the experimental animals , feeding and management : the experimental 
 procedures complied with the guide for the care and use of agricultural animals of obihiro 
 university .
the experiment was carried out at the field center of animal science and agriculture , 
 obihiro university of agriculture and veterinary medicine .
fifty multiparous holstein cows , 
 in which parity was from 1st to 5th at dry period , were used in this study and had calved 
 between september 2011 and august 2012 .
parity and body condition score ( bcs ) of the 
 experimental cows at initiation of the study were 2.4  0.2 and 3.41  0.04 , respectively . 

the study was performed from 3 weeks before the expected parturition to 100 days pp .
cows 
 close to the dry period , about 1 month before the expected calving date , were moved to a 
 paddock and fed a limited total mixed ration [ dry matter ( dm ) basis : 127 g of crude protein 
 ( cp)/kg and 6.6 mj of net energy for lactation ( nel)/kg ] consisting of grass silage ( 3.5 kg , 
 dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , maize silage ( 5.1 kg , dm basis : 86 g of 
 cp / kg and 6.0 mj of nel / kg ) , concentrate for dry cows ( 2.0 kg , dm basis : 170 g of cp / kg and 
 6.8 mj of nel / kg ) and grass hay ( dm basis : 125 g of cp / kg and 5.7 mj of nel / kg ) ad 
 libitum until parturition .
after parturition , cows were housed in a free - stall 
 barn and fed a lactation diet , which was a mixed ration ( dm basis : 155 g of cp / kg and 6.2 mj 
 of nel / kg ) consisting of grass ( 6.5 kg , dm basis : 165 g of cp / kg and 5.5 mj of nel / kg ) , 
 maize silage ( 12.5 kg , dm basis : 84 g of cp / kg and 6.4 mj of nel / kg ) and concentrate for 
 dairy cows ( 8.0 kg , dm basis : 180 g of cp / kg and 7.1 mj of nel / kg ) ad 
 libitum .
in addition , the diets were supplemented with minerals , and the dairy 
 cow concentrate was prepared according to each cow s specific requirements for milk 
 production .
grass hay ( dm basis : 104 g of cp / kg and 5.2 mj of nel / kg ) and water were 
 available ad libitum .
cows were milked twice daily between 05:00 and 06:30 
 hr and between 17:00 and 18:30 hr .
the experimental itt and sampling : the itt was performed 3 weeks before 
 the expected calving date .
the cows were weighed the day before the initiation of the itt , 
 and bw was used to determine the doses of insulin for the itt .
immediately before the itt , an extension catheter was inserted into the 
 right or left jugular vein .
the itt was performed by intravenously administering 0.05 iu / kg 
 bw of insulin ( novolin r 100 iu / ml ; novo nordisk pharma , tokyo , japan ) , 
 followed by administration of 5 ml heparinized saline ( 100 
 iu / ml ) .
blood samples were 
 collected via the jugular vein at 0 ( before insulin injection ) , 30 , 45 and 60 min relative 
 to the administration of insulin via caudal venipuncture to measure glucose and insulin .
bcs was assessed twice a week from 3 weeks before the expected parturition to 3 weeks after 
 calving by the same operator by using a 1 to 5 scale with 0.25 intervals , where 1=thin and 
 5=very fat .
blood samples were obtained by 
 caudal venipuncture twice a week from 3 weeks before the expected parturition to 3 weeks 
 after calving .
blood samples were collected via the jugular vein from the calves immediately 
 after birth .
nonheparinized and silicone - coated 9-ml tubes ( venoject , 
 autosep , gel + clot . act .
vp - as109k ; terumo corporation , tokyo , japan ) were used for 
 biochemical analysis , and sterile 10-ml tubes containing 200 
 l of stabilizer solution ( 0.3 m edta and 1% acetyl salicylic acid , ph 
 7.4 ) were used for hormonal analysis .
serum was obtained by centrifuging the blood samples 
 for 15 min at 38c in an incubator .
all the tubes were centrifuged at 2,000  
 g for 20 min at 4c , and plasma samples were maintained at 30c until 
 analysis .
in addition , milk samples were collected twice a week after milking until the 
 onset of luteal activity .
the milk samples were centrifuged at 1,500  g 
 for 15 min at 4c , and the skim milk samples were stored at 30c until analysis for 
 progesterone concentration .
daily milk yield was recorded until 100 days pp . peripartum 
 diseases , such as milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , 
 lameness , retained placenta , endometritis and mastitis , were recorded when that has been 
 diagnosed from 3 weeks prepartum to 3 weeks postpartum by veterinarian in the experimental 
 farm .
the experimental measurement of hormones and metabolites : plasma and skim 
 milk progesterone concentrations were determined using enzyme immunoassay ( eia ) after 
 extraction with diethyl ether , as described previously ; the extraction efficiency was 90% .
the standard curve ranged from 0.05 to 50 
 ng / ml , and the 50% effective dose ( ed50 ) of 
 the assay was 0.66 ng / ml . the mean intra - assay and 
 inter - assay coefficients of variation ( cvs ) were 6.0% and 9.2% , respectively .
the total 
 plasma insulin - like growth factor 1 ( igf-1 ) concentration was determined using eia by using 
 the biotin 
streptavidin amplification technique 
 after protein extraction by using acid ethanol ( 87.5% ethanol and 12.5% 2 n hydrochloric 
 acid ) to obtain igf-1 free from binding proteins .
intra- and inter - assay cvs were 5.9% and 6.1% , 
 respectively , and the ed50 of this assay system was 7.2 
 ng / ml . the plasma gh concentrations were determined 
 using eia as described previously ; the standard 
 curve ranged from 0.78 to 100 ng / ml , and the 
 ed50 was 21 ng / ml . intra- and inter - assay cvs 
 were 3.1% and 8.2% , respectively .
the plasma insulin concentrations were determined using an 
 enzyme - linked immunosorbent assay ( elisa ) kit ( bovine insulin elisa 10 - 1201 - 01 ; mercodia , 
 uppsala , sweden ) .
the serum concentrations of glucose , non - esterified fatty acids ( nefa ) , -hydroxybutyrate 
 ( bhba ) , total protein ( tp ) , albumin ( alb ) , blood urea nitrogen ( bun ) and total cholesterol 
 ( t - cho ) and the activities of aspartate aminotransferase ( ast ) were measured using a 
 clinical chemistry automated analyzer ( tba120fr ; toshiba medical systems co. , ltd . , tochigi , 
 japan ) .
the experimental identification of the onset of luteal activity : when the 
 progesterone concentration in the plasma or skim milk had increased to more than 1 
 ng / ml , the cows were considered to show luteal activity 
 .
the experimental statistical analysis : sixteen cows were excluded from 
 data analysis , because of the following reasons : a pregnancy period of more than 287 or less 
 than 273 days ( n=6 ) , severe mastitis ( n=3 ) , twin calving ( n=1 ) , blood collection loss at itt 
 ( n=3 ) and mistakes in insulin injection at itt ( n=3 ) .
cows were divided into two groups 
 based on the time required for glucose to reach the minimum levels after insulin injection . 
 cows with a minimum glucose at 60 min after insulin injection were considered to have lower 
 insulin sensitivity and/or lower glucose metabolism compared to cows with a minimum glucose 
 level by 45 min after insulin injection .
therefore , cows with a minimum glucose level at 60 
 min after insulin injection were defined as the insulin resistant group ( ir group ) , whereas 
 those with a minimum glucose level by 45 min after insulin injection were defined as the 
 non - insulin resistant ( nir group ) in this study . before data analysis ,
bcs , plasma igf-1 , gh 
 and insulin concentrations , and serum metabolite concentrations were averaged weekly . the 
 period of 06 days after calving was considered as the parturient week ( 0 week pp ) , and the 
 kolmogorov 
smirnov test ( sas enterprise guide version 4.3 ; sas institute inc . , cary , nc , 
 u.s.a . ) was used for statistical testing of normality . in addition , the data were analyzed 
 separately for the prepartum and pp periods .
stat view ( stat view 5.0 software ; abacus 
 concepts inc . , berkeley , ca , u.s.a . ) was used for data analysis by using the repeated 
 measures of the anova procedure , including time ( week ) , group ( nir or ir ) and their 
 interaction in the model as fixed effects .
diagnosis of peripartum diseases and sex of calves in the nir and ir groups were analyzed 
 using the chi - square test , and other data , including results for calves between nir and ir , 
 were analyzed using the student s t - test or wilcoxon s signed rank test 
 ( sas enterprise guide version 4.3 ; sas institute inc . ) .
results are presented as mean  
 standard error of the mean ( sem ) ; differences with p<0.05 were 
 considered significant .
in 28 of the 34 experimental cows , the time required for glucose to reach the minimum level 
 was 45 min after insulin injection with one exception ( 30 min ; n=1 , 45 min ; n=27 , nir 
 group ) . the remaining experimental cows ( n=6 ) required 60 min after insulin injection to 
 attain the minimum glucose levels ( ir group ) .
serum glucose concentrations at 60 min after 
 insulin injection were higher in the nir group than in the ir group , although glucose levels 
 at the other time points did not differ between the nir and ir groups ( fig .
1.the change in serum glucose concentration after insulin injection at insulin 
 tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups .
p<0.05 ) . the change in serum glucose concentration after insulin injection at insulin 
 tolerance test ( itt ) in the nir ( n=28 ) and ir ( n=6 ) groups .
table 1table 1.parity , calving difficulty , sex of calves , peripartum disease , luteal activity 
 onset and milk yield in the nir and ir groupsnir groupir groupp - value(n=28)(n=6)parity at the onset of experiment2.4  0.32.2  0.70.460calving difficulty1.1  0.11.0  0.00.700sex of calves ( male / female)14/143/31.000diagnosis of peripartum disease9/28 ( 32%)1/6 ( 17%)0.645days to the onset of luteal activity ( days)38.3  3.820.3  3.60.039average of daily milk yield between days 7 and 100 pp ( kg)41.4  0.935.9  2.00.013total milk yield from days 7 to 100 pp ( kg)3,888.1  81.23,375.5  185.90.013values are the mean  sem .
a ) nir group ; cows with a minimum glucose level by 45 min 
 after insulin injection .
ir group ; cows with a minimum glucose level at 60 min after 
 insulin injection .
b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy 
 calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia 
 ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of 
 cow .
c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , 
 lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 
 weeks postpartum . shows the parity , calving difficulty , sex of calves , peripartum disease 
 diagnosis , luteal activity onset and milk yield until 100 days pp in the nir and ir groups . 

days until the onset of luteal activity in the ir group were fewer than those in the nir 
 group ( p<0.05 ) .
in addition , the average ( p<0.05 ) 
 and total ( p<0.05 ) milk yields until 100 days pp were lower in the ir 
 group than in the nir group .
peripartum diseases were diagnosed as mastitis ( n=6 ) , 
 hypocalcemia ( n=1 ) and milk fever ( n=2 ) in nir group , and as mastitis ( n=1 ) in ir group , and 
 there was no significant difference in the number of cows with the peripartum diseases 
 between nir and ir groups .
no significant difference was noted in other factors between the 
 nir and ir groups .
a ) nir group ; cows with a minimum glucose level by 45 min 
 after insulin injection .
ir group ; cows with a minimum glucose level at 60 min after 
 insulin injection .
b ) 1 , unassisted birth ( natural , without human assistance ) ; 2 , easy 
 calving with human assistance ; 3 , difficult calving with a few humans ; 4 , dystocia 
 ( requiring considerably more force than normal ) ; and 5 , surgical treatment or death of 
 cow .
c ) milk fever , hypocalcemia , ketosis , ruminal acidosis , displaced abomasum , 
 lameness , retained placenta , endometritis and mastitis from 3 weeks prepartum to 3 
 weeks postpartum .
2.serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , 
 and bcs during the experimental period [ mean  sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) 
 groups ] .
* indicates differences of p<0.05 , and  indicates 
 differences of p<0.1 between the nir and ir groups . shows the circulating serum metabolite concentrations , enzyme levels , plasma 
 metabolic concentrations and bcs during the experimental period . during the prepartum 
 period , bcs ( p<0.05 ) , and serum bun concentrations 
 ( p<0.05 )
were lower , whereas serum glucose ( p=0.05 ) and 
 alb concentrations ( p=0.10 ) tended to be lower in the ir group than in the 
 nir group . during the pp period ,
cows of the nir group had higher serum nefa 
 ( p<0.05 ) and bhba ( p=0.09 ) concentrations than those 
 in the ir group .
in addition , treatment and time effects were observed 
 ( p<0.05 ) for bcs during the pp period : bcs at 0 
 ( p=0.08 ) and 1 ( p<0.05 ) week pp were lower in the ir 
 group than in the nir group .
no significant differences were noted in the other factors 
 between the nir and ir groups in each period .
serum metabolite concentrations , activities of enzymes and plasma metabolic hormones , 
 and bcs during the experimental period [ mean  sem : solid , nir ( n=28 ) ; open , ir ( n=6 ) 
 groups ] .
* indicates differences of p<0.05 , and  indicates 
 differences of p<0.1 between the nir and ir groups .
bw , plasma metabolic hormone levels and serum glucose concentrations at birth in the calves 
 of cows of the nir and ir groups are shown in table 
 2table 2.bw and plasma metabolic hormones and serum glucose concentrations at birth in the 
 calves of the nir and ir groupscalves of nircalves of irp - value(n=28)(n=6)bw at the birth ( kg)47.2  0.942.1  1.70.020plasma gh concentration 
 ( ng / ml)13.6  1.315.2  4.80.653plasma igf-1 concentration 
 ( ng / ml)121.5  6.369.8  5.60.001plasma insulin concentration 
 ( ng / ml)0.3
 0.00.7  0.20.061serum glucose concentration 
 ( mg / dl)77.4  5.272.1  14.80.684values are the mean  sem .
a ) nir group ; cows with a minimum glucose level by 45 min 
 after insulin injection .
ir group ; cows with a minimum glucose level at 60 min after 
 insulin injection .. bw at birth in the calves of the ir group was lower than that in the calves 
 of the nir group ( p<0.05 ) .
furthermore , the calves of the ir group 
 showed lower plasma igf-1 concentration ( p<0.001 ) and higher plasma 
 insulin concentration ( p=0.06 ) .
no significant differences were noted in 
 the plasma gh and serum glucose levels at birth between the calves of the nir and ir 
 groups .
a ) nir group ; cows with a minimum glucose level by 45 min 
 after insulin injection .
ir group ; cows with a minimum glucose level at 60 min after 
 insulin injection .
in this study , the six cows that reached the minimum glucose levels at 60 min after insulin 
 injection were considered to be ir ; the reason for ir was thought to be the slow recovery of 
 glucose after insulin injection , which is consistent with the findings of a previous study 
 by lee et al . .
in general , bcs 
 and blood glucose and bun concentrations are known to be associated with energy status and 
 feed intake [ 7 , 8 , 38 ] . during the prepartum period ,
ir 
 cows showed lower energy status and feed intake owing to the lower bcs and glucose and bun 
 concentrations .
although ir by itt was confirmed at 3 weeks before calving , a difference in 
 energy status between ir and nir cows was noted . in particular ,
bcs can not be evaluated on 
 the basis of the change in energy status of the real - time feed intake ; therefore , in this study , ir cows might have become insulin 
 resistant during an earlier time .
malnutrition causes imbalance in glucose homeostasis , and 
 the decrease of insulin in circulation induces the reduction of feed intake in dairy cows 
 .
therefore , the feed intake reduction in ir 
 cows might have inhibited the volatile fatty acid production in the rumen and thus 
 suppressed gluconeogenesis in the liver .
lower 
 energy status , such as lower bcs , before calving of ir group in this study might be caused 
 by long - term malnutrition from previous lactation .
however , in this study , the reasons for 
 the lower energy status in the ir cows were not clear ; thus , further studies are warranted 
 to confirm the onset of insulin resistance in pregnant dairy cows .
bcs at 0 and 1 week pp were lower in the ir group than in the nir group .
nir cows showed 
 higher serum nefa and bhba concentrations than those in ir cows , although the levels of 
 metabolic hormones did not differ between the ir and nir cows .
furthermore , the average and 
 total milk yield until 100 days pp were lower in the ir group than in the nir group .
higher 
 nefa and bhba indicate greater mobilization of adipose tissue and failure of lipid 
 metabolism in the liver [ 14 , 15 ] .
however , cows with lower bcs had sustained reduced plasma nefa and 
 bhba concentrations after calving compared to cows with higher bcs [ 28 , 33 ] . cows with lower bcs 
 produce milk by protein mobilization , because of the limited body fat ; thus , fat - corrected 
 milk yield in those cows was lower than moderate and fat cows .
conversely , it was indicated that higher bcs cows have ability to 
 mobilize fat to maintain energetic homeostasis after feed restriction .
additionally , roche et al .   have concluded that bcs at calving had positive effect on milk yield , 
 and optimal bcs at calving was 3.5 in the 5-point scale . in the present study ,
greater bcs 
 and better gluconeogenesis in nir group might produce greater milk yield compared with ir 
 group , although the differences of them between nir and ir groups were not so greater .
days 
 to the onset of luteal activity in the ir group were fewer than in the nir group
. in dairy 
 cows , lowered energy status during the peripartum period is known to delay the first 
 ovulation after parturition [ 2 , 19 ] .
butler and smith   showed 
 that a negative energy balance was directly related to the pp interval to the first 
 ovulation and that the differences in the energy balance were reflected in the milk yield . 

in addition , cows with a delayed first ovulation showed higher nefa and bhba concentrations 
 after parturition [ 21 , 31 , 39 ] .
therefore , in this study , 
 higher nefa and bhba concentrations of nir cows during the pp period might have delayed the 
 onset of luteal activity , and the lowered milk yield of ir cows might induce earlier 
 resumption of ovarian activity .
the maternal endocrine and metabolic milieu transferred through the placenta during late 
 pregnancy affects the environment of the fetus [ 17 , 
 24 , 30 ] . in 
 humans ,
ir of the mother is associated with low birth weight of the infant ; in cattle , maternal malnutrition during gestation is 
 related to the lowered development of both the placenta and the fetus [ 24 , 30 ] .
further , in ewes , 
 restricted maternal feeding during gestation was related to lower bw and plasma igf-1 , 
 insulin and glucose concentrations in the fetus , although maternal igf-1 concentrations were 
 not affected . in the present study ,
calves of 
 the ir cows showed lowered bw at birth and a lower plasma igf-1 concentration , supporting 
 the findings of previous studies .
in addition , they showed higher insulin levels than those 
 of nir cows , despite the similar glucose levels . in the late gestation , fetal growth
is 
 mainly regulated by igf-1 , and the dominant regulator of igf-1 production in the fetus is 
 fetal glucose and insulin .
thus , the differences 
 in blood metabolic hormones and glucose concentrations between the calves of the nir and ir 
 groups might be attributed to the fetal nutritional condition that was affected by maternal 
 endocrine and metabolic milieu . in humans ,
lower bw at birth is known to be associated with 
 a wide range of adverse outcomes later in life , including diabetes ; further , obese children with low birth weight have higher blood 
 insulin to glucose concentration and show higher insulin resistance as revealed by the 
 homeostasis model assessment compared with obese children with normal birth weight .
therefore , calves of ir cows might develop insulin 
 resistance in the future . in conclusion , the findings of the present study suggest that ir at 3 weeks before 
 parturition in dairy cows is related to the pp metabolic status , milk production and 
 resumption of ovarian activity along with growth , as well as the metabolic status of their 
 calves .
therefore , ir evaluated on the basis of the recovery of glucose after an injection 
 of a small dose of insulin during the dry period might be an indication of the pp 
 performance of pregnant dairy cows , as well as the growth , fertility and milk production of 
 their calves .
in addition , the reason for ir in the present study was thought to be the slow 
 recovery of glucose after insulin injection as well as the previous study .
therefore , the enhancement of the gluconeogenesis in 
 the liver by energy supplementation , such as glycerol , or hepatic stimulant , such as amino 
 acids , should be confirmed in order to improve the ir .
 this study aimed to investigate the effects of insulin resistance ( ir ) during the 
 close - up dry period on the metabolic status and performance of dairy cows as well as to 
 determine the effects on body weight ( bw ) and metabolic status of their calves . an insulin 
 tolerance test ( itt ) was conducted by administering 0.05 iu / kg bw of insulin to 34 
 multiparous holstein cows at 3 weeks prepartum . 
 blood samples were collected at 0 , 30 , 45 
 and 60 min after insulin injection , and cows were divided into two groups based on the 
 time required for glucose to reach the minimum levels [ non - ir ( nir ) , 45 min ( n=28 ) ; and 
 ir , 60 min ( n=6 ) ] . 
 blood or milk sampling and body condition score ( bcs ) estimation were 
 performed twice weekly during the experimental period . 
 blood samples from calves were 
 collected immediately after birth . 
 cows with ir showed lower bcs 
 ( p<0.05 ) and serum urea nitrogen ( p<0.05 ) and 
 glucose concentration ( p=0.05 ) before calving , and lower serum 
 non - esterified fatty acid concentration ( p<0.05 ) and milk yield 
 ( p<0.05 ) and earlier resumption of luteal activity 
 ( p<0.05 ) after calving ; their calves showed lower bw 
 ( p<0.05 ) and plasma insulin - like growth factor - i concentration 
 ( p<0.001 ) and higher plasma insulin concentration 
 ( p<0.05 ) . in conclusion , ir at 3 weeks prepartum in dairy cows is 
 related to postpartum metabolic status and performance along with growth and metabolic 
 status of their calves . 
MATERIALS AND METHODS
RESULTS
DISCUSSION