hb _ S2, and Hb _ S3, respectively. The sample is analyzed and the blood hemoglobin concentration (Hb) is determined.

Selected sows were randomly assigned to two groups:

1. control group (C)

2. Treatment group (T)

Randomization was performed using a polar numbering with the ear tag of the sow. Each group included 50 sows.

Sows in the treatment group will deliver a first dose of 12.5ml of iron dextran equivalent to 2500mg of iron intramuscularly in the neck area (Uniferon, 20%) on day 70 of pregnancy (6 weeks before farrowing). The control group received 12.5mL of isotonic saline intramuscularly in the neck region on the same day. Any reactions due to iron injection were commented on and treated accordingly.

On day 84, sows received a second dose of 12.5mL (2,500mg) of iron injection (Uniferon) intramuscularly in the neck area two weeks after the first dose of iron (4 weeks before farrowing). In addition, control sows will receive a second dose of isotonic saline.

Table 1: hb in sows

All fully developed, dead piglets were collected in litters at farrowing. Dead piglets were necropsied and lungs were tested. If the lungs sink in water, the piglets are considered to be stillborn.

Mummified fetuses were recorded but were not included in the trial. If practical, the gender was recorded and individual body weights were obtained for each of the stillborn and live piglets.

Table 2: total number of stillbirth piglets

It is noted that the hemoglobin concentration in the sows shown in table 1 increased for sows with baseline Hb below 105 g/l. Specifically, anemic sows with a low baseline Hb of <105g/l experienced an increase of 1.40g/l compared to 0.28g/l for the control group from the start of the trial to one week before the expected farrowing and an increase of 1.66g/l compared to 0.22 for the control group from the start to 4 weeks after farrowing. The results show that anemic pregnant sows benefit from administration of the iron carbohydrate complex. For sows with baseline Hb above 105g/l, Δ (Hb _ S1, Hb _ S2) and Δ (Hb _ S1, Hb _ S3) were negative, indicating that these animals did not benefit from treatment.

When sows with a baseline of <105g/l were treated with the iron carbohydrate complex, the dead-birth rate in table 2 decreased from 7.9% in the control group to 6.6% in the treated group. The results show that the stillbirth offspring rate is reduced by administration of the iron carbohydrate complex. In contrast, when baseline Hb was above 105g/l, the mortality rate decreased from 6.6% in the control group to 6.4% in the treatment group, indicating that no improvement in mortality rate was obtained for this group of sows.

Reference to the literature

Archibald,R.,Hancock,E.E.I.,1939.Iron Deficiency-Stillbirth of Swine.Canadian Journal of Comparative Medicine 3,134.

Bille,N.,Nielsen,N.C.,Larsen,J.L.,Svendsen,J.,1974.Preweaning mortality in pigs.2.The perinatal period.Nordisk veterinaermedicin,26,294-313.

Graat,E.A.M.,Frankena,K.,Bos,H.,2001.Principles and methods of sampling in animal disease surveys,In:Noordhuizen,J.P.T.M.,Frankena,K.,Thrusfield,M.V.,Graat,E.A.M.(Eds.)Application of quantitative methods in veterinary epidemiology.Wageningen Pers,Wageningen,The Netherlands,pp.45-46.

Jensen,A.K.,Pedersen,K.S.,Nielsen,J.P.,2013.Association between blood haemoglobin concentration in sows and neonatal piglets.In Proceedings of the5th ESPHM,Edinburgh,UK.

Jensen,A.K.,Nielsen,J.P.,2014.Association between stillborn piglets and haemoglobin concentration in sows at farrowing.In Proceedings of the 6th ESPHM,Sorrento,Italy.

Moore,R.,Redmond,H.,Livingston Jr,C.,1965.Iron deficiency anemia as a cause of stillbirths in swine.Journal of the American Veterinary Medical Association 147,746.

Morris,C.J.,Earl,J.R.,Trenam,C.W.,Blake,D.R.,1995.Reactive oxygen species and iron—a dangerous partnership in inflammation.The international journal of biochemistry and cell biology 27,109-122.

Randall,G.C.B.,Penny,R.H.C.,1967.Still birth in pigs:the possible role of anoxia.Veterinary Record 81,359-361.

Rootwelt,V.,Reksen,O.,Farstad,W.,Framstad,T.,2012.Associations between intrapartum death and piglet,placental,and umbilical characteristics.Journal of Animal Science 90,4289-4296.

Svetina,A.,Vrabac,L.,M.,Turk,R.,2006.Relation between erythrocyte parameters and stillbirth in piglets.Veterinarski arhiv 76,297-303.

Thorn,C.2010.Hematology of the pig.In Schalm's Veterinary Hematology 6th Edition,Wiley-Blackwell,Ames,Iowa,Weiss,D.,Wardrop,K.,eds.(Iowa,Wiley-Blackwell),p.848.

Vinther J.,2013.National average productivity in pig production in 2012(Landsgennemsnit for productivitet i svineproduktionen 2012).Videncenter for svineproduktion

Zaleski,H.M.,Hacker,R.R.,1993.Variables related to the progress of parturition and probability of stillbirth in swine.The Canadian Veterinary Journal 34,109.

Auvigne,V.,et al.,2010.Anaemia in the hyperprolific sow:Effect of injectable iron administration and relation with fattering score.