Patent Application: US-201515522466-A

Abstract:
the present invention relates to an iron carbohydrate complex for use in a method of increasing the blood haemoglobin concentration in a pregnant non - human mammal , wherein the pregnant non - human mammal having a blood haemoglobin level of 105 g / l or less , is administered one or more doses of iron carbohydrate complex comprising an amount of elemental iron of 1800 mg or more per dose . the method relates to the further effects of decreasing the rate of stillborn offsprings from a pregnant non - human mammal having a blood haemoglobin level of 105 g / l or less , increasing the blood haemoglobin concentration of offspring litters within 3 days from birth and / or weaning , or increasing the litter size in a subsequent parity of a non - human mammal having a blood haemoglobin level of 105 g / l or less .

Description:
the present invention relates to an iron carbohydrate complex for use in a method of increasing the blood haemoglobin concentration , for use in a method of decreasing the rate of stillborn 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 the litter size . the study is carried out in a commercial sow herd , which is representative of intensive sow herds in denmark . the study population consists of pregnant sows with low haemoglobin values and their offspring litters until weaning . the pregnant sows are selected for the study between 60 - 80 days of gestation . the study is a randomised clinical trial . approval from the danish animal experiments inspectorate and from the danish medical agency is obtained before the start of the study . the study is carried out according to the protocol . a prevalence study of anaemia in danish sows is carried out in 5 herds selected in cooperation with specialised veterinary pig practices . a herd with high prevalence of anaemia is selected for the study . 4 . iron supplementation of piglets by injection of 200 mg iron dextran at day 4 only 1 . provision of extra iron to the sows either orally or injectably ( except in feed formulations ) 4 . obvious infectious disease or management problems that may affect stillbirths the sample size necessary to estimate a prevalence is calculated by the equation : n =( z ( 1 − α / 2 ) 2 σ 2 )/ l 2 ( j . p . t . m et al ., 2001 ) in a herd with 50 % of sows suffering from anaemia (& lt ; 100 g hb per litre ) a sample size of 50 animals is sufficient to determine the prevalence with an allowable error of +/− 13 %. all multiparous sows from a sufficient number of weekly batches corresponding to 200 animals will be identified at day 60 - 80 day of gestation . first parity sows will be excluded . initial measurement of haemoglobin in the 200 sows will be performed by haemocue measurement . healthy sows with haemoglobin lower than 100 g / l will be included in the study . the sows are restrained and 5 ml blood is sampled in edta and plain vacutainer tubes from the jugular vein at the start of the trial ( week eleven of gestation ) and one week before expected farrowing . the samples will be analysed by 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 . all 200 edta stabilized blood samples will be tested initially for haemoglobin concentration by haemocue . the equipment necessary for this testing will be provided by pharmacosmos . selection of 100 anaemic sows will be based on this testing . samples from the 100 selected sows will subjected to complete haematology ( baseline measurement ) at the university of copenhagen . the blood samples are analysed for erythrocyte count ( rbc ), leucocyte count ( total leucocyte count and differential leucocyte count ), platelets , mean platelet volume ( mpv ), red blood cell distribution width ( rdw ), haemoglobin concentration ( hb ), haemoglobin distribution width ( hdw ), haematocrit ( hct ), mean cell volume ( mcv ), mean corpuscular haemoglobin ( mch ) and mean cell haemoglobin concentration ( mchc ). reticulocyte indices will also be analysed , which include reticulocyte count ( absolute and relative ), reticulocyte haemoglobin content ( chr ), mean reticulocyte corpuscular haemoglobin concentration ( chcmr ), reticulocyte cellular volume ( mcvr ), reticulocyte red cell distribution width ( rdwr ) and reticulocyte haemoglobin distribution width ( hdwr ). the serum samples are stored until study sows for clinical trial are identified . for each sow the following recordings are made : age of sow , age of first insemination , number of dead born and live born piglets in previous parity , date of insemination , date of farrowing and parity of the sow . the sample size in each group necessary to calculate differences between groups of animals is calculated by the equation ( graat et al ., 2001 ) n = 2 ×( z α + z β ) 2 * sd 2 / a 2 z α , z β = values of standard normal distribution at specified levels of confidence and power the constant 2 implies that the sd is equal in both groups . we assume a two sided test with α of 0 . 05 , z 0 . 05 = 1 . 96 and a power of 80 %, z 0 . 20 = 0 . 84 . the haemoglobin difference between two groups ( a ) is assumed to be 10 g / l and the sd is set to 15 g / l based on previous study . when assuming a haemoglobin concentration difference of 9 g / l between treatment and control groups , 50 sows are required per group . serum samples obtained from the selected 100 sows ( 50 in treatment and 50 in control groups ) are analysed for serum iron , total iron binding capacity ( tibc ) and serum ferritin , if possible . serum iron and total iron binding capacity are analysed at the central laboratory , university of copenhagen whereas serum ferritin measurement is performed at professor ritzmanns laboratory , tierärztliche fakultät , ludwig - maximilians - universität , münchen . transferrin saturation is calculated for all the blood samples using the formula : tfs (%)= serum iron / tibc × 100 . baseline back fat measurement of the sows is done using ultrasonic device . the measurement is taken at the last floating rib , 7 cm on either side of the midline of the back according to the guidelines of the manufacturer . back fat measurement will be performed at start of the trial , at farrowing and at immediately before weaning . randomisation is done using lottery with ear tag numbers of sows . preparations of test and control substance and equipment for injection will be provided by pharmacosmos . sows in the treatment group will receive 12 . 5 ml ( 2 , 500 mg ) iron injection ( uniferon ) intramuscularly in the neck region at day 70 of gestation ( 6 weeks before farrowing ). the control group will receive 12 . 5 ml of isotonic saline intramuscularly in the neck region in the same day . any reactions due to the iron injection are noted and treated accordingly . two weeks after the first dose of iron at day 84 ( 4 weeks before farrowing ), the sows will receive second dose of 12 . 5 ml ( 2 , 500 mg ) iron injection ( uniferon ) intramuscularly in the neck region intramuscularly . also , the control sows will receive second dose of isotonic saline . all sows are managed according to standard procedures at the farm during the entire study period . all fully developed dead piglets in a litter are collected at farrowing . the dead piglets are necropsied and lungs are tested . if the lungs sink in water , the piglet is considered as stillborn . stillborn piglets are classified as follows : 1 . non - fresh stillbirths : showing signs of degeneration , brown skin colour — these piglets probably die more than a week before onset of farrowing ( randall and penny 1967 ) 2 . pre - partum stillbirths : showing no external signs of decay but with the same brick - red colour of all their abdominal organs due to haemolysis and autolysis — these pigs die in utero in the days closely preceding farrowing ( bille et al ., 1974 ) 3 . intra - partum stillbirths : normal colour of the abdominal organs but presence of mucus and / or meconium in the trachea indicating piglets dying during farrowing mummified foetus is recorded but not included in the trial . for each stillborn and live born piglets , the sex is recorded and individual bodyweight obtained , if practically feasible . blood is collected from the anterior vena cava from a subset of two live born piglets per litter . these piglets are selected randomly among all the piglets in that particular litter . the blood from two dead born piglets per litter is collected according to the guidelines of rootwelt et al ., 2012 . the blood is subjected to complete haematology including serum iron , tibc and serum ferritin as described in sections 1 . 8 and 2 . 1 . lactate in each piglet will also be measured . during the study it is expected that 20 % of sows are culled at weaning . among these sows 10 animals are selected by convenience for studies on injection site reactions . the injection site is assessed macroscopically to determine the extent of tissue damage caused due to injection . samples for histological examination will be fixed in formalin . the number of total born piglets in treated animals will be compared to those of controls . the back fat thickness of sows close to weaning will be recorded . sas 9 . 3 is employed for the data analysis . the haematology of sows before and after iron injection are compared using general linear model using proc glm procedure in sas . the differences in haematology in sows between control and treatment sows are calculated using general linear model with proc glm procedure . explanatory factors will include baseline hb values , number of total born piglets and parity of the sow . the probability of piglet being stillborn in each control and treatment sows is calculated using generalised linear models using proc logistic procedure . in this procedure , hb at baseline measurement , number of total born piglets , parity of the sow , sex of the piglet are explanatory variables that are considered for analysis . the differences in haematology between live and stillborn piglets are carried out using linear mixed model with proc mixed procedure with sow as the random factor . other explanatory variables will include litter size , parity of the sow and haemoglobin of the sow . 100 sows were selected from a herd showing a high prevalence of anaemia . for practical reasons the study was performed in two batches . all data was pooled . 5 ml blood was sampled in edta from the jugular vein at the start of the trial , i . e . week 8 of gestation , about one week before expected farrowing , and 4 week after farrowing . the blood samples were marked hb_s1 , hb_s2 , and hb_s3 , respectively . the samples were analysed and the blood haemoglobin concentration ( hb ) was determined . randomisation is done using lottery with ear tag numbers of sows . each group included 50 sows . sows in the treatment group was delivered a first dose of 12 . 5 ml iron dextran ( uniferon , 20 %), corresponding to 2500 mg iron , intramuscularly in the neck region at day 70 of gestation ( 6 weeks before farrowing ). the control group received 12 . 5 ml of isotonic saline intramuscularly in the neck region in the same day . any reactions due to the iron injection are noted and treated accordingly . two weeks after the first dose of iron at day 84 ( 4 weeks before farrowing ), the sows received a second dose of 12 . 5 ml ( 2 , 500 mg ) iron injection ( uniferon ) intramuscularly in the neck region . also , the control sows will received second dose of isotonic saline . all fully developed dead piglets in a litter are collected at farrowing . the dead piglets are necropsied and lungs are tested . if the lungs sink in water , the piglet is considered as stillborn . mummified foetus is recorded but not included in the trial . for each stillborn and live born piglet , the sex is recorded and individual bodyweight obtained , if practically feasible . it is noted that the haemoglobin concentration in sows shown in table 1 is increased for the sows having a baseline hb below 105 g / l . specifically , the anaemic sows having a low baseline hb of & lt ; 105 g / l experience an increase of 1 . 40 g / l , compared to 0 . 28 g / l for the control group , from the start of the trial to one week before expected farrowing and an increase of 1 . 66 g / l , compared to 0 . 22 for the control group , from the start to 4 weeks after farrowing . the results show that anaemic pregnant sows benefit from the administration of the iron carbohydrate complex . the δ ( hb_s1 , hb_s2 ) and δ ( hb_s1 , hb_s3 ) for sows having baseline hb above 105 g / l are negative indicating that these animals are not benefitting from the treatment . the stillborn rate in table 2 is decreasing from 7 . 9 % in the control group to 6 . 6 % in the treated group , when the sows having a baseline & lt ; 105 g / l are treated with the iron carbohydrate complex . the result indicates that the rate of stillborn offspring is decreased by the administration of an iron carbohydrate complex . conversely , the stillborn rate is decreasing from 6 . 6 % in the control group to 6 . 4 % in the treated group when the baseline hb is above 105 g / l , indicating that an improvement in the stillborn rate for this group of sows is not obtained . 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 ., 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( 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 the 5th 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 ., belić , 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 &# 39 ; 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 .