induced parturition in cows may have several technological and medical advantages over spontaneous delivery , such as avoiding unattended night - time calving , providing colostrum to the newborn in time , preventing dystocia caused by relative fetal oversize , dealing with fetal deformities , and hydroallantois .
calving can be successfully induced by an intramuscular injection of prostaglandin f2 ( pgf2 ) .
it acts on the corpus luteum to cause luteolysis and stops the production of progesterone .
time required from treatment to calf delivery ranges from 24 to 72 hours , on average 48 hours .
however , the major problem arising from this procedure is a dramatic rise in the incidence of retained placenta ( rp ) , up to 80% in some cases , although no other serious harmful effects on cows and calves have been reported .
retained placenta ( rp ) is a reproductive disorder seen in cows and water buffalos .
it is characterized by the inability of the animal to expel fetal membranes within 12 hours after parturition .
its incidence varies from 1.3 to 39.2% of overall parturitions , resulting in serious economic losses in the dairy industry throughout the world .
the etiology of rp is complex and not yet fully understood . in the majority of cases , it is directly caused by a disturbance of the prepartal loosening mechanism in the placentomes .
furthermore , cows with rp are at high risk of developing metritis and silent heat , resulting in prolonged service period and low fertility rate [ 6 , 7 ] .
various factors , such as age , breed , heredity , environment , hormonal status , nutrition , and immunity had been implied to contribute to the development of rp . however , no single factor was sufficient to provide a satisfactory explanation for the mechanism underlying the development of this disorder .
it is well established that innate and acquired immune defense mechanisms are lowest in the peripartum period due to metabolic strain during pregnancy , calving , and early lactation .
two immune mechanisms had been proposed to explain the detachment of the fetal membranes during parturition . both were based on the hypothesis that upon parturition , when blood supply to the placenta begins to cease , the placenta becomes a 
foreign body  that the maternal immune system must recognize , attack , and expel .
gunnink   demonstrated that leukocytes were less able to recognize cotyledon tissue harvested from cows with rp compared non - rp .
however , kimura et al .   argued that 38 hours needed to expel fetal membranes was a too short time for lymphocytes to mount an effective rejection process ; hence , they focused on the role of innate immunity mediated by neutrophils through their oxygen - dependent killing capabilities .
pregnancy in dairy cows is considered to induce oxidative stress , which in turn can be a significant underlying factor to dysfunctional host immune and inflammatory responses that can increase the incidence of perinatal disorders during the transition period [ 11 , 12 ] .
proposed a pathway of rp development starting with an imbalance of the antioxidant capacity , followed by a decrease in estrogen production , resulting in decreased pgf2 and accumulation of arachidonic and linoleic acids in the placental tissue .
therefore , it is necessary that the animal organism should be able to maintain its antioxidative / oxidative processes in balance during peripartal period .
methods for quantifying oxidative stress mostly include direct or indirect measures of antioxidants and oxidants , or their metabolic products present in animal tissues .
malondialdehyde ( mda ) is one of the several low - molecular weight end - products that arise from oxidative decomposition of polyunsaturated fatty acids .
mda readily reacts with thiobarbituric acid producing a red pigment that can be measured spectrophotometrically .
selenium is well established as one of the key elements in the antioxidative defense of a living organism . in the active form , it is incorporated in glutathione peroxidases ( gpx ) , a family of enzymes that use the reductive potential of glutathione ( gsh ) to convert hydroperoxides to corresponding alcohols . according to bernabucci et al .
[ 16 , 17 ] , plasma gpx activity in cows raises several days prior to parturition and gradually declines afterwards , while in the erythrocytes , the activity remains constant .
however , in its reaction with the peroxyl radicals vitamin e produces hydroperoxides that are still toxic and must be removed   by gpx , hence the functional interdependence of selenium and vitamin e. the first report on the relationship between se and vitamin e status of cows and retention of placenta was published by trinder et al .   drawing substantial attention to this subject .
experiments conducted by jaskowski   and wentink et al .   established a minimal selenium concentration in cow 's blood plasma of 30  ng / ml , below which the incidence of rp would significantly rise , namely , from 5 - 6% to 2022% .
preventive effects of different doses of se and/or vitamin e against rp prepartally supplemented to cows , have been reported over the years by a number of authors [ 2025 ] .
all three known types of iodothyronine deiodinases ( i d ) , enzymes which activate and/or inactivate thyronines through deiodination , contain se in the form of selenocysteine in their catalytic sites
. however , i d activities are not as tightly linked to se status as the activity of gpx .
therefore , serum thyroxine ( t4 ) and triiodothyronine ( t3 ) levels as well as t4/t3 ratio may only be slightly affected by marginal se deficiency . according to nixon
the stage of lactation and season of the year have a strong influence on changes in serum thyronines .
.   reported that serums t4 and t3 concentrations in periparturient cows start to decrease 14 days prior to delivery and recover slowly within the first few weeks after parturition .
high incidence of retained placenta , together with a wide variability in the intervals between prostaglandin f2 treatment and calf delivery , remains the primary limiting factor for the practical use of pgf2 in the synchronization of parturition on dairy farms .
the aim of this trial was to determine to which extent a preparturient supplementation with different doses of se and vitamin e would influence the incidence of rp , as well as selenium , mda , and thyronines status in cows treated with pgf2 for the induction of parturition .
thirty - three ( 33 ) holstein - frisian cows included in this investigation were randomly distributed to 3 groups and supplemented with sodium selenite ( ss ) and tocopherol acetate ( tac ) as follows : control group ( n = 9 ) was not supplemented , and it served as a negative control;group a ( n = 11 ) was supplemented 10  mg ss and 400  mg tac;group b ( n = 13 ) was supplemented 20  mg ss and 800  mg tac .
control group ( n = 9 ) was not supplemented , and it served as a negative control ; group a ( n = 11 ) was supplemented 10  mg ss and 400  mg tac ; group b ( n = 13 ) was supplemented 20  mg ss and 800  mg tac .
the supplement was administered by a single intramuscular injection between days 250 and 255 of gestation ; parturition was induced using a single intramuscular injection of pgf2 ( 2  ml , 500  g of cloprostenol ) not before day 275 of gestation ; venous blood samples for analysis were taken 12 hours postpartum .
all animals were clinically healthy , multiparous , and single calve , with no previous record of retained placenta or other disorders .
determination of selenium in whole blood samples of cows was carried out using atomic absorption spectrometry - hydride technique .
samples of whole blood ( 0.5  g ) were accurately weighted using analytical balance denver instrument , model tb-215d ( denver instruments , usa ) , transferred into teflon microwave vessels , and digested with 8  ml of 69% nitric acid ( sigma - aldrich , usa ) and 2  ml of 30% hydrogen peroxide ( fluka analytical , usa ) . microwave oven ( milestone , germany , model touch control ) was set to the following program : temperature ramp from ambient temperature at 180c followed by 15  min of holding time and 20  min of cooling time .
digested samples were transferred to volumetric flasks and diluted using 5  m hydrochloric acid ( sigma - aldrich , usa ) to the final volume of 25  ml .
determination of selenium concentration was carried out using solaar , series 4 spectrometer equipped with vp70 hydride module and ec90 electrical furnace for precise temperature control of the analytical cuvette ( thermo electron , uk ) . measuring absorption at 196 
nm was performed after seh4 formation in the hydride system with 5% nabh4 ( j. t. baker , the netherlands ) and 0.6% naoh ( merck , germany ) .
stabilization and baseline delay times were 40 and 60 seconds , respectively , and reading time was 7 seconds in three replicates .
quantification of se content was performed using five - point calibration curve ( 1040  g / kg , including zero ) of reference standard solutions ( merck , germany ) .
quality control was achieved using blank samples fortified in 20  g / kg of se and certified reference material ( bcr 189 ) .
good linearity was obtained from the calibration curve ( r = 0.998 ) , and the measured concentration of the reference material was in the range of the reference value .
briefly , a 3  ml of 0.1% orthophosphoric acid , 1  ml of 0.6% thiobarbituric acid , and 0.1  ml of 0.28% hydrated ferrous sulfate solution were added to 0.3  ml of serum .
the produced chromogen was extracted with n - butyl alcohol ( 4  ml ) .
after centrifugation ( 2200  g , 10 minutes ) , the butanol layer was separated for spectrophotometric measurement at 535  nm .  
glutathione peroxidase activity was measured in whole blood samples by the coupled test described by gnzler et al . .
blood samples were hemolyzed using drabkin 's reagent ( 1.6  mm kcn , 1.2  mm k2fe ( cn)6 , and 0.023  m nahco3 ) .
glutathione ( gsh ) as the donor of hydrogen becomes oxidized to gs - sg . in the second phase of this coupled reaction
, gs - sg is reduced to gsh by nadph and glutathione reductase ( gr ) .
final concentrations of used reagents were 100  mm phosphate buffer ( ph 7.4 ) , 4  mm edta , 6  mm gsh , 0.375  iu / ml gr , 0.3  mm nadph and 1.575  mm tbh .
low concentration of tbh ( under 2.32  mm ) as used in this method determines only the activity of se - dependent gpx .
the reduction of nadph was followed for 3  min at 366  nm using a cecil ce2021 spectrophotometer ( uk ) with a peltier thermostat unit .
absorbance ( a ) values were taken at 30 seconds intervals , and the results were expressed in microkatals per liter ( kat / l ) .
concentrations of t3 and t4 were measured in heparinized plasma samples using commercial standard ria kits ( inep , zemun ) .
the assay is based on the competition between unlabelled t3 and t4 and a fixed quantity of l labeled t3 and t4 for a limited number of binding sites on t3 and t4 specific antibodies ( bound to the tubes ) . allowing a fixed amount of tracer and antibody to react with different amounts of unlabelled ligand
, the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand .
antigen - antibody complex is bound to the tubes , and the supernatant is then separated by decantation . counting the radioactivity of the bound phase
obtained results were arranged and analyzed from two aspects : table 1 displays experimental results according to se and vitamin e treatments as previously described , and table 2 displays the results according to the absence / presence of retained placenta in cows , diagnosed 12 hours postpartum , independently from se and vitamin e treatment .
analysis was performed using ms excel 2007 and graph pad prism 5 statistical software packages .
the treatment of cows with 10  mg sodium selenite ( ss ) and 400  mg tocopherol acetate ( tac ) reduced the incidence of retained placenta from 66.7% in the control group to 38.2% in group a , while treatment with 20  mg ss and 800  mg tac further reduced rp to 30.8% in group b ( table 1 ) .
blood selenium concentrations and gpx activities in both treated groups ( a or b ) were significantly higher compared to the control ; however , there were no significant differences between groups a and b. blood plasma mda content was lowest in group b ( 3.95  0.88  m ) , significantly lower than both control ( 5.70  0.94  m ) and group a ( 4.59  1.20  m ) .
total plasma t4 levels were highest in group a ( 59.55  13.00  nm ) and lowest in group b ( 43.2  16.2  nm ) , while mean t3 concentrations ranged from 1.1  0.4  nm in group b to 1.7  0.6  nm in control group .
comparison between cows with and without the diagnosed rp ( independently from ss and tac treatment ) , revealed the following ( table 2 ) : cows with rp had significantly lower blood se content and gpx activity and higher plasma mda concentrations compared to those free of rp .
plasma t3 did not differ significantly , while plasma t4 concentration was significantly higher in cows with rp .
the primary aim of this study was to test the assumption that prepartum supplementation of cows with se and vitamin e should exhibit the protective effect against the onset of retained placenta in cows with prostaglandin f2 induced parturition in a similar fashion as in animals with spontaneous parturition . to our best knowledge , this is the first such attempt ; therefore , the only available frame of reference for our data is the research done on animals with spontaneous labour . in cows supplemented with se and vitamin e , 20 days prior to parturition induced by pgf2 , the incidence of retained placenta was effectively halved , from 66.7% in the control group to 38.2% and 30.8% in groups a and b , respectively ( table 1 ) .
similarly , julien et al .   demonstrated that a single 20-day prepartum injection of 50  mg of sodium selenite and 680  iu of -tocopherol acetate effectively reduced the incidence of rp from 51.2% in the control group of cows to 8.8% in the treated group .
selenium alone was at least as effective as a combination of selenium and vitamin e .
the higher the dose of selenium administered , the lower the incidence of rp was registered . in all these experiments ,
the percentage of rp in herds was reduced to values close to zero , while in our trial the lowest value remained at 30.8% .
therefore , we estimate that further increase in se dosage would not produce a linear but rather asymptotic decrease in rp incidence
the supplement dosage should be considered with great care , balancing between sufficient supply and avoiding toxicity .
marked discrepancies in dose / effect ratios described in the literature may be the result of different basal se status of animals , which is highly dependable on naturally occurring se levels in locally produced feedstuffs . according to investigations conducted by jovanovi et al .   in the region surrounding the experimental farm , se content in cereals and hay ranged from 40 to 62  gse / kg .
furthermore , as the retention of placenta is known to be a multifactorial disorder , our results demonstrate that certain effects of pgf2 may not fall into the domain of oxidant - antioxidant balance in the body and can not be overcome solely by the antioxidative actions of se and vitamin e. blood selenium concentration is considered to be a good indicator of the long - term se status in most animal species .
selenium concentration was significantly elevated ( table 1 ) in groups a ( 163  28  ng / ml ) and b ( 188  30  ng / ml ) supplemented with 10  mg and 20  mg sodium selenite , respectively , compared to unsupplemented control animals ( 129  18  ng / ml ) .
cows suffering from retained placenta ( table 2 ) had significantly lower ( p < 0.05 ) blood se content ( 138  40  ng / ml ) compared to non - rp animals ( 176  33  ng / ml ) .
collected blood samples from 254 se unsupplemented dairy herds in norway and found that their blood se concentrations ranged from 60 to 120  ng / g and proposed the content of 100150  ngse / g as a delimiting range below which a higher incidence of rp could be expected .
blood selenium concentrations measured in their study were slightly below the range of the values found in unsupplemented cows in our experiment .
this reflects the fact that norwegian crops are slightly more selenium deficient   than those in the northern part of serbia .
however , both sets of data corroborate that the blood concentration of 150  ngse / ml could represent the boundary between higher and lower probability for the onset of rp .
the activity of selenium - dependent glutathione peroxidase ( gpx ) in the blood of dairy cows from groups a and b ( 182  32 and 186  33  kat / l , resp . ) was twice as high as in the control ( 91  15  kat / l ) ; however , activities did not significantly differ between groups a and b ( table 1 ) .
blood gpx activity was significantly lower in cows with rp than in non - rp cows ( table 2 ) .
comparable results were presented in studies conducted by wischral et al .   and kankofer et al . .
plasma mda concentrations were 5.70  0.94  m in control , 4.59  1.20  m in group a , and 3.95  0.88  m in group b. mda was significantly lower in treated animals ( p < 0.05 in group a and p < 0.01 in group b ) compared to control ( table 1 ) . however , all of these values are generally substantially higher than those published by wischral et al .
we assume that high levels of mda may be due to elevated oxidative stress caused by premature parturition induced by pgf2. animals with rp had significantly higher ( p < 0.01 ) plasma mda than non - rp cows ( table 2 ) , which is in accordance with the findings of the above - mentioned authors .
concentrations of plasma thyroid hormones , t4 and t3 , in our experiment ( table 1 ) were below those published by jovanovi et al .   for 12 months old heifers fed se - adequate feed but corresponded to the levels published by pethes et al .   and djokovi et al
however , t4 and t3 did not apparently depend upon blood selenium supplementation of the animals . on the other hand ,
plasma t4 was significantly higher in cows with diagnosed rp , compared to non - rp ( table 2 ) .
our study demonstrated that in cows treated with single intramuscular injection of 20  mg sodium selenite and 800  mg tocopherol acetate three weeks prepartum there was a significant increase in plasma gpx activity and decrease in mda concentration .
consequently , incidence of retained placenta in supplemented compared to nonsupplemented animals was reduced in half .
this indicates that oxidative stress may be the principal , but not the only , cause of increased rp in animals with induced parturition .
further investigation is needed , using reasonably higher supplement doses of antioxidants , to show whether the incidence of rp can be brought to values closer to zero in a similar fashion as in cows with spontaneous calving .
 the incidence of retained placenta ( rp ) in cows increases in cases of parturition induced by prostaglandin f2. we analyzed the effects of different doses of supplemental selenium and vitamin e on the incidence of rp , blood selenium , plasma thyronines , and malondialdehyde concentration . 
 thirty - three clinically healthy , multiparous holstein - frisian cows were assigned to 3 groups and supplemented with a single intramuscular injection of sodium selenite ( ss ) and tocopherol acetate ( tac ) between days 250 to 255 of gestation : control  unsupplemented ; group a10  mg ss + 400  mg tac ; group b20  mg ss + 800  mg tac . 
 parturition was induced using pgf2 not before day 275 of gestation . 
 the rp incidence was reduced from 66.7% in the control to 38.2 and 30.8% in groups a and b , respectively . 
 blood selenium and glutathione peroxidase activity in treated groups were significantly higher compared to control , with no significant difference between groups a and b. plasma malondialdehyde in group b was significantly lower than that in control and group a , while thyronines levels were not affected . 
 comparison of rp and non - rp cows , independently of supplement treatment , revealed higher blood selenium and glutathione peroxidase activity and lower mda and thyroxine in non - rp animals , while triiodothyronine level did not differ . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion