Digestibility of amino acids in fish meal and blood-derived protein sources fed to pigs

Objective An experiment was conducted to determine the standardized ileal digestibility (SID) of amino acids (AA) in fish meal (FM) and blood-derived protein sources including spray-dried porcine plasma (SDPP), porcine red blood cells (PRBC), and blood meal (BM) fed to growing pigs. Methods Ten barrows (mean initial body weight of 22.1±1.54 kg) surgically fitted with T-cannulas at the distal ileum were allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Four experimental diets were prepared to contain FM, SDPP, PRBC, or BM as the sole source of nitrogen. A nitrogen-free diet was prepared and included to estimate the basal ileal endogenous losses of AA. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7. Results The SID of crude protein in BM (48.0%) was less (p<0.05) than in FM, SDPP, and PRBC (83.4%, 83.9%, and 87.3%, respectively). Pigs fed the diet containing BM had less (p<0.05) SID of AA, except isoleucine and proline, than those fed the diet containing FM, SDPP, or PRBC. Among FM, SDPP, and PRBC, there was no difference in the SID of crude protein and all AA, except isoleucine. The SID of isoleucine in PRBC and BM (62.7% and 48.3%, respectively) was less (p<0.05) than in FM and SDPP (88.0% and 84.9%, respectively). The SID of lysine in FM, SDPP, PRBC, and BM was 85.4%, 84.9%, 89.7%, and 51.9%, respectively. Conclusion The SID of most AA was not different among FM, SDPP, and PRBC, but BM had lower SID of most AA than FM, SDPP, and PRBC.


INTRODUCTION
Protein sources derived from animal byproducts have been used in the modern swine production system, especially for weanling pigs because of their high concentrations of digestible crude protein (CP) and amino acids (AA) as well as functional components. In addition, weanling pigs are susceptible to anti-nutritional factors in soybean meal such as glycinin and β-conglycinin, leading to a limited inclusion in diets immediately after weaning [1]. Thus, fish meal (FM) has been widely used in diets for weanling pigs which contains high concentrations of CP and AA as well as macrominerals such as calcium and phosphorus [2]. However, the concentration of AA as well as their digestibility vary among sources of FM depending on the quality of fish materials and processing conditions [3].
During the meat processing, blood from slaughtered animals can be collected, processed, and dried to produce blood meal (BM), which can be used as a protein source in swine diets. On the other hand, collected blood can be treated with anticoagulant and centrifuged to separate plasma, which can be further processed to produce spray-dried animal plasma (SDAP) [4]. It has been reported that dietary supplementation of SDAP improves growth performance by providing immunoglobulins in the intestinal lumen, which bind to the pathogens and reduce the proinflammatory responses and post-weaning diarrhea of pigs [5]. Red blood cells are the byproduct from the production of SDAP, which can be also used in weanling pig diets as a protein source [6]. Red blood cells contain high concentration of CP and lysine but low concentration of isoleucine [7]. Because the major components consisting of blood-derived protein sources are different from each other, digestibility of AA may vary among blood-derived protein sources. However, there is a limited information regarding the comparison of AA digestibility among blood-derived protein sources. Therefore, this study aimed to determine the standardized ileal digestibility (SID) of CP and AA in FM, spray-dried porcine plasma (SDPP), porcine red blood cells (PRBC), and BM fed to growing pigs. The null hypothesis of this study was that the SID of CP and AA are not different among FM and 3 blood-derived protein sources.

Animal care
Protocols of the animal experiment were reviewed and ap-proved by the Purdue University Animal Care and Use Committee (West Lafayette, IN, USA).

Animals, housing, and experimental design
A total of 10 barrows were surgically fitted with T-cannulas at the distal ileum based on the method reported by Dilger et al [8], followed by 7 days of recovery period. Thereafter, pigs with initial body weight (BW) of 22.1±1.54 kg were moved to metabolism crates (1.22×1.22 m 2 ). Pigs were divided into 2 replicates based on BW (i.e., heaviest 5 pigs and lightest 5 pigs) and allotted to a duplicated 5×4 incomplete Latin square design with 5 experimental diets and 4 periods. Pigs had free access to water via nipple drinkers.

Experimental diets, feeding, and sample collection
Five experimental diets were prepared based on cornstarch and sucrose (Table 1). Four diets were prepared to contain FM, SDPP, PRBC, or BM as the sole source of nitrogen (N) with providing 160 g/kg CP in each diet. Fish meal used in this experiment was mechanically extracted from menhaden and purchased from a local supplier. Blood-derived protein sources in this experiment were obtained from Darling Ingredient Inc. (Cold Spring, KY, USA). Nitrogen-free diet (NFD) was prepared to estimate the basal ileal endogenous losses (BEL) of CP and AA in pigs. Soybean oil and cellulose Table 1. Ingredient composition of experimental diets containing fish meal, spray-dried porcine plasma, porcine red blood cells, and blood meal (g/kg as-fed basis)  were each added at 50 g/kg to supply energy and dietary fiber, respectively. Experimental diets were formulated to meet or exceed the vitamin and mineral requirement estimates suggested in National Research Council (NRC) [9]. Chromic oxide was added to all diets at 5 g/kg as an index marker. Individual BW of pigs were measured at the beginning of each experimental period to calculate the daily amount of feed offered which was 4% of mean BW within replicates. Pigs were fed twice a day at 0800 and 1700 hours. For the 7-day experimental period, pigs were fed for 5 days as adaptation, and ileal digesta samples were collected for 9 hours on days 6 and 7. Ileal digesta samples were collected via Tcannulas by attaching plastic sample bags (Whirl-Pak bag; NASCO, Fort Atkinson, WI, USA) containing 10 mL of 10% formic acid. Attached plastic samples bags were changed every 30 minutes and immediately stored at -20°C. At the end of each experimental period, frozen ileal digesta samples were slightly thawed, pooled within pigs, and subsampled. Collected subsamples were stored at -20°C before further analyses.

Chemical analyses
Frozen ileal digesta samples were freeze dried before the chemical analyses. Test ingredients, experimental diets, and freeze-dried ileal digesta samples were finely ground (<0.75 mm) using a centrifugal grinder (ZM 200; Retsch GmbH, Haan, Germany). The concentrations of dry matter (DM) in ground ingredients, diets, and freeze-dried ileal digesta samples were analyzed by drying at 105°C for 24 h in a forcedair drying oven (Precision Scientific Co., Chicago, IL, USA; method 934.0) [10]. A combustion method was used to analyze the concentrations of N in ground samples (TruMac N; LECO Corp., St. Joseph, MI, USA; method 990.03) [11], and the concentration of CP was calculated by multiplying the N concentration by 6.25. To digest samples for the analysis of AA, samples of test ingredients, experimental diets, and ileal digesta were hydrolyzed by 6 M HCl (or BaOH for tryptophan analysis) at 110°C for 24 h under N atmosphere. An oxidation of samples using performic acid was conducted for the analysis of methionine and cysteine before digestion. High-performance liquid chromatography was used to determine the concentrations of AA in digested samples after postcolumn derivatization ( Experimental diets and ileal digesta were analyzed for the concentration of chromium (Cr) using a spectrophotometer (Spark 10M; Tecan Group Ltd., Männedorf, Switzerland) at 450 nm of absorbance after the wet digestion in nitric acid and 70% perchloric acid [12].

Calculations
The apparent ileal digestibility (AID) of CP and AA in test ingredients were calculated by the index method suggested in Kong and Adeola [13]: where Cr in and Cr out represent the concentration of Cr (g/kg DM) in experimental diets and ileal digesta, respectively; AA in and AA out represent the concentration of CP or AA (g/kg DM) in experimental diets and ileal digesta, respectively. Data from pigs fed NFD were used to estimate the BEL [g/kg DM intake (DMI)] of CP and AA using the following equation: BEL (g/kg DMI) = AA out ×(Cr in /Cr out ).
To calculate the SID of CP and AA in test ingredients, the AID of CP and AA were corrected for the estimated BEL of CP and AA using the following equation:

Statistical analyses
Data were tested for normality by univariate procedure of SAS (version 9.4; SAS Inst. Inc., Cary, NC, USA), and data outside of 2.5 times interquartile range were considered outliers. Thereafter, data were analyzed by mixed linear models procedure of SAS. Model included experimental diet as fixed variable and replicate, period within replicate, and pig within replicate as random variables. Pairwise comparison with the Tukey's adjustment was conducted to separate the estimated least squares means among experimental diets. Experimental unit was the individual pig. Significance of the model and difference was determined by p<0.05.

RESULTS
All pigs were healthy throughout except one pig fed NFD during the last experimental period, which was removed from experiment. Also, data from 2 pigs fed NFD and 1 pig fed the diet containing PRBC were detected as outliers and therefore treated as missing observations in the dataset.
On an as-fed basis, the concentration of CP in test ingredients ranged from 658 g/kg for FM to 968 g/kg for PRBC ( Table 2). The concentration of AEE in SDPP was 26.2 g/kg, whereas that in PRBC was 8.3 g/kg. Fish meal had the greatest concentration of ash at 201.4 g/kg. The analyzed concentrations of AA in experimental diets are close to the calculated values based on the analyzed concentrations of AA in test ingredients ( Table 3).
The AID of CP and AA, except isoleucine, in BM were less (p<0.05) than in the other test ingredients (Table 4). There was no difference in the AID of AA among FM, SDPP, and PRBC, except isoleucine, methionine, and cysteine. Pigs fed the diets containing PRBC and BM had less (p<0.05) AID of isoleucine than those fed the diets containing FM and SDPP. The AID of methionine in FM was greater (p<0.05) than in SDPP, but not different from the value in PRBC. Pigs fed the SDPP diet had greater (p<0.05) AID of cysteine than those fed the PRBC diet, which was not different from FM.
The BEL of CP in pigs fed NFD was 19.9 g/kg DMI ( Table  5). The BEL of indispensable AA ranged from 89 mg/kg DMI for methionine to 666 mg/kg DMI for valine. The SID of CP in BM was less (p<0.05) than in FM, SDPP, and PRBC (Table 6). Pigs fed the diet containing BM had less (p<0.05) SID of AA, except isoleucine and proline, than those fed the diet containing FM, SDPP, or PRBC. Among FM, SDPP, and PRBC, there was no difference in the SID of CP and all AA, except isoleucine. The SID of isoleucine in PRBC and BM was less (p<0.05) than in FM and SDPP.

DISCUSSION
The analyzed CP, AA, and ash concentrations in FM are consistent with the previously reported values [9,14,15]. In addition, the concentration of AEE in FM also agrees with the values reported in Casas et al [14] and Lagos and Stein [15]. The FM used in the current study is representative due to similarity in AA concentration for FM reported in previous studies [9,14,15]. The concentration of AA in SDPP are comparable to the values for plasma protein [9] and the values for SDAP [16] despite the differences in source of blood plasma. Torrallardona [5] reported that the concentration of AA in SDPP was not substantially different from that in spraydried bovine plasma (SDBP). However, SDPP used in the current study has greater indispensable AA and lower ash concentrations compared to SDPP used in Wu et al [17], which may be due to the differences in processing conditions [5]. Compared to nutrient composition of SDAP in previous studies [9,16,17], SDPP used in the current study contains slightly greater CP and lower ash. This observation is consistent with Torrallardona [5] who suggested that the concentration of CP in SDAP is negatively related to the concentration of ash. The concentration of indispensable AA in PRBC agrees with the values for spray-dried blood cells [6,16].  Because blood cells are almost exclusively composed of hemoglobin [4], it is assumed that nutrient compositions of blood cells products are relatively consistent among sources regardless of species origins. The concentration of CP in BM is slightly greater than previously reported CP concentrations ranging from 887 to 917 g/kg as-fed basis, whereas the concentration of indispensable AA, except isoleucine and leucine, is close to the previously reported values [9,16,18]. Compared to the reference values, the concentration of isoleucine was greater, but that of leucine was lower in BM used in the current study. This may be due to the reduced inclusion rate of blood cells because blood cells contain low concentration of isoleucine and high concentration of leucine. In addition, BM used in the current study was produced using a mixture of blood collected from both beef and pork productions, and therefore, variations in AA concentrations might be related to the ratio between bovine and porcine blood. Fish meal and SDPP have been widely used in diets for weanling pigs due to their high quality of protein and favorable AA contents for animal growth [2,19]. Although the use of PRBC in swine diets is not as common as FM or SDPP, practical applications in weanling pig diets have been reported in previous publications [6,7,20]. Even though animal protein sources have been generally used for weanling pigs, they have been also used in growing pig diets depending on the availability of feed ingredients or functional purposes. Therefore, Table 4. Apparent ileal digestibility (%) of crude protein and amino acids in fish meal, spray-dried porcine plasma, porcine red blood cells, and blood meal fed to growing pigs 1)   the current experiment was conducted to determine the SID values using growing pigs to provide necessary information required to properly use FM and blood-derived protein sources in diets for growing pigs. The SID of CP and AA, except isoleucine, were not different among FM, SDPP, and PRBC, which may indicate that FM, SDPP, and PRBC tested in this experiment have similar AA availability for pigs. However, it should be noted that the functional properties of FM, SDPP, and PRBC are different when fed to weanling pigs despite the similarity of SID of AA. Fish meal has been used in weanling pig diets mainly because it contains highly digestible and balanced AA as well as poly unsaturated fatty acids [2] and because it is a cost-effective protein source compared to SDPP. On the other hand, SDPP has been added in diets for weanling pigs to improve the immune status of pigs by providing exogenous immunoglobulins, leading to the reduction of post-weaning diarrhea and the increase in growth performance [5]. Therefore, together with the SID of AA in FM and SDPP, their functional properties should be considered when formulating diets containing FM and SDPP. The reason for less SID of isoleucine in PRBC compared to FM and SDPP remains unclear; perhaps this is partly due to the low concentration of isoleucine (5.8 g/kg as-fed basis) in PRBC, which translates to less than 1 g/kg of diet. Such a low dietary concentration is susceptible to attendant analytical errors. This observation implies that crystalline isoleucine or isoleucine-rich feed ingredient is required to prevent potential isoleucine deficiency when feeding PRBC to pigs.
The BEL of CP and AA observed in this study are comparable to the values summarized in previous studies [9,21,22]. The BEL of proline was relatively greater than the BEL of other AA, which may be due to the altered AA metabolism in the gastrointestinal tract caused by deficiency of AA in pigs fed NFD [22]. The SID of CP and AA in FM are close to the SID values of menhaden FM reported in Rojas et al [23] and Casas et al [14], but somewhat greater than SID values of menhaden FM presented by Lagos and Stein [15]. This inconsistency may be due to the differences in BW of pigs used in experiments or FM products such as processing conditions or oxidation [3]. In addition, Jones et al [24] reported that the inclusion rate of fish solubles in FM may influence the nutritional values of FM, although growth performance was not affected by the inclusion of fish solubles.
The SID of CP and AA in SDPP observed in the current study are in agreement with NRC [9] and Wu et al [17]; however, the SID values in SDPP are less than the values reported in Gottlob et al [25] and Almeida et al [16], in which SDAP was used as a test ingredient. In a meta-analysis study reported by Balan et al [19], there was no difference between Table 6. Standardized ileal digestibility (%) of crude protein and amino acids in fish meal, spray-dried porcine plasma, porcine red blood cells, and blood meal fed to growing pigs 1)