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1 Document downloded from: This pper must be cited s: Mrtinez Predes, EM.; Ródens Mrtínez, L.; Mrtínez Vllespín, B.; Cerver Frs, MC.; Bls Ferrer, E.; Brecchi, G.; Boiti, C... (2012). Effects of feeding progrmme on the performnce nd energy blnce of nulliprous rbbit does. Animl. 6(7): doi: /s The finl publiction is vilble t Copyright Cmbridge University Press (CUP): STM Journls Additionl Informtion

2 Editoril Mnger(tm) for Animl: An Interntionl Journl of Animl Bioscience Mnuscript Drft Mnuscript Number: Title: Effects of rering feeding progrmme on the performnce nd energy blnce of young rbbit does Short Title: Feeding progrmmes for young rbbit does Article Type: Full Reserch Pper Section/Ctegory: 2. Nutrition Keywords: rbbit femles, rering, pubertl development, body condition, metbolic sttus Corresponding Author: Jun José Pscul, Ph.D. Corresponding Author's Institution: Universidd Politécnic de Vlenci First Author: Eugenio Mrtínez-Predes Order of Authors: Eugenio Mrtínez-Predes;Luis Ródens;Betriz Mrtínez-Vllespín;Conch Cerver;Enrique Bls;Gbriele Brecchi;Cristino Boiti;Jun José Pscul, Ph.D. Suggested Reviewers: Luis Flço e Cunh Proffesor, INSTITUTO SUPERIOR DE AGRONOMÍA. DEPARTAMENTO DE PRODUÇÂO, UNIVERSIDADE TECNICA DE LISBOA lflco@is.utl.pt Member of the Europen Group on Rbbit Nutrition with wide experience in rbbit femle metbolism. Gerolmo Xiccto Full Proffesor, DIPARTAMENTO DI SCIENZE ZOOTECNICHE, UNIVERSITÀ DI PADOVA gerolmo.xiccto@unipd.it Actul President of World Rbbit Science Assocition hs dediquted min of his reserch ctivity to rbbit femle nutrition. Nuri Nicodemus Proffesor, Deprtmento de Producción Animl, Universidd Politécnic de Mdrid nuri.nicodemus@upm.es This resercher is one of the persons working in rering diets for rbbit femles.

3 Mnuscript 1 2 Running hed: feeding progrmmes for young rbbit does 3 4 Effects of rering feeding progrmme on the performnce nd energy blnce of young rbbit does E. Mrtínez-Predes 1, L. Ródens 1., B. Mrtínez-Vllespín 1, C. Cerver 1, E. Bls 1, G. Brecchi 2, C. Boiti 2, J.J. Pscul 1, Instituto de Cienci y Tecnologí Animl, Universitt Politècnic de Vlènci, Cmino de Ver s/n, 46022, Vlenci, Spin 2 Diprtimento di Scienze Bioptologiche ed Igiene delle Produzioni Animli e Alimentri, Vi S. Costnzo 4, Perugi, Università degli Studi di Perugi, Itly Corresponding uthor: J.J. Pscul. E-mil: jupscu@dc.upv.es Phone: Fx:

4 20 ABSTRACT A totl of 228 young rbbit femles ged 9 weeks were used to evlute five rering feeding progrmmes: CAL, fed d libitum with control diet [C: 11.0 MJ digestible energy (DE) nd 114 g digestible protein (DP) per kg dry mtter (DM)] until first prturition; CR, receiving the C diet restricted (140 g/d) from 12 weeks of ge to first prtum; F, fed d libitum with moderte energy fibrous diet [F: 8.7 MJ DE nd 88 g DP per kg DM] until first prtum; nd finlly, FC nd FCF, fed with F diet d libitum until 16 weeks of ge, whereupon FC group received the C diet d libitum until first prtum, while FCF group received the C diet d libitum until 20 weeks of ge nd then the F diet d libitum until first prtum. CAL group hd higher mortlity rte compred to the other groups between 9 nd 12 weeks of ge (34 vs. 3%; P<0.05) nd during the lst 3 weeks of first pregnncy (14 vs. 3%; P<0.05). CAL nd FC femles presented higher BW nd perirenl ft thickness (PFT) thn CR femles t week 20 (+0.41 kg nd +0.6 mm; P<0.05), with F femles showing medium vlues. The type of feeding procedure did not ffect the fertility rte of young femles t first AI. Differences in BW disppered t prturition, when only CAL femles presented greter PFT thn CR nd FC femles (+0.3 mm; P<0.05). In comprison to FCF, CAL femles hd smller nd thinner live born litters (-2.5 kits nd -139 g, respectively; P<0.05), with CR, F nd FC femles showing medium vlues. The low number of kits born live for CAL femles ws due to their lesser totl number of kits born (-1.7 kits; P<0.05) nd the greter mortlity of their litters t birth (+13.9 %; P<0.05) compred to FCF. NEFA ws higher in the blood of femles fed C diet (CAL nd CR) thn in others t prtum dy (on verge mmol/l; P<0.05). In conclusion, the d libitum use of diets for lctting rbbit does throughout the rering

5 period could led young rbbit femles to present higher risk of erly deth nd smller litter size t first prturition. Feed restriction or erlier use of suitbly fibrous diets led femles to chieve the criticl BW nd ft mss t first mting to ensure reproduction Key words: rbbit femles, rering, pubertl development, body condition, metbolic sttus

6 54 IMPLICATIONS Obtining well-developed rbbit femles tht produce lrge number of helthy, mrketble litters per mting over multiple prities is still one of the min priorities for rbbit production. This objective not only involves the use of dequte mngement progrmmes during reproduction, but lso pproprite mngement of nutrition during pre- nd post-pubertl growth to ensure better development of future reproductive femles. The correct design of rering progrmmes tht tke into ccount the young rbbit femle s requirements nd priorities, while ensuring both dequte pubertl development nd future reproductive performnce, is pressing need

7 79 INTRODUCTION The negtive effects of underfeeding on pubertl mturtion hve long been known in numerous species (Frisch, 1984). However, overfeeding during rering period hs been lso relted with lower reproductive performnce in diry heifers (Sejrsen et l., 1982), pullets (Whitehed, 1988) nd gilts (Klindt et l., 1999). Young rbbit femles fed d libitum until first prturition usully suffer similr problems to those mentioned for other species. For this reson, in the lst decde some works ssessed the possible impct of different mngement nd feeding plns for rering period on femle development nd reproduction: feed restriction (Rommers et l., 2004), BW t wening or t the first rtificil insemintion (AI) (Rommers et l., 2001, 2001b, 2002), or the use of fibrous diets (Xiccto et l., 1999; Pscul et l., 2002; Quevedo et l., 2005). However, some of these works hve shown n ntgonism between proper development nd improvement of reproductive response. The erlier the introduction of the restriction progrmme nd the lower the energy supply, the higher the voluntry feed intke of primiprous does with improved milk yield or reduced body reserves mobilistion during first lcttion (Nizz et l., 1997; Xiccto et l., 1999; Pscul et l., 2002), but the lter or the lower their pubertl mturtion (Pscul et l., 2002; Rommers et l., 2004). On the bsis of this previous informtion, in this work we evluted the effects on nulliprous rbbit does development of diet for reproductive rbbit does provided during the rering period both d libitum nd restricted, compred with three different feeding progrmmes bsed on the use of moderte-energy fibrous diet designed

8 for young rbbit femles nd provided: i) until first prturition, ii) until first mting nd iii) until first prturition pplying flushing round first mting

9 107 MATERIAL AND METHODS The experimentl procedure ws pproved by the niml welfre ethics committee of the Universitt Politècnic de Vlènci (UPV) nd crried out following the Europen Union recommendtions on cre nd protection of nimls used for experimentl purposes (2003) nd the dvice for pplied nutrition reserch in rbbits ccording to the Europen Group on Rbbit Nutrition (Fernández-Crmon et l., 2005) Diets Ingredients nd chemicl composition of the experimentl pelleted diets used in this tril re summrised in Tble 1. A control diet (C), similr to commercil diet for reproductive rbbit does [11.0 MJ digestible energy (DE) nd 114 g digestible protein (DP) per kg dry mtter (DM)], ws formulted following the recommendtions of De Bls nd Mteos (2010). In ddition, moderte energy diet with high fibre content (F) ws lso formulted [8.7 MJ DE nd 88 g DP per kg DM], including some minor ingredients nd supplements to prtilly correct obvious deficiencies in mino cids nd minerls. (Tble1) Apprent digestibility coefficients of energy nd CP were determined for ech diet, using totl of 30 three-wy crossbred rbbits, ged 42 dys with n verge BW of 1.32 (s.d. 0.07) kg ccording to Perez et l. (1995). Chemicl nlysis of diets nd feces were performed following the AOAC (1999) methods for DM, sh, ether extrct, CP, nd crude fibre (934.01, , , nd , respectively). Ether extrct ws determined fter cid hydrolysis.

10 NDF, ADF nd ADL were nlysed sequentilly (Vn Soest et l., 1991) using thermo-stble mylse (Thermmyl L120, Novo Nordisk, Gentofte, Denmrk) pretretment nd expressed exclusive of residul sh. Gross energy ws determined by dibtic bomb clorimetry (Gllenkmp Autobomb, Loughborough, UK) following the recommendtion of EGRAN (2001) Animls nd experimentl procedure A totl of 228 young rbbit does (line A from UPV, selected over 36 genertions for litter size t wening) were used from 9 weeks of ge to first prturition. The nimls were housed in trditionl building under controlled environmentl conditions, with light lternting on cycle of 16 h light nd 8 h drk. The experiment ws crried out from Jnury to June Until 9 weeks of ge, young rbbit femles were cged collectively, receiving the sme commercil diet d libitum (185 g crude fibre nd 175 g CP per kg DM), nd subsequently housed in individul cges with ccess to one of the experimentl 148 diets. Combining two diets nd three different feeding schemes, five feeding 149 progrmmes were formed (Figure 1). The CAL group included femles which received the C diet d libitum until first prturition. The CR group included femles which received the C diet d libitum until 12 weeks of ge nd then 140 g per dy until first prturition, with 7-dy flushing period (C diet d libitum) round AI. The F group included femles which received the F diet d libitum until first prturition. Finlly, FC nd FCF groups were femles tht received F diet d libitum until 16 weeks of ge, whereupon the FC group received the C diet d libitum until first

11 prturition, while FCF group received the C diet d libitum until 20 weeks of ge nd then the F diet d libitum until first prturition. (Figure 1) While nimls from different experimentl groups kept the sme feeding progrmme, dt were nlysed nd presented s whole (CAL nd CR until 12 weeks of ge, F, FC nd FCF until 16 weeks of ge, nd then FC nd FCF until 20 weeks of ge). Does were rtificilly inseminted t the end of the 18 th week of ge. As of this dte, successive AI were crried out every 21 dys, s necessry. After the 28th dy of pregnncy, mternl cges were provided with nest equipped for the litter. The trits mesured for ll does were BW nd food intke t 9, 12, 16, 18 (AI), 20 nd 23 (prturition) weeks of ge, s well s perirenl ft thickness (PFT) by ultrsounds t 9, 12, 18, 20 nd 23 weeks of ge. Totl nd live litter size nd weight t prtum were lso recorded. From 12 rbbit does per group, blood smples were collected t 9, 12, 18 nd 23 weeks of ge. On smpling dy, feeders were closed t 07:00 h nd blood smples were tken from the centrl er rtery into EDTAcontining tubes from 11:00 to 13:00 h. Blood smples were centrifuged immeditely fter smpling (3000 g, 4ºC nd 10 minutes) nd plsm ws stored t 20ºC before being ssyed for insulin, glucose, non-esterified ftty cids (NEFA), leptin, cortisol nd tri-iodothyroxine (T3) concentrtions. Controls t 9, 12, 16 nd 20 weeks of ge were done on Mondys, nd those t 18 weeks of ge (AI) on Fridy Ultrsound mesurements The PFT of does ws mesured by ultrsound to evlute body condition, s 180 described by Pscul et l. (2000 nd 2004). Imges were obtined with n

12 181 ultrsound unit (JustVision 200 SSA-320A rel-time mchine; Toshib) equipped 182 with imge nlyser softwre to determine distnces. Estimted body energy content (EBE; MJ/kg) ws determined t AI nd prturition from BW nd PFT dt s described by Pscul et l. (2004) Hormone nd metbolite ssys Plsm insulin concentrtions were determined by the double ntibody/peg technique using porcine insulin rdioimmunossy (RIA) kit (Linco Reserch Inc., St Chrles, MO, USA). The ntiserum ws guine pig nti-porcine insulin, while both 191 lbelled ntigen nd stndrds used purified recombinnt humn insulin. Leptin concentrtions were determined by double ntibody RIA using the multi-species leptin kit (Linco Reserch Inc.) s previously reported (Brecchi et l. 2006). Totl T3 ws ssyed by RIA ccording to the procedure provided by the mnufcturer (Immunotech, Mrseille, Frnce). The ssy sensitivity ws 0.13 ng/ml, nd the mjor nlogues of T3 did not interfere with the ssy. Plsm cortisol ws ssyed by RIA, using the CORT kit (ICN Biomedicls Inc., Cost Mes, CA, USA). CORT ssy sensitivity ws 0.15 ng/ml. Dilution nd recovery tests done on insulin, leptin, T3 nd corticosterone using five different smples of rbbit plsm showed linerity. Glucose ws nlysed by the glucose oxidse method using the Glucose Infinity kit from Sigm (Sigm Dignostic Inc., St. Louis, MO, USA). NEFA concentrtions were nlysed using enzymtic colorimetric ssy from Wko (Wko Chemicls GmbH, Neuss, Germny) s previously reported (Brecchi et l., 2006) Sttisticl Anlysis

13 The model used to nlyse performnce, hormonl nd metbolic dt of young rbbit does during rering nd first gesttion ws mixed model (PROC MIXED by SAS, Sttisticl Anlysis System, 2002), in repeted mesure design tht took into 210 ccount the vrition between nimls nd covrition within them. Covrince structures were objectively compred using the most severe criteri (Schwrz Byesin criterion), s suggested by Littell et l. (1998). The model included the feeding progrmme (CAL, CR F, FC nd FCF), the week of ge (9, 12, 18, 20 nd 23 weeks; dt for week 16 ws lso included for consumption nd BW), nd their interction s fixed effects. Rndom terms in the model included permnent effect of ech niml (p) nd the error term (e), both ssumed to hve n verge of zero, nd vrince σ 2 p nd σ 2 e. Different contrsts were computed to test the significnce of the differences between tretments while nimls of different experimentl groups received the sme feeding progrmme t 12 weeks [(CAL+CR)/2 vs. (F+FC+FCF)/3], t 16 weeks [CAL vs. CR vs. (F+FC+FCF)/3], t 18 nd 20 weeks [CAL vs. CR vs. F vs. (FC+FCF)/2] nd prturition [CAL vs. CR vs. F vs. FC vs. FCF]. To nlyse the litter dt t first prturition, fixed effects model (PROC GLM of SAS, 2002) ws used tht included only the feeding progrmme (CAL, CR F, FC nd FCF). Dt concerning mortlity of femles during the rering nd first pregnncy were nlysed ccording to nonprmetric procedure (PROC NPAR1WAY of SAS, 2002), using chi-squre test for men seprtion. 228

14 229 RESULTS Animl Performnce A high mortlity rte ws observed in the CAL group (34%) between 9 nd 12 weeks of ge (Figure 2) compred to the F group (3%; P<0.05), probbly due to n outbrek of epizootic rbbit enteropthy (ERE). Mortlity ws low nd similr in groups under different feeding progrmmes from 12 to 20 weeks of ge. However, the CAL group gin presented significntly higher mortlity (14%; P<0.05) compred to the other groups (on verge 3%) during the lst 3 weeks of pregnncy. (Figure 2) Dily intke, BW nd PFT of young rbbit does during rering nd pregnncy re presented in Tble 2 nd Figures 3 nd 4. The BW nd PFT t 9 weeks of ge ws 1.97±0.03 (stndrd error) kg nd 6.9±0.1 mm, respectively. Femles d libitum fed with C diet (CAL) showed significntly higher DE nd DP intke between 9 nd 12 weeks of ge (+89 kj nd +11 g per dy, respectively; P<0.05) nd BW t week 12 (+0.11 kg; P<0.05) thn those with F diet. From 12 to 16 weeks, DE nd DP intke were similr for CAL nd F femles (on verge 841 kj nd 86 g per dy, respectively), but significntly lower for those restricted (CR; 699 kj nd 72 g per dy; P<0.05). Thus, BW t week 16 ws significntly higher for CAL thn for F group (3.69 nd 3.47 kg, respectively; P<0.05), nd higher for both thn for CR (3.24 kg; P<0.05). (Tble 2) From 16 to 20 weeks of ge, DE nd DP intke of F group ws even higher (on verge 792 kj nd 80 g per dy, respectively) thn tht observed for CAL group (742 kj nd 76 g per dy; P<0.05) nd higher for both thn for CR group (690 kj nd

15 g per kg per dy; P<0.05). In fct, F femles going on to d libitum C diet t 16 weeks (FC) showed the highest intke vlues (on verge 883 kj nd 91 g per dy; P<0.05). In consequence, CAL nd FC femles t week 20 presented higher BW nd PFT (4.34 kg nd 7.3 mm, respectively) thn CR femles (3.93 nd 6.7 mm; P<0.05), with F femles showing medium vlues (4.14 kg nd 7.1 mm). The type of feeding progrmme did not ffect the fertility rte of young femles t first AI (85.2, 84.0, 89.7 nd 85.0% for CAL, CR, F nd FC femles, respectively). During the lst 3 weeks of pregnncy, F nd FC femles presented higher DE nd DP intke (on v. 630 kj nd 65 g per dy, respectively) thn CR nd FCF femles (on v. 586 kj nd 60 g per dy; P<0.05), with CAL femles showing the lowest intke vlues (537 kj nd 55 g per dy; P<0.05). Thus, differences in BW between feeding progrmmes disppered t prturition (Figure 3), while only CAL femles presented greter PFT thn CR nd FC femles (6.4 vs. 6.1 mm, respectively; P<0.05). (Figures 3 nd 4) Tble 3 shows the effect of the feeding progrmme dopted during doe rering on litter trits t the first prturition. In comprison to the CAL group, FCF femles hd lrger (7.7 vs. 5.2 kits; P<0.05) nd hevier live born litters (419 vs. 280 g; P<0.05), with CR, F nd FC femles showing medium vlues (on v. 6.1 kits nd 349 g). The smll number of kits born live t first prturition to CAL femles ws due to their lower number of totl kits born (6.6 vs. 8.3 kits P<0.05) nd the greter mortlity of their litters t birth (20.6 vs. 6.7 %; P<0.05) compred to FCF. (Tble 3) 278

16 Metbolic nd hormonl prmeters The plsm profiles of insulin, glucose, NEFA, leptin, cortisol nd T3 during rering nd first pregnncy in the different feeding progrmmes re shown in Figure 5. An increse in circulting insulin concentrtions ws observed with dvncing ge in ll 284 the groups, lthough it decresed t prturition (Figure 5). CAL group nimls presented lower men plsm insulin concentrtion thn F femles t 18 weeks of ge ( 19.5 µui/ml; P<0.05). Both glucose nd NEFA plsm concentrtions showed the highest vlues t weeks of ge nd dropped therefter. Glucose concentrtion in plsm ws opposite to insulin, being lower for CAL ( 31.4 mg/dl; P<0.05) thn for F femles t 12 weeks of ge (Figure 5b). At prtition dy, glucose ws lower in CAL, CR nd 291 FCF thn F nd FC femles (on v mg/dl; P<0.05). Although NEFA levels were similr for ll the groups t 18 weeks of ge (Figure 5c), femles receiving the C diet (CAL, CR nd FC) presented the highest NEFA vlues in plsm t prturition, only being significntly higher in CAL nd CR compred to F femles (on v mmol/l; P<0.05). Leptin levels were similr for ll groups t 12 weeks of ge nd t prtum dy (Figure 297 5d). An increse in plsm leptin concentrtion ws observed t 18 weeks, especilly in CR femles (6.6 ng/ml; P<0.05), where plsm hd higher leptin levels thn CAL (5.1 ng/ml), nd both F nd FC femles (on verge 3.3 ng/ml; P<0.05). Plsm cortisol incresed from 9 to 12 nd 18 weeks of ge, lthough it decresed t prturition (Figure 5e). No significnt differences between feeding progrmmes on cortisol in plsm were observed throughout the experiment. Plsm concentrtions of T3 t 12 weeks of ge were similr for ll the groups (Figure 5f). Femles given C diet d libitum t 18 weeks (CAL nd FC) hd higher levels of plsm T3 thn CR

17 femles (on v mmol/l; P<0.05). However, CAL femles showed higher T3 levels thn FC femles t prturition ( 0.96 mmol/l; P<0.05). (Figure 5)

18 DISCUSSION No previous work evluting the use of rering diets described the high mortlity rte observed in the present work from 9 to 12 weeks of ge when young femles were 314 fed the control diet. This fct seems to be relted to ERE incidence when no medicted diets re used. Under these conditions, insufficient level or indequte qulity of dietry fibre cn increse the risk of digestive disorders in young rbbits (Gidenne, 1997; Gidenne nd Grci, 2006). In the current work, lthough higher soluble fibre ws expected for the F diet (from lflf nd beet pulp), both diets were designed to meet fibre recommendtions to prevent digestive problems from 9 to 12 weeks of ge (ADL>50, ADF>190 nd NDF-ADF>80 g/kg). However, recent review (Bls nd Gidenne, 2010) highlighted tht, even if requirements proposed to prevent digestive disorders re met, replcing strch with low or high digestible fibre reduces mortlity rte, especilly in the context of ERE. Young rbbit femle needs from 9 to 12 weeks of ge (pprox MJ per dy, considering their men live weight nd dily gin; Xiccto nd Trocino, 2010) were met with both C nd F diets (1.74 nd 1.54 MJ per dy, respectively). Although the lower DE intke led femles receiving the F diet to rech 12 weeks with smller BW, s in previous work (Pscul et l., 2002), the min metbolic nd hormonl prmeters here exmined were not gretly ffected. Rebollr et l. (2011) lso found similr concentrtions of leptin (2.8 ng/ml) nd NEFA (0.22 mmol/l) in the blood of young femles t 11 weeks of ge when compring d libitum supplying of control nd fibre-rich diets. However, when higher feed restriction is sserted (even below niml needs 1.03 MJ per dy; Rommers et l., 2004), the blood levels of glucose, leptin, insulin, nd T3 of young femles (from 6 to 12 weeks of ge) were clerly reduced.

19 As consequence of feed intke restriction from 12 to 18 weeks of ge, CR femles reched 18 weeks of ge with dely in their development, showing lower BW nd PFT thn those fed d libitum. These results gree with those reported in previous works where feed restriction reduced BW s well s body ft nd protein content of young rbbit femles t first AI (Rommers et l., 2001, 2004), nd even cused dely in the effectiveness of this AI (Rebollr et l., 2011). In the present work, CR femles presented slight reduction of T3 blood levels t 18 weeks of ge together with n unexpected higher concentrtion of leptin compred to those with free ccess to the control diet. Severl studies hve shown tht fsting reduces leptin, minly synthesised nd secreted by dipocytes, circulting in blood t levels proportionl to body ft stores in humns (Weigle et l., 1997), gilts (Brb et l., 2001), ruminnts (Chillird et l., 2000), nd lso in rbbits (Rommers et l., 2004; Brecchi et l., 2006; Rebollr et l., 2011). However, the mechnisms whereby feeding restriction ffects circulting leptin levels re still uncler, nd different responses were observed depending on type nd length of fsting nd blood smpling protocols. In this respect, Brecchi et l. (2006) described higher leptin levels in the plsm of 48- h fsted thn in 24-h fsted does. In ny cse, it might be considered tht CR femles were subject to 4-dy flushing period prior to AI, where nimls hd free ccess to the C diet, which could hve conditioned the plsm metbolic profile for these dys. On the other hnd, femles with free ccess to the F diet were ble to compenste for the lower nutritive dietry concentrtion with greter feed intke from 12 to 18 weeks of ge. Thus, they chieved DE nd DP intkes similr to those of rbbits receiving the C diet d libitum nd, consequently, reduced their gps in BW nd PFT t 18 weeks of ge. In fct, these differences disppered when femles of F group

20 hd free ccess to C diet s of 16 weeks of ge. Pscul et l. (2002) described how young rbbit femles fed with low-energy diet (8 MJ DE/kg DM) s of 10 weeks of ge presented, during lte rering, greter DE intke thn those fed 150 g per dy of stndrd diet (11 MJ DE/kg DM). The greter feed intke, however, did not compenste erlier differences in BW nd these rbbits chieved first AI 10 dys lter. However, lter introduction of low-energy diet t 13 weeks of ge (Quevedo et l., 2005) or the use of moderte-energy diets (9.5 MJ DE/kg DM; Xiccto et l., 1999) enbled young rbbit femles to chieve first mting t n dequte ge nd BW. In this sense, lthough the use of moderte low-energy diet during the rering period led femles to rech first mting with lower energy body reserves (Figure 6) nd lower blood leptin levels thn those fed with conventionl diet for reproductive 373 does, no consequence on fertility t first AI ws reported. It is well-known tht nutrient restriction my dely the onset of puberty, leding to the hypothesis tht criticl som must be chieved before puberty cn occur (Frisch, 1980). Furthermore, Aris-Álvrez et l. (2009) recently proposed tht reching the permissive leptin threshold should be necessry for pubertl reproductive ctivity, nd my be ssocited with inhibition of reproduction if the criticl som is insufficient to trigger gesttion (Moschos et l., 2002). In fct, when the reltionship between fertility nd blood leptin levels of young rbbit femles round first insemintion is drwn (Figure 7), the hypothesis of leptin threshold for initition of puberty nd reproductive success which is not improved by dditionl provision of this hormone seems to be confirmed. Consequently, these results revel tht in terms of d libitum feeding during rering, both feed restriction nd erlier use of moderte low-energy diet (8.7 MJ/kg DM) led femles to chieve the criticl BW nd

21 ft mss t first AI to ensure reproduction, in spite of their lower ftness nd leptin content in blood. (Figures 6 nd 7) After the first AI, lthough young femles receiving the C diet d libitum mintined greter consumption thn those restricted until 20 weeks of ge, the ftness ccumulted by CAL femles throughout rering llowed them to reduce their feed intke s pregnncy progressed, llowing CR femles to diminish the differences in BW, PFT, nd EBE observed up to this point with the CAL group during lte pregnncy. In previous work (Rommers et l., 2004), where development between young femles fed d libitum nd erly restricted (restriction: from 5 to 10 weeks of ge; recovery: 10 to 17.5 weeks of ge) ws compred, lthough compenstory growth of the restricted group ws lso observed during pregnncy, the erly differences chieved in BW of femles were mintined throughout the 3 reproductive cycles controlled by the uthors. In gilts, where feed restriction of young femles hs been studied extensively, most works (Sørensen et l., 1998; Klindt et l., 1999 nd 2001b) show tht moderte feed restriction during the rering period helps femles void excessive ftness, while more intense restriction (erlier nd/or stronger) leds to smller development nd sometimes even to lower reproductive 404 performnce. Therefore, these results seem to confirm the effectiveness of moderte restrictive feeding in preventing excessive ftness in young femles, lthough the strting ge nd restriction level should be controlled to void n indequte pre-pubertl body development. A prcticl lterntive to restriction could be the use of fibrous diets. Severl works found in the literture showed tht the use of fibrous diets during rering led nulliprous rbbit femles to greter DE intke fter first mting, independently of

22 their previous growth rte during development. Even so, when low-energy fibrous diets re used (<8.5 MJ DE/kg DM), femles re not ble to compenste the previous developmentl dely (Pscul et l., 2002; Quevedo et l., 2005). However, when femles hve the chnce of receiving moderte-energy fibrous diet (pprox. 9 MJ DE/kg DM), they rech the first prturition with development nd BW similr to those of rbbit does fed d libitum diet for reproductive does (>10.5 MJ DE/kg DM), but with lower ftness (Xiccto et l., 1999; Rebollr et l., 2011). In the present work, nd independently of the fibrous feeding systems used (F, FC or FCF), femles reched first prturition in n intermedite developmentl sitution to tht observed in femles fed with the C diet d libitum or restricted. Similr results were lso obtined by Rebollr et l. (2011), where the use of fibrous diet (9.4 MJ DE/kg DM) from 11 weeks of ge to first prturition led young rbbit femles to rech the end of first pregnncy with body energy nd protein content hlfwy between d libitum nd restricted dministrtion of control diet (11.6 MJ DE/kg DM). The use of fibrous diet with 8.5 to 9.5 MJ DE/kg DM should therefore llow young rbbit femles to rech first prturition in n dequte stte of development, voiding excessive ftness without the need for feeding restriction. In fct, the possible negtive effects of excessive ftness could be behind the problems detected round first prturition in the CAL group. Compred to the other feeding systems evluted here, femles fed the C diet d libitum during rering showed the lowest DE intke nd the highest body energy mobilistion recorded during lte pregnncy. In fct, the plsm of these femles t prtum dy ws chrcterised by higher NEFA nd lower glucose levels. The forementioned profile is frequently relted to pregnncy toxemi risk (Mrtenink nd Herdt, 1988; Bezille, 1995; Rosell, 2000), nd could explin the higher mortlity in lte pregnncy for the

23 436 femles of this group nd the smller size of their litters t first birth cused by both 437 lower totl litter size nd higher mortlity t birth. Rommers et l. (2002) lso observed tht hevier young femles t first AI (more thn 4 kg BW) hd higher percentge of stillborn t first prturition (13.4%) thn smller femles (5%). In gilts, Klindt et l. (2001 nd 2001b) relted n excessive energy intke during rering with lower number of corpor lute nd live embryos per gilt, nd lso observed tendency towrds the reduction of litter size t first birth ( 0.8 piglets born) nd the increse of gilts removed until this time (+13%). In this sense, the highest prolificcy nd the lowest mortlity t birth were recorded for femles given the F diet with flushing of 4 weeks with the C diet pplied round first mting. In recent revision, Theu-Clement (2007) concluded tht feed flushing fter nutritive restriction could improve the reproduction performnce, t lest t the beginning of the reproductive creer. From the results of the present work it could be concluded tht the d libitum use of diets formulted to cover the needs of lctting rbbit does for the whole rering period could led young rbbit femles to present higher risk of erly deth nd smller litter size t first prturition. As n lterntive, either feed restriction or erlier use of n dequte fibrous diet could led femles to chieve the criticl BW nd ft mss t first AI to ensure reproduction. However, under these feeding progrmmes for young femles, the strting ge nd nutritive level of the fibrous diet should be controlled to void n indequte pre-pubertl development ACKNOWLEDGEMENT The uthors thnk the Spnish Ministry of Eduction nd Science (Project AGL ) for the economic support to conduct this study. 461

24 REFERENCES Assocition of Officil Anlyticl Chemist Officil methods of nlysis, 18th edition. AOAC, Githersburg, MD, USA (5th revision). Aris-Álvrez M, Grcí-Grcí RM, Rebollr PG, Nicodemus N, Revuelt L, Millán P, Lorenzo PL Effects of lignin-rich fibre diet on productive, reproductive nd endocrine prmeters in nulliprous rbbit does. Livestock Science 123, Brb CR, Brrett JB, Kreling RR, Rmpcek GB Serum leptin concentrtions, luteinizing hormone nd growth hormone secretion during feed nd metbolic fuel restriction in the prepuberl gilt. Domestic Animl Endocrinology 20, Bezille P Toxémie de gesttion et hypoclcémie chez l brebis. In: Mldies métboliques des rumints. Le Point Vétérinrie 27, specil number, Bls E., Gidenne T Digestion of sugrs nd strch. In: Nutrition of the Rbbit (eds C De Bls nd Wisemn), pp CABI Publishing, Wllingford, UK. Brecchi G, Bonnno A, Gleti G, Federici C, Mrnesi M, Gobbetti A, Zerni M, Boiti C Hormonl nd metbolic dpttion to csting: Effects on the hypothlmicpituitry-ovrin xis nd reproductive performnce of rbbit does. Domestic Animl Endocrinology 31, Chillird Y, Ferly A, Fulconnier Y, Bonnet M, Rouel J, Bocquier F Adipose tissue metbolism nd its role in dpttions to undernutrition in ruminnts. Proceedings of the Nutrition Society 59, De Bls C, Mteos GG Feed formultion. In: Nutrition of the Rbbit (eds C De Bls nd Wisemn), pp CABI Publishing, Wllingford, UK. EGRAN Attempts to hrmonize chemicl nlyses of feeds nd feces for rbbit feed evlution. World Rbbit Science 9, Europen Union Protection of nimls used for experimentl purposes. Directive 86/609/EEC of 24th November 1986, mended 16th September 2003.

25 Fernández-Crmon J, Bls E, Pscul JJ, Mertens L, Gidenne T, Xiccto G nd Grcí J Recommendtions nd guidelines for pplied nutrition experiments in rbbits. World Rbbit Science 13, Frisch RE Pubertl dipose tissue: is it necessry for norml sexul mturtion? Evidence from the rt nd humn femle. Federtion Proceedings 39, Frisch RE Body ft, puberty nd fertility. Biologicl Reviews 59, Gidenne T Ceco-colic digestion in the growing rbbit: impct of nutritionl fctors nd relted disturbnces. Livestock Production Science 51, Gidenne T, Grcí J Nutritionl strtegies improving the digestive helth of the wened rbbit. In: Recent Advnces in Rbbit Science (eds L Mertens nd P Coudert), pp COST. ILVO, Merelbeke, Belgium. Klindt J, Yen JT, Christenson RK Effect of prepubertl feeding regimen on reproductive development of gilts. Journl of Animl Science 77, Klindt J, Yen JT, Christenson RK Effect of prepubertl feeding regimen on reproductive development nd performnce of gilts through the first pregnncy. Journl of Animl Science 79, Klindt J, Yen JT, Christenson RK 2001b. Level of dietry energy during prepubertl growth nd reproductive development of gilts. Journl of Animl Science 79, Littell RC, Henry PR nd Ammermn CB Sttisticl nlysis of repeted mesures dt using SAS procedures. Journl of Animl Science 76, Mrtenink JV, Herdt T.H Pregncy toxemi nd ketosis of ewes nd does. Veterinry Clinics of North Americ: Food Animl Prctice 4, Moschos S, Chn JL, Mntzoros CS Leptin nd reproduction: review. Fertility nd Sterility 77, Pscul JJ, Cstell F, Cerver C, Bls E, Fernández-Crmon J The use of ultrsound mesurement of perirenl ft thickness to estimte chnges in body condition of young femle rbbits. Animl Science 70,

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29 Tbles nd Figures Click here to downlod Tble: REV_Tbles nd Figures-6.doc Tble 1 Ingredients nd chemicl composition of experimentl diets Ingredient (g/kg) C Diets Brley Alflf hy Sunflower mel Soy mel 85 - Sugr beet pulp Cerel strw Soy oil HCl L-lysine, DL-methionine, L-threonine, L-tryptophn, L-Arginine, Diclcium phosphte Monosodium phosphte Slt 5 5 Vitmin-minerl mixture Chemicl composition (g/kg DM) Dry Mtter (DM, g/kg) Ash Strch Ether Extrct Crude Protein Neutrl Detergent Fibre Acid Detergent Fibre Acid Detergent Lignin Gross Energy (MJ/kg DM) Digestible energy (DE; MJ/kg DM) Digestible protein (DP; g/kg DM) DP/DE (g/mj) Per Kg of feed: Vitmin A: 8,375 IU; Vitmin D3: 750 IU; Vitmin E: 20 mg; Vitmin K3: 1 mg; Vitmin B1: 1 mg; Vitmin B2: 2 mg; Vitmin B6: 1 mg; Nicotinic cid: 20 mg; Choline chloride: 250 mg; Mg: 290 mg; Mn: 20 mg; Zn: 60 mg; I: 1.25 mg; Fe: 26 mg; Cu: 10 mg; Co: 0.7; Butyl hydroxylnysole+ethoxiquin: 4 mg. F

30 Tble 2 Dily dry mtter (g DM per kg metbolic weight (BW 0.75 )), digestible energy (kj DE per kg BW 0.75 ) nd digestible protein (g DP per kg BW 0.75 ) intke of young rbbit does during rering nd first pregnncy (men ± stndrd error) Feeding progrmme 1 CAL F 9-12 wk DM intke ± b ±0.94 DE intke b ± ±9.5 DP intke b ± ±0.99 CAL CR F wk DM intke b ± ± c ±0.91 DE intke b ± ± b ±9.2 DP intke b ± ± b ±0.96 CAL CR F FC wk DM intke b ± ± d ± c ±1.03 DE intke b ± ± b ± c ±10.6 DP intke b ± ± b ± c ±1.09 Erly pregnncy; wk DM intke b ± ± d ± c ±1.03 DE intke b ± ± c ± d ±10.6 DP intke b ± ± c ± d ±1.09 CAL CR F FC FCF Lte pregnncy; wk DM intke ± b ± d ± b ± c ±1.55 DE intke ± b ± c ± bc ± b ±15.9 DP intke ± bc ± c ± c ± b ± Feeding progrmme: CAL group received the C diet d libitum until 1 st prtum; CR group received the C diet d libitum until 12 wk nd then, 140 g per dy until 1 st prtum; F group received the F diet d libitum until 1 st prtum; FC nd FCF group received F diet d libitum until 16 wk nd then, FC group received the C diet d libitum until 1 st prtum nd FCF group the C diet d libitum until 20 wk nd then the F diet d libitum until 1 st prtum.,b,c,d Mens within row not shring ny superscript re significntly different t P<0.05.

31 Tble 3 Litter size nd weight t first prtum (men ± stndrd error) Feeding progrmme 1 CALminusc ul? CR F FC FCF Litter size t prtum Totl 6.6 ± b ± b ± b ± b ±0.6 Alive 5.2 ± b ± b ± b ± b ±0.6 Mortlity t birth 20.6 c 8.3 b 12.1 b 12.4 b 6.7 Litter weight t prtum (g) Totl 340 ± b ± b ± ± b ±24.5 Alive 280 ± bc ± bc ± b ± b ± Feeding progrmme: Abbrevitions s in Tble 2.,b Mens within row not shring ny superscript re significntly different t P<0.05.

32 Figure 1 Digrm of the different feeding progrmmes crried out during rering nd first pregnncy for the 5 experimentl groups (CAL, CR, F, FC nd FCF) 9wk 12wk 16wk 18wk 20wk 23wk CAL CAL CR CAL CR CAL* CR F F FC F CAL FCF F CAL FAL 1 st AI 1 st prturition *Flushing 4 dys before insemintion CAL: C diet d libitum; CR: C diet restricted t 140 g per dy; F: F diet d libitum AI: Artificil Insemintion

33 Mortlity (%) Figure 2 Percentge of does ded during the rering nd first pregnncy (from 9 to 23 week of ge) with the different feeding progrmmes (bbrevitions s in Tble 2). Brs within period not shring ny superscript re significntly different t P<0.05. AI: Artificil Insemintion b CAL CR F FC FCF b 6 b 4 b 0 3 b 9-12 wk wk wk AI- 20 wk 20 wk- first prturition

34 Live weight (g) Figure 3 Live weight evolution of young rbbit does during rering nd first pregnncy (9 to 23 wk of ge) with the different feeding progrmmes (bbrevitions s in Tble 2). Dt t 9 week of ge re presented s whole. Brs not shring ny superscript re significntly different t P<0.05 CAL CR F FC FCF f ij hi g g k h j k ij hi hi ij ij 3400 d e 2900 c b prturition Age (week)

35 Perirenl ft thickness (mm) Figure 4 Perirenl ft thickness evolution of young rbbit does during rering nd first pregnncy (9 to 23 wk of ge) with the different feeding progrmmes (bbrevitions s in Tble 2). Dt t 9 week of ge re presented s whole. Brs not shring ny superscript re significntly different t P<0.05 8,0 CAL CR F FC FCF 7,5 f ef ef ef e ef 7,0 6,5 d bc bc d cd b b b 6,0 5,5 5, prturition Age (week)

36 Leptin (ng/ml) Cortisol (µg/dl). T3 (mmol/l). Insulin (µui/ml) Glucose (mg/dl). NEFA (mmol/l). Figure 5 Evolution of blood plsm () insulin, (b) glucose, (c) non esterified ftty cids (NEFA), (d) leptin, (e) cortisol nd (f) tri-iodothyroxine (T3) concentrtions in young rbbit does during rering nd first pregnncy (9 to 23 wk of ge) with the different feeding progrmmes ( ; bbrevitions s in Tble 2). Dt t 9 week of ge re presented s whole. Brs not shring ny superscript re significntly different t P< bc b d cd cd bc bc b b b b c b b b b b b b 2,1 1,8 1,5 1,2 0,9 0,6 0,3 c b b b b b b 0 0 0, prtum prtum Age (wk) Age (wk) () (b) (c) prtum Age (wk) b b d e bc c bc bc bc bc b bc cd d cd cd bcd bcd bc b b b b b d c d cd b b b b prtum prtum Age (wk) Age (wk) (d) (e) (f) prtum Age (wk)

37 Estimted body energy (MJ per kg) Figure 6 Estimted energy content of young rbbit does t effective rtificil insemintion (AI) nd prturition dys with the different feeding progrmmes (bbrevitions s in Tble 2). Brs not shring ny superscript re significntly different t P<0.05 9,0 CAL CR F FC FCF 8,5 8,0 c b bc 7,5 7,0 6,5 6,0 Effective AI Prtum

38 Fertility (%). Figure 7 Reltionship between leptin levels in the blood of young rbbit does t first mting (16-18 wks of ge) nd the fertility observed during the first reproductive cycle. Dt obtined from the present results nd three previous works of the literture Present work Aris-Álvrez et l., 2009 Nicodemus et l., 2007 Brecchi et l., Leptin (ng/ml)