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Chemical characterization, dry matter and crude protein ruminal degradability and in vitro intestinal digestion of acid and fermented silage from tilapia filleting residue
Animal Feed Science and Technology 136 (2007) 226–239
Chemical characterization, dry matter and crude protein ruminal degradability and in vitro intestinal digestion of acid and fermented silage from tilapia filleting residue Luiz Juliano Val´erio Geron a , L´ucia Maria Zeoula a , Rose Meire Vidotti b,∗ , Makoto Matsushita a , Ricardo Kazama a , Saul Ferreira Caldas Neto a , Fernanda Fereli a a
Animal Science Department, Laboratory of Analysis of Food and Animal Nutrition, Experimental Farm Iguatemi, Maringa State University, Maringa, Av. Colombo, 5790-CEP, 87020-900 Parana, Brazil b Sao Paulo State Fishing Institute, CP: 1052, S˜ ao Jos´e do Rio Preto CEP: 15025-970, Brazil Received 15 April 2005; received in revised form 31 August 2006; accepted 5 September 2006
Abstract The objective of the present study was to evaluate the chemical characteristics, amino acids and fatty acids profiles, dry matter (DM) and crude protein (CP) ruminal degradability and rumen-undegraded protein digestible in the intestine (RUPD ) of the tilapia filleting residue (TFR) and of the acid and fermented silage of tilapia filleting residue (ASTFR and FSTFR, respectively). The ASTFR was obtained by an acidification process with the addition of 0.02 L/kg TFR of sulfuric acid and 0.02 L/kg TFR of formic acid and the FSTFR was obtained by fermentation with the addition of 0.05 kg/kg TFR of natural yogurt, 0.15 kg/kg TFR of molasses and 0.003 kg/kg TFR of sorbic acid. A completely randomized design was used to characterize the fatty acids profile of the ASTFR and FSTFR and to determine ruminal degradability of DM and CP of ASTFR, FSTFR and fishmeal (FIME). The amino acids profiles of the ASTFR and FSTFR differed from TFR, but ASTFR and FSTFR were similar to each other. The fatty acids profiles of the ASTFR and FSTFR were similar to the TFR Abbreviations: ASTFR, acid silage tilapia filleting residue; CP, crude protein; DM, dry matter; ED, effective degradability; FIME, fishmeal; FSTFR, fermented silage tilapia filleting residue; RUP, rumen undegraded protein; TFR, tilapia filleting residue; v/w, volume/weight; w/w, weight/weight ∗ Corresponding author. Tel.: +55 17 32166769. E-mail address: [email protected] (R.M. Vidotti). 0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.09.006
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profile. The ASTFR showed a higher (P<0.05) saturated fatty acid content in comparison to the FSTFR. The effective degradability (ED) of the CP at solid passage rate of 0.05 h−1 of the ASTFR and FSTFR were of 0.76 and 0.81, respectively, which are higher (P<0.05) than the TFR value of 0.44. To the ASTFR and FSTFR lower values (P<0.05) were observed to RUPD of 13.5 and 18.8 g/kg, respectively, in comparison the FIME of 224.4 g/kg RUP. The ASTRF and FSTRF showed alteration composition chemical and amino acids profile without altered the nutritive value of TFR. The processes of acidification or fermentation are methodologies that can be used as a conservation procedure of tilapia filleting residue. © 2006 Elsevier B.V. All rights reserved. Keywords: Amino acids; Fatty acids; Fish silage; Digestible protein in the small intestine
1. Introduction The utilization of low cost and good quality alternative feed sources can decrease production costs and increase profits. Among several alternative sources, tilapia-filleting residue, presents great nutritional quality and high potential for animal production. Brazil and Mexico, both populous countries with great water resources, will be the two main producers of the tilapia industry in the Western Hemisphere (Fitzsmmons, 2000). The largest markets of tilapia in Brazil form the filleting industry, which is found in the south and southeast of Brazil. Fish silage is a noble protein, a product of high biological value for animal feeding, which can be produced from dead fish, species that are sub-utilized in the fish industry, or from marine fishing, commercial fish waste and industrial residues. These are considered low quality raw materials, that if not used may cause environmental, sanitary and economical problems (Vidotti et al., 2003). The processes used for conservation of tilapia filleting residue (TFR) consist of two distinct methodologies: acidification and fermentation. The acidification process begins with a triturated mass of fish, allowing free enzyme activity to liquefy the tissues. Acidification is conducted by adding mineral or organic acids such as: formic, sulfuric, chloride, propionic, etc. (Disney and James, 1979; Wignall and Tatterson, 1997; Vidotti et al., 2002). The anaerobic fermentation process was initiated by adding microorganisms and a carbohydrate source (Lindgren and Pleje, 1983; Van Wyk and Heyderych, 1985; Vidotti et al., 2002). Fish silage is a source similar to fishmeal with respect to composition profile; however, it behaves differently in the digestive tract, which in spite of the high protein content is a poor source of RUP (Ouellet et al., 1997). Effective degradability of CP in the rumen depends on the feed characteristics, ingestion level, type of processing which the feed was undergone and possible limitations in the rumen fermentation process (pH, ammonia concentration, fatty acids) according to Orskov (1998). Available information about the chemical and biological characteristics of the tilapia filleting residue silage to feed ruminants is scarce. Therefore, the objective of this study was to evaluate chemical composition, amino acids profile, fatty acids profile of acid (ASTFR)
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and fermented (FSTFR) silage of tilapia filleting residue, besides to estimate the protein fraction, DM and CP ruminal degradability and intestinal digestion of rumen undegraded protein of ASTFR and FSTFR compared to fishmeal.
2. Material and methods 2.1. Conservation of tilapia filleting residue The tilapia filleting residue (TFR) was obtained at a tilapia filleting factory located in the town of Euclides da Cunha in Sao Paulo state, Brazil. The TFR contained head, carcass, but no viscera. The TFR was triturated using a commercially available meat grinder and placed in sealed plastic buckets (3 L capacity), and it was, submitted to the two processes: acidification or fermentation. The acid silage of tilapia filleting residue (ASTFR) was produced by adding 0.02 L/kg TFR of sulfuric acid and 0.02 L/kg TFR. of formic acid. The fermented silage of tilapia filleting residue (FSTFR) was produced by adding 0.05 kg/kg TFR of Lactobacillus ssp. (Nestle® , natural yogurt), 0.15 kg/kg TFR of sugar cane molasses and 0.003 kg/kg TFR of sorbic acid. The treatments were conducted with three replications each. The pH measurements were conducted using a pH meter (Tecnal® , TEC3MP) on the following days: 1, 3, 5, 7, 9, 11, 15, 21, 30, 60, 90 and 180 after the silage process had begun. A commercial fishmeal (Alisul Alimentos S/A Parana, Brazil) was analyzed in this experiment in order to compare with ASTFR and FSTFR. 2.2. Protein fraction, rumen degradability and intestinal digestion of rumen undegraded protein It was calculated the nitrogen fractions: A, B1 , B2 , B3 and C of TFR, ASTFR, FSTFR and FIME. The fractions A was obtained using the methodology of Krishnamoorthy et al. (1983) and fraction B1 , B2 and B3 according to Sniffen et al. (1992). The fraction C was obtained by acid detergent insoluble nitrogen (ADIN) (Van Soest et al., 1991). Ruminal degradability of dry matter (DM) and crude protein (CP) of ASTFR, FSTFR and fishmeal (FIME) was determined using three Holstein steers with initial body weight (BW) of 380 ± 61 kg fitted with ruminal cannula distributed in a completely randomized desing with three repetitions. The animals were daily fed with 17.0 kg of corn silage (fresh weight) and 2.5 kg of concentrated (fresh weight). The composition of the ration on dry matter basis in g/kg was as follows: 640.0 corn silage, 223.0 corn, 87.0 soybean meal and 50.0 fishmeal. The animals were adapted to the experimental diet for a period of 14 days, in which they were fed twice a day. First, the ASTFR, FSTFR samples were dried at 55 ◦ C over 72 h, ground through a 2 mm screen (Wiley mill). Subsequently 6.0 g of sample were weighed in 10 cm × 17 cm nylon bags made of nitrogen free polyester (Ankon Technology Co. Fairport, NY, USA) with a mean pore size of 53 m and heat sealed. Bags were placed into the rumen for 2, 4, 6, 8,
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12, 24, 48 and 72 h. The FIME sample, as fed, was also weighed (6.0 g) in nylon bags and incubated as mentioned above. Three samples bags/feed were taken and incubated at each sampling time, for provide sufficient residue for subsequent chemical analysis. Bags were placed in the rumen at the same time and removed sequentially. The bags were removed, rinsed briefly in bucket of cold water, then rinsed three cycles of 5 min with cold water in a semi automatic washing machine and the bags residues were dried at 55 ◦ C over 72 h. The soluble fraction (time 0) was measured in two bags per sample but without rumen incubation and the bags were placed in water-bath at 37 ◦ C over 1 h. The bags residues were analyzed for DM and nitrogen. Intestinal digestibility (ID) of rumen undegraded protein of ASTFR, FSTFR and FIME was determined using the three-step in vitro procedure following the methodology described by Calsamiglia and Stern (1995). 2.3. Calculations Dry matter and crude protein ruminal degradability were determined using the equation described by Mehrez. and Orskov (1977): p = a + b × (1 − e−ct ) where: p = potential degradability in time t; a = intercept representing the fraction soluble; b = insoluble but potentially degradable fraction; c = constant rate of degradability of fraction b; t = incubation time; a + b ≤ 1. The non-linear parameters “a”, “b” and “c” were estimated using the iterative least squares procedure SAS (1987). Effective degradability (ED) of dry matter and crude protein in the rumen was calculated using the model of Orskov and Mcdonald (1979): ED = a + ((b × c)/(c + k)), where: k = solids outflow rate: “a”, “b” and “c” were previously defined. The solid outflow rates (k) used for each feed were 0.02, 0.05 and 0.08 h−1 , which are attributed to low, medium and high levels of feed ingestion, respectively (AFRC, 1993). 2.4. Laboratory analyses All chemical analyses of samples of TFR, ASTFR, FSTFR and FIME were made on duplicated according to AOAC (1984). Dry matter content was determined at 105 ◦ C over 24 h (procedure ID 7.007) in a forced air oven. Nitrogen content was determined by the micro-Kjeldahl method (ID 7.015) and crude protein (CP) was determined as nitrogen × 6.25. Ash was analysed by incineration in a muffle furnace (ID 7.009) at 550–600 ◦ C for 4 h and ether extract (EE) was determined using a Sohxlet extractor, and washing with petroleum ether (ID 920.39) according to AOAC (1990). Amino acid profile of TFR, ASTFR and FSTFR (average samples) were determined except for triptophan, at the High Technology laboratory (LAB TEC, Mogiana Alimentos S.A. Campinas, SP, Brazil). Gas chromatography analysis to determine fatty acid content was conducted following methodology described by Bligh and Dyer (1959); and procedures described by Hartmam and Lago (1973). The content phosphorus of TFR, ASTFR and FSTRF were determined in colorimetric spectrophotometer (Chimadzu® , model UV 1601). The content calcium of feeds
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Table 1 Content of dry matter (DM), organic matter (OM), crude protein (CP), ash, ether extract (EE), calcium (Ca) and phosphorus (P) of the tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR) and of the fishmeal (FIME) Feeds
DM
OM (g/kg DM)
CP (g/kg DM)
Ash (g/kg DM)
EE (g/kg DM)
Ca (g/kg DM)
P (g/kg DM)
TFR ASTFR FSTFR FIME
397.7 370.2 366.9 932.7
797.3 765.3 812.7 736.8
406.9 370.1 316.4 551.0
202.7 234.7 187.3 263.2
355.1 353.7 321.9 19.2
49.4 48.6 49.7 83.9
45.3 42.5 35.8 69.4
was realized used nitric (HNO3 ) and percloric (HClO4 ) acid in atomic absorption spectrophotometer (GBC® , model 932 AA). 2.5. Statistical analysis A completely randomized design with three replications was used to characterize the fatty acids profile and intestinal digestion of rumen undegraded protein for both processing methods. The average data were compared by Tukey test at a significance level of 0.05. The TFR which had just one sample (no replication) was not analyzed statistically. For DM and CP degradability, the data obtained were submitted to ANOVA procedure using a SAS in completely randomized design, with three replications. The averages were compared by Tukey test at a significance level of 0.05.
3. 3.Results 3.1. Chemical composition of feeds The chemical composition of the TFR, ASTFR, FSTFR and FIME are in Table 1. In general, it can be seen that chemical composition of the silages contained variation in relation to TFR. The DM values of TFR, ASTFR and FSTFR were, respectively, 397.7, 370.2 and 366.9 g/kg. The CP contents of ASTFR and FSTFR reduced 9% and 22%, respectively, compared to the CP content of TFR, (406.9 g/kg). Fat content also reduced 9% for FSTFR in relation of TFR and similar values were found for ASTFR and FSTFR. The TFR initial pH was 6.3 and at the end of the ensilage process, the ASTFR and FSTFR pH was 3.2 and 4.2, respectively. Both were considered satisfactory to avoid proliferation of undesirable microorganisms (Van Wyk and Heyderych, 1985). Vidotti et al. (2002) reported that initial pH of tilapia residue was 6.7 and after 31 days of storage through the acidification process it reduced to 2.8. 3.2. Amino acid and fatty profile of feeds The amino acids profile of TFR, ASTFR and FSTFR are presented in Table 2. The amino acids profile of ASTFR and FSTFR varied slightly compared to TFR. The amino
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Table 2 Amino acids profile of the tilapia filleting residue (TFR) of the acid silage of tilapia filleting residue (ASTFR) and of the fermented silage of tilapia filleting residue (FSTFR) Amino acids Alanine Arginine Aspartic acid Glycine Isoleucine Leucine Glutamic acid Lysine Cystine Methionine Phenylalanine Tyrosine Threonine Proline Valine Histidine Serine CP (g/kg)
TFR (g/100 g CP)
ASTFR (g/100 g CP)
FSTFR (g/100 g CP)
7.6 7.5 9.6 8.1 4.9 9.4 13.7 9.3 1.5 3.2 3.8 1.3 2.1 7.4 6.2 2.8 1.6
6.8 8.5 8.8 10.2 4.7 8.4 13.3 8.8 1.2 3.5 3.6 1.8 3.4 6.2 5.8 2.6 2.8
7.4 5.8 10.9 9.4 4.5 8.1 13.8 8.1 1.5 4.3 3.0 1.3 3.1 7.3 6.4 2.5 2.7
406.9
370.1
316.4
CP, crude protein.
acids profile was not statistically analyzed since the final sample of feeds (TFR, ASTFR and FSTFR) was a composition of the three replications by feed. The ASTFR contained higher levels of arginine, glycine, tyrosine, threonine and serine and lower levels of alanine, leucine, cystine and proline compared to TFR (Table 2). The FSTFR showed higher concentration of aspartic acid, glycine, methionine, threonine and serine, than TFR. However, the contents of leucine, lysine, phenylalanine and histidine were lower. The highest glutamic acid concentration was found in TFR (13.7 g/100 g CP), ASTFR (13.3 g/100 g CP) and FSTFR (13.8 g/100 g CP), as it has been reported by Morales-Ulloa and Oetterer (1997) and Vidotti et al. (2003). ASTFR contained higher concentration of glycine, tyrosine, lysine, phenylalanine and arginine, than in the FSTFR and this showed higher contents of alanine, valine, proline methionine, aspartic acid and cystine than ASTFR. Fatty acid profiles and the concentrations the fatty acid saturated and unsaturated of TFR, ASTFR and FSTFR are presented in Table 3. The lower values for the saturated fatty acids: myristic, palmitic and stearic acids; higher values for long chain unsaturated fatty acids: linoleic, linolenic, di-homo linolenic and docosahexaenoic acids were observed for TFR than ASTFR. However the FSTFR contained similar values for saturated and unsaturated fatty acids when compared to TFR. The FSTFR contained higher (P<0.05) concentrations of linoleic (C18:2); linolenic (C18:3), eicosatrienoic (20:3n9); docosatrienoic (20:3n3) acid when compared to ASTFR, but the ASTFR showed higher levels (P<0.05) of palmitic (16:0), stearic (16:0), oleic (18:1).
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Fatty acids
Common nomenclature
TFR (g/100 g fat)
ASTFR (g/100 g fat)
ASTFR (g/100 g fat)
S.E.M.
14:0 16:0 16:1 18:0 18:19 18:17 18:26 18:36 18:33 20:0 20:111 20:39 20:33 22:63
Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Vacenic acid Linoleic acid Linolenic acid ␣ linolenic acid Araquidic acid Gadoleic acid Mead acid Di-homo linolenic acid Docosahexaenoic acid
2.8 26.3 4.4 6.5 37.8 3.0 14.5 0.6 1.9 0.3 0.3 0.7 0.6 0.4
3.6a 31.2a 4.9a 8.1a 41.7a 4.0a 3.5b 0.3b 1.9a 0.2a 0.2a 0.2b 0.2b 0.1b
2.7a 23.1b 4.4b 6.1b 37.2b 3.3b 17.9a 0.8a 1.7b 0.2a 0.3a 1.0a 0.8a 0.5a
0.20 0.04 0.05 0.03 0.03 0.08 0.03 0.15 0.05 0.16 0.24 0.10 0.14 0.33
55.6 44.4 0.80
43.1 56.9 1.32
32.1 67.9 2.12
– – –
Fatty acids saturated (g/100 g fat) Fatty acids unsaturated (g/100 g fat) Ratio unsaturated/saturated
Averages in the same line, followed by different letters are different (P<0.05) by the Tukey test. S.E.M. standard error mean.
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Table 3 Fatty acids profile of the tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR)
Feeds
TFR ASTFR FSTFR FIME
Nitrogen fractions A (g CP/kg DM)
B1 (g CP/kg DM)
B2 (g CP/kg DM)
B3 (g CP/kg DM)
C (g CP/kg DM)
A (g CP/kg total CP)
B1 (g CP/kg total CP)
B2 (g CP/kg total CP)
B3 (g CP/kg total CP)
C (g CP/kg total CP)
10.0 216.0 250.0 62.0
34.0 0.1 2.0 −12.0
197.0 137.0 79.0 183.0
119.0 8.0 5.0 326.0
2.0 2.0 1.0 7.0
26.0 595.0 744.0 110.0
95.0 0.4 3.0 −22.0
545.0 376.0 234.0 323.0
329.0 23.0 15.0 576.0
5.0 5.0 3.0 13.0
A: fast available fraction (non-protein nitrogen); B1 : Rumen rapidly degraded fraction (peptides and oligopeptides); B2 : intermediary degradation fraction (cytoplasmatic proteins); B3 : slow degradation fraction (proteins insoluble in neutral detergent); C: insoluble fraction (unavailable in the rumen and in the intestine).
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Table 4 Nitrogen fractions of tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR) and of the fishmeal (FIME)
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The conservation processes (acidification and fermentation) of TFR increased concentration of unsaturated fatty acids, which resulted in a higher ratio of unsaturated:saturated relation to TFR. This ratio was 38% higher in ASTFR than in FSTFR. 3.3. Nitrogen fractions of crude protein and ruminal degradability of DM and CP of feeds The nitrogen fractions of TFR, ASTFR, FSTFR and FIME are presented in Table 4. The fraction A (non-protein nitrogen) of CP, was 26.0, 595.0, 744.0 and 110.0 g/kg of CP, respectively for TFR, ASTFR, FSTFR and FIME. The Fraction B1 of CP (constituted of soluble proteins and rapidly degraded, peptides and oligopept´ıdeos) was lower than 3.0 g/kg for ASTFR, FSTFR and FIME, and represented 95.0 g/kg of CP for TFR. The fraction B2 (constituted of insoluble proteins of intermediated ruminal degradability) and B3 (proteins insoluble in neutral detergent, of slow degradation in the rumen) was 399.0 g/kg of CP for ASTFR and 249.0 g/kg of CP for FSTFR. These values were lower compared to TFR (874.0 g/kg of CP). The fraction C, (considered unavailable in the rumen and intestines) was 13.0 g/kg of CP to the FIME and less than 10.0 g/kg of CP to the other feeds. It can be seen from Table 5 that the soluble fraction “a” of DM of FSTFR was higher (P<0.05) than that found for ASTFR and FIME, the latter showed the lowest “a” value (0.18). These results are in agreement with those observed for the fraction A of protein observed in Table 4. Table 5 Soluble (a) and potentially degraded insoluble fractions (b), degradation rate (c), potential degradability (PD) and effective degradability (ED) of dry matter g/kg DM and crude protein in g/kg CP of the feed at passage rates of 0.02, 0.05 and 0.08 h−1 Variables
ASTFR
FSTFR
FIME
S.E.M.
Dry matter (DM) (g/kg DM) a b c (h−1 ) PD ED (0.02 h−1 ) ED (0.05 h−1 ) ED (0.08 h−1 )
330.0b 590.0a 0.49 920.0 890.0a 80.05a 820.0a
700.0a 240.0c 0.15 940,0 910.0a 880.0a 850.0a
180.0c 340.0b 0.09 530.0 460.0b 400.0b 360.0b
0.08 0.05 – – 0.07 0.08 0.08
Crude protein (CP) (g/kg CP) a b c (h−1 ) PD ED (0.02 h−1 ) ED (0.05 h−1 ) ED (0.08 h−1 )
350.0b 590.0a 0.37 940.0 830.0a 760.0a 730.0a
470.0a 490.0b 0.14 960.0 880.0a 810.0a 760.0a
160.0c 480.0b 0.07 640.0 630.0a 440.0b 380.0b
0.05 0.04 – – 0.05 0.07 0.07
Averages in the same line, followed by different letters are different (P<0.05) by the Tukey test. ASTFR: acid silage of the tilapia filleting residue; FSTFR: fermented silage of tilapia filleting residue and FIME: fishmeal. S.E.M.: standard error mean.
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Table 6 Content of crude protein (CP), rumen-degraded protein (RDP) and rumen-undegraded protein (RUP) for 16 h of ruminal incubation, in vitro intestinal digestibility coefficient of RUP (IDRUP), RUP digestible in the small intestine (RUPD ) and in vitro intestinal digestibility coefficient of crude protein (IDCP) without ruminal incubation of acid (ASTFR) and fermented (FSTFR) silages of tilapia filleting residue and of the fishmeal (FIME) Feed
CP g/kg DM
RDP (g/kg DM)
RUP (g/kg DM)
IDRUP
RUPD (g/kg DM)
ASTFR FSTFR FIME
370.1 316.4 551.0
350.5a 300.3a 251.8b
19.6b 16.1b 299.2a
0.94a 0.84a 0.75b
18.4b 13.5b 224.4a
S.E.M.
–
0.05
0.12
0.15
0.11
Feed
CP (g/kg DM)
IDCP
Feed without ruminal incabation ASTFR FSTFR FIME S.E.M.
370.1 316.4 551.0 –
0.98a 0.97a 0.88b 0.04
Averages in the same column, followed by different letters are different (P<0.05) by the Tukey test. S.E.M. standard error mean.
The DM fraction “b” (insoluble, potentially degradable) was highest (P<0.05) for ASTFR, than FSTFR and FIME, but the fraction “b” of FIME was higher (P<0.05) than FSRT. However, for both ensiled were observed similar values of ED for DM, but the fermentation process reduced ruminal degradation rate of the fraction “b” (0.15 h−1 versus 0.49 h−1 ). The potential degradability (PD) of DM of ASTFR and FSTFR was in average 43% higher than that of FIME. Also, ED of DM of ASTFR and FSTFR was higher than that of FIME, independent of the solid outflow rate (0.02, 0.05 and 0.08 h−1 ). It can be seen that fraction “a” of CP of FSTFR was 0.47, and therefore higher (P<0.05) than ASTFR (0.35) and FIME, and the latter contained the lowest (P<0.05) value (0.16). The fraction “b” of CP was higher (P<0.05) to ASTFR (0.59) compared to FSTFR (0.49) and FIME (0.48). Similarly to the dry matter, the lowest degradability rate of fraction “b” (c) of CP was observed in the fermentation process. Silage acidification process releases and makes more readily available CP. FIME contained lower degradation rate of fraction “b” than the silage and lower PD of CP. The ED of CP with outflow rates of 0.05 and 0.08 h−1 was higher (P<0.05) for FSTFR and ASTFR compared to FIME; however, at 0.02 h−1 (low consumption) no difference was observed. 3.4. In vitro digestibility coefficient of crude protein of feeds The values of rumen degraded proteins (RDP), rumen undegraded protein (RUP), in vitro intestinal digestibility coefficient of RUP (IDRUP) and RUP digestible in the small intestine (RUPD ) are presented in Table 6. The values found for RUP of ASTFR and FSTFR were 19.6 g/kg of DM and 16.1 g/kg of DM, respectively; lower (P<0.05) than observed for FIME (299.2 g/kg of DM). The ASTFR (0.94) and FSTFR (0.84) had high IDRUP (P<0.05), than FIME (0.75).
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The content of RUPD of FIME was higher (224.4 g/kg DM) compared to the ASTFR and FSTFR (18.4 and 13.5 g/kg DM, respectively). The values of CP and in vitro intestinal digestibility coefficient of protein (IDCP) of ASTFR, FSTFR and FIME, without being submitted to ruminal incubation period are found in Table 6. The IDCP for ASTFR and FSTFR were, respectively, 0.98 and 0.97 higher (P<0.05) than 0.88 found for FIME.
4. Discussion 4.1. Chemical composition of feeds Both ensiling processes affected the chemical composition of the TFR. The fermentation reduced the contents of CP and EE compared to the original values found in TFR. Probably molasses addition to FSTFR diluted CP and fat content, which was also observed by Morales-Ulloa and Oetterer (1995). Reduced CP levels observed for ASTFR compared to TFR were also reported by Vidotti et al. (2002). The authors found CP levels of 396.0 (g/kg), for acid silage, compared to 429.0 g/kg of TFR. This reduction in CP levels of silage is a consequence of protein hydrolysis, since the autolysis effect on the degradation of proteins and nucleoproteins may transform them in more simple compounds, such as, amino acids (Baraquet and Lindo, 1985) and ammonia that can volatilize during storage of ASTFR and FSTFR. Vidotti et al. (2003) reported for DM, CP, EE, and mineral matter (MM) of TFR the following values 314.0; 429.0; 346.0; 166.0 g/kg DM, respectively; values near to the ones reported in the present study to CP and EE of 406.7 and 355.1 g/kg DM, respectively. 4.2. Amino acid and fatty acids profile of feeds The variation of the amino acids contents was observed in TFR compared to the both silage. There was an increase in the levels of glycine, threonine and serine and decrease in the contents of leucine for both, fermentation and acidification processes. The fermentation process increased the level of methionine in 25% and reduced lysine in 15%, compared to TFR. Such increase in the levels of glycine, tyrosine, lysine, phenylalanine and arginine of ASTFR compared to FSTFR is probably due to the processing method. In a similar shape, Vidotti et al. (2003) reported higher contents of glycine and tyrosine to the ASTFR than FSTFR. In general, the differences observed in the chemical composition and amino acid profiles of silages in this work compared to that reported in the literature may be due to different places of origin, processing method, freshness of the raw material, storage conditions, previous contamination and residue composition. That is, the presence or not of either one of the following parts like: heads, gills, scales, dorsal column, intestine contents and muscle may influence the feed composition. Still, according to Yone et al. (1986), Hossaain et al. (1987) and Baraquet and Lindo (1985) the microbe fermentation alters the composition of CP and MM and increases volatile nitrogen losses.
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The higher concentration of unsaturated fatty acids in FSTFR and the higher levels (P<0.05) of saturated fatty acids in ASTFR showed that both, acidification and fermentation processes altered the fatty acids profile of the silages. Fermentation increased the concentration of unsaturated fatty acids, such as linoleic acid, which in its conjugated forms is highly beneficial. It is anti carcinogenic, anti diabetic and can alter the lipids metabolism and body composition of the animals (Pariza et al., 2001). Berez (1997) while studying sardine residue silage in ration for chicken reported a fatty acid profile containing 13.7 g/100 g of fat of oleic acid and 2.0 g/100 g of fat of acid linoleic. In the present work using tilapia residue, the acid oleic concentrations was 41.7 and 37.2 g/100 g of fat, while linoleic acid was 3.5 and 17.9 g/100 g of fat for ASTFR and FSTFR, respectively. 4.3. Nitrogen fraction of crude protein and ruminal degradability of DM and CP of feeds The ensiling processes of TFR (acid and fermented) changed the nitrogen fraction of the crude protein, increasing considerably fraction A. The fermentation increased the fraction A of CP compared to acidification. Similarly, studies in situ conducted, the FSTFR contained higher value of fraction “a” (soluble fraction of CP, quickly available in the rumen) compared to ASTFR. According to Baraquet and Lindo (1985) autolysis effects on protein degradation, may transform it into more simple compounds, such as amino acids, peptides, pyrimidine and purine basis. The fermentation process and the molasses addition increased the fraction A. The fraction A + B1 indicates higher supply of NPN, peptides and oligopeptides all degradable in the rumen, and a higher amount of available nutrients to ruminal microorganisms. Considering this, it is recommended to use an energetic source that have a quickly available energy to minimize the ruminal nitrogen losses while feeding ASTFR and FSTFR. In this way, the use of these feeds is linked to the characteristics of their protein fractions, and may be supplied as a source of less soluble proteins. The ASTFR and FSTFR protein fraction profiles changed greatly compared to TFR. The fractions B2 + B3 , that is, insoluble proteins of intermediary and slow degradation, that would release dietetic amino acids to the intestinal were converted to the fraction more soluble (A + B1 ) when TFR was undergone either fermentation or acidification process. The FSTFR had less of the B2 + B3 fraction compared to ASTFR and B2 was higher than B3 for both feeds. Similar behavior was observed in our studies in situ conducted to evaluate ruminal degradability of CP, where fraction “b” of CP of FSTFR was lower than the results observed for ASTFR. The FIME is characterized as a protein source different from the FSTFR and ASTFR, due to different processing method. FIME showed CP content of 551.0 g/kg DM and the B2 + B3 fraction of 899.0 g/kg CP. The thermal treatment of FIME, reduces ruminal degradation, partially because it blocks the reactive sites for microbe proteolitic enzymes and, partially, because it reduces protein solubility (Waltz and Loerch, 1986). This means that FIME presents itself as ruminal escape protein, due to low ruminal degradability, and should be used to feed ruminants in the presence of nitrogen sources available to ruminal microorganisms.
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4.4. In vitro digestibility coefficient of feeds The high values of RDP found for ASTFR and FSTFR compared to the FIME were due to processing method. This is confirmed by the adopted methodologies in this study, which showed a higher presence of soluble protein in the ASTFR and FSTFR. According to Ouellet et al. (1997), the acid silage of fish residue has a high protein contents, with a high ruminal degradability. The feeds that contained high rumen degradation also resulted in low RUPD levels, from 13.5 and 18.4 g/kg DM for FSTFR and ASTFR, respectively. Therefore, the use of these foods as silages for high production ruminants does not supply an amino acid profile correspondent to the ingested food. It means that the ASTFR and FSTFR supply low bypass protein. For this reason, ASTFR and FSTFR shall be supplied together with less soluble protein sources, while the FIME must be supplied with soluble protein. The high levels of RUP and RUPD of FIME compared to ASTFR and FSTFR were probably due to the higher escape in the rumen and also, to the higher intestinal digestion, which releases dietary amino acids available for absorption and used by the animal metabolism. The high IDCP, without ruminal incubation, of ASTFR and FSTFR compared to FIME, showed that the fermentation and acidification processes resulted in high intestinal digestibility. Thus, ASTFR and FSTFR contained high ruminal or intestinal digestion, and if processed in the presence of heat it may be possible to reduce ruminal degradation, similar to FIME, and in such manner they could be dietary protein sources that could alter amino acid composition available in the intestine for the animal.
5. Conclusions The conservations processes (fermentation and acidification), of tilapia filleting residue altered the chemical composition, the amino acid and fatty acid profiles, without altering the nutritious value of the tilapia filleting residue. The fermentation and acidification processes can be used to conserve the tilapia filleting residue. The fermentation and acidification process increased the feed soluble protein.
Acknowledgments The authors thanks to the CNPq, Conselho Nacional de Desenvolvimento Desenvolvimento Cient´ıfico e T´ecnologico, for the financial support.
References AFRC, 1993. Energy and protein requirements of ruminants. In: Agric. Food Res. Council. Tech. Commun. on responses to nutrients. CAB International, Wallingford, Oxon, UK. AOAC, 1984. Official Methods of Analysis, 14th ed. Association of Official Analytical Chemists, Washington, DC.
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AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA. Baraquet, N.J., Lindo, M.M.K., 1985. Transformac¸o˜ es bioqu´ımicas “post mortem”em pescado. Bol. Intit. Tecnol. Alim. 22, 169–192. Berez, Z., 1997. Utilizaci´on del ensilado de residuos de pescado en pollos. In: Figueroa, V., S´anchez, M. (Eds.), Tratamiento y utilizaci´on de residuos de origen animal, pesquero y alimenticio en la alimentaci´on animal. FAO, Habana, Roma, pp. 15–29. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Calsamiglia, S., Stern, M.D., 1995. A three-step in vitro procedure for estimating intestinal digestion of protein ruminants. J. Anim. Sci. 73, 1459–1465. Disney, J.G., James, D., 1979. Fish silage production and its use FAO/WHO (1985). Fish Reprod. 230, 105– 109. Fitzsmmons, K., 2000. Furete trends of tilapia aquacture in tehe Americas. In: Barry, A., Costa-Pierce, James, E.R. (Eds.), Tilapi Aquaculture in the Americas, vol. 2. The World Aquaculture Society, Botton Rouge, pp. 252–264. Hartmam, L., Lago, R., 1973. A raoid preparation of fatty acid ethy esters from lipids. Lab. Pract. 22, 475–476. Hossaain, M.A., Furuichi, M., Yone, Y., 1987. TBA and VBN values and proximate, aminoacid and fatty acid composition of scrap meals fermented with fungi. Nippon Suisan, Gakkaishi 53, 1629–1632. Krishnamoorthy, U., Sniffen, C.J., Stern, M.D., 1983. Evaluation of a mathematical model of rumen digestion and in vitro simulation of rumen proteolysis to estimate the rumen-undegraded nitrogen content of feedstuffs. Br. J. Nutr. 50, 555–568. Lindgren, S., Pleje, M., 1983. Silage fermentation of fish or fish waste products with lactic acid bacteria. J. Sci. Food Agric. 34, 1057–1067. Mehrez, A.Z., Orskov, E.R., 1977. A study of the artificial fiber bag tecnique for determmining the digestibility of feeds in the rumen. J. Agric. Sci. 88, 6450–6550. Morales-Ulloa, D.F., Oetterer, M., 1995. Bioconvervac¸a˜ o de res´ıduos da industria pesqueira. Sci. Technol. Food 15, 206–214. Morales-Ulloa, D.F.E., Oetterer, M., 1997. Composic¸a˜ o em amino´acidos de silagens qu´ımicas, biol´ogicas e enzim´aticas preparadas com res´ıduos de sardinha. Sci. Technol. Food 3, 203–342. Orskov, E.R., Mcdonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92, 499–504. Orskov, E.R., 1998. Nutrici´on proteica de los rumiantes. Ed: Acribia, S.A. Zaragoza, pp. 178. Ouellet, D.R., Seoane, J.R., Veira, D.M., 1997. Effect of supplementation with fishmeal or fish protein hydrolysate on growth, nutrient digestibility and rumen fermentation of growing cattle fed grass silage. Anim. Feed Sci. Technol. 68, 307–326. Pariza, M.W., Park, Y., Cook, M.E., 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40, 283–298. SAS, 1987. SAS Users Guide. Statistical Analysis Systems Institute, Cary, NC, USA. Sniffen, C.J., O’connor, J.D., Van Soest, P.J., 1992. A net carbohydrate and protein system for evaluating cattle diets. II. Carbohydrate and protein availability. J. Anim. Sci. 70, 3562–3577. 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, 3583–3597. Van Wyk, H.J., Heyderych, C.M.S., 1985. The production of naturally fermented fish silage using various Lactobaclli and deferent carbohydrate sources. J. Sci. Food Agric. 36, 103–109. Vidotti, R.M., Carneiro, D.J., Viegas, E.M.M., 2002. Acid and fermented silage characterization and determination of apparent digestibility coefficient of crude protein for pacu (Piaractus meopotamicus). J. World Aquat. Soc. 33, 57–62. Vidotti, R.M., Viegas, E.M.M., Carneiro, D.J., 2003. Amino acid composition of processed fish silage using different raw materials. Anim. Feed Sci. Technol. 105, 199–204. Waltz, D.M., Loerch, S.C., 1986. Effect of acid and alkali treatment of soybean meal on nitrogen utilization by ruminants. J. Anim. Sci. 63, 879–887. Yone, Y., Hossain, M.A., Furuichi, M., 1986. Effect of fermented and fermented-resteamed serap meais on growth and feed efficiency of red sea bream. B. Jpn. Soc. Fish. 3, 549–552. Wignall, J., Tatterson, I., 1997. Fish silage 1997. Process. Biochem..
Chemical characterization, dry matter and crude protein ruminal degradability and in vitro intestinal digestion of acid and fermented silage from tilapia filleting residue Luiz Juliano Val´erio Geron a , L´ucia Maria Zeoula a , Rose Meire Vidotti b,∗ , Makoto Matsushita a , Ricardo Kazama a , Saul Ferreira Caldas Neto a , Fernanda Fereli a a
Animal Science Department, Laboratory of Analysis of Food and Animal Nutrition, Experimental Farm Iguatemi, Maringa State University, Maringa, Av. Colombo, 5790-CEP, 87020-900 Parana, Brazil b Sao Paulo State Fishing Institute, CP: 1052, S˜ ao Jos´e do Rio Preto CEP: 15025-970, Brazil Received 15 April 2005; received in revised form 31 August 2006; accepted 5 September 2006
Abstract The objective of the present study was to evaluate the chemical characteristics, amino acids and fatty acids profiles, dry matter (DM) and crude protein (CP) ruminal degradability and rumen-undegraded protein digestible in the intestine (RUPD ) of the tilapia filleting residue (TFR) and of the acid and fermented silage of tilapia filleting residue (ASTFR and FSTFR, respectively). The ASTFR was obtained by an acidification process with the addition of 0.02 L/kg TFR of sulfuric acid and 0.02 L/kg TFR of formic acid and the FSTFR was obtained by fermentation with the addition of 0.05 kg/kg TFR of natural yogurt, 0.15 kg/kg TFR of molasses and 0.003 kg/kg TFR of sorbic acid. A completely randomized design was used to characterize the fatty acids profile of the ASTFR and FSTFR and to determine ruminal degradability of DM and CP of ASTFR, FSTFR and fishmeal (FIME). The amino acids profiles of the ASTFR and FSTFR differed from TFR, but ASTFR and FSTFR were similar to each other. The fatty acids profiles of the ASTFR and FSTFR were similar to the TFR Abbreviations: ASTFR, acid silage tilapia filleting residue; CP, crude protein; DM, dry matter; ED, effective degradability; FIME, fishmeal; FSTFR, fermented silage tilapia filleting residue; RUP, rumen undegraded protein; TFR, tilapia filleting residue; v/w, volume/weight; w/w, weight/weight ∗ Corresponding author. Tel.: +55 17 32166769. E-mail address: [email protected] (R.M. Vidotti). 0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.09.006
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profile. The ASTFR showed a higher (P<0.05) saturated fatty acid content in comparison to the FSTFR. The effective degradability (ED) of the CP at solid passage rate of 0.05 h−1 of the ASTFR and FSTFR were of 0.76 and 0.81, respectively, which are higher (P<0.05) than the TFR value of 0.44. To the ASTFR and FSTFR lower values (P<0.05) were observed to RUPD of 13.5 and 18.8 g/kg, respectively, in comparison the FIME of 224.4 g/kg RUP. The ASTRF and FSTRF showed alteration composition chemical and amino acids profile without altered the nutritive value of TFR. The processes of acidification or fermentation are methodologies that can be used as a conservation procedure of tilapia filleting residue. © 2006 Elsevier B.V. All rights reserved. Keywords: Amino acids; Fatty acids; Fish silage; Digestible protein in the small intestine
1. Introduction The utilization of low cost and good quality alternative feed sources can decrease production costs and increase profits. Among several alternative sources, tilapia-filleting residue, presents great nutritional quality and high potential for animal production. Brazil and Mexico, both populous countries with great water resources, will be the two main producers of the tilapia industry in the Western Hemisphere (Fitzsmmons, 2000). The largest markets of tilapia in Brazil form the filleting industry, which is found in the south and southeast of Brazil. Fish silage is a noble protein, a product of high biological value for animal feeding, which can be produced from dead fish, species that are sub-utilized in the fish industry, or from marine fishing, commercial fish waste and industrial residues. These are considered low quality raw materials, that if not used may cause environmental, sanitary and economical problems (Vidotti et al., 2003). The processes used for conservation of tilapia filleting residue (TFR) consist of two distinct methodologies: acidification and fermentation. The acidification process begins with a triturated mass of fish, allowing free enzyme activity to liquefy the tissues. Acidification is conducted by adding mineral or organic acids such as: formic, sulfuric, chloride, propionic, etc. (Disney and James, 1979; Wignall and Tatterson, 1997; Vidotti et al., 2002). The anaerobic fermentation process was initiated by adding microorganisms and a carbohydrate source (Lindgren and Pleje, 1983; Van Wyk and Heyderych, 1985; Vidotti et al., 2002). Fish silage is a source similar to fishmeal with respect to composition profile; however, it behaves differently in the digestive tract, which in spite of the high protein content is a poor source of RUP (Ouellet et al., 1997). Effective degradability of CP in the rumen depends on the feed characteristics, ingestion level, type of processing which the feed was undergone and possible limitations in the rumen fermentation process (pH, ammonia concentration, fatty acids) according to Orskov (1998). Available information about the chemical and biological characteristics of the tilapia filleting residue silage to feed ruminants is scarce. Therefore, the objective of this study was to evaluate chemical composition, amino acids profile, fatty acids profile of acid (ASTFR)
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and fermented (FSTFR) silage of tilapia filleting residue, besides to estimate the protein fraction, DM and CP ruminal degradability and intestinal digestion of rumen undegraded protein of ASTFR and FSTFR compared to fishmeal.
2. Material and methods 2.1. Conservation of tilapia filleting residue The tilapia filleting residue (TFR) was obtained at a tilapia filleting factory located in the town of Euclides da Cunha in Sao Paulo state, Brazil. The TFR contained head, carcass, but no viscera. The TFR was triturated using a commercially available meat grinder and placed in sealed plastic buckets (3 L capacity), and it was, submitted to the two processes: acidification or fermentation. The acid silage of tilapia filleting residue (ASTFR) was produced by adding 0.02 L/kg TFR of sulfuric acid and 0.02 L/kg TFR. of formic acid. The fermented silage of tilapia filleting residue (FSTFR) was produced by adding 0.05 kg/kg TFR of Lactobacillus ssp. (Nestle® , natural yogurt), 0.15 kg/kg TFR of sugar cane molasses and 0.003 kg/kg TFR of sorbic acid. The treatments were conducted with three replications each. The pH measurements were conducted using a pH meter (Tecnal® , TEC3MP) on the following days: 1, 3, 5, 7, 9, 11, 15, 21, 30, 60, 90 and 180 after the silage process had begun. A commercial fishmeal (Alisul Alimentos S/A Parana, Brazil) was analyzed in this experiment in order to compare with ASTFR and FSTFR. 2.2. Protein fraction, rumen degradability and intestinal digestion of rumen undegraded protein It was calculated the nitrogen fractions: A, B1 , B2 , B3 and C of TFR, ASTFR, FSTFR and FIME. The fractions A was obtained using the methodology of Krishnamoorthy et al. (1983) and fraction B1 , B2 and B3 according to Sniffen et al. (1992). The fraction C was obtained by acid detergent insoluble nitrogen (ADIN) (Van Soest et al., 1991). Ruminal degradability of dry matter (DM) and crude protein (CP) of ASTFR, FSTFR and fishmeal (FIME) was determined using three Holstein steers with initial body weight (BW) of 380 ± 61 kg fitted with ruminal cannula distributed in a completely randomized desing with three repetitions. The animals were daily fed with 17.0 kg of corn silage (fresh weight) and 2.5 kg of concentrated (fresh weight). The composition of the ration on dry matter basis in g/kg was as follows: 640.0 corn silage, 223.0 corn, 87.0 soybean meal and 50.0 fishmeal. The animals were adapted to the experimental diet for a period of 14 days, in which they were fed twice a day. First, the ASTFR, FSTFR samples were dried at 55 ◦ C over 72 h, ground through a 2 mm screen (Wiley mill). Subsequently 6.0 g of sample were weighed in 10 cm × 17 cm nylon bags made of nitrogen free polyester (Ankon Technology Co. Fairport, NY, USA) with a mean pore size of 53 m and heat sealed. Bags were placed into the rumen for 2, 4, 6, 8,
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12, 24, 48 and 72 h. The FIME sample, as fed, was also weighed (6.0 g) in nylon bags and incubated as mentioned above. Three samples bags/feed were taken and incubated at each sampling time, for provide sufficient residue for subsequent chemical analysis. Bags were placed in the rumen at the same time and removed sequentially. The bags were removed, rinsed briefly in bucket of cold water, then rinsed three cycles of 5 min with cold water in a semi automatic washing machine and the bags residues were dried at 55 ◦ C over 72 h. The soluble fraction (time 0) was measured in two bags per sample but without rumen incubation and the bags were placed in water-bath at 37 ◦ C over 1 h. The bags residues were analyzed for DM and nitrogen. Intestinal digestibility (ID) of rumen undegraded protein of ASTFR, FSTFR and FIME was determined using the three-step in vitro procedure following the methodology described by Calsamiglia and Stern (1995). 2.3. Calculations Dry matter and crude protein ruminal degradability were determined using the equation described by Mehrez. and Orskov (1977): p = a + b × (1 − e−ct ) where: p = potential degradability in time t; a = intercept representing the fraction soluble; b = insoluble but potentially degradable fraction; c = constant rate of degradability of fraction b; t = incubation time; a + b ≤ 1. The non-linear parameters “a”, “b” and “c” were estimated using the iterative least squares procedure SAS (1987). Effective degradability (ED) of dry matter and crude protein in the rumen was calculated using the model of Orskov and Mcdonald (1979): ED = a + ((b × c)/(c + k)), where: k = solids outflow rate: “a”, “b” and “c” were previously defined. The solid outflow rates (k) used for each feed were 0.02, 0.05 and 0.08 h−1 , which are attributed to low, medium and high levels of feed ingestion, respectively (AFRC, 1993). 2.4. Laboratory analyses All chemical analyses of samples of TFR, ASTFR, FSTFR and FIME were made on duplicated according to AOAC (1984). Dry matter content was determined at 105 ◦ C over 24 h (procedure ID 7.007) in a forced air oven. Nitrogen content was determined by the micro-Kjeldahl method (ID 7.015) and crude protein (CP) was determined as nitrogen × 6.25. Ash was analysed by incineration in a muffle furnace (ID 7.009) at 550–600 ◦ C for 4 h and ether extract (EE) was determined using a Sohxlet extractor, and washing with petroleum ether (ID 920.39) according to AOAC (1990). Amino acid profile of TFR, ASTFR and FSTFR (average samples) were determined except for triptophan, at the High Technology laboratory (LAB TEC, Mogiana Alimentos S.A. Campinas, SP, Brazil). Gas chromatography analysis to determine fatty acid content was conducted following methodology described by Bligh and Dyer (1959); and procedures described by Hartmam and Lago (1973). The content phosphorus of TFR, ASTFR and FSTRF were determined in colorimetric spectrophotometer (Chimadzu® , model UV 1601). The content calcium of feeds
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Table 1 Content of dry matter (DM), organic matter (OM), crude protein (CP), ash, ether extract (EE), calcium (Ca) and phosphorus (P) of the tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR) and of the fishmeal (FIME) Feeds
DM
OM (g/kg DM)
CP (g/kg DM)
Ash (g/kg DM)
EE (g/kg DM)
Ca (g/kg DM)
P (g/kg DM)
TFR ASTFR FSTFR FIME
397.7 370.2 366.9 932.7
797.3 765.3 812.7 736.8
406.9 370.1 316.4 551.0
202.7 234.7 187.3 263.2
355.1 353.7 321.9 19.2
49.4 48.6 49.7 83.9
45.3 42.5 35.8 69.4
was realized used nitric (HNO3 ) and percloric (HClO4 ) acid in atomic absorption spectrophotometer (GBC® , model 932 AA). 2.5. Statistical analysis A completely randomized design with three replications was used to characterize the fatty acids profile and intestinal digestion of rumen undegraded protein for both processing methods. The average data were compared by Tukey test at a significance level of 0.05. The TFR which had just one sample (no replication) was not analyzed statistically. For DM and CP degradability, the data obtained were submitted to ANOVA procedure using a SAS in completely randomized design, with three replications. The averages were compared by Tukey test at a significance level of 0.05.
3. 3.Results 3.1. Chemical composition of feeds The chemical composition of the TFR, ASTFR, FSTFR and FIME are in Table 1. In general, it can be seen that chemical composition of the silages contained variation in relation to TFR. The DM values of TFR, ASTFR and FSTFR were, respectively, 397.7, 370.2 and 366.9 g/kg. The CP contents of ASTFR and FSTFR reduced 9% and 22%, respectively, compared to the CP content of TFR, (406.9 g/kg). Fat content also reduced 9% for FSTFR in relation of TFR and similar values were found for ASTFR and FSTFR. The TFR initial pH was 6.3 and at the end of the ensilage process, the ASTFR and FSTFR pH was 3.2 and 4.2, respectively. Both were considered satisfactory to avoid proliferation of undesirable microorganisms (Van Wyk and Heyderych, 1985). Vidotti et al. (2002) reported that initial pH of tilapia residue was 6.7 and after 31 days of storage through the acidification process it reduced to 2.8. 3.2. Amino acid and fatty profile of feeds The amino acids profile of TFR, ASTFR and FSTFR are presented in Table 2. The amino acids profile of ASTFR and FSTFR varied slightly compared to TFR. The amino
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Table 2 Amino acids profile of the tilapia filleting residue (TFR) of the acid silage of tilapia filleting residue (ASTFR) and of the fermented silage of tilapia filleting residue (FSTFR) Amino acids Alanine Arginine Aspartic acid Glycine Isoleucine Leucine Glutamic acid Lysine Cystine Methionine Phenylalanine Tyrosine Threonine Proline Valine Histidine Serine CP (g/kg)
TFR (g/100 g CP)
ASTFR (g/100 g CP)
FSTFR (g/100 g CP)
7.6 7.5 9.6 8.1 4.9 9.4 13.7 9.3 1.5 3.2 3.8 1.3 2.1 7.4 6.2 2.8 1.6
6.8 8.5 8.8 10.2 4.7 8.4 13.3 8.8 1.2 3.5 3.6 1.8 3.4 6.2 5.8 2.6 2.8
7.4 5.8 10.9 9.4 4.5 8.1 13.8 8.1 1.5 4.3 3.0 1.3 3.1 7.3 6.4 2.5 2.7
406.9
370.1
316.4
CP, crude protein.
acids profile was not statistically analyzed since the final sample of feeds (TFR, ASTFR and FSTFR) was a composition of the three replications by feed. The ASTFR contained higher levels of arginine, glycine, tyrosine, threonine and serine and lower levels of alanine, leucine, cystine and proline compared to TFR (Table 2). The FSTFR showed higher concentration of aspartic acid, glycine, methionine, threonine and serine, than TFR. However, the contents of leucine, lysine, phenylalanine and histidine were lower. The highest glutamic acid concentration was found in TFR (13.7 g/100 g CP), ASTFR (13.3 g/100 g CP) and FSTFR (13.8 g/100 g CP), as it has been reported by Morales-Ulloa and Oetterer (1997) and Vidotti et al. (2003). ASTFR contained higher concentration of glycine, tyrosine, lysine, phenylalanine and arginine, than in the FSTFR and this showed higher contents of alanine, valine, proline methionine, aspartic acid and cystine than ASTFR. Fatty acid profiles and the concentrations the fatty acid saturated and unsaturated of TFR, ASTFR and FSTFR are presented in Table 3. The lower values for the saturated fatty acids: myristic, palmitic and stearic acids; higher values for long chain unsaturated fatty acids: linoleic, linolenic, di-homo linolenic and docosahexaenoic acids were observed for TFR than ASTFR. However the FSTFR contained similar values for saturated and unsaturated fatty acids when compared to TFR. The FSTFR contained higher (P<0.05) concentrations of linoleic (C18:2); linolenic (C18:3), eicosatrienoic (20:3n9); docosatrienoic (20:3n3) acid when compared to ASTFR, but the ASTFR showed higher levels (P<0.05) of palmitic (16:0), stearic (16:0), oleic (18:1).
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Fatty acids
Common nomenclature
TFR (g/100 g fat)
ASTFR (g/100 g fat)
ASTFR (g/100 g fat)
S.E.M.
14:0 16:0 16:1 18:0 18:19 18:17 18:26 18:36 18:33 20:0 20:111 20:39 20:33 22:63
Myristic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Vacenic acid Linoleic acid Linolenic acid ␣ linolenic acid Araquidic acid Gadoleic acid Mead acid Di-homo linolenic acid Docosahexaenoic acid
2.8 26.3 4.4 6.5 37.8 3.0 14.5 0.6 1.9 0.3 0.3 0.7 0.6 0.4
3.6a 31.2a 4.9a 8.1a 41.7a 4.0a 3.5b 0.3b 1.9a 0.2a 0.2a 0.2b 0.2b 0.1b
2.7a 23.1b 4.4b 6.1b 37.2b 3.3b 17.9a 0.8a 1.7b 0.2a 0.3a 1.0a 0.8a 0.5a
0.20 0.04 0.05 0.03 0.03 0.08 0.03 0.15 0.05 0.16 0.24 0.10 0.14 0.33
55.6 44.4 0.80
43.1 56.9 1.32
32.1 67.9 2.12
– – –
Fatty acids saturated (g/100 g fat) Fatty acids unsaturated (g/100 g fat) Ratio unsaturated/saturated
Averages in the same line, followed by different letters are different (P<0.05) by the Tukey test. S.E.M. standard error mean.
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Table 3 Fatty acids profile of the tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR)
Feeds
TFR ASTFR FSTFR FIME
Nitrogen fractions A (g CP/kg DM)
B1 (g CP/kg DM)
B2 (g CP/kg DM)
B3 (g CP/kg DM)
C (g CP/kg DM)
A (g CP/kg total CP)
B1 (g CP/kg total CP)
B2 (g CP/kg total CP)
B3 (g CP/kg total CP)
C (g CP/kg total CP)
10.0 216.0 250.0 62.0
34.0 0.1 2.0 −12.0
197.0 137.0 79.0 183.0
119.0 8.0 5.0 326.0
2.0 2.0 1.0 7.0
26.0 595.0 744.0 110.0
95.0 0.4 3.0 −22.0
545.0 376.0 234.0 323.0
329.0 23.0 15.0 576.0
5.0 5.0 3.0 13.0
A: fast available fraction (non-protein nitrogen); B1 : Rumen rapidly degraded fraction (peptides and oligopeptides); B2 : intermediary degradation fraction (cytoplasmatic proteins); B3 : slow degradation fraction (proteins insoluble in neutral detergent); C: insoluble fraction (unavailable in the rumen and in the intestine).
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Table 4 Nitrogen fractions of tilapia filleting residue (TFR), of the acid silage of tilapia filleting residue (ASTFR), of the fermented silage of tilapia filleting residue (FSTFR) and of the fishmeal (FIME)
233
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The conservation processes (acidification and fermentation) of TFR increased concentration of unsaturated fatty acids, which resulted in a higher ratio of unsaturated:saturated relation to TFR. This ratio was 38% higher in ASTFR than in FSTFR. 3.3. Nitrogen fractions of crude protein and ruminal degradability of DM and CP of feeds The nitrogen fractions of TFR, ASTFR, FSTFR and FIME are presented in Table 4. The fraction A (non-protein nitrogen) of CP, was 26.0, 595.0, 744.0 and 110.0 g/kg of CP, respectively for TFR, ASTFR, FSTFR and FIME. The Fraction B1 of CP (constituted of soluble proteins and rapidly degraded, peptides and oligopept´ıdeos) was lower than 3.0 g/kg for ASTFR, FSTFR and FIME, and represented 95.0 g/kg of CP for TFR. The fraction B2 (constituted of insoluble proteins of intermediated ruminal degradability) and B3 (proteins insoluble in neutral detergent, of slow degradation in the rumen) was 399.0 g/kg of CP for ASTFR and 249.0 g/kg of CP for FSTFR. These values were lower compared to TFR (874.0 g/kg of CP). The fraction C, (considered unavailable in the rumen and intestines) was 13.0 g/kg of CP to the FIME and less than 10.0 g/kg of CP to the other feeds. It can be seen from Table 5 that the soluble fraction “a” of DM of FSTFR was higher (P<0.05) than that found for ASTFR and FIME, the latter showed the lowest “a” value (0.18). These results are in agreement with those observed for the fraction A of protein observed in Table 4. Table 5 Soluble (a) and potentially degraded insoluble fractions (b), degradation rate (c), potential degradability (PD) and effective degradability (ED) of dry matter g/kg DM and crude protein in g/kg CP of the feed at passage rates of 0.02, 0.05 and 0.08 h−1 Variables
ASTFR
FSTFR
FIME
S.E.M.
Dry matter (DM) (g/kg DM) a b c (h−1 ) PD ED (0.02 h−1 ) ED (0.05 h−1 ) ED (0.08 h−1 )
330.0b 590.0a 0.49 920.0 890.0a 80.05a 820.0a
700.0a 240.0c 0.15 940,0 910.0a 880.0a 850.0a
180.0c 340.0b 0.09 530.0 460.0b 400.0b 360.0b
0.08 0.05 – – 0.07 0.08 0.08
Crude protein (CP) (g/kg CP) a b c (h−1 ) PD ED (0.02 h−1 ) ED (0.05 h−1 ) ED (0.08 h−1 )
350.0b 590.0a 0.37 940.0 830.0a 760.0a 730.0a
470.0a 490.0b 0.14 960.0 880.0a 810.0a 760.0a
160.0c 480.0b 0.07 640.0 630.0a 440.0b 380.0b
0.05 0.04 – – 0.05 0.07 0.07
Averages in the same line, followed by different letters are different (P<0.05) by the Tukey test. ASTFR: acid silage of the tilapia filleting residue; FSTFR: fermented silage of tilapia filleting residue and FIME: fishmeal. S.E.M.: standard error mean.
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Table 6 Content of crude protein (CP), rumen-degraded protein (RDP) and rumen-undegraded protein (RUP) for 16 h of ruminal incubation, in vitro intestinal digestibility coefficient of RUP (IDRUP), RUP digestible in the small intestine (RUPD ) and in vitro intestinal digestibility coefficient of crude protein (IDCP) without ruminal incubation of acid (ASTFR) and fermented (FSTFR) silages of tilapia filleting residue and of the fishmeal (FIME) Feed
CP g/kg DM
RDP (g/kg DM)
RUP (g/kg DM)
IDRUP
RUPD (g/kg DM)
ASTFR FSTFR FIME
370.1 316.4 551.0
350.5a 300.3a 251.8b
19.6b 16.1b 299.2a
0.94a 0.84a 0.75b
18.4b 13.5b 224.4a
S.E.M.
–
0.05
0.12
0.15
0.11
Feed
CP (g/kg DM)
IDCP
Feed without ruminal incabation ASTFR FSTFR FIME S.E.M.
370.1 316.4 551.0 –
0.98a 0.97a 0.88b 0.04
Averages in the same column, followed by different letters are different (P<0.05) by the Tukey test. S.E.M. standard error mean.
The DM fraction “b” (insoluble, potentially degradable) was highest (P<0.05) for ASTFR, than FSTFR and FIME, but the fraction “b” of FIME was higher (P<0.05) than FSRT. However, for both ensiled were observed similar values of ED for DM, but the fermentation process reduced ruminal degradation rate of the fraction “b” (0.15 h−1 versus 0.49 h−1 ). The potential degradability (PD) of DM of ASTFR and FSTFR was in average 43% higher than that of FIME. Also, ED of DM of ASTFR and FSTFR was higher than that of FIME, independent of the solid outflow rate (0.02, 0.05 and 0.08 h−1 ). It can be seen that fraction “a” of CP of FSTFR was 0.47, and therefore higher (P<0.05) than ASTFR (0.35) and FIME, and the latter contained the lowest (P<0.05) value (0.16). The fraction “b” of CP was higher (P<0.05) to ASTFR (0.59) compared to FSTFR (0.49) and FIME (0.48). Similarly to the dry matter, the lowest degradability rate of fraction “b” (c) of CP was observed in the fermentation process. Silage acidification process releases and makes more readily available CP. FIME contained lower degradation rate of fraction “b” than the silage and lower PD of CP. The ED of CP with outflow rates of 0.05 and 0.08 h−1 was higher (P<0.05) for FSTFR and ASTFR compared to FIME; however, at 0.02 h−1 (low consumption) no difference was observed. 3.4. In vitro digestibility coefficient of crude protein of feeds The values of rumen degraded proteins (RDP), rumen undegraded protein (RUP), in vitro intestinal digestibility coefficient of RUP (IDRUP) and RUP digestible in the small intestine (RUPD ) are presented in Table 6. The values found for RUP of ASTFR and FSTFR were 19.6 g/kg of DM and 16.1 g/kg of DM, respectively; lower (P<0.05) than observed for FIME (299.2 g/kg of DM). The ASTFR (0.94) and FSTFR (0.84) had high IDRUP (P<0.05), than FIME (0.75).
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The content of RUPD of FIME was higher (224.4 g/kg DM) compared to the ASTFR and FSTFR (18.4 and 13.5 g/kg DM, respectively). The values of CP and in vitro intestinal digestibility coefficient of protein (IDCP) of ASTFR, FSTFR and FIME, without being submitted to ruminal incubation period are found in Table 6. The IDCP for ASTFR and FSTFR were, respectively, 0.98 and 0.97 higher (P<0.05) than 0.88 found for FIME.
4. Discussion 4.1. Chemical composition of feeds Both ensiling processes affected the chemical composition of the TFR. The fermentation reduced the contents of CP and EE compared to the original values found in TFR. Probably molasses addition to FSTFR diluted CP and fat content, which was also observed by Morales-Ulloa and Oetterer (1995). Reduced CP levels observed for ASTFR compared to TFR were also reported by Vidotti et al. (2002). The authors found CP levels of 396.0 (g/kg), for acid silage, compared to 429.0 g/kg of TFR. This reduction in CP levels of silage is a consequence of protein hydrolysis, since the autolysis effect on the degradation of proteins and nucleoproteins may transform them in more simple compounds, such as, amino acids (Baraquet and Lindo, 1985) and ammonia that can volatilize during storage of ASTFR and FSTFR. Vidotti et al. (2003) reported for DM, CP, EE, and mineral matter (MM) of TFR the following values 314.0; 429.0; 346.0; 166.0 g/kg DM, respectively; values near to the ones reported in the present study to CP and EE of 406.7 and 355.1 g/kg DM, respectively. 4.2. Amino acid and fatty acids profile of feeds The variation of the amino acids contents was observed in TFR compared to the both silage. There was an increase in the levels of glycine, threonine and serine and decrease in the contents of leucine for both, fermentation and acidification processes. The fermentation process increased the level of methionine in 25% and reduced lysine in 15%, compared to TFR. Such increase in the levels of glycine, tyrosine, lysine, phenylalanine and arginine of ASTFR compared to FSTFR is probably due to the processing method. In a similar shape, Vidotti et al. (2003) reported higher contents of glycine and tyrosine to the ASTFR than FSTFR. In general, the differences observed in the chemical composition and amino acid profiles of silages in this work compared to that reported in the literature may be due to different places of origin, processing method, freshness of the raw material, storage conditions, previous contamination and residue composition. That is, the presence or not of either one of the following parts like: heads, gills, scales, dorsal column, intestine contents and muscle may influence the feed composition. Still, according to Yone et al. (1986), Hossaain et al. (1987) and Baraquet and Lindo (1985) the microbe fermentation alters the composition of CP and MM and increases volatile nitrogen losses.
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The higher concentration of unsaturated fatty acids in FSTFR and the higher levels (P<0.05) of saturated fatty acids in ASTFR showed that both, acidification and fermentation processes altered the fatty acids profile of the silages. Fermentation increased the concentration of unsaturated fatty acids, such as linoleic acid, which in its conjugated forms is highly beneficial. It is anti carcinogenic, anti diabetic and can alter the lipids metabolism and body composition of the animals (Pariza et al., 2001). Berez (1997) while studying sardine residue silage in ration for chicken reported a fatty acid profile containing 13.7 g/100 g of fat of oleic acid and 2.0 g/100 g of fat of acid linoleic. In the present work using tilapia residue, the acid oleic concentrations was 41.7 and 37.2 g/100 g of fat, while linoleic acid was 3.5 and 17.9 g/100 g of fat for ASTFR and FSTFR, respectively. 4.3. Nitrogen fraction of crude protein and ruminal degradability of DM and CP of feeds The ensiling processes of TFR (acid and fermented) changed the nitrogen fraction of the crude protein, increasing considerably fraction A. The fermentation increased the fraction A of CP compared to acidification. Similarly, studies in situ conducted, the FSTFR contained higher value of fraction “a” (soluble fraction of CP, quickly available in the rumen) compared to ASTFR. According to Baraquet and Lindo (1985) autolysis effects on protein degradation, may transform it into more simple compounds, such as amino acids, peptides, pyrimidine and purine basis. The fermentation process and the molasses addition increased the fraction A. The fraction A + B1 indicates higher supply of NPN, peptides and oligopeptides all degradable in the rumen, and a higher amount of available nutrients to ruminal microorganisms. Considering this, it is recommended to use an energetic source that have a quickly available energy to minimize the ruminal nitrogen losses while feeding ASTFR and FSTFR. In this way, the use of these feeds is linked to the characteristics of their protein fractions, and may be supplied as a source of less soluble proteins. The ASTFR and FSTFR protein fraction profiles changed greatly compared to TFR. The fractions B2 + B3 , that is, insoluble proteins of intermediary and slow degradation, that would release dietetic amino acids to the intestinal were converted to the fraction more soluble (A + B1 ) when TFR was undergone either fermentation or acidification process. The FSTFR had less of the B2 + B3 fraction compared to ASTFR and B2 was higher than B3 for both feeds. Similar behavior was observed in our studies in situ conducted to evaluate ruminal degradability of CP, where fraction “b” of CP of FSTFR was lower than the results observed for ASTFR. The FIME is characterized as a protein source different from the FSTFR and ASTFR, due to different processing method. FIME showed CP content of 551.0 g/kg DM and the B2 + B3 fraction of 899.0 g/kg CP. The thermal treatment of FIME, reduces ruminal degradation, partially because it blocks the reactive sites for microbe proteolitic enzymes and, partially, because it reduces protein solubility (Waltz and Loerch, 1986). This means that FIME presents itself as ruminal escape protein, due to low ruminal degradability, and should be used to feed ruminants in the presence of nitrogen sources available to ruminal microorganisms.
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4.4. In vitro digestibility coefficient of feeds The high values of RDP found for ASTFR and FSTFR compared to the FIME were due to processing method. This is confirmed by the adopted methodologies in this study, which showed a higher presence of soluble protein in the ASTFR and FSTFR. According to Ouellet et al. (1997), the acid silage of fish residue has a high protein contents, with a high ruminal degradability. The feeds that contained high rumen degradation also resulted in low RUPD levels, from 13.5 and 18.4 g/kg DM for FSTFR and ASTFR, respectively. Therefore, the use of these foods as silages for high production ruminants does not supply an amino acid profile correspondent to the ingested food. It means that the ASTFR and FSTFR supply low bypass protein. For this reason, ASTFR and FSTFR shall be supplied together with less soluble protein sources, while the FIME must be supplied with soluble protein. The high levels of RUP and RUPD of FIME compared to ASTFR and FSTFR were probably due to the higher escape in the rumen and also, to the higher intestinal digestion, which releases dietary amino acids available for absorption and used by the animal metabolism. The high IDCP, without ruminal incubation, of ASTFR and FSTFR compared to FIME, showed that the fermentation and acidification processes resulted in high intestinal digestibility. Thus, ASTFR and FSTFR contained high ruminal or intestinal digestion, and if processed in the presence of heat it may be possible to reduce ruminal degradation, similar to FIME, and in such manner they could be dietary protein sources that could alter amino acid composition available in the intestine for the animal.
5. Conclusions The conservations processes (fermentation and acidification), of tilapia filleting residue altered the chemical composition, the amino acid and fatty acid profiles, without altering the nutritious value of the tilapia filleting residue. The fermentation and acidification processes can be used to conserve the tilapia filleting residue. The fermentation and acidification process increased the feed soluble protein.
Acknowledgments The authors thanks to the CNPq, Conselho Nacional de Desenvolvimento Desenvolvimento Cient´ıfico e T´ecnologico, for the financial support.
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