Contents. Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag

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1 Reprod Dom Anim 44, (29); doi: /j x ISSN Effect of Preprtum Energetic Supplementtion on Productive nd Reproductive Chrcteristics, nd Metolic nd Hormonl Profiles in Diry Cows under Grzing Conditions D Cvestny 1,2, M Kulcs r 3, D Crespi 1, Y Chillird 4, A L Mnn 1, O Blogh 3, M Keresztes 3, C Delvud 4, G Huszenicz 3 nd A Meikle 2 1 Ntionl Agriculturl Reserch Institute (INIA L Estnzuel), Coloni, Uruguy; 2 Fculty of Veterinry Sciences, Montevideo, Uruguy; 3 Szent Isv n University, Fculty of Veterinry Science, Budpest, Hungry; 4 Unite de Recherche sur les Herivores, INRA-Theix, Sint-Gene`s- Chmpnelle, Frnce Contents The effect of crcked corn grin supplementtion (3.5 kg dy) during 3 weeks efore the expected clving dte on milk production nd composition, ody condition score (BCS), metolic nd hormonl profiles nd length of postprtum noestrus ws evluted in multiprous Holstein diry cows under grzing conditions ( supplemented group, n = 1; group, n = 1). Body condition score ws weekly recorded during the periprtum period, from dys )21 to +35 (prturition = dy ). Non-esterified ftty cids, -hydroxyutyrte, cholesterol, ure, insulin, insulin-like growth fctor I (IGF-I), leptin, thyroxine (T 4 ) nd 3,3 5-triiodothyroinine (T 3 ) were weekly determined in plsm from dys )21 to +35. The reinitition of ovrin cyclicity ws twice weekly determined y ovrin ultrsonogrphy nd confirmed y plsm progesterone concentrtions. Cows fed energy concentrte preprtum hd higher BCS during the preprtum nd postprtum nd produced more milk. Nonesterified ftty cids plsm concentrtions were significntly higher in the energy group, while cholesterol ws higher in the control group. Treted cows hd higher levels of plsm insulin, IGF-I nd leptin pre-clving. IGF-I, leptin nd T 4 were diminished during the erly postprtum period in oth groups. Insulin levels were lso diminished in the control group, ut levels remined high in the energy-supplemented group. Treted cows ovulted sooner fter prturition thn controls. We conclude tht Energetic supplementtion preprtum in cows under grzing conditions incresed milk production nd reduced the reinitition of ovrin ctivity, consistent with etter EB (BCS), higher preprtum levels of IGF-I, leptin nd insulin, nd higher insulin levels during erly postprtum. Introduction During the trnsition period (3 weeks efore to 3 weeks fter clving), cows must dpt their metolism to the high demnds of lcttion nd to different diet to meet the requirements of milk production (Drckley 1999), nd the energy required for hrvesting their feed in psture-sed diry system (Acost 1997). As the mount of energy required for mintennce nd milk production exceeds the mount of energy otined from dietry sources, the cow is in negtive energy lnce (NEB; Chillird 1999). Thus, to chieve high levels of milk production in the erly postprtum period, the cow must moilize ody reserves tht in turn, re relted to the energy intke in the dry period. Ft stores re primrily used for lcttion nd mintennce, with reproductive processes receiving lower priority (Butler 23). In previous studies, we found tht diry cows under grzing system lost etween.5 nd 1 point of ody condition score (BCS) in the month efore clving (Cvestny et l. 25), nd BCS t clving ws relted to the first ovultion postprtum (Meikle et l. 24). The min endocrine signls tht hve een suggested to inform the reproductive xis of the energy lnce sttus re insulin, insulin-like growth fctor I (IGF-I) nd leptin, oth in indoor nd grzing production systems (Butler 23; Meikle et l. 24). Insulin-like growth fctor I nd insulin ct s promoters of folliculr growth nd thus hve n effect on the resumption of ovrin cyclicity (Spicer nd Echternkmp 1995). Leptin lso my ct s mjor modultor of reproduction y regulting feed intke nd through locl effect in the ovry to regulte follicle size, nd possily oocyte qulity (O Cllghn nd Bolnd 1999). Nutritionl nd metolic sttus in the erly postprtum period cn e predicted y IGF-I levels (Zulu et l. 22) nd y insulin tht is importnt in the homeosttic control of energy metolism. Insulin is positively correlted with energy intke (Chillird 1999), lthough different feedstuffs my ffect plsm levels of this hormone differently (Relo et l. 21; Roinson et l. 22). Leptin is involved in the regultion of energy metolism; it signls the mount of white dipose tissue deposits within the ody (O Cllghn nd Bolnd 1999 ; Chillird et l. 25). These metolic hormones (IGF-I, leptin nd insulin) re higher in cows in positive energy lnce (EB; Lucy 23). Lcttion hypoleptinemi is proly not due only to negtive EB nd could ply role to increse the efficiency of energy utiliztion during oth erly nd lte lcttion phses (Chillird et l. 25). Thyroid hormones re mjor modultors of metolism. In diry cows, plsm T 3 nd T 4 concentrtions were negtively correlted with dily milk yield (Tiirts 1997). Besides, thyroid hormones hve een reported to stimulte ovrin function cting directly on grnulos nd thecl cells y incresing progesterone production nd romtse ctivity (Spicer et l. 21). Feeding higher quntity of fermentle crohydrtes during the preprtum trnsition period prepres the microil popultion to lcttion diets, promotes development of ruminl ppille, increses sorptive cpcity of the rumen epithelium nd reduces lipolysis y delivering more glycogenic precursor to the liver (Grummer 1995; Relo et l. 25). Metolic response to cows fed high-energy density diets depends

2 664 D Cvestny, M Kulcs r, D Crespi, Y Chillird, A L Mnn, O Blogh, M Keresztes, C Delvud, G Huszenicz nd A Meikle on the form on which energy is given. While some reported tht these diets resulted in incresed leptin, IGF-I nd insulin concentrtions during the preprtum period ut not during postprtum period, ut hd no effect on milk production (Holtenius et l. 23), others did not find this response (Roinson et l. 22). Similrly, under grzing conditions, Roche et l. (25) showed tht the level of feeding not referring to energy density nd or concentrte supplementtion during the lst month efore clving incresed preprtum leptin, IGF-I nd glucose, ut not during postprtum period nd hd no effect on milk production. There re mny reports in the literture on the reltionships of energy lnce nd fertility, lmost ll of them relted to confined diry systems with TMR feeding; we did not find in the literture reports tht link energy lnce with hormonl nd metolic profiles in Holstein cows under psture-sed production system. In ccordnce with this, in this study, we imed to modify the signls tht link preprtum EB with postprtum milk production nd reproductive performnce in grzing Holstein cows, y chieving etter EB through preprtum energy supplementtion concentrte. Our working hypothesis ws to determine whether 3-week preprtum 3.5 kg crcked corn grin supplementtion could influence the periprturient metolic nd endocrine chnges, milk production nd length of postprtum noestrus in psture-fed diry cows with low BCS efore clving. Mterils nd Methods Experimentl design From the experimentl herd of the diry frm reserch sttion of INIA L Estnzuel (Coloni, Uruguy), 2 multiprous Holstein cows with n verge ody weight of 578 ± 14 kg nd BCS of ±.1 (scle 1 5, Edmonson et l. 1989) were used. The cows were expected to clve t the eginning of the ustrl utumn (Mrch) nd 28 dys efore the individul expected clving dte, cows were seprted from the herd nd llocted in different pddock of nturl pstures. At dy 21 efore the expected clving dte, cows were ssigned to two preprtum tretment groups, considering BCS nd dys dry. Tretments were: group (n = 1) tht received (t 8: AM) 3.5 kg dy of crcked corn grin [82% orgnic dry mtter digestiility (ODMD), 9% neutrl detergent fire (NDF) nd 7.7% cid detergent fire (ADF); 13.5 MJ] ech cow in individul feeders, nd group (n = 1) with no concentrte. Cows were offered lso les of hy of improved psture [legumes (white clover, Trifolium repens), nd Grminee (rye grss, Lolium multiflorum; 54.5% ODMD, 12.4% crude protein (CP), 6.1% NDF nd 47.2% ADF] d liitum, nd oth groups were kept in seprte pddocks of improved nturl pstures (11.8% CP, 58.2% NDF nd 34.7% ADF; 9.9 MJ). After prturition, oth groups were group fed nd received the sme diet, tht consisted in 4. kg dy of commercil concentrte (19.3% CP, 28.3% NDF nd 14.1% ADF; 7.1 MJ) dministered twice dy individully during milking time. This ws complemented with 12 kg dy (fresh se) of corn silge (73.9% ODMD, 6.2% CP, 41.1% NDF nd 28.3% ADF; 1.6 MJ) nd the nimls hd ccess to dily strip of improved pstures consisting in mixture of lflf (Medicgo stiv), white clover nd tll fescue (Festuc rundince) (62.5% ODMD, 17.7% CP, 52.4% NDF nd 38.3% ADF; 9.2 MJ). Nutrient composition ws clculted on dry mtter sis. Under this psture-sed system, grzing is done in different pddocks, which re mnged under rottionl system. The estimted distnce wlked per dy ws 2 km during the preprtum nd km during the postprtum period, depending on the loction of the psture strip (this distnce does not include the one corresponding to grzing). Body condition score ws determined weekly from the eginning of the experiment until week 5 postprtum y the sme opertor. Milk production ws recorded dily nd then verged for ech week. For determintions of milk composition (ft nd protein content), composite smple of four consecutive milking of ech week ws tken during the first 5 weeks postprtum. Milk ft production ws nlysed y the Mojonnier method nd milk protein production ws nlysed y the Kjeldhl method (with Bentley 2 equipment; Bentley Instruments Inc., Chsk, MN, USA). Determintion of ovrin cyclicity The reinitition of ovrin cyclicity ws monitored twice week y trnsrectl ovrin ultrsonogrphy using 5. MHz liner proe (Alok 5; Alok, Tokyo, Jpn). Ovultion ws determined y disppernce of the lrgest follicle followed y the formtion of corpus luteum, which ws confirmed y plsm progesterone concentrtions, from smples tken twice week strting t the second week postprtum. The reinitition of ovrin cyclicity ws defined s the dy when progesterone incresed from sl concentrtions in two consecutive smples of >1.6 nm or one smple of >3.2 nm (Meikle et l. 24). Blood iochemistry Blood smples were otined weekly in the morning efore the dministrtion of concentrte y jugulr venipuncture in heprinized vcuum tues, from dy )28 to dy 35 (prturition: dy ), centrifuged nd then stored t )2 C until nlysis. Smples nlysed were those corresponding to dys )21, )7, +7, +21 nd +35. Ure ws nlysed y the Urese UV method with Weiner (Weiner Lortories, Rosrio, Argentin) kit # ; totl cholesterol with the CHOD-PAP method with Weiner kit # ; non-esterified ftty cids (NEFA) were nlysed y the ACS-ACOD (cil-coa sinthetse & cil-coa oxidse) method with NEFA-C kit # of Wko Chemicls (Richmond, VA, USA); -hydroxyutyrte (BHB) with 3-HBDH-NAD+3-hydroxyutyrte dehydrogense- NAD+ method (Rnut, Rndox Lortories Ltd, Crumlin, Co., Antrim, UK). For qulity controls, it ws used Lyotrol N nd P, s well s internl controls of the veterinry lortory Miguel Ruino (DILAVE, Uruguy). The intr-ssy coefficient of vrition ws 3.7% for ll the prmeters, nd the interssy CV ws 9.6%.

3 Preprtum Supplementtion in Grzing Diry Cows 665 Hormone determintion Progesterone ws determined y rdioimmunossy using commercil kit (Cot--Count; DPC, Dignostic Products Co., Los Angeles, CA, USA). The intr- nd inter-coefficients of vrition were 6% nd 11%. The sensitivity ws.1 nm. Thyroxine (T 4 ) nd 3,3,5-tri-iodothyronine (T 3 ) were determined y 125 I-Spec RIA-coted tue kits (Institute of Isotopes Co., Ltd, Budpest, Hungry). The sensitivity ws.5 nm (T 4 ) nd.19 nm (T 3 ). The intr-ssys CV were % for T 4 nd % for T 3. The interssys CV were 5.8% nd 6.5%, respectively. The plsm insulin content ws quntified s free insulin with commercil 125 I-lelled rdioimmunometric sndwich ssy kit (BI-Insulin IRMA kit; CIS Bio Interntionl Ltd, Susidiry of Schering S.A., Gif- Sur-Yvette, Frnce; sensitivity:.86 pm; intr-ssy nd interssy CV: from 1.3% to 5.6% nd 8.5%, respectively). The plsm IGF-I concentrtions were determined with 125 I-lelled two-site immunordiometric method, which includes preceding extrction of IGF-I with n ethnolic HCl solution, nd efore-ssy neutrliztion of extrcts (DSL-56 Active IGF-I-coted Tue IRMA Kit; Dignostic Systems Lortories Inc., Wester, TX, USA; sensitivity:.11 nm; intr-ssy nd interssy CV: from 3.4% to 6.6% nd 7.%, respectively). The ssy ws run in ccordnce with the mnufcturer s instruction, with the exception of n overnight incution of the neutrlized extrct on +4 C (rther thn its 3 h incution on room temperture). Both the insulin nd IGF-I ssys were vlidted for ovine plsm nd serum smples. In oth ssy systems, the inding pttern of serilly diluted ovine plsm smples ws prllel to tht of the stndrd curves. The recovery rtes of dded known quntity of insulin (35.9, 89.8 nd pmol per smple) nd IGF- I (.78, 2.6 nd 7.8 nmol per smple, given either efore or fter the extrction) to stndrd ovine plsm smples (n = 3; ll with predetermined low insulin nd IGF-I content) were the following: 94 16% (insulin), 78 88% (IGF-I given efore extrction) nd 89 17% (IGF-I given fter extrction). Plsm leptin concentrtion ws quntified with ruminnt-specific 125 I-RIA (Delvud et l. 22) dpted nd modified y Kulcs r et l. (26). This version of ssy ws vlidted for ovine plsm s defined previously (Meikle et l. 24; sensitivity:.32 nm; interssy nd intrssy CV: 12.2%, 5.6%, 6.1%, nd 1.1%, 4.6% nd 5.3% in rnges of qulity control smples with known low, medium nd high leptin content, respectively). Sttisticl nlysis Milk production, BCS, metolites nd hormones concentrtions were nlysed y the Proc Mixed (Sttisticl Anlysis System, SAS Institute, Cry, NC, USA) nd the model included tretment, weekly oservtions nd their interctions. The covrince structure ws utoregressive order 1 nd cow within tretment ws set s rndom effect. Lest-squre mens were compred with LSD with significnce level of 5%. Previous lcttion milk production, BCS t the eginning of the experiment nd numer of dys dry were tested s covriles ut the effect ws not significnt, therefore, they were not included in the nlysis. Ovrin cyclicity ws nlysed y Proc GLM (SAS) with the preprtum tretment s the fixed effect. Proportion of cows ovulting in the erly postprtum ws nlysed y chisqure procedure. Results re presented s lest-squre mens ± pooled stndrd error. Person s correltion coefficients were clculted to study reltionships etween vriles. Results Body condition score Body condition score ws similr in oth groups t the eginning of the tril, ut from 7 dys preprtum through remining the experimentl period, cows in the group hd higher BCS thn cows in the group (Tle 1, p <.1), except t clving nd t the first week postprtum, where BCS ws similr in oth groups. In the group, cows incresed their BCS during the first 2 weeks of tretment nd there ws decrese from the week prior to prturition to 1 week postprtum (p <.1; Fig. 1). Therefter, it remined stedy during the remining of the tril, ut from weeks 3 to 5 postprtum, it ws higher thn in the Tle 1. Fixed effects included in the model for mesured prmeters in cows under grzing conditions (fixed effects re tretment, experimentl week nd their interction) Vrile Tretment Week Tretment*week Body condition score ** *** * Milk production * *** NS Non-esterified ftty cids * ***.15 -Hydroxyutyrte NS ** *** Cholesterol * *** NS Ure.15 * ** Insulin ** * *** IGF-I.9 *** * Leptin.8 *** NS 3,3 5-Triiodothyroinine.12 *** NS Thyroxine NS *** NS *p <.5; **p <.1; ***p <.1; NS = p >.2. BCS Dys ( = prturition) Fig. 1. Evolution of ody condition score in nd (crcked corn preprtum supplementtion) groups in diry cows under grzing conditions (, : different letters etween tretments t the sme point differ, p <.5)

4 666 D Cvestny, M Kulcs r, D Crespi, Y Chillird, A L Mnn, O Blogh, M Keresztes, C Delvud, G Huszenicz nd A Meikle group. BCS in the group decresed only in the first week postprtum nd did not recover to their initil level during the remining of the experimentl period. In the group, BCS score ws positively correlted with IGF-I (r =.41, p <.1, n = 43), leptin (r =.49, p <.1, n = 43) nd T 4 (r =.35, p <.5, n = 43) nd negtively correlted with BHB nd ure (r = ).25, p =.8 nd r = ).26, p =.7, respectively, n = 43). BCS ws positively correlted with IGF-I, nd leptin (r =.37 nd.3, p <.5, n = 41) nd T 4 (r =.45, p <.5, n = 41) in the group. Milk production nd composition Milk production ws higher for the group t weeks 2 nd 4 postprtum (p <.5; Tle 1, Fig. 2). In the group, milk ws negtively correlted with milk protein per cent, IGF-I nd leptin (r = ).47, ).42 nd ).49, respectively, p <.5, n = 26). In the group, milk ws positively correlted with T 4 (r =.37, p <.5, n = 3), nd to cholesterol (r =.49, p <.1, n = 29). Milk protein nd ft percentge showed decresing pttern s the lcttion progressed, nd there were no tretment differences (p >.5; Fig. 2,c). In the group, ft ws negtively correlted with cholesterol (r =.49, p <.4, n = 2). In the group ws positively correlted to IGF-I nd ure (r =.48 nd.56, () 35 Milk (l) () 3.8 Ft % (c) 4. Protein % Dys postprtum Fig. 2. Milk production (), milk ft percentge () nd milk protein percentge (c) in nd (crcked corn preprtum supplementtion) groups in diry cows under grzing conditions (, : different letters etween tretments t the sme point differ, p <.5) p <.5, n = 19) nd negtively correlted with insulin (r = ).43, p =.6, n = 19). Metolites nd hormones There ws tretment effect on plsm NEFA levels (p <.5; Tle 1), nd while preprtum concentrtions were similr in oth groups, postprtum levels were higher during the first 2 weeks postprtum in the group. Cows in this group hd mrked increse in NEFA fter clving nd presented decrese from the first week postprtum. Cows in the group did not hve postprtum increse, ut lso showed decrese during the postprtum period (Fig. 3). In the group, NEFA ws negtively ().8 NEFA mmol/l ().9 BHB mmol/l (c) Cholesterol mmol/l (d) 1 Ure mmol/l Dys (Prturition = ) Fig. 3. Levels of () non-esterified ftty cids, () -hydroxyutyrte, (c) cholesterol nd (d) ure in nd (crcked corn preprtum supplementtion) groups in diry cows under grzing conditions (, : different letters etween tretments t the sme point differ, p <.5)

5 Preprtum Supplementtion in Grzing Diry Cows 667 correlted to, T 3 nd T 4 (r = ).3 nd ).58, p <.5 nd p <.1, n = 49) nd positively correlted to BHB (r =.58, p <.1, n = 5). The BHB concentrtions were ffected y the interction tretment*week (p <.1; Tle 1). While group hd significnt increse fter clving nd mintined this level for the remining of the tril, the group mintined lower BHB levels until the first week postprtum nd incresed y the second week postprtum. BHB levels were different in oth groups only t the first week postprtum (p <.1; Fig. 3). In the group, BHB ws positively correlted to T 3 nd T 4 (r =.56 nd r =.57, p <.1, n = 5). In the group, BHB ws negtively correlted with T 4, IGF-I nd leptin (r = ).6, r = ).54 nd r = ).46, p <.1, n = 49). The plsm cholesterol levels were different etween groups (p <.5). There ws significnt effect of week, cholesterol levels in the group were higher in weeks 3 nd 5 postprtum (p <.5), ut no significnt interction mong them (Tle 1). Both groups hd constnt nd similr levels during the preprtum, nd rose fter clving. Tretment differences were found fter the third week postprtum, where the group hd higher levels (p <.5), nd so remined up to the end of the tril (Fig. 3c). In the group, cholesterol ws positively correlted to BHB, ure, T 3 nd T 4 (r =.56, p <.1, r =.37, p <.1, r =.49, p <.1 nd r =.29, p =.6, n = 44) nd negtively correlted to leptin (r = ).32, p <.5, n = 44). In the group, BHB ws positively correlted to milk, nd T 3 (r =.49, p <.1, n = 29 nd r =.38, p <.1, n = 48), nd negtively correlted to IGF-I nd leptin (r = ).33 nd r = ).36, p <.5, n = 48). There ws significnt effect of week nd week*tretment interctions (p <.1; Tle 1) in plsm ure tht incresed mrkedly in the group during the postprtum period while in the group there ws n increse up to the first week postprtum (p <.5), to return to initil levels t the end of the tril. Ure ws higher in the group t weeks 3 nd 5 postprtum (p <.5), nd decresed in oth groups from the third week postprtum to the end of the tril (Fig. 3d). In the group, ure ws positively correlted to BHB nd cholesterol (r =.54 nd r =.37, p <.1, n = 5). In the group, ure ws positively correlted to milk ft per cent (r =.56, p <.5, n = 29), nd negtively correlted to leptin (r = ).28, p <.5, n = 48). While similr t the eginning of the tril, plsm insulin levels were consistently higher in group (p <.5), nd lso showed tretment*week interction (Tle 1). This tretment difference ws due to mrked decrese efore prturition in the group compred with the group tht mintined high levels during the postprtum period (Fig. 4). Men IGF-I levels throughout the experimentl period differed, there ws lso n effect of week (p <.1) nd significnt tretment*week interction (p <.5) (Tle 1). Tretment difference ws ecuse of the higher levels found during the lst week preprtum in the group (p <.1, Fig. 4). Both groups decresed fter clving, nd the levels remined low throughout the experimentl period. In () 25 IGF-I nmol/l Insulin pmol/l () (c).4 Leptin nmol/l Dys ( = prturition) (d) 4 2 T 3 nmol/l (e) 5 T 4 nmol/l Dys ( = prturition) Fig. 4. Concentrtions of () insulin, () IGF-I, (c) leptin, (d) T 3 nd (e) T 4 in nd (crcked corn preprtum supplementtion) groups in diry cows under grzing conditions (, : different letters etween tretments t the sme point differ, p <.5 for insulin nd leptin, nd p <.1 for IGF-I)

6 668 D Cvestny, M Kulcs r, D Crespi, Y Chillird, A L Mnn, O Blogh, M Keresztes, C Delvud, G Huszenicz nd A Meikle the group, IGF-I ws positively correlted to BCS, leptin nd T 4 (r =.41, p <.1, r =.95, nd r =.56, nd p <.1, n = 44) nd negtively correlted to milk (r = ).42, p <.5, n = 29). In the group, IGF-I ws positively correlted to milk ft per cent, BCS, T 4 nd leptin (r =.48, p <.5, n = 19, r =.36, p <.5, n = 41, r =.6, p <.1, n = 49, r =.94, p <.1, n = 49), nd negtively correlted to BHB, NEFA nd cholesterol (r = ).54, p <.1, r = ).26, p =.6, r = ).33, p <.5, n = 49). Leptin ws high during the preprtum period, dropped drsticlly fter clving in oth groups, nd remined low for the rest of the tril, lthough it ws not higher in the group thn in the group (Tle 1). Leptin ws higher in treted cows during the first week preprtum (p <.5), ut levels were similr in oth groups during the postprtum period (Fig. 4c). In the group, leptin ws positively correlted to BCS, T 4 nd IGF-I (r =.49, r =.6 nd r =.94, p <.1, n = 44), nd negtively correlted to milk production nd cholesterol (r = ).49, p <.5, n = 26 nd r = ).32, p < 5, n = 44). In the group, leptin ws positively correlted to BCS, T 4 nd IGF-I (r =.29, p =.5, n = 41; r =.59, p <.1, n = 49 nd r =.94, p <.1, n = 49) nd negtively correlted to BHB, nd ure (r = ).46, p <.1, n = 49 nd r = ).28, p =.5, n = 48). The T 3 vlues were not influenced y tretment (Tle 1), nd incresed throughout the experimentl period (Fig. 4d). There were no tretment differences in the T 4 levels (Tle 1), ut oth groups decresed until the first week postprtum, nd then incresed until the end of the tril. The group tend to present higher T 4 levels during the preprtum (p =.12), ut not during the postprtum period (Fig. 4e). T 3 ws positively correlted to T 4 (.34, p <.5, n = 44). Reproductive prmeters Intervl from clving to first ovultion ws 25. ± 3.7 dys for the group nd 37.4 ± 3.7 dys for the group (men ± SEM, p <.5). Cows in the group ovulted 12 dys sooner thn the group (p <.5). While ll the cows in the energy group ovulted efore 35 dys postprtum, only 6% of those in the control group hd the first ovultion efore this period (Mntel- Henszel chi-squre: 4.75, p <.5). Discussion The dministrtion of n energy supplement to grzing cows incresed milk production, BCS during the preprtum nd postprtum period, nd decresed the postprtum cyclic period. These results re ssocited with incresed preprtum levels of insulin, IGF-I nd leptin nd postprtum insulin concentrtions in the supplemented cows. The supplementtion resulted in n increse in BCS during the first 2 weeks of tretment nd until 1 week prior to clving, proly ecuse of the energy content in the corn grin, which proly resulted in higher estimted energy intke. The etter digestion of the nutrients is likely to e produced y higher ctivity of the ruminl flor dpted to the high concentrte diet fed in the postprtum period (NRC 21). The higher BCS of the group during postprtum period could e explined y the etter ruminl dpttion to the postprtum diets nd higher dry mtter intke (Curtis et l. 1985) s mentioned ove. supplementtion incresed y 12% the milk production during the experimentl period, which could e ecuse of the etter dpttion to the postprtum diet. Informtion regrding the effect of energy supply during preprtum on milk production is conflicting; some studies found n increse (Ingvrtsen nd Andersen 2; Overton nd Wldron 24) while others found no effect (Mshek nd Beede 2; Holtenius et l. 23; Roche et l. 25) in milk production. These differences might e ttriuted to the type of supplement, the wy of dministrtion nd the quntity, s well s the potentil production of the cows used in the different experiments. Another fctor susceptile to increse milk production in energy group is the higher BCS nd the resulting higher ody ft moiliztion (see plsm NEFA t week 1 in Fig. 3) s reported efore (Chillird 1999; Kokkonen et l. 25). The increse in the NEFA levels in the group ws ssocited with drop in the BCS efore nd fter prturition. This could hve een ecuse of n effect of the higher ody ftness per se (Chillird et l. 2), nd or to higher milk production, s lso reported y Ingvrtsen nd Andersen (2), ut opposed to Stockdle nd Roche (22). As the group did not show n increse in this metolite, we suggest tht in this group, there ws not n NEB tht is consistent with the lower milk production nd loss of BCS. The decrese in NEFA t the third week postprtum in the group ws the consequence of n increse in feed intke, which cn e lso oserved in the BCS improvement. In the energy-supplemented cows, the elevtion of NEFA ws prlleled with n incresed production of BHB. At this moment, the cows in the group were in deeper NEB, ecuse of higher milk production nd insufficient intke. The increse in the cholesterol levels in the postprtum period could e ecuse of the mount of milk production, to the gret energetic demnds for lcttion tht result in n increse of the synthesis of lipoproteins in the liver, ut BCS nd NEFA profiles suggest tht the cholesterol increse is ecuse of etter energy lnce s suggested efore (Cvestny et l. 25). The ure increse in cows ws ecuse of higher protein ctolism cused y lower content of glucogenic sustrtes tht forced the cows to use their protein reserves s suggested previously (Jorritsm et l. 23). These uthors found tht cows with ruminl flor not dpted to lcttion rtions might lso fce higher plsm ure concentrtions ecuse of mismtch etween energy nd protein t the level of the rumen. The most interesting finding in our tril ws tht insulin levels remined consistently higher in the group through ll the experimentl period. This suggests tht feeding more fermentle crohydrtes during the

7 Preprtum Supplementtion in Grzing Diry Cows 669 preprtum trnsition period my deliver more glucogenic precursor to the liver nd enhnce insulin synthesis. The higher plsm preprtum insulin concentrtions with respect to those in the postprtum period in the control group gree with previous works (Holtenius et l. 23; Meikle et l. 24; Kokkonen et l. 25). Furthermore, incresed propionte production s result of corn-sed diet results in n incresed secretion of insulin (Brockmn nd Lrveld 1986). Insulin plys centrl role in the homeosttic control of energy metolism (Chillird 1999). Dietry chnges cuse n immedite nd rpid stimulus in metolic hormones nd metolites; the most importnt of these re glucose nd insulin; if the dietry intke is low, insulin concentrtions cn e reduced (O Cllghn nd Bolnd 1999), nd this ws oserved in the group tht hd lso lower levels of insulin in the sme period. The higher postprtum NEFA together with higher insulinemi in the group could reflect higher insulin resistnce s result of higher ody ftness (Chillird 1999); lthough this is true in very oese cows tht ws not the sitution in this study. In contrst to pre-clving IGF-I profiles tht were higher in group, this feeding did not mintin postprtum IGF-I levels s hs een previously reported y Lucy (23) nd Roche et l. (25). After prturition IGF-I decresed despite incresing feed intke (Rhods et l. 24). Low plsm IGF-I levels in erly lcttion re generlly consequence of hypoinsulinemi (McGuire et l. 1995; Butler 23); however, in the group insulin levels re high in tht period. Cows in poor energy sttus hve low circulting concentrtions of IGF-I (Pushpkumr et l. 23). We do not find n explntion regrding to why the group, with incresed insulin concentrtions during the postprtum period when compred to s, did not differ in IGF-I levels. Chillird (1999) stted tht erly lcttion cows in negtive EB re resistnt to GH nd tht this decresed plsm IGF- I postprtum is independent of insulinemi. More recently, Butler et l. (23) reported tht the liver is refrctory to GH during NEB nd this uncoupling of the GH-IGF xis results in diminished plsm concentrtions of IGF-I. Mshek nd Beede (2) hypothesized tht nutrition-medited decline in IGF-I during the periprtum period my prevent full metolic dpttion to the nutrient demnds of lcttion, nd consequently reduce milk production. Preprtum higher levels of plsm leptin in the group gree with Holtenius et l. (23) nd Kokkonen et l. (25) in cows fed confined, nd with Roche et l. (25), who worked with cows under grzing conditions. Leptin cts s n energy reserve signl, secreted y the white dipose tissue (Chillird et l. 25) nd this is consistent with the BCS increse found in the group. Furthermore, the higher energy intke cts s short-term stimulus for plsm leptin (Delvud et l. 22). The decrese in postprtum leptin levels hs lso een reported (Chillird et l. 25). Leptin levels remined low nd constnt throughout the rest of the experimentl period for oth groups, which is in greement with findings of Holtenius et l. (23) nd Roche et l. (25), who found tht preprtum levels of feeding did not ffect lood leptin concentrtions postprtum, ut not with the oservtion of Kokkonen et l. (25) who found tht preprtum supplemented cows hd higher postprtum plsm leptin. Plsm leptin content hd positive correltion with BCS nd ws good indictor of level of ody ft in periprtum diry cows in greement with Meikle et l. (24). The peripherl tissues try to fit their current locl energy metolism to the postprtum ctolic condition, for exmple, y interfering in the pthwy of thyroid hormones, resulting in diminished circultion s oserved here, s previously reported (Meikle et l. 24). Thyroxine nd T 3 re the lowest in erly lcttion when production of milk is the gretest (Meikle et l. 24). In our results, this decrese ner clving ws found only in T 4, while T 3 remined constnt ll over the experimentl period. In diry cttle, peripherl thyroid hormone metolism plys mjor role in regulting the homeosttic nd teleophoretic responses involved in the mintennce of high priority functions (Chillird 1999). The supplementtion on preprtum cows shortened the intervls from prturition to first ovultion. In disgreement with our findings, Kedy et l. (21) under confined conditions, found tht energetic supplementtion on the dry period delyed the reinitition of cyclicity. The onset of postprtum ovrin cyclicity ws reported to hve negtive correltion with the ndir of the NEB (Butler 23); the fster the cows recover from NEB the sooner they will strt cycling. In our study, energetic supplementtion incresed the hormonl levels, which hve een reported to e relted with reproduction in the preprtum period (Spicer nd Echternkmp 1995), lthough in the postprtum period, only insulin levels were higher in the group. In greement with our results, Gong et l. (22) reported tht the diet resulting in higher insulin concentrtion reduced the intervl from clving to first ovultion. It is likely tht incresed insulin concentrtions promoted the differentition nd mturtion of dominnt follicles during erly lcttion, therey incresing the chnce of these dominnt follicles ovulting in response to the LH surge (Butler 23). Insulin stimultes ovrin progesterone nd oestrdiol production (Spicer nd Echternkmp 1995). Britt (1992) hypothesized tht ovrin follicles re detrimentlly ffected y exposure to NEB during their erly growth nd development. Roth et l. (21) reported tht n cute stress (e.g. NEB) ffected folliculr steroidogenesis 24 dys lter. According to this, we ssumed tht lower insulin nd IGF-I levels in the group on the week pre-clving ffected steroidogenesis nd follicle size, nd tht could e the cuse of the longer postprtum cyclic period on tht group. Dt regrding leptin nd reproductive performnce re complex nd sometimes contrdictory. It hs een reported tht cows with decresed leptin present delyed onset of cyclicity on the postprtum (Kdokw et l. 2). On the other hnd, leptin my ply permissive role, when incresed ove criticl threshold ( nm ccording to the review y Chillird et l. 25), in the ctivtion of the hypothlmus pituitry xis nd consequent reinitition of ovrin

8 67 D Cvestny, M Kulcs r, D Crespi, Y Chillird, A L Mnn, O Blogh, M Keresztes, C Delvud, G Huszenicz nd A Meikle ctivity (Meikle et l. 24). This would llow cows tht hd higher BCS in the preprtum nd postprtum nd thus, higher leptin plsm levels in the preprtum nd or the postprtum period (Meikle et l. 24; Kokkonen et l. 25) to shorten the intervl clving to first ovultion. Conclusion Energetic supplementtion dministered 3 weeks preclving in grzing conditions incresed milk production nd hd positive effect on the reinitition of ovrin ctivity, which is consistent with etter EB (BCS), higher preprtum levels of IGF-I, leptin nd insulin, nd higher insulin levels during erly postprtum period. It must e stressed tht prmeters used to estimte energy lnce (i.e. BCS, NEFA, BHB nd insulin) re not lwys univocl, nd thus they cnnot e used isolted; evlution of severl of these metolites nd or hormones should give more cler informtion on the energy sttus of the high producing diry cow. Acknowledgements The uthors re thnkful to Julin Medín, nd Mcren Pin, for their prticiption in the field work. The help of Alejndro Mendoz in the preprtion of the dt for the nlysis is lso pprecited. The tril ws finnced prtilly y INIA, y grnt to D. Cvestny of the CSIC, University of Uruguy, y the ECOS progrm (ECOS-Sud N U 3BO2) of the French government, nd y the Ntionl Reserch Fund of Hungry (project numer: OTKA T ). References Acost Y, 1997: Utilizcio n de ensiljes, concentrdos y psturs pr produccio n de leche. In: Restino E, Indrte E (eds), Psturs y Produccio n Animl en A res de Gnderı Intensiv. Serie Te cnic No 15. INIA, Uruguy, pp Britt JH, 1992: Influence of nutrition nd weight loss on reproduction nd erly emryonic deth in cttle. Proc XVII World Buitrics Congr, Brockmn RP, Lrveld B, 1986: Hormonl regultion of metolism in ruminnts: review. Livest Prod Sci 14, Butler WR, 23: lnce reltionships with folliculr development, ovultion nd fertility in postprtum diry cows. 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