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  Twin-pregnancy increases susceptibility of ewes to hypoglycaemicstress and pregnancy toxaemia Christina Schlumbohm, J. Harmeyer  * Department of Physiology, School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hannover, Germany Accepted 8 May 2007 Abstract Pregnancy toxaemia is a metabolic disorder with a high mortality rate and occurs in twin-bearing ewes in late gestation. Maternalhypoglycaemia is a characteristic symptom of the disease and has been attributed to an increase in glucose uptake by the twin-bearinguterus. The possibility that a reduced maternal glucose production rate might cause hypoglycaemia, has received little attention in thepast. It was the aim of this study to investigate this explanation as a possible alternative. Six ewes were sequentially subjected to two typesof hypoglycaemic stress, firstly by fasting for 14 h and secondly through induction of moderate hyperketonaemia. Glucose kinetics wereassessed in each animal during the dry non-gestational period, during late gestation, and during early lactation. Application of thesestress factors was associated with variation of plasma glucose concentration from 4.9 to 0.87 mmol L  1 .The plasma glucose concentration was always significantly related to the glucose production rate. The greatest stress-induced reduc-tions in glucose concentration and glucose production rate were seen during late gestation in twin-bearing ewes. The decline in the glu-cose production rate after an overnight fast and during induced hyperketonaemia was greater in twin-bearing ewes than in single-bearingewes (59% and 43%, respectively,  p  < 0.05). The stress conditions resulted in the lowest levels of glucose concentration and glucose turn-over rates in the stressed, hyperketonaemic, late gestation twin-bearing ewes. This illustrates that the glucose homoeostatic system of ewes bearing twins is significantly more susceptible to hypoglycaemic stress than that of ewes bearing single lambs. These findings alsoshow that the primary cause of hypoglycaemia in late gestation twin-pregnant ewes is an increased susceptibility to a stress related reduc-tion in glucose production rate. This metabolic condition leaves the twin-pregnant ewe predisposed for the development of pregnancytoxaemia.   2007 Elsevier Ltd. All rights reserved. Keywords:  Glucose production; Twin-gestation; Twin-pregnancy; Accelerated starvation; Ketones; Betahydroxybutyrate; Glucose turnover; Isotopedilution; Sheep 1. Introduction Pregnancy toxaemia is a well-known metabolic disorderof glucose and fat metabolism in sheep and goats, whichusually occurs spontaneously in the last three weeks beforeparturition. Ewes with multiple foetuses are mostly affected(Reid, 1968). The disease is associated with low plasmaconcentrations of glucose and markedly increased plasmaconcentrations of ketone bodies (Bickhardt et al., 1993;Scott et al., 1995; Sigurdsson, 1988; Van Saun, 2000). Itis believed that the disorder is caused by an inability of the twin-pregnant ewe to meet the increased glucosedemand of the uteroplacental unit (Reid, 1968; Rook,2000). However, this theory does not explain why the dis-order occurs during late gestation rather than during earlylactation? Energy intake and glucose turnover are 40–100%greater during early lactation than during late gestation(Bergman et al., 1974; Perry et al., 1994; Wilson et al.,1983). This should make early lactating ewes more predis- 0034-5288/$ - see front matter    2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.rvsc.2007.05.001 Abbreviations:  BHB,  b -hydroxybutyrate;  D -BHB or  DL -BHB,  D - or  DL - b -hydroxybutyrate. * Corresponding author. Tel.: +49 5103 2692; fax: +49 5103 2693. E-mail address: (J. Harmeyer).  Available online at Research in Veterinary Science 84 (2008) 286–299  posed to ketosis than periparturient gestating ewes, as isthe case with early-lactating and pregnant dairy cows. Sim-ilarly, the mortality rate from ovine pregnancy toxaemia isconsiderably higher than that of bovine ketosis and the effi-cacy of treatment is much lower even though the haemato-logic and metabolic features of the two conditions aresimilar (Henze et al., 1995; Sargison et al., 1994). Anothernoteworthy aspect of pregnancy toxaemia in sheep is thatlong-term underfeeding of twin-bearing ewes during lategestation is often tolerated and does not necessarily resultin pregnancy toxaemia (Bergman et al., 1974; Leng,1965). Under field conditions, the disease occurs sporadi-cally and unpredictably even if good quality feed shouldensure the availability of sufficient nutrients (Bickhardtet al., 1993; Reid, 1968). Still there is no doubt that a rela-tive shortage of glucose availability accompanies preg-nancy toxaemia. However, the general accepted view,that the ewe is over strained and develops pregnancy toxa-emia due to an increasing supply of glucose to the growinguterus and conceptus appears to be a conclusion, drawn byanalogy to ketotic dairy cows. From experimental datathere is good evidence that foetal energy consumptionand growth are related to the availability of nutrients, espe-cially glucose (Clapp, 2006). It is also worth noting that thesupply of glucose to the late-pregnant uterus is not increas-ing as dramatically as to the lactating mammary gland dur-ing early lactation. These observations lead to the questionwhether an impaired maternal glucose production contrib-utes to the onset of hypoglycaemia and development of pregnancy toxaemia rather than an increased supply of glu-cose to the pregnant uterus. This assumption implies thatthe primary cause of pregnancy toxaemia is not due toan inadequately high foetal glucose consumption butrather a shortage of maternal glucose production possiblycaused by an impaired maternal glucose homoeostatic sys-tem. In our study, isotope dilution techniques with labelledglucose were used to estimate glucose production and dis-posal in sheep pregnant with single or twin-lambs. 2. Materials and methods  2.1. Experimental animals Six healthy, non-ketotic ewes (80 ± 8.0 kg) were pur-chased from a local breeder. The breeder was chosen,because of the superior general health status of his herd,as known from the Clinics for Pigs and Small Ruminantswhich is also located at the School of Veterinary Medicinein Hannover. The herd was farmed for mutton productionfor which a seasonal reproductive cycle was enforced.Selection criteria for the ewes were a good body conditionscore and an age of three years or older. The selection cri-terion ‘‘age’’ was chosen because we assumed that olderanimals were more susceptible to metabolic stress. Owingto the fact that the breeds ‘‘Texel’’ and ‘‘German Black-face’’ do not differ with respect to breeding performanceand incidence rates of pregnancy toxaemia, breed of theanimals was not a selection criterion. The mean numberof lambs born per ewe for the German Blackface and Texelewes were 1.68 and 1.69, respectively and their mean bodyweights at 105 days of age were 44.9 kg and 39.5 kg, respec-tively (Mendel and Zindath, 2004). There was also no dif-ference in the incidence of pregnancy toxaemia betweenGerman Blackface and Texel ewes (9.5% and 8.6%, respec-tively; Bickhardt et al., 1998). Three of the selected animalswere Texels and three were German Blackfaces. The gesta-tional ages and numbers of foetuses were determined byultrasonography before the beginning of the study. Bychance, the three Texel ewes carried twins and the threeGerman Blackface ewes carried singletons. The sheep wereheld in two group pens (3.5 m · 4 m, one pen for eachbreed) on straw bedding. Good quality hay containing10.9% crude protein, 8.2% ash, and 8.7 megajoules metab-olizable energy per kg dry matter was available  ad libitum .Water was available at all times. During the last six weeksof pregnancy, the feed was supplemented twice daily withconcentrate (Club-Schafkraft, Club KraftfutterwerkeNord, 21107 Hamburg, Germany) containing 17% crudeprotein, 1.5% Ca, 0.5% P, and 10.2 megajoules metaboliz-able energy per kg of feed. Ewes bearing twins received1000 g of concentrate per day, and ewes bearing singletonsreceived 500 g of concentrate per day. The supplementswere offered in two equal portions at 09:00 and 15:00. Foodwas withheld from about 17:00 on the day before the mainexperiments started until the end of the experiment (about13:00 on the next day). The ewes had fasted for about 15 hbefore the experiments began (at 8:00).Experimental animals, each with a companion animal,were transferred from their pens to the laboratory 1 hbefore the beginning of the experiments. Each experimentconsisted of two phases in which glucose metabolism wasmeasured, i.e., after an overnight fast and during inducedhyperketonaemia. Hyperketonaemia depressed the plasmaglucose concentration as shown by other investigators(Bergman et al., 1963; Kammula, 1976; Radcliffe et al.,1983). Six experiments of this kind were carried out witheach animal; two during the dry non-gestating period (fourweeks after their lambs were weaned at 100 d or more afterparturition); two during late pregnancy (10 ± 8 d ante-par-tum; term = 147 ± 3 d); and two during early lactation(two to four weeks after lambing). This resulted in 36experiments: 12 in the dry season, 12 during late gestation,and 12 in early lactation.Inthemorningonthedaybeforetheexperimentsstarted,two indwelling polyethylene catheters (ID = 0.86 mm) wereplaced into both jugular veins. A venous blood sample wastaken at about 17:00. The catheters were flushed, filled withsaline (0.9% w/v NaCl) containing 750 U ml  1 heparin(U.S.P. XXI, Carl Roth, 76185 Karlsruhe, Germany),closed, and then secured to a bandage with adhesive tapethat had been placed around the neck. One catheter wasused for sampling and the other was used for infusion.The animal experiments were approved by the ethical com-mittee on animal rights protection of the Hannover District C. Schlumbohm, J. Harmeyer / Research in Veterinary Science 84 (2008) 286–299  287  Government in accordance with German legislation onanimal rights and welfare.  2.2. Experimental design The objective of the study was to measure glucose con-centration in plasma and glucose turnover after moderatehypoglycaemic stress consisting of an overnight (15–16 h)fast and subsequent induction of hyperketonaemia.Hyperketonaemia was induced by a continuous intrave-nous infusion of a  DL - b -hydroxybutyrate ( DL -BHB) solu-tion for 3 h, which increased the  D -BHB concentration to3.7–5.7 mmol L  1 .The rates of BHB infusion required to increase  D -BHBlevels varied between animals and between reproductivestates. The required doses were determined from the resultsof a BHB tolerance test carried out on the day before themain experiment (from 10:00 to 13:00). For the tolerancetest, a  DL -BHB racemate solution containing 1.2 mol L  1 D -BHB was infused for 3 h, during which the infusion ratewas increased eight times in a stepwise manner every20 min. For the late gestation ewes, the initial  D -BHB infu-sion rate was 8  l mol kg  1 min  1 and was incremented insteps of 2.5  l mol kg  1 min  1 . For the dry and lactatingewes, the initial  D -BHB infusion rate was 11  l mol kg  1 min  1 and the increments were 3.0  l mol kg  1 min  1 . Aftereach 20 min infusion period, a 4.0 ml blood sample was col-lectedintoheparinizedtesttubesformeasurementof  D -BHBconcentration. Plots of plasma  D -BHB concentrationsagainsttheir corresponding D -BHB infusion rates were usedto determine the  D -BHB infusion rates required to elevateplasma  D -BHB concentrations to the desired level for themain experiment.Each main experiment started with a 60 min adaptationperiod and lasted for 300 min (Fig. 1). All blood samples(5.0 ml) were collected into heparinized tubes (Na-HeparinMonovetten  , Fa. Sarstedt, 51588 Nu¨rmbrecht, Germany).Two blood samples were drawn via the catheters at thebeginning and end of the adaptation period ( t  = 0, t  = 60 min) for measurement of plasma glucose concentra-tions. For measurement of glucose turnover, a bolus of 1.85 MBq  D -2-[ 3 H]-glucose in 5 ml sterile 10% glucose solu-tion was administered 60 min after the start of the experi-ment. Six serial blood samples (5.0 ml) were thencollected at 70, 80, 90, 100, 110, and 120 min after the startof the experiment.At the end of this period ( t  = 120 min), hypoglycaemiawas induced by a continuous 3 h ( t  = 120–300 min) intrave-nousinfusionof1.2 mol L  1 D -BHBusingaperistalticpump(Ismatic MV-CA4, 97877 Wertheim Monfeld, Germany).The previous day the solution had been autoclaved, filteredand the pH adjusted to 7.4. The solution was stored at 4  Cuntil used. The infusion rate was monitored by continuousweighing of the reservoir containing the infusate. Becauseof differences in BHB tolerances and body weights betweenanimals, infusion volumes varied from 0.9 to 2.9 ml min  1 .After 2 h of   DL -BHB infusion (at  t  = 240 min), glucose con-centrations were decreased significantly. At this time, a sec-ond dose of 1.85 MBq of 2-[ 3 H]-glucose was administeredto determine glucose turnover during the induced hypo-glycaemia. In this second phase of the experiment glucoseand  D -BHB concentrations were determined in samples at120, 240, 260, 280, and 300 min after the start of theexperiment. Radioactivity of the glucose tracer was deter-mined in plasma samples at 250, 260, 270, 280, 290, and300 minafterthestartoftheexperiment.Bloodsampleswereimmediately placed on crushed ice and the plasma wasseparated at the end of the experiment by centrifugationat 3000  g   for 10 min at 4  C. The plasma samples weredivided into three sub-samples and stored at   20  C untilanalysis.  2.3. Laboratory methods The concentration of   D -BHB was determined using themethod of  Williamson et al. (1962) after deproteinizationwith 1 M HClO 4 , centrifugation, neutralization with 1 MK 3 PO 4 , and subsequent centrifugation. The intra-assaycoefficient of variation of this method was 4.6 ± 3.9%(mean ± SD,  n  = 160) for duplicate measurements. Theinter-assay coefficient of variation for the standard solutionwas 5.5 ± 4.6% (mean ± SD,  n  = 53). The glucose concen-tration in the plasma was determined with a reflection pho-tometer (Reflolux  , Roche, 68305 Mannheim, Germany).Test experiments in our laboratory showed that glucoseconcentrations estimated using this method differed by<5% ( n  = 40) from those estimated using the conventionalphotometric Glucose-Perid  method (Roche Diagnostics,68305 Mannheim, Germany). For measurement of   D -2-[ 3 H]-glucose radioactivity, 1 ml plasma was deproteinizedfollowing the Somogyi (1952) procedure. This involvedthe addition of 0.25 M Ba(OH) 2 , followed by 5.65% ZnSO 4 and centrifugation at 4  C for 20 min at 3000  g  . The super-natants were decanted into scintillation vials, lyophilized toremove [ 3 H]-H 2 O, dissolved in 2.0 ml deionized water, andmixed with 14 ml of a scintillation cocktail (Hydroluma  ,Baker, 64521 Groß-Gerau, Germany). The emission of radioactivity from  D -2-[ 3 H]-glucose in 10 min was mea-sured using a liquid scintillation counter (Tri-Carb  2500TR, Canberra-Packard, 63303 Dreieich, Germany). minutes 060120180240300 Continuous i.v. infusion of a 1.2 molar DL-BHB solution at a rate of 0.9 to 2.9 ml/min)  Adaptation period BloodsamplesBolus of D-2-[ 3 H]-glucose Bolus of D-2-[ 3 H]-glucose Fig. 1. Schematic representation of the experimental design.288  C. Schlumbohm, J. Harmeyer / Research in Veterinary Science 84 (2008) 286–299   2.4. Chemicals D -2-[ 3 H]-glucose (specific activity: 0.74–1.11 TBqmmol  1 ) was purchased from New England Nuclear(NEN, 63303 Dreieich, Germany, now PerkinElmer). Thesodium salt of the  DL -BHB racemate was synthesized asdescribed previously (Schlumbohm and Harmeyer, 1999).The proportion of D isomer in the  DL -BHB racemate solu-tion was 42% (quantified enzymatically according to Wil-liamson et al., 1962). Concentrations of   D -BHB in theinfusate and in blood plasma were measured using NAD, D -BHB dehydrogenase, and  D - b -hydroxybutyric acid as astandard (Schlumbohm and Harmeyer, 2003). These chem-icals were obtained from Roche Diagnostics (68305 Mann-heim, Germany). All other chemicals were purchased fromVWR (VWR International GmbH, 64295 Darmstadt,Germany).  2.5. Calculation of glucose kinetics Glucose turnover and the rate constant of glucose turn-over were calculated from the decay curve of the 2-[ 3 H]-glucose concentration in the plasma vs time after tracerinjection, the injected dose of tracer, and the mean concen-tration of glucose in the plasma for 60 min after injectionof the tracer. The non-compartmental approach describedby Shipley and Clark (1972), and modified by Schlumbohm and Harmeyer (1999), was used. The decay curve of thespecific radioactivity of glucose in plasma (SA) t  after thebolus injection of tracer was fitted (SigmaPlot TM , SystatSoftware GmbH, 40688 Erkrath, Germany) to a two-expo-nential equation of the form: ð SA Þ t  ½ Bq mmol  1  ¼  E  1 exp ð k  1    t  Þ þ  E  2 exp ð k  2    t  Þ where (SA) t  is the specific radioactivity of glucose at time  t after the bolus injection [Bq mmol  1 glucose],  E  1 ,  E  2  thecoefficients [Bq mmol  1 glucose],  k  1 ,  k  2  the exponents[min  1 ] and  t  the min after injection of tracer.To prevent high (SA) t  values from dominating the iter-ation process, the squares of the deviations of the measureddata from the curve were weighted by dividing them by thesquares of the measured data. The specific radioactivity of glucose at time  t  after the bolus injection (SA) t  was calcu-lated by dividing the glucose radioactivity per litre plasmaby the glucose content per litre plasma: ð SA Þ t  ½ Bq mmol  1  ¼ ð  A Þ t  ½ Bq L  1 ð C  Þ t  ½ mmol L  1  where ( A ) t  is the [ 3 H]-glucose radioactivity per litre plasmaat time  t  relative to the time of the bolus injection ( t  = 0)[Bq L  1 ] and ( C  ) t  the concentration of glucose in bloodat time  t  relative to the time of the bolus injection ( t  = 0)[mmol L  1 ].Because plasma glucose concentrations of samplestaken during the 50 min sampling periods varied by<5%, mean glucose concentrations were used in the calcu-lation of (SA) t . The sampling periods after the two tracerinjections were relatively short compared with the dura-tion of the decay curve, which theoretically extends fromthe time of tracer injection to infinity. For calculation of the two areas under the curves (AUC), the radioactivityvs time curves were extrapolated over a time period of 4 h, i.e., from the time of injection of tracer ( t  = 0 min)to 4 h after marker injection ( t  = 240 min) (cut-off). Thesetimes corresponded to  t  = 60 min and  t  = 240 min (start-ing points) and  t  = 300 min and  t  = 480 min (cut-offs) of the experimental program. The latter points were chosenas cut-offs of the curves since (SA) t  was <1% of (SA) 0 at this time. The AUC for each decay curve was calcu-lated using the measured data from 10 to 60 min after tra-cer injection and the areas under the extrapolated timesegments for the time interval from tracer injection until10 min and from 60 min to 240 min after each tracerinjection. Glucose radioactivity in plasma ( A ) t  after2-[ 3 H]-glucose injection was extrapolated using the sameprocedure.The turnover of glucose per litre of the glucose distribu-tion volume ((TO) n , normalized glucose turnover) was cal-culated by dividing ( A ) 0  by the AUC as follows: ð TO Þ n ½ mmol L  1 min  1  ¼ ð  A Þ 0 ½ Bq L  1  AUC ½ Bq mmol  1 min  where (TO) n  is the normalized glucose turnover, ( A ) 0  theconcentration of [ 3 H]-glucose at the time of tracer injection[Bq L  1 ]; corresponding to  t  = 60 min and  t  = 240 min of the experimental program and AUC the area under curve.Plasma glucose radioactivity after the second 2-[ 3 H]-glucose injection was confounded by carryover of radioac-tivity from the first injection of [ 3 H]-glucose. Carryover of radioactivity, which amounted to 5–11% of the AUCderived from the second tracer injection, was estimatedfrom extrapolation of the SA vs time curve after the firsttracer injection and subtracted from the second AUC.The turnover of glucose per kg body weight, TO[ l mol kg  1 min  1 ] was estimated by multiplying TO n  bythe fractional distribution volume of glucose ( V  ) [L kg  1 ].According to Bergman and Hogue (1967), the fractionaldistribution volume of glucose is relatively constant, rang-ing from 0.24 to 0.27. We estimated  V   by dividing thedose of tracer radioactivity [Bq] by the extrapolated con-centration of tracer radioactivity at time zero ( A ) 0 [Bq L  1 ] and the body weight of the ewe [kg]. The mean V   of 0.24 was used for calculation of TO. The rate con-stant of glucose turnover,  k  , was calculated by dividing(SA) 0  by the AUC: k  ½ min  1  ¼ ð SA Þ 0 ½ Bq mmol  1  AUC ½ Bq mmol  1 min  where (SA) 0  is the specific radioactivity of [ 3 H]-glucose atthe time of bolus injection [Bq mmol  1 ]; corresponding to t  = 60 min and  t  = 240 min of the experimental program,Fig. 1. C. Schlumbohm, J. Harmeyer / Research in Veterinary Science 84 (2008) 286–299  289
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