2. Material and methodsThe details

2. Material and methods
The details of animals and treatments of this study were
described elsewhere [9]. In brief, the study was carried out
in 19 nonpregnant Holstein dairycows at a parity of 3.00.1
and at 28.0  0.2 (mean  SD) wk after parturition. Cows in
later lactationwere selected to allowthe investigation of the
direct effects of treatment infusion without interference
with a negative energy balance and the characteristic
endocrine and metabolic changes during the transition
period. Starting at 2 wk before and throughout the experiment,
animals were fed with hay ad libitum and with a
concentrate rich in protein and energy twice daily according
to the individual milk production. Cows were randomly
assigned to 3 treatment groups: an adjusted intravenous
insulin infusion to induce hypoglycemia at a plasma glucose
concentrations of 2.5 mmol/L (HypoG, n ¼ 5), a
hyperinsulinemic-euglycemic clamp to maintain plasma
glucose concentrations at the preinfusion concentrations to
study effects of insulin at simultaneously normal glucose
concentrations (EuG, n¼6), and a 0.9% saline infusion (NaCl,
n¼8) as a control with normal concentrations of insulin and
glucose. Infusions and/or clamps started at 9 AM and were
continued until 9 AM2 d later through one of the indwelling
intravenous catheters (Cavafix Certo Splittocan, B. Braun
Melsungen AG, Germany), whereas the second catheterwas
used for blood sampling.
Hyperinsulinemic hypoglycemia was induced by infusion
of bovine insulin (Sigma-Aldrich, Saint Louis, MO,
#I4011, USA). Insulin infusion rates were adjusted according
to plasma glucose concentrations, which was measured
every 5-min in the first 2 h and hourly during the further
course of infusion. Hyperinsulinemic-euglycemic clamp
was performed by insulin infusion at a constant infusion
rate of 0.6 mU/kg BW/min (according to the average infusion
rate in HypoG) and an additional continuously regulated
glucose infusion (Dr G. Bichsel AG, Interlaken,
Switzerland) to maintain plasma glucose at the preinfusion
concentrations. Control (NaCl) cows were continuously
infused with a 0.9% saline solution at an infusion rate of
20 mL/h administered through an automatic pump (Perfuser,
B. Braun Melsungen AG).
The reference samples were taken 1wk and immediately
before the start of infusions. Plasma glucose concentrations
were measured enzymatically in duplicate with an automated
analyzer (Cobas Mira 2, Hoffmann-La Roche, Basel,
Switzerland) by commercial kit (BioMerieux no. 61270,
Marcy l’Etoile, France, intra- and interassay coefficient of
variation (CV) both

ผลลัพธ์ (อังกฤษ) 1:

2. Material and methodsThe details of animals and treatments of this study weredescribed elsewhere [9]. In brief, the study was carried outin 19 nonpregnant Holstein dairycows at a parity of 3.0 0.1and at 28.0 0.2 (mean SD) wk after parturition. Cows inlater lactationwere selected to allowthe investigation of thedirect effects of treatment infusion without interferencewith a negative energy balance and the characteristicendocrine and metabolic changes during the transitionperiod. Starting at 2 wk before and throughout the experiment,animals were fed with hay ad libitum and with aconcentrate rich in protein and energy twice daily accordingto the individual milk production. Cows were randomlyassigned to 3 treatment groups: an adjusted intravenousinsulin infusion to induce hypoglycemia at a plasma glucoseconcentrations of 2.5 mmol/L (HypoG, n ¼ 5), ahyperinsulinemic-euglycemic clamp to maintain plasmaglucose concentrations at the preinfusion concentrations tostudy effects of insulin at simultaneously normal glucoseconcentrations (EuG, n¼6), and a 0.9% saline infusion (NaCl,n¼8) as a control with normal concentrations of insulin andglucose. Infusions and/or clamps started at 9 AM and werecontinued until 9 AM2 d later through one of the indwellingintravenous catheters (Cavafix Certo Splittocan, B. BraunMelsungen AG, Germany), whereas the second catheterwasused for blood sampling.Hyperinsulinemic hypoglycemia was induced by infusionof bovine insulin (Sigma-Aldrich, Saint Louis, MO,#I4011, USA). Insulin infusion rates were adjusted accordingto plasma glucose concentrations, which was measuredevery 5-min in the first 2 h and hourly during the furthercourse of infusion. Hyperinsulinemic-euglycemic clampwas performed by insulin infusion at a constant infusionrate of 0.6 mU/kg BW/min (according to the average infusionrate in HypoG) and an additional continuously regulatedglucose infusion (Dr G. Bichsel AG, Interlaken,Switzerland) to maintain plasma glucose at the preinfusionconcentrations. Control (NaCl) cows were continuouslyinfused with a 0.9% saline solution at an infusion rate of20 mL/h administered through an automatic pump (Perfuser,B. Braun Melsungen AG).The reference samples were taken 1wk and immediatelybefore the start of infusions. Plasma glucose concentrationswere measured enzymatically in duplicate with an automatedanalyzer (Cobas Mira 2, Hoffmann-La Roche, Basel,Switzerland) by commercial kit (BioMerieux no. 61270,Marcy l'Etoile, France, intra- and interassay coefficient ofvariation (CV) both <4%). Plasma insulin was measured induplicate by radioimmunoassay as described previously[10] with intra- and interassay CV 8.2% and 15.5%, respectively,and plasma glucagon concentrations were measuredin duplicate by using a commercial radioimmunoassay kit(cat. # GL-32K, MILLIPORE, Zug, Switzerland, intra- andinterassay CV 4.0% and 12.5%, respectively).The area under the curve (AUC) of the measured variablesduring day 2 of the experiment was calculated by thetrapezoidal rule (combination of rectangular and triangulararea compartments). AUC was calculated of the whole timespan and then divided by the number of hours to achievevalues which are similar to a single measurement concentration(AUC/h). Data were evaluated by using a generallinear models procedure of SAS (SAS Institute Inc, Cary, NC,USA, 2002–2008, Release 9.2), including treatments(HypoG, EuG, and NaCl) as fixed effect. Data obtained fromthe reference samples were included as covariates into themodel to compensate for initial differences between individuals.Differences between means were determined bythe Tukey test. Data are presented as means standarderror of the mean, and differences were considered significantif P < 0.05.

ผลลัพธ์ (อังกฤษ) 2:

2. Material and methods
The details of this Study of Animals and Treatments were
described Elsewhere [9]. In brief, The Study was carried out
in 19 Nonpregnant Holstein Dairycows at a parity of 3.0? 0.1
and at 28.0? 0.2 (mean? SD) wk after parturition. Cows in
later Lactationwere Selected to Allowthe Investigation of The
Direct effects of infusion Treatment Without interference
with a Negative Energy Balance and The characteristic
endocrine and metabolic The Changes during transition
period. Starting at 2 WK Before and throughout The Experiment,
Fed Animals were with Hay Ad libitum and with a
concentrate rich in protein and Energy Twice Daily according
to The Individual Milk production. Cows were Randomly
assigned to 3 Treatment groups: an adjusted intravenous
insulin infusion to induce Hypoglycemia at a Plasma glucose
concentrations of 2.5 mmol / L (HypoG, n ¼ 5), a
Hyperinsulinemic-Euglycemic Clamp to Maintain Plasma
glucose concentrations at The Preinfusion concentrations to.
Simultaneously Study effects of insulin at normal glucose
concentrations (Eug, N¼6), and a 0.9% SALINE infusion (NaCl,
N¼8) As a Control with normal concentrations of insulin and
glucose. Infusions and / or clamps Started at 9 AM and were
later Through Continued until 9 AM2 D One of The indwelling
intravenous catheters (Cavafix Certo Splittocan, B. Braun
Melsungen AG, Germany), whereas The Second Catheterwas
Used for Blood sampling.
Hyperinsulinemic Hypoglycemia was. induced by infusion
of bovine insulin (Sigma-Aldrich, Saint Louis, MO,
# I4011, USA). Insulin infusion Rates were adjusted according
to Plasma glucose concentrations, which was measured
Every 5-min in The First 2 h and hourly during The Further
course of infusion. Hyperinsulinemic Clamp-Euglycemic
insulin infusion was performed by Constant infusion at a
rate of 0.6 mU / kg BW / min (according to The Average infusion
rate in HypoG) and an Additional continuously regulated
glucose infusion (Dr G. Bichsel AG, Interlaken,
Switzerland). to Maintain at The Preinfusion Plasma glucose
concentrations. Control (NaCl) Cows were continuously
infused with a 0.9% SALINE Solution at an infusion rate of
20 mL / h Administered Through an Automatic Pump (Perfuser,
B. Braun Melsungen AG).
The reference samples were 1wk and immediately Taken
Before The start of. infusions. Plasma glucose concentrations
were measured enzymatically in Duplicate with an Automated
Analyzer (Cobas Mira 2, Hoffmann-La Roche, Basel,
Switzerland) by Commercial Kit (BioMerieux no. 61270,
Marcy L'Etoile, France, intra-and Interassay coefficient of
variation (. CV) both <4%). Plasma insulin was measured in
Duplicate by Radioimmunoassay As described previously
[10] with intra-and Interassay CV 8.2% and 15.5%, respectively,
and Plasma glucagon concentrations were measured
by using a Duplicate in Commercial Radioimmunoassay Kit
(Cat. # GL-32K,. MILLIPORE, Zug, Switzerland, and intra-
Interassay CV 4.0% and 12.5%, respectively).
The Area under The Curve (AUC) of The measured variables
during Day 2 of The Experiment was calculated by The
trapezoidal Rule (Combination of rectangular and triangular.
area compartments). AUC was calculated of The Whole time
span and then divided by number of hours to Achieve The
values ​​which are similar to a single Concentration Measurement
(AUC / h). Data were evaluated by using a general
Linear models Procedure of SAS (SAS Institute Inc, Cary, NC,
USA, from 2,002 to 2008, Release 9.2), including Treatments
(HypoG, Eug, and NaCl) As Fixed Effect. Data obtained from
The reference samples were Included As covariates Into The
Model to compensate for Initial Differences between Individuals.
Differences between means were Determined by
The Tukey test. Data are presented as means? standard
of Error The mean, and Differences were considered significant
IF P <.05.

ผลลัพธ์ (อังกฤษ) 3:

2. Material and methods
The details of animals and treatments of this study were
described elsewhere []. In, 9 brief the. Study was carried out
in 19 nonpregnant Holstein dairycows at a parity of 3.0  0.1
and at 28.0  0.2 (mean  SD) wk after. Parturition. Cows in
later lactationwere selected to allowthe investigation of the
direct effects of treatment infusion. Without interference
.With a negative energy balance and the characteristic
endocrine and metabolic changes during the transition
period. Starting. At 2 wk before and throughout the experiment
animals, were fed with hay ad libitum and with a
concentrate rich in protein. And energy twice daily according
to the individual milk production. Cows were randomly
assigned to 3 treatment groups: an. Adjusted intravenous
.Insulin infusion to induce hypoglycemia at a plasma glucose
concentrations of 2.5 mmol / L (HypoG n ¼, 5), a
hyperinsulinemic-euglycemic. Clamp to maintain plasma
glucose concentrations at the preinfusion concentrations to
study effects of insulin at simultaneously. Normal glucose
concentrations (EuG n ¼, 6), and a 0.9% saline infusion (NaCl
n, ¼ 8) as a control with normal concentrations. Of insulin and
.Glucose. Infusions and / or clamps started at 9 AM and were
continued until 9 AM2 d later through one of the indwelling
intravenous. Catheters (Cavafix, Certo Splittocan B. Braun
Melsungen, AG Germany), whereas the second catheterwas
used for blood sampling.
Hyperinsulinemic. Hypoglycemia was induced by infusion
of bovine insulin (Sigma-Aldrich Saint Louis,,,, MO
# I4011 USA).Insulin infusion rates were adjusted according
to plasma, glucose concentrations which was measured
every 5-min in the. First 2 h and hourly during the further
course of infusion. Hyperinsulinemic-euglycemic clamp
was performed by insulin infusion. At a constant infusion
rate of 0.6 mU / kg BW / min (according to the average infusion
rate in HypoG) and an additional continuously. Regulated
.Glucose infusion (Dr G. Bichsel, AG Interlaken
Switzerland), to maintain plasma glucose at the preinfusion
concentrations.? Control (NaCl) cows were continuously
infused with a 0.9% saline solution at an infusion rate of
20 mL / h administered through. An automatic pump (Perfuser
B. Braun, Melsungen AG).
The reference samples were taken 1wk and immediately
before the start. Of infusions.Plasma glucose concentrations
were measured enzymatically in duplicate with an automated
analyzer (Cobas, Mira 2 Hoffmann-La. ,, Roche Basel
Switzerland) by commercial kit (BioMerieux No. 61270
Marcy, l ', Etoile France intra - and, interassay coefficient. Of
variation (CV) both < 4%). Plasma insulin was measured in
duplicate by radioimmunoassay as described previously
.[] with 10 intra - and interassay CV 8.2%, and 15.5% respectively
and, plasma glucagon concentrations were measured
in duplicate. By using a commercial radioimmunoassay kit
(cat. # GL-32K MILLIPORE Zug,,,, Switzerland intra - and
interassay CV 4.0%, and 12.5% respectively).
The. Area under the curve (AUC) of the measured variables
during day 2 of the experiment was calculated by the
.Trapezoidal rule (combination of rectangular and triangular
area compartments). AUC was calculated of the whole time
span. And then divided by the number of hours to achieve
values which are similar to a single measurement concentration
(AUC / h).? Data were evaluated by using a general
linear models procedure of SAS (SAS Institute Inc Cary NC,,,,, 2002
USA - 2008 Release. 9.2), including treatments
.,, (HypoG EuG and NaCl) as fixed effect. Data obtained from
the reference samples were included as covariates into the
model. To compensate for initial differences between individuals.
Differences between means were determined by
the Tukey, test. Data are presented as means  standard
error of the mean and differences, were considered significant
if P < 0.05.