BACKGROUND
lactation is a physiological state which in most animal species is associated with a strong increase in energy and nutrient requirement for milk production.
this demand is usually met by marked increase in feed intake and by utilization of body’s energy stores.
in some animal species such as rats or mice, additionally metabolic adaptations occur during lactation which aim to conserve energy for milk production  <cit> .
down-regulation of proteins involved in fatty acid uptake and oxidation in liver and skeletal muscle and a decreased thermogenesis contribute to these energy sparing effects  <cit> .
we have observed that these effects in lactating rodents are mediated by a suppression of peroxisome proliferator-activated receptor α , a transcription factor which controls the expression of many genes involved in lipid catabolism  <cit> .
in lactating rats, it has been moreover observed that an activation of pparα by feeding an oxidized fat causes a disturbance of those metabolic adaptations.
it has been found that there was an increased uptake of fatty acids into the liver and an increased oxidation of fatty acids in the lactating rats fed the oxidized fat, which in turn led to a reduced availability of fatty acids for milk production, resulting in a reduced milk triacylglycerol  concentration and reduced growth rates of the suckling pups  <cit> .
in lactating sows, milk production is important for the post-natal growth of their litters.
however, in contrast to rodents, the role of pparα in lactating sows has not yet been determined.
according to rodent studies, an activation of pparα during lactation could suppress milk fat synthesis due to a reduced availability of fatty acids as a result of an increased utilization of fatty acids by liver and muscle.
the question whether activation of pparα could affect milk fat synthesis is of relevance as dietary components such as polyunsaturated fatty acids  are able to activate pparα  <cit> .
moreover, it is known that a negative energy balance which is common in lactating sows causes an activation of pparα due to increased concentrations of nefa in plasma released from adipose tissue  <cit> .
indeed, recently an activation of pparα in liver of lactating sows with a negative energy balance has been observed  <cit> .
however, metabolic effects of activation of pparα in lactating sows have not been investigated so far.
in order to investigate the hypothesis that activation of pparα could impair milk production, we performed an experiment with lactating sows which were fed a diet supplemented with clofibrate as a synthetic agonist of pparα and determined milk composition and gains of litters.
methods
animals and diets
twenty second parity sows  were artificially inseminated and kept in single crates until day  <dig> of pregnancy.
from day  <dig> to  <dig> of pregnancy, the sows were kept in groups in pens equipped with fully slatted floors, nipple drinkers and feeders with a temperature of 19 ± 1°c with a 12:12 h light–dark cycle and 60–80% relative humidity by means of an air conditioning system.
on day  <dig> of pregnancy, they were housed individually in farrowing crates.
after parturition, the sows were randomly assigned into two feeding groups of  <dig> animals each with an equalized litter size of  <dig> piglets per sow.
infrared heaters were used to provide a surrounding temperature of 35°c for newborn pigs.
the duration of the suckling period was 21 days.
during lactation all sows were fed a nutritionally adequate basal diet consisting mainly of : wheat , barley , soybean meal with 43% crude protein , a mixture of soybean oil and palm oil   and a mineral supplement  .
the basal diet contained  <dig>  mj metabolizable energy /kg; concentrations of crude nutrients were : crude protein, 154; crude fat, 74; crude fiber, 45; crude ash, 43; starch,  <dig>  major fatty acids of the basal diet were : palmitic acid ,  <dig> ; stearic acid ,  <dig> ; oleic acid ,  <dig> ; linoleic acid ,  <dig> ; α-linolenic acid   <dig> .
the control group received the basal diet without any further supplement.
the second group  received the basal diet supplemented with 2 g of clofibrate  per kg of diet.
at the day of parturition, the sows were given 3 kg of the diet.
from day  <dig> of lactation to later lactation, the amount of diet administered was calculated in order to meet the energy requirement of the sows   <cit> .
water was given ad libitum by nipple drinkers.
feed intakes were recorded daily and body weights weekly.
the energy balance of the sows during lactation was estimated as the difference of me intake and total energy requirement for maintenance and energy requirement for milk production.
it was assumed that milk production for 1 kg of litter gain is equivalent to an energy demand of  <dig>  mj me  <cit> .
the piglets were offered a creep feed  from day  <dig> until weaning.
all experimental procedures were carried out in accordance with established guidelines for the care and handling of laboratory animals and were approved by the local animal welfare authorities .
sample collection
on day  <dig> of lactation, blood from vena jugularis was collected 3 h after feed intake into heparinized polyethylene tubes  and plasma was subsequently obtained by centrifugation of the blood  and stored at −20°c.
on day  <dig>  liver and muscle biopsy samples were taken percutaneously after anaesthesia by intramuscular injection of  <dig> mg azaperon  per kg body mass, 20 mg ketamine  per kg body mass and up to  <dig>  mg thiopental  per kg body mass as required for maintenance of anaesthesia.
the pigs were placed in the left lateral recumbency and the surgical area was infiltrated with 1 ml of 2% lidocaine hydrochloride  for local anaesthesia.
a percutaneous liver biopsy was performed with a 16 g/ <dig>  mm biopsy needle  on a histocore® system  on expected anatomical location of the liver under constant ultrasound guidance.
the intramuscular biopsy  was performed with a spirotome .
after removal of the samples, the sites for biopsy were subsequently closed with synthetic absorbable surgical suture.
for collection of milk samples, sows were given 20 i.u.
oxytocin  by intramuscular injection.
80–100 ml of milk was expressed manually from all active teats of each sow.
liver and muscle samples were stored at −80°c and milk samples were stored at −20°c pending analysis.
concentrations of nutrients in milk
measurements of protein and fat concentrations in the milk were performed by the official methods of verband deutscher landwirtschaftlicher untersuchungs- und forschungsanstalten   <cit> .
the concentration of lactose in the milk was determined using a commercial kit  according to the manufacturer’s instructions.
the gross energy content of the milk was calculated using the following gross energy values: fat = 39 kj/g, protein = 24 kj/g, lactose = 17 kj/g.
for analysis of the fatty acid composition of milk total lipids, lipids were extracted from the milk with a hexane:isopropanol  mixture  <cit> .
fatty acids were analyzed by capillary gas chromatography after converting fatty acids into their methyl esters by trimethyl sulfonium hydroxide  <cit> .
concentrations of retinol and α-tocopherol in milk samples were determined by hplc  using a modification of the method of balz et al.
<cit> .
samples of  <dig>  ml of milk were mixed with 2 ml of a 10 g/l pyrogallol solution  and 300 μl of a saturated sodium hydroxide solution.
after flushing with nitrogen, this mixture was heated for 30 min at 70°c in closed glass tubes.
retinol and α-tocopherol were then extracted by addition of 2 ml of n-hexane and 2 ml of bidest.
water.
after centrifugation, an aliquot of the hexane phase was evaporated to dryness under nitrogen and re-dissolved in methanol containing  <dig> % of butylated hydroxytoluene.
retinol and α-tocopherol were separated isocratically by hplc using a mixture of methanol and water  as mobile phase and a lichrosher  <dig> rp <dig> column  and detected by fluorescence .
concentration of tag and nefa in plasma
plasma concentrations of tag and nefa were measured using enzymatic kits .
rna isolation and qpcr
rna isolation from frozen liver and muscle biopsies and quantitative real-time pcr  analysis were performed as described recently in detail  <cit> .
in brief, total rna from frozen liver and muscle samples was isolated using trizol® reagent  according to the manufacturer’s protocol and purified using the rneasy minikit .
concentration and purity of the rna were estimated from the optical density at  <dig> and 280 nm, respectively.
the integrity of total rna was verified using  <dig> % agarose gel electrophoresis.
cdna was synthesized using  <dig>  μg of total rna,  <dig> pmol oligo <dig> primer ,  <dig>  μl 10 mm dntp mix , 5 μl 5x rt reaction buffer , and  <dig> units m-mulv reverse transcriptase  at 42°c for 60 min and a final inactivating step at 70°c for 10 min in a thermo cycler .
qpcr runs were performed with a rotorgene  <dig> system  using kapa sybr fast qpcr universal mastermix  and gene-specific primer pairs from eurofins mwg operon .
ct-values of target and reference genes were obtained using rotorgene software  <dig> .
determination of relative expression levels was calculated using genorm normalization factor including the three most stable out of six reference genes  <cit> .
gene-specific primer pairs were designed intron-spanning if possible using primer <dig> and blast.
characteristics of primers used for qpcr analysis are shown in table  <dig> table  <dig>
characteristics of gene specific primers used for qpcr
gene
forward primer
pcr product size
ncbi genbank
slope
r
2
efficiency
m-value
reverse primer
reference genes
actb
atp5g1
gsr
rps9
pparα target genes
acox1
cd36
cpt1a
cpt1b
cyp4a24
fabp1
fabp3
lpl
slc27a1
ucp3
genes of the ubiquitin proteasome system
fbxo32
trim63
ubb
abbreviations: acox1 acyl-coa oxidase, actb  actin, beta, atp5g1 atp synthase, h+ transporting, mitochondrial fo complex, subunit c <dig>  cd36 fatty acid translocase, cpt1 carnitine-palmitoyl-transferase, cyp4a24 cytochrome p <dig> a <dig>  fabp fatty acid binding protein, fbxo32 f-box protein  <dig>  lpl lipoprotein lipase, gsr glutathione reductase, pparα peroxisome proliferator- activated receptor α, rps9 ribosomal protein s <dig>  slc27a1 solute carrier family  <dig> , member  <dig>  trim63 tripartite motif containing  <dig>  e <dig> ubiquitin protein ligase, ubb ubiquitin b, ucp3 uncoupling protein  <dig>
statistical analysis
statistical analysis was performed by student’s t test using the minitab statistical software rel.
<dig>  .
differences were considered statistically significant for p <  <dig> .
RESULTS
daily feed intake, body weights and energy balance of the lactating sows
the daily feed intake during the three weeks of lactation was not different between control sows and sows administered clofibrate .
body weights of the sows at day  <dig> post partum and day  <dig> post partum and body weight losses of the sows from day  <dig> to day  <dig> were similar in both groups of sows .
the calculated energy balance of the sows from day  <dig> post partum to day  <dig> post partum, the time period in which piglets did not receive any additional feed, was also similar in both groups of sows .table  <dig>
daily feed intake, body weights and energy balance of lactating control sows and sows administered clofibrate
control
clofibrate
p
-value
values are mean ± sd ; p.p.
= post partum.
mrna concentrations of pparα target genes in tissues of the lactating sows
in overall, administration of clofibrate had only a moderate effect on the expression of the pparα target genes in liver and muscle of sows on day  <dig> of lactation .
in the liver, mrna concentrations of only  <dig> out of the  <dig> pparα target genes considered  were significantly up-regulated by administration of clofibrate .
the extent of up-regulation for these genes, with the only exception of cpt1a was moderate.
in muscle, there was a significant  up-regulation of  <dig> out of the  <dig> pparα target genes considered .
as in the liver, the extent of up-regulation of these genes, being in the range between 43% and 68%, however, was relatively moderate .table  <dig>
relative mrna concentrations of pparα target genes in liver and muscle of lactating control sows and sows administered clofibrate on day  <dig> of lactation
control
clofibrate
p
-value
acox1
cd36
cpt1a
cyp4a24
fabp1
lpl
slc27a1
aco
cd36
cpt1b
cyp4a24
fabp3
lpl
slc27a1
ucp3
values are mean ± sd ; abbreviations: see footnote of table  <dig>
concentrations of tag and nefa in plasma
the concentrations of tag and nefa in plasma on day  <dig> of lactation were significantly lower in the clofibrate group than in the control group .table  <dig>
concentrations of tag and nefa in plasma of lactating control sows and sows administered clofibrate on day  <dig> of lactation
control
clofibrate
p-
value
values are mean ± sd .
milk composition of the lactating sows and gains of litters
concentrations of fat, lactose and the calculated gross energy content of the milk on day  <dig> of lactation did not differ between control sows and sows administered clofibrate.
however, the concentration of protein in the milk was higher in the clofibrate group than in the control group .
the fatty acid composition of milk total lipids was slightly different between the two groups of sows.
in the milk of sows administered clofibrate, the proportion of palmitic acid  was slightly higher and proportions of linoleic acid  and α-linoleic acid  were slightly lower than in milk of control sows .
proportions of all the other fatty acids in milk total lipids were unchanged between the two groups of sows .
according to the differences in the proportions of palmitic acid, linoleic acid and α-linoleic acid, milk of sows administered clofibrate had a higher proportion of total saturated fatty acids and a lower proportion of total polyunsaturated fatty acids  than milk of control sows .
the concentrations of the fat soluble vitamins retinol and α-tocopherol in the milk, expressed per g of fat, were also similar in both groups of sows .
gains of litters during the first 14 days  and the whole 21 days suckling period were also not different between control sows and sows supplemented with clofibrate .table  <dig>
composition of milk of lactating control sows and sows administered clofibrate on day  <dig> of lactation
control
clofibrate
p-
value
nutrients
gross energy #
fatty acids
fat soluble vitamins
values are mean ± sd ; abbreviations: sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids.
#calculated value.
weights of litters from lactating control sows and sows administered clofibrate at birth and gains of litters during the period from day  <dig> to day  <dig> of lactation
control
clofibrate
p
-value
values are mean ± sd .
litters were standardized to  <dig> piglets per sow.
relative mrna concentrations of components of the ubiquitin proteasome system in muscle
in order to find out whether an enhanced proteolysis in muscle could be the reason for an increase of milk protein content found in sows treated with clofibrate, mrna concentrations of ubb , fbox <dig>  and trim <dig>  in muscle were determined.
relative mrna concentrations of these three genes were higher in sows treated with clofibrate than in control sows .
discussion
this study was performed to investigate the effect of activation of pparα on the expression of genes involved in the utilization of fatty acids in liver and muscle of lactating sows.
moreover, we aimed to find out whether alterations in fatty acid utilization could affect milk fat synthesis.
to induce activation of pparα, lactating sows were fed a diet supplemented with clofibrate, a well-known synthetic agonist of pparα belonging to the group of fibrates  <cit> .
we found that sows fed the diet supplemented with clofibrate indeed showed an up-regulation of several classical pparα target genes involved in fatty acid uptake and oxidation in liver and muscle which in its extent, however, was weaker than in previous studies with young pigs treated with clofibrate  <cit> .
the changes in mrna concentrations of pparα target genes indicate that the clofibrate treatment of the sows caused a moderate activation of pparα in liver and muscle.
the main reason for the relatively weak up-regulation of pparα target genes might be that the dose of clofibrate given to the sows in this study  was lower than the doses given to young pigs in the recent studies   <cit> .
moreover, it should be noted that pigs have generally a relatively low expression of pparα in comparison to some other species such as rodents and target genes are less responsive against agonists  <cit> .
it has been shown in sows that a negative energy balance commonly observed during lactation could induce activation of pparα due to increased concentrations of nefa released from adipose tissue  <cit> .
as the control sows considered in this study had only a slight negative energy balance which was reflected by low plasma nefa concentrations, it is unlikely that there was a pronounced activation of pparα in tissues of the control sows.
moreover, it is unlikely that there was an activation of pparα by dietary fatty acids as the dietary fat, a mixture of soybean oil and palmoil , provided low amounts of highly unsaturated fatty acids with a high affinity to pparα.
the finding that plasma tag and nefa concentrations were reduced by clofibrate treatment suggests that the moderate up-regulation of pparα target genes led to an increased utilization of tag and fatty acids in tissues.
reduced plasma tag concentrations which have been reported as a typical feature of activation of pparα in rats and pigs are mainly due to a reduced secretion of lipid from the liver into the blood and an increased clearance of plasma tag by lipoprotein lipase   <cit> .
as sows treated with clofibrate showed an up-regulation of lpl in muscle, it is likely that the reduction of plasma tag concentration was at least in part due to an increased lipolysis.
a reduction of plasma nefa concentration which has also been reported in humans or animals treated with fibrates  <cit>  might be mainly due to the up-regulation of fatty acid transporters which are direct targets of pparα  <cit> .
in the present study, we observed an up-regulation of cd <dig>  one of the key fatty acid transporters, in muscle of sows treated with clofibrate which might contribute to the reduction of plasma nefa concentration observed in the sows treated with clofibrate.
the finding that fat and energy content of the milk and weight gains of litters during the suckling period  were not different between the two groups of sows shows that the moderate up-regulation of pparα target genes in liver and muscle by clofibrate did not cause an impairment of milk synthesis in the mammary gland.
sows’ milk fat consists mainly of two sources of fatty acids, namely those deriving from de-novo fatty acid synthesis in the mammary gland and those released from tag-rich lipoproteins by lpl and taken up into the mammary gland.
in contrast, plasma nefa contribute less to the production of milk tag as the uptake of nefa into the mammary gland is relatively low in sows in comparison to other species such as cattle  <cit> .
fatty acids deriving from de-novo synthesis are mainly saturated and monounsaturated, while those transported via chylomicrons to the mammary gland are reflecting the dietary fatty acid profile  <cit> .
a reduced plasma tag concentration in sows treated with clofibrate indicates that there was a lower availability of fatty acids from tag-rich lipoproteins in the mammary gland which might provide an explanation for the finding that milk of these sows had slightly lower concentrations of pufa.
nevertheless, this study shows that a moderate reduction of plasma tag concentration by administration of a strong pparα agonist does not impair milk fat synthesis.
an unexpected finding of this study was that sows treated with clofibrate showed an increased protein content in the milk.
as milk protein synthesis depends on the availability of amino acids in the mammary gland  <cit> , an increased flux of amino acids via blood to the mammary gland could provide a potential explanation for this observation.
recently, we have observed that treatment of rodents with clofibrate stimulates the ubiquitin proteasome system and enhances protein breakdown in muscle  <cit> .
in the present study we observed that treatment of sows with clofibrate causes an up-regulation of ubiquitin and the two e <dig> ligases atrogin- <dig> and murf- <dig> which are-rate limiting for the ubiquitin proteasome system.
this finding suggests, although we do not have direct evidence for this, that treatment with clofibrate stimulates protein breakdown in muscle of sows which in turn could increase the availability of amino acids for milk protein synthesis.
however, this remains a matter of speculation.
we moreover observed that concentrations of retinol and α-tocopherol in the milk were not different between the two groups indicating that clofibrate did not influence the metabolism of fat soluble vitamins, i.e.
their delivery from tag-rich lipoproteins into the mammary gland.
it has been established that dietary fatty acids such as n- <dig> pufa can act as agonists of pparα  <cit> .
based on the fact that the pparα activating potential of those fatty acids is lower than that of synthetic pparα agonists such as clofibrate, the findings of the present study clearly indicate that fatty acids in sow diets acting as native pparα agonists might be uncritical with respect to milk production and gains of litters of lactating sows.
the study moreover suggests that a moderate up-regulation of pparα target genes involved in fatty acid uptake and oxidation in liver and muscle as a consequence of a negative energy balance might not interfere with milk production of sows.
this suggestion is supported by the observation that lactating sows are able to maintain their milk production even under the condition of a negative energy balance as long as their body fat reserves are not depleted to a large extent  <cit> .
CONCLUSIONS
in overall, the present study shows that treatment of lactating sows with clofibrate, a strong synthetic pparα agonist, induces only a moderate activation of pparα in liver and muscle and in turn had limited effect on whole body fatty acid utilization.
this may be the reason why clofibrate treatment did not influence milk composition and gains of litters during the suckling period.
thus, the present study indicates that activation of pparα induced either by native agonists such as dietary pufa or a by negative energy balance might be largely uncritical in lactating sows with respect to milk production and litter gains in lactating sows.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
dkg conceived of the study, supervised the analyses and drafted the manuscript.
bg performed the analyses and the statistical analyses.
sr conducted the animal experiment.
em supervised the analysis of vitamins.
sh and gr established and supervised the liver and muscle biopsy sampling procedure.
sb conducted liver and muscle biopsy sampling.
ge was responsible for animal keeping.
ke conceived of the study, participated in its design and coordination and helped to draft the manuscript.
all authors read and approved the final manuscript.

 lactation is a physiological state which in most animal species is associated with a strong increase in energy and nutrient requirement for milk production. this demand is usually met by marked increase in feed intake and by utilization of body’s energy stores. in some animal species such as rats or mice, additionally metabolic adaptations occur during lactation which aim to conserve energy for milk production. down-regulation of proteins involved in fatty acid uptake and oxidation in liver and skeletal muscle and a decreased thermogenesis contribute to these energy sparing effects. we have observed that these effects in lactating rodents are mediated by a suppression of peroxisome proliferator-activated receptor α, a transcription factor which controls the expression of many genes involved in lipid catabolism. in lactating rats, it has been moreover observed that an activation of pparα by feeding an oxidized fat causes a disturbance of those metabolic adaptations. it has been found that there was an increased uptake of fatty acids into the liver and an increased oxidation of fatty acids in the lactating rats fed the oxidized fat, which in turn led to a reduced availability of fatty acids for milk production, resulting in a reduced milk triacylglycerol concentration and reduced growth rates of the suckling pups. in lactating sows, milk production is important for the post-natal growth of their litters. however, in contrast to rodents, the role of pparα in lactating sows has not yet been determined. according to rodent studies, an activation of pparα during lactation could suppress milk fat synthesis due to a reduced availability of fatty acids as a result of an increased utilization of fatty acids by liver and muscle. the question whether activation of pparα could affect milk fat synthesis is of relevance as dietary components such as polyunsaturated fatty acids are able to activate pparα. moreover, it is known that a negative energy balance which is common in lactating sows causes an activation of pparα due to increased concentrations of nefa in plasma released from adipose tissue. indeed, recently an activation of pparα in liver of lactating sows with a negative energy balance has been observed. however, metabolic effects of activation of pparα in lactating sows have not been investigated so far. in order to investigate the hypothesis that activation of pparα could impair milk production, we performed an experiment with lactating sows which were fed a diet supplemented with clofibrate as a synthetic agonist of pparα and determined milk composition and gains of litters. methods animals and diets twenty second parity sows were artificially inseminated and kept in single crates until day of pregnancy. from day to of pregnancy, the sows were kept in groups in pens equipped with fully slatted floors, nipple drinkers and feeders with a temperature of 19 ± 1°c with a 12:12 h light–dark cycle and 60–80% relative humidity by means of an air conditioning system. on day of pregnancy, they were housed individually in farrowing crates. after parturition, the sows were randomly assigned into two feeding groups of animals each with an equalized litter size of piglets per sow. infrared heaters were used to provide a surrounding temperature of 35°c for newborn pigs. the duration of the suckling period was 21 days. during lactation all sows were fed a nutritionally adequate basal diet consisting mainly of: wheat, barley, soybean meal with 43% crude protein, a mixture of soybean oil and palm oil and a mineral supplement. the basal diet contained  mj metabolizable energy /kg; concentrations of crude nutrients were: crude protein, 154; crude fat, 74; crude fiber, 45; crude ash, 43; starch, major fatty acids of the basal diet were: palmitic acid,; stearic acid,; oleic acid,; linoleic acid,; α-linolenic acid. the control group received the basal diet without any further supplement. the second group received the basal diet supplemented with 2 g of clofibrate per kg of diet. at the day of parturition, the sows were given 3 kg of the diet. from day of lactation to later lactation, the amount of diet administered was calculated in order to meet the energy requirement of the sows. water was given ad libitum by nipple drinkers. feed intakes were recorded daily and body weights weekly. the energy balance of the sows during lactation was estimated as the difference of me intake and total energy requirement for maintenance and energy requirement for milk production. it was assumed that milk production for 1 kg of litter gain is equivalent to an energy demand of  mj me. the piglets were offered a creep feed from day until weaning. all experimental procedures were carried out in accordance with established guidelines for the care and handling of laboratory animals and were approved by the local animal welfare authorities. sample collection on day of lactation, blood from vena jugularis was collected 3 h after feed intake into heparinized polyethylene tubes and plasma was subsequently obtained by centrifugation of the blood and stored at −20°c. on day liver and muscle biopsy samples were taken percutaneously after anaesthesia by intramuscular injection of mg azaperon per kg body mass, 20 mg ketamine per kg body mass and up to  mg thiopental per kg body mass as required for maintenance of anaesthesia. the pigs were placed in the left lateral recumbency and the surgical area was infiltrated with 1 ml of 2% lidocaine hydrochloride for local anaesthesia. a percutaneous liver biopsy was performed with a 16 g/  mm biopsy needle on a histocore® system on expected anatomical location of the liver under constant ultrasound guidance. the intramuscular biopsy was performed with a spirotome. after removal of the samples, the sites for biopsy were subsequently closed with synthetic absorbable surgical suture. for collection of milk samples, sows were given 20 i.u. oxytocin by intramuscular injection. 80–100 ml of milk was expressed manually from all active teats of each sow. liver and muscle samples were stored at −80°c and milk samples were stored at −20°c pending analysis. concentrations of nutrients in milk measurements of protein and fat concentrations in the milk were performed by the official methods of verband deutscher landwirtschaftlicher untersuchungs- und forschungsanstalten. the concentration of lactose in the milk was determined using a commercial kit according to the manufacturer’s instructions. the gross energy content of the milk was calculated using the following gross energy values: fat = 39 kj/g, protein = 24 kj/g, lactose = 17 kj/g. for analysis of the fatty acid composition of milk total lipids, lipids were extracted from the milk with a hexane:isopropanol mixture. fatty acids were analyzed by capillary gas chromatography after converting fatty acids into their methyl esters by trimethyl sulfonium hydroxide. concentrations of retinol and α-tocopherol in milk samples were determined by hplc using a modification of the method of balz et al.. samples of  ml of milk were mixed with 2 ml of a 10 g/l pyrogallol solution and 300 μl of a saturated sodium hydroxide solution. after flushing with nitrogen, this mixture was heated for 30 min at 70°c in closed glass tubes. retinol and α-tocopherol were then extracted by addition of 2 ml of n-hexane and 2 ml of bidest. water. after centrifugation, an aliquot of the hexane phase was evaporated to dryness under nitrogen and re-dissolved in methanol containing % of butylated hydroxytoluene. retinol and α-tocopherol were separated isocratically by hplc using a mixture of methanol and water as mobile phase and a lichrosher rp column and detected by fluorescence. concentration of tag and nefa in plasma plasma concentrations of tag and nefa were measured using enzymatic kits. rna isolation and qpcr rna isolation from frozen liver and muscle biopsies and quantitative real-time pcr analysis were performed as described recently in detail. in brief, total rna from frozen liver and muscle samples was isolated using trizol® reagent according to the manufacturer’s protocol and purified using the rneasy minikit. concentration and purity of the rna were estimated from the optical density at and 280 nm, respectively. the integrity of total rna was verified using % agarose gel electrophoresis. cdna was synthesized using  μg of total rna, pmol oligo primer,  μl 10 mm dntp mix, 5 μl 5x rt reaction buffer, and units m-mulv reverse transcriptase at 42°c for 60 min and a final inactivating step at 70°c for 10 min in a thermo cycler. qpcr runs were performed with a rotorgene system using kapa sybr fast qpcr universal mastermix and gene-specific primer pairs from eurofins mwg operon. ct-values of target and reference genes were obtained using rotorgene software. determination of relative expression levels was calculated using genorm normalization factor including the three most stable out of six reference genes. gene-specific primer pairs were designed intron-spanning if possible using primer and blast. characteristics of primers used for qpcr analysis are shown in table  table characteristics of gene specific primers used for qpcr gene forward primer pcr product size ncbi genbank slope r 2 efficiency m-value reverse primer reference genes   actb   atp5g1   gsr   rps9 pparα target genes   acox1   cd36   cpt1a   cpt1b   cyp4a24   fabp1   fabp3   lpl   slc27a1   ucp3 genes of the ubiquitin proteasome system   fbxo32   trim63   ubb abbreviations: acox1 acyl-coa oxidase, actb  actin, beta, atp5g1 atp synthase, h+ transporting, mitochondrial fo complex, subunit c cd36 fatty acid translocase, cpt1 carnitine-palmitoyl-transferase, cyp4a24 cytochrome p a fabp fatty acid binding protein, fbxo32 f-box protein lpl lipoprotein lipase, gsr glutathione reductase, pparα peroxisome proliferator- activated receptor α, rps9 ribosomal protein s slc27a1 solute carrier family, member trim63 tripartite motif containing e ubiquitin protein ligase, ubb ubiquitin b, ucp3 uncoupling protein statistical analysis statistical analysis was performed by student’s t test using the minitab statistical software rel.. differences were considered statistically significant for p < .  daily feed intake, body weights and energy balance of the lactating sows the daily feed intake during the three weeks of lactation was not different between control sows and sows administered clofibrate. body weights of the sows at day post partum and day post partum and body weight losses of the sows from day to day were similar in both groups of sows. the calculated energy balance of the sows from day post partum to day post partum, the time period in which piglets did not receive any additional feed, was also similar in both groups of sows.table daily feed intake, body weights and energy balance of lactating control sows and sows administered clofibrate control clofibrate p -value values are mean ± sd; p.p. = post partum. mrna concentrations of pparα target genes in tissues of the lactating sows in overall, administration of clofibrate had only a moderate effect on the expression of the pparα target genes in liver and muscle of sows on day of lactation. in the liver, mrna concentrations of only out of the pparα target genes considered were significantly up-regulated by administration of clofibrate. the extent of up-regulation for these genes, with the only exception of cpt1a was moderate. in muscle, there was a significant up-regulation of out of the pparα target genes considered. as in the liver, the extent of up-regulation of these genes, being in the range between 43% and 68%, however, was relatively moderate.table relative mrna concentrations of pparα target genes in liver and muscle of lactating control sows and sows administered clofibrate on day of lactation control clofibrate p -value   acox1   cd36   cpt1a   cyp4a24   fabp1   lpl   slc27a1   aco   cd36   cpt1b   cyp4a24   fabp3   lpl   slc27a1   ucp3 values are mean ± sd; abbreviations: see footnote of table  concentrations of tag and nefa in plasma the concentrations of tag and nefa in plasma on day of lactation were significantly lower in the clofibrate group than in the control group.table concentrations of tag and nefa in plasma of lactating control sows and sows administered clofibrate on day of lactation control clofibrate p- value values are mean ± sd. milk composition of the lactating sows and gains of litters concentrations of fat, lactose and the calculated gross energy content of the milk on day of lactation did not differ between control sows and sows administered clofibrate. however, the concentration of protein in the milk was higher in the clofibrate group than in the control group. the fatty acid composition of milk total lipids was slightly different between the two groups of sows. in the milk of sows administered clofibrate, the proportion of palmitic acid was slightly higher and proportions of linoleic acid and α-linoleic acid were slightly lower than in milk of control sows. proportions of all the other fatty acids in milk total lipids were unchanged between the two groups of sows. according to the differences in the proportions of palmitic acid, linoleic acid and α-linoleic acid, milk of sows administered clofibrate had a higher proportion of total saturated fatty acids and a lower proportion of total polyunsaturated fatty acids than milk of control sows. the concentrations of the fat soluble vitamins retinol and α-tocopherol in the milk, expressed per g of fat, were also similar in both groups of sows. gains of litters during the first 14 days and the whole 21 days suckling period were also not different between control sows and sows supplemented with clofibrate.table composition of milk of lactating control sows and sows administered clofibrate on day of lactation control clofibrate p- value nutrients gross energy # fatty acids fat soluble vitamins values are mean ± sd; abbreviations: sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids. #calculated value. weights of litters from lactating control sows and sows administered clofibrate at birth and gains of litters during the period from day to day of lactation control clofibrate p -value values are mean ± sd. litters were standardized to piglets per sow. relative mrna concentrations of components of the ubiquitin proteasome system in muscle in order to find out whether an enhanced proteolysis in muscle could be the reason for an increase of milk protein content found in sows treated with clofibrate, mrna concentrations of ubb, fbox and trim in muscle were determined. relative mrna concentrations of these three genes were higher in sows treated with clofibrate than in control sows. discussion this study was performed to investigate the effect of activation of pparα on the expression of genes involved in the utilization of fatty acids in liver and muscle of lactating sows. moreover, we aimed to find out whether alterations in fatty acid utilization could affect milk fat synthesis. to induce activation of pparα, lactating sows were fed a diet supplemented with clofibrate, a well-known synthetic agonist of pparα belonging to the group of fibrates. we found that sows fed the diet supplemented with clofibrate indeed showed an up-regulation of several classical pparα target genes involved in fatty acid uptake and oxidation in liver and muscle which in its extent, however, was weaker than in previous studies with young pigs treated with clofibrate. the changes in mrna concentrations of pparα target genes indicate that the clofibrate treatment of the sows caused a moderate activation of pparα in liver and muscle. the main reason for the relatively weak up-regulation of pparα target genes might be that the dose of clofibrate given to the sows in this study was lower than the doses given to young pigs in the recent studies. moreover, it should be noted that pigs have generally a relatively low expression of pparα in comparison to some other species such as rodents and target genes are less responsive against agonists. it has been shown in sows that a negative energy balance commonly observed during lactation could induce activation of pparα due to increased concentrations of nefa released from adipose tissue. as the control sows considered in this study had only a slight negative energy balance which was reflected by low plasma nefa concentrations, it is unlikely that there was a pronounced activation of pparα in tissues of the control sows. moreover, it is unlikely that there was an activation of pparα by dietary fatty acids as the dietary fat, a mixture of soybean oil and palmoil, provided low amounts of highly unsaturated fatty acids with a high affinity to pparα. the finding that plasma tag and nefa concentrations were reduced by clofibrate treatment suggests that the moderate up-regulation of pparα target genes led to an increased utilization of tag and fatty acids in tissues. reduced plasma tag concentrations which have been reported as a typical feature of activation of pparα in rats and pigs are mainly due to a reduced secretion of lipid from the liver into the blood and an increased clearance of plasma tag by lipoprotein lipase. as sows treated with clofibrate showed an up-regulation of lpl in muscle, it is likely that the reduction of plasma tag concentration was at least in part due to an increased lipolysis. a reduction of plasma nefa concentration which has also been reported in humans or animals treated with fibrates might be mainly due to the up-regulation of fatty acid transporters which are direct targets of pparα. in the present study, we observed an up-regulation of cd one of the key fatty acid transporters, in muscle of sows treated with clofibrate which might contribute to the reduction of plasma nefa concentration observed in the sows treated with clofibrate. the finding that fat and energy content of the milk and weight gains of litters during the suckling period were not different between the two groups of sows shows that the moderate up-regulation of pparα target genes in liver and muscle by clofibrate did not cause an impairment of milk synthesis in the mammary gland. sows’ milk fat consists mainly of two sources of fatty acids, namely those deriving from de-novo fatty acid synthesis in the mammary gland and those released from tag-rich lipoproteins by lpl and taken up into the mammary gland. in contrast, plasma nefa contribute less to the production of milk tag as the uptake of nefa into the mammary gland is relatively low in sows in comparison to other species such as cattle. fatty acids deriving from de-novo synthesis are mainly saturated and monounsaturated, while those transported via chylomicrons to the mammary gland are reflecting the dietary fatty acid profile. a reduced plasma tag concentration in sows treated with clofibrate indicates that there was a lower availability of fatty acids from tag-rich lipoproteins in the mammary gland which might provide an explanation for the finding that milk of these sows had slightly lower concentrations of pufa. nevertheless, this study shows that a moderate reduction of plasma tag concentration by administration of a strong pparα agonist does not impair milk fat synthesis. an unexpected finding of this study was that sows treated with clofibrate showed an increased protein content in the milk. as milk protein synthesis depends on the availability of amino acids in the mammary gland, an increased flux of amino acids via blood to the mammary gland could provide a potential explanation for this observation. recently, we have observed that treatment of rodents with clofibrate stimulates the ubiquitin proteasome system and enhances protein breakdown in muscle. in the present study we observed that treatment of sows with clofibrate causes an up-regulation of ubiquitin and the two e ligases atrogin- and murf- which are-rate limiting for the ubiquitin proteasome system. this finding suggests, although we do not have direct evidence for this, that treatment with clofibrate stimulates protein breakdown in muscle of sows which in turn could increase the availability of amino acids for milk protein synthesis. however, this remains a matter of speculation. we moreover observed that concentrations of retinol and α-tocopherol in the milk were not different between the two groups indicating that clofibrate did not influence the metabolism of fat soluble vitamins, i.e. their delivery from tag-rich lipoproteins into the mammary gland. it has been established that dietary fatty acids such as n- pufa can act as agonists of pparα. based on the fact that the pparα activating potential of those fatty acids is lower than that of synthetic pparα agonists such as clofibrate, the findings of the present study clearly indicate that fatty acids in sow diets acting as native pparα agonists might be uncritical with respect to milk production and gains of litters of lactating sows. the study moreover suggests that a moderate up-regulation of pparα target genes involved in fatty acid uptake and oxidation in liver and muscle as a consequence of a negative energy balance might not interfere with milk production of sows. this suggestion is supported by the observation that lactating sows are able to maintain their milk production even under the condition of a negative energy balance as long as their body fat reserves are not depleted to a large extent.  in overall, the present study shows that treatment of lactating sows with clofibrate, a strong synthetic pparα agonist, induces only a moderate activation of pparα in liver and muscle and in turn had limited effect on whole body fatty acid utilization. this may be the reason why clofibrate treatment did not influence milk composition and gains of litters during the suckling period. thus, the present study indicates that activation of pparα induced either by native agonists such as dietary pufa or a by negative energy balance might be largely uncritical in lactating sows with respect to milk production and litter gains in lactating sows. competing interests the authors declare that they have no competing interests. authors’ contributions dkg conceived of the study, supervised the analyses and drafted the manuscript. bg performed the analyses and the statistical analyses. sr conducted the animal experiment. em supervised the analysis of vitamins. sh and gr established and supervised the liver and muscle biopsy sampling procedure. sb conducted liver and muscle biopsy sampling. ge was responsible for animal keeping. ke conceived of the study, participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. 
thus, the present study indicates that activation of pparα induced either by native agonists such as dietary polyunsaturated fatty acids or a by negative energy balance might be largely uncritical in lactating sows with respect to milk production and litter gains in lactating sows. in the clofibrate group, mrna concentrations of various pparα target genes involved in fatty acid utilization in liver and skeletal muscle were moderately up-regulated. it is shown that treatment with clofibrate induces only a moderate up-regulation of pparα target genes in liver and muscle of lactating sows and in turn might have limited effect on whole body fatty acid utilization. twenty lactating sows received either a basal diet or the same diet with supplementation of 2 g of clofibrate per kg of diet. therefore, the present study aimed to investigate the effect of treatment with clofibrate, a strong synthetic agonist of pparα, on milk composition and litter gains in lactating sows. fat and energy content of the milk and gains of litters during the suckling period were not different between the control group and the clofibrate group. this may be the reason why clofibrate treatment did not influence milk fat content and gains of litters during the suckling period. it has not yet been investigated whether agonists of pparα are impairing milk production of lactating sows in a similar manner as in rats.
 in rats, it has been observed that treatment with activators of peroxisome proliferator-activated receptor α disturbs metabolic adaptations during lactation, which in turn lead to a reduction of milk fat content and gains of litters during the suckling period. it has not yet been investigated whether agonists of pparα are impairing milk production of lactating sows in a similar manner as in rats. therefore, the present study aimed to investigate the effect of treatment with clofibrate, a strong synthetic agonist of pparα, on milk composition and litter gains in lactating sows.  twenty lactating sows received either a basal diet or the same diet with supplementation of 2 g of clofibrate per kg of diet. in the clofibrate group, mrna concentrations of various pparα target genes involved in fatty acid utilization in liver and skeletal muscle were moderately up-regulated. fat and energy content of the milk and gains of litters during the suckling period were not different between the control group and the clofibrate group.  it is shown that treatment with clofibrate induces only a moderate up-regulation of pparα target genes in liver and muscle of lactating sows and in turn might have limited effect on whole body fatty acid utilization. this may be the reason why clofibrate treatment did not influence milk fat content and gains of litters during the suckling period. thus, the present study indicates that activation of pparα induced either by native agonists such as dietary polyunsaturated fatty acids or a by negative energy balance might be largely uncritical in lactating sows with respect to milk production and litter gains in lactating sows. issue-copyright-statement© the author 2015 