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Effects of diets with increasing levels of Spirulina platensis on the performance and apparent digestibility in growing rabbits
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Livestock Science 118 (2008) 173 – 177
www.elsevier.com/locate/livsci
Short communication
Effects of diets with increasing levels of Spirulina platensis on the performance and apparent digestibility in growing rabbits P.G. Peiretti a,⁎, G. Meineri b b
a Institute of Science of Food Production, National Research Council, Grugliasco (TO), Italy Department of Animal Production, Epidemiology and Ecology, University of Torino, Grugliasco (TO), Italy
Received 18 February 2008; received in revised form 15 April 2008; accepted 22 April 2008
Abstract The efficiency of diets with the inclusion of Spirulina platensis (SP) for rabbit growing has been tested. The trial was carried out on 40 crossbred rabbits that were randomly allocated to four groups each with ten animals (five male and five female rabbits each), kept in individual cages. Three isoproteic and isoenergetic diets were formulated with an increasing level of SP (5%, 10% and 15%); the diets were tested against a control diet without microalgae. The experimental period lasted 24 days and the faeces were collected during the last week. No obvious health problems were encountered during the experiment and no rabbits died during the feeding trial. The measured parameters were growth performance and digestibility of dry matter (DM), organic matter (OM), crude protein (CP), crude fibre (CF), ether extract (EE), neutral detergent fibre (NDF), acid detergent fibre (ADF) and gross energy (GE). The results show that the final weight, weight gain and feed efficiency did not differ significantly (P N 0.05) among the dietary treatments, but an SP inclusion level of 10% gave the highest feed intake. The DM, OM, CP, GE, NDF and ADF digestibilities of the control diet were higher than those of the SP-containing diets. © 2008 Elsevier B.V. All rights reserved. Keywords: Microalgae; Growth performance; Digestibility; Rabbit; Acid insoluble ash
1. Introduction The main protein supplement of choice for rabbits rations is soybean meal. However, due to the scarcity and escalating costs of soybean meal and its widespread diffusion as a genetically modified organism (GMO), research on alternative protein sources is gaining importance. Production costs currently prevent extensive use of microalgae as an animal feed, but numerous nutritional ⁎ Corresponding author. Tel.: +39 011 6709230; fax: +39 011 6709297. E-mail address: [email protected] (P.G. Peiretti). 1871-1413/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.04.017
experiments clearly demonstrate the high value of some species of microalgae as a protein supplement for fish, cattle, pigs, and chickens (Soeder, 1986). Simplified production techniques have been set up to obtain large quantities of plant biomass at competitive prices (Molina Grima et al., 1994). The dietary use of algae has also been proposed by many authors as a protein feed supplement for swine (Yap et al.; 1982; Hugh et al., 1985; Grinstead et al., 2000) or as a protein replacement for soybean meal in rabbit diets (Battaglini, 1979; Raju and Sreemannarayana, 1995; Sreemannarayana et al., 1995). Blue–green algae are considered to be a good source of protein and energy (Harel et al., 2002). Among these
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P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177
Table 1 Ingredients of the diets (%) Spirulina powder a Soybean seed meal Corn Dehydrated alfalfa meal Pelleted ground hay Corn oil Lignosulphite Vitamin–mineral premix b
0 15 27 38 12 4 2 2
5 7 30 39 11 4 2 2
10 1 30 31 20 4 2 2
15 – 30 – 46 4 3 2
a Proximate composition: 7.3% moisture, 61.8% crude protein, 1.7% fat, 9.8% ash, 0.4% crude fibre, 26.3% nitrogen free extract, and 20.1 MJ/kg DM gross energy. b Per kg of the complete diet: Vit. A 200 UI; α-tocopheryl acetate 16 mg; Niacin 72 mg; Vit. B6 16 mg; Choline 0.48 mg; DL-methionine 600 mg; Ca 500 mg; P 920 mg; K 500 mg; Na 1 g; Mg 60 mg; Mn 1.7 mg; Cu 0.6 mg.
microalgae, Spirulina platensis (SP) is protein rich (Nandeesha et al., 2001), easily digestible and also a rich source of essential fatty acids (e.g. γ-linolenic acid; Belay et al., 1996), chlorophyll, carotenoids, minerals, vitamins (especially Vitamin B12 and provitamin A), carbohydrates, sterols (Henrikson, 1997) and a few pigments (phycocyanin and allophycocyanin), which are mainly responsible for antioxidant activities (Miranda et al., 1998). SP is commercially cultivated in open ponds throughout the world, primarily as a human food supplement due to its potential antiviral, antioxidant, hepato protective, antiallergenic and immunomodulator activities (Khan et al., 2005) but also for animal feed, due to its protein content of over 60% (Richmond, 1990). S. platensis, which is easily harvested, provides a superior natural source of carotenoids and has been used throughout the world as a feed component in quality broiler and layer diets to enhance yolk colour and flesh (Colas et al., 1979; Ross and Dominy, 1990). However, to the authors' knowledge, there are no published reports on the feeding value of S. platensis for rabbits. The aim of the present research was to evaluate the performance of growing rabbits and the apparent digestibility of SP in growing rabbits fed diets containing increasing levels of SP as a protein replacement for soybean meal. 2. Methods 2.1. Experimental The study was carried out at the CISRA (Centro Interdipartimentale Servizio Ricovero Animali) experimental rabbitry of the University of Turin. Forty weaned crossbred rabbits aged nine weeks with a mean weight of 2034 ± 174 g were randomly assigned to four groups of 10 (five male and five female rabbits each) with equal initial weight variability. The
animals were housed individually under standard conditions at a temperature of 22 °C ± 2 °C in wire cages at a height of 90 cm from the concrete floor. Four isoproteic and isoenergetic diets were formulated with increasing levels of Spirulina (0%, 5%, 10% and 15%), which was obtained from the Ornitalia Product Service s.a.s. (Colleredo di Monte Albano (UD), Italy). All diets were pelleted and stored in darkness to avoid auto-oxidation of the lipid sources. The ingredients and the chemical composition of the four diets are shown in Tables 1 and 2, respectively. After 1 week for adaptation to the diets and cages, the animals were fed ad libitum for 24 days. The rabbits had free access to clean drinking water. Feed was weighed and distributed two times a week. The unconsumed feed was weighed and discarded before offering the new diet to determine the feed consumption. The rabbits were weighed individually at 7, 14, 20 and 24 days to determine the weight gain, feed intake and feed efficiency during the experimental period. 2.2. Digestibility trial The apparent digestibilities of the four diets were determined within the last week. The faeces were collected over a period of five days at approximately 0900 h before the next daily ration was provided. The faeces were collected using a nylon net placed under the cages of each trial, to avoid urine contamination. Each pooled fecal sample was taken and placed in a two-layer plastic bag to prevent the loss of moisture and immediately frozen at −20 °C. The frozen samples were individually mixed thoroughly and pooled, ground in a homogenizer (Tecator, Herndon, VA, USA) and the representative samples were then weighed on an aluminium foil pan, dried in a draft oven at 80 °C to constant weight and stored for chemical analysis. 2.3. Analytical methods All the analyses were carried out on duplicate samples. The proximate composition of the diets and faeces was determined according to the AOAC method (Association of Official Table 2 Chemical composition of the diets (%) Spirulina powder (% of diet)
Dry matter (% as fed) Organic matter Crude protein Crude fibre Ether extract Ash Neutral detergent fibre Acid detergent fibre Acid insoluble ash Gross energy (MJ/kg DM) Digestible energy (MJ/kg DM) a a
0
5
10
15
89.9 92.1 18.8 14.5 4.8 7.9 30.1 17.0 1.0 18.1 12.2
90.3 91.7 19.1 15.8 7.0 8.3 29.8 19.3 1.2 18.9 11.8
90.8 91.8 19.6 15.9 6.9 8.2 34.2 19.4 1.2 18.9 11.8
88.2 92.0 19.1 15.7 7.5 8.0 32.5 18.6 1.4 19.8 11.9
The digestible energy content of the diets was calculated using the regression proposed by Fernandez-Carmona et al. (1996).
P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177 Table 3 Productive performance (means ± S.E.) of rabbits fed four levels of Spirulina powder Spirulina powder (% of diet)
Number of rabbits Mortality (%) Initial weight (g) Final weight (g) Weight gain (g/day) Feed intake (g/day) Feed efficiency
0
5
10
15
10
10
10
10
0
0
0
0
2047 ± 67
2024 ± 44
2030 ± 58
2032 ± 56
2983 ± 138
3150 ± 65
3184 ± 73
3050 ± 76
39.0 ± 3.9
46.9 ± 2.1
48.1 ± 3.7
42.4 ± 2.7
182.6 ± 8.1a 210.4 ± 5.2bc 227.0 ± 8.2c 198.6 ± 8.6ab 4.7 ± 0.4
4.5 ± 0.2
4.7 ± 0.3
4.7 ± 0.2
Values in the same row with unlike superscripts differ (P b 0.05).
Analytical Chemists, 1990). The dietary and faecal samples were analysed to determine dry matter (DM), total N content, ash by ignition to 550 °C, and ether extract (EE) using the Soxlet method, crude fibre according to the Weende method, neutral detergent fibre (NDF) without sodium sulfite sulphite and α-amylase, and acid detergent fibre (ADF), as described by Van Soest et al. (1991) expressed exclusive of residual ash. The gross energy (GE) was determined by means of an adiabatic bomb calorimeter (IKA C7000, Staufen, Germany). The acid insoluble ash content of the diets and faeces was determined according to the method of Van Keulen and Young (1977). 2.4. Calculation and statistical analysis The digestibility coefficients were calculated following the indirect digestibility method (Furuichi and Takahashi, 1981) using acid insoluble ash (AIA) as an inert marker. The calculation of the digestibility of DM was as follows: DM digestibility (%) = (1 −A/B) ⁎ 100, where A and B are the acid insoluble ash concentrations in the feed and faeces, respectively. The digestibility of the other nutrients (X) was calculated as follows: Digestibility (X in %) = (1 −A/B ⁎XB/XA) ⁎ 100, where XA and XB are the concentrations of X in the feed and faeces, respectively. Statistical analyses were performed using the SPSS software package (version 11.5.1 for Windows, SPSS Inc., USA). The analysis of variance was used to evaluate the effects of different concentrations of SP on growth performance and the digestibility of the four different diets in rabbits. The means were compared with a Duncan test and the significance level was set to a level of 5%.
3. Results and discussion The performance data are given in Table 3. No rabbits died during the trial and there was good consumption of
175
the SP diets. The feed intake of the group fed the 10% SP diet was the highest while the lowest feed intake was obtained in the control group. Feed efficiency was not affected by SP. The apparent digestibility coefficients are reported in Table 4. The DM, OM, CP, GE, NDF and ADF digestibilities of the control diet were higher than those of the SP diets. Inclusion of 15% SP in the diet resulted in very low digestibilities compared to the control, and compared to diets containing 5% and 10% SP and the differences were significant in each case. The only exception was the digestibility of the CP which differed only from that of the control diet. The EE digestibility of the group fed the 10% SP diet was similar to that of the control group and was higher than the other two groups. Previous studies have evaluated higher inclusions of microalgae (up to 33%) as a protein replacement for soybean meal in weanling pigs (Yap et al., 1982; Hugh et al., 1985). Yoshida and Hoshi (1980) and Becker and Venkataraman (1982) fed varying levels of spirulina to growing chickens with satisfactory results at the lower levels of 5 to 10%; growth was depressed at levels above 20%. The current study was conducted using SP inclusion levels of between 5 and 15%, because the low level used in pig trials by Grinstead et al. (2000) showed inconsistent and minimal improvement in growth performance when the pigs were fed diets containing 0.2, 0.5 and 2% SP. The same authors found that feed intake was significantly lower for the pigs fed SP in pelleted diets than for the pigs fed meal diets. Conversely, Yap et al. (1982) found that pigs fed high levels of Spirulina maxima (14%), as a protein replacement for dried skimmed milk, had similar performance to those fed the control diet containing skimmed milk. Tamiya (1961) found that the weight gain of rabbits fed a 5% Chlorella supplemented diet was higher than those Table 4 Percent of the apparent digestibility coefficients (mean ± S.E.) obtained using AIA (acid insoluble ash) as the internal marker Spirulina powder (% of diet) 0 Dry matter Organic matter Crude protein Ether extract Gross energy Crude fibre Acid detergent fibre Neutral detergent fibre
5 a
10 b
15 b
56.8 ± 0.1c 57.2 ± 0.1c 68.0 ± 0.3b 85.4 ± 0.1c 58.9 ± 0.3c 13.2 ± 2.0c 4.0 ± 0.6d
69.7 ± 0.4 70.3 ± 0.4a 71.9 ± 0.6a 88.5 ± 0.4ab 69.2 ± 0.2a 29.3 ± 1.9a 28.6 ± 0.7a
62.2 ± 0.3 62.8 ± 0.3b 66.9 ± 0.1c 88.1 ± 0.2b 62.5 ± 0.6b 23.8 ± 2.0ab 21.7 ± 1.2b
61.9 ± 0.4 62.5 ± 0.4b 68.8 ± 0.1b 89.2 ± 0.3a 62.7 ± 0.6b 21.0 ± 1.6b 21.2 ± 0.5c
38.1 ± 0.9a
25.4 ± 1.1c
32.1 ± 1.5b 19.2 ± 0.6d
Values in the same row with unlike superscripts differ (P b 0.05).
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P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177
fed a 5% soybean meal supplemented diet, while Battaglini et al. (1979) showed that inclusion of 12% of a green alga (Scenedesmus acutus), as a protein replacement for soybean meal, in rabbit feed decreased the total feed consumption and weight gain. Ross and Dominy (1990) evaluated growth performance of growing chickens fed diets that contained 0, 5, 10, 15, and 20% SP to replace dehulled soybean meal. Significant growth depression occurred with the diet containing 20% SP as early as the first week. By the third week, the chicks receiving 10 and 20% SP grew significantly slower than those in the control group, while the feed efficiency was not affected by the treatment. The digestibility results of the present study are generally in agreement with Battaglini et al. (1979) who found that, with the exception of ether extract, the nutritive component digestibilities of rabbits fed diets with an inclusion of 12% of a green alga (S. acutus) were lower than those of rabbits fed diets with the same inclusion of soybean meal. The method of preparation of the algal sample bears heavily on the efficiency of utilization of the protein contained in the sample. Moreover, heat stability problems have been observed in pig diets with SP supplements. In this context, Grinstead et al. (2000) suggested that high temperatures associated with the pelletting process may have damaged or inactivated the SP and consequently reduced the digestibility of the SP diets. Battaglini et al. (1979) instead suggested that a thermal shock would improve the digestibility of microalgae in rabbit diets. SP digestibility in the rabbits was higher than in other monogastric species. The rabbit, due to its caecal microbial activity and the nutritional contribution of caecotrophy can, in fact, perform a microbial fermentation action, when microalgae are included in the mixed feed as a protein concentrate, that is similar to that of ruminants, which, among all the animals that have been fed with microalgae, have given the best performance (Battaglini et al., 1979). 4. Conclusion The main result of the present investigation is that SP can be used in rabbit diets, without any adverse effects on growth performance, because the lower digestibility of the SP diets was balanced by an increased feed consumption. This conclusion is valid only for the fattening period (from 9 to 13 weeks of age) observed in this study, particularly considering the diet with 15% SP. The effect of lower digestibility might not be apparent in that period or during 24 days in rabbit, contrary to chickens which grew more slowly following just one week on a diet containing 20%
SP. In the future a fattening experiment between weaning at 35 days age and slaughter at 70–84 days should be performed to rule out the possibility of adverse effect. Acknowledgements The research was supported by the National Research Council; thanks are due to Mrs. K.I. Ricci for her technical support and Mr. G. Cerato for the preparation of the pelleted diets. References Association of Official Analytical Chemists (AOAC), 1990. In: Helrich, K. (Ed.), Official Method of Analysis, 15th Ed. Arlington, VA. Battaglini, M., 1979. Algae and yeasts for rabbits. Coniglicolt. 16, 39–40. Battaglini, M., Grandi, A., Paletti, C., Pushparaj, B., 1979. Use of biomass of Scenedesmus acutus 8 M as an alternative protein concentrate in diets for growing rabbits. Zoot. Nutr. Anim. 5, 211–218. Becker, E., Venkataraman, L., 1982. Biotechnology and Exploitation of Algae — the Indian Approach. German agency for technical cooperation, Eschborn, Germany, pp. 96–176. Belay, A., Kato, T., Ota, Y., 1996. Spirulina (Arthrospira): potential application as an animal feed supplement. J. Appl. Phycol. 8, 303–311. Colas, B., Sauvageot, F., Harscoat, J.P., Sauveur, B., 1979. Proteines alimentaires et qualite de l'oeuf. II. — influence de la nature des proteines destribuees aux poules sur les caracteristiques sensorielles de l'oeuf et la teneur en acides amines libres du jaune. Ann. Zootech. 28, 297–314. Fernandez-Carmona, J., Cervera, C., Blas, E., 1996. Prediction of the energy value of rabbit feeds varying widely in fibre content. Anim. Feed Sci. Technol. 64, 61–75. Furuichi, Y., Takahashi, T., 1981. Evaluation of acid insoluble ash as a marker in digestion studies. Agric. Biol. Chem. 45 (10), 2219–2224. Grinstead, G.S., Tokach, M.D., Dritz, S.S., Goodband, R.D., Nelssen, J.L., 2000. Effects of Spirulina platensis on growth performance of weanling pigs. Anim. Feed Sci. Technol. 83, 237–247. Harel, M., Koven, W., Lein, I., Bar, Y., Beherens, P., Stubbleffeld, J., Zohar, Y., Place, A.R., 2002. Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213, 347–362. Henrikson, R., 1997. Earth Food Spirulina: How This Remarkable Blue–Green Algae Can Transform Your Health and Our Planet. Renore Enterprises, Kenwood, CA. Hugh, W.I., Dominy, W., Duerr, E., 1985. Evaluation of Dehydrated Spirulina (Spirulina platensis) as a Protein Replacement in Swine Starter Diets. University of Hawaii Research and Extension Series, Honolulu. ID — ISSN 0271-9916, 10 pp. Khan, Z., Bhadouria, P., Bisen, P.S., 2005. Nutritional and therapeutic potential of Spirulina. Curr. Pharm. Biotechnol. 6, 373–379. Molina Grima, E., García Camacho, F., Sanchez Perez, J.A., Urda Cardona, J., Acién Fernández, F.G., Fernández Sevilla, J.M., 1994. Outdoor chemostat culture of Phaeodactylum tricornutum UTEX 640 in a tubular photobioreactor for the production of eicosapentaenoic acid. Biotechnol. Appl. Biochem. 20, 279–290. Miranda, M.S., Cintra, R.G., Barros, S.B., Mancini Filho, J., 1998. Antioxidant activity of the microalga Spirulina maxima. Braz. J. Med. Biol. Res. 31, 1075–1079.
P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177 Nandeesha, M.C., Gangadhara, B., Manissery, J.K., Venkataraman, L.V., 2001. Growth performance of two Indian major carps, catla (Catla catla) and rohu (Labeo rohita) fed diets containing different levels of Spirulina platensis. Biores. Technol. 80, 117–120. Raju, K.V.S., Sreemannarayana, O., 1995. Feeding of Ulva fasciata to rabbits — feed efficiency and carcass characteristics. Indian Vet. J. 72, 1331–1332. Richmond, A., 1990. Large scale microalgal culture and applications. Progress in Phycological Research, vol. 7. Biopress, Bristol, pp. 1–62. Ross, E., Dominy, W., 1990. The nutritional value of dehydrated, blue–green algae (Spirulina platensis) for poultry. Poult. Sci. 69, 794–800. Soeder, C.J., 1986. An historical outline of applied algology. In: Richmond, A. (Ed.), Handbook of Microalgal Mass Cultures. CRC Press, Boca Raton., FL. Sreemannarayana, O., Raju, K.V.S., Ramaraju, G.V.A.N.S., Prasad, J.R., 1995. The use of Ulva fasciata, a marine alga as rabbit feed: growth and conversion efficiency. Indian Vet. J. 72, 989–991.
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Tamiya, H., 1961. Chemical composition and applicability as food and feed of mass cultured unicellular algae. Final Report No. 1, Contract DA 92-551-FEC 33129 U.S. Army Research and Development Group. Office of the Chief of Research and Development, U.S. Army. Van Keulen, J., Young, B.A., 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44, 282–287. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3591. Yap, T.N., Wu, J.F., Pond, W.G., Krook, L., 1982. Feasibility of feeding Spirulina maxima, or Chlorella sp. to pigs weaned to a dry diet at 4 to 8 days of age. Nutr. Rep. Int. 25, 543–552. Yoshida, M., Hoshi, H., 1980. Nutritive value of spirulina, green algae, for poultry feed. Jpn. Poult. Sci. 17, 27–30.
Livestock Science 118 (2008) 173 – 177
www.elsevier.com/locate/livsci
Short communication
Effects of diets with increasing levels of Spirulina platensis on the performance and apparent digestibility in growing rabbits P.G. Peiretti a,⁎, G. Meineri b b
a Institute of Science of Food Production, National Research Council, Grugliasco (TO), Italy Department of Animal Production, Epidemiology and Ecology, University of Torino, Grugliasco (TO), Italy
Received 18 February 2008; received in revised form 15 April 2008; accepted 22 April 2008
Abstract The efficiency of diets with the inclusion of Spirulina platensis (SP) for rabbit growing has been tested. The trial was carried out on 40 crossbred rabbits that were randomly allocated to four groups each with ten animals (five male and five female rabbits each), kept in individual cages. Three isoproteic and isoenergetic diets were formulated with an increasing level of SP (5%, 10% and 15%); the diets were tested against a control diet without microalgae. The experimental period lasted 24 days and the faeces were collected during the last week. No obvious health problems were encountered during the experiment and no rabbits died during the feeding trial. The measured parameters were growth performance and digestibility of dry matter (DM), organic matter (OM), crude protein (CP), crude fibre (CF), ether extract (EE), neutral detergent fibre (NDF), acid detergent fibre (ADF) and gross energy (GE). The results show that the final weight, weight gain and feed efficiency did not differ significantly (P N 0.05) among the dietary treatments, but an SP inclusion level of 10% gave the highest feed intake. The DM, OM, CP, GE, NDF and ADF digestibilities of the control diet were higher than those of the SP-containing diets. © 2008 Elsevier B.V. All rights reserved. Keywords: Microalgae; Growth performance; Digestibility; Rabbit; Acid insoluble ash
1. Introduction The main protein supplement of choice for rabbits rations is soybean meal. However, due to the scarcity and escalating costs of soybean meal and its widespread diffusion as a genetically modified organism (GMO), research on alternative protein sources is gaining importance. Production costs currently prevent extensive use of microalgae as an animal feed, but numerous nutritional ⁎ Corresponding author. Tel.: +39 011 6709230; fax: +39 011 6709297. E-mail address: [email protected] (P.G. Peiretti). 1871-1413/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2008.04.017
experiments clearly demonstrate the high value of some species of microalgae as a protein supplement for fish, cattle, pigs, and chickens (Soeder, 1986). Simplified production techniques have been set up to obtain large quantities of plant biomass at competitive prices (Molina Grima et al., 1994). The dietary use of algae has also been proposed by many authors as a protein feed supplement for swine (Yap et al.; 1982; Hugh et al., 1985; Grinstead et al., 2000) or as a protein replacement for soybean meal in rabbit diets (Battaglini, 1979; Raju and Sreemannarayana, 1995; Sreemannarayana et al., 1995). Blue–green algae are considered to be a good source of protein and energy (Harel et al., 2002). Among these
174
P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177
Table 1 Ingredients of the diets (%) Spirulina powder a Soybean seed meal Corn Dehydrated alfalfa meal Pelleted ground hay Corn oil Lignosulphite Vitamin–mineral premix b
0 15 27 38 12 4 2 2
5 7 30 39 11 4 2 2
10 1 30 31 20 4 2 2
15 – 30 – 46 4 3 2
a Proximate composition: 7.3% moisture, 61.8% crude protein, 1.7% fat, 9.8% ash, 0.4% crude fibre, 26.3% nitrogen free extract, and 20.1 MJ/kg DM gross energy. b Per kg of the complete diet: Vit. A 200 UI; α-tocopheryl acetate 16 mg; Niacin 72 mg; Vit. B6 16 mg; Choline 0.48 mg; DL-methionine 600 mg; Ca 500 mg; P 920 mg; K 500 mg; Na 1 g; Mg 60 mg; Mn 1.7 mg; Cu 0.6 mg.
microalgae, Spirulina platensis (SP) is protein rich (Nandeesha et al., 2001), easily digestible and also a rich source of essential fatty acids (e.g. γ-linolenic acid; Belay et al., 1996), chlorophyll, carotenoids, minerals, vitamins (especially Vitamin B12 and provitamin A), carbohydrates, sterols (Henrikson, 1997) and a few pigments (phycocyanin and allophycocyanin), which are mainly responsible for antioxidant activities (Miranda et al., 1998). SP is commercially cultivated in open ponds throughout the world, primarily as a human food supplement due to its potential antiviral, antioxidant, hepato protective, antiallergenic and immunomodulator activities (Khan et al., 2005) but also for animal feed, due to its protein content of over 60% (Richmond, 1990). S. platensis, which is easily harvested, provides a superior natural source of carotenoids and has been used throughout the world as a feed component in quality broiler and layer diets to enhance yolk colour and flesh (Colas et al., 1979; Ross and Dominy, 1990). However, to the authors' knowledge, there are no published reports on the feeding value of S. platensis for rabbits. The aim of the present research was to evaluate the performance of growing rabbits and the apparent digestibility of SP in growing rabbits fed diets containing increasing levels of SP as a protein replacement for soybean meal. 2. Methods 2.1. Experimental The study was carried out at the CISRA (Centro Interdipartimentale Servizio Ricovero Animali) experimental rabbitry of the University of Turin. Forty weaned crossbred rabbits aged nine weeks with a mean weight of 2034 ± 174 g were randomly assigned to four groups of 10 (five male and five female rabbits each) with equal initial weight variability. The
animals were housed individually under standard conditions at a temperature of 22 °C ± 2 °C in wire cages at a height of 90 cm from the concrete floor. Four isoproteic and isoenergetic diets were formulated with increasing levels of Spirulina (0%, 5%, 10% and 15%), which was obtained from the Ornitalia Product Service s.a.s. (Colleredo di Monte Albano (UD), Italy). All diets were pelleted and stored in darkness to avoid auto-oxidation of the lipid sources. The ingredients and the chemical composition of the four diets are shown in Tables 1 and 2, respectively. After 1 week for adaptation to the diets and cages, the animals were fed ad libitum for 24 days. The rabbits had free access to clean drinking water. Feed was weighed and distributed two times a week. The unconsumed feed was weighed and discarded before offering the new diet to determine the feed consumption. The rabbits were weighed individually at 7, 14, 20 and 24 days to determine the weight gain, feed intake and feed efficiency during the experimental period. 2.2. Digestibility trial The apparent digestibilities of the four diets were determined within the last week. The faeces were collected over a period of five days at approximately 0900 h before the next daily ration was provided. The faeces were collected using a nylon net placed under the cages of each trial, to avoid urine contamination. Each pooled fecal sample was taken and placed in a two-layer plastic bag to prevent the loss of moisture and immediately frozen at −20 °C. The frozen samples were individually mixed thoroughly and pooled, ground in a homogenizer (Tecator, Herndon, VA, USA) and the representative samples were then weighed on an aluminium foil pan, dried in a draft oven at 80 °C to constant weight and stored for chemical analysis. 2.3. Analytical methods All the analyses were carried out on duplicate samples. The proximate composition of the diets and faeces was determined according to the AOAC method (Association of Official Table 2 Chemical composition of the diets (%) Spirulina powder (% of diet)
Dry matter (% as fed) Organic matter Crude protein Crude fibre Ether extract Ash Neutral detergent fibre Acid detergent fibre Acid insoluble ash Gross energy (MJ/kg DM) Digestible energy (MJ/kg DM) a a
0
5
10
15
89.9 92.1 18.8 14.5 4.8 7.9 30.1 17.0 1.0 18.1 12.2
90.3 91.7 19.1 15.8 7.0 8.3 29.8 19.3 1.2 18.9 11.8
90.8 91.8 19.6 15.9 6.9 8.2 34.2 19.4 1.2 18.9 11.8
88.2 92.0 19.1 15.7 7.5 8.0 32.5 18.6 1.4 19.8 11.9
The digestible energy content of the diets was calculated using the regression proposed by Fernandez-Carmona et al. (1996).
P.G. Peiretti, G. Meineri / Livestock Science 118 (2008) 173–177 Table 3 Productive performance (means ± S.E.) of rabbits fed four levels of Spirulina powder Spirulina powder (% of diet)
Number of rabbits Mortality (%) Initial weight (g) Final weight (g) Weight gain (g/day) Feed intake (g/day) Feed efficiency
0
5
10
15
10
10
10
10
0
0
0
0
2047 ± 67
2024 ± 44
2030 ± 58
2032 ± 56
2983 ± 138
3150 ± 65
3184 ± 73
3050 ± 76
39.0 ± 3.9
46.9 ± 2.1
48.1 ± 3.7
42.4 ± 2.7
182.6 ± 8.1a 210.4 ± 5.2bc 227.0 ± 8.2c 198.6 ± 8.6ab 4.7 ± 0.4
4.5 ± 0.2
4.7 ± 0.3
4.7 ± 0.2
Values in the same row with unlike superscripts differ (P b 0.05).
Analytical Chemists, 1990). The dietary and faecal samples were analysed to determine dry matter (DM), total N content, ash by ignition to 550 °C, and ether extract (EE) using the Soxlet method, crude fibre according to the Weende method, neutral detergent fibre (NDF) without sodium sulfite sulphite and α-amylase, and acid detergent fibre (ADF), as described by Van Soest et al. (1991) expressed exclusive of residual ash. The gross energy (GE) was determined by means of an adiabatic bomb calorimeter (IKA C7000, Staufen, Germany). The acid insoluble ash content of the diets and faeces was determined according to the method of Van Keulen and Young (1977). 2.4. Calculation and statistical analysis The digestibility coefficients were calculated following the indirect digestibility method (Furuichi and Takahashi, 1981) using acid insoluble ash (AIA) as an inert marker. The calculation of the digestibility of DM was as follows: DM digestibility (%) = (1 −A/B) ⁎ 100, where A and B are the acid insoluble ash concentrations in the feed and faeces, respectively. The digestibility of the other nutrients (X) was calculated as follows: Digestibility (X in %) = (1 −A/B ⁎XB/XA) ⁎ 100, where XA and XB are the concentrations of X in the feed and faeces, respectively. Statistical analyses were performed using the SPSS software package (version 11.5.1 for Windows, SPSS Inc., USA). The analysis of variance was used to evaluate the effects of different concentrations of SP on growth performance and the digestibility of the four different diets in rabbits. The means were compared with a Duncan test and the significance level was set to a level of 5%.
3. Results and discussion The performance data are given in Table 3. No rabbits died during the trial and there was good consumption of
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the SP diets. The feed intake of the group fed the 10% SP diet was the highest while the lowest feed intake was obtained in the control group. Feed efficiency was not affected by SP. The apparent digestibility coefficients are reported in Table 4. The DM, OM, CP, GE, NDF and ADF digestibilities of the control diet were higher than those of the SP diets. Inclusion of 15% SP in the diet resulted in very low digestibilities compared to the control, and compared to diets containing 5% and 10% SP and the differences were significant in each case. The only exception was the digestibility of the CP which differed only from that of the control diet. The EE digestibility of the group fed the 10% SP diet was similar to that of the control group and was higher than the other two groups. Previous studies have evaluated higher inclusions of microalgae (up to 33%) as a protein replacement for soybean meal in weanling pigs (Yap et al., 1982; Hugh et al., 1985). Yoshida and Hoshi (1980) and Becker and Venkataraman (1982) fed varying levels of spirulina to growing chickens with satisfactory results at the lower levels of 5 to 10%; growth was depressed at levels above 20%. The current study was conducted using SP inclusion levels of between 5 and 15%, because the low level used in pig trials by Grinstead et al. (2000) showed inconsistent and minimal improvement in growth performance when the pigs were fed diets containing 0.2, 0.5 and 2% SP. The same authors found that feed intake was significantly lower for the pigs fed SP in pelleted diets than for the pigs fed meal diets. Conversely, Yap et al. (1982) found that pigs fed high levels of Spirulina maxima (14%), as a protein replacement for dried skimmed milk, had similar performance to those fed the control diet containing skimmed milk. Tamiya (1961) found that the weight gain of rabbits fed a 5% Chlorella supplemented diet was higher than those Table 4 Percent of the apparent digestibility coefficients (mean ± S.E.) obtained using AIA (acid insoluble ash) as the internal marker Spirulina powder (% of diet) 0 Dry matter Organic matter Crude protein Ether extract Gross energy Crude fibre Acid detergent fibre Neutral detergent fibre
5 a
10 b
15 b
56.8 ± 0.1c 57.2 ± 0.1c 68.0 ± 0.3b 85.4 ± 0.1c 58.9 ± 0.3c 13.2 ± 2.0c 4.0 ± 0.6d
69.7 ± 0.4 70.3 ± 0.4a 71.9 ± 0.6a 88.5 ± 0.4ab 69.2 ± 0.2a 29.3 ± 1.9a 28.6 ± 0.7a
62.2 ± 0.3 62.8 ± 0.3b 66.9 ± 0.1c 88.1 ± 0.2b 62.5 ± 0.6b 23.8 ± 2.0ab 21.7 ± 1.2b
61.9 ± 0.4 62.5 ± 0.4b 68.8 ± 0.1b 89.2 ± 0.3a 62.7 ± 0.6b 21.0 ± 1.6b 21.2 ± 0.5c
38.1 ± 0.9a
25.4 ± 1.1c
32.1 ± 1.5b 19.2 ± 0.6d
Values in the same row with unlike superscripts differ (P b 0.05).
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fed a 5% soybean meal supplemented diet, while Battaglini et al. (1979) showed that inclusion of 12% of a green alga (Scenedesmus acutus), as a protein replacement for soybean meal, in rabbit feed decreased the total feed consumption and weight gain. Ross and Dominy (1990) evaluated growth performance of growing chickens fed diets that contained 0, 5, 10, 15, and 20% SP to replace dehulled soybean meal. Significant growth depression occurred with the diet containing 20% SP as early as the first week. By the third week, the chicks receiving 10 and 20% SP grew significantly slower than those in the control group, while the feed efficiency was not affected by the treatment. The digestibility results of the present study are generally in agreement with Battaglini et al. (1979) who found that, with the exception of ether extract, the nutritive component digestibilities of rabbits fed diets with an inclusion of 12% of a green alga (S. acutus) were lower than those of rabbits fed diets with the same inclusion of soybean meal. The method of preparation of the algal sample bears heavily on the efficiency of utilization of the protein contained in the sample. Moreover, heat stability problems have been observed in pig diets with SP supplements. In this context, Grinstead et al. (2000) suggested that high temperatures associated with the pelletting process may have damaged or inactivated the SP and consequently reduced the digestibility of the SP diets. Battaglini et al. (1979) instead suggested that a thermal shock would improve the digestibility of microalgae in rabbit diets. SP digestibility in the rabbits was higher than in other monogastric species. The rabbit, due to its caecal microbial activity and the nutritional contribution of caecotrophy can, in fact, perform a microbial fermentation action, when microalgae are included in the mixed feed as a protein concentrate, that is similar to that of ruminants, which, among all the animals that have been fed with microalgae, have given the best performance (Battaglini et al., 1979). 4. Conclusion The main result of the present investigation is that SP can be used in rabbit diets, without any adverse effects on growth performance, because the lower digestibility of the SP diets was balanced by an increased feed consumption. This conclusion is valid only for the fattening period (from 9 to 13 weeks of age) observed in this study, particularly considering the diet with 15% SP. The effect of lower digestibility might not be apparent in that period or during 24 days in rabbit, contrary to chickens which grew more slowly following just one week on a diet containing 20%
SP. In the future a fattening experiment between weaning at 35 days age and slaughter at 70–84 days should be performed to rule out the possibility of adverse effect. Acknowledgements The research was supported by the National Research Council; thanks are due to Mrs. K.I. Ricci for her technical support and Mr. G. Cerato for the preparation of the pelleted diets. References Association of Official Analytical Chemists (AOAC), 1990. In: Helrich, K. (Ed.), Official Method of Analysis, 15th Ed. Arlington, VA. Battaglini, M., 1979. Algae and yeasts for rabbits. Coniglicolt. 16, 39–40. Battaglini, M., Grandi, A., Paletti, C., Pushparaj, B., 1979. Use of biomass of Scenedesmus acutus 8 M as an alternative protein concentrate in diets for growing rabbits. Zoot. Nutr. Anim. 5, 211–218. Becker, E., Venkataraman, L., 1982. Biotechnology and Exploitation of Algae — the Indian Approach. German agency for technical cooperation, Eschborn, Germany, pp. 96–176. Belay, A., Kato, T., Ota, Y., 1996. Spirulina (Arthrospira): potential application as an animal feed supplement. J. Appl. Phycol. 8, 303–311. Colas, B., Sauvageot, F., Harscoat, J.P., Sauveur, B., 1979. Proteines alimentaires et qualite de l'oeuf. II. — influence de la nature des proteines destribuees aux poules sur les caracteristiques sensorielles de l'oeuf et la teneur en acides amines libres du jaune. Ann. Zootech. 28, 297–314. Fernandez-Carmona, J., Cervera, C., Blas, E., 1996. Prediction of the energy value of rabbit feeds varying widely in fibre content. Anim. Feed Sci. Technol. 64, 61–75. Furuichi, Y., Takahashi, T., 1981. Evaluation of acid insoluble ash as a marker in digestion studies. Agric. Biol. Chem. 45 (10), 2219–2224. Grinstead, G.S., Tokach, M.D., Dritz, S.S., Goodband, R.D., Nelssen, J.L., 2000. Effects of Spirulina platensis on growth performance of weanling pigs. Anim. Feed Sci. Technol. 83, 237–247. Harel, M., Koven, W., Lein, I., Bar, Y., Beherens, P., Stubbleffeld, J., Zohar, Y., Place, A.R., 2002. Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213, 347–362. Henrikson, R., 1997. Earth Food Spirulina: How This Remarkable Blue–Green Algae Can Transform Your Health and Our Planet. Renore Enterprises, Kenwood, CA. Hugh, W.I., Dominy, W., Duerr, E., 1985. Evaluation of Dehydrated Spirulina (Spirulina platensis) as a Protein Replacement in Swine Starter Diets. University of Hawaii Research and Extension Series, Honolulu. ID — ISSN 0271-9916, 10 pp. Khan, Z., Bhadouria, P., Bisen, P.S., 2005. Nutritional and therapeutic potential of Spirulina. Curr. Pharm. Biotechnol. 6, 373–379. Molina Grima, E., García Camacho, F., Sanchez Perez, J.A., Urda Cardona, J., Acién Fernández, F.G., Fernández Sevilla, J.M., 1994. Outdoor chemostat culture of Phaeodactylum tricornutum UTEX 640 in a tubular photobioreactor for the production of eicosapentaenoic acid. Biotechnol. Appl. Biochem. 20, 279–290. Miranda, M.S., Cintra, R.G., Barros, S.B., Mancini Filho, J., 1998. Antioxidant activity of the microalga Spirulina maxima. Braz. J. Med. Biol. Res. 31, 1075–1079.
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