1 Comparative Biochemistry and Physiology Part A 134 (2003) Effects of a fiber-rich diet on physiology and survival of farm-reared red-legged partridges (Alectoris rufa) a, a a b Javier Millan *, Christian Gortazar, Francisco J. Buenestado, Pablo Rodrıguez, b a Francisco S. Tortosa, Rafael Villafuerte a Instituto de Investigacion en Recursos Cinegeticos (IREC, CSIC-UCLM), P.O. Box 535, Ciudad Real, Spain b Departamento de Zoologıa, Universidad de Cordoba, Cordoba, Spain Received 12 February 2002; received in revised form 2 July 2002; accepted 4 July 2002 Abstract The sudden shift from a commercial energy-rich, fiber-poor diet to natural food is assumed as one of e causes of e low success of gamebird restocking. We fed farmed red-legged partridge (Alectoris rufa) chicks wi a diet rich in fiber from eir first mon of life to e time of eir release to assess e possible effects on morphometric characteristics and plasma biochemistry and wheer ose effects enhance survival after release into e wild. At e time of release, treated partridges showed heavier gizzards and bursas of Fabricius and longer small intestines an control birds fed commercial feed. Treated birds had also smaller pectoral muscles, lighter spleens and lower plasma levels of proteins, glucose, cholesterol and triglycerides. After release and radiotracking in a hunting area, we found no differences in e survival between bo groups. Survival at e end of e study (day 39) was 58.3"14.2% for e control group and 24.2"13.8% for e treated group. The fiber-rich diet seems to have important effects on e physiology of e redlegged partridge, not always taken into account in previous studies Elsevier Science Inc. All rights reserved. Keywords: Alectoris rufa; Farm-rearing; Feed; Fibre; Gamebird; Management; Red-legged partridge; Spain; Survival 1. Introduction Farm-reared galliforms fed commercial feed often show physiological differences in comparison to wild individuals (Moss, 1972; Liukkonen- Anttila et al., 2000). These fiber-poor, energy-rich diets in farmed birds are associated wi shorter intestines and lighter gizzards an in wild birds or even an in captive birds fed natural food (Liukkonen-Anttila et al., 1999). These physiological differences are claimed as one of e reasons for e poor results of e restockings (Dowell, 1992). *Corresponding auor. Tel.: q ; fax: q address: (J. Millan). Alough oer factors such as hormones, rhym of feeding, composition and amount of diet, parasite load and gut microbes affect gut size, e fiber content of e diet seems to have a major role in determining e leng of e intestine (Leopold, 1953; Moss, 1972). Fiber-rich diets improve reproductive parameters in rock partridges (Alectoris graeca, Bergero et al., 1995), stimulate larger intestinal villi and improve e immune capacity of e intestine of broiler chickens (Hedge et al., 1978), or increase e energy uptake in galliforms (McBee and West, 1969). In addition, Paganin et al. (1993) showed at rock partridges wi larger intestines survived longer after release. The red-legged partridge (Alectoris rufa) is a medium-sized galliform bird native from e Ibe /03/$ - see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S Ž
2 86 J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) rian peninsula. More an 4 million farm-reared partridges are released in autumn yearly to supplement stocks for shooting. It has been pointed out at ese individuals have lighter viscera and shorter guts an wild ones (Millan et al., 2001) and released red-legged partridges survive only a short period in e wild after release (Gortazar et al., 2000). Since fiber-rich items constitute e bulk of e autumn winter diet in red-legged partridges in e Iberian peninsula (Jimenez et al., 1991), e stress resulting from e sudden shift from commercial to natural food could be one of e reasons for ese findings, especially taking into account at acclimatization of released gamebirds is not usually done. In order to know e effects of a fiber-rich diet on e survival of released red-legged partridges, we fed two groups of farm-reared chicks wi a feed of 4% and 8% fiber-content, respectively, and we tested wheer (i) e high amount of fiber in e diet would enlarge e partridge s gut size, (ii) if e fiber content affects oer aspects of e bird s physiology, such as e amount of energy reserves, and (iii) e changes would increase e partridge s survival after release. Therefore, we measured morphometry, splanchnometry and plasma biochemistry. We also studied eir survival after release into e wild and compared eir survival wi at of farmed partridges fed commercial feed. 2. Material and meods 2.1. Partridge rearing nd Four hundred partridges hatched on June 2, 2000, were kept indoors and fed a commercial starter feed wi 30% protein until July 5. From en onwards, ey were divided into two separate groups (200 per group) and kept outdoors at a 2 density of 0.5 birdsym. Control partridges were given a commercial pelleted feed wi a metabolisable energy (ME) content of 2750 kcalykg ( kjykg), 21% protein, 3.3% fat and 4.1% fiber. Following red-legged partridge traditional breeding management, in addition to e commercial pellets, an increasing amount of whole wheat was provided, decreasing e initial fiber content from 3.9% (only pellets) to nearly 2% (final ird of e rearing only wheat). Treated partridges were given a feed wi e same ME content, 21% protein, 8.2% fat and 8% fiber (Piensos Nanta, Madrid, Spain). Main fiber sources in bo feeds were bran, soy and barley. The increase in fiber was achieved by a higher proportion of bran. According to e European Union directive 92y89, crude fiber content was determined in feeds wi e Weende meod. Bo partridge groups were fed ad libitum and routinely treated against coccidia (Amprolium, Bayer, Germany) and helmins (Flubenol, Laboratorios Esteve, Spain) Sampling Seventy partridges of each group were ringed. st nd On July 21 (sampling 1), August 22 (sampling 2), September 28 (sampling 3) and October 25 (sampling 4), ese partridges were weighed wi a portable dinamometer to e nearest gram and e tarsus leng was measured to e nearest millimeter. Pectoral angle, a measure of e wid of bo pectoral muscles, was obtained wi e aid of a protractor adapted to an angle measurer. A 0.5-ml blood sample was obtained from e brachial vein wi a capillary tube to measure e haematocrit and plasma proteins. On December 4 (sampling 5), 31 ringed birds of e treated group and 19 of e control group were humanely euanised and necropsied. They were weighed, measured and sexed. The heart, liver (wiout gallbladder), gizzard (emptied and wiout e inner cuticle), bursa of Fabricius and spleen of each bird were weighed to e nearest milligram after resection of any fat or connective tissue and after drying for a few seconds on filter paper. We also measured one ceca and e small intestine from e pylorus to e rectum. All measurements were taken by e same observer (JM). On samplings 3 and 5, we obtained 1 ml of blood of 28 ringed birds (14 treated and 14 nontreated). Heparinised samples were centrifuged and plasma was stored at y70 8C. We measured, by spectrophotometry, e following parameters: glucose (glucose oxidase meod), cholesterol (combined cholesterol esterase, cholesterol oxidase and peroxidase meod), triglycerides (combined lipase, glycerol kinase, glycerol phosphate oxidase and peroxidase meod), uric acid (uricase meod) and total proteins (biuret meod) wi an autoanalyser Cobas Mira (Productos Roche Espana, Madrid, Spain). At each sampling, we performed a coprological analysis of 20 partridges per group selected at
3 J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) random to confirm at antiparasitic treatment had been carried out properly Radio-tracking At sampling 5, 28 non-ringed birds (14 of each group) were fitted wi 9-g radio-transmitters (Biotrack, Dorset, UK) and released in a 1200 ha privately-owned hunting area in Medina Sidonia (Cadiz, Souern Spain, W, N). The landscape is characterized by large beetroot, wheat and sunflower fields, surrounded by only 5% narrow hedges of Termomediterranean scrub of Olea europaea and Pistacia lentiscus. Game management practices include artificial watering and supplementary feeding of wheat from June to October and intense predator persecution. Partridges were released wiout acclimatization pens and radiotracked for 39 days Statistical analysis Differences in pectoral angle, tarsus leng and haematocrit among groups were tested using twoway ANOVA (factors: sex and feed). Differences in organ size were tested using two-way ANCOVA (factors: sex and feed; covariates: body mass for organ mass, tarsus leng for organ leng, see Millan et al., 2001). Differences in biochemical parameters were tested using a Mann Whitney U test, analyzing bo sexes independently. Survival of radio-tagged partridges was estimated by aid of e Kaplan Meier product limit estimate (see Church, 1993) and log-rank tests were used to test for differences in e survival between e two release groups. Five animals at survived less an 5 days were excluded from e analyses because ey could have died due to causes related to handling (Tsai et al., 1999). 3. Results Average values and standard deviations of e studied parameters at each sampling are summarized in Tables 1 and 2. During e four first samplings, bo groups seemed to follow a similar development pattern. However, at necropsy, 6- mon-old partridges fed wi fiber-rich feed had heavier gizzards (16% heavier in males, 45% in females) and longer small intestines (7% longer in males, 10% in females). Caeca leng was similar in bo groups. In contrast, treated partridges had lighter spleens, but heavier bursas. We also found differences in e plasma biochemistry parameters at is time: treated partridges showed lower plasma levels of proteins, glucose, cholesterol and triglycerides. Periodical coprological analysis confirm e absence of helmins and very low levels of coccidia oocyst shedding until e day of release. However, we found no differences in e survival between groups (Log Rank Test, Wald Wsy 2.52, Test statisticsy1.44 P)0.05). Survival was 58.3"14.2% for e control group and 24.2"13.8% for e treated group (Fig. 1). Predation (ns4), trauma (ns3) and coccidiosis (ns 3) were identified as presumed mortality causes of e released partridges. Ten birds eier survived or were lost due to transmitter failures. In e remaining eight cases e cause of dea could not been identified. 4. Discussion 4.1. Survival As in previous studies (Birkan, 1971; Gortazar et al., 2000), e survival of released farm-reared red-legged partridges was very low. In contrast to Paganin et al. (1993) and our expectations, a higher amount of fiber in e diet did not improve e chance of survival. However, it is difficult to draw definitive conclusions considering e small sample size and e lack of replication Morphometry and plasma chemistry We found differences in e gut characteristics between treated and control groups, mainly in gizzard weight and small intestine leng. Causes known to affect gut size were reviewed by Moss (1972). Since bo groups belonged to e same flock and e differences were only in feed composition, e observed changes were due to e diet. Nevereless, even in e treated group, e leng of e small intestine was smaller an reported in wild individuals (Millan et al., 2001). We also found no differences in e leng of e caeca in opposition to Paganin et al. (1993) or Mussa et al. (1997). These auors compared rock partridges fed wi feed containing 6% and 17% fiber, respectively. We gave e red-legged partridges 4% and 8% crude fiber-content diets.
4 Table 1 Studied measures (mean"s.d.) in juvenile red-legged partridges (Alectoris rufa), fed wi food containing 8% fiber (treated) or 4% fiber (control) Sampling Group Treated Control Treated Control Treated Control Treated Control Treated Control Body mass (g) 231.4" " " " " " " " " "91.9 (70) (68) (66) (56) (64) (56) (63) (55) (31) (19) Tarsus leng 50.8" " " " " " " " " "3.3 (mm) (70) (68) (67) (56) (63) (56) (63) (55) (31) (19) Pectoral angle 15.5" " " " " " " " " "0.8 (8) (69) (68) (67) a (56) a (63) (56) (63) (55) (31) a (19) a Haematocrit 40.9" " " " " " " " " "4.9 (%) (69) (67) (64) (54) (60) c (52) c (55) (46) (19) (14) Heart (g) 1.65" "0.2 (31) (19) Spleen (g) 0.13" "0.06 (31) a (19) a Liver (g) 6.63" "1.0 (31) (19) Gizzard (g) 9.88" "1.2 (31) c (19) c Bursa (g) 0.29" "0.1 (31) a (19) a Small intestine 72.9" "5.4 (cm) (29) b (19) b Cecum (cm) 15.3" "1.3 (31) (19) a b c Values from each sampling wi e same superscript indicate e statistical significance in e effect of treatment (ANCOVAS and ANOVAS: P-0.05, P-0.01, P-0.001; d e Mann Whitney U: males P-0.05, females P-0.05). Sample size is given in pareneses. 88 J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 85 91
5 Table 2 Studied biochemical parameters (mean"s.d.) in juvenile red-legged partridges (Alectoris rufa), fed wi food containing 8% fiber (treated) or 4% fiber (control) Sampling Group Treated Control Treated Control Treated Control Proteins (gyl) 60.2"6 (58) 59.0"5 (52) 59.6"6 (60) a 62.9"6 (40) a 61.2"8 (20) a 72.5"8 (14) a Glucose (mmolyl) 21.24"3.06 (14) 22.39"2.67 (15) 21.78"4.4 (14) d 23.39"4.05 (15) d Cholesterol (mgyl) 1725"322 (15) 1804"402 (15) 1383"413 (14) d,e 2768"1056 (15) d,e Triglycerids (mgyl) 1694"515 (15) 1537"796 (15) 1121"525 (14) e 2123"828 (15) e Uric acid (mmolyl) " (12) " (14) " (14) " (15) Calcium (meqyl) 9.35"1.95 (3) 8.3"2.85 (12) 9" 2.85 (14) 8.7"3.3 (8) a d Values from each sampling wi e same superscript indicate e statistical significance in e effect of treatment (ANOVAS: P-0.01; Mann Whitney U: males P-0.05, e females P-0.05). Sample size is given in pareneses. J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003)
6 90 J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) Fig. 1. Kaplan Meier survival functions of radio-tagged redlegged partridges released, depending on e amount of fiber in e diet. Control 4% fiber (solid line) and treated 8% fiber (dashed line). Log-Rank Test, Wald Wsy2.52, Test statistics y1.44 P)0.05. Since e amount of fiber in e diet of wild partridges may be higher but has not been quantified, is increase may not be sufficient to obtain relevant differences in caeca leng, but oer reasons could also explain is finding. Millan et al. (2001) noted greater differences in e ceca an in small intestine leng between wild and farmed partridges. The explanation could be related wi e findings of Moss (1972): he observed a continuous decrease in gut leng in captive red grouse (Lagopus lagopus scoticus) generations during 4 years. Since no change in diet occurred in at time, he postulated some adaptive advantage in having short guts in captivity. Therefore, if e same phenomenon happened in e red-legged partridges during e last 30 years (when partridge farm-rearing began in Spain), e treatment of one generation of partridges may not be enough to obtain longer ceca. In addition, oer diet compounds apart from fiber, such as tannins or oer secondary compounds, are also involved in determining gut size (i.e. Liukkonen-Anttila et al., 2001). Treated partridges had lighter spleens and heavier bursas. These findings are unclear. Bo organs have different roles in e immune system: e bursa is a primary site of differentiation and proliferation of B lymphocytes and antibody diversification until e bird has reached e age of approximately 4 6mons (when it begins to atrophy). The spleen is a secondary lymphoid organ, important for antigen processing and antibody production after hatching (Sharma, 1998). Those are only two among more an 10 different tissues involved in e immune defense. In eir ontogeny, many aspects remain unclear (Sharma, 1998). Since diseases are one of e problems of restockings (Dowell, 1992), ese differences in immune organ weights could be relevant for future research. Pectoral muscles were wider in control animals. Galliforms have relatively short wings and large flight muscles and escape predation by a rapid take-off followed by short flights (Butler and Bishop, 2000). Thus, ese partridges wi wider pectoral muscles may be more capable to perform stronger flights to avoid predators. In addition, levels of glucose were higher in control partridges. Galliforms have a majority of fast glycolytic fibers in eir flight muscles restricted to anaerobic metabolism of carbohydrates (Butler and Bishop, 2000). Therefore, high plasma glucose levels may also help control partridges to escape from predators. Plasma levels of proteins, cholesterol and triglycerides were also higher in control partridges. Plasma proteins have a wide range of functions, including resistance to infection, maintenance of homeostasis or being a source of amino acids to e tissues, among oers (see Rogers, 2000). Lipid depots are e major energy reserve for birds, which begin to metabolize fat reserves after a short period of starvation (Blem, 2000). For example, e shift from a seed-rich diet to a less energetic one is associated wi a fall in plasma cholesterol levels (Fudge, 1991). The final extreme stages of starvation may be sustained by breakdown of proteins (Blem, 1990). Thus, ese greater levels of proteins and energetic reserves may help partridges to resist weaer, diseases or starvation and, erefore, to survive e first days after release, when e highest number of deas occurs (Gortazar et al., 2000), independently of eir ability to obtain nutrients from natural food Conclusions We did not obtain gut measures in e intermediate samplings. Following Redig (1989), e enlargement of gut may require from 4 8 weeks in galliforms. We did not find differences in plasma biochemistry or e pectoral angle when e partridges were almost 4 mons old, as we did at e time of release. Therefore, a better management
7 J. Millan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) scheme could consist of a fiber-rich diet during e first mons of rearing in order to enlarge eir digestive tract and, erefore, improve eir nutrient absorption effectiveness and from en afterwards, a more energetic andyor protein-rich diet to make partridges ready for e release. We conclude at a diet wi higher fiber content affects not only gut size, as shown in previous studies, but also affects oer physiological parameters of farm-reared red-legged partridges, such as e immune organ size, blood chemistry, or muscle size. Apparently, is diet was not sufficient to improve partridge survival after release in e wild. More research is needed in order to develop a diet at combines an optimal development of e intestine wi an adequate storage of energy reserves in farm-reared partridges. Acknowledgments This is a contribution to FEDER-UE IF 1FD and a project by CSIC-Junta de Andalucıa. We wish to ank Junta de Andalucıa, P. Prieto from e breeding facility, J. Lizaso and J. Rosell (Piensos Nanta) for e composition and manufacture of e experimental feed, J.A. Blanco, J. Vicente, M. Castillo, M. Munoz, X. Gerrikago- itia and P. Tizzani for field and laboratory assistance and J. Vinuela for helpful comments to e manuscript. References Bergero, D., Debernardi, M., Mussa, P.P., Paganin, M., Effetto di diete a diverso contenuto in fibra ed in energia sulle prestazioni riproduttive delle coturnici (Alectoris graeca). Rivista di Avicoltura 9, Birkan, M.G., Reussite des lachers ˆ de perdrix grise (Perdix perdix L.) et de perdrix rouge (Alectoris rufa L.) d9elevage. Proceedings of e Ten Congress of e International Union of Game Biologists, Paris, France, pp Blem, C.R., Avian energy storage. Curr. Orniol. 7, Blem, C.R., Energy balance. In: Whittow, G.C. (Ed.), Sturkie s Avian Physiology. 5 ed.. Academic Press, San Diego, CA, USA, pp Butler, P.J., Bishop, C.M., Flight. In: Whittow, G.C. (Ed.), Sturkie s Avian Physiology. 5 ed.. Academic Press, San Diego, CA, USA, pp Church, K.E., Survival and nesting biology of translocated grey partridge (Perdix perdix) in New York State, USA. Gibier Faune Sauvage 10, Dowell, S.D., Problems and pitfalls of gamebird reintroduction and restocking: an overview. Gibier Faune Sauvage 9, Fudge, A.M., Avian practice tips. Vet. Clin. N. Am. Small. 21,. Gortazar, C., Villafuerte, R., Martın, M., Success of traditional restocking of red-legged partridge for hunting purposes in areas of low density of noreast Spain. Aragon. Z. Jagdwiss. 46, Hedge, S.N., Rolls, B.A., Turvey, A., Coates, M.E., The effect on chicks of dietary fibre from different sources: a grow factor in wheat bran. Br. J. Nutr. 40, Jimenez, R., Hodar, J.A., Camacho, I., La alimentacion de la perdiz comun, Alectoris rufa, en otono-invierno en el sur de Espana. Gibier Faune Sauvage 8, Leopold, A.S., Intestinal morphology of gallinaceus birds in relation to food habits. J. Wildlife Manage. 17, Liukkonen-Anttila, T., Putaala, A., Hissa, R., Does shifting from a commercial to a natural diet affect e nutritional status of e hand-reared grey partridges Perdix perdix? Wildlife Biol. 5, Liukkonen-Anttila, T., Saartoala, R., Hissa, R., Impact of hand-rearing on morphology and physiology of e capercaillie (Tetrao urogallus). Comp. Biochem. Physiol. A 125, Liukkonen-Anttila, T., Kentala, A., Hissa, R., Tannins-a dietary problem for hand-reared grey partridge Perdrix perdrix after release? Comp. Biochem. Physiol. C 130, McBee, R.H., West, G.C., Cecal fermentation in e willow ptarmigan. Condor 71, Millan, J., Gortazar, C., Villafuerte, R., Marked differences in e splanchnometry of farm-bred and wild redlegged partridges (Alectoris rufa L.). Poult. Sci. 80, Moss, R., Effects of captivity on gut leng in red grouse. J. Wildlife Manage. 36, Mussa, P.P., Debernardi, M., Paganin, M., Bergero, D., Modulazione della lunghezza intestinale di coturnici delle Alpi (Alectoris graeca) mediante l incremento della componente fibrosa della dieta. Suppl. Ric. Biol. Selvaggina 27, Paganin, M., Dondini, G., Vergari, S., Dessi-Fulgheri, F., La dieta e l esperienza influenzano la sopravivenza di coturnici (Alectoris graeca) liberate in natura. Suppl. Ric. Biol. Selvaggina 21, Redig, P.T., The avian ceca, obligate combustion chambers or facultative afterburners? The conditioning influence of diet. J. Exp. Zool. Suppl. 3, Rogers, K.S., Plasma proteins. In: Feldman, B.F., Zinkl, J.G., Jain, N.C. (Eds.), Schalm s Veterinary Hematology. 5 ed.. Lippincott, Williams & Wilkins, Philadelphia, Pennsylvania, USA, pp Sharma, J.M., Avian immunology. In: Pastoret, P.P., Griebel, P., Bazin, H., Govaerts, A. (Eds.), Handbook of Vertebrate Immunology. Academic Press, San Diego, CA, USA, pp Tsai, K., Pollock, K.H., Brownie, C., Effects of violation of assumptions for survival analysis meods in radiotelemetry studies. J. Wildlife Manage. 63,