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October - December 2018 World Rabbit Sci. 26 (4) 269 - 333 EDITOR IN CHIEF World Rabbit Science is the o§cial journal of the World J.J. Pascual Rabbit Science Association (WRSA). One of the main Universitat Politècnica de València, Instituto de Ciencia y Tecnología Animal. objectives of the WRSA is to encourage communication and P.O. Box 22012, 46071 Valencia. Spain. collaboration among individuals and organisations associated [email protected] with rabbit production and rabbit science in general. World Rabbit Science is the only international peer-reviewed ASSOCIATE EDITORS journal included in the ISI Thomson Scientific devoted to F. Agnoletti publishing original research in the field of rabbit science. Istituto Zooprofilattico Sperimentale delle Venezie. Sezione diagnostica di Treviso World Rabbit Science is indexed and abstracted in the Vicolo Mazzini, 4. 31020 Villorba, Treviso. Italy. SciSearch®, Current Contents® and Focus On (Veterinary [email protected] Science & Medicine) since the beginning of 2005. Papers or J.M. Corpa reviews of the literature submitted to World Rabbit Science Instituto CEU de Ciencias Biomédicas, Departamento PASAPTA. Facultad de must not have been published previously in an international Veterinaria. Universidad Ceu Cardenal Herrera refereed scientific journal. Previous presentations at a C/ Tirant lo Blanch, 7. 46115 Alfara del Patriarca, Valencia. Spain. scientific meeting, field day reports or similar documents can [email protected] be published in World Rabbit Science, but they will be also subjected to peer-review process. P. García World Rabbit Science is published in English four times a Dpto. Producción Agraria.- Campos de Prácticas. Escuela de Ingeniería year in a single volume. Authors may publish in World Rabbit Agronómica, Alimentaria y de Biosistemas Science regardless of the membership in the World Rabbit Universidad Politécnica de Madrid. Cdad. Universitaria, s/n. 28040 Madrid. Spain. Science Association, although joining the WRSA is highly [email protected] encouraged. Views expressed in papers published in World H. Garreau Rabbit Science represent the opinion of the author(s) and do UMR 1388 GenPhySE : Génétique, Physiologie et Systèmes d'Elevage not necessarily reflect the o§cial policy of the WRSA or the 24 Chemin de Borde Rouge. Auzeville Tolosane, CS 52627. Editor in Chief. 31326 Castanet Tolosan Cedex. France. [email protected] G. González-Mariscal Centro de Investigación en Reproducción Animal, CINVESTAV-UAT. EDITORIAL OFFICE Apdo Postal 62, Tlaxcala, Tlax. 90000, Mexico. World Rabbit Science [email protected] Instituto de Ciencia y Tecnología Animal P. Hernández Universitat Politècnica de València. Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Camino de Vera s/n. 46071 Valencia. Spain Camino de Vera s/n. 46071 Valencia. Spain. EDITORIAL SECRETARY [email protected] C. Lario, Managing Editor L. Lamothe Contact for questions on relations with Section Editors, UMR 1388 GenPhySE: Génétique, Physiologie et Systèmes d'Elevage. submissions and proofs. 24 Chemin de Borde Rouge. Auzeville Tolosane, CS 52627. Universitat Politècnica de València. Editorial UPV 31326 Castanet Tolosan Cedex. France. Camino de Vera s/n. 46071 Valencia. Spain. [email protected]. [email protected]

M. Petracci SUBSCRIPTION INFORMATION Dipartimento di Scienze e Tecnologie Agro-Alimentari Alma Mater Studiorum Volume 26, 2018, 4 issues: 80 € Università di Bologna Piazza Goidanich, 60. 47521 Cesena. Italy. For more information, contact the Editorial UPV: [email protected] [email protected] Z. Volek Department of Nutrition Physiology and Animal Product Quality. Institute of Animal PUBLISHED BY Science. Přátelství 815. CZ-104 00, Prague-Uhříněves. Czech Republic. Universitat Politècnica de València. [email protected] Camino de Vera s/n. 46071 Valencia. Spain. Instructions to authors are available at: ASSOCIATE DIRECTOR www.wrs.upv.es S. Calvet Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Camino de Vera s/n. 46071 Valencia. Spain. [email protected] World Rabbit Science 2nd Series PRINTED IN SPAIN BY: Byprint percom, s.l. LEGAL DEPOSIT: nº V-1162-2003, Marzo 2003. COVER DESIGN: A. Climent. Universitat Politècnica de València. LAYOUT: Enrique Mateo. Triskelion Disseny Editorial. ISSN: 1257-5011 EISSN: 1989-8886 W orld World Rabbit Sci. 2018, 26: 269-276 R abbit doi:10.4995/wrs.2018.10061 Science © WRSA, UPV, 2003

MILK DIGESTION IN THE YOUNG RABBIT: METHODOLOGY AND FIRST RESULTS GIDENNE T., BANNELIER C., GALLOIS M., SEGURA M., LAMBRECHT V.

GenPhySE, Université de Toulouse, INRA, ENVT, CASTANET TOLOSAN, France.

Abstract: This study aims to determine the digestibility of milk by the young rabbit (21-25 d old), taking into account the increment of digesta content and urine excretion. Nineteen litters of 9 young rabbits 21 to 25 d old were used: 12 litters (S group) fed exclusively with milk using controlled suckling, and 7 litters (Control group) with free suckling and access to the pelleted feed of the doe. The faecal digestibility of milk dry matter (DM) was measured between 21 and 25 d of age, for S litters housed from 15 d of age in a metabolism cage separately from their mother. Between 21 and 25 d, the milk intake, faeces and urine excretion were controlled daily, and the mean increment in digesta content was measured by comparing digesta weight of the whole tract at 21 and 25 d of age (one kit per litter). The increment in digesta content from 21 to 25 d averaged 77% (+8.5 g), sourcing mainly from stomach and caecum contents increase (+57 and +120% respectively). The mean increase for the dry content of the gut (from 21 to 25 d) was 1.75 g DM/kit, and was considered as non-digested to calculate the digestibility coefficient of the milk. The milk intake averaged 30 g/d/kit (7.9 g DM/d kit). No faecal excretion was recorded between 21 and 25 d. From the milk intake and increment in digesta content, the corrected digestibility of the milk dry matter reached 94% (minimum=92.9%, maximum=95.6%). The daily urine excretion averaged 5.1 mL/kit, corresponding to 1.2 g DM/kit. Therefore, the corrected DM retention coefficient of the milk was 79.5%. The quantity of nitrogen excreted in urine was low (0.06 g/d kits), thus the corrected nitrogen retention coefficient for milk reached 82% and the nitrogen retained (corrected) reached 0.44 g/d kit. Accordingly, the amount in metabolisable protein for the milk was 90 g/kg (fresh). The corrected energy retention coefficient was estimated to 95.8%, for a crude energy concentration estimated at 28.14 MJ/kg DM for the milk. Thus, the energy retained (corrected) reached 223 kJ/d kit and the content in metabolisable energy for the milk was 26.94 MJ/kg DM. Key Words: young rabbit, digestion, milk, methodology.

INTRODUCTION

As in other mammals, the weaning period is crucial for the young rabbit, as the gastrointestinal functions develop sharply from 3rd week of age when the rabbit starts to eat solid feed. However, the digestive capacity of the young rabbit, essential to determine the nutritional requirements and to further prevent digestive diseases, has been extensively studied after weaning (Carabaño et al., 2010), and only a few studies look at the digestion before weaning, when young rabbits consume milk and solid pelleted feed (Gallois et al., 2005, 2008). For instance, the whole digestive tract e§ciency increased by 10% before weaning (32 d old) and remained steady (organic matter) or decreased (lipids, protein) after weaning (Gidenne et al., 2007). Only the study by Parigi-Bini et al. (1991) assessed the digestion for suckling rabbit, and, by comparative slaughter technique and multiple regression method, they estimated that the milk dry matter was totally digested. However, in this study, the suckling rabbits also consumed solid feed (from doe feeder), and no results were obtained for the digestion of exclusively milk-fed rabbits. Furthermore, around weaning the digestive tract is developing sharply, and Gallois et al. (2005) found a 72% increase in digesta content between

Correspondence: T. Gidenne, [email protected]. Received April 2018 - Accepted July 2018. https://doi.org/10.4995/wrs.2018.10061

World Rabbit Sci. 26: 269-276 269 GIDENNE et al.

21 and 28 d of age (+38.2 g) for suckling rabbits also consuming solid feed. This increment in digesta content must be taken into account in the digestibility calculation. Our study thus aimed to determine the digestion of the milk only, for 3 wk-old rabbits exclusively milk-fed and housed in metabolism cages separately from the doe. We also aimed to adjust a procedure to correct the calculation of digestibility coe§cient by measuring the increment in digesta content during the digestibility period (21-25 d of age).

MATERIALS AND METHODS

Experimental design, animals and housing The study was conducted in accordance with the French legislation on animal experimentation and ethics, and the senior researchers were authorised by the French Ministry of Agriculture to conduct experiments on living animals at the INRA facilities of PECTOUL, Auzeville, France. This study used 19 litters of 9 young each, obtained with a 3-way cross (female INRA1067×Grimaud line PS19, male Zika maternal quality) and from multiparous does (parity between 2 and 4). Does and litters were housed in metabolism cages (H: 29 cm, L: 47 cm), in an indoor breeding unit (light schedule: 07:00-19:00; 21°C). Does were freely fed a commercial pelleted feed and artiﬁcial insemination was performed 11 d after delivering. At 17 d of age, the litters were divided into 2 groups. Seven litters were assigned to the control group (C) and remained classically housed in the doe cage with their mother and with free nursing and access to the doe feed. Twelve litters were assigned to the “suckling only” group (S), and housed in a metabolism cage (same model as the control group but without nest box) without access to solid feed, separately from their mother (housed adjacent). Daily suckling was achieved at 9:00 am by introducing the mother into the litter cage, from 17 d to 25 d of age. The cages were equipped with a 1 mm mesh to collect the faeces, and with a stainless funnel adapted under the cage to collect urine from 21 to 25 d of age. The growth performances were measured at 15, 21 and 25 d in the 2 groups. Health status was checked through a daily clinical examination of the animals. This consisted of monitoring the animals for clinical signs of digestive disorders such as diarrhoea, caecal impaction, suspicion of ERE (Epizootic Rabbit Enteropathy) or other pathologies (respiratory problems, injuries …).

Procedure for urine collection and milk digestibility measurement A period of adaptation to controlled nursing was managed between 17 and 21 d of age. This also should allow the removal of digesta sourced from a potential solid feed intake before 17 d of age. The digestibility period covered 4 consecutive days: from 21 to 25 d of age. Each morning, milk intake was ﬁrst measured by weighing the doe before and after milking. Then, potential hard faeces excretion was checked and the urine was quantitatively collected in a beaker containing 40 mL of sulphuric acid (10% v/v) to prevent ammonia volatilisation (Udert et al., 2003), as follows: before urine collection, the litter was removed from the cage and the funnel was rinsed with 50 mL of sulphuric acid solution (10% v/v). The weight and volume of urine collected were measured daily, and 10% of the urine daily collected was stored at –18°C for subsequent N analysis. Milk was sampled from 3 does of the control group at the 18th day of lactation, then stored at –18°C, for further analysis in dry matter, crude fat and nitrogen.

Procedure to correct the digestibility coecient and calculations The digestive content is increasing sharply when the dry feed intake starts (around 17 d of age), for instance the stomach content tripled from 17 to 35 d (Orengo and Gidenne, 2007). This increment in digesta weight must be taken into account when calculating the digestibility coe§cient to respect the balance between intake and excretion. Therefore, one kit of each litter of the S group was sacriﬁced (at 09:00 before milking) at 21 d of age and at 25 d (after the last urine collection) to determine the weight of the digesta content (stomach, small intestine, caecum and colon). Then, for each rabbit, a pooled sample (from each segment) was dried (24 h, 103°C) to calculate the dry matter (DM) content of the whole gut (DCG) at 21 and 25 d of age. The mean variation in DCG between 25 and 21 d

270 World Rabbit Sci. 26: 269-276 MILK DIGESTION IN THE YOUNG RABBIT was considered as excreta to calculate the corrected digestibility coe§cient of the milk. As we did not detect any faecal excretion between 21 and 25 d, the formula to calculate the milk DM digestibility is: (DMim-DCG)/DMim, with DMim corresponding to the DM intake from milk only. Nitrogen concentration of urine sample (one per litter) was analysed to calculate the nitrogen excretion over the 4-d collection periods (21-25 d of age). The equation of Parigi-Bini and Cesselli (1976) was used to calculate the urinary energy excretion using nitrogen excretion: Y=−0.72+12.37X, where Y is the urinary energy excretion (kcal/d) and X is the urinary nitrogen excretion (g/d). The energy concentration in milk was estimated during the 4th week of lactation to 28.14 MJ/kg DM (7.32 MJ/kg), according to Maertens et al. (2006), while the energy concentration of digesta was estimated to 15.06 MJ/kg DM, similar to that in faeces of 4-wk-old rabbit (Gallois et al., 2008). Therefore, coe§cients of retention for nitrogen and energy were calculated as: intake−(urine excretion+digesta content increment).

Chemical analysis The following chemical analyses were performed on milk, digesta content and urine according to ISO methods and considering the recommendations of the European Group on Rabbit Nutrition (EGRAN, 2001): dry matter (DM) (ISO 6496, 1999), nitrogen (N) (Dumas method, ISO 16634, 2004) using a Leco auto-analyser (model FP-428, Leco Corp., St Joseph, MI, USA). Crude fat was determined in milk samples using Soxtec system H+ (after acid hydrolysis pre-treatment) according to the method described by Alstin and Nilsson (1990).

Data Analysis Two litters from the S group were removed from data analysis due to milking problems with the doe. Another litter showed a digesta content weight 2 times higher than the other 9 values of the S group, at 21 and 25 d of age, and this was the only litter with a small faecal excretion during 2 d after trial start (2.4 g DM at 21 d, and 0.6 g DM at 22 d old). We assume that this litter already consumed pelleted feed with the doe at 17 d of age. We thus removed it from digesta content analysis and from the milk digestion calculation. Live weight data for litter and doe and data of digestive segment weight of kits were compared for age eÄect, according to a mono-factorial variance analysis (procedure GLM under SAS). Milk intake and milk digestion data were original and obtained only for the S group, thus we give the mean value associated with the variation coe§cient and the minimum and maximum values observed.

RESULTS AND DISCUSSION

Growth of litters and doe live weight variation during milking period In the control group, the live weight of the litters ranged within the classical values observed for this rabbit line (Table 1). They were similar to that of S group at the start of the trial (mean=240 g/kit at 15 d of age), but were 10% heavier at 21 d and 18% heavier at 25 d (P<0.01). The growth rate was 33% higher for the control group during the

Table 1: Live weight and growth of the young rabbits, from 15 to 25 d of age, according to the suckling procedure. Group S C rVC (%) P-value Live weight (g/kit) 15 d 238 243 9.1 0.616 21 d 296 325 9.1 0.057 25 d 370 436 8.9 <0.01 Weight gain (g/d) 15-21 d 9.8 13.6 27.4 0.024 21-25 d 18.4 27.7 28.7 <0.01 15-25 d 13.2 19.3 14.6 <0.001 S, suckling only, separate housing for the litter and the doe, without access to solid feed (10 litters); C, control, free suckling and access to solid feeds (7 litters). rVC: Residual variation coe§cient, calculated as (root mean square error)/mean, and expressed in %.

World Rabbit Sci. 26: 269-276 271 GIDENNE et al.

Table 2: Doe live weight variation between 15 and 25 d of lactation. Group S C rVC (%) P-value Live weight (g) 15 d 4534 4701 9.3 0.401 21 d 4163 4632 9.0 0.017 25 d 4284 4563 10.1 0.17 Weight gain (g/d) 15-21 d –61.8 –5.4 60 <0.001 21-25 d 30.2 –20.0 312 <0.01 15-25 d –25.0 –11.2 77 0.079 S, suckiling only, separate housing for the litter and the doe, without access to solid feed (10 litters); C, control, free suckling and access to solid feeds (7 litters). rVC: Residual variation coe§cient, calculated as (root mean square error)/mean, and expressed in %.

21-25 d period (P<0.01). This result was expected, as control group kits were able to access the solid feed in their mother’s feeder. According to the literature, the solid intake ranged between 1 and 5 g/d (Gidenne et al., 2015) for freely nursed litters. In parallel, the weight of the doe decreased by 20 g/d during the 21-25 d period for the control group (Table 2), while it increased by 30 g/d in S group. This may suggest that the nursing behaviour of the S does was impaired by our procedure to control the milk intake of the litter (daily transfer from their cage to the litter cage and back). Similar eÄects of controlled milking on litter growth have already been described (Ubilla et al., 2000; Zhang et al., 2018).

Development of the digestive tract during the digestibility period At 21 d of age, the whole fresh content of the gut corresponded to 3.7% of the live weight, and 5.3% at 25 d of age (Table 3). Accordingly, between 21 and 25 d the whole digesta content rose by 77% (+8.5 g), although the rabbits were fed only with milk. This increase was sourced mainly from stomach and caecum contents increase: +57% and +120%, respectively. Within 4 d the relative weight of the stomach content decreased from 55 to 49% (P<0.001), while for the caecum it increased from 33% to 40% (P<0.001). Similarly, Gallois et al. (2005) described a similar increase for the stomach content fresh weight (+53%) between 21 to 28 d of age, but a sharper increase for the caecum (+350%), as the young rabbit consumed a significant amount of solid feed after 21 d of age. However, a large inter-individual variability (25 to 50%) was observed for all criteria, although the live weight variability of those sacrificed kits was low (5.7%). The dry matter concentration of the whole digestive content (pooled sample of the diÄerent segment) averaged 20.6%, with no significant diÄerence between 21 and 25 d (P= 0.151). The total dry content of the gut averaged 2.3 g per kit at 21 d of age and reached 4.0 g at 25 d. Thus, the mean increase for the dry content of the gut (DCGi)

Table 3: Changes in digestive content weight of the exclusively milk-fed young rabbit1 gut (group S), during the digestibility measurement period. 21 d old 25 d old rVC (%) P-value Live weight (g/kit) 291 371 5.7 <0.01 Weight of content (g fresh matter) Stomach 6.1 9.6 23.6 <0.01 Small intestine 0.7 1.4 43.5 >0.01 Cæcum 3.6 7.9 38.7 <0.001 Colon 0.6 0.6 53.3 0.970 Total fresh content 11.0 19.5 23.0 <0.001 Total dry content (g) 2.3 4.0 31.7 <0.001 1Mean value for 9 kits sacriﬁced at 21 d, and 9 kits at 25 d (after digestibility measurements), only suckling and without access to solid feed (group S). rVC: Residual variation coe§cient, calculated as (root mean square error)/mean, and expressed in %.

272 World Rabbit Sci. 26: 269-276 MILK DIGESTION IN THE YOUNG RABBIT

Table 4: Milk consumption of exclusively milk-fed litters (group S), from 21 to 25 d of age, and digestibility of the dry matter from the milk1. Mean* VC (%) Min Max Period 21 to 25 d old Milk intake (g/d kit) 29.6 4.2 23.8 38.0 Milk intake (g dry matter/d kit) 7.9 12.8 6.2 9.9 Dry matter digestibility, corrected for DCGi2 (%) 94.4 0.8 92.9 95.6 1No faecal excretion was detected for the 9 litters between 21 and 25 d. 2Mean increase for the dry content of the gut (DCGi) from 21 to 25 d old (g DM/kit)=1.75 g (see Table 3). *Mean calculated on 9 litters of 8 kits. VC: Variation coe§cient (standard deviation/mean) expressed in %. from 21 to 25 d of age (g DM/kit) was 1.75 g, and was considered as non-digested to calculate the digestibility coe§cient. During digesta sampling, we found some hard faecal material in the distal colon for 7 out of 9 kits at 21 or 25 d of age. These hard faeces were not retrieved on the collection sieve, but some faecal material was found in the stomach in 2 kits at 21 d. This suggested that the young rabbit would still practice coprophagy at 3 wk of age, under our controlled milking procedure with a separate housing for doe and litter.

Milk intake and digestion The chemical composition of the milk was as classically reported (Lebas, 1971; Maertens et al., 2006), with a dry matter level of 260 g/kg, ash content of 85 g/kg, nitrogen concentration of 66.2 g N/kg DM corresponding to 422 g/kg DM of crude protein (using a conversion coe§cient of 6.38), and a crude fat content of 424 g/kg DM. Therefore, the sum of protein and lipid corresponded to almost 85% of the milk composition. The crude energy concentration was calculated from the chemical composition and literature data (Maertens et al., 2006) and estimated at 28.85 Kj/kg DM, corresponding to 7.50 Kj/kg. The milk intake averaged 30 g/d and per kit (7.9 g DM/d kit, Table 4), which corresponded to a total milk production of 240 g/d (8 kits per litter). This ranged within the value reported in the literature for European commercial doe lines (Maertens et al., 2006; Savietto et al., 2014). From the dry matter milk intake and increment in digesta content, we calculated that the corrected digestibility of the milk reached 94.4%, with a minimum value of 92.9% and a maximum at 95.6%, thus corresponding to a relatively low variability (0.8%). In contrast, for young rabbits (between 21 and 26 d old) fed milk and solid feed, Parigi-Bini et al. (1991) estimated (multiple regression) that milk was totally digested (100.2%), while the solid feed was digested to 64.4%. The urine excretion averaged 5.1 mL/d kit and had 50% variability among litters, although the DM urine excretion had a 11% variability and averaged 1.2 g DM/d/kit (Table 5). Besides, no signiﬁcant relationship was found between the urine volume (21-25 d) and the milk intake (R²=0.12, n=9, P=0.75), while weight gain was logically correlated

Table 5: Urine excretion of the exclusively milk-fed litters (group S), from 21 to 25 d of age, and dry matter retention of the milk1. Mean* VC (%) Min Max Period 21 to 25 d old Urine excretion (mL/d kit) 5.1 48 1.7 10.2 Urine excretion (g dry matter/d kit) 1.2 11 1.0 1.4 Dry matter retention (corrected for DCGi2) (%) 79.5 2.8 77.1 83.1 1No faecal excretion was detected for the 9 litters between 21 and 25 d. 2Mean increase for the dry content of the gut (DCGi) from 21 to 25 d old (g dry matter/kit)= 1.75g (see Table 3). *Mean calculated on 8 litters of 8 kits (for one litter, the urine excretion was unavailable). VC: Variation coe§cient (standard deviation/ mean) expressed in %.

World Rabbit Sci. 26: 269-276 273 GIDENNE et al.

Table 6: Digestibility and retention of nitrogen from the milk for the 3-wk-old rabbit exclusively milk-fed (group S). Mean* VC (%) Min Max Nitrogen balance, period 21 to 25 d Nitrogen intake from the milk*1 (g/d kit) 0.53 10.4 0.49 0.65 Nitrogen excretion from urine*1 (g/d kit) 0.06 33.5 0.04 0.10 Nitrogen digestion and retention Corrected digestibility of nitrogen** (%) 94.1 0.6 93.5 95.2 Nitrogen retention coe§cient (%) 87.8 5.5 79.2 93.0 Corrected nitrogen retention coe§cient (%)** 81.9 5.5 72.7 88.1 Nitrogen retained (g/d kit) (corrected)** 0.44 15.9 0.35 0.58 N retained: N digestible coe§cient** (%) 87.0 5.9 77.8 92.6 Metabolisable protein2 of the milk (g/kg) 90 7 80 97 Metabolisable protein2 of the milk (g/kg dry) 346 7 307 372 *Mean calculated on 8 litters of 8 kits (for one litter, the urine excretion was unavailable). **Correction for the increase in the gut nitrogen content, from 21 to 25 d of age= 0.13 g N/kit. 1No faecal excretion was detected for the 8 litters between 21 and 25 d. 2Nitrogen was converted in protein using a coe§cient of 6.38. VC: Variation coe§cient (standard deviation/mean) expressed in %.

with milk intake (R²=0.70, n=9, P<0.010). When accounting for the urine excretion, the corrected DM retention coe§cient of the milk was 79.5%, with a 2.8% variation. As the quantity of nitrogen excreted in urine was low (0.06 g/d/kits), the corrected nitrogen digestibility of the milk reached 94% (Table 6) and varied very little (0.6%). The corrected nitrogen retention coe§cient for milk was much lower (82%) and more variable, and the nitrogen retained (corrected) reached 0.44 g/d kit. In comparison, Parigi-Bini et al. (1991) calculated higher coe§cients, with a nitrogen digestion of 98.6%, and a nitrogen retention of 94%. Accordingly, we calculated that the content in metabolisable protein for the milk was 90 g/kg (fresh), with a variation coe§cient of 7%. We only estimated the energy digestion and retention, as the mass of digesta and milk samples were too low to perform precise calorimetric measurements. Thus, energy content of milk was assessed from the literature (Maertens et al., 2006), while we assumed that energy content was similar to that of faeces (Gallois et al., 2008). Accordingly,

Table 7: Estimation of the digestion and retention of energy from the milk, for the 3-wk-old rabbit exclusively milk-fed (group S). Mean* VC (%) Min Max Energy balance, period 21 to 25 d Energy intake from the milk*1 (kJ/d kit) 233 10 211 284 Variation in gut energy content, between 25 and 21 d of age (kJ/kit) 26 52.0 1.8 10.8 Energy excretion from urine*1 (kJ/d kit) 3.2 34 1.9 5.1 Energy digestion and retention Corrected digestibility of energy** (%) 97.2 1.5 94.8 98.5 Energy retention coe§cient (%) 98.6 0.6 97.6 99.3 Corrected energy retention coeÄ.** (%) 95.8 0.8 94.6 97.0 Energy retained (kJ/d kit) (corrected)** 223 11.4 199 276 Energy retained: digestible energy coeÄ.** (%) 98.5 0.6 97.6 99.2 Metabolisable energy of the milk (MJ/kg) 7.00 0.8 6.91 7.09 Metabolisable energy of the milk (MJ/kg dry matter) 26.94 0.8 26.58 27.26 *Mean calculated on 8 litters of 8 kits (for one litter, the urine excretion was unavailable). **Correction for the variation in the content of energy in the gut, between 21 and 25 d of age =26.1 kJ/kit. 1No faecal excretion was detected for the 8 litters between 21 and 25 d. VC: Variation coe§cient (standard deviation/mean) expressed in %.

274 World Rabbit Sci. 26: 269-276 MILK DIGESTION IN THE YOUNG RABBIT the corrected energy digestibility of the milk was very high 97% (Table 7), as well as the corrected energy retention coe§cient of the milk (95.8%); hence, the energy retained (corrected) reached 223 kJ/d kit. In comparison, Parigi- Bini et al. (1991) estimated a slightly higher coe§cient for milk energy digestion (99.7%), but a much lower energy retention (86%). It should be noted that the amount of energy excreted in urine (3.2 kJ/d kit) was 50% lower than energy “retained” from digesta increment (6.5 kJ/d kit). Therefore, we suspected that the energy concentration in urine may be underestimated, as it was calculated according to the equation of Parigi-Bini and Cesselli (1976) adapted to growing rabbit at 2 kg live weight. In perspective, energy concentration of urine and digesta (and milk) should be measured in several samples to improve the precision of the energy balance in milk-fed young rabbit. However, we estimated that the content in metabolisable energy for the milk was 7.00 MJ/kg (fresh), with a variation coe§cient of 0.8%.

CONCLUSION

Our study presented original results about milk digestion by the young rabbit. Our methodology took into account the increment of digesta content in the 3-wk-old rabbit. Initially recognised as fully digested, we found that after correction for digesta increment, the milk DM digestion however reached 94% and DM retention was 90%. The milk nitrogen retention was relatively high (82%), corresponding to a metabolisable protein concentration of 90 g/kg. In perspective, our first results on milk digestion could be confirmed by increasing the sample size (involving a higher number of sacrificed kits) to allow further chemical analysis and address the digestion of lipid. The milk digestion here was measured in 3-wk-old rabbits fed exclusively with milk, and this will allow us to calculate digestibility coe§cient for feed before weaning, taking into account this value. However, we cannot exclude digestive interactions between solid feed and milk for rabbits beginning to consume pellets and starting caecotrophy (between 3 and 5 wk of age).

Acknowledgments: The authors thank INRA PHASE division for the ﬁnancial support. The authors would also like to thank the technicians involved in the experiment at the INRA UE PECTOUL (Patrick Aymard, Jacques De Dapper & Jean De Dapper) and in the GenPhySE laboratory (Véronique Tartié).

REFERENCES

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Savietto D., Cervera C., Blas E., Baselga M., Larsen T., Udert K.M., Larsen T.A., Biebow M., Gujer W.P. 2003. Friggens N.C., Pascual J.J. 2014. Environmental Urea hydrolysis and precipitation dynamics in a urine- sensitivity diÄers between rabbit lines selected for collecting system. Water Res., 37: 2571-2582. reproductive intensity and longevity. Animal, 7: 1969-1977. https://doi.org/10.1016/S0043-1354(03)00065-4 https://doi.org/10.1017/S175173111300178X Zhang Y.K., Cui H.X., Sun D.F., Liu L.H., Xu X.R. 2018. EÄects of Ubilla E., Rebollar P.G., Pazo D., Esquiﬁno A., Alvariño J.M.R. doe-litter separation on intestinal bacteria, immune response 2000. EÄects of doe–litter separation on endocrinological and and morphology of suckling rabbits. World Rabbit Sci., 26: productivity variables in lactating rabbits. Livest. Prod. Sci., 67: 71-79. https://doi.org/10.4995/wrs.2018.5917 67-74. https://doi.org/10.1016/S0301-6226(00)00196-2

276 World Rabbit Sci. 26: 269-276 W orld World Rabbit Sci. 2018, 26: 277-285 R abbit doi:10.4995/wrs.2018.10182 Science © WRSA, UPV, 2003

PERFORMANCE, HAEMATO-BIOCHEMICAL INDICES AND ANTIOXIDANT STATUS OF GROWING RABBITS FED ON DIETS SUPPLEMENTED WITH MUCUNA PRURIENS LEAF MEAL OLORUNTOLA, O.D.*, AYODELE, S.O.†, ADEYEYE, S.A.‡, AGBEDE, J.O.§

*Department of Animal Science. Adekunle Ajasin University. AKUNGBA-AKOKO. Nigeria. †Department of Agricultural Technology. The Federal Polytechnic. ADO EKITI. Nigeria. ‡Department of Animal Health and Production. The Federal College of Agriculture. AKURE. Nigeria. §Department of Animal Production and Health. The Federal University of Technology, AKURE, Nigeria.

Abstract: The effects of dietary Mucuna pruriens leaf meal (MLM) supplementation on rabbits’ performance, haemato-biochemical indices and antioxidant status outside their thermal neutrality zone (21 to 25°C) were evaluated. One hundred and twenty 35-d old crossbreed (Chinchilla×New Zealand) rabbits weighing 694±5 g were allotted to 4 treatments (30 rabbits/treatment; 3 rabbits/replicate). A basal diet (crude protein: 16.9%, crude fibre: 17.6%, digestible energy: 2671 kcal/kg) was divided into 4 equal portions i.e. diets 1, 2, 3 and 4, supplemented with 0, 4, 8 and 12 g MLM/kg, respectively, and pelleted. The average body weight in rabbits fed on diets 3 and 4 was higher compared to those fed on diet 1 (control) at 91 d of age (+228 and +262 g, respectively; P=0.01). Within 35 to 91 d, the average daily weight gain in rabbits fed on diets 3 and 4 was higher compared to those fed on the control diet (+4.1and +4.8 g/d, respectively; P=0.01). The dressing-out percentage of rabbits fed on diets 3 and 4 increased (P=0.05) compared to those fed the control diet. At 63 d and 91 d of age, the white blood cell level of rabbits fed on diet 4 increased significantly compared to those fed the control diet (+5.05×109 and +5.32×109/L, respectively). At 63 and 91 d of age, the cholesterol level of rabbits fed on diets 3 (–1.0 and –1.16 mmol/L, respectively) and 4 (–1.10 and –1.21 mmol/L, respectively), were significantly lower compared to those fed on the control diet. The aspartate aminotransferase (AST) concentration in rabbits fed on diet 4 was reduced compared to those on control diet at 63 d of age (–33.68 IU/L; P=0.02). At 63 d and 91 d of age, compared to control, the activities of glutathione peroxidase in rabbits fed on diets 3 (+35.77 and +49.09 mg protein, respectively) and 4 (+54.52 and +55.02 mg protein, respectively) increased significantly, while catalase activities in rabbits fed diet 4 (+217.7 and +209.5 mg/g, respectively) also increased significantly. It could be concluded that dietary MLM supplementation enhanced the rabbits’ performance, reduced serum AST and cholesterol and improved the antioxidant status. Key Words: Mucuna pruriens, rabbits, performance, antioxidant status, slaughter traits, health status.

INTRODUCTION

Changes in climate and rising ambient temperatures in various African and sub-Saharan regions have been reported as one of the causes of economic losses to livestock farmers (Tawfeek et al., 2014). Ambient temperature outside the thermal neutrality zone (21 to 25°C) for rabbits represents a heat-stress condition and high heat stress could cause a reduction in growth performance, reproductive rates and meat quality (Marai et al., 2002; Attia et al., 2017; El-Desoky et al., 2017 , Marco-Jiménez et al., 2017). Heat stress also promotes increased free radical generation, which leads to the formation of reactive oxygen species (ROS) and induced cellular oxidative stress (Tawfeek et al., 2014). Excessive free radical production in the animal’s body can produce a negative eÄect on biological activities (Halliwell and Gutteridge, 1989). Dietary manipulation has been identiﬁed as the most aÄordable method of alleviating the negative eÄect of high ambient temperature in the tropics, as the high cost of cooling animal pens is unaÄordable

Correspondence: O.D. Oloruntola, [email protected]. Received May 2018 - Accepted August 2018. https://doi.org/10.4995/wrs.2018.10182

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for farmers (Konca et al., 2009). Therefore, the use of diets rich in natural antioxidants as a means of removing excessive free radicals from the animal’s body and consequent alleviation of high-temperature negative eÄects on animal production is becoming popular (Liu et al., 2010; Kone et al., 2016; Zeweil and Elgindy, 2016; Li et al., 2018). Mucuna pruriens belongs to the family Fabaceae. The plant is among the under-utilised wild legumes and is widespread in the world’s tropical and subtropical regions. Mucuna has bioactive compounds which make it useful for activity against bacteria (Kumar et al., 2009). Mucuna pruriens has been reported to have antivenom, antidiabetic, antioxidant and neuroprotective properties (Kumar and Muthu, 2010; Suresh et al., 2013; Yadav et al., 2017). However, Mucuna pruriens leaf meal (MLM), like most other leaf meals, contains some phytochemicals (Duke, 1995; Yadav et al., 2017) which may alter blood proﬁles of animals fed diets containing MLM (Ansari et al., 2012, El-Gindy and Zeweil, 2017). Studies on the eÄect of dietary Mucuna pruriens leaf meal on performance, blood proﬁle and anti-oxidative status of growing rabbits are rare. Therefore, this work investigated the performance, blood indices and antioxidant status of growing rabbits fed on diets supplemented with 0, 4, 8 or 12 g of MLM/kg.

MATERIALS AND METHODS

Experimental site The experiment was carried out at the Rabbit Unit, Agricultural Technology Department Teaching and Research Farm (ATDT&RF), The Federal Polytechnic, Ado Ekiti, Nigeria from October to December in 2017. The rabbit house had a mean temperature and mean relative humidity of 29.6°C and 77.5%, respectively.

Feeds, chemical analyses, animals and experimental design Mucuna pruriens leaves harvested fresh within the premises of ATDT&RF, The Federal Polytechnic, Ado Ekiti, Nigeria were chopped, air-dried under the shed for 7 d, milled with a 2 mm hammer mill to MLM and analysed for tannin (Van-Burden and Robinson, 1981), terpenoid (Ferguson, 1956), cardiac glycoside (Ferguson, 1956), saponin (Shad et al., 2013), steroid (Edeoga et al., 2005), alkaloid (Harborne, 1973), flavonoid (Shad et al., 2013), Ferric reducing antioxidant property (FRAP) (Pulido et al., 2002) and 2, 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) (Gyamfi et al., 1999) (Table 1). A basal diet was formulated to support optimal rabbit growth (de Blas and Mateos, 2010) (Table 2). The basal diet was divided into 4 equal portions and designated diets 1, 2, 3 and 4. Diets 1 to 4 contained 0, 4, 8 and 12 g MLM/kg, respectively. The diets were pelleted to 4 mm diameter and 8 mm in length and then analysed for chemical composition. AOAC (1990) procedures were used to determine crude protein (988.05), crude fibre (962.09) and acid detergent fibre (973.18) in diets. Neutral detergent fibre and acid detergent lignin level were determined as described by Mertens (2002) and Robertson and Van Soest (1981), respectively. Gross energy was determined Table 1: Chemical constituents of Mucuna pruriens using a combustion calorimeter (Model: e2k combustion leaf meal. calorimeter, www.cal2k.com). Digestible energy was Parameters (mg/g) Quantity estimated at 0.65 of the gross energy (Xiccato and Phytochemicals Trocino, 2010). The recommendations and guidelines Tannin 3.08±0.01 for applied nutrition and experiments in rabbits were Terpenoid 12.40±0.04 followed in management of the rabbits (Fernández- Cardiac glycoside 9.91±0.01 Carmona et al., 2005). One hundred and twenty 35-d Saponin 30.72±0.52 old crossbreed (Chinchilla×New Zealand) weaner rabbits Steroid 9.51±0.01 of equal sexes and weighing 964±5 g were allotted to Alkaloid 12.12±0.38 4 dietary treatments on (30 rabbits/treatment; 3 rabbits/ Flavonoid 91.15±0.33 replicate). The rabbits were housed in wire meshed Antioxidant parameters cages, accommodated in a well-ventilated pen, oÄered FRAP 38.35±0.04 water and experimental diet ad libitum and the pen was DPPH (%) 21.17±0.52 cleaned and disinfected daily for 56 d of the experiment. FRAP: Ferric reducing antioxidant property; DPPH: 2, 2-diphenyl-1-picrylhydrazyl hydrate.

278 World Rabbit Sci. 26: 277-285 MUCUNA LEAF MEAL AS PHYTOGENIC RABBIT FEED SUPPLEMENT

Experimental procedure Table 2: Ingredients and chemical composition (%) of the basal diet. The rabbits and feed were weighed at 7-d intervals after the beginning of the experiment and the average Ingredients daily weight gain (ADWG), average daily feed intake Maize 8.00 (ADFI) and the feed conversion ratio (FCR) were Wheat oÄal 8.00 Soybean meal 16.1 calculated, respectively, for post-weaning period (35- Maize husk 22.0 63 d), finishing period (64-91 d) and whole fattening Cassava peels 22.0 period (35-91 d). Blood samples were collected from Brewers dried grain 21.7 10 rabbits/experimental group at 63 and 91 d of age from Bone meal 1.10 overnight fasted selected rabbits, for determination of Premix 0.25 haematological indices, serum biochemical indices and Methionine 0.20 serum concentration of lipid peroxidase (LPx), superoxide Lysine 0.10 dismutase (SOD), glutathione peroxidase (GPx) and Salt 0.25 catalase (CAT). The method described by Burnett et al. Vegetable oil 0.30 (2003) was used for the blood collection. The dorsal Chemical composition surface of the pinna was swabbed with cotton wool Crude protein 16.88 impregnated into 70% isopropanol and then rubbed with Crude fibre 17.59 petroleum jelly. A brooding bulb was placed at a height Neutral detergent fibre 39.17 of approximately 45 cm above the ear to supply heat to Acid detergent fibre 17.54 enhance the ear vein dilation. Thereafter, about 10 mL of Acid detergent lignin 3.68 blood were collected from the prominent ear vein of the Gross energy (kcal/kg) 4108 rabbits using a nineteen gauge 3.8 cm needle. The blood Digestible energy (kcal/kg) 2671 samples meant for serum antioxidant enzymes/serum biochemical and haematological indices determination were dispensed into plain (no anticoagulant) and anticoagulant (ethylenediaminetetra-acetic acid) bottles, respectively. At 91 d, after the blood sample collection, one rabbit was selected from each replicate, weighed, tagged, stunned and euthanised as described by Blasco et al. (1993). The skin, legs, head, and intestines were removed and the dressing- out percentage (DP) was then calculated. The weights of major internal organs (liver, heart, lung, kidney, and spleen) were determined separately and expressed as the percentage of slaughter weight.

Blood analysis The haematological indices (WBC, white blood cells; RBC, red blood cells; Hbc, haemoglobin concentration; and PCV, packed cell volume) were determined on the day of collection by Shenzhen Mindray Auto Haematology Analyzer (Model Bc-3200, Shenzhen Mindray Biomedical Electronics Co. Hamburg 20537, Germany). The serum biochemical (TP, total protein; creatinine; cholesterol; AST, aspartate aminotransferase; and bilirubin) were determined with a Reﬂectron® Plus 8C79 (Roche Diagnostic, GonbH Mannheim, Germany), using Reﬂectron kits. Concentrations of serum GPx and SOD activity were determined as described by Rotruck et al. (1973) and Misra and Fridovich (1972), respectively. The method described by Aebi (1974) was used to determine CAT activity, while lipid peroxidase was determined with the Ohkawa et al. (1979) method.

Statistical analysis

A completely randomised design with the following model: Xij=µ+αi+Єij was adopted in this study. Where Xij=any of the th response variables; µ=the overall mean; αi=eÄect of the i treatment (i=diets 1, 2, 3 and 4) and Єij=random error due to experimentation. All data collected in this study were subjected to analysis of variance using SPSS statistical software package 2011, version 20. The diÄerences between treatment groups were determined by Duncan’s multiple range, while statistical signiﬁcance was assessed at P<0.05.

World Rabbit Sci. 26: 277-285 279 OLORUNTOLA et al.

RESULTS

Growth performance and carcass traits of rabbits The eÄects of dietary MLM supplementation on growth performance of growing rabbits are shown in Table 3. At post- weaning period (days 35 to 63), MLM supplementation did not significantly (P>0.05) influence the rabbits’ average body weight (ABW), ADWG, ADFI, and FCR. At the finishing period (days 64 to 91), only the ABW at 91 d was aÄected by the MLM supplementation, such that ABW in rabbits fed on diets 3 and 4 was significantly higher (+228 and +262 g, respectively; P=0.01) when compared to ABW recorded for rabbits fed on diet 1 (control). Over the entire feeding trial (days 35 to 91), compared to control, the average body weight gain (ABWG) was higher in the rabbits fed diet 3 and diet 4 (+231 and +267 g, respectively; P<0.01). Similarly, the ADWG in rabbits fed on diets 3 and diet 4 was significantly higher when compared to those fed on the control diet (+4.1 and +4.8 g/d, respectively; P=0.01). The ADFI were not aÄected by the MLM supplementation, while FCR values tend (P=0.09) to be reduced with MLM supplementation. Table 4 shows the eÄect of dietary MLM supplementation on carcass and relative internal organs of growing rabbits at day 91 of age. The slaughter weight (SW) of rabbits fed on diets 3 and 4, significantly increased compared to the rabbits fed on the control diet (+228 and +262 g, respectively; P=0.04). Similarly, the DP of rabbits fed on diets 3 and 4 was higher compared to the rabbits fed on the control diet (+4.4 and +5.0 percentage points, respectively; P=0.05). MLM supplementation did not significantly (P>0.05) influence the relative weights of the liver, heart, lung, kidney, and spleen.

Haematological indices The eÄect of including MLM in rabbit diets on haematological indices is presented in Table 5. At 63 d of age, the WBC level of rabbits fed on diet 4 was significantly higher compared with the rabbits fed on the control diet (+5.05×109/L; P=0.05). At 91 d of age, the WBC level of rabbits fed on diet 4 significantly higher when compared to those rabbits fed on the control diet (+5.32×109/L; P=0.05). However, the RBC, Hbc, and PCV were not (P>0.05) influenced by the MLM supplementation at 63 d and 91 d of age.

Table 3: EÄects of Mucuna pruriens leaf meal (MLM) supplementation on performance of growing rabbits. Diet 1 Diet 2 Diet 3 Diet 4 0 g MLM/kg 4 g MLM/kg 8 g MLM/kg 12 g MLM/kg SEM P-value No. of rabbits 30 30 30 30 Post-weaning period (35-63 d) Average body weight at 35 d (g) 697 694 694 691 5 0.99 Average body weight at 63 d (g) 1346 1438 1489 1536 29 0.09 Average daily weight gain (g/d) 23.2 26.6 28.4 30.1 1.0 0.07 Average daily feed intake (g/d) 62.2 64.7 63.5 63.6 1.5 0.96 Feed conversion ratio 2.70 2.44 2.24 2.12 0.09 0.11 Finishing period (64-91 d) Average body weight at 91 d (g) 2093a 2151a 2321b 2354b 37 0.01 Average daily weight gain (g/d) 26.7 25.5 29.7 29.2 1.0 0.45 Average daily feed intake (g/d) 75.1 75.8 76.8 78.0 0.5 0.26 Feed conversion ratio 2.89 2.99 2.58 2.70 0.10 0.52 Whole fattening period (35-91 d) Average body weight gain (g) 1396a 1458a 1627b 1663b 39 0.01 Average daily weight gain (g/d) 24.9a 26.0a 29.1b 29.7b 0.7 0.01 Average daily feed intake (g/d) 68.7 70.3 70.2 70.8 0.8 0.83 Feed conversion ratio 2.76 2.70 2.41 2.38 0.06 0.09 Means within a row with diÄerent letters are signiﬁcantly diÄerent (P<0.05); SEM: Standard error of the mean.

280 World Rabbit Sci. 26: 277-285 MUCUNA LEAF MEAL AS PHYTOGENIC RABBIT FEED SUPPLEMENT

Table 4: EÄects of Mucuna pruriens leaf meal (MLM) supplementation on carcass and relative internal organs (% slaughter weight) of growing rabbits at 91 d of age. Diet 1 Diet 2 Diet 3 Diet 4 0 g MLM/kg 4 g MLM/kg 8 g MLM/kg 12 g MLM/kg SEM P-value No. of rabbits 10 10 10 10 Slaughter weight (g) 1977a 2036a 2206b 2239b 37 0.04 Dressing-out percentage 57.5a 58.7a 61.9b 62.5b 0.7 0.05 Liver 2.69 2.69 2.72 2.58 0.14 0.96 Heart 0.25 0.23 0.24 0.24 0.02 0.98 Lung 0.42 0.42 0.42 0.42 0.02 0.97 Kidney 0.45 0.46 0.44 0.44 0.02 0.99 Spleen 0.03 0.03 0.04 0.04 0.01 0.98 Means within a row with diÄerent letters are signiﬁcantly diÄerent (P<0.05); SEM: Standard error of the mean; Dressing-out percentage=Hot carcass weight/slaughter weight×100.

Serum metabolites The eÄect of dietary MLM supplementation on serum metabolites of growing rabbits is shown in Table 6. At 63 d, the cholesterol level of rabbits fed on diets 3 and 4, was lower compared to those rabbits fed on the control diet (–1.0 and –1.1 mmol/L, respectively; P=0.05). The AST concentration in rabbits fed on diet 4 was signiﬁcantly lower compared to those rabbits on the control diet (–33.68 IU/L; P=0.02). At 91 d, the serum cholesterol level was lower in rabbits fed on diets 3 and 4 when compared to those rabbits fed the control diet (–1.16 and –1.21 mmol/L, respectively; P=0.03). The TP, creatinine, and bilirubin were not signiﬁcantly (P>0.05) aÄected by MLM supplementation at 63 d and 91 d of age.

Activities of antioxidant enzymes The eÄect of including MLM in rabbit diets on activities of antioxidant enzymes are presented in Table 7. At 63 d, compared with the control, the activities of GPx in rabbits fed on diets 3 (+35.77 mg protein) and 4 (+54.52 mg protein) significantly (P=0.01) increased, while CAT activities in rabbits fed diet 4 (+217.66 mg/g) increased significantly (P=0.02). At 91 d of age, compared with the control, GPx activities in rabbits fed diets 3 (+49.09 mg protein) and 4 (+55.02 mg protein) increased significantly (P=0.03). The lipid peroxidase and superoxide dismutase were not (P>0.05) influenced by MLM supplementation at 63 d and 91 d of age. The CAT activities in rabbits fed diet 4 (+209.46 mg/g) significantly (P=0.03) increased compared with the control.

Table 5: EÄects of Mucuna pruriens leaf meal (MLM) supplementation on haematological indices of growing rabbits. Diet 1 Diet 2 Diet 3 Diet 4 0 g MLM/kg 4 g MLM/kg 8 g MLM/kg 12 g MLM/kg SEM P-value No. of rabbits 10 10 10 10 Observations at 63 d old White blood cells (×109/L) 7.04a 9.71ab 9.24ab 12.09b 0.70 0.05 Red blood cells (×1012/L) 3.88 4.98 5.29 5.83 0.42 0.45 Haemoglobin conc. (g/dL) 14.52 13.98 14.20 13.38 0.60 0.95 Packed cell volume (%) 45.00 44.00 46.33 43.00 1.67 0.93 Observations at 91 d old White blood cells (×109/L) 7.10a 9.98ab 9.84ab 12.42b 0.69 0.02 Red blood cells (×1012/L) 4.11 5.49 5.69 6.28 0.42 0.37 Haemoglobin conc. (g/dL) 14.67 15.00 15.20 15.38 0.48 0.97 Packed cell volume (%) 44.33 46.67 49.33 50.68 1.44 0.46 Means within a row with diÄerent letters are signiﬁcantly diÄerent (P<0.05); SEM: Standard error of the mean.

World Rabbit Sci. 26: 277-285 281 OLORUNTOLA et al.

Table 6: EÄects of Mucuna pruriens leaf meal (MLM) supplementation on serum metabolites of growing rabbits. Diet 1 Diet 2 Diet 3 Diet 4 0 g MLM/kg 4 g MLM/kg 8 g MLM/kg 12 g MLM/kg SEM P-value No. of rabbits 10 10 10 10 Observations at 63 d old Total protein (g/dL) 7.09 7.79 7.85 7.80 0.53 0.96 Creatinine (µmol/L) 100.40 95.85 93.87 99.01 6.14 0.99 Cholesterol (mmol/L) 2.21b 1.51ab 1.19a 1.11a 0.17 0.05 Aspertate aminotransferase (IU/L) 95.27c 84.09bc 67.32ab 61.59a 4.90 0.02 Bilirubin total (µmol/L) 9.20 9.35 9.19 9.06 0.74 0.94 Observations at 91 d old Total protein (g/dL) 6.89 7.84 8.08 8.10 0.56 0.89 Creatinine (µmol/L) 98.24 93.17 96.47 100.01 6.88 0.99 Cholesterol (mmol/L) 2.24b 1.47ab 1.08a 1.03a 0.18 0.03 Aspertate aminotransferase (IU/L) 91.34 90.09 80.68 82.21 3.01 0.54 Bilirubin total (µmol/L) 9.69 9.46 8.92 9.01 0.79 0.98 Means within a row with diÄerent letters are signiﬁcantly diÄerent (P<0.05); SEM: Standard error of the mean.

DISCUSSION

The consequences of exposure of rabbits to ambient temperature outside their thermal neutrality zone (21 to 25°C) are impaired growth, feed intake and utilisation (Brewer and Cruise, 1994; Marai et al., 2002), and heat stress is also one of the factors causing oxidative stress in the tropics (Kumar et al., 2011). Previous studies had identified the intestinal mucosa damage and increased muscle protein hydrolysis being caused by temperature-induced free radicals as the possible cause of thermal stress-induced impaired growth performance (Yuan et al., 2007; Jimoh et al., 2017, Li et al., 2018). In this study, dietary supplementation of rabbit diets with MLM at 8 or 12 g/kg caused increased average body weight gain and average daily weight gain of the rabbits when compared to the control. This suggests that dietary MLM supplementation of diets could serve as a growth performance enhancer in rabbits raised under tropical high ambient temperature. The phenolic compounds present in MLM as detected in this study might have demonstrated high antioxidant and free radical scavenging activities, which help to maintain the intestinal mucosa integrity, thereby promoting growth performance (Yuan et al., 2007). Furthermore, the use of leaf meal as the phytogenic growth promoter in the animal is on the increase (Valenzuela-Grijalva et al., 2017); in particular, the antioxidant and anti-inflammatory activity of some phytochemicals in the leaf meal is of great interest due to their ability to suppress the metabolism of inflammatory prostaglandins. Some of the phytochemicals (e.g. flavonoids and terpenoids) detected in MLM in this study

Table 7: EÄects of Mucuna pruriens leaf meal (MLM) supplementation on antioxidant enzyme activities of growing rabbits. Diet 1 Diet 2 Diet 3 Diet 4 0 g MLM/kg 4 g MLM/kg 8 g MLM/kg 12 g MLM/kg SEM P-value No. of rabbits 10 10 10 10 Observations at 63 d old Lipid peroxidase (nmol/mol x106) 15.18 16.93 16.64 16.06 1.08 0.96 Superoxide dismutase (m/mg) 81.33 92.00 91.33 94.66 2.73 0.37 Glutathione peroxidase (mg protein) 84.8a 99.6ab 120.6bc 139.3c 7.2 0.01 Catalase (mg/g) 379.8a 500.3ab 499.7ab 597.5b 27.9 0.02 Observations at 91 d old Lipid peroxidase (nmol/mol x106) 14.88 16.36 15.94 15.68 0.90 0.96 Superoxide dismutase (m/mg) 84.33 92.00 92.67 94.33 2.64 0.61 Glutathione peroxidase (mg protein) 90.3a 105.3ab 139.4bc 145.4c 8.5 0.03 Catalase (mg/g) 388.8a 506.0ab 515.1ab 598.2b 27.8 0.03 Means within a row with diÄerent letters are signiﬁcantly diÄerent (P<0.05); SEM: Standard error of the mean.

282 World Rabbit Sci. 26: 277-285 MUCUNA LEAF MEAL AS PHYTOGENIC RABBIT FEED SUPPLEMENT possess anti-inflammatory activity (Muanda et al., 2011). These phytochemicals may over-express antioxidant enzyme, thereby down-regulating the inflammatory process. The reactive oxygen radicals produced during the process of food digestion in the digestive tract can attack the intestinal mucosa surface and thereby aÄect the normal process of nutrient absorption. Therefore, the anti-inflammatory and antioxidant activities in the intestinal mucosa may result in maintenance of healthy gut morphology (Kamel, 2000), improved increased nutrient absorption and enhanced growth (Cardoso et al., 2012) as recorded in this study. Previously, Li et al. (2018) reported improved daily weight gain, average feed intake in rabbits’ whose diets were supplemented with 1 or 5 g/kg Eucommia ulmoides leaves, while Ayodele et al. (2016) reported improved feed conversion ratio in rabbits fed on 5 or 10% alchornea leaf meal inclusion diets. However, further studies are required to study the eÄect of MLM on the morphology of the rabbits’ intestinal mucosa. Phytogenetic additives can produce antioxidant eÄects, which may result in health conditions for the animals and improved growth of target tissues (Valenzuela-Grijalva et al., 2017). In addition, relative weights of the internal organ of animals may increase abnormally in response to the presence of toxins in their diet (Ayodele et al., 2016). In this study, the increased SW and DP observed in rabbits fed on 8 and 12 g/kg MLM supplemented diets suggests that MLM promoted the growth of edible portion of the rabbits more than oÄal, and that some of the phytochemicals present in MLM exert direct or indirect eÄects on animal metabolism, probably by modulating animal metabolism in favour of increasing the development of edible portions of the rabbits (Jiang et al., 2007; Devi et al., 2015; Valenzuela-Grijalva et al., 2017). The stability of the rabbits’ relative weight of internal organs (liver, heart, lung, kidney, and spleen) across the varying levels of dietary MLM supplementation as observed in this study also suggests the uncompromised health status of the rabbits. Oxidative stress caused as a result of high ambient temperature could result in pathological changes such as tissue damage and adverse eÄects on blood indices (Avellini et al., 1995; Adekonla and Ayo 2009). The stability of blood indices such as RBC, Hbc, and PCV in rabbits across the various dietary treatments in this study is an indication that MLM supplementation supports or does not interfere with normal haemopoiesis processes. However, the observed rise in WBC in rabbits fed MLM supplemented diets, especially 12 g/kg supplemented diet in this study, may be the product of immunostimulatory activities of MLM. The WBC counts possess phagocytic function and biomarkers for immune functions. An immunostimulatory activity in animals is one of the biological activities being associated with phytogenic feed additives (Valenzuela-Grijalva et al., 2017). Similar results were reported in broiler chickens fed 2.5 g/kg Azadirachta indica leaf meal (Ansari et al., 2012) and in rabbits fed 1 g/kg alchornea leaf meal (Oloruntola et al., 2016a). The reduction of serum cholesterol levels in rabbits fed on 8 and 12 g/kg in this study suggests that MLM supplementation interferes with the uptake and catabolism of cholesterol in the rabbits. According to Lording and Friend (1991), decreased uptake of cholesterol or increased loss or cholesterol catabolism is among the causes of hypocholesterolaemia. Phytochemicals in the phytogenic feed additives were reported to exhibit various biological activities that can influence the functions of the intestinal tract (Valenzuela-Grijalva et al., 2017). In particular, saponin, one of the detected phytochemicals in MLM, was linked to the reduction of cholesterol uptake in the gut (Yilkal, 2015). Recently, Oloruntola et al. (2016a,b) recorded reduction of serum cholesterol level in rabbits and broiler chickens fed on diets containing 50 or 100 g/kg alchornea leaf meal. The reduction of serum AST in this study suggests that MLM has protective and therapeutic properties, as abnormally rising AST concentration indicates liver and biliary system disease, skeletal muscle disease, myocardial injury/diseases, haemolytic disorder and haemolysis (Lording and Friend, 1991). This is further supported by the stability of the TP, creatinine and bilirubin concentration in the rabbits fed the various experimental diets supplemented with varying levels of MLM. The antioxidant property of MLM and the presence of some phytochemicals at the therapeutic level (Sies, 1997; Yadav et al., 2017) may be responsible for these observations in this study. High ambient temperature and, consequently, heat stress promote increased free radical generation and lipid oxidation. This condition enhances the generation of free radicals, which if not adequately removed could cause irreversible damage to cells (Tawfeek et al., 2014; Meineri et al., 2017). The importance of antioxidant enzymes, particularly in animals being raised under high ambient temperature, in eliminating the oxygen free radicals being induced by excessive heat has been reported (Masella et al., 2005; Li et al., 2018). Results from this study showed that dietary MLM supplementation could increase the levels of blood GPx and CAT in rabbits, implying that heat stress-induced oxidative destruction could be reduced in rabbits by dietary MLM supplementation. This observation

World Rabbit Sci. 26: 277-285 283 OLORUNTOLA et al.

suggests that dietary MLM is rich in antioxidant and could play a signiﬁcant role in improving the health of rabbits. The antioxidant potential of this supplement used in this study may be related to the concentration of phenolic substances (tannin, ﬂavonoids), ferric reducing antioxidant property and 2,2-diphenyl-1-1-picrylhydrazyl hydrate in MLM.

CONCLUSION The dietary MLM supplementation increased the ﬁnal live weight of rabbits, average daily weight gain and dressing- out percentage, exerted the immunomodulatory eÄect by increasing white blood cells, reduced the serum aspartate aminotransferase and cholesterol and increased serum glutathione peroxidase and catalase.

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W orld World Rabbit Sci. 2018, 26: 287-298 R abbit doi:10.4995/wrs.2018.7644 Science © WRSA, UPV, 2003

ANALYSIS OF THE IMPACT OF CYTOPLASMIC AND MITOCHONDRIAL INHERITANCE ON LITTER SIZE AND CARCASS IN RABBITS NGUYEN N.T.*, BRAJKOVIC V.†, CUBRIC-CURIK V.†, RISTOV S.‡, VEIR Z.§, SZENDRŐ Z.*, NAGY I.*, CURIK I.†

*Kaposvár University, Faculty of Animal Science, Guba S. 40, 7400 KAPOSVÁR, Hungary. †Univeristy of Zagreb, Svetošimunska 25, 10000 ZAGREB, Croatia. ‡Ruđer Bošković Institute, Bijenička cesta 54, 10000 ZAGREB, Croatia . §University of J.J. Strossmayer, Faculty of Medicine, Kralja Petra Svačića 1d, 31000 OSIJEK, Croatia.

Abstract: The effects of mitogenome variation on economically important traits have been reported in a number of domestic animal species. In this study, the first of its kind on rabbits, we have performed the estimation of the contribution of cytoplasmic and D-loop mitochondrial DNA (mtDNA) sequence effects on the litter size and carcass traits in three Pannon rabbit breeds (Pannon Ka, Pannon Large and Pannon White). The observed effects of both estimates, coming from cytoplasmic or D-loop mtDNA variation, were negligible. The most likely explanation for the results obtained is the lack of mitogenome polymorphism in all three populations, as suggested from the analysis performed on the D-loop mtDNA sequence, here assigned to the two most frequent rabbit haplotypes. The extent of potential benefits of the introduction, or alteration, of mitogenome variation in rabbit breeding remains an open question for future research. Key Words: mitochondrial DNA, breeding value, litter size, carcass, rabbit.

INTRODUCTION

Understanding the relationship between DNA sequence and economically important production traits is one of the major challenges in modern animal breeding. The e§ciency of breeding programmes is based on an accurate estimation of genetic parameters such as heritability, genetic correlation and breeding value. Quantitative traits are generally assumed to be under the control of infinitely linked or unlinked genes, each of infinitesimal additive eÄects, and with a considerable influence of non-genetic (environmental) components (Hill, 2010). On the other hand, other genetic factors such as mitogenome variation can also influence inheritance of quantitative traits and consequently have an impact on the estimation of the genetic parameters. The mitochondrial genome (mitogenome) is a closed circular DNA molecule. In rabbits, the length of the molecule is approximately 17245 nucleotides, varying by repeated motifs placed in the control region, encoding for the synthesis of 13 proteins essential for the oxidative phosphorylation system and responsible for regulation of the cellular energy metabolism (Wallace, 1999). Mitogenome is inherited only through the maternal lineage, thus providing a genetic mechanism for cytoplasmic inheritance with a potential impact on the quantitative traits and the estimation of genetic parameters important in animal breeding (Van Vleck, 2000). In a simulation study, Boettcher et al. (1996c) observed that ignoring cytoplasmic eÄects will lead to biased estimates of heritability. Starting with the study of Bell et al. (1985), the cytoplasmic eÄects were most comprehensively covered in cattle populations, analysed as the eÄects of maternal lineages present in individuals (cows) on milk production (Kennedy 1986; Boettcher et al., 1996b; Boettcher et al., 1997), as well as on the growth traits (Pun et al., 2012). The estimated eÄects in all those studies varied from negligible to the impact of up to 5% of phenotypic variation (Gibson

Correspondence: I. Nagy., [email protected]. Received May 2017 - Accepted September 2018. https://doi.org/10.4995/wrs.2018.7644

World Rabbit Sci. 26: 287-298 287 NGUYEN et al.

et al., 1997). Similar models were also performed in poultry (Szwaczkowski et al., 1999) and sheep (Hanford et al., 2003; Snowder et al., 2004). In all those studies, cytoplasmic eÄects were analysed under assumption that maternal lineages derived from the pedigree reﬂect the actual mitogenome polymorphism. However, this is quite a relaxed assumption and, in order to obtain more accurate estimates, quantitative cytoplasmic genetic models have to be further extended to the association analyses of the mitogenome variation (polymorphism), from D-loop mitochondrial DNA (mtDNA) to complete mitogenome, with production traits. Good examples are provided in studies related to cattle (Boettcher et al., 1996a; Mezzadra et al., 2005), poultry (Li et al., 1998; Zhao et al., 2015), swine (Yen et al., 2007; Fernández et al., 2008; Yu et al., 2015, Tsai et al., 2016) and sheep (Chen et al., 2017) populations, as well as to humans (Ruiz-Pesini et al., 2000; Liu et al., 2012), where the association of certain mitogenome polymorphisms with quantitative traits have been analysed. To date, no single analysis has evaluated the impact of the cytoplasmic eÄects, either of maternal lineages or of mitogenome sequence variation, on any traits that are important in rabbit production. Thus, the main objective of this paper was to estimate the eÄects of cytoplasmic and mitochondrial inheritance on litter size traits such as the number of kits born alive (NBA), number of kits born dead (NBD) and the total number of kits born (TNB) in Pannon Ka (PK), Pannon Large (PL) and Pannon White (PW) rabbit breeds, as well as on a carcass trait: the thigh muscle volume (TMV) measured in vivo by computer tomography (CT), but only in PW.

MATERIAL AND METHODS

Data information Data in this study was collected in 3 Pannon breeds; PW (established in 1992), PK (established in 1999) and PL (established in 2004) over a period of 24 yr (1992 to 2016) at the experimental rabbit farm of the Kaposvár University. General development and management of the Pannon rabbit breeds was described by Matics et al. (2014). In Table 1 we present the kindling records, the size of the pedigree, and the number of does and mating bucks included in the analysis. The litter size traits analysed were the number of kits born alive (NBA), number of kits born dead (NBD) and the total number of kits born (TNB). Thigh muscle volume (TMV cm3) was obtained by summing the surface of 11-12 CT scans (Nagy et al., 2013a). Due to the fact that records showed highly unbalanced frequencies for litter kindling, parities were combined into 4 categories (parities 1, 2, 3-10, >10). Descriptive statistics of the examined traits are also presented in Table 1.

Pedigree and molecular analyses Sampling for molecular analysis Prior to molecular analyses, the authors performed a pedigree analysis (deﬁning) maternal lineage in order to avoid examining a lot of animals with the same female founder. To determine maternal (founder) lineages from the pedigree

Table 1: Descriptive statistics for the litter size and carcass traits analyses in Pannon rabbit breeds. Breed Trait Animals in pedigree Does Bucks Records Mean SD Range Pannon NBA 7832 2941 1241 20227 8.54 3.04 1-19 White NBD 7832 2941 1241 20227 0.43 1.10 0-15 TNB 7832 2941 1241 20227 8.97 3.08 1-19 TMV 8001 2000 1017 6724 374.50 42.50 230-570 Pannon NBA 5198 899 1896 13847 9.27 3.12 1-20 Ka NBD 5198 899 1896 13852 0.43 1.14 0-15 TNB 5198 899 1896 13847 9.70 3.16 1-21

Pannon NBA 3714 935 1737 5913 8.69 3.16 1-20 Large NBD 3714 935 1737 5990 0.82 1.68 0-15 TNB 3714 935 1737 5988 9.40 3.32 1-21 NBA: number of kits born alive, NBD: number of kits born dead, TNB:s total number of kits born, TMV: thigh muscle volume (cm3).

288 World Rabbit Sci. 26: 287-298 IMPACT OF CYTOPLASMIC INHERITANCE ON RABBIT PRODUCTION TRAITS and to choose samples for molecular analysis we used mag_sampl module implemented in the MaGelLan 1.0 (Maternal Genealogy Lineage Analyser) software (Ristov et al. 2016; https://github.com/sristov/magellan). Analysis was performed on the previously corrected pedigree utilising the same software. In total, there were 2, 6 and 4 maternal lineages in PK, PL and PW breeds, respectively. The blood of several rabbits per each maternal lineage was further taken for molecular analyses. In this way, we were able to analyse maternal lineage segregation consistency throughout the pedigree. Molecular analysis The DNA was extracted from 31 (27 and 4 from maternal lineages 1-2) PK, 25 (1, 1, 4, 14, 4 and 1 from maternal lineages 1-6) PL and 22 (2, 1, 12 and 7 from maternal lineages) PW blood samples using commercially available NucleoSpin Blood Kit according to manufacturer’s protocol (Macherey-Nagel GmbH & Co. KG, Germany). A 332-base pairs (bp) fragment of the mitochondrial D-loop region was amplified by polymerase chain reaction (PCR) using forward (5'-CACCATCAGCACCCAAAG-3') (Melo-Ferreira et al., 2009) and reverse primers (5'-ATTTAAGAGGAACGTGTGGG-3') (Pierpaoli et al., 1999). PCRs were performed in a 25 μL volume containing 0.2 μM of each primer and using Emerald AMP GT PCR Master Mix (Takara Bio Inc, Japan) according to the manufacturer’s protocol. The amplification reactions were performed on a iCycler (Biorad, Germany), comprised of an initial denaturation at 95°C for five min, 38 cycles of denaturation at 95°C for 45 s, annealing at 52°C for 45 s, extension at 72°C for 1 min and final extension at 72°C for 1 min. After purifying the first round of PCR products, PCR was performed again using the Big Dye-terminator method in a thermal cycler. This PCR products/sequences was then read (sequenced) using a capillary electrophoresis ABI PRISM® 3100-Avant Genetic Analyzer. The sequences were visualised and aligned using MEGA 7 (Kumar et al., 2016), together with sequences taken from the GeneBank. Haplotypes were constructed using DNA Sp 5.10 (Librado and Rozas, 2009) and Median-joining network (Bandelt et al., 1999) was constructed by PopART (Leigh and Bryant, 2015). D-loop mtDNA sequences of Pannon rabbits were deposited in GenBank under the accession numbers KY977609-KY977686. A detailed description of all sequences used in analyses is provided in Supplement Table S1 (available at the end of the document). Maternal pedigree verification (Maternal lineage segregation pedigree consistency) Originally, only 2 haplotypes (D-loop mtDNA sequences), hereafter named H1 and H2, were found in PL and PW, while only H1 was found in PK population. We further imputed (assigned) obtained mtDNA sequences to the maternal lineages (Mag_stat module from MaGelLan) and consequently verified the consistency of maternal lineage segregation through the pedigree (Mag_verif module from MaGelLan). A single conflict was found in PL pedigree, where H2, present in individual 13-20188 (YOB 2013), was not consistent with the pedigree of three sequenced individuals in the same maternal lineage. After the identification, the utilisation of Mag_con_demo module as described in Čačić et al., (2014), and the exclusion of non-consistent individual from the dataset, only H1 was present in the PL breed. Thus, the diÄerence between two haplotypes (H1 vs. H2) for the litter size and carcass traits was tested only in PW breed. The final number of descendants for each lineage and each haplotype (H1 & H2) is shown in Table 2. Calculation of inbreeding coe§cients

Inbreeding coe§cient of dams (FDam) and litters (FLitter) were calculated with ENDOG 4.8 software (Gutiérrez et al., 2010; http://webs.ucm.es/info/prodanim/html/JP_Web.htm#_Endog_3.0:_A). The pedigree ﬁles did not contain all

Table 2: The ﬁnal number of descendants for each lineage and each haplotype. Breed PK PL PW D-loop mt DNA Haplotype H1 H1 Total H1 H2 Maternal lineage 1 2429 94 386 0 386 Maternal lineage 2 638 73 258 0 258 Maternal lineage 3 - 227 1078 1078 0 Maternal lineage 4 - 600 440 440 0 Maternal lineage 5 - 238 - - - Maternal lineage 6 - 35 - - - Total numbers 3067 1267 2162 1518 644 PK, Pannon Ka; PL, Pannon Large; PW, and Pannon White. World Rabbit Sci. 26: 287-298 289 NGUYEN et al.

progeny of the does presented in the data set. Thus, before calculation of litter inbreeding coe§cients, the dummy progeny was created according to the unique combinations of their parents (does and related mating bucks) and then litter inbreeding coe§cients were calculated.

Quantitative genetic analyses To analyse the impact of cytoplasmic and D-loop mtDNA eÄects on the analysed traits, we employed 10 diÄerent models, described in detail in Table 3. The ﬁrst 7 models (see Table 3) referred to the litter size traits. All these models had the same ﬁxed eÄects known to have impact on their variability (Nagy et al., 2013a, Nagy et al.,

2013b). Thus, as a ﬁxed eÄect we modelled: parity (4), year-month (101 in PL, 246 in PW and 185 in PK), F Dam

and FLitter. In the first 7 models, permanent environment and additive genetic eÄects were treated as random eÄects while models were diÄerent due to the presence/absence of dam or sire or both cytoplasmic or D-loop mtDNA eÄects, all treated as random eÄects. Here, in addition to the models with maternal lineage of dam (does) eÄect, which is a classical approach used in a large number of studies (Boettcher et al., 1996; Boettcher et al., 1997; Snowder et al., 2004), we also modelled the maternal lineage of sire (bucks) eÄect. This decision was based on the established evidence that certain mitogenome mutations have strong impact on human male fertility (Ruiz- Pesini et al., 2000; John et al., 2005) and, consequently, can aÄect the litter size. We applied models 5, 6 and 7 to estimate the variance of the contribution of the diÄerence between H1 and H2, applicable only in PW breed. The last three models (see Table 3) referred to the thigh muscle volume and all had the same fixed eÄects known to have impact on their variability (Gyovai et al. 2012). After the same logic, the following fixed eÄects were modelled

in all three models (Table 3): year-month (75), F Dam, body weight at CT-scan (1), sex (2) and Pixel (3). Additive genetic eÄects and random litter eÄects were treated as random eÄects, while three models were extended with D-loop mtDNA eÄects. Mathematical description and general structure referred to NBA, NBD and TNB (equation 1) and TMV (equation 2) basic models are: y =Xb + Za + Wp + e (1) y=Xb + Za + Wc + e (2)

Table 3: Description of models used in estimating cytoplasmic and D-loop mtDNA eÄects. Litter size traits models (1-7) 1 2 3 4 5* 6* 7* 8 9 10* Carcass traits models (8-10) 1 2 3 4 5* 6* 7* 8 9 10* Fixed eÄects Parity XXXXXXX Year-month XXXXXXXXXX

FDam XXXXXXXXXX

FLitter XXXXXXX Body weight at CT-scan XXX

Sex XXX Pixel XXX Random eÄects Permanent XXXXX X Additive XXXXX XXXX Maternal lineages of dames X X X Maternal lineages of sires XX D-loop mtDNA of mothers X X X D-loop mtDNA of sires XX Litter XXX

*Reduced dataset as the number of known haplotypes following maternal segregation was smaller. FDam and FLitter are inbreeding coe§cients of dam and litter, respectively.

290 World Rabbit Sci. 26: 287-298 IMPACT OF CYTOPLASMIC INHERITANCE ON RABBIT PRODUCTION TRAITS where y=vector of phenotypic observations, b=vector of fixed eÄects, a=vector of additive genetic eÄects, p=vector of permanent environmental eÄects (random, only considered in equation 1), c=vector of common litter eÄects (random, only considered in equation 2), e=vector of residuals, while X, Z and W are incidence matrices relating the records to the fixed, animal and random permanent environmental or common litter eÄects, respectively. Additionally, the extended models accounted for cytoplasmic eÄects to NBA, NBD and TNB (equation 3) and TMV (equation 4) y =Xb + Za + Wp + Km + e (3) y=Xb + Za + Wc + Km + e (4) where m=vector of random cytoplasmic eÄects, and K is the incidence matrix relating records to random cytoplasmic eÄects. In addition to estimating the variance of the contribution of the diÄerence between H1 and H2 (as in models 5, 6, 7, and 10), we also analysed the significance of the diÄerence between H1 and H2 haplotypes. Conclusions from results obtained were the same as in models 5, 6, 7 and 10, and are not presented. Variance components and genetic parameters were estimated by REML method using the PEST (for data coding) (Groeneveld, 1990: ftp://ftp.tzv.fal.de/pub/pest/doc/pest-manual-Apr-2006.pdf) and VCE6 software (Groeneveld, et al., 2008; ftp://ftp.tzv.fal.de/pub/vce6/doc/vce6-manual-3.1-A4.pdf) applying the single-trait animal models. In order to compare the fit of the models, the PREDICTION procedure of PEST (Groeneveld, 1990) was applied to calculate mean squared error (MSE), bias and correlation between the observed and predicted values of NBA, NBD, TNB and TMV.

RESULTS

Descriptive statistics Means and standard deviations of TNB and NBA of the analysed breeds (Table 1) showed the highest value for Pannon Ka breed (maternal line), which was expected as this population is more intensively selected for the litter size traits than are Pannon White or Pannon Large population. However, the observed litter size values were close to those reported previously (Al-Saef et al., 2008; Nagy et al., 2011; Nagy et al., 2013; Nagy et al., 2014). On the other side, PW breed is intensively selected for the lean production (Matics et al., 2014); as a result, TMV might be higher in comparison to the previous studies (Gyovai et al., 2011).

D-loop mtDNA diversity The variability of D-loop mtDNA polymorphism in 3 Pannon Rabbit breeds was extremely low, as in PK and PL only 1 haplotype (H1) was found, while in PW only 2 haplotypes, H1 (76%) and H2 (24%), were identiﬁed. The phylogenetic position of 2 haplotypes (H1 and H2) found in Pannon Rabbits is presented in Figure 1. While separated by 13 mutations, haplotypes H1 and H2 were grouped within 2 most common rabbit haplotypes (see Figure 1). This

Table 4: Estimated variance components (V) and genetic parameters for additive genetic (A), cytoplasmic (cyt), D-loop mtDNA (Hcyt) and environmental eÄects (E) for thigh muscle volume (in cm3; TMV) of Pannon White rabbits. 2 2 2 2 2 Model VA h Vcyt cyt VHcyt Hcyt VC c VE e MSE 8 246.86 0.275 - - 68.52 0.076 583.19 0.649 419.88 9 247.00 0.275 0.000 0.000 68.54 0.076 583.14 0.649 419.76 10 280.02 0.302 0.000 0.000 55.07 0.059 592.57 0.639 418.05

Model 10 refers to the reduced dataset as the number of known haplotypes following maternal segregation was smaller; VA, Vcyt, 2 VHcyt, Vc and VE are additive, cytoplasmic maternal, D-loop mtDNA haplotype, random litter, and residual variances, respectively; h is the narrow sense heritability; cyt2 is the contribution of cytoplasmic maternal variance to the phenotypic variance; Hcyt2 is the contribution of D-loop mtDNA haplotype variance to the phenotypic variance; c2 is the contribution of common litter variance to the phenotypic variance; e2 is the contribution of residual variance to phenotypic variance; MSE is mean squared error.

World Rabbit Sci. 26: 287-298 291 NGUYEN et al.

Figure 1: Median-joining network diagram showing phylogenetic positions of haplotypes found in Pannon Ka, Pannon Large and Pannon White rabbits with respect to haplotypes found in other wild and domestic rabbit populations (see the legend for a detailed description). Diagram was constructed based on mtDNA haplotypes identiﬁed by polymorphism analysis of a 332-bp fragment of mtDNA (RRS: NC001913 positions 15492–15824). Circles are proportional to haplotype frequency, the black points represent hypothetical sequences that were not observed, while the number of mutations separating nodes are given near branches in parentheses. Names of the presented haplotypes contain identiﬁcation, accession number and origin (abbreviation) with the exception of Pannon breeds that are given by full name.

heterogeneity of maternal origin is in accordance with the formation history of the breed, as PW is a synthetic breed derived from 2 breeds, the Californian and New Zealand rabbits. H1 is by far the most represented haplotype in rabbits, and a number of very diverse rabbit populations share this haplotype (Asian domestic, Australian wild, European domestic and European wild). H2 is the second most frequent haplotype, with sequences found in Australian wild, European domestic and European wild populations. More detailed description of the haplotype origin is presented in Supplement Table S1.

Cytoplasmic and mtDNA haplotype eects

Carcass trait The estimated contribution of cytoplasmic maternal variance to the phenotypic variance (cyt2) and the contribution of D-loop mtDNA haplotype variance to the phenotypic variance (Hcyt2) were zero (models 9 and 10), indicating that neither maternal lineage nor D-loop mtDNA haplotype eÄects were present in PW (Table 4). There was no signiﬁcant

292 World Rabbit Sci. 26: 287-298 IMPACT OF CYTOPLASMIC INHERITANCE ON RABBIT PRODUCTION TRAITS

Table 5: Estimated variance components (V) and genetic parameters for additive genetic (A), cytoplasmic (maternal and paternal) and environmental eÄects (E) for litter size traits in Pannon rabbit breeds. 2 2 2 2 2 Breed Traits Model VA h Vmcyt mcyt Vpcyt pcyt Vpe p VE e MSE Pannon White 1 0.674 0.076 - - - 0.713 0.080 7.529 0.844 6.812 2 0.674 0.076 0.000 0.000 - 0.712 0.080 7.529 0.844 6.812 NBA 3 0.672 0.075 - - 0.012 0.001 0.710 0.080 7.523 0.844 6.802 4 0.672 0.075 0.000 0.000 0.012 0.001 0.711 0.080 7.523 0.844 6.802 1 0.024 0.020 - - - 0.024 0.020 1.135 0.960 1.086 2 0.024 0.020 0.000 0.000 - 0.024 0.020 1.135 0.960 1.086 NBD 3 0.024 0.020 - - 0.000 0.000 0.024 0.020 1.135 0.960 1.086 4 0.024 0.020 0.000 0.000 0.000 0.000 0.024 0.020 1.135 0.960 1.086 1 0.695 0.076 - - - 0.760 0.083 7.652 0.840 6.910 2 0.695 0.076 0.000 0.000 - 0.760 0.084 7.652 0.840 6.910 TNB 3 0.694 0.076 - - 0.005 0.001 0.759 0.083 7.649 0.840 6.906 4 0.694 0.076 0.000 0.000 0.005 0.001 0.759 0.083 7.649 0.840 6.906 Pannon Ka 1 0.754 0.087 - - - 0.647 0.075 7.270 0.838 6.545 2 0.755 0.087 0.000 0.000 - 0.647 0.075 7.270 0.838 6.544 NBA 3 0.760 0.087 - - 0.000 0.000 0.647 0.075 7.270 0.838 6.544 4 0.760 0.087 0.000 0.000 0.027 0.003 0.644 0.074 7.266 0.835 6.539 1 0.060 0.047 - - - 0.000 0.000 1.217 0.953 1.163 2 0.060 0.047 0.000 0.000 - 0.000 0.000 1.217 0.953 1.163 NBD 3 0.060 0.047 - - 0.000 0.000 0.000 1.217 0.953 1.163 4 0.060 0.047 0.000 0.000 0.000 0.000 0.000 1.217 0.953 1.163 1 0.885 0.100 - - - 0.638 0.072 7.288 0.827 6.542 2 0.887 0.101 0.000 0.000 - 0.637 0.072 7.289 0.827 6.542 TNB 3 0.887 0.101 - - 0.000 0.638 0.072 7.289 0.827 6.542 4 0.887 0.101 0.000 0.000 0.000 0.638 0.072 7.288 0.827 6.542 Pannon Large 1 0.852 0.088 - - - 1.220 0.126 7.630 0.786 6.694 2 0.853 0.088 0.000 0.000 - 1.219 0.126 7.630 0.786 6.694 NBA 3 0.853 0.088 - - 0.000 1.219 0.126 7.630 0.786 6.694 4 0.853 0.088 0.000 0.000 0.000 1.219 0.126 7.630 0.786 6.694 1 0.088 0.032 - - - 0.036 0.013 2.624 0.955 2.492 2 0.088 0.032 0.000 0.000 - 0.036 0.013 2.624 0.955 2.492 NBD 3 0.087 0.032 - - 0.004 0.001 0.034 0.012 2.624 0.954 2.491 4 0.087 0.032 0.000 0.000 0.004 0.001 0.037 0.012 2.624 0.954 2.491 1 0.877 0.083 --- 1.250 0.118 8.466 0.799 7.466 2 0.880 0.083 0.000 0.000 - 1.250 0.118 8.464 0.799 7.466 TNB 3 0.878 0.083 - - 0.000 1.250 0.118 8.465 0.799 7.465 4 0.878 0.083 0.000 0.000 0.000 1.250 0.118 8.465 0.799 7.465

NBA is number of kits born alive, NBD is number of kits born dead and TNB is total number of kits born; VA, Vmcyt, Vpcyt, VPe and VE are additive, cytoplasmic maternal, cytoplasmic paternal, permanent environmental, and residual variances, respectively; h2 is the narrow sense heritability; mcyt2 is the contribution of cytoplasmic maternal variance to the phenotypic variance; pcyt2 is the contribution of cytoplasmic paternal variance to the phenotypic variance; p2 is the contribution of permanent environmental variance to the phenotypic variance; e2 is the contribution of residual variance to phenotypic variance; MSE is mean squared error. eÄect of haplotype when haplotypes were compared as ﬁxed eÄect. The estimated heritabilities (h2) of TMV, the trait introduced as a selection criterion in 2004, were moderate, ranging from 0.275±0.02 to 0.302±0.02. Ratios of the random litter eÄects to the phenotypic variance were small for TMV, ranging from 0.059±0.01 to 0.076±0.01. The smallest MSE values, representing goodness of ﬁt, were obtained in models with cytoplasmic eÄects. However, this latter result has no importance, as the eÄects of these components were equal to zero.

World Rabbit Sci. 26: 287-298 293 NGUYEN et al.

Table 6: Estimated variance components (V) for additive genetic (A), D-loop mtDNA (maternal and paternal) and environmental eÄects (E) for litter size traits in Pannon White rabbit. 2 2 2 2 2 Traits Model VA h VHma Hma VHpa Hpa Vpe p VE e MSE NBA 5 0.606 0.071 0.000 0.000 - - 0.736 0.086 7.252 0.844 6.463 6 0.617 0.071 - - 0.006 0.001 0.716 0.083 7.310 0.845 6.402 7 0.652 0.074 0.009 0.001 0.000 0.000 0.739 0.084 7.415 0.841 6.459 NBD 5 0.038 0.031 0.000 0.000 - - 0.008 0.006 1.193 0.963 1.131 6 0.029 0.023 - - 0.000 0.000 0.020 0.016 1.211 0.962 1.140 7 0.035 0.027 0.000 0.000 0.000 0.000 0.015 0.012 1.251 0.961 1.168 TNB 5 0.602 0.068 0.000 0.00000 - - 0.817 0.093 7.377 0.839 6.559 6 0.628 0.070 - - 0.002 0.000 0.818 0.092 7.474 0.838 6.518 7 0.637 0.070 0.005 0.001 0.000 0.000 0.833 0.092 7.601 0.838 6.611

NBA is number of kits born alive, NBD is number of kits born dead and TNB is total number of kits born; VA, VHma, VHpa, VPe and VE are additive, D-loop mtDNA maternal haplotype, D-loop mtDNA paternal haplotype, permanent environmental, and residual variances, respectively; h2 is narrow sense heritability; Hma2 is the contribution of D-loop mtDNA maternal haplotype variance to the phenotypic variance; Hpa2 is the contribution of D-loop mtDNA paternal haplotype variance to the phenotypic variance; p2 is the contribution of permanent environmental variance to the phenotypic variance; e2 is the contribution of residual variance to phenotypic variance; MSE is mean squared error.

Litter size traits Estimated additive genetic variances, cytoplasmic or D-loop mtDNA (analysed from maternal and paternal side), permanent environmental and residual variances by the magnitude and ratios (compared to the phenotypic variance) are summarised in Tables 5 and 6 for litter traits. The best model ﬁts (smallest MSE values) were obtained in models with cytoplasmic eÄects. Note that in Model 5, 6 and 7 in PW rabbits sample sizes were reduced. However, both estimated cytoplasmic eÄects (maternal - mcyt2 and paternal - pcyt2) ranged from zero (0.0%) to negligible (0.3%±0.003), obtained for p2 for NBA in Pannon Ka (see Table 5. for the details). Similar results, with negligible contribution to phenotypic variance, from 0.1%±0.001 (maternal - Hma 2) to 0.02-0.1%±0.003-0.001 (paternal - Hpa2), were obtained for D-loop mtDNA haplotypes. The estimated heritabilities of Pannon rabbits for NBA ranged from 0.071±0.0078-0.088±0.0167, 0.020-0.047±0.0037-0.0078 (NBD) and 0.068-0.101±0.0075-0.0161 (TNB). A similar trend was observed for variance components of permanent environmental eÄect, ranging for NBA in 0.075-0.126±0.0062-0.0149, for NBD in 0.000-0.020±0.0000-0.0094 and for TNB in 0.072-0.118±0.0066-0.0145.

DISCUSSIONS AND CONCLUSION

Although encoding for a small number of genes, the eÄects of mitogenome variation on production traits are reported in a number of animal domestic species. Until now, the impact of mitogenome on production traits had never been analysed in rabbits. In this study, we analysed the eÄects of mitogenome variation on litter size traits (NBA, NBD and TNB) and on one carcass trait (TMV) measured in vivo by computer tomography (CT). We began our analysis by testing cytoplasmic eÄects on production traits, where the impact of maternal lineages was analysed for both does and mating bucks. For all traits and breeds the impact of cytoplasmic inheritance was absent or negligible. Moreover, in all 3 Pannon breeds we determined D-loop mtDNA haplotypes for each maternal lineage. Overall, there were only 2 diÄerent haplotypes present in PW (H1 and H2), while in the other 2 breeds (PK and PL) only H1 was present - if we exclude a small number of rabbits in PK breed with non-consistent mtDNA segregation in a pedigree. We also found no signiﬁcant contribution of D-loop mtDNA sequence polymorphism on any of production traits analysed in PW. The lack of information on complete mitogenome polymorphism is the most likely explanation for the observed results. Our molecular analysis was restricted to only 332 bps and it is possible that complete mitogenome sequence variation is higher than that observed for D-loop mtDNA sequence (332 bps). On a 332 bp long sequence, haplotypes H1 and H2 do belong to the most common haplotypes in rabbits.

294 World Rabbit Sci. 26: 287-298 IMPACT OF CYTOPLASMIC INHERITANCE ON RABBIT PRODUCTION TRAITS

However, we are not able to say that segregating mitogenome variation is optimal with respect to production traits, or that they are free of detrimental mutations. Thus, the magnitude of the potential beneﬁts of the introduction or alteration of mitogenome variation using gene editing techniques (Hickey et al., 2016), in rabbit breeding programmes remains an open question for future research.

Acknowledgments: This study was supported by the EFOP-3.6.1-16-2016-00007 project, the CEEPUS scholarship for the year 2016 and the Croatian Science Foundation under Project IP-11-2013_9070 (“Utilisation of the whole mitogenome in cattle breeding and conservation genetics”, http://mitotauromics.agr.hr/). This research was partly supported by the European Regional Development Fund under the grant KK.01.1.1.01.0009 (DATACROSS).

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Genetic parameters and genetic trends of reproduction mitochondrial DNA haplotype and the reproductive capacity of traits in synthetic Pannon rabbits using repeatability and domestic pigs (Sus scrofa domesticus). BMC Genetics, 17: multi-trait animal models. Archiv Tierzucht, 54: 297-307. 67. https://doi.org/10.1186/s12863-016-0375-4 https://doi.org/10.5194/aab-54-297-2011 Van Vleck L.D. 2000. Selection index and introduction to mixed Nagy I., Gyovai P., Radnai I., Kiszlinger H., Farkas J., Szendrő models methods. CRC Press In., Boca Raton, Florida 33431. Zs. 2013a. Genetic parameters, genetic trends and 19: 225-231. inbreeding depression of growth and carcass traits in Yen N.T., Lin C.S., Ju C.C., Wang S.C., Huang M.C. 2007. Pannon terminal line rabbits. Archiv Tierzucht, 56: 191-199. Mitochondrial DNA polymorphism and determination of eÄects https://doi.org/10.7482/0003-9438-56-018 on reproductive trait in pigs. Reprod. Domest. Anim. 42: 387- Nagy I., Gorjanc G., Curik I., Farkas J., Kiszlinger H., Szendrő 92. https://doi.org/10.1111/j.1439-0531.2006.00797.x Zs. 2013b. The contribution of dominance and inbreeding Yu G., Xiang H., Tian J., Yin J., Pinkert C.A., Li Q., Zhao X. depression in estimating variance components for litter size in 2015. Mitochondrial Haplotypes Inﬂuence Metabolic Traits in Pannon White rabbits. J. Anim. Breed. Genet., 130: 303-311. Porcine Transmitochondrial Cybrids. Sci. Rep., 19: 13118-10. https://doi.org/10.1111/jbg.12022 https://doi.org/10.1038/srep13118 Nagy, I., Farkas, J., Curik, I., Gorjanc, G., Gyovai, P., Szendrő, Zs., Wallace D. C. 1999. Mitochondrial diseases in man and mouse. 2014 - Estimation of additive and dominance variance for litter Science, 283: 1482-1488. https://doi.org/10.1126/ size components in rabbits. Czech J. Anim. Sci., 59: 182-189. science.283.5407.1482 https://doi.org/10.17221/7342-CJAS Zhao X., Wu N., Zhu Q., Gaur U., Gu T., Li D. 2015. High- Pierpaoli M., Riga F., Trocchi V., Randi E. 1999. Species altitude adaptation of Tibetan chicken from MT-COI and distinction and evolutionary relationships of the Italian ATP-6 perspective. Mitochondrial DNA, Early Online: 1-9. hare (Lepus corsicanus) as described by mitochondrial https://doi.org/10.3109/19401736.2015.1015006 DNA sequencing. Mol. Ecol., 8: 1805-1817. https://doi.org/10.1046/j.1365-294x.1999.00766.x

296 World Rabbit Sci. 26: 287-298 IMPACT OF CYTOPLASMIC INHERITANCE ON RABBIT PRODUCTION TRAITS

Supplement Table S1: Description of rabbit (Oryctolagus cuniculus) haplotype sequences presented in Figure 1. Accession # Origin Haplotype Reference Description AJ293831 France H6 Bolet et al., 2000. Domestic Fauve de Bourgogne AJ293832 Belgium H7 Bolet et al., 2000. Domestic Belgian hare AJ293833 France H8 Bolet et al., 2000. Domestic Fauve de Bourgogne AJ293834 France H9 Bolet et al., 2000. Domestic Argente de Champagne AJ293835 Great Britain H10 Bolet et al., 2000. Domestic English AJ293836 Belgium H11 Bolet et al., 2000. Domestic Flemish giant AJ293837 France H12 Bolet et al., 2000. Domestic Fauve de Bourgogne AJ293838 Hungary H13 Bolet et al., 2000. Domestic Hungarian Giant AJ293839 France H14 Bolet et al., 2000. Domestic French Lop AJ293840 France H15 Bolet et al., 2000. Domestic French Lop AJ293841 France H16 Bolet et al., 2000. Domestic French Lop AJ293843 Austria H17 Bolet et al., 2000. Domestic Vienna White AJ293844 Belgium H18 Bolet et al., 2000. Domestic Flemish Giant U62924 Australia H4 Fuller et al., 1997.. Wild rabbit U62925 Australia H1 Fuller et al., 1997 Wild rabbit U62926 Australia H16 Fuller et al., 1997 Wild rabbit U62927 Australia H2 Fuller et al., 1997 Wild rabbit NC_001913 Unknown H1 Gissi et al., 1998. Rabbit reference sequence AF534080 China H1 Long, et al., 2003. Qixing AF534081 China H1 Long, et al., 2003 Haerbin White AF534082 China H1 Long, et al., 2003 Zhenhai thick-hair Angora AF534083 China H1 Long, et al., 2003 Big ear brown rabbit AF534085 Belgium H1 Long, et al., 2003 Belgium AF534092 China H1 Long, et al., 2003 Sichuan White AF534094 Germany H1 Long, et al., 2003 Rex AF534095 Germany H1 Long, et al., 2003 Angora AF534096 Germany H1 Long, et al., 2003 Zika AF534097 China H1 Long, et al., 2003 Fujian Brown AF534098 China H1 Long, et al., 2003 Taihang Moutain AF534099 China H3 Long, et al., 2003 Yufeng Brown AF534100 Germany H2 Long, et al., 2003 Zika (Germany great line) AF534101 Germany H2 Long, et al., 2003 Rex AF534103 China H4 Long, et al., 2003 Zhenhai thick-hair Angora AF534104 Japan H1 Long, et al., 2003 Japanese White AF534105 China H1 Long, et al., 2003 Yufeng Brown AF534107 Germany H5 Long, et al., 2003 Zika KY977609 Hungary H1 This study Pannon Large KY977634 Hungary H1 This study Pannon Ka KY977665 Hungary H1 This study Pannon White KY977670 Hungary H2 This study Pannon White Z83340 Spain/Portugal H19 van der Loo et al., 1997. Wild rabbit Z83341 Spain/Portugal H20 van der Loo et al., 1997. Wild rabbit Z83342 Spain/Portugal H21 van der Loo et al., 1997. Wild rabbit Supplement Table S1 continues on next page

World Rabbit Sci. 26: 287-298 297 NGUYEN et al.

Supplement Table S1 continues from previous page

Accession # Origin Haplotype Reference Description Z83343 Spain/Portugal H22 van der Loo et al., 1997. Wild rabbit Z83344 Spain/Portugal H23 van der Loo et al., 1997. Wild rabbit Z83346 Spain H24 van der Loo et al., 1997. Wild rabbit Z83347 Spain H25 van der Loo et al., 1997. Wild rabbit Z83348 Spain H26 van der Loo et al., 1997. Wild rabbit Z83350 Spain H27 van der Loo et al., 1997. Wild rabbit Z83352 Spain H16 van der Loo et al., 1997. Wild rabbit Z83354 Spain H28 van der Loo et al., 1997. Wild rabbit Z83355 Spain H29 van der Loo et al., 1997. Wild rabbit Z83356 Spain H30 van der Loo et al., 1997. Wild rabbit Z83357 Spain H31 van der Loo et al., 1997. Wild rabbit Z83358 Spain H32 van der Loo et al., 1997. Wild rabbit Z83359 Spain H33 van der Loo et al., 1997. Wild rabbit Z83360 Spain H34 van der Loo et al., 1997. Wild rabbit Z83361 Spain H35 van der Loo et al., 1997. Wild rabbit Z83362 Spain H36 van der Loo et al., 1997. Wild rabbit Z83363 Spain H37 van der Loo et al., 1997. Wild rabbit Z83364 Spain H38 van der Loo et al., 1997. Wild rabbit Z83365 Spain H2 van der Loo et al., 1997. Wild rabbit Z83366 Spain H4 van der Loo et al., 1997. Wild rabbit Z83367 Spain H1 van der Loo et al., 1997. Wild rabbit Bolet G., Brun J.M., Monnerot M., Abeni F., Arnal C., Arnold J., Bell D., Bergoglio G., Besenfelder U., Bosze S., Boucher S., Chanteloup N., Ducourouble M.C., Durand-Tardif M.H., Esteves P.J., Ferrand N., Gautier A., Haas C., Hewitt G., Jehl N., Saleil G. 2000. Evaluation and conservation of European rabbit (Oryctolagus cuniculus) genetic resources. First results and inferences. World Rabbit Science, 8 (sp): 281-315. Fuller S.J., Wilson J.C., Mather P.B. 1997. Patterns of diÄerentiation among wild rabbit populations Oryctolagus cuniculus L. arid and semiarid ecosystems of north-eastern Australia. Molecular Ecology, 2: 145-153. https://doi.org/10.1046/j.1365-294X.1997.00167.x Gissi C., Gullberg A., Arnason U. 1998. The complete mitochondrial DNA sequence of the rabbit, Oryctolagus cuniculus. Genomics, 2: 161-169. https://doi.org/10.1006/geno.1998.5282 Long J.R., Qiu X.P., Zeng F.T., Tang L.M., Zhang Y.P. 2003. Origin of rabbit (Oryctolagus cuniculus) in China: evidence from mitochondrial DNA control region sequence analysis. Animal Genetics, 34: 82-87. https://doi.org/10.1046/j.1365-2052.2003.00945.x van der Loo W., Mougel F., Sánchez M.S., Bouton C., Castien E., Soriguer R., Hamers R., Monnerot M. 1997. Evolutionnary patterns at the antibody constant region in rabbit (Oryctolagus cuniculus): characterisation of endemic b-locus allotypes and their frequency correlation with major mitochondrial gene types in Spain. Gibier Faune Sauvage, 14: 427-449.

298 World Rabbit Sci. 26: 287-298 W orld World Rabbit Sci. 2018, 26: 299-305 R abbit doi:10.4995/wrs.2018.9587 Science © WRSA, UPV, 2003

ARE COMBI PARKS JUST AS USEFUL AS REGULAR PARKS FOR FATTENERS FOR PART-TIME GROUP HOUSING OF RABBIT DOES? ROMMERS J., DE GREEF K.H.

Wageningen Livestock Research, P.O. Box 338, 6700 AH WAGENINGEN, The Netherlands.

Abstract: Group housing of lactating rabbit does is desirable from a welfare standpoint, but agonistic behaviour can cause severe skin injuries, which are undesired for animal welfare. Park layout, creating hiding places and escape possibilities, may help redirect attention away from fighting, which could in turn help prevent skin damage. An experiment was performed to test whether more damaging behaviour would occur in a combi park (with nest box panels) after mixing, compared to a regular park for fatteners, as nest box panels would obstruct does when escaping aggressive interactions. In addition, the position of the PVC pipe underneath the platform differed between parks (longitudinal or transversal), resulting in different escape routes. Twenty- two parks were used, in which 5 does per park were grouped at 23 d of lactation until weaning at 36 d of lactation. Skin injuries were scored at 4 d after grouping and on the day before weaning. The presence of nest box panels in a combi park and the position of the PVC pipe underneath the platform did not affect the level of skin damage. Moderate to severe injuries were observed, mostly at the hind quarters, ears and head. Five to 6% of the does were severely injured (wounds). There was a positive relationship between the average skin injury per park at 4 d after grouping and at weaning. On average, average injury score per park increased from grouping to weaning, but there are differences between individual parks. From this experiment it can be concluded that group housing of lactating rabbit does involves animals getting injured. In fact, 5 to 6% of the does were severely injured (wounds). Social dynamics of group housed does are insufficiently understood and might be important to reduce damaging behaviour in group housing. Key Words: rabbits, lactating does, group housing, skin injuries, social behaviour.

INTRODUCTION

In the Netherlands, there is high public concern about animal welfare in livestock farming. In commercial rabbit production, most fatteners are now housed in welfare improved housing systems, either in welfare cages or in so- called parks. The latter initiative is rewarded with a price premium by the retail. It is foreseen that group housing of rabbit does will be demanded by politicians and NGOs in the future, and might be rewarded by market partners (de Greef et al., 2016). Aggression between lactating does is the key issue that needs to be tackled before group housing of rabbit does is ready to be implemented in rabbit farms (Rommers and de Jong, 2005, 2010; Hoy and Verga, 2006; Andrist et al., 2013; Szendro et al., 2016). Fights between does mainly take place in the first days after grouping of unfamiliar does, when a dominance hierarchy needs to established. Fights to establish the hierarchy in a group can be seen as adaptive social behaviour of does. However, excessive fighting between rabbits leading to severe injuries and increased mortality is unacceptable in terms of animal welfare. Enrichment in the form of hiding places and escape possibilities helps redirect attention away from fighting, which could help prevent injury. Previous experiments by our group (Rommers et al., 2013) showed that panels and PVC pipes seemed to provide good opportunities for escape.

Correspondence: J. Rommers., [email protected]. Received February 2018 - Accepted July 2018. https://doi.org/10.4995/wrs.2018.9587

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Parks equipped with wooden panels and PVC pipes could enable group housing of rabbit does with fewer injuries. In a previous pilot scheme at 2 commercial farms in the Netherlands (Rommers et al., 2014b), does were part-time group housed successfully in parks for fatteners from 18 d or 21 d of lactation onwards. Only some superficial to moderate injuries were observed. However, mortality of rabbits during the fattening period was high (up to 25%). In 5 successive pilot studies at a commercial rabbit farm in 2016, a combi park system was used, in which does can be housed individual until mixing. At mixing, the side walls were removed to create a park. Kits performed well in the group housing, as well as after weaning. However, the finding of previous experiments, demonstrating low levels of aggression between does, could not be confirmed. Substantial skin damage events were observed after mixing (Rommers and de Greef, 2017). In the fifth pilot, the combi park was adapted to a layout that is more comparable to a regular park for fatteners, as used in the study in 2014 (Rommers et al., 2014b). This final pilot suggested that the level of aggression in the earlier pilots was caused by the layout of the combi park, and that combi parks thus needed to be adapted to prevent high levels of aggression (Rommers and de Greef, 2017). In all pilots, skin injuries were less severe at weaning than 4 d after mixing, and mortality of the rabbits after weaning was limited (Rommers and de Greef, 2017). Thus, aggression between does after mixing, resulting in skin injuries, seems the main technical issue to be resolved for successful part-time group housing of does with kits. The current experiment was performed to test whether or not the layout of the combi park was indeed the bottleneck for aggression among rabbit does after mixing at 23 d of lactation. We hypothesised that removing the nest box panels would reduce aggression. In the former nest box areas, does could get stuck and nest box panels would obstruct does in escaping from aggressive interactions. Two diÄerent types of parks were tested: parks with nest box panels and parks without nest box panels (park model for meat rabbits)

MATERIALS AND METHODS

Ethical statement Mixing of animals is known to elicit agonistic interactions, in which animals can cause damage (scratches, biting wounds) to each other. In an earlier pilot in comparable conditions, no substantial damage was observed (Rommers and de Greef, 2014b). Husbandry optimisation is regarded as one of the key factors in minimising negative consequences of agonistic interactions. The housing of the grouped animals provided shelter for animals to evade each other. Management instructions contained directives to monitor problems among animals and instructions to remove damaged animals when necessary. The work in present project series was classiﬁed (23-3-2015) by the Animal Ethical Committee Welfare Body as not being an animal experiment under the law ‘Wet op de Dierproeven’ (Directive 2010/63/EU).

Animals and housing The experiment was performed from April until June 2017 using 110 multiparous lactating does (Hycole), which were artificially inseminated 11 d postpartum. The experiment was conducted at a commercial rabbit farm with 600 does and oÄspring in one compartment. The compartment contained 528 individual welfare pens for does. After weaning, these individual pens were merged into 88 so-called combi parks for fatteners. Parks were set up in the compartment in 8 rows of 11 cages. In the experiment, 2 rows (22 parks) were used. A combi park is composed of 6 individual pens for lactating does. Each pen was 38 cm wide and 100 cm deep and was open at the top. A slatted platform of 40 cm width was mounted at 25 cm above the floor. The slatted floor and platform were made out of plastic (Meneghin, Italy). A nest box (25×38 cm; long×wide) could be placed in the front part of the pen. Each pen had 2 drinking nipples and a feeder. Roughage was provided by a pressed straw roll that was put in the pen. A combi park was created by taking out the side walls, creating a floor surface of 2.28 × 1.00 m. The combi park was open at the top. Two wooden panels and a PVC pipe were placed beneath the platform, providing the does with possibilities to escape. In the current experiment, we housed 5 does per combi park. At mixing, all does were transferred to another combi park. This was done to avoid aggression among does that may be related to does defending the range where their nest had been, although earlier work did not confirm this behaviour (Rommers et al., 2014).

300 World Rabbit Sci. 26: 299-305 PART-TIME GROUP HOUSING OF RABBIT DOES IN PARKS

Treatments 1. EÄect of nest box panels: The nest box panels remained in or were removed from the combi park (see Figure 1a and 1b). When the panels were removed, the combi park stood as model for a regular park for fatteners, as used in a previous study (Rommers et al., 2014b). 2. EÄect of the location of the PVC pipe beneath the platform: The PVC pipe was placed in transversal or longitudinal direction underneath the platform in between the 2 wooden panels. The idea behind this is that the position of the pipe inﬂuences escape/ hide possibilities. Figure 1a: Combi parks with nest box panels. Table 1 gives an overview of the diÄerent treatments and number of replicates. The experiment was concluded at weaning (36 d of lactation).

Measurements Skin injuries: all does were scored for skin injuries the day before mixing, at 4 d after being mixed and at weaning (at 36 d of lactation). A scoring system (Kalle, 1994) was used diÄerentiating: 0=none, 1=superﬁcial, 2=moderate and 3=severe (wound) skin injuries on diÄerent parts of the animal (head, ears, body, limbs, tail and genitals). For each doe, the highest score of the rabbit was used for Figure 1b: Combi parks without nest box panels, further analysis. standing model for fattening park.

Statistical analysis

The combi park was the experimental unit Skin injuries: the average injury score per park was calculated and used for statistical analysis. First, we tested whether or not the average injury score per park was normally distributed using the UNIVARIATE procedure of SAS (version 9.4). This was indeed the case and average injury score was analysed using the GLM procedure of SAS (version 9.4) with presence of nest box panels and position of the PVC pipe as factors. To test whether or not there was a relationship between the average skin injury per park at four days after mixing and at weaning, the Regressive procedure of SAS (version 9.4) was used.

RESULTS

Two does died in the period between mixing and weaning (one doe in the combi park with nest box panels and transversal position of the PVC pipe, and one doe at the combi park without nest box panels and longitudinal position

Table 1: Overview of the treatments tested and number of replicates (group pens with 5 animals each). Presence of nest box panels/Position of PVC pipe Absent Present Total Transversal 6 6 12 Longitudinal 5 5 10 Total 11 11 22

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100 of the PVC pipe). No visual signs of aggression were noticed on their bodies. No dissection was applied, so 80 the reason for death is unclear. 60

40 Skin injuries % of injuries of %

20 Most of the injuries (71%) are observed on the body, mostly at the hindquarters, 22% at the head and 7% at 0 the ears (Figure 2). At the legs, tail and genitals almost head ears body legs tail genitals no injuries were observed. The injuries at the ears were 4d-mixing weaning only observed in does housed in the combi parks with Figure 2: Position of skin injuries on the does. nest box panels, whereas at the combi parks without 4d-mixing; weaning. nest box panels no injuries at the ears were observed. For the other limbs, there were no diÄerences observed between treatments. 2,5 Table 2 presents the analysis of the average injury score 2 y = 0,7138x + 0,4009 per park. There were no diÄerences between treatments. 1,5 R² = 0,4535 So, no eÄect of nest box panels or position of the PVC pipe underneath the platform on skin injuries was found. 1 A signiﬁcant (P<0.001) relationship between the 0,5 average injury score per park at 4 days after mixing

average average at injury score weaning 0 and at weaning was found (Figure 3). The trend line 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 (y=0.7138 x+0.4009) indicates that higher scores at average injury score 4 d after mixing mixing generally coincide with higher scores at weaning. Comparing this trend line with the y=x-line reveals that Figure 3: Relationship between average injury score per the trend line is higher than the y=x-line for the whole park four days after mixing and at weaning based on the data range. This indicates that later damage scores (at highest injury score of the rabbit. weaning) are systematically higher than earlier scores (4 d after mixing).

Injured does per park Besides the average skin injury scores, the number of does that were injured per pen was also calculated. Table 3 shows the number of does with skin injuries, 4 d after mixing (see Table 3a) and at weaning (Table 3b). Four days after mixing, 65% of the does were uninjured, 20% had only superﬁcial injuries (scratches), 8% had moderate injuries, and 5% had severe injuries (wounds) as the highest injury score. At weaning these percentages were increased. Thirteen per cent of the does were moderately injured and 6% had wounds.

DISCUSSION

The hypothesis of this experiment was that part-time group housing in combi parks results in more agonistic interactions by does getting stuck in the former nest box areas and this would obstruct does in escaping from aggressive interactions. Therefore, in parks with nest box panels more skin damage would occur than in group

Table 2: Average injury score (least squares means±standard error) for parks with or without nest box panels and PVC pipe transversal (trans) or longitudinal (long) placed underneath the platform. P-value P-value P-value Nest box panels with with without without panels pipe Panels×pipe PVC pipe trans long trans long 4 d after mixing 0.63±0.19 0.44±0.21 0.49±0.19 0.64±0.21 0.93 0.91 0.29 At weaning 0.89±0.20 0.60±0.22 0.66±0.20 0.95±0.22 0.77 0.98 0.18

302 World Rabbit Sci. 26: 299-305 PART-TIME GROUP HOUSING OF RABBIT DOES IN PARKS

Table 3a: Number and percentage of injured does for the diÄerent treatments at 4 d after mixing according to the severity of injury (based on the highest injury score per doe). Nest box panels with without Position PVC pipe transversal longitudinal transversal longitudinal Total Number of does: Score 0 17 (56%) 17 (68%) 23 (77%) 16 (64%) 73 (66%) Score 1 9 (30%) 6 (24%) 3 (10%) 4 (16%) 22 (20%) Score 2 2 (7%) 1 (4%) 3 (10%) 3 (12%) 9 (8%) Score 3 2 (7%) 1 (4%) 1 (3%) 2 (8%) 6 (5%) housing in regular fattener parks. However, in the present experiment we found that neither the nest box panels nor position of the PVC pipe reduced skin damage. In both park types, moderate to severe skin injuries were observed. In a former pilot study, removal of the nest box panels in a combi park, creating a layout more comparable to that in regular fattening parks, reduced the severity of the skin injuries. Presence of the nest box panels walls in the combi park was thought to hinder does escaping from aggressive interactions after mixing, resulting in more skin damage. According to the former study, in which we studied the agonistic behaviour among does during group housing (Rommers et al., 2011), we observed that does could seek each other out to fight. Somehow the ranking order needs to be established, even though there are escape possibilities present. This implies that damage due to social conflicts will occur. Previous experiments at our institute (Rommers et al., 2005, 2010, 2014), as well as from others (Andrist et al., 2013; Maertens and Buijs, 2016), show that group housing repeatedly resulted in moderate to severe skin injuries. Minor injuries like scratches will heal within a couple of days (Kalle, 1994) and do not need that much attention. However, this is not the case with moderate and severe injuries, as deeper tissue is damaged. In our study, 5 to 6% of the does were severely injured (score 3, wounds). According to Andrist et al. (2013), skin injuries can vary between farms; our data were collected at one farm. In an earlier work, less moderate to severe injuries were observed at weaning compared to few days after mixing (Rommers and de Greef, 2017), which is in agreement with other studies (Andrist et al., 2012; Zomeño, 2017). However, in the current experiment the severity of the skin injuries increased from four days after mixing until weaning, as shown by the significant trend lines. There is no obvious explanation for this. From an ethological point of view, this is not expected, as damaging aggressive interactions should be reduced after a social hierarchy is set. It also contrasts with the results of the mentioned 5 earlier pilots (Rommers and de Greef, 2017). In this dataset, the later scores were generally lower than the scores 4 d after mixing, with a trend line amounting to 0.4168 x+0.4124 (r2=0.22). Thus, there is considerable variation among parks, but the general (significant) trend in this variability is diÄerent in the present experiment (increasing damage in time) than expected from social interactions and observed in earlier work (decrease of damage in time). Social dynamics in the parks with relatively high incidences of wounds deserve deeper analysis. The question arises of whether management measures can be taken to minimise negative social interactions. Buijs et al. (2016) tried to access the character of a doe by using the ano-genital distance in young rabbits and studied its relation to adult agonistic behaviour. Female rabbits with a greater ano-genital distance at birth showed more

Table 3b: Number and percentage of injured does for the diÄerent treatments at weaning according to the severity of injury (based on the highest injury score per doe). Nest box panels with without Position PVC pipe transversal longitudinal transversal longitudinal Total Number of does: Score 0 10 (35%) 14 (56%) 16 (53%) 11 (46%) 51 (47%) Score 1 13 (45%) 8 (32%) 9 (30%) 7 (29%) 37 (34%) Score 2 5 (17%) 2 (8%) 4 (13%) 2 (8%) 13 (12%) Score 3 1 (3%) 1 (4%) 1 (3%) 4 (17%) 7 (6%)

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oÄensive agonistic behaviour upon grouping as adults. They concluded that the ano-genital distance measurements may be useful when selecting less aggressive breeding rabbits, which may be helpful to breed rabbits suitable for group housing systems. There are indications that group stability has an eÄect on agonistic interactions and skin lesions (Andrist et al., 2012). Group composition could be another factor providing an intervention tool to minimise agonistic interactions. In our experiment we used a homogeneous population of lactating does. They were all multiparous and pregnant of the following litter. This might not be the optimal group composition compared to a stable group hierarchy. To our knowledge, there is only little information about group composition. Pilots performed by Maticz et al. (2017) indicated that mixing young does with a multiparous doe resulted in fewer skin injuries as compared to mixing does of similar age. Earlier grouping before kindling, so that does become acquainted, might also be a factor that can be taken into account. According to Zomeño et al. (2017), aggressive interactions were reduced when does were grouped beforehand, from eight to 2 d before kindling. Group housing during rearing, in order to prepare the does for group housing in production, might be worthwhile investigating. Finally, there could be genetic diÄerences in social behaviour which are as yet unknown. Part-time group housing of lactating rabbit does also has some positive eÄects. Positive social interactions can be observed, such as grooming of group mates and resting in close body contact with each other (Rommers et al., 2011). In group housing systems, animals have more space to move and can perform normal locomotion, like hopping and running. In addition, more enrichment material, such as pipes and panels can be oÄered. Nevertheless, it seems inevitable that with the current rabbit breeds, group housing will result in moderate to severe injuries. It remains an ethical discussion whether or not the positive eÄects of group housing outweigh the presence of severe injuries that have a clear negative impact on rabbit welfare.

CONCLUSIONS

1. Part-time group housing of lactating rabbit does is associated with occurrence of moderate to severe skin damage. In our study, 5 to 6% of the does were severely injured and had wounds. 2. There was a positive relationship between the average skin injury per park at four days after grouping and at weaning. On average, the mean injury score per park increased from grouping to weaning, but there were diÄerences between individual parks. 3. The presence of nest box panels in a combi park as well as the position of the shelter pipe underneath the platform did not aÄect the level of skin damage. 4. Social dynamics of group housed does is insu§ciently understood to further reduce damaging behaviour.

Acknowledgement: This study was ﬁnanced by the PPS “Duurzame konijnenhouderij” (TKI-AF-15234) and the Ministry of Economic AÄairs. We thank Mr and Mrs Slegh, at whose farm the experiment was conducted. Without their help this experiment would not have been possible.

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Kalle, G. 1994. Kaninchen in Gruppenhaltung. DGS, 25, 16-20. Rommers J.M., Reuvekamp B.J.F., Gunnink H., de Jong, I.C. 2014. Maertens L., Buijs S. 2016. Impact of housing system (cage vs. EÄect of hiding places, straw and territory on aggression in part-time housing) and ﬂoor type on rabbit doe welfare. In: group-housed rabbit does. Appl. Anim. Behav. Sci.,157: 117- Proceedings of 11th World Rabbit Congress, Qingdao (China), 126. https://doi.org/10.1016/j.applanim.2014.05.011 707-710. Rommers, J.M., de Jong, I.C., Reuvekamp, B., de Greef, K.H. Matics Zs., Sendrö Zs., Radnai I., Farkas T.P., Kasza R. et al. 2017. 2014b. Onderzoek naar groepshuisvesting van voedsters in ANIWA- Experimental results at Kapsvár Univeristy. In: Proc. parken binnen de PPS duurzame konijnenhouderij. Rapport 20th International Symposium on Housing and Diseases of 749, Wageningen UR Livestock Research, Lelystad, The Rabbits, Fur providing animals and Pet animals. Celle, 17-18 Netherlands. March 2017, 27-36. Rommers J., de Greef K. 2017. Towards part-time group housing th Rommers, J.M., de Jong, I.C. 2005. De haalbaarheid van of lactating rabbit does? In Proc.: 20 International Symposium groepshuisvesting voor voedsters in de praktijk. ASG rapport on Housing and Diseases of Rabbits, Fur providing animals ASG 05/102047. Wageningen UR Livestock Research, and Pet animals. Celle, 17-18 March 2017, 3-13. Lelystad, The Netherlands. Rommers J., de Greef K. 2018. Zijn combi-parken even geschikt Rommers, J.M., de Jong, I.C. 2010. Haltung von Häsinnen: als parken voor parttime groepshuisvesting van voedsters? Gruppen- und Einzelnhaltung kombiniert. DGS Magazin, 22, KonijnenWijzer, in press. 55-58. Szendrő, Z., McNitt, J., Matics, Z., Mikó, A., Gerencsér, Z. Rommers J., Gunnink H., Klop A., de Jong I. 2011. Dynamics in 2016. Alternative and enriched housing systems for aggressive behaviour of rabbit does in a group housing system: breeding does: a review. World Rabbit Sci., 24, 1-14. a descriptive study. In Proc.: 17th International Symposium on https://doi.org/10.4995/wrs.2016.3801 Housing and Diseases of Rabbits, Furproviding Animals and Vastrade F.M. 1987. Spacing behaviour of free-ranging domestic Pet animals, 11-12 May, Celle, 75-85. rabbits, Oryctolagus cuniculus L. Appl. Anim. Behav. Sci., 18: Rommers J.M., Reuvekamp B.F.J., Gunnink H., de Jong I.C. 2013. 185-195. https://d oi.org/10.1016/0168-1591(87)90192-4 EÄect of diÄerent hiding places on aggression among does in Zomeño C., Birolo M., ZuÄellatro A., Xiccato G., Trocino A. 2017. a group-housing system: a pilot. In Proc.: of 18th International Aggressiveness in group-housed rabbit does: Inﬂuence of group Symposium on Housing and Diseases of Rabbits, Furproviding size and pen characteristics. Appl. Anim. Behav. Sci., 194: Animals and Pet animals, 22-23 May, Celle, 59-68. 79-85. https://doi.org/10.1016/j.applanim.2017.05.016

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W orld World Rabbit Sci. 2018, 26: 307-312 R abbit doi:10.4995/wrs.2018.10225 Science © WRSA, UPV, 2003

MOLECULAR FEATURES OF HEPATITIS E VIRUS FROM FARMED RABBITS IN SHANDONG PROVINCE, CHINA ZHANG H.*, ZHOU Y.†, LIU J.‡

*Department of Teaching Affairs, Hebei University of Economics and Business, SHIJIAZHUANG CITY 050000, China. †Center for Disease Control, Daiyue Bureau of Animal Husbandry, TAI’AN CITY 271000, China. ‡Institute of Microbiology Research, Baolai-Leelai Bio-Tech. Ltd., TAI’AN CITY 271000, China.

Abstract: This study was undertaken to investigate the genetic variability of hepatitis E virus (HEV) from farmed rabbits in Shandong province, China. A total of 50 fresh faecal samples from 5 rabbit farms were collected and subjected to reverse transcription and nested polymerase chain reaction (RT-nPCR) for a fragment sequence of HEV capsid gene. The results demonstrated that HEV RNA was observed in 6 faecal samples (6/50, 12.0%). In addition, the result of phylogenetic analysis showed that the 6 HEV isolates were classified into HEV-3 genotype with other rabbit HEV isolates from other countries, and shared 85.2-87.2%, 81.5-83.1%, and 77.0-78.6% nucleotide similarities with rabbit HEV isolates from Korea, the United States and France, respectively. To sum up, the HEV isolated in this study from farmed rabbits belongs to the HEV-3 genotype, and the zoonotic ability and pathogenesis of the rabbit HEV merit further study due to the fact that HEV-3 genotype has the potential to trigger zoonotic infections. Key Words: HEV, farmed rabbit, RT-nPCR, phylogenetic analysis, nucleotide identity.

INTRODUCTION

Hepatitis E virus falls into the genus Orthohepevirus of the family Hepeviridae. The genus Orthohepevirus is composed of 4 species (Orthohepevirus A to Orthohepevirus D), and Orthohepevirus A comprises 7 genotypes (HEV-1 to HEV-7) (Johne et al., 2014; Smith et al., 2014; Krzowska-Firych et al., 2018). Among the 7 genotypes, HEV-1 and HEV-2 can only infect people and the infection route is mainly associated with drinking water contaminated by HEV (Purcell and Emerson, 2008; Pérez-Gracia et al., 2014; King et al., 2018). In comparison, HEV-3 and HEV-4, recognised as zoonotic pathogens (Meng, 2010a, 2011), not only infect people but can also infect many animal species, such as pigs, rabbits and deer, and the main infection route may be related with HEV-contaminated animal meats or internal organs (Tei et al., 2003; Yazaki et al., 2003; Zhao et al., 2009; Meng, 2010b; Pavio et al., 2015; Li et al., 2017). Wild boars have become an important reservoir for HEV-5 and HEV-6; HEV-7 has been isolated from camels, and has the potential to infect people (Woo et al., 2014; Lee et al., 2016; Sridhar et al., 2017). Although hepatitis caused by HEV is usually self-limiting, the HEV infection can result in chronic hepatitis among patients with immunosuppression (Emerson and Purcell, 2003; Kamar et al., 2008; Fujiwara et al., 2014; Frias et al., 2018). So far, HEV infections among rabbits have been detected in several countries, such as China (Zhao et al., 2009), the United States (Cossaboom et al., 2011, 2012), France (Izopet et al., 2012), Germany (Eiden et al., 2016), Italy (Di Bartolo et al., 2016) and Korea (Ahn et al., 2017). For example, the detection rates of HEV RNA in farmed rabbit serum samples in China, the United States and Mongolia were 2-7.5%, 16%, and 71.6%, respectively (Cossaboom et al., 2011; Geng et al., 2011; Jirintai et al., 2012; Zhao et al., 2009). The prevalence of HEV RNA in wild rabbit

Correspondence: H Zhang, [email protected]. Received May 2018 - Accepted September 2018. https://doi.org/10.4995/wrs.2018.10225

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liver samples was 5-23% and 60% in France and the Netherlands, respectively (Izopet et al., 2012; Lhomme et al., 2015; Burt et al., 2016). HEV isolates from rabbits have a genome of approximately 7.2 kb, containing 3 open reading frames, genetically close to HEV-3 (Cossaboom et al., 2012; Smith et al., 2014). Rabbit HEV can not only cause mild hepatitis in rabbits (Liu et al., 2013), but can also replicate in A549 and PLC/PRF/5 cells of human origin (Jirintai et al., 2012). In addition, scientists have found a rabbit HEV isolate (HEV-3 genotype) genetically close to human HEV in France, indicating that zoonotic transmission of the rabbit HEV-3 may be possible (Izopet et al., 2012). As an endemic country for hepatitis E (Syed et al., 2018), many epidemiological investigations into anti-HEV antibody in various animal species have been carried out in China, including pigs, deer, chickens, goats, ferrets, minks and cows (Zheng et al., 2006; Geng et al., 2011; Zhang et al., 2015; Huang et al., 2016; Li et al., 2017; Shuai et al., 2017; Wang et al., 2018). However, to our best knowledge, relatively little information about genetic variability of HEV isolated in farmed rabbits has been reported in China to date. Therefore, the current study aimed to investigate whether HEV RNA was present or diÄuse in farmed rabbits in Shandong province, China, and to further investigate the molecular features.

MATERIALS AND METHODS

Collection and treatment of rabbit faeces From March to July, 2017, fresh faecal samples were randomly obtained from 50 New Zealand white breed rabbits from 5 rabbit farms in Shandong province, China (10 rabbits from each farm). The rabbits were raised for meat consumption. The rabbits were between 8 and 10 wk (mean 9 wk) of age, and the animals reared in the 5 farms appeared to be healthy. A sample of 2 grams of fresh faeces taken from an individual rabbit was mixed with 20 mL of phosphate-buÄered saline (PBS) and then centrifuged for 30 min at 1200×g. The supernatant was centrifuged for 10 min at 16000×g. The supernatant was stored at −70°C for RNA extraction.

RT-nPCR and sequencing Based on the manufacturer’s instructions, 200 µL of supernatant was used to extract HEV RNA with a RNA kit (Invitrogen, USA). Subsequently, a specific primer targeting the HEV capsid gene was used to amplify cDNA using a commercial RT-PCR kit (Invitrogen, USA). The generated cDNA was stored at −20°C for further study. A reverse transcription and nested polymerase chain reaction (RT-nPCR) method was used to detect the partial fragment of capsid gene of HEV (Ahn et al., 2017). The first PCR reaction condition with specific external primers (Table 1) was as follows: 95°C (5 min), 40 cycles of 95°C (30 s), 50°C (30 s), and 72°C (30 s), followed by an extension for 10 min at 72°C. Subsequently, the first PCR product was subjected to the second PCR with specific internal primers (Table 1), and the amplification condition was as follows: 95°C (5 min), 35 cycles of 95°C (30 s), 56°C (30 s), and 72°C (30 s), followed by an extension for 10 min at 72°C. The second PCR products were first cloned into a Cloning Vector, and then transformed into Competent Cells DH-5α. The positive plasmid was selected and sequenced.

Table 1: Primers used in this study for detecting a fragment sequence of Rabbit HEV. Primers Sequence (5’→3’) Location* External forward primer CCG ACA GAA TTG ATT TCG GC 6,376-6,398 External reverse primer CAR AGT GAC YTT AGA CCA ATC AAG 6,766-6,789 Internal forward primer GTC TCA GCC AAT GGC GAG CC 6, 427-6,429 Internal reverse primer GCR CCT GTT GCS ACA TTR ACA AAT 6,723-6,746 *Primer location was expressed according to the genome of reference strain HEV FJ906895.

308 World Rabbit Sci. 26: 307-312 MOLECULAR FEATURES OF HEPATITIS E VIRUS FROM FARMED RABBITS IN SHANDONG PROVINCE, CHINA

Phylogenetic tree construction and identity analysis A phylogenetic tree was constructed in this study using the neighbour-joining method, following the instructions of the MEGA 7.0 software package. Reference nucleotide sequences of diÄerent HEV genotypes were obtained from GenBank. The nucleotide sequence identity of HEV was also analysed using Bioedit 7.0 software.

RESULTS

A total of 50 fresh manure samples were obtained from 5 rabbit farms in Shandong province, China. The RT-nPCR results showed that 6 out of 50 samples (6/50, 12.0%) were positive for the fragment of HEV capsid gene (243 bp). The 6 HEV positive samples came from 3 rabbit farms (3/5, 60.0%) (Table 2).

Figure 1: Phylogenetic analysis of a fragment of capsid gene of rabbit HEV. The 6 rabbit HEV isolates in this study are indicated as black triangles.

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Table 2: Positive HEV RNA rates in faecal samples of farmed rabbits. Rabbit farms Number of faecal sample Number of HEV RNA-positive samples Positive rate (%) A 10 2 20.0 B 10 1 10.0 C 10 0 - D 10 3 30.0 E 10 0 - Total 50 6 12.0

Phylogenetic Analysis and nucleotide identity showed that these 6 HEV isolates, designated as SD/China/Rabbit/1-6, were classiﬁed into HEV genotype 3 (Figure 1), and shared 85.2–87.2%, 81.5–83.1%, and 77.0–78.6% nucleotide similarities with rabbit HEV isolates from Korea, the United States and France, respectively.

DISCUSSION

Numerous studies have been conducted in several countries to investigate the genetic variability of HEV RNA in rabbits (Zhao et al., 2009; Cossaboom et al., 2011; Izopet et al., 2012; Jirintai et al., 2012), and the HEV RNA shared close similarity with the HEV-3 genotype (Zhao et al., 2009; Ahn et al., 2017; Kaiser et al., 2018; Ryll et al., 2018). Similarly, HEV RNA isolated in this study can be classified into HEV genotype 3. In addition, a study carried out in Germany showed that HEV RNA was detected in the archived sera in 1989 (Eiden et al., 2016). The research result indicated that HEV infections in rabbits may have occurred long before rabbit HEV was first detected in China (Zhao et al., 2009). In the current study, the occurrence of HEV in farmed rabbits was detected by RT-nPCR. The results showed that 3 out of 5 sampled rabbit farms (60.0%) were positive for HEV and 12.0% of the collected faecal samples carried HEV RNA. In the United States and Korea, HEV strains have also been found in 15.0 and 17.0% of manure samples of farmed rabbits, respectively (Cossaboom et al., 2011; Ahn et al., 2017). In addition, a previous study conducted in Beijing, China reported that 6 out of 492 faecal samples of farmed rabbits (5.0%) were positive for HEV RNA (Xia et al., 2015). The discrepancy of detection rate of HEV RNA in faecal samples in diÄerent studies may be associated with many factors, such as rabbit breed, rabbit age and rearing environments (Cossaboom et al., 2011). The sampling size in this study was relatively small (only 50 samples from 5 rabbit farms), which was a major limitation of this study, but the findings to some extent reflected the occurrence of HEV in farmed rabbit faeces in Shandong province, China. In addition, it is noteworthy that rabbit HEV isolated in this study belonged to genotype 3 HEV, which has the potential to cause zoonotic infection (Lhomme et al., 2013). Thus, the risk for cross-species and zoonotic infections with rabbit HEV should be evaluated, and the need to carry out extensive molecular epidemiological research into rabbit HEV merits serious consideration.

Acknowledgements: This study was supported by the Intramural Fund of Hebei University of Economics and Business in 2018 (2018PY17) and the Development Plan of Science and Technology of Tai’an, China (2016NS1051).

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312 World Rabbit Sci. 26: 307-312 W orld World Rabbit Sci. 2018, 26: 313-320 R abbit doi:10.4995/wrs.2018.9823 Science © WRSA, UPV, 2003

HAEMATOLOGICAL RESPONSE IN THE TREATMENT OF NATURALLY ACQUIRED ECTOPARASITE INFESTATIONS IN RABBITS MOONARMART W.*, TANSAKUL M.†, KIEWSIRI C.‡, WATANABOONCHAI R.‡, SOMRITH W.§, YINHARNMINGMONGKOL C.*, TUNHIKORN M.*

*Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Salaya, NAKHON PATHOM 73170, Thailand. †Graduate Program in Translational Medicine, Research Center, Faculty of Medicine Ramathibodi Hospital, 270 Rama VI Road, Ratchatewi, BANGKOK 10400, Thailand. ‡Veterinary students, Faculty of Veterinary Science, Mahidol University, Salaya, NAKHON PATHOM 73170, Thailand. §Pasupalan Livestock & Wildlife Hospital, Faculty of Veterinary Science, Mahidol University Kanchanaburi Campus, Sai Yok, KANCHANABURI 71150, Thailand.

Abstract: The objective of this study was to determine changes in haematological values of topical administration of selamectin in rabbits with at least one species of naturally acquired ectoparasite infestation (Sarcoptes scabiei var. cuniculi, Psoroptes cuniculi, or Cheyletiella spp). Thirty-five rabbits were enrolled. They underwent physical examination and assessment of ectoparasite infestations on days 0, 14, 28, 56 and 86. Blood for haematology and serum biochemistry was collected on days 0, 14, 28 and 56. Selamectin was topically applied at a dose of 15 mg/kg onto the skin on days 0, 14 and 28, respectively. No ectoparasites or eggs were found in these rabbits from day 28 onwards by skin scraping and/or tape cytology. Haematology and serum biochemistry values were within normal limit throughout the study. However, the neutrophil to lymphocyte ratio significantly decreased post-treatment from 2.89 (0.90-5.47) on day 0 to 1.38 (0.56-3.09), and 1.44 (0.42-4.47) on days 14 and 56, respectively. There were no adverse drug reactions or treatment- related mortalities during the study. This study indicated that selamectin was effective and safe in the treatment and prevent re-infestation for at least 58 d post-treatment. Moreover, the neutrophil to lymphocyte ratio could be used for monitoring of inflammatory response in rabbits. Key Words: ectoparasite, haematology, lymphocyte, neutrophil, rabbit, selamectin.

INTRODUCTION

Rabbits have not only been used as pets or laboratory animals, but also for meat production. Most commercial rabbit farms in Thailand are small in size, where appropriate wellness care programmes and farm management are neglected. Control of ectoparasite infestations in rabbits has been overlooked, which leads to reduction in production and increase husbandry costs (Mikled et al., 2008). To date, reports on the prevalence of ectoparasites in rabbits in Thailand and Southeast Asia are limited. However, anecdotal ﬁndings of ectoparasites in veterinary practices included Psoroptes cuniculi, Sarcoptes scabiei var. cuniculi, and Cheyletiella parasitovorax. Selamectin (Revolution®, Zoetis Inc., Kalamazoo, MI, USA), a macrocyclic lactone of the avermectin compound, is available as a spot-on formulation. It is a safe, broad-spectrum parasiticide approved for topical use in dogs and cats. However, selamectin is not licensed for treating ectoparasites in rabbits (Fisher et al., 2007). The extra-label use of selamectin for the treatment of ectoparasites and endoparasites in rabbits has been reviewed and suggested (Fisher

Correspondence: M. Tunhikorn, [email protected]. Received March 2018 - Accepted July 2018. https://doi.org/10.4995/wrs.2018.9823

World Rabbit Sci. 26: 313-320 313 MOONARMART et al.

et al., 2007), including Psoroptes cuniculi at a dose of 6 or 18 mg/kg, given once or twice with an interval of 28 d between treatments (McTier et al., 2003), or at doses of 6 to 18 mg/kg in one application (Kurtdede et al., 2007); Sarcoptes scabiei var. cuniculi at doses of 8 to 14 mg/kg twice, 30 d apart (Farmaki et al., 2009) or at doses of 8 to 14 mg/kg once (Kurtdede et al., 2007); and Cheyletiella spp. at a dose of 12 mg/kg once (Kim et al., 2008) or at doses of 6.2-20.0 mg/kg, 1-3 times with an interval of 2-4 wk (Mellgren and Bergvall, 2008). Selamectin at a dose of 20 mg/kg given every week for 3 wk was e§cient for the treatment of ﬂea infestation in rabbits, with no adverse reaction (Carpenter et al., 2012). Data on haematological and serum biochemical parameters in healthy rabbits and rabbits with clinical diseases have increased, together with the increasing popularity of rabbits as pets (Hinton et al., 1982; Archetti et al., 2008; Özkan et al., 2012), but also in rabbit farmed for meat production (Belhassen et al., 2016; Stancu et al., 2017; Sun et al., 2018). The aim of the study was to investigate changes in haematology and selected biochemistry in the use of topical administration of selamectin for the treatment of naturally acquired ectoparasite infestations in farm rabbits.

MATERIALS AND METHODS

Animal care The protocol used in the present study was approved by the Faculty of Veterinary Science Animal Care and Use Committee, no. MUVS-2015-18, Mahidol University, Thailand. This was a prospective, non-controlled study carried out from June to September 2015.

Animals, farm and management To be eligible for inclusion, a rabbit had to be older than 2 mo, had at least one of these ectoparasite infestations (Psoroptes cuniculi, Sarcoptes scabiei var. cuniculi, and Cheyletiella spp), and had not been treated with ectoparasiticides, antibiotics, steroidal anti-inflammatory drugs, or non-steroidal anti-inflammatory drugs, in the last 90 d before the study. Rabbits were excluded from the study if they were pregnant or suÄering from severe systemic illness. Power calculations for study population size were performed by G*Power version 3.1.7 (Heinrich-Heine- Universität Düsseldorf, http://www.gpower.hhu.de). For the power analysis on a repeated measures ANOVA with 4 measurements, a power of 0.80, an alpha level of 0.05, and a medium eÄect size (f=0.25) (Faul et al., 2013), the required sample size was 24. Thirty-five rabbits recruited into this study were obtained from a commercial rabbit farm in the rural area of Karnchanaburi province, the western region of Thailand. There were 13 males and 22 females, mixed breed, the average±standard deviation (SD) of age was 15.57±5.42 mo (ranged from 5 to 24 mo), and the average±SD of body weight in the first assessment on day 0 (pre-treatment) was 2.54±0.60 kg (ranged from 1.16 to 3.6 kg). These rabbits were housed in wire cages individually with natural atmosphere and ventilation. They were given ad libitum access to commercial diet of approximately 14-16% protein and water. Rabbits underwent physical examination from head to tail, starting from observation of their attitude, mental alertness, skin and hair coat, respiration, and posture. Hydration status was evaluated by skin tent and capillary refill time (CRT). Mucous membrane was assessed by examination of the conjunctiva or oral cavity. Palpation of the thoracic cage, abdomen, and extremities was carried out. Auscultation of the heart and lungs was also performed. All rabbits underwent physical examination and their body weight was weighed in the same way on days 0, 14, 28, 56 and 86.

Assessment of ectoparasite infestations The rabbits enrolled in the present study had at least one ectoparasite infestation assessed by detection of live mites from both skin scrapings and tape cytology between 4 to 8 sites. Skin scrapings and tape cytology were taken from each rabbit from sites showing the most likelihood for ectoparasite infestation; for example, ear margins, feet, and pruritic and/or lesioned skin (any crusts, scales, papules, erythema, and alopecia). Each skin scraping or tape cytology was carried out over an area of about one square inch. Skin scraping was performed to an approximately constant depth, following one or two drops of mineral oil placed on the skin. The material obtained was transferred to

314 World Rabbit Sci. 26: 313-320 HAEMATOLOGY IN ECTOPARASITOSIS TREATMENT IN RABBITS mineral oil on microscope slides. These slides and tape cytology were examined by microscopic examination under low power magniﬁcation (×4 or ×10). The severity of the clinical manifestations of aÄected areas were classiﬁed to be “absent” if no abnormality detected; “mild” if the intensity of the manifestation was low and only a small area of the body was aÄected; “moderate” if the manifestation was great intensity over a small area or a lower intensity over a large area of the body; and “severe” if the manifestation was great intensity over a large area of the body. AÄected areas of the body were recorded as ear (including head and neck), body (thorax and abdomen) and extremities.

Blood collections and evaluations A total of 2 mL of blood was collected from the lateral saphenous vein of each rabbit into tripotassium ethylenediaminetetraacetic acid (K3-EDTA)-treated and plain tubes to evaluate haematology and selected serum biochemistry parameters (alanine aminotransferase [ALT], aspartate aminotransferase [AST], blood urea nitrogen [BUN], and creatinine) on days 0, 14, 28 and 56. All blood samples were collected in the afternoon between 12:00 pm to 6:00 pm. A 24-gauge sterile needle was used in every rabbit. Serum was separated from blood within 30 min of collection. Blood in K3-EDTA-treated and serum samples were transported to the laboratory at 4°C within 18 h for haematological and biochemical analyses. Haematological parameters were evaluated by Animal Blood Counter ABCTM (Horiba ABX Diagnostic (Thailand) Ltd., Bangkok, Thailand). Recommended settings and calibration for rabbit haematology were applied according to the manufacturer’s protocol. Serum biochemistry parameters were assessed by a Sapphire 400 Auto-Chemistry Analyzer (D.A.P. Siam Group Ltd, Bangkok, Thailand) using kits by the same company. The parameters and the respective methods employed are as follows: ALT/AST–kinetic 37°C/kinetic without pyridoxal method, BUN–enzyme kinetic method, and creatinine–JaÄe kinetic method.

Treatment Selamectin (Revolution® Green for dogs, each tube containing 2 mL of 12% selamectin (120 mg/mL), Zoetis Inc, Kalamazoo, MI, USA) was topically applied onto the skin at the base of the neck at a dose of 15 mg/kg body weight following physical examination, skin scrapings, tape cytology and blood collection. Repeated treatments were performed in the same manner on days 14 and 28 in order to kill all adults of Psoroptes cuniculi, Sarcoptes scabiei var. cuniculi, and Cheyletiella parasitovorax and to prevent re-infestation from egg to adult of these ectoparasites.

Statistical analysis Computerised statistical software (SPSS 18.0 for Windows, Chicago, IL, USA) was used for analyses. Probabilities <0.05 were considered statistical signiﬁcant. The numerical parameters obtained from each day of examination were tested for normality by the Shapiro-Wilk test. Comparisons of numerical variables on days 0, 14, 28, 56 and 86 were performed within rabbits by repeated measure ANOVA with a Bonferroni comparison for post-hoc analyses. The Friedman test was carried out to compare the severity of the clinical manifestations of each aÄected area within the subjects on days 0, 14, 28, 56 and 86.

RESULTS

Distribution of number of rabbits with ectoparasite infestations and percentage of parasitological cure rate obtained from day 0, 14, 28, 56 and 86 are illustrated in Table 1. There were no adverse drug reactions or treatment-related mortalities during the study. Ectoparasites were still found in three rabbits on day 14 with either of Psoroptes cuniculi, Sarcoptes scabiei var. cuniculi, or both Sarcoptes scabiei var. cuniculi and Cheyletiella spp. No ectoparasites or eggs were found from skin scrapings or tape cytology in all rabbits from day 28 onwards. No evidence of recurrent infestation on day 58 following the third treatment on day 28 was observed. The severity of the clinical manifestations of aÄected areas was signiﬁcantly decreased with time (Table 2, all P<0.01). Friedman test was used to compared the severity of the clinical manifestations within the subjects on days 0, 14, 28, 56 and 86. There were signiﬁcantly diÄerent obtained from all areas between day 0 and day 14, day 0 and day 28,

World Rabbit Sci. 26: 313-320 315 MOONARMART et al.

Table 1: E§cacy of selamectin against natural infestation of ectoparasites in 35 rabbits. Number of rabbits with ectoparasite infestations and (%) of parasitological cure rate Type of infestations Day 0 Day 14 Day 28 Day 56 Day 86 Psoroptes cuniculi 1 (0) 1 (0) 0 (100) 0 (100) 0 (100) Sarcoptes scabiei var. cuniculi 16 (0) 1 (93.75) 0 (100) 0 (100) 0 (100) Cheyletiella spp. 1 (0) 0 (100) 0 (100) 0 (100) 0 (100) Psoroptes cuniculi & Cheyletiella spp. 1 (0) 0 (100) 0 (100) 0 (100) 0 (100) Sarcoptes scabiei var. cuniculi & Cheyletiella spp. 16 (0) 1 (93.75) 0 (100) 0 (100) 0 (100) Selamectin 15 mg/kg was administered topically in a single spot following physical examination, skin scrapings and tape cytology on day 0, 14 and 28, respectively. Results display numbers of rabbits with ectoparasites present and percentages (in brackets) of parasitological cure rate obtained from days 0, 14, 28, 56 and 86, respectively.

day 0 and day 56, day 0 and day 86, day 14 and day 28, day 14 and day 56 and day 14 and day 86 (all P<0.01). No diÄerences were obtained from any areas between day 28 and 56, day 28 and 86, and day 56 and day 86) (P>0.05). Haematological values and serum biochemistry values; ALT, AST, BUN and creatinine on days 0, 14, 28 and 56 are shown in Table 3. Haematological parameters obtained from day 0, 14, 28 and 56 were in accordance with those of healthy rabbits in previous studies (Hinton et al., 1982; Archetti et al., 2008; Özkan et al., 2012). However, the total white blood cell (WBC) count measured on day 0 was higher than on day 28 (P=0.005). Neutrophils, monocytes, and neutrophil/lymphocyte ratio were higher on day 0 when compared to days 14 (P<0.01), 28 (P=0.03) and 56 (P<0.01), whereas lymphocytes, erythrocytes, haemoglobin, and packed cell volume (PCV) were lower on day 0. The AST, BUN and creatinine values in the present study overlapped those reported by other authors in healthy rabbits (Özkan et al., 2012; Varga, 2014). However, serum ALT levels obtained from 25% of these rabbits were higher than those reported by Özkan et al. (2012) and Varga (2014). Interestingly, BUN was lower on day 56 when compared to days 0 (P=0.002), 14 (P=0.04) and 28 (P=0.001) respectively, which was similar to the changes in creatinine levels. The body weight of the rabbits was signiﬁcantly increased with time during the study (P<0.01, Figure 1). Mean±SD of the body weight of the rabbits on days 0, 14, 28, 56 and 86 were 2.54±0.60 kg, 2.72±0.62 kg, 2.87±0.56 kg, 3.01±0.53 kg, and 3.13±0.53 kg, respectively.

Table 2: Severity of the clinical manifestations, number of rabbits and percentages (in braquets), on days 0, 14, 28, 56 and 86. AÄected area Day 0 Day 14 Day 28 Day 56 Day 86 P Ear (including head and neck) <0.01 Absent 3 (8.6) 14 (40.0) 35 (100.0) 35 (100.0) 35 (100.0) Mild 11 (31.4) 18 (51.4) 0 (0) 0 (0) 0 (0) Moderate 8 (22.9) 2 (5.7) 0 (0) 0 (0) 0 (0) Severe 13 (37.1) 1 (2.9) 0 (0) 0 (0) 0 (0) Body (thorax and abdomen) <0.01 Absent 9 (25.7) 23 (65.7) 35 (100.0) 35 (100.0) 35 (100.0) Mild 22 (62.9) 12 (34.3) 0 (0) 0 (0) 0 (0) Moderate 2 (5.7) 0 (0) 0 (0) 0 (0) 0 (0) Severe 2 (5.7) 0 (0) 0 (0) 0 (0) 0 (0) Extremities <0.01 Absent 1 (2.9) 18 (51.4) 34 (97.1) 35 (100.0) 35 (100.0) Mild 19 (54.3) 16 (45.7) 1 (2.9) 0 (0) 0 (0) Moderate 6 (17.1) 1 (2.9) 0 (0) 0 (0) 0 (0) Severe 9 (25.7) 0 (0) 0 (0) 0 (0) 0 (0) The severity of the clinical manifestations of aÄected areas were classiﬁed as absent, mild, moderate, and severe on days 0, 14, 28, 56 and 86.

316 World Rabbit Sci. 26: 313-320 HAEMATOLOGY IN ECTOPARASITOSIS TREATMENT IN RABBITS 0.001 <0.01 d 0.001 0.001 <0.01 c c c 0.04 b 0.001 b c=d=e <0.01 <0.01 <0.01

0.04 0.03, 0.04, 0.006, 0.01 0.03, c 0.005 b a b b a a=b 0.04, <0.01, b P (post-hoc) a=b=c=d a=b=c=d a=b=c=d <0.01, a=c a=c=d 0.002, <0.01, <0.01, <0.01, a a a 0.01, a a,b 0.11 P (model) 0 (7-21) <0.01 (35-51) <0.01 (49-632) <0.01 a (212-662) 0.005 (0.7-1.4) <0.01 (3.6-13.0) 0.008 a b (5.54-8.37) <0.01 (0.42-4.47) <0.01 (11.1-16.7) <0.01 (16.8-22.9) (31.6-34.4) <0.01 <0.01 a Day 56 (1568-8060) 0.02 (1440–5200) <0.01 c c c a b ab a ab c 14 2.2 (0 -77) 0.38 25 (0-154) 0.31 62 (53-67) 0.06 14 (5-192) 0.23 43 69 (21-209) 0.37 0 (36-528) 212 (10-32) (30-47) (9.6-15.5) 14.1 (0.7-1.3) 1.0 a (16.7-22.8) (31.4-33.6) 20.5 33.0 (159-680) 411 (462-4736) 2954 a b (1064-9424) 3770 (5.09-7.76) 6.90 (3.5-12.4) 7.0 b b Day 28 a a (0.40-12.14) 1.44 a a bc bc 21 (8-53) a ab 0.06 (0-1) 16 (0-189) 62 (53-70) 39 17 63 (28-208) 1.0 137 6.2 396 12.6 6.26 2270 20.2 32.6 2.03 3823 0 0 (35-50) (10-20) (199-920) (0.7-1.5) (4.0-13.5) (65-1080) b b (5.60-8.30) (10.5-15.0) (15.6-20.7) (28.0-31.0) (0.56-3.09) c Day 14 (1300-4860) (1628-7560) b 8 (0-85) a a a b b ab a b ab 62 (54-68) 32 (8-128) 43 16 70 (33-129) 1.2 320 7.0 463 18.2 29.6 1.38 7.00 12.8 3770 2882 0 0 (11-30) (28-44) (8.8-14.7) (0.3-1.7) Day 0 (160-887) (60-1443) a b (16.2-22.1) (29.9-34.1) (3.5-12.6) (0.90-5.47) (4.77-7.25) c a (2065-9828) (1007-3999) b b a b b b b a b 22 (0-204) 61 (54-65) 60 (8-113) 26 (3-100) 37 17 1.2 7.4 484 354 12.0 6.09 32.5 19.8 5070 /L) 6 /L) /L) 1960 6 12 /L) /L) /L) 6 6 6 /L) 6 /L) 9 /L) 9 Monocytes (10 PCV (%) Neutrophils (10 Neutrophils WBC (10 Neutrophil/lymphocyte ratioNeutrophil/lymphocyte 2.89 Lymphocytes (10 Lymphocytes Creatinine (mg/dL) Creatinine Haematological parameters, ALT, AST, BUN, and creatinine obtained from day 0, 14, 28 and 56. obtained from BUN, and creatinine AST, ALT, 3: Haematological parameters, Table Parameters Results are illustrated in mean (minimum-maximum). Statistical diÄerences between day 0, 14, 28 and 56 were tested alanine by repeated measure ANOVA with at P <0.05. signiﬁcantly diÄerent not sharing the same letter were a analyses. Means in a row ALT, Bonferroni count; comparison cell blood for white post-hoc total WBC, concentration; haemoglobin corpuscular mean MCHC, haemoglobin; corpuscular mean MCH, volume; corpuscular mean MCV, volume; cell packed PCV, nitrogen. BUN, blood urea aspartate aminotransferase, AST, aminotransferase; Haemoglobin (g/dL) MCV (fL) MCHC (g/dL) Platelets (10 ALT (IU/L) ALT Eosinophils (10 Band neutrophils (10 Band neutrophils Basophils (10 Erythrocytes (10 Erythrocytes MCH (pg) AST (IU/L) AST BUN (mg/dL)

World Rabbit Sci. 26: 313-320 317 MOONARMART et al.

DISCUSSION

Assessment of e§cacy of selamectin in the present study was based on ectoparasites detected by skin scrapings and tape cytology and its safety was evaluated from general examination, evidence of adverse reaction, haematological values, and selected parameters of serum biochemistry. The present study showed that 100% of the rabbits in our sample with natural ectoparasite infestations were cured after 2 consecutive treatments of selamectin topical application on day 0 and day 14, whereas one topical application was eÄective to cure 91.4%. The life Figure 1: Average of body weight of the rabbits. cycles from egg to adult of Sarcoptes scabiei var. cuniculi, Psoroptes cuniculi and Cheyletiella spp. are completed within 2-3 wk, 3 and 5 wk, respectively (Wall and Shearer, 2001). As the course of this study was extended to 58 d after the last topical application, this would allow the mite numbers to increase based on their life cycle from egg to adult. This study also suggested that three consecutive applications of selamectin at dose of 15 mg/kg body weight every 2 wk could prevent re-infestation by ectoparasites in rabbits for at least 58 d after the last application. Selamectin has been shown to be eÄective and safe against ectoparasites in rabbits as well as in dogs and cats (Shanks et al., 2000; Six et al., 2000; McTier et al., 2003; Curtis, 2004; Kurtdede et al., 2007). To date, there has been no report of adverse reaction or toxicity regarding the usage of a single spot-on of selamectin in rabbits (McTier et al., 2003; Kurtdede et al., 2007; Carpenter et al., 2012). We also found no adverse reactions associated with selamectin in the present study following each episode of 3 consecutive doses of 15 mg/kg body weight. This was the first study reporting changes in haematological parameters in the use of selamectin for the treatment of naturally acquired ectoparasite infestations in rabbits. The post-treatment haematological findings were significantly diÄerent from pre-treatment, including the increase in erythrocytes, PCV, haemoglobin, and lymphocytes, and the decrease in the total WBC count, neutrophils and monocytes. Reference ranges of normal haematological parameters reported from previous studies have varied (Etim et al., 2014). We therefore interpreted haematological parameters with reference to these reports (Hinton et al., 1982; Bortolotti et al., 1989; Archetti et al., 2008; Özkan et al., 2012; Varga, 2014; Cray, 2015; Moore et al., 2015) in conjunction with the normal hydration status obtained from physical examination. The total WBC count pre- and post- treatments from most of the rabbits in the present study was within normal reference range (Hinton et al., 1982; Bortolotti et al., 1989; Archetti et al., 2008), but it was significantly lower on day 28 when compared to pre-treatment. Leucocytosis in uncommon in rabbits, even in the presence of inflammation or infection (Toth and Krueger, 1988; Toth and January, 1990; Harcourt-Brown and Baker, 2001). These reports were in agreement with the present study, where total WBC count was not increased even in the pre-treatment rabbits. However, the neutrophil to lymphocyte ratio significantly decreased post-treatment, with nearly 1:1 on day 56 of the study. Monocyte counts obtained from every examination were within the normal limit of 2-10% of total WBC (Varga, 2014), but they decreased significantly post-treatment. This may suggest that chronic inflammation aÄected these rabbits before treatment. Interestingly, eosinophils did not change over the period of the study and values were within the normal limit of 0-5% for total WBC. Eosinophilia is linked to parasitic disease in other species (Melillo, 2007; Varga, 2014), but not in this study. Taken together, we would suggest that the neutrophil to lymphocyte ratio is the most predictive value for the inflammation response and ectoparasite infestations in rabbits. It was reported that the normal ratio of neutrophils to lymphocytes in rabbit over one year of age was approximately 1:1 (Varga, 2014). Stress and diseases can influence this ratio, as it was a method suggested to predict health of rabbits and can be applied to predict diseased status in these rabbits. The published normal reference ranges for PCV varies between studies, with values between 30% to 50% (Hinton et al., 1982; Melillo, 2007; Archetti et al., 2008; Marshall, 2008; Özkan et al., 2012; Cray, 2015; Moore et al., 2015), but pet rabbits frequently have lower PCV of 30% to 40% (Harcourt-Brown and Baker, 2001). Before treatment, approximately 25% of rabbits in the present study had PCV less than 30% together with lower RBC and haemoglobin

318 World Rabbit Sci. 26: 313-320 HAEMATOLOGY IN ECTOPARASITOSIS TREATMENT IN RABBITS levels, which may suggest that these rabbits had regenerative anaemia due to ectoparasite infestations, as these parameters increased significantly post-treatment. Serum biochemistry parameter; AST, BUN and creatinine were within the normal range (Melillo, 2007; Archetti et al., 2008; Özkan et al., 2012) throughout the study, although BUN and creatinine measured on day 56 were lower than on days 0, 14 and 28. Moreover, ALT was higher than the normal range in a quarter of these rabbits. This would be a normal result, as slightly increased ALT levels are a common finding in healthy rabbits (Marshall, 2008; Varga, 2014). Taken together, this study suggested that ectoparasite infestations and selamectin treatment did not aÄect liver or renal function of rabbits. The present study also demonstrated that ectoparasite infestations in rabbits aÄected their quality of life. The body weight noticeably increased after the treatments. Taken together with improvement of skin lesions and haematological changes, it indicated that the rabbits were free to consume ad libitum diet when their illness had gradually resolved. This study has several limitations. First, we conducted the study in only one commercial rabbit farm where we were requested to provide a veterinary service. However, this farm was isolated from other farms and rabbits were reared in the same environment, with good ventilation and welfare. This study appeared more similar to experimental conditions rather than a field study. Second, this study was conducted in a prospective, non-controlled fashion which provided limited evidence to compare treatment and placebo. Third, we evaluated hydration status from physical examination and did not find dehydration in these rabbits. Therefore, we assumed that the haematological and serum biochemistry values obtained in this study were measured from rabbits with normal hydration status.

CONCLUSIONS

In this field study, topical selamectin application of 15 mg/kg at two-week interval was completely eÄective in the treatment of naturally acquired infestations of Sarcoptes scabiei var. cuniculi, Psoroptes cuniculi and Cheyletiella spp. in rabbits. One more dose of 15 mg/kg application on day 28 proved to be eÄective in the treatment and prevent re-infestation for at least 58 d post-treatment. This study has shown selamectin to be safe for field use in rabbits. Moreover, the neutrophil to lymphocyte ratio can be used for monitoring of inflammatory response in rabbits.

Conflict of interest: This project was funded by Zoetis (Thailand) Limited, the company that distributed the intervention used in this study. The research was performed independently, with Zoetis (Thailand) Limited having no influence over study design, data acquisition, analyses, results, manuscript preparation or scientific publication. None of the authors has any other financial or personal relationship that could inappropriately influence or bias the content of the manuscript.

Acknowledgements: We would like to thank the owner of the commercial rabbit farm in Thong Pha Phum district, Karnchanaburi province, in the western region of Thailand, who willingly participated in this study. This project was fully supported by Zoetis (Thailand) Limited, 323 United Center Building, 46th ﬂoor, Silom Road, Bangrak, Bangkok 10500, Thailand.

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320 World Rabbit Sci. 26: 313-320 W orld World Rabbit Sci. 2018, 26: 321-333 R abbit doi:10.4995/wrs.2018.10435 Science © WRSA, UPV, 2003

CONSUMER BEHAVIOUR RELATED TO RABBIT MEAT AS FUNCTIONAL FOOD PETRESCU D.C.*, PETRESCU-MAG R.M.†

*Faculty of Business, Babeș-Bolyai University, 400174 CLUJ-NAPOCA,7 Horea St., Romania. †Faculty of Environmental Science and Engineering, Babeș-Bolyai University, 400294 CLUJ-NAPOCA, 30 Fantanele St., Romania.

Abstract: Rabbit is one of the most versatile livestock species, responding successfully to bio-economic principles, which promote a clever use of resources and their conversion into added value products, such as functional foods (FFs). The excellent nutritive and dietetic properties of rabbit meat justify granting it the attributes of FFs. Based on the premise that it is the consumer who ultimately decides what kind of meat to buy, a consumer focus should be a core factor in private or public meat sector strategies. Following this assumption, the aim of this study is to contribute to understanding Romanian consumer behaviour related to rabbit meat as a functional food and to provide information on how to better market rabbit meat as FF in the domestic market. As far as the authors know, this study is the first one to assess and report on Romanian consumer perceptions, knowledge and behaviours related to rabbit meat. Consequently, the variables investigated reflect rabbit meat consumption habits. They also reveal preferences related to the acquisition of rabbit meat, perceptions on future rabbit meat consumption, perceptions of rabbit meat characteristics, perceptions of rabbit meat main characteristics compared to other types of meat, and rabbit meat consumption deterrents. The survey results show that rabbit meat is perceived as lean and low cholesterol, healthier and tastier than other meats, but more expensive, that its consumption is low, being 2.2 times lower than chicken and 1.8 times lower than pork, and that 29.6% of people surveyed have never eaten rabbit meat. The findings ascertain that the understanding of Romanian consumer behaviour related to rabbit meat as FF is an optimal tool for changing behaviour patterns towards a more sustainable market. The transfer of this knowledge towards marketers mainly focusing on how to increase consumer satisfaction for FFs, especially meat, is the leverage for designing successful businesses regarding market re-orientation, development or even reduction of health cost. Key Words: consumer behaviour, rabbit meat, functional food, sustainability.

INTRODUCTION

In recent decades, the concept of food quality has undergone important changes backed by market, cultural, and political globalisation. We are now dealing with consumers who face changes in their lifestyle and consumption habits (Dinu et al., 2010) and who are more informed and concerned about what they eat or where their food comes from. They are the smart consumers, more committed to environmental protection, animal welfare or health concerns, which obviously has an impact on their perception of quality. The International Standardisation Organisation (ISO) provides one of the most popular definitions of quality, which is often taken for granted, but which bears an elusive meaning: “degree to which a set of inherent characteristics fulfils requirement” (International Standardisation Organisation, 2015). Despite the several definitions of quality, more or less accepted, one thing remains undeniable: quality is a term which largely depends on socio-economic and cultural factors, such as ethics, religious beliefs and traditions (Font-i-Furnols and Guerrero, 2014).

Correspondence: D.C. Petrescu, [email protected]. Received June 2018 - Accepted September 2018. https://doi.org/10.4995/wrs.2018.10435

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Within the food sector, the term meat quality traditionally entails meat properties related to suitability of the meat for eating, further processing and storage, including retail display (Andersen et al., 2005). More recently, fat and protein content, texture and even vanity products (e.g., clams, insects), as Arnold (2009) called them, have increased their weighting in defining meat quality. The quality of meat and meat products varies according to intrinsic and extrinsic parameters that can sometimes be shaped to make a product more desirable (Font-i-Furnols and Guerrero, 2014). This becomes a mandatory requirement in an extremely competitive market. It is generally agreed that a market’s competitive edge depends on the ability to develop new, diÄerentiated products which are able to exploit and satisfy consumer preferences (Grunert et al., 2004). This permanent search for food quality extends, among other concepts, to that of functional foods (FFs), a term coined in Japan in the 1980s. It refers to “food products fortified with special constituents that possess advantageous physiological eÄects” (Kubomura, 1998). On the one hand, meat and meat products in general are considered functional foods, as they contain numerous beneficial compounds such as proteins, amino acids or fatty acids (omega-3 fatty acids, GLA, CLA) (Dalle Zotte and Szendrő, 2011). On the other, consumption of red meat and processed meat, in particular, is often blamed for an increased risk of certain chronic diseases, such as cardiovascular disease and cancer, and scientific evidence has already revealed the risks of red meat consumption in cardiovascular disease (McAfee et al., 2010). This is why consumers nowadays are increasingly asking for functional meat products which have reduced salt, nitrites and nitrates, cholesterol and fat contents (Khan et al., 2011). Based on rabbit meat’s excellent nutritive and dietetic properties, it can be successfully included in the FFs category. Worldwide, rabbit meat is valued for its high nutritional properties, with a lower-fat content, less saturated fatty acids and lower cholesterol contents than other meats (Dalle Zotte and Szendrő, 2011). Compared to other meat types (chicken, beef, and pork), it was found that rabbit meat was richer in calcium (21.4 mg/100 g) and phosphorus (347 mg/100 g) and lower in fat (9.2 g/100 g) and cholesterol (56.4 mg/100 g) (Nistor et al., 2013; Grădinaru, 2017). Unfortunately, rabbit meat consumption has less tradition in Romanian food culture. However, this drawback can become an opportunity for the domestic market by reorienting the oÄer towards rabbit meat and rabbit meat products as “new products”, “new entry”, or FFs (Petracci et al., 2018). The authors are realists and acknowledge that product development is risky, and that launching new products requires constant input from the market, especially from consumers (Grunert et al., 2004). Therefore, the aim of this study is to contribute to understanding Romanian consumer behaviour related to rabbit meat as FF and consequently to provide information on how to manage better delivery of rabbit meat as FF in the domestic market. To the best of our knowledge, this study is the first one to assess and report on Romanian consumer perceptions, knowledge and behaviours related to rabbit meat. A better understanding of consumer behaviour is recognised as a sine qua non condition for developing successful businesses regarding market re-orientation, development (Annunziata and Vecchio, 2010) and even public health cost reduction. The authors’ review of the international literature found a dearth of data on consumers’ attitudes to rabbit meat, mainly for Europe (Bodnar and Horvath, 2008a; Szakaly et al., 2009; Wang et al., 2013; Bodnar and Bodnar Skobra, 2014; Buitrago-Vera et al., 2016).

MATERIAL AND METHODS

The research is based on a survey using a sample of 216 persons from Cluj-Napoca and from its surrounding localities (belonging to Cluj county, North-West of Romania), both from rural and urban areas, interviewed face-to-face. The limit of 216 interviews was determined by time and budget constraints. The interviews were held with people who went shopping at hypermarkets and supermarkets (Carrefour, Lidl, Kaufland, and Profi), five small neighbourhood stores and peasant markets. Rural areas were selected at random from a list of Cluj-Napoca neighbouring localities. Weekdays and interview starting times throughout day were also selected at random and every fourth person who came out of the shop/market was asked for an interview. A filter question was asked at the beginning of the questionnaire, asking if they have an influence on what type of food is bought for at least 25% of the food eaten by them and the people who live with them (either because they buy at least 25% of the food they eat or because they influence those who buy it). Answer options were “Yes” and “No”. The positive response rate was 17%, which means that 17% of contacted persons agreed to answer, they had an influence on at least 25% of the food bought by their family, and they finalised the questionnaire. Considering that the sampling procedure ensured the selection of a random sample, for a sample of this size (216 persons, out of a population of around 350 000 inhabitants), the

322 World Rabbit Sci. 26: 321-333 RABBIT MEAT CONSUMER BEHAVIOUR confidence level was 95% and the sample error was 6.65%. Even though the usually accepted error in social science is 4%-5%, higher levels are used and accepted as long as their level is acknowledged. Thus, there are numerous studies in social sciences, focused on food or other topics, with results associated to higher error. For example, an error of 7% was accepted in a study on consumers’ willingness to pay for nutritional claims (Rhormens et al., 2017); an investigation on perceptions regarding Community Based Marine Ecotourism relied on results with an error of 7% (De-Magistris and Lopéz-Galán, 2016); in a study by Pérez López et al. (2005) on organisational learning, an error of 6.9% was used. The structure of the sample by gender and living environment was: 53% men, 47% women, 84% people from urban areas and 26% people from rural areas. Considering age and education, sample shares were: 28% between 18-25 yr old, 19% between 26-45 yr old, 53% over 45 yr old; moreover, 58% had a higher education (faculty, master, PhD, ongoing or finished) and 42% had a lower level of education. It should be mentioned that Cluj-Napoca is a university city, with over 60000 students and with 46% of its stable population holding a higher education degree (based on 2011 census; INSSE, 2018, apud Ecoduri, 2018). Data analysis was carried out using Excel software and SPSS version 21. For comparison of diÄerences regarding an ordinal variable between two groups, we ran the Mann-Whitney U test. Wilcoxon signed-rank test was used to compare two repeated measurements on a single sample to assess whether their mean ranks diÄered. The level of statistical significance was set at P<0.05. The variables investigated in this study were selected in order to create a complex image of the rabbit meat consumer behaviour and focused on purchasing habits, buying preferences and perceptions of rabbit meat characteristics. They include the following: 1. Rabbit meat consumption habits (frequency, adoption of consumption by others in the family, location, type of meat from the processing point of view); 2. Preferences related to the acquisition of rabbit meat; 3. Perceptions on future rabbit meat consumption; 4. Perceptions of rabbit meat characteristics; 5. Perceptions of main rabbit meat characteristics (healthiness, taste, and price) compared to other types of meat; 6. Rabbit meat consumption deterrents (more details are included in Annex 1). The questionnaire was structured as follows. Firstly, a short introduction was presented: greetings, presentation of the study and request to participate. Secondly, the filter question was asked and the interview continued with people who answered “yes”. Thirdly, the questionnaire set of questions followed, grouped in six chapters as described in the previous paragraph and in Annex 1. The selected variables are key points both for sustainable production and marketing by the meat sector and for consumer information-education measures by public decision makers. As it is the consumer who ultimately decides what kind of meat to buy, a consumer focus should be at the core of private or public meat sector strategies.

BACKGROUND

Clever use of resources and the rabbit meat market The domestic rabbit (Oryctolagus cuniculus) is recognised as one of the most suitable animals for sustainable farming for a multitude of reasons. One is its capacity to use various cheap vegetal matters, including cellulose from wood, or sundry scraps from industry or agriculture which are poor in macronutrients (Ibrahim et al., 2011; Petrescu-Mag et al., 2014; Sima and Sim, 2015), so its competition with humans or other domestic animals for food is limited. Others refer to its high rate of reproduction, early maturity, rapid growth rate, high genetic selection potential and e§cient land space utilisation (Cheeke, 1980). Moreover, rabbit manure can be used as a garden fertiliser and compost ingredient, due to its high content in nitrogen, phosphorus, and potassium ((Lukefahr et al., 1998), or it can be converted into methane gas for household fuel needs (Lukefahr and Cheeke, 1990). Arguments put forward by Lukefahr (1998) reveal the opportunity for rabbit production to become an instrument for fostering human development through alleviation of poverty. Additionally, evidence from western countries, such as Spain, indicates that various farming alternative can be successful, from wild rabbit game farms to intensive meat rabbit farms (González-Redondo and Sánchez-Martínez, 2014). All these make rabbit a versatile livestock species (Lukefahr and Cheeke, 1990), able to respond successfully to the principles of bio-economy, which promotes a clever use of resources and their conversion into value added products (European Commission, 2012), such as FFs.

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Starting from the fact that meat intake per capita is a relevant indicator for the economic welfare of a population (Stanciu et al., 2015), a study by Petrescu et al. (2017) revealed that meat is dominant in Romanian food culture (but at much lower levels compared to Western countries), as it is frequently (at least 4 d/wk) consumed by two thirds of the people surveyed and very frequently consumed (6-7 d/wk) by almost one third. The meat consumption behaviour of Romanians citizen is still oriented towards pork and chicken, and the local rabbit production in Romania is supported only by small farms which are not competitive (Blaga and Burny, 2014). Lack of tradition of rabbit meat consumption, a dearth of local rabbit breeds or the high price of rabbit meat are among the main obstacles for rabbit meat consumption in Romania (Petrescu et al., 2013). According to Blaga and Burny (2014), the price varies between 4.48-9.01 €/kg, which is similar to the price in France, where in 2015 the average price per kg was 9.31 € (STATISTA, 2108a), while in Spain the average price per kg was 5.11 Euros (STATISTA, 2018b), but it can also be bought at lower prices (from 3.99 €/kg) if the consumer resorts to traditional purchasing places (e.g. street markets), an aspect highlighted in a study by Baviera-Puig et al. (2017) through commercial observation at point of sale. Based on empirical observation and analysis of available data (e.g. FAO, Romanian National Institute of Statistics), there are no reliable statistics and reports for Romania on rabbit production and/or consumption (Petrescu et al., 2013). National statistics worldwide do not generally include rabbit production. Even the European Union meat production statistics include data only for beef and veal, sheep meat and goat meat, poultry meat and pig meat (EUROSTAT, 2015). This is because, globally, per capita consumption of these meats is the highest: in 2013 the average person consumed around 16 kg of pig meat, followed by 15 kg of poultry; 9 kg of beef/buÄalo meat, 2 kg of mutton & goat; and only a fraction of other meat types (among which is rabbit meat) (Ritchie and Roser, 2018). Worldwide, more than 1.2 billion rabbits are slaughtered for meat every year and China accounts for 40% of global production, while EU-27 is responsible only for 28% of global production, with Italy, France and Spain leading the ﬁeld (FAOSTAT, 2012).

Consumer behaviour related to FFs The meaning of food behaviour accepted in this paper is that of a set of reactions to internal and external factors that stimulate or hinder food intake (Popescu et al., 2015). Understanding the factors that influence consumer behaviour in buying FFs, in general, is a current concern for academic research. Thus, poring over the most relevant studies dedicated to FFs will contribute to a better understanding of consumer choice and behaviour. Health consciousness was among the most frequently mentioned motives for consuming FFs. Thus, Verbeke (2005) found that the acceptance of FFs is higher within families with an ill member. Socio-cultural determinants are also on the list of the factors that influence FF consumer behaviour. For example, a study in Malaysia examined how three ethnic groups manage their values in terms of FF consumption and it was observed that participants started to pay attention to their food choices only when these were inconsistent with cultural or physical characteristics that were familiar to them (Hasnah Hassan, 2011). Pappalardo and Lusk (2016) utilised food values in conjunction with willingness to pay (WTP) measures to identify Italian consumers’ subjective beliefs about FFs. The results indicated that the surveyed consumers’ WTP for FFs varied with food features related to origin, naturalness, or price, which revealed that those consumers had diÄerent subjective beliefs about FFs and non-FFs. DiÄerent studies argue that gender, age, education, marital status and health situation are the main predictors of FF consumption (Annunziata and Vecchio 2010; Hung et al., 2016; Vecchio et al., 2016). Regarding the factors aÄecting rabbit meat consumption, there is scant scientific knowledge. In our neighbouring country, a research on 1274 Hungarians showed that those who refused consumption were vegetarian or that their attitude was caused by emotional reasons; another obstacle in rabbit meat consumption was the lack of rabbit meat and rabbit meat products in supermarkets (Bodnar and Horvath, 2008a). In an investigation carried out with 304 South African consumers, it was shown that 47% of the respondents had eaten rabbit meat prior to the study; this experience in rabbit meat consumption contributed positively to the desire to eat it again; they declared that they preferred to purchase rabbit meat in portions; 49% of respondents who consumed rabbit meat would definitely have considered price when making their purchase choice; 8% indicated that price would not influence their purchase; 61% of respondents who had already consumed rabbit meat would have liked to pay less for it than for chicken (HoÄman et al., 2004). Indeed, in many cases, one of the major obstacles to higher rabbit meat consumption was the

324 World Rabbit Sci. 26: 321-333 RABBIT MEAT CONSUMER BEHAVIOUR price, and Dalle Zotte (2002) argued that the higher production cost of rabbit meat in developed countries compared to other meats was prohibitive. Catalans consumers revealed the importance of the Catalonian identity in food consumer behaviour, as they preferred rabbit meat of local origin to other sources of production (Kallas and Gil, 2012).

RESULTS AND DISCUSSION

Rabbit meat consumption habits As expected, according to the present study, rabbit meat consumption in a comparative context is low, being 2.2 times lower than chicken and 1.8 times lower than pork (Table 1). Among the people surveyed, 29.6% have never eaten rabbit meat, which is a very high percentage, but understandable given the fact that its availability is limited to self- production, small producers and to some of the supermarkets located in big cities. A similar share of respondents (31%) declared that other members of their family also did not eat rabbit meat. The dominant consumption frequency was “less than one day per month” (56% of the sample), followed at a long distance by “between 1 and 3 d per month” (12.5% of the sample), while only extremely low shares declared frequencies “between 1 and 4 d per week” (1.4%) and “between 5 and 7 d per week” (0.5%). Similarly, in Hungary, 46% of the interviewees bought rabbit meat only once or twice a year (Bodnar and Horvath, 2008b). Fresh meat is the most frequently purchased type, probably due to its greater availability (compared to frozen or semi-cooked meats). The fact that 23.1% of those surveyed purchased/received cooked rabbit meat is explained by its consumption location –at friends’ house or restaurants. However, the most common consumption place remains “at home” (Table 2). In this context, it is very important to stimulate restaurants to include rabbit in their menus and to diversify their recipes in order to promote rabbit meat consumption and make it attractive for consumers.

Consumer preferences on the acquisition of rabbit meat There may be a diÄerence between what one desires to do and what he/she is allowed/able to do, for instance a consumer may want to buy rabbit meat but cannot because it is not for sale on the market or because the price is too high for their budget. In this context, where current behaviour is shaped not only by preference, but also by restrictions (for example, lack of availability of a preferred purchasing location would force the consumer to use a source which he/she does not like), in order to highlight what they want, it was necessary to investigate consumer preferences regarding rabbit meat along with their current practices. Preferences regarding rabbit meat type, supplier, purchasing location and producer’s country of origin are the main variables to characterise consumer behaviour in the case of meat, providing marketers with reference points for their strategies. Romanians prefer to buy already slaughtered animals, whole, from small Romanian producers (Table 3). However, most of them prefer to buy it from supermarkets (32.9%), thus revealing supermarkets as a suitable vector for the promotion of FFs, rabbit meat in particular (Table 3). Unlike Romanian people, in Hungary, consumers prefer to buy rabbit directly from the farmer (70% of Hungarians surveyed), as they consider it to be fresher and not so expensive as it is in supermarkets and because they declare themselves unsatisﬁed with the distribution of rabbit meat in supermarkets (Bodnar and Horvath, 2008b).

Consumer perceptions on their future rabbit meat consumption The survey disclosed a relatively reduced self-estimated future consumption, with 32.4% believing they would not eat rabbit meat in the near future, 44% considering they would eat it less than 1 d per month, 19.9% estimating a consumption frequency between 1 and 3 d per month, 3.7% between 1 and 4 d per week, and nobody envisaging

Table 1: Consumption frequency of diÄerent meat types (average scores for the sample). Chicken Pork Beef/Veal Sheep Rabbit Fish Other Score* 4.0 3.3 2.6 1.6 1.9 2.9 1.2 * Average sample score, calculated using the individual estimations provided by consumers, coded on the scale: 1=never (minimum level), 2=less than 1 d per month, 3=between 1 and 3 d per month, 4=between 1 and 4 d per week, 5=between 5 and 7 d per week (maximum level).

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Table 2: Type of rabbit meat purchased and type of consumption location (percentage of total sample). Type of processing of purchased/received rabbit meat Fresh Frozen Semi-cooked Cooked In the form of meat products None, because I do not None, because (sausages, salami, ham, consume / consume I get / grow my pastrami etc.) it rarely own rabbits 38.4% 14.4% 0.5% 23.1% 6.5% 36.6% 3.7 Type of consumption location At home At the restaurant At friends’ house I did not eat/ I do not remember 51.9% 8.3% 31.0% 32.4%

a consumption between 5 and 7 d per week. However, a statistically signiﬁcant diÄerence was found between the two consumption frequencies – current and future, with a higher frequency of intended rabbit meat consumption compared to past consumption frequency (Z=–2.068, P=0.039) (Table 4).

Consumer perceptions of rabbit meat characteristics The list of information used to investigate consumer perceptions on rabbit meat characteristics had 24 components (see section 3. Research methodology and Annex 1, point 4). To test the reliability of the scale, the internal consistency was measured using the Cronbach’s coe§cient alpha. The average correlation among all the items that made up the scale was 0.696, lower than the recommended level of 0.7. One item was removed –the habit of eating rabbit meat in the ﬁrst part of life (as a child and teenager)– and the new value of the Cronbach’s coe§cient alpha was 0.725, an acceptable one. Taste was rewarded with the highest score among all tested characteristics, which is a very good and important result, as taste is the main driver of food consumption. Through various studies, taste was proven to be a very important factor for food choice, dietary behaviours and intake; for example, 82% of tested Australian consumers rated taste as very/extremely important factor for food choice (Kourouniotis et al., 2016). In Spain, also, consumers nominated good taste (72.4%), healthiness (35.9%) and having low fat content (14.6%) as the 3 main reasons for eating rabbit meat (Buitrago-Vera et al., 2016). In the described survey herein, 64.4% of people consider taste is good and very good, thus revealing that the fundamental requirement for the consumption of a food item is already fulﬁlled. In second place was texture, which received good and very good ratings from 58.5% of interviewees. Texture in food preferences has recently gained remarkable importance as a consequence of its intensive promotion for a variety of foods (e.g., crunchy, creamy and soft) (Jeltema et al., 2015). On the one hand, the fact that the next best scores were

Table 3: Consumer preferences regarding rabbit meat (percentage of total sample). Preferred type of meat From animal bought alive Whole animal, Certain IndiÄerent I do not want to I do not want to buy at all and cut by our family already parts buy because I do because I receive it or I slaughtered not eat it often grow the rabbits 20.8% 32.1% 18.5% 7.4% 35.2% 9.3% Preferred type of supplier Small producers Large farms From wilderness IndiÄerent None 36.6% 2.8% 23.6% 15.7% 32.9% Preferred purchasing location Supermarket Specialised Peasant Directly Hunting Self- Restaurant IndiÄerent None shops market from production because I farms do not eat it often 32.9% 21.3% 6.0% 19.9% 5.1% 5.6% 0.5% 8.8% 0% Preferred producer country of origin Romania Other EU countries Non- EU IndiÄerent 67.6% 1.4% 0% 31.0%

326 World Rabbit Sci. 26: 321-333 RABBIT MEAT CONSUMER BEHAVIOUR

Table 4: Results of Wilcoxon signed-rank test concerning the diÄerence between the mean ranks of the current frequency consumption of rabbit meat and the future one. Ranks Test Statisticsd Future consumption - N Mean Rank Sum of Ranks Z –2.068e Current consumption Negative Ranks 21a 30.38 638.00 Asymp. Sig. (2-tailed) 0.039 Positive Ranks 38b 29.79 1132.00 Ties 157c Total 216 N: Number of cases aFuture consumptionCurrent consumption. cFuture consumption=Current consumption. dWilcoxon Signed Ranks Test. eBased on negative ranks. gained by cholesterol level and leanness supports rabbit meat promotion as a FF (Figure 1). On the other, when the characteristic of rabbit meat being a FF was specifically tested, the average score was low, as only 6% agreed (totally or mostly with this fact) and 30.6% did not have an opinion. In other words, most people recognised certain attributes of rabbit meat, but they were not able to connect them with a positive impact on their health. The influence of gender on rabbit meat consumption frequency variables -past and future- and the perception of rabbit meat characteristics was investigated and significant diÄerences (P>0.05) between men and women were observed in some cases (Table 5). The results indicated that men seemed to appreciate rabbit meat more than women. For the latter category, disgust and ethical concerns were stronger. Similarly, women in a Spanish study, had stronger perception of rabbit as a companion animal compared to men (González-Redondo and Contreras- Chacón, 2012). French women also mentioned disgust as a generator of low meat consumption, regardless of its type (Rousset et al., 2005).

Perceptions of rabbit meat main characteristics (healthiness, taste, and price) compared to other types of meat Healthiness, taste, and price were indicated by the consumers surveyed as the most important attributes that inﬂuence meat choice. Therefore, rabbit meat was compared with the most commonly consumed types of meat on

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Taste 3.9 Texture 3.8 Aspect 3.6 Smell 3.5 Easiness/Di§culty in cooking 3.4 Availability on the market 2.6 Versatility in cooking 3.5 Price 2.8 Price-quality ratio 2.9 It is disgusting 2.2 It is a FFs 2.0 It is about the same as any other meat 2.7 It is a food and its purpose is to be eaten 3.3 Rabbit is a being that feels and lives, so it should not be eaten 2.3 The rabbit is a cute animal, a friend, a pet, so it should not be eaten 2.4 How natural it is compared to other types of meat 3.5 The number of people in Romania who eat rabbit meat 3.3 Number of rabbit meat consumers in other EU countries compared to Romania 3.2 It is a special food, suitable only for special occasions 3.3 Cholesterol level 3.7 Leanness 3.6 Risk for your health due to hormones, antibiotics etc. compared to other types of meat 2.8 Risk for your health due to parasites, animal diseases compared to other types of meat 2.7 Figure 1: Consumer evaluation of rabbit meat characteristics (average scores of the sample) behaviour. Source: Prepared by the authors. (To interpret the scores, see the explanations for answer options for each question in section Material and Methods).

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Table 5: Man-Whitney U test results for diÄerences between women and men regarding rabbit meat frequency consumption (past and future) and perception of rabbit meat characteristics. P-value (for Higher frequency/ Variable signiﬁcant diÄerence) Stronger agreement Men Women Frequency of past rabbit meat consumption P=0.008 X Frequency of future intended rabbit meat consumption P=0.006 X Perception: taste P=0.001 X Perception: texture P=0.003 X Perception: smell P=0.017 X Perception: easiness to cook P=0.010 X Perception: versatility in cooking P=0.013 X Perception: it is disgusting P=0.035 X Perception: rabbit is a living creature that feels and has its own life, so it P=0.006 X should not be eaten Perception: rabbit is a cute animal, a friend, a pet, so it should not be eaten P=0.004 X Perception: it is more natural than then types of meat P=0.024 X Perception: it presents a lower risk for your health due to hormones, P=0.006 X antibiotics etc. than other types of meat Perception: it presents a lower risk for your health due to parasites, rabbit P=0.015 X diseases than other types of meat

these characteristics. The fact that interviewed consumers perceived rabbit meat as healthier than all tested meats and ﬁsh supports its promotion as a FF. Also, its evaluation as tastier than all the rest is an encouraging ﬁnding for producers and sellers considering bringing it into the market (Table 6). The perception of rabbit meat price as more expensive than the most popular meats in Romania –chicken and pig meat– is a real obstacle in increasing its consumption (Table 6). Accordingly, the weight of other advantages, such as being a FF (healthy) and tasty, must be potentiated to overcome the hindering eÄect of a high price.

Hindering factors in front of rabbit meat consumption The most important obstacles to rabbit meat consumption stated by the interviewees were high price and disgust (each named by 31.9% of sample), lack of availability on the market (mentioned by 30.6% of sample), empathy with another living creature which is deprived of freedom and slaughtered (mentioned by 26.4% of the sample) and the fact that the rabbit is perceived as a pet, and as a cute animal (nominated by 25.5% of the sample). An ethical obstacle to meat eating was also mentioned by Bastian et al. (2012) for Australian consumers, who showed that many people liked eating meat and, in order to maintain their eating habits, they denied that the animals they consumed had

Table 6: Comparative perceptions (average scores of the sample). Healthiness* Rabbit compared to: Chicken Pork Beef Sheep Fish 2.3 2.5 2.3 2.2 2.3 Taste** Rabbit compared to: Chicken Pork Beef Sheep Fish 2.1 2.3 2.1 2.2 2.2 Price*** Rabbit compared to: Chicken Pork Beef Sheep Fish 1.5 1.6 2.0 2.2 2.3 *1=rabbit meat is less healthy than…, 2=rabbit meat is equally healthy as…, 3=rabbit meat is healthier than… **1=rabbit meat is less tasty than…, 2=rabbit meat is equally tasty as…, 3=rabbit meat is more tasty than… ***1=rabbit meat is more expensive than…, 2=rabbit meat is the same price as…, 3=rabbit meat is less expensive than…

328 World Rabbit Sci. 26: 321-333 RABBIT MEAT CONSUMER BEHAVIOUR minds, thus reducing dissonance between loving eating meat and caring about animals. According to Buitrago-Vera et al. (2016), a similar situation is present among Spanish consumers, who declared that the main obstacles facing rabbit meat consumption were the fact that they were not used to it (28.3%) and that they disliked the taste (26.7%). A balanced perspective on the current study requires mentioning its limitations. Thus, it should be observed that a more in-depth investigation of each of the 6 sections of the questionnaire (Rabbit meat consumption habits; Consumer preferences regarding the acquisition of rabbit meat; Consumer perceptions on their future rabbit meat consumption; Consumer perceptions of rabbit meat characteristics; Perceptions of rabbit meat main characteristics compared to other types of meat; and Hindering factors in front of rabbit meat consumption) is possible in a follow- up study considering additional variables and perspectives. Thus, diÄerences not only according to demographics (e.g., gender), but also within them, can be explored and variables such as lifestyle can be added (e.g., health and environmental concerns, social inﬂuences on consumption). The fact that the error was slightly higher than the habitually used level of 4-5% for Social Sciences, reaching 6.65% is a limitation that must be considered in terms of practical use of the results. Moreover, the sample can be extended beyond the local area to national level and its representativeness can be increased.

CONCLUSIONS

Population growth, urban expansion and increasing standards of living in the developing world are responsible for dietary transformation and rapid growth in human consumption of animal proteins (Boland et al., 2013). In the context of human preference for healthy and high-quality meat, and the current trend towards reducing captures in the wild (Petrescu-Mag et al., 2014), rabbit meat production should be valued as an optimal tool for endorsing sustainable food consumption. Thus, rabbit meat may contribute to a "balanced diet" with possible preventive eÄects on non- communicable diseases (e.g., cardiovascular diseases, cancer and diabetes) (Corpet, 2011). Similar to poultry, the nutritional profile and technological traits of rabbit meat make it suitable for inclusion in added value products which respond both to modern consumer demands for healthy food and to industry requirements in terms of flexibility (Petracci and Cavani, 2013). Rabbit meat production and consumption is a possible solution worldwide, where economic growth is mandatory to sustain human progress by reducing poverty, hunger and malnutrition and providing safety and aÄordable food. Raising awareness of what we eat and how the food arrived on our plate is not only a matter of being informed, but also of ethics. It also has to do with environmental protection, animal welfare and food equity. The contribution of this study is the creation of the Romanian rabbit meat consumer profile. From a practical perspective, the information presented here can guide decision makers towards developing new marketing strategies to raise consumer interest in meat quality in general and in rabbit meat as FF in particular. The consumers surveyed ate rabbit meat less than once a month and appreciated its taste, texture, leanness and low cholesterol content the most among all rabbit meat characteristics. Compared to chicken, pork, beef, sheep meat and fish, rabbit meat was perceived as healthier, highlighting the existence of the premises for its promotion as FF. The main deterrents to rabbit meat consumption were price, disgust, lack of availability and ethical concerns. The results of this study shed light on the chance to evolve towards a market richer in FFs, thus revealing opportunities for marketers to adjust their interest to consumers’ needs and, at the same time, to respond to economic e§ciency and environmental protection needs.

Acknowledgments: This study was partially developed through the research programme “The creation of a model for the evaluation of food quality from the point of view of consumer health and environmental protection”, selected within the bilateral cooperation between the Romanian Academy and Wallonia – WBI, FRS-FNRS. “La présente publication a été rendue possible grâce à l’Accord qui lie WBI, le FRS-FNRS et l’Académie Roumaine.”

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ANNEX 1. VARIABLES USED IN THE STUDY

1. Rabbit meat consumption habits (frequency, adoption of consumption by others in the family, location, type of meat from the processing point of view) 1.1. Consumption frequency in a comparative context: 1.a. Consumption frequency: chicken; 1.b. Consumption frequency: pork; 1.c. Consumption frequency: beef/veal; 1.d. Consumption frequency: sheep; 1.e. Consumption frequency: rabbit; 1.f. Consumption frequency: ﬁsh; 1.g. Consumption frequency: other. Answer options: Never; Less than one day per month; Between 1 and 3 d per month; Between 1 and 4 d per week; Between 5 and 7 d per week. 1.2. Rabbit meat consumption by other family members. Answer options: Yes; No.

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1.3. Type of location for rabbit meat consumption. Answer options: At home; At the restaurant; At friends’ house; I did not eat/ I do not remember. 1.4. Rabbit meat purchasing habits: 1.4.a. Type of processing of purchased/received rabbit meat. Answer options: Fresh; Frozen; Semi-cooked; Cooked; In the form of meat products (sausages, salami, ham, pastrami, etc.); None, because I do not consume/ consume it rarely; None, because I get / grow my own rabbits. 2. Preferences related to the acquisition of rabbit meat 2.1. Preferred type of meat. Answer options: From animals bought alive and cut by our family; Whole animal, already slaughtered; Certain parts; IndiÄerent; I do not want to buy because I do not eat it often; I do not want to buy because I receive it or I grow the rabbits. 2.2. Preferred type of purchasing location. Answer options: Supermarket; Specialised shops; Peasant market; Directly from the farm; Hunting; Self-production; Restaurant; IndiÄerent; No preference because I do not buy it often. 2.3. Preferred type of supplier. Answer options: Small producers; Large farms; From wilderness; IndiÄerent; No preference because I do not buy it often. 2.4. Preferred producer country of origin. Answer options: Romania; Other EU countries; Non-EU countries; IndiÄerent. 3. Perception on future rabbit meat consumption 3.1. Perception of near future (next 12 months) rabbit meat consumption frequency. Answer options: Never; Less than one day per month; Between 1 and 3 d per month; Between 1 and 4 d per week; Between 5 and 7 d per week. 4. Perception of rabbit meat characteristics 4.1. Taste; 4.2. Texture; 4.3. Aspect; 4.4. Smell. Answer options: Very good (coded 5); Good; Average; Bad; Very bad (coded 1). 4.5. Easiness/Di§culty in cooking. Answer options: Very easy (coded 5); Easy; Average easiness; Di§cult; Very di§cult (coded 1). 4.6. Availability on the market. Answer options: Very good (coded 5); Good; Average; Bad; Very bad (coded 1). 4.7. Versatility in cooking (it can be prepared in many ways). Answer options: Very good (coded 5); Good; Average; Bad; Very bad (coded 1). 4.8. Price (compared to your budget). Answer options: Very cheap (coded 5); Cheap; Average price; Expensive; Very expensive (coded 1). 4.9. Price-quality ratio. Answer options: Very good (coded 5); Good; Average; Bad; Very bad (coded 1). 4.10. It is disgusting (it makes you sick if you eat it). Answer options: Very disgusting (coded 1); Disgusting; IndiÄerent; Appetising; Very appetising (coded 5). 4.11. It is a FF (food that claims to improve health or well-being by providing beneﬁts beyond that of the traditional nutrients it contains); 4.12. It is about the same as any other meat; 4.13. Rabbit is a food source and its purpose is to be eaten; 4.14. Respect for the life of another creature (rabbit feels and it has its own life) and empathy with a living creature which is deprived of freedom and slaughtered, leading to the conclusion that rabbit should not be eaten; 4.15. Rabbit is a cute animal, a friend, a pet, so it should not be eaten; Answer options: Total agreement (coded 5); Mostly agreement; Not agreement, nor disagreement; Mostly disagreement; Total disagreement (coded 1); 4.16. How natural it is compared to other types of meat (the animals received more natural feed and lived in more natural conditions). Answer options: Much more natural (coded 5); More natural; The same; Less natural; Much less natural (coded 1). 4.17. The number of people in Romania who eat rabbit meat. Answer options: Very high number of people (coded 5); High number of people; Moderate number; few people; Very few people (coded 1). 4.18. Number of rabbit meat consumers in other EU countries compared to Romania. Answer options: Much more people compared to Romania (coded 5); More people compared to Romania; The same as in Romania; Fewer people compared to Romania; Much fewer people compared to Romania (coded 1). 4.19. It is a special food, suitable only for special occasions. Answer options: Total agreement (coded 5); Mostly agreement; Not agreement, nor disagreement; Mostly disagreement; Total disagreement (coded 1); 4.20. Cholesterol level. Answer options: Very low (coded 5); Low; Average; High; Very high (coded 1). 4.21. Leanness. Answer options: Very lean (coded 5); Lean; Average; Fat; Very fat (coded 1). 4.22. Risk for your health due to the use of hormones, antibiotics etc. compared to other types of meat; 4.23. Risk for your health due to parasites, animal diseases compared to other types of meat. Answer options: Very low (coded 5); Low; Average; High; Very high (coded 1). 4.24. How used you are to eating rabbit meat (based on how much you eat it as a child or teenager).

332 World Rabbit Sci. 26: 321-333 RABBIT MEAT CONSUMER BEHAVIOUR

Answer options: Not at all used to eating it (coded 1); Mostly not used to eating it; Average level of habit to eat it; Mostly used to eating it; Very used to eating it (coded 5). 5. Perceptions of rabbit meat main characteristics (healthiness, taste, and price) compared to other types of meat 5.1. Healthiness of rabbit meat: a. compared to chicken meat; b. compared to pork; c. compared to beef/veal meat; d. compared to sheep meat; e. compared to fish. Answer options: Less healthy; The same; Healthier. 5.2. How tasty is rabbit meat: a. compared to chicken meat; b. compared to pork; c. compared to beef/veal meat; d. compared to sheep meat; e. compared to fish. Answer options: Less tasty; The same; Tastier. 5.3. How is the price of rabbit meat: a. compared to chicken meat; b. compared to pork; c. compared to beef/veal meat; d. compared to sheep meat; e. compared to fish. Answer options: More expensive; The same; Cheaper. 6. Rabbit meat consumption deterrents All variables listed at point 4 above were tested as consumption deterrents. People were asked about their eÄect as rabbit meat consumption deterrents and the eÄect was presented on a 3-point scale (strong eÄect, average eÄect, and weak eÄect).

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INSTRUCTIONS TO AUTHORS

Results of work contained in manuscripts submitted to WRS must not have been published previously in an international refereed scientific journal. Previous presentation at a scientific meeting or the use data in field day reports or similar documents, including local technical press, does not preclude the publication of such data in WRS. Views expressed in papers published in WRS represent the opinion of the author(s) and do not necessarily reflect the o§cial policy of the World Rabbit Science Association or the Editor-in-Chief.

TYPE OF ARTICLES Research articles: Data of research articles must have been statistically analysed using approved statistical methods. Treatment means must be accompanied by standard errors. If Bayesian analyses are performed, posterior means or modes must be accompanied by credibility intervals or high posterior density intervals. Review articles: The journal only publishs reviews ordered from the Editorial Board (Associate Editors or Editor in Chief). These revisions are demanded in “hot topics” and to authors with a large career in this specific topic. Reviews should include the term “Review” in the title. Technical notes: A technical note is a vehicle to report a field study or a new method, technique or procedure of interest to WRS readers. Letters to the Editor: Letters judged suitable for publication by the Section Editor, will be printed in a special section of the journal. The purpose of this section is to encourage scientific debate and discussion among those interested in rabbit production and biology. When these letters refer to published articles they must provide supporting evidence based on published data for the points made, or must develop logical scientific hypotheses. Letters based on conjectures or on unsubstantiated claims will not be published.

PREPARATION OF MANUSCRIPTS

Papers must be written in English, following current usage. Spelling should follow that of the Oxford Dictionary. The only word admitted by the WRSA for young rabbits is ‘kit’ (plural= ‘kits’). Words such ‘pups’, ‘young rabbits’ or ‘bunnies’ should be avoided. Manuscripts should be written with wide margins and be double spaced. Pages should be numbered. Lines should be numbered to help the refereeing procedure. Font: Use Times New Roman font with a type size of 12 points. Units: The International System of Units should be used. Temperatures should be given in degrees Celsius. First Page: Should bear the Running Head, the title of the paper, the names of the authors, the complete postal address of the authors and phone, fax and e-mail of the corresponding author. Running Head: A short title, to serve as a running head and consisting of not more than 50 letters and spaces must also be given on the ﬁrst page after the mention “Running Head”. Headings: Major headings (Abstract, Introduction, Materials and Methods, Results, Discussion, Conclusions and References) are centered and appear in bold type capitalized. First subheadings appear at the left margin on a separate line in bold type and are followed by punctuation. Second subheadings appear in italics at the beginning of the ﬁrst line of a paragraph.

World Rabbit Sci. 335 INSTRUCTIONS TO AUTHORS

Illustrations: All the illustrations (ﬁgures and tables) should be presented each on a separate sheet and referred to in the text by their number. Tables should be numbered with Arabic numerals and be accompanied by adequate titles and, if necessary, table footnotes. Figures should also be numbered with Arabic numerals, and the title given on the same sheet. Figure legends should be explicit so that the illustrations are comprehensible without reference to the text. Figures can be sent in Word, PDF, TIFF, BMP, JPG, JPEG or GIF format, but XLS format is recommended for its subsequent homogenisation. If there are no possibilities of sending ﬁgures by e-mail, they can be sent by post, with a copy of the paper. Citations: Citations should be made in lower case. Apart from reviews, the number of citations should be minimised; select only the most pertinent ones. When two or more citations are included in a grouping within a sentence, the citations must be arranged in chronological order, and if needed, alphabetically within the year. For two authors “and” (e.g. Blasco and Ouhayoun, 1996) has to be employed; but for three and more authors cited “et al.”, has to be used (e.g. Coudert et al., 1992). If, two papers abbreviate identically in the text, place a diÄerent letter after the date for each paper, both in the text and in the references list (e.g. Lebas et al., 1992a).

Paper Sections Abstract: The abstract should be written in a single paragraph. It should be informative, containing the main numeric results. The abstract should be understandable without reference to the paper. No references should be given in the abstract. The abstract will have a maximum of 350 words. Key Words: List up to a maximum of six key words at the end of the abstract. Introduction: The introduction briefly justifies the research and specifies the hypotheses to be tested. Extensive discussion of the relevant literature should be included in the discussion of the results, not in the introduction. To minimise length and avoid redundancy, no more than three references should be cited to support a specific concept. Materials and Methods used should be given in enough detail to permit the reader to repeat the experiment. If some methods refer to other published papers, they should be accessible by the normal reader. Some harmonised methods, recommendations and guidelines for rabbit science experiments (nutrition, meat, reproduction...) have been published in WRS, and the Editorial O§ce encourage their use. Results (may be combined with discussion) should be presented in graphics or tabular form when feasible. The text should explain or elaborate on the presented data, but numbers should not be normally repeated within the text. Figures should not repeat the information given in tables. Mean and standard error (or standard deviation) must be expressed with the same degree of accuracy. The same applies for credibility intervals in Bayesian analyses. Some examples are listed below: 2452±43; 0.732±0.021; 7.500±0.015; 9750±240; 9.75±0.24 In a normal situation, the standard error, or the credibility intervals in the Bayesian case, are expressed by two significant digits, e.g. 35 or 0.35 or 0.0035. Examples for a rabbit live weight: 1756±25 g or 1.756±0.025 kg. Discussion (may be combined with results and with conclusions) should interpret the results integrating literature results with the research findings to provide the reader with a basis on which to accept or reject the hypotheses tested. At the end, the discussion may also include technical or economical implications when suitable. Conclusions (may be combined with discussion): Main technical or economical implications can be written separately in a paragraph of conclusions when suitable. Acknowledgements: When appropriate, names of technical assistants, funding organisations, research grants, and other thanks must be included, in Acknowledgements. References list: The references should be given in full with the name and forename initial(s) of the author(s), year, full title of the article, and journal of publication with indication of the volume, first and last page of the article. In the list of references, the order should be alphabetical with papers by the same authors arranged in the order 1) single author, 2) two authors alphabetically according to the name of the second author, and 3) three or more authors chronologically with a,b,c

336 World Rabbit Sci. WORLD RABBIT SCIENCE 2018 etc. for papers published in the same year. References should be abbreviated in accordance with the rules of Biosciences Information Service (Biosis). In uncertainty about the correct abbreviation, the full journal title should be employed. Names of authors are in lower case, name of the journal and number of the journal in italics. Transcriptions from non-Latin alphabets must be written between square brackets. Some examples are given below.

Adamson I., Fisher H. 1973. Aminoacid requirements of the growing rabbit: an estimate of quantitative needs. J. Nutr., 103: 1306-1310. Colin M., 1993. Rabbit production in East European countries. World Rabbit Sci., 1: 37-52. EC Council. 2002. Regulation laying down health rules concerning animal by-products not intended for human consumption. No. 1774/2002/ EC, 3 October 2002. Oº. J. Eur. Comm., 10 October 2002, L 273, 1-95. Gallot S., Magdelaine P. 2007. Évolution des performances techniques et des coûts de production en volailles de chair. Rapport ITAVI. Available at: http://www.itavi.asso.fr/economie/references/PerformancesTechniquesEtCoutsProduction2006.pdf. Accessed February 2009. Italian Law. 1993. Attuazione della direttiva 93/119/CE relativa alla protezione degli animali durante la macellazione e l’abbattimento. Decreto Legislativo No 33, 1 September 1988. Gazzetta U·ciale, 28 September 1998, No 226. Koehl P.F. 1988. The performances of rabbit production units followed through technical and economical management. In Proc.: 4th World Rabbit Congress, 10-14 October, 1988. Budapest, Hungary. 1, 318-325. Lebas F., Coudert P., Rouvier R., de Rochambeau H. 1986. The rabbit, breeding and pathology. F.A.O., Rome, Italy. SAS. 1988. SAS/STAT User’s Guide (Release 6.03). SAS Inst. Inc., Cary NC, USA. Smith J.E., Lang G.H. 1992. Composition of rabbit blood. in: Foster R.P., Manners G.P.R. (ed). Biology of mammals. Boº Inc., Corronsac SD, USA, 789-792. Yu B., Chio P.W.S., Young C.L., Huang H.H. 1987.[A study of rabbit T-type canula and ileal digestibility]. J. Chin. Soc. Anim. Sci., 16: 73-81.

Manuscript Submission Submit the ﬁle to the Manuscript Submission Centre of WRS website. This platform will manage your paper throughout whole evaluation process by one of the Associate Editors, according to the main subject of the paper. http://www.wrs.upv.es

Review process The manuscript received will be submitted to two referees and, returned to the author(s) for final writing. After modifications according to the referees’ comments, the revised paper should be sent to the Section Editor attached to an e-mail. The Section Editor proposes the publication of the paper to the Editor-in-Chief. Exceptionally, authors can receive comments or suggestions from the Editor-in-Chief. Proof-prints are sent to the authors by e-mail in a PDF file. Authors should examine them carefully and send by e-mail the misprints observed. Modifications of the paper’s content are not allowed in print- proofs.

Copyright and charges If the manuscript is accepted for publication, copyrights will be assigned exclusively to the Publisher. No part of this publication (except summaries) may be reproduced without the prior written consent of the Publisher. There will be no page charge. Authors will receive a pdf ﬁle containing their article, but no oÄ-prints are provided.

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