Basal diet and indigestible marker influence apparent digestibilities of nitrogen and amino acids of cottonseed meal and soybean meal in pigs.

This study was to determine the apparent ileal nitrogen (N) and amino acid digestibilities of cottonseed meal (CSM) and soybean meal (SBM) in simple carbohydrate-based and more complex wheat-based diets. Twenty five Large White × Landrace boars (40.9 kg) were randomly allocated to one of 5 dietary treatments: a wheat diet, 40% CSM in either a sugar:starch (1:1) or wheat-based diet, or 40% SBM in either a sugar:starch or wheat-based diet for 14 d. All diets contained vitamins, minerals, and contained acid-insoluble ash (AIA) and Cr2O3 as indigestible markers. Rations were offered (1,800 g/pig per d) in 3 meals/d on d 1 - 11 and 8 meals/d on d 12 - 13. On d 14, the pigs were fed hourly for 8 h. After the 8th meal, pigs were anaesthetised with isoflurothane, and ileal and faecal digesta sampled from the terminal ileum and rectum before lethal injection of barbiturate. Apparent ileal digestibility (AID) of N was greater (12.1%) when CSM was fed in the wheat-based diet as compared to the simple carbohydrate-based diet, whereas apparent ileal N digestibility of SBM was slightly lower (-4.5%) in the wheat-based diet as compared to the sugar:starch-based diet. Apparent ileal amino acid digestibility generally respondeds similarly to N. Therefore, while there was a wide difference in apparent N and amino acid digestibilities of SBM and CSM when they were fed in the sugar:starch-based diets, these differences were less apparent when they were fed the wheat-based diet. There was an apparent net release of N into the hindgut of pigs fed CSM in both base diets. Conversely, there was quite substantial apparent digestion of N in the hindgut of pigs fed SBM in both base diets. The use of Cr2O3 as an indigestible marker resulted in lower apparent ileal and faecal digestibilities than using AIA, particularly for diets containing CSM. These data demonstrate that the basal diet and choice of indigestible marker can substantially influence the estimate of apparent N digestibility, this response can differ for different protein meals.

Introduction

Cotton is grown in many parts of the world with global production of cotton seeds being 44 million tonnes per year which, after removal of n class="Chemical">oil, hulls and lint, provides approximately 15 million tonnes of cottonseed meal available for livestock (Soy and n class="Chemical">Oilseed Bluebook, 2015). However, cottonseed meal (CSM) can be of variable quality and in general has a poorer amino acid digestibility and availability than other oilseed meals (Prawirodigdo et al., 1998, Gonzalez-Vega and Stein, 2012, Ma et al., 2018), although the use of CSM in pig diets has been little researched (Stein et al., 2016).

Digestibility of dietary protein may be affected by several factors including the amino acid profile of the protein source, the concentration of the protein source and the nutrienpan>t profile of other feeds consumed with the protein source. However, data are equivocal with respect to the absolute effect of these factors, or any interactions, on the digestibility of dietary protein sources. For instance, protein digestibility in n class="Species">piglets was more dependent upon the amino acid profile of the protein source rather than on other components in the diet including carbohydrates (Turlington et al., 1989). Moreover, wide variations in dietary protein content may influence the apparent ileal digestibility (AID) or total tract (ATTD) of nitrogen (N) or amino acids in growing pigs (Fan et al., 1994, Ma et al., 2018). Variation in processing technique can also contribute to variation in AID. For instance, Ma et al. (2018) found that cottonseed co-products treated using a range of different processing technologies also resulted in widely varying AID. In addition, using a meta-analysis approach, Messad et al. (2016) found that the AID of amino acids in oilseeds was inversely related to their neutral detergent fibre content and that this is partially related to increased endogenous losses of amino acids (Stein et al., 2007). Leterme et al. (1994) observed substantial differences in AID of N in pigs fed different carbohydrate fractions, and suggested that this was due to an effect of specific carbohydrate fractions and the rate of endogenous protein loss. Furthermore, Wigan et al. (1995) found that digestible energy of lupins was higher when they were fed in a wheat-based diet compared to a sugar-based diet; it was suggested that the higher digestibility when pigs were fed the wheat-based diet may have been due to more of the complex carbohydrates passing to the hindgut and providing increased substrate for hindgut fermentation. Thus, carbohydrates and fibre present in the basal diet may substantially influence the AID of amino acids (Messad et al., 2016). Therefore, the present study was conducted to determine AID of N and amino acids in pigs fed cottonseed meal (CSM) or soya bean meal (SBM) in a simple (sugar + starch) and a complex (wheat) carbohydrate-based diet. Soybean and cottonseed meals were chosen as test oilseed protein meals to provide additional comparative information on CSM and because the oilseeds meals differ widely in their apparent amino acid digestibilities. A further objective was to compare chromic oxide (Cr2O3) and acid-insoluble ash (AIA) as indigestible markers.

Materials and methods

Diets, animals and experimental design

The protocol used in this experiment conformed to all Animal Experimentation Ethics Cn class="Chemical">ommittee regulations concerning the health and care of experimental animals and was approved by the Victorian Institute of Animal Science Animal Ethics Cn class="Chemical">ommittee.

The measured nutrient contents of SBM, CSM and n class="Species">wheat and estimated enpan>ergy contenpan>t are presenpan>ted in Table 1. The presenpan>t study used 5 formulated diets (Table 2). The basal control diet contained 926 g n class="Species">wheat/kg with wheat providing the sole source of dietary carbohydrate and protein. The other 4 diets contained 400 g/kg CSM in a sugar + starch (1:1 wt/wt) based or a wheat-based and 400 g/kg SBM in both basal diets. Iron sulphate heptahydrate (FeSO4·7H2O) was added (2 g/kg) to the CSM diets to inactivate the gossypol present in CSM (Knabe et al., 1979). Chromic oxide and acid insoluble ash (Celite, a diatomaceous earth produced by Celite Cooperation Lompoc, California, USA) were included in the diets (2 g/kg) as indigestible markers.

Table 1

Protein and amino acids profile of the cottonseed meal (CSM), soybean meal (SBM) and wheat used in the present experiment (g/kg, air-dry basis).

Item	CSM	SBM	Wheat
Dry matter	911	887	892
Organic matter	849	828	881
Crude protein (N × 6.25)	371	443	133
Indispensable amino acids
Arginine	38.1	31.5	6.9
Histidine	11.4	13.8	3.0
Isoleucine	12.4	22.0	5.0
Leucine	20.6	32.9	9.1
Lysine	15.9	27.2	3.6
Phenylalanine	18.9	24.0	7.1
Threonine	11.8	20.2	3.9
Tyrosine	8.9	16.6	4.6
Valine	17.5	22.6	6.3
Dispensable amino acids
Alanine	13.8	20.1	4.7
Aspartic acid	30.9	48.5	6.6
Glutamic acid	67.0	74.0	37.8
Glycine	14.6	18.4	5.4
Proline	13.4	23.4	13.6
Serine	13.4	24.9	13.6

Table 2

Composition of the experimental diets (g/kg, air-dry basis).

Item	Diet
	Wheat	Cottonseed meal		Soybean meal
		Simple	Wheat	Simple	Wheat
Ingredient
Cottonseed meal	–	400	400
Soybean meal	–	–	–	400	400
Wheat	926	–	526	–	526
Cane sugar (sucrose)	–	263	–	263	–
Starch (wheat starch)	–	263	–	263	–
FeSO4.7H2O	–	2.0	2.0	–	–
K2SO4	1.5	–	–	1.5	1.5
Minerals + Vitamins premix1	5.0	5.0	5.0	5.0	5.0
Dicalcium phosphate	30.0	30.0	30.0	30.0	30.0
Cr2O3	2.0	2.0	2.0	2.0	2.0
Acid-insoluble ash (Celite)	2.0	2.0	2.0	2.0	2.0
Tallow	33.5	33.5	33.5	33.5	33.5
Determined analysis
Dry matter	894	917	896	908	888
Organic matter	854	871	828	860	820
Crude protein (N × 6.25)	123	149	219	177	246
Indispensable amino acids
Arginine	5.5	13.8	16.6	12.4	14.5
Histidine	2.8	3.4	5.6	4.5	6.5
Isoleucine	4.8	4.5	7.2	8.6	11.2
Leucine	8.8	7.8	1.3	13.6	18.0
Lysine	7.0	6.0	7.9	10.9	12.7
Phenylalanine	6.2	7.6	10.5	9.3	12.1
Threonine	5.4	4.0	6.8	7.0	10.7
Tyrosine	4.1	4.0	5.8	6.2	8.7
Valine	6.0	6.3	9.8	9.0	12.3
Dispensable amino acids
Alanine	4.7	5.1	7.8	7.8	10.5
Aspartic acid	6.8	11.3	15.8	19.5	22.8
Glutamic acid	37.3	24.1	44.4	31.7	49.8
Glycine	5.4	5.4	8.4	7.4	10.1
Proline	3.4	4.6	12.5	8.8	16.4
Serine	6.9	5.6	9.7	9.3	13.5
Calculated analysis
Digestible energy, MJ/kg2	14.4	14.3	14.1	14.9	14.7

The minerals and vitamins premix was added to contribute (mg/kg air dry diet): retinol, 6.4; cholecalciferol, 0.083; α-tocopherol, 22; menadione, 0.60; riboflavin, 3.3; nicotinic acid, 16.5; pantothenic acid, 5.5; pyridoxine, 1.1; biotin, 0.056; choline, 1,100; cyanocobalamin, 0.017; Fe, 88; Zn, 55; Mn, 22; Cu, 6.6; I, 0.22; Se, 0.1.

Estimated from ingredients using AUS pig (Black et al., 1986).

Protein and amino acids profile of the cottonseed meal (CSM), n class="Species">soybean meal (SBM) and n class="Species">wheat used in the present experiment (g/kg, air-dry basis).

Cn class="Chemical">omposition of the experimental diets (g/kg, air-dry basis).

The minerals and vitamins premix was added to contribute (n class="Chemical">mg/kg air dry diet): retinol, 6.4; cholecalciferol, 0.083; α-tocopherol, 22; menadione, 0.60; riboflavin, 3.3; nicotinic acid, 16.5; pantothenic acid, 5.5; pyridoxine, 1.1; biotin, 0.056; choline, 1,100; cyanocobalamin, 0.017; Fe, 88; Zn, 55; Mn, 22; Cu, 6.6; I, 0.22; Se, 0.1.

Estimated frn class="Chemical">om ingredients using AUS n class="Species">pig (Black et al., 1986).

Twenty-five n class="Species">Large White × Landrace enpan>tire male n class="Species">pigs (40.9 ± 2.6 kg, mean ± standard deviation) were individually penned and randomly assigned to one of the 5 experimental diets (Table 2) in a completely randomised design. The experimental diets were offered to pigs in a mash form for 14 d. Water was constantly available via nipple drinkers during the experimental period. The diets were introduced to the pigs over a 3-d period. Rations were offered (1,800 g/pig per day) in 3 meals/d (at 06:00, 12:00 and 18:00) from d 1 to 11 and in 8 meals/d (every 3 h) from d 12 to 13. There were no feed refusals at this level of feed offered.

Cn class="Chemical">ommenpan>cing at 01:00 on d 14, the n class="Species">pigs received their daily allocation of feed in 8 portions that were fed hourly. After the 8th meal, each pig was sedated using 5 mL of Stresnil (Janssen Pharmaceutical, Beerse, Belgium) injected intramuscularly and left undisturbed in their pens for 15 min. The sedated pigs were then anaesthetised by inhalation of isoflurane (Rhone Merieux, Australia). After each pig was anaesthetized, a ventral abdominal midline incision was made, the caecum was located and then a 150 cm portion of the terminal ileum and the rectum were excised to enable simultaneous collection of the ileal and faecal digesta. The ileal digesta was gently expelled, collected and stored at −20 °C until analysed. Faecal digesta was also expelled, collected and stored frozen until analysis. Finally, the anaesthetised pigs were killed with a lethal injection (15 mL) of pentobarbitone sodium (Valabarb 300 mg/mL; Boehringer Ingelheim Pty. Ltd, NSW, Australia) administered directly into the vena cava. Ileal digesta from all pigs were freeze-dried, finely ground and analysed for amino acids, N, organic matter (OM), Cr2O3 and AIA contents. Faecal digesta were freeze-dried and ground for total N, OM, Cr2O3 and AIA determination.

Chemical analyses

n class="Disease">Dry matter contenpan>t of n class="Species">wheat, CSM, SBM and experimental diets was determined by drying for 16 hr in a forced-air oven at 100 °C. Organic matter was measured in the wheat, CSM and SBM, experimental diets, and ileal and faecal digesta by combustion of the samples in a furnace chamber at 650 °C for 4 h. Total N content of the samples was determined using a Micro Kjeldahl method (AOAC, 1990). The amino acid contents of the diet and ileal digesta were determined following acid hydrolysis in 6 mol/L hydrochloric acid. The mixture of amino acids was separated on an HPLC ion exchange (strong cation exchange) column (Waters Australia Pty. Ltd., Box Hill, Victoria, Australia) using post column derivatization with ninhydrin (Rayner, 1985).

Concentrations of chromium in the diets, ileal and faecal digesta were determined using a modification of the atomic absorption spectrophotometric method of Williams et al. (1962) and the chromium contents used to calculate AID and ATTD as described by Saha and Gilbreath (1993). The concentration of AIA in the diets and digesta was determined using a gravitation technique, modified from the technique described by McCarthy et al. (1974). Since Cr2O3 is insoluble in 4 mol/L hydrochloric acid (McCarthy et al., 1974), when AIA is simultaneously used as an inert marker with Cr2O3 in a diet, the non-Cr2O3 AIA values should be obtained by difference (McCarthy et al., 1974). Therefore, non-Cr2O3 AIA was calculated as total AIA concentration minus Cr2O3 concentration.

Statistical analyses

For the analyses of the AID of individual amino acids, the data were analysed by analysis of variance (An class="Chemical">NOVA) with the model being a (2 base diets × 2 protein sources) + n class="Species">wheat diet treatment structure split for marker within each pig. The respective factors were protein source (CSM and SBM), basal diets (sugar + starch and wheat) and markers (Cr2O3 and AIA). In studying the effects of basal diets on the AID and ATTD of N, OM and DM, the statistical analyses were performed so that the differences between site of sampling (ileum versus rectum) and their interactions could be determined. Thus, site of sampling (ileal and faecal) was used as an additional factor. All statistical analyses were performed using GENSTAT version 18 (VSN International Ltd, Hertfordshire, UK).

Results

Apparent ileal digestibility of indispensable amino acids

The AID of indispensable amino acids (n class="Chemical">IAA) are presenpan>ted in Table 3. The AID of n class="Chemical">IAA was significantly (P < 0.05) higher in SBM diets compared to CSM diets, respectively, for arginine (81% vs. 73%), histidine (73% vs. 59%), isoleucine (83% vs. 57%), leucine (77% vs. 55%), lysine (82% vs. 48%), phenylalanine (74% vs. 70%), threonine (77% vs. 46%), tyrosine (76% vs. 59%) and valine (79% vs. 60%). The AID was significantly (P < 0.05) higher in the sugar and starch base diet compared to the wheat-based diet, respectively, for arginine, isoleucine, phenylalanine, tyrosine, but was lower for threonine. There was no effect (P > 0.05) of base diet on the AID for histidine, leucine, lysine and valine. The AID was significantly (P < 0.05) lower when determined using Cr2O3 compared to AIA, respectively, for arginine, histidine, isoleucine, leucine, lysine, phenylalanine, threonine, tyrosine and valine (Table 4).

Table 3

Effect of marker, protein source and basal diets and on the apparent ileal digestibility of indispensable amino acids of the experimental diets (%).1

Marker	Chromium					Acid insoluble ash					SED2	SED3	Significance4
Protein source	Wheat	Cottonseed meal		Soybean meal		Wheat	Cottonseed meal		Soybean meal
Basal diet		Simple	Wheat	Simple	Wheat		Simple	Wheat	Simple	Wheat
Arginine	65.1	73.9	67.8	84.0	74.6	76.0	80.8	71.0	87.5	75.9	1.52	0.95	D∗∗∗; P∗∗∗; B∗∗∗; M∗∗∗; P×M∗∗; B×M∗∗
Histidine	66.2	49.0	55.8	77.4	65.3	76.7	62.3	67.4	82.2	69.0	3.43	1.63	D∗; P∗∗∗; M∗∗∗; P×B∗∗∗; P×M∗∗
Isoleucine	75.2	54.8	49.3	82.2	75.4	82.7	66.9	57.8	86.1	88.2	2.74	1.48	D∗∗; P∗∗∗; B∗∗∗; P×B∗∗; M∗∗∗; P×B×M∗∗∗
Leucine	73.0	46.5	49.8	78.3	68.9	83.1	60.8	64.3	83.3	78.9	2.80	1.71	D∗∗∗; P∗∗∗; M∗∗∗; P×B∗∗; PxM∗∗∗
Lysine	66.2	36.7	41.4	82.0	77.0	76.7	54.9	57.2	86.0	81.0	2.89	1.83	D∗∗; P∗∗∗; M∗∗∗; P×B∗; P×M∗∗∗
Phenylalanine	83.0	70.2	63.7	81.7	63.6	88.4	78.1	69.3	83.3	67.8	2.07	1.16	D∗∗∗; P∗; B∗∗∗; M∗∗∗; P×B∗∗; P×M∗∗; P×B×M∗
Threonine	71.3	32.2	46.1	72.5	75.8	80.2	50.1	57.2	78.5	81.3	3.59	1.96	D∗∗∗, P∗∗∗; B∗∗; M∗∗∗; P×M∗∗∗
Tyrosine	69.3	55.7	51.7	76.9	69.8	78.9	67.6	59.5	81.9	73.7	3.85	1.67	D∗; P∗∗∗; B∗; M∗∗∗; P×M∗∗
Valine	73.6	52.0	55.4	79.4	74.5	81.8	64.7	68.2	83.9	78.7	2.45	1.70	D∗∗∗; P∗∗∗; M∗∗∗; P×B∗; P×M∗∗∗

SED = standard error of difference.

Basal diets were either a 1:1 sugar and starch mix (simple) or pure wheat (wheat); protein sources were either 400 g/kg cottonseed meal or soybean meal mixed with the basal diet or diet containing 926 g/kg wheat.

When comparing different basal diet means.

When comparing same basal diet means.

Significance of effects of pure (926 g/kg) wheat vs. other diets (D), protein source (P), basal diet (B) or indigestible marker (M) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Table 4

Effect of marker, protein source and basal diets and on the apparent ileal digestibility of dispensable amino acids of the experimental diets (%).1

Marker	Chromium					Acid insoluble ash					SED2	SED3	Significance4
Protein source	Wheat	Cottonseed meal		Soybean meal		Wheat	Cottonseed meal		Soybean meal
Basal diet		Simple	Wheat	Simple	Wheat		Simple	Wheat	Simple	Wheat
Alanine	62.3	39.3	46.3	76.6	59.1	74.0	55.5	60.6	81.7	66.2	3.50	2.07	D∗∗; P∗∗∗; B∗; M∗∗∗; P×B∗∗∗; P×M∗∗∗
Aspartic acid	63.6	54.1	62.0	77.2	67.5	74.9	66.3	69.5	82.2	73.2	2.73	1.48	P∗∗∗; M∗∗∗; P×B∗∗∗; P×M∗
Glutamic acid	88.6	68.0	62.8	78.0	61.9	92.2	76.4	68.4	82.8	68.8	2.56	1.18	D∗∗∗; P∗; B∗∗∗; P×B∗
Glycine	49.3	22.1	48.3	58.9	47.4	65.1	42.6	56.7	67.8	55.8	5.43	2.62	P∗∗∗; P×B∗∗∗; M∗∗∗; P×M∗; B×M∗; P×B×M∗
Proline	78.9	−63.3	55.2	69.5	74.4	85.4	−19.6	63.1	76.0	77.6	15.0	4.94	D∗∗; P∗∗∗; B∗∗∗; M∗∗∗; P×B∗∗∗; P×M∗∗∗; B×M∗∗∗; P×B×M∗∗
Serine	74.4	46.8	61.1	78.2	75.7	82.4	60.9	69.4	82.9	80.5	2.99	1.61	D∗∗∗; P∗∗∗; B∗; M∗∗∗; P×B∗∗; P×M∗∗∗

SED = standard error of difference.

Basal diets were either a 1:1 sugar and starch mix (simple) or pure wheat (wheat); protein sources were either 400 g/kg cottonseed meal or soybean meal mixed with the basal diet or diet containing 926 g/kg wheat.

When comparing different basal diet means.

When comparing same basal diet means.

Significance of effects of pure (926 g/kg) wheat vs. other diets (D), protein source (P), basal diet (B) or indigestible marker (M) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

When cn class="Chemical">omparing different basal diet means.

When cn class="Chemical">omparinpan>g same basal diet means.

Significance of effects of pure (926 g/kg) n class="Species">wheat vs. other diets (D), protein source (P), basal diet (B) or indigestible marker (M) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

When cn class="Chemical">omparing different basal diet means.

When cn class="Chemical">omparinpan>g same basal diet means.

Significance of effects of pure (926 g/kg) n class="Species">wheat vs. other diets (D), protein source (P), basal diet (B) or indigestible marker (M) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

There were also several interactions between protein sources, basal diets and indigestibility marker. For example, AID determined using Cr2O3 was decreased more significantly (P < 0.01) than that determined using AIA in diets containing CSM compared to SBM for arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine and valine. For arginine, the AID was decreased (P < 0.01) to a greater extent in a sugar and starch diet compared with a wheat-based diet when calculated using Cr2O3 and AIA as digestibility markers, respectively. The AID was significantly higher for SBM in a simple carbohydrate-based diet compared to a complex carbohydrate-based diet, respectively, for all IAA, but variable for CSM when comparing the base diets.

Apparent ileal digestibility of total nitrogen and dispensable amino acid

Apparent ileal digestibility of dispensable amino acids (n class="Chemical">DAA) was significantly (P < 0.001) higher in SBM diets compared to CSM diets, respectively, for alanine (71% vs. 50%), aspartic acid (75% vs. 63%), glutamic acid (73% vs. 69%), glycine (58% vs. 42%), proline (74% vs. 9%) and serine (79% vs. 60%). Digestibility was significantly higher in the simple base diet compared to the complex base diet for alanine (63 vs. 58%) and glutamic acid (76% vs. 58%), but was lower for proline (16% vs. 68%) and serine (67% vs. 72%). There was no effect (P > 0.10) of base diet on AID for aspartic acid and glycine. The AID was significantly (P < 0.001) lower when digestibility was determined using Cr2O3 compared to AIA, respectively, for alanine (57% vs. 68%), aspartic acid (65% vs. 73%), glycine (45% vs. 58%), proline (43% vs. 57%) and serine (67% vs. 75%).

There were also several interactions between protein sources, base diets and indigestibility marker. For example, AID was significantly (P < 0.001) higher for SBM in a simple base diet compared to a complex base diet, respectively, for all DAA, except glycine and proline, whereas for CSM digestibility was lower in a simple base diet compared to the complex base diet for all DAA except glutamate. For proline, AID was substantially less and indeed negative. The AID determined using Cr2O3 was decreased significantly (P < 0.001) more than for digestibility determined using AIA in diets containing CSM compared to SBM. There was also an interaction such that AID was decreased to greater extent in a simple base diet compared with a complex base diet for glycine and proline when calculated using Cr2O3 and AIA digestibility markers, respectively.

Apparent ileal and faecal digestibilities of nitrogen, organic matter and dry matter

The AID and ATTD of n class="Chemical">N, n class="Chemical">OM and dry matter are given in Table 5. The apparent digestibility of N was higher in SBM than in CSM (81% vs. 68%), was lower when digestibility was determined with Cr2O3 as compared to AIA (71% vs. 79%) and was lower when measured in the terminal ileum compared to the rectum (74% vs. 76%) (Table 5). However, there was an interaction (P < 0.001) such that the AID of N in CSM was higher than the ATTD (69% vs. 67%) whereas the converse was true for SBM (79% vs. 83%). The apparent digestibility of OM was higher in SBM than in CSM (84% vs. 74%), lower when digestibility was determined with Cr2O3 as compared to AIA (75% vs. 83%) and when measured in the terminal ileum compared to the rectum (74% vs. 76%) and higher when measured using a simple basal diet (84% vs. 74%). However, there was an interaction (P < 0.001) such that digestibility of organic matter in CSM increased to a lesser degree between the ileum and rectum (71% vs. 77%) than it did in SBM (78% vs. 87%). While there were a number of other interactions, these were mainly related to different magnitudes of response rather than any differences in direction of response. Responses in apparent digestibility of dry matter were similar to organic matter (Table 5).

Table 5

Effect of marker, protein source, basal diets and site of sampling on the apparent digestibility (%) of nitrogen (N), dry matter and organic matter of the experimental diets.1

Marker	Chromium					Acid insoluble ash					SED2	SED3	Significance4
Protein source	Wheat	Cottonseed meal		Soybean meal		Wheat	Cottonseed meal		Soybean meal
Basal diet		Simple	Wheat	Simple	Wheat		Simple	Wheat	Simple	Wheat
N
Ileal	73.4	57.1	73.0	79.0	71.4	81.6	67.3	75.9	82.7	78.8	1.20	1.08	D∗; P∗∗∗; M∗∗∗; S∗; P×S∗∗∗
Faecal	77.2	56.5	63.9	81.5	78.0	82.4	67.7	76.2	86.3	85.7
Dry matter
Ileal	65.8	64.4	53.7	77.4	60.0	77.2	75.0	64.5	84.5	68.5	1.37	1.21	D∗: P∗∗∗; B∗∗∗; M∗∗∗; S∗∗∗; B×M∗∗;
Faecal	78.9	73.7	61.1	89.2	77.1	83.3	80.5	74.4	92.0	84.9			P×M∗∗; B×S∗∗∗; M×S∗∗; P×S∗∗∗; B×M×S∗∗
Organic matter
Ileal	69.4	69.2	61.8	81.1	67.0	82.6	80.3	70.4	87.9	74.8	1.29	1.15	P∗∗∗; B∗∗∗; M∗∗∗; S∗∗∗; B×M∗∗∗; M×S∗;
Faecal	82.0	77.3	64.8	92.4	81.3	86.7	84.6	79.2	94.9	88.7			P×S∗∗∗; B×M×S∗∗∗

SED = standard error of difference.

Basal diets were either a 1:1 sugar and starch mix (Simple) or pure wheat (Wheat); protein sources were either 400 g/kg cottonseed meal or soybean meal mixed with the basal diet or diet containing 926 g/kg wheat.

When comparing different basal diet means.

When comparing same basal diet means.

Significance of effects of pure (926 g/kg) wheat vs other diets (D), protein source (P), basal diet (B), indigestible marker (M) or site (S) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

When cn class="Chemical">omparing different basal diet means.

When cn class="Chemical">omparinpan>g same basal diet means.

Significance of effects of pure (926 g/kg) n class="Species">wheat vs other diets (D), protein source (P), basal diet (B), indigestible marker (M) or site (S) and interactions: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Discussion

These data demonstn class="Species">rate that the AID of amino acids and n class="Chemical">N of both CSM and SBM diets differ when these protein sources are offered to the pigs in different basal diets. The trends in the AID changes were consistent regardless of whether Cr2O3 and AIA were used as indigestible markers, although the latter yielded consistently higher values. In addition, the responses of the pigs to the different basal diets were variable as indicated by significant interactions between dietary protein sources, basal diets and markers. These interactions have important implications for how digestibility of N and amino acids may differ from experimental values and may be influenced by the complex diets that are used commercially.

These data clearly show that the choice of indigestible marker can have a profound effect upon the estimates of the AID of amino acids. The use of Cr2O3 resulted in lower estimates of the AID of amino acids than n class="Chemical">AIA, particularly for most of the amino acids in CSM. The AID of amino acids and N of SBM in a simple basal diet determined using Cr2O3 as a marker in the present study were more similar to those determined in our laboratory in an earlier study (Prawirodigdo et al., 1998), again using Cr2O3 as a marker, than estimates obtained using AIA. However, the AID of amino acids and N of CSM in a simple basal diet determined using AIA as an indigestible marker were very similar to those determined previously (Prawirodigdo et al., 1998). The concentration of Cr2O3 in the diets and digesta samples was measured by X-ray spectrometry in the previous study and by atomic absorption spectrophotometry in the present study and this may have contributed to differences. A drawback of the atomic absorption spectrophotometry technique to measure Cr2O3 content is that it appears to not always provide an accurate determination for Cr2O3 content (Jagger et al., 1992, Kozloski et al., 1998), possibly because other minerals such as Ca, P or Mg can interfere with Cr analyses (Saha and Gilbreath, 1991a, Saha and Gilbreath, 1991b). It is also likely that at least some of the Cr is soluble in the alkaline or acidic fluids in the gastrointestinal tract and that these become volatile during sample digestion prior to atomic absorption analyses (Saha and Gilbreath, 1991a, Saha and Gilbreath, 1991b). Hence, AIA was also included as an indigestible marker in the present study.

A number of researchers have shown that estimation of AID and endogenous losses of amino acids could be impacted by the choice of indigestible marker. For example, Favero et al. (2014) found that both AID and endogenous losses of amino acids were lower when determined using Cr2O3 as an indigestible marker as compared to both AIA or titanium oxide (TiO2) which is consistent with the present findings. On the other hand, Fan and Sauer (1995) found that AID was higher in high fibre diets when determined using Cr2O3 as compared to AIA which they attributed to possible separation of the markers with Cr2O3 being associated with the liquid phase. Yin et al., 2000b, Yin et al., 2000a compared the use of total collection via the post-valve "T" caecal (PVTC) cannula and the use of Cr2O3 and TiO2 as indigestible markers and found that Cr2O3 underestimated the ATTD of crude protein and DM. On the other hand, the ATTD assessed using TiO2 as a marker agreed well with the total collection method using PVTC. Furthermore, Yin et al., 2000b, Yin et al., 2000a found that both markers, but in particular Cr2O3, were poorly recovered from ileal digesta collected using PVTC, from pigs consuming high fibre diets. Finally, Moughan et al. (1991) reported quantitative recovery of both endogenous and exogenous AIA but relatively poor recovery of Cr2O3 in pigs fed barley-based diets. However, separation of markers into solid and liquid phases shouldn't be an issue when collecting total digesta as in the present study so there may be other reasons for the discrepancies between the methods.

To further investigate the effect of marker on the ATTD of n class="Chemical">N, n class="Chemical">OM and DM, Prawirodigdo (1999) compared Cr2O3 and AIA with total faecal collection in pigs fed similar diets to those used in the present study. Prawirodigdo (1999) found that the ATTD of N determined using AIA as a marker was highly correlated (R2 = 0.95) to that determined by total faecal collection and that the slope (0.99) and intercept (0.01) were not significantly different from 1 to 0, respectively. On the other hand, the relationship between ATTD of N determined using Cr2O3 was less well correlated (R2 = 0.87) with total faecal collection and both the slope (0.75) and intercept (0.21) were significantly different than 1 and 0, respectively. Prawirodigdo (1999) reported that for diets with a high digestibility the three methods gave similar estimates of digestibility, whereas for diets with lower digestibilities, Cr2O3 seriously underestimated N digestibility determined using total faecal collection.

Since, for reasons outlined above, we have greater confidence in digestibilities determined using n class="Chemical">AIA as a marker, the following discussion about the effect of basal diet on digestibilities will use the n class="Chemical">AIA marker method as the standard measurement. The present data show a generally higher AID of N and the majority of amino acids in CSM when fed in a complex wheat-based diet compared to when CSM is provided in a simple base diet, possibly due to the different protein and/or carbohydrate profiles of the diets. However, the general response with diets containing SBM was for either unchanged or a small decrease in apparent ileal digestibility of most amino acids when a complex rather than simple base diet was used. In this context, the alloxan-induced diabetic neuromast (AIDN) of whole commercial diets containing 25% CSM and SBM in a predominantly sorghum base were similar suggesting some synergy between cereal and CSM with respect to the AID of N (Power, 2000). Also, other workers who have determined the AID of amino acids in CSM using complex cereal basal diets have found most values near the upper end of the range of normally encountered values (Batterham et al., 1990, Li et al., 2000). Conversely, the AID of amino acids obtained using simple carbohydrate basal diets have been towards the lower end of the range (Knabe et al., 1989, Prawirodigdo et al., 1998).

Most recent ileal digestibility studies of protein meals or protein sources in Australia have used a cereal base diet to simulate the conditions under which they are fed cn class="Chemical">ommercially (King et al., 2000, Collins et al., 2005, Collins et al., 2006). A possible reason for the increase in the AID of amino acids when CSM is mixed with a wheat diet may be related to the increase in protein content of the whole diet. Some workers have found that AID of amino acids and N increased as the dietary protein content increased (Fan et al., 1994, Fan and Sauer, 1995, Langer and Fuller, 1996), possibly because of a dilution of endogenous protein losses (Power, 2000). In the present experiment the increase in the AID of proline, threonine, glycine and serine in CSM in a wheat-based diet appeared to be the major amino acids contributing to the significant improvement in the AID of N compared to when CSM was offered in a simple base diet. It is possible that the pigs given CSM in a sugar + starch-base diet secreted a much greater proportion of endogenous protein relative to dietary protein into the small intestine, than the pigs consuming CSM protein in a wheat-based diet. Other investigators (Holmes et al., 1974, Sauer et al., 1977) have also found high levels of glycine and threonine in the ileal digesta of growing pigs given a protein-free diet. In addition, glycine is a major component of bile salt conjugates, and accounts for more than 90% of the amino acids secreted in bile juice of pigs (Souffrant, 1991, Fan and Sauer, 1995). Moreover, the endogenous secretions of the small intestine contain mucins that are rich in threonine, serine and proline (Neutra and Forstner, 1987). An increased mucin secretion may explain why the AID of these amino acids in CSM in a simple basal diet were relatively low compared to the AID of other amino acids in the present experiment. In this context, if there was any free gossypol present in the CSM diets this could also increase mucin secretion as was the case in the rat proximal colon (Kuhn et al., 2002).

There was a very low AID for n class="Chemical">proline (−40%) in n class="Species">pigs fed CSM in a simple base diet, as was the case in our earlier study (Prawirodigdo et al., 1998). It is possible that the secretion of endogenous proteins high in proline caused a negative balance of proline input (dietary origin): proline output ratio (endogenous origin). Furthermore, when CSM was fed to pigs as the sole dietary protein source in a simple base diet, the AID of N of CSM was markedly lower than that in a pure wheat diet. It is possible that interaction between the protein in CSM and that in wheat may have increased the AID of N for CSM in the wheat-based diet. Finally, the sugar and starch in the simple diet would have been rapidly digested and absorbed in the proximal small intestine (Beech et al., 1991, Noah et al., 1999) resulting in the digesta being comprised of relatively undigested and fibrous CSM in much of the small intestine. This could also increase the endogenous losses of amino acids and decrease the AID of amino acids.

Data frn class="Chemical">om the presenpan>t study confirm those of previous studies (Batterham et al., 1990, Prawirodigdo et al., 1998) where there was no further digestion of n class="Chemical">N in the hindgut of pigs fed CSM diets. There are two possible reasons for this. Firstly, the bound N of the CSM may have been resistant to microbial attack due to Maillard reaction during processing of CSM. A second alternative is that while hindgut microbes may have broken down the bound protein, gossypol released at the same time may have been toxic to hind gut microbes and possibly mucosal cells (Berardi and Goldblatt, 1980) thereby increasing N recovery in the faecal digesta. This free gossypol may also have stimulated the pigs to protect their body tissue by secretion of endogenous N from goblet cells of hindgut. Low and Zebrowska (1989) stated that the goblet cells secrete a protective sulphated carbohydrate–protein complex. In the present experiment the secretion may have contributed N to the total N content of the faecal digesta of the pigs. Thus, the AFDN value of CSM was similar to the AIDN.

There was further N digestion in the hind gut of pigs fed SBM in both basal diets, which is again consistent with the observations of Prawirodigdo et al. (1998). In addition, the further degradation of N in the hindgut of pigs given pure wheat diets suggests that there was no anti-nutritive effect of wheat on digestibility of N in pig. Consistent with the AFDOM values found in our previous experiment (Prawirodigdo et al. (1998), the AFDOM of CSM and SBM diets in the present experiment was also higher than the IDOM. These data also show that there was further OM digestion in the hindgut of pigs given the pure wheat diet. Similarly, the AFDDM of CSM, SBM and wheat was higher than AIDDM.

Cottonseed meal contains n class="Chemical">gossypol which is a polyphenpan>olic compound that is toxic to monogastric animals, and this has lead producers to limit the presence of CSM in feeds for both poultry and pigs (Berardi and Goldblatt, 1980, Tanksley, 1990, Swiatkiewicz et al., 2016, Duodu et al., 2018). Even though the acute oral toxicity of gossypol is relatively low, if even small amounts of free gossypol are ingested over a prolonged period, it can be fatal (Berardi and Goldblatt, 1980). Also, gossypol can reduce protein digestibility by inhibition of pepsin and trypsin activity in the intestine and binding dietary iron. Consequently, to increase the utilization of CSM as a dietary protein source for pigs, any free gossypol must be inactivated. Although heat treatment can inactivate gossypol, the use of these thermal processes may produce inert and indigestible complexes between gossypol and protein (Berardi and Goldblatt, 1980). Gossypol toxicity can also be prevented by the addition of iron salts to the diet, such as ferrous sulfate, iron forms an insoluble and irreversible complex with gossypol in the intestinal tract, avoiding its absorption (Knabe et al., 1979) and this was the approach taken in the present study. While it is possible that the addition of iron sulphate may have an effect on amino acid digestibility, there is little data to support this. Indeed, there was a very small increase (ca. <1%) in the amino acid digestibility in rats receiving ferrous sulphate compared to ferrous lactate although there was no control diet without the addition of iron with which to compare (Perez-Llamas et al., 2001).

Conclusions

In conclusion, the present study suggests that the basal diet used can influence the AID of amino acids and n class="Chemical">N and the response can differ for differenpan>t protein sources. Thus, the AID of amino acids in CSM appears to be higher in a complex basal diet than when fed in a simple basal diet. Given that CSM is generally fed to pigs in a complex diet under conventional production systems, the AID of amino acids may be higher than sometimes assumed. Also, AIA appears to be a more reliable digestibility indicator than Cr2O3.

Conflicts of interest

The authors declare that we have no financial and personal relationships with other n class="Species">people or organizations that can inappropriately influenpan>ce our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.