Ruminal and post-ruminal phytate degradation of diets containing rapeseed meal or soybean meal

ABSTRACT

This study aimed to investigate ruminal and post-ruminal degradation of phytic acid (InsP₆) in diets containing either rapeseed meal (RSM) or soybean meal (SBM). In Experiment 1, the effective degradability of crude protein (CPED) and InsP₆ (InsP₆ED) was evaluated by incubating RSM and SBM in situ in three rumen-fistulated lactating Jersey cows for 2, 4, 6, 8, 16, 24, 48 and 72 h, and calculating effective degradability at rumen passage rates of 2% and 5%/h. In Experiment 2, eight wethers were assigned for 8 weeks to two dietary treatments (Diet RSM and Diet SBM) containing 150 g of either meal and 100 g of maize silage per feeding time and had free access to hay and water. Titanium dioxide (TiO₂) was added to the diets for the last 5 days of the study. The wethers were then stunned, exsanguinated and digesta from the reticulo-rumen, omasum, abomasum, jejunum, colon, and rectum were sampled. In Experiment 1, the InsP₆ED of RSM (InsP₆ED₂: 83%; InsP₆ED₅: 64%) decreased almost identically to that of CPED with increasing passage rate (CPED₂: 78%; CPED₅: 63%) and was significantly lower than that of SBM (InsP₆ED₂: 93%; InsP₆ED₅: 85%). In Experiment 2, ruminal InsP₆ disappearance was significantly higher in wethers fed Diet SBM (89%) than in those fed Diet RSM (76%). Total post-ruminal InsP₆ degradation was 6% for Diet RSM and 4% for Diet SBM (p = 0.186). The total tract InsP₆ disappearance was higher in Diet SBM (93%) than in Diet RSM (82%). Considering higher InsP₆ contents in RSM, Diet RSM resulted in significantly higher amounts of ruminally (Diet RSM: 4.5 g/d; Diet SBM: 3.4 g/d) and total tract (Diet RSM: 4.9 g/d; Diet SBM: 3.5 g/d) degraded InsP₆. InsP₅ was quantified in most of the digesta samples after feeding Diet RSM but was not detectable in the majority of digesta samples for Diet SBM. Concentrations of myo-inositol (MI) tended to be higher (p = 0.060) in the blood plasma of wethers fed Diet RSM. The consistency between ruminal InsP₆ disappearance in wethers and in situ calculated InsP₆ED₂, along with the very low extent of post-ruminal InsP₆ degradation, suggests that at a low rumen passage rate, InsP₆-P from the feed becoming available to ruminants is almost entirely from InsP₆ degradation in the rumen.

KEYWORDS:

• InsP₆ degradation
• rapeseed meal
• soybean meal
• inositol phosphates
• myo-inositol
• ruminants

1. Introduction

Phytate [any salt of myo-inositol 1,2,3,4,5,6 hexakis (dihydrogen phosphate), InsP₆] represents the predominant phosphorus (P) source in plant seeds and their processing by-products. Phosphorus must be cleaved from InsP₆ before absorption. The cascade of P cleavage can proceed completely such that myo-inositol (MI), which can also be absorbed by animals, is released (Huber 2016). Such hydrolysis is catalysed by phytases and other phosphatases found in plants, microorganisms, and the mucosa of the intestine (Bitar and Reinhold 1972; Eeckhout and De Paepe 1994; Yanke et al. 1998).

Ruminants have been reported to be able to utilise almost all P bound to InsP₆ (Nelson et al. 1976; Morse et al. 1992) owing to the phytase activity of the rumen microbiota (Raun et al. 1956; Yanke et al. 1998). According to Nelson et al. (1976), degradation of InsP₆ is initiated in the rumen and proceeds to near completion before the digesta reaches the lower parts of the digestive tract. However, some recent studies have indicated that ruminal InsP₆ degradation is not complete, and the values vary between and within studies. In an in situ study by Haese et al. (2022), the ruminal escape of InsP₆ from rapeseed meal (RSM) was calculated to range from 26% to 52% at a rumen passage rate of 5%/h depending on the desolventising/toasting conditions that had been applied to the RSM. In a study by Park et al. (2002), more than 20% of dietary InsP₆ left the sheep rumen. With different levels of dietary InsP₆, Ray et al. (2013) observed 85–94% ruminal InsP₆ disappearance in first-lactation cows. Factors such as concentrate type (Haese et al. 2017a), feed processing (Haese et al. 2022), dietary InsP₆ concentration (Ray et al. 2013), and forage-to-concentrate ratio (Yanke et al. 1998) may affect the activity or accessibility of rumen microbial phytase and consequently the rate and extent of ruminal InsP₆ degradation.

Regarding the total digestive tract, faecal InsP₆ disappearance was reported to be 69–98% by Kincaid et al. (2005) and 85–94% by Haese et al. (2014). Nevertheless, in vivo studies describing ruminal and post-ruminal InsP₆ degradation, including the formation of lower inositol phosphate (InsP) isomers post-ruminally, are scarce. If InsP₆ from the feed cannot be completely degraded in the rumen, the relevance of post-ruminal InsP₆ degradation may be higher. The small and large intestines have been suggested to be relevant to post-ruminal InsP₆ degradation (Park et al. 2002; Ray et al. 2012, 2013; Jarrett et al. 2014).

The objective of this study was to investigate ruminal and post-ruminal InsP₆ degradation in wethers fed RSM- and soybean meal (SBM)-based diets, respectively. Previous in situ studies have shown a slower progression of InsP₆ disappearance in RSM compared to SBM (Haese et al. 2017a) and consequently a lower ruminal disappearance of InsP₆ for RSM than for SBM (Haese et al. 2020). Hence, we hypothesised lower ruminal and higher post-ruminal InsP₆ disappearance in wethers fed an RSM-containing diet than in those fed an SBM-containing diet. In addition, in situ ruminal InsP₆ disappearance was studied in rumen-fistulated cows in an attempt to link the data from the wether study with preliminary data from in situ studies conducted in cows.

2. Materials and methods

2.1. Experiment 1

An in situ study approved by the Animal Welfare Authority (Regierungspräsidium Stuttgart, Germany, approval code: V352/18 TE) was conducted according to German animal welfare regulations. Rapeseed meal and SBM were incubated in three rumen-fistulated lactating Jersey cows. The cows were fed a total mixed ration (TMR) consisting of 35% concentrate mixture (16% winter barley, 21% maize, 27% faba beans, 16% peas, and 20% rapeseed cake), 2% RSM, 23% maize silage, 14% grass silage, 17% hay, and 2.5% straw on a dry matter (DM) basis. The content of P, net energy for lactation, and crude protein (CP) in the TMR was 4.43 g/kg DM, 6.7 MJ, and 149 g/kg DM, respectively. The average daily DM intake and milk yield of cows were 20 kg and 23 kg, respectively. Feed and water were provided for ad libitum consumption.

Rapeseed meal and SBM containing 21.6 g/kg DM and 14.2 g/kg DM InsP₆, respectively (Table 1), were incubated based on the recommended protocol of the GfE [Gesellschaft für Ernährungsphysiologie] (2022) and all details as described by Seifried et al. (2017). Briefly, SBM and RSM were ground to pass through a 2-mm sieve. An amount of 8 g (± 0.015 g) was weighed and placed into polyester bags (10 × 20 cm, pore size of 50 μm, ANKOM Technology, USA) and subsequently incubated in the rumen for 2, 4, 6, 8, 16, 24, 48, and 72 h. To acquire a sufficient amount of incubation residue, three replications for 2, 4, 6 and 8 h, four replications for 16 h, and six replications for 24, 48, and 72 h were incubated. The bags were immersed in warm water (approximately 39°C) for 10 min before placement in the rumen. Three bags of each meal were washed in a washing machine (Miele & Cie. KG, Gütersloh, Germany) without ruminal incubation to obtain the value at 0 h.

Table 1. Analysed composition of single feeds and diets containing rapeseed meal (Diet RSM) or soybean meal (Diet SBM) [g/kg DM^#].

	Rapeseed meal	Soybean meal	Maize silage	Hay
Crude protein	371	526	76	54
Crude ash	88	84	30	59
NDFom^†	325	176	444	696
ADFom^§	264	73	246	462
P	11.6	7.1	2.0	2.0
InsP6	21.6	14.2	n.d^.◊	n.d.
InsP5^¶	3.9	2.6	n.d.	n.d.
InsP4^‡	0.10	n.d.	n.d.	n.d.
Myo-inositol	0.30	0.65	1.88	0.44
	Diet RSM		Diet SBM
InsP6	17.7		11.6
InsP5	3.2		2.1
Myo-inositol	0.57		0.86
P	3.3		2.0

Notes: ^#DM, dry matter; ^†NDFom, neutral detergent fibre expressed exclusive of residual ash; ^§ADFom, acid detergent fibre expressed exclusive of residual ash; ^¶sum of InsP₅ isomers; ^‡sum of InsP₄ isomers; ^◊n.d., not detectable.

Following withdrawal from the rumen, bags were immediately soaked in ice-cold water to suppress microbial fermentation. The bags were then rinsed with cold tap water to remove adherent particles and subsequently washed in a washing machine (Miele & Cie. KG, Gütersloh, Germany) for 20 min without detergent and spinning. All the bags were then dried for 24 h at 60°C. The residues from the dried bags were weighed and pooled per cow and incubation time. Pooled samples were pulverised using a mixer mill (Type MM400, Retsch GmbH, Haan, Germany) and stored at 4°C until further processing.

2.2. Experiment 2

2.2.1. Animals and diets

The experiment was approved by the Animal Welfare Officer of the University of Hohenheim (approval code: T/192/19 TE) and was conducted in accordance with the German Animal Welfare Legislation. Eight 10-year-old Merino wethers (body weight: 110 ± 8 kg) were randomly assigned to two dietary treatments and kept in a pen with straw bedding.

Two experimental diets using RSM and SBM from the same batch as in Experiment 1 were freshly prepared (Diet RSM: 100 g maize silage + 150 g RSM; Diet SBM: 100 g maize silage + 150 g SBM) and fed twice daily at 7:30 and 16:00 h right after mixing. The concentration of InsP₆ was calculated to be 17.7 g/kg DM in Diet RSM and 11.6 g/kg DM in Diet SBM based on the concentrations in the single feeds except for hay (Table 1). To reduce selection and ensure complete feed intake, 30 ml water was added during mixing. The wethers had free access to hay and water. The diets were provided for 8 weeks to ensure sufficient adaptation time. In the first 6 weeks of adaptation, the wethers were kept in two separate groups according to their treatment and fed on a group basis. From week 7 onwards, the wethers were individually fed their respective diets. At the end of the adaptation period, titanium dioxide (TiO₂) was added to the diets (1.3 g per feeding time per animal).

Rapeseed meal, SBM, and hay were collected, stored at 4°C. Maize silage was frozen and lyophilised. Maize silage and hay samples were pooled over time to obtain one sample for each feed. The hay sample was first ground through a 2-mm sieve to obtain smaller feed particles (Type SM1, Retsch GmbH, Haan, Germany). Subsequently, all feed samples were ground to pass through a 0.5-mm sieve (Ultra Centrifugal Mill ZM 200, Retsch GmbH, Haan, Germany), and a portion of each was further pulverised using a mixer mill (Type MM400, Retsch GmbH, Haan, Germany).

2.2.2. Sampling and sample preparation

Samples were obtained on four separate days. On each sampling day, two wethers (one from each treatment group) were fed their respective diet 2 h before they were sacrificed and had no access to hay in that time period to achieve a similar rumen fill. The animals were stunned with a captive bolt gun and exsanguinated by cutting the jugular vein.

Blood from the vena jugularis was collected immediately after exsanguination into Sarstedt® collectors [Glucose FH; Sarstedt AG & Co., Nümbrecht, Germany] containing sodium fluoride and centrifuged at 2,000 × g for 10 min to separate the plasma. The left flank and abdominal wall musculature of the wethers were incised using a commercial blade. Digestive tract sections were separated and clamped using forceps. The reticulo-rumen contents were collected as rumen pools and pressed through a coarse Hessian bag. Particulate matter retained in the bag was weighed and defined as large particulate matter (LPM). Five percent of the total weight of the LPM was collected for analysis. The filtrate was collected in a bucket and immediately homogenised using a magnetic stirrer. Approximately 800 g of the filtrate was transferred to centrifuge cups and centrifuged at 10,000 × g at 4°C for 30 min. The supernatant was collected and defined as the fluid phase (FP), whereas the remaining pellet was resuspended in double-distilled water and centrifuged again. The supernatant from the second centrifugation step was discarded and the residue was harvested as small particulate matter (SPM). Digesta from the omasum, abomasum, colon (from half of the spiral colon to 2 m before the entrance of the rectum), and rectum were quantitatively collected. Contents from 6 m posterior to the transition of the small intestine and 2 m prior to the transition of the large intestine were collected as jejunum samples, which were then rinsed with cold double-distilled water. Digesta samples were immediately frozen at −20°C after sampling or centrifugation. The frozen digesta samples were then lyophilised. Eight to 14 g of each dried digesta sample were pulverised using a mixer mill (Type MM400; Retsch GmbH, Haan, Germany) prior to chemical analysis.

2.3. Chemical analyses

Analysis of crude nutrients followed the official methods in Germany (Verband Deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten (VDLUFA) 2012). The feed samples were analysed for DM (method 3.1), crude ash (method 8.1), CP (method 4.1.1), crude fibre (method 6.1.1), neutral detergent fibre without residual ash (NDFom; method 6.5.1), and acid detergent fibre without residual ash (ADFom; method 6.5.2).

In the bag residues from Experiment 1, DM and CP were determined as described above. To determine the concentrations of InsP₆ in the concentrates and bag residues, samples were extracted using the method of Zeller et al. (2015) with modifications described by Sommerfeld et al. (2018). In brief, samples were extracted with a solution of 0.2 M EDTA and 0.1 M sodium fluoride at 4°C (pH = 8) for 30 min. After centrifugation following two extractions at 12,000 × g for 15 min, 1 ml sample was collected and centrifuged at 14,000 × g for 15 min and filtered prior to measurement by high-performance ion chromatography (ICS-3000 system, Dionex, Idstein, Germany).

In Experiment 2, concentrations of TiO₂ were determined in the pulverised digesta samples using a modified sulphuric and nitric acid wet digestion method of Boguhn et al. (2009), followed by measurement on an inductively coupled plasma optical emission spectrometer as described in detail by Zeller et al. (2015). To determine the concentrations of InsP_3-6 in the feed and digesta, samples were extracted using the method described in Experiment 1. To determine InsP_1-2 in the feed and digesta samples, a buffer solution containing 50 mM Tris, 50 mM glycine, and 0.2 M sodium fluoride at pH 9 was used for the extraction; otherwise, the same procedure for determining InsP_3-6 was performed. The concentrations of MI in the feed, blood plasma, and digesta samples were measured based on the method described by Sommerfeld et al. (2018) using gas chromatography/mass spectrometry after derivatisation of the samples. The analysed values of the omasum and abomasum were combined as omasum + abomasum based on their DM fractions for further calculations, as the complete separation of the omasal and abomasal digesta during sampling was not possible due to the exchange of fluids between these two sections.

2.4. Calculations and statistics

In Experiment 1, the equations suggested by Ørskov and McDonald (1979) [Equation (1)(1) $Degradation\left[\mathrm{\%}\right]=a+b\cdot \left(1-e^{-ct}\right)$(1) ] and McDonald (1981) [Equation (2)(2) $Degradation\left[\mathrm{\%}\right]=a+b\cdot \left(1-e^{-c\left(t-L\right)}\right)fort>L$(2) ] were used to describe ruminal degradation kinetics, and calculations were performed using GraphPad Prism software (version 5.0, GraphPad Software Inc., CA, USA):

(1) $Degradation\left[\mathrm{\%}\right]=a+b\cdot \left(1-e^{-ct}\right)$(1)

(2) $Degradation\left[\mathrm{\%}\right]=a+b\cdot \left(1-e^{-c\left(t-L\right)}\right)fort>L$(2)

where a [%] is the rapidly degradable fraction acquired from the 0 h incubation time, b [%] is the potentially degradable fraction, c [%/h] is the degradation rate, and L [h] represents the lag time. The best-fitting model for each feed was selected using the Akaike Information Criterion (AIC). The effective degradability (ED) of CP and InsP₆ was calculated using either the equation of McDonald (1981) [Equation (3)(3) $ED\left[\mathrm{\%}\right]=a+\left[\left(b\cdot c\right)/\left(c+k\right)\right]$(3) ] or the modified equation of Wulf and Südekum (2005) [Equation (4)(4) $ED\left[\mathrm{\%}\right]=a+\left[\left(b\cdot c\right)/\left(c+k\right)\right]\cdot e^{-kL}$(4) ], assuming rumen passage rate (k) = 0.02 and 0.05 per h.

(3) $ED\left[\mathrm{\%}\right]=a+\left[\left(b\cdot c\right)/\left(c+k\right)\right]$(3)

(4) $ED\left[\mathrm{\%}\right]=a+\left[\left(b\cdot c\right)/\left(c+k\right)\right]\cdot e^{-kL}$(4)

In Experiment 2, InsP₆ disappearance was calculated based on the concentrations of InsP₆ and TiO₂ in the diet and digesta, using the following equation:

$Ins{P}_{6}disappearance\left[\mathrm{\%}\right]=100-100\cdot \left(\frac{\mathrm{T}\mathrm{i}\mathrm{O}{\hspace{0.17em}}_2^{\hspace{0.17em}}indiet\left[\frac{g}{kg}DM\right]}{\mathrm{T}\mathrm{i}{\mathrm{O}}_{2}indigesta\left[\frac{g}{kg}DM\right]}\right)\cdot \left(\frac{Ins{P}_{6}indigesta\left[\frac{g}{kg}DM\right]}{Ins{P}_{6}indiet\left[\frac{g}{kg}DM\right]}\right)$

where the TiO₂ concentration in the diet was calculated as the amount of TiO₂ provided [g/d] divided by the DM intake of the diet [kg/d].

Statistical analysis of the data was performed using the software program SAS (version 9.4, SAS Institute Inc., Cary, USA) using the SAS statement PROC MIXED with the following model:

$y_i=\mu +{\alpha }_i+e_i$

where y_i is the target trait, α_i is the fixed treatment effect, µ is the overall mean, and e_i is the residual error of y_i. Statistical significance was set at p ≤ 0.05. Data are presented as the mean and standard error of the mean.

3. Results

3.1. Experiment 1

Fraction a of InsP₆ averaged 1.9% for RSM and 32% for SBM. Fraction b was 98% and 68% for RSM and SBM, respectively (Table 2). A lag time for InsP₆ degradation was observed at 3.6 h for RSM and 1.0 h for SBM. The degradation rate c was lower for RSM (16%/h) than for SBM (23%/h). Fractions a and b of CP were 13% and 82% for RSM and 10% and 90% for SBM, respectively. No lag phase in CP degradation was observed for either meal. The degradation rate c of CP was 7.9%/h for RSM and 8.4%/h for SBM, with no significant differences. CPED was higher for SBM than for RSM (CPED₂: 83 vs. 78%; CPED₅: 66 vs. 63%). The RSM values of InsP₆ED resembled those of CPED (InsP₆ED₂: 83%; InsP₆ED₅: 64%) and were significantly lower than those of SBM (InsP₆ED₂: 93%; InsP₆ED₅: 85%).

Table 2. Estimated ruminal degradation parameters and effective degradability of phytate (InsP₆) and crude protein (CP) from in situ incubation of rapeseed meal (RSM) and soybean meal (SBM).

	RSM	SBM	SEM^#	p-Value
CP degradation
a^† [%]	13	10	-	-
b^§ [%]	82	90	-	-
c^¶ [%/h]	7.9	8.4	0.40	0.349
CPED2^‡ [%]	78	83	0.8	0.006
CPED5 [%]	63	66	0.9	0.038
InsP6 degradation
Lag^◊ [h]	3.6	1.0	0.30	0.005
a [%]	1.9	32	-	-
b [%]	98	68	-	-
c [%/h]	16	23	1.4	0.007
InsP6ED2^♦ [%]	83	93	0.5	0.003
InsP6ED5 [%]	64	85	1.1	0.005

Notes: Data are presented as treatment means; n = 3 animals; ^#SEM, standard error of the mean; ^†a, rapidly degradable fraction; ^§b, potentially degradable fraction; ^¶c, degradation rate; ^‡CPED, effective degradability of CP at rumen passage rates of 2 (CPED₂) and 5 (CPED₅) %/h; ^◊Lag, lag time; ^♦InsP₆ED, effective degradability of InsP₆ at rumen passage rates of 2 (InsP₆ED₂) and 5 (InsP₆ED₅) %/h.

3.2. Experiment 2

3.2.1. Concentrations of inositol phosphate isomers and myo-inositol

In the FP of the rumen, InsP₆ concentration was lower than the limit of quantification (<0.13 g/kg DM) and InsP₅ was not detectable (Table 3). Feeding RSM resulted in significantly higher InsP₆ concentrations in the SPM (1.53 vs. 0.59 g/kg DM) and LPM (2.21 vs. 0.83 g/kg DM) of the rumen pool, omasum + abomasum (1.52 vs. 0.47 g/kg DM), colon (1.54 vs. 0.43 g/kg DM), and rectum (1.57 vs. 0.43 g/kg DM). InsP₅ was quantified in the SPM and LPM of the rumen pool, omasum + abomasum, jejunum, colon, and rectum of wethers fed Diet RSM but was not detectable in the majority of digesta samples from those fed Diet SBM. InsP_1-4 were not detected in any digesta samples.

Table 3. Concentrations of inositol phosphate (InsP) isomers in digestive tract contents of wethers fed diets containing rapeseed meal (Diet RSM) or soybean meal (Diet SBM) [g/kg DM^#].

	Diet RSM	Diet SBM	SEM^†	p-Value
InsP6
Rumen pool
Fluid phase	<LOQ^‡	<LOQ	-	-
Small particulate phase	1.53	0.59	0.226	0.026
Large particulate phase	2.21	0.83	0.292	0.015
Omasum + abomasum^§	1.52	0.47	0.206	0.011
Jejunum	0.67	0.23	0.177	0.131
Colon	1.54	0.43	0.236	0.016
Rectum	1.57	0.43	0.256	0.020
InsP5^¶
Rumen pool
Fluid phase	n.d.^◊	n.d.	-	-
Small particulate phase	0.30	n.d.	-	-
Large particulate phase	0.36	n.d.	-	-
Omasum + abomasum	0.22	n.d.	-	-
Jejunum	0.05	n.d.	-	-
Colon	0.13	n.d.	-	-
Rectum	0.07	n.d.	-	-

Notes: Data are presented as treatment means, n = 4 animals; ^#DM, dry matter; ^†SEM, standard error of the mean; ^§Calculated with respective fractions [%] of omasal and abomasal digesta on a dry matter basis; ^¶The sum of InsP₅ isomers; ^‡<LOQ, not quantifiable; ^◊n.d., not detectable.

Myo-inositol was not quantifiable (≤0.05 g/kg DM) in the majority of the samples from the rumen pool, colon, and rectum. Only traces of MI were determined, without significant differences in the omasum + abomasum (Table 4). The MI concentration in the jejunal content was not significantly different but higher for Diet RSM than Diet SBM by trend in the blood plasma (4.6 vs. 3.8 µg/ml).

Table 4. Concentrations of myo-inositol in digestive tract contents [g/kg DM^#] and blood plasma [µg/ml] of wethers fed diets containing rapeseed meal (Diet RSM) or soybean meal (Diet SBM).

	Diet RSM	Diet SBM	SEM^†	p-Value
Omasum + abomasum^§	0.05	0.02	0.018	0.386
Jejunum	0.99	0.72	0.120	0.162
Blood plasma	4.6	3.8	0.24	0.060

Notes: Data are presented as treatment means, n = 4 animals; ^#DM, dry matter; ^†SEM, standard error of the mean; ^§Calculated with respective fractions [%] of omasal and abomasal digesta on a dry matter basis.

3.2.2. InsP₆ disappearance and degraded amount of InsP₆

Ruminal InsP₆ disappearance measured at the omasum + abomasum was 89% for Diet SBM, which was significantly higher than that in Diet RSM (76%; Table 5). Up to the jejunum, 88% and 94% of ingested InsP₆ disappeared in wethers fed Diet RSM and Diet SBM, respectively. InsP₆ disappearance up to the colon and rectum was higher in the Diet SBM group than in the Diet RSM group (p = 0.046 and 0.057, respectively). Total post-ruminal InsP₆ disappearance, calculated as the difference between ruminal and total tract InsP₆ disappearance, did not differ between Diet RSM and Diet SBM (6 vs. 4%). A significantly higher amount of InsP₆ was degraded ruminally and in total tract for Diet RSM (ruminal: 4.5 g/d; total tract: 4.9 g/d) in comparison with Diet SBM (ruminal: 3.4 g/d; total tract: 3.5 g/d; Figure 1).

Figure 1. Amount of ruminal (p = 0.007) and total tract (p = 0.002) degraded InsP₆ from feed in wethers fed diets containing rapeseed meal (Diet RSM) or soybean meal (Diet SBM) [g/d]; data are presented as treatment means (n = 4 animals) with error bars (standard error of the mean) and calculated with differences between intake and omasum + abomasum (ruminal), and intake and rectum (total tract), respectively.

Table 5. Ruminal and post-ruminal InsP₆ disappearance in wethers fed diets containing rapeseed meal (Diet RSM) or soybean meal (Diet SBM) [%].

	Diet RSM	Diet SBM	SEM^#	p-Value
Ruminal^†	76	89	3.2	0.033
Post-ruminal
Jejunum	88	94	2.9	0.297
Colon	83	92	2.8	0.046
Rectum	82	93	3.0	0.057
Total post-ruminal^§	6	4	1.2	0.186

Notes: Data are presented as treatment means, n = 4 animals; ^#SEM, standard error of the mean; ^†Calculated with respective InsP₆ disappearance in fractions of omasum and abomasum on a dry matter basis; ^§Calculated as the difference between total tract (rectum) and ruminal disappearance.

4. Discussion

4.1. Degradation characteristics of experimental feed determined in situ

The significantly lower InsP₆ED for RSM compared to SBM is consistent with the results from previous in situ studies (Park et al. 1999; Konishi et al. 1999; Haese et al. 2020). As suggested by the previous authors, this observation may be attributed to the differences in the location and binding form of InsP₆ in rapeseed and soybean, as these can influence the solubility of InsP₆ as well as the accessibility of microbial phytase to its substrate. Thus, the aggregation of InsP₆ with proteins inside protein storage vacuoles (Gillespie et al. 2005) and a higher amount of less soluble Mg- and Ca-phytate (Gillberg and Törnell 1976) compared to K-phytate may slow the degradation process in RSM, resulting in a greater rumen outflow of InsP₆ in comparison with SBM. An almost identical InsP₆ED and CPED for RSM against a difference of approximately 20% points between InsP₆ED₅ and CPED₅ for SBM in the current study also confirms the close relationship between InsP₆ and CP degradation in RSM, as previously reported (Konishi et al. 1999; Haese et al. 2017a, 2022).

4.2. Ruminal InsP₆ disappearance

Voluntary hay intake of wethers may have been different and straw intake due to housing on straw bedding might have occurred. However, the hay did not contain InsP₆ (Table 1) and a previous study of our department found an InsP₆ concentration below 0.1 g/kg DM in straw (unpublished data). Assuming that the straw used in the present study contained InsP₆ at a similarly low level as in the previous study, the calculations of InsP₆ disappearance can be regarded as not affected by variable straw and hay intake of the wethers. Consistent with the relatively high InsP₆ content of Diet RSM compared to Diet SBM, InsP₆ concentrations determined in the particulate matter of the rumen pool and omasum + abomasum were also higher. However, the amount of ruminally degraded InsP₆ was greater for Diet RSM (4.5 g/d) than for Diet SBM (3.4 g/d) despite the lower ruminal disappearance for Diet RSM (76%) compared to Diet SBM (89%). This is consistent with the study by Ray et al. (2013), where a high content of InsP₆ in the feed stimulated microbial phytase activity in the rumen, leading to increased ruminal InsP₆ degradation. In the present study, the wethers were assumed to have a low rumen passage rate, as they were fed to meet maintenance energy requirements, and the measured ruminal InsP₆ disappearance was almost identical to the in situ calculated InsP₆ED₂ in Experiment 1. The extent of ruminal InsP₆ disappearance seems to be very high if the retention time of the digesta in the rumen is long. Accordingly, the relatively low ruminal InsP₆ disappearance (55–66%) reported by Kebreab et al. (2005) may be ascribed to a high passage rate in lactating cows fed high levels of whole crop wheat, as insufficient time of digesta retention in the rumen renders microbial hydrolysis of InsP₆ less complete. However, in the present study, despite a long ruminal retention time, InsP₆ disappearance was not complete, and a greater portion of InsP₆ from RSM than SBM remained undegraded by rumen microorganisms, as also characterised by Experiment 1. This is inconsistent with the finding that nearly 100% of InsP₆ is ruminally degradable (Nelson et al. 1976). In that study, the calves were fed a diet based on corn and SBM. A much faster disappearance (Haese et al. 2017a) and a higher InsP₆ED for corn than for SBM have been reported (Haese et al. 2020), which may explain the differences between the current study and Nelson et al. (1976).

Notably, no InsP₆ was detected in the FP of the rumen pool in either treatment. In an in vitro study, the InsP₆ concentration in the fermenter fluid markedly decreased between 0 and 3 h of incubation (Haese et al. 2017b), indicating that the fraction of InsP₆ dissolved in the rumen fluid was rapidly degraded. This supports the assumption made in the in situ study that the soluble fraction a of InsP₆ determined by the washing procedure, is rapidly degraded. The fact that only traces of InsP₅ and no less phosphorylated InsP isomers were detected is consistent with the assertions of in situ (Haese et al. 2020) and in vitro studies (Brask-Pedersen et al. 2011) that the initial degradation step of InsP₆ determines the extent of degradation. Once the first phosphate group is cleaved from the molecule, degradation proceeds quickly and completely in ruminants, which deviates from non-ruminants, where the addition of exogenous phytase to the feed increases the concentrations of InsP₄ and less phosphorylated InsP isomers (Rodehutscord et al. 2022).

4.3. Post-ruminal InsP₆ disappearance

Total post-ruminal InsP₆ disappearance was 6% (Diet RSM) and 4% (Diet SBM) when expressed as a percentage of feed InsP₆ (equivalent to 25% for Diet RSM and 35% for Diet SBM as percentage of ruminal InsP₆ outflow). These values are relatively low compared to the values reported by Park et al. (2002) (17%) and Kebreab et al. (2005) (11–27%). If InsP₆ is not degraded, even after a long retention time of digesta in the rumen, it may not undergo substantial post-ruminal degradation. The greater extent of post-ruminal InsP₆ disappearance observed in the aforementioned studies may be a result of the degradation of potential rumen degradable InsP₆ which was not entirely degraded before leaving the rumen due to an insufficient retention time of digesta. Ruminally undegraded InsP₆ was presumably mainly degraded in the upper part of the small intestine in the current study, as InsP₆ disappearance and increased MI concentrations were observed in the jejunum compared to the omasum + abomasum in both treatments. This is similar to the result of the study by Ray et al. (2013), where approximately 78% of post-ruminal InsP₆ degradation occurred between the omasum and ileum. In contrast, Park et al. (2002) reported that almost no InsP₆ was degraded between the abomasum and the jejunum. In that study, 88% of the post-ruminal InsP₆ degradation occurred posterior to the jejunum. Although intestinal CP degradation was not measured, it is highly possible that some of the ruminally undegraded InsP₆ was released from protein-phytate complexes through the enzymatic digestion of rumen undegradable protein (RUP) in the abomasum and upper part of the small intestine, which was subsequently subjected to degradation by phosphatases and phytases of microorganisms and the intestinal mucosa (Bitar and Reinhold 1972). A relatively higher extent of InsP₆ disappearance (0.7 vs. 0.2 g/d) observed between the omasum + abomasum and jejunum upon feeding Diet RSM may be attributed to a higher amount of InsP₆ entering from the rumen (1.4 vs. 0.4 g/d) or that released from RUP degradation through the intense association between InsP₆ and CP.

In total, 18% and 7% of dietary InsP₆ reached the rectum of wethers fed Diet RSM and Diet SBM, respectively. Apparently, rumen undegradable InsP₆ from the used meals is also resistant to microbial degradation in the large intestine. Similarly, Brask-Pedersen et al. (2013) found no further reduction of InsP₆-P posterior to the ileum (2.6 g/d in the ileum and 2.7 g/d in the faeces) in lactating cows fed TMR comprising 20% DM of rapeseed cake. Although the large intestine is the second most common site of microbial fermentation in ruminants, and certain phytase-producing microbes in the large intestine, similar to those in the rumen, are considered to contribute to large intestinal InsP₆ degradation (Ray et al. 2012, 2013; Jarrett et al. 2014), the large intestine is characterised by a lower fill capacity, a much shorter retention time, and a more homogenous digesta composition compared to the rumen (Mambrini and Peyraud 1997). A less efficient microbial phytase has been reported in the large intestine than in the rumen (Ray et al. 2012). In that study, less than 20% of the InsP₆ infused into the ileum was degraded in the large intestine, and this did not differ between the infusion rates of InsP₆. Wang et al. (2020) found that the number of bacterial genera significantly correlated with P digestibility was lower in the jejunum, followed by the colon, than in the forestomach of goats. This is reinforced by the results of Park et al. (2002), in which 76% of InsP₆ disappeared in the rumen, followed by 13% InsP₆ disappearance between the jejunum and colon. Combined with the results from this study and other studies, it can be concluded that a certain portion of potentially rumen degradable InsP₆ is degraded in the large intestine, whereas rumen undegradable InsP₆ remains mostly undegraded until it is excreted. Despite the great capability of ruminants to digest high amounts of InsP₆ from feed, as the total tract degraded amount of InsP₆ was 4.9 g/d for Diet RSM and 3.5 g/d for Diet SBM, processing of oilseeds leads to the formation of RUP, which may render InsP₆-P unavailable for ruminants.

4.4. Myo-inositol

Trace amounts of MI were detected in the omasum + abomasum, and no MI was detected in the rumen pool, colon, or rectum in either treatment group. In pigs, the digesta MI concentration was markedly reduced in the hindgut when they were fed corn-SBM- and corn-SBM-rapeseed cake-based diets with or without phytase supplementation (Rosenfelder-Kuon et al. 2020). The authors suggested the possibility of absorption by the large intestine or microbial degradation. It is not known whether MI can also be absorbed by the epithelial cells of the forestomach. However, it is very likely that the microbiota in the rumen metabolises the MI released from InsP₆. For archaea, bacteria, and eukaryotes, such as protozoa and fungi which are present in the forestomach (Castillo-González et al. 2014; Liu et al. 2021), MI serves as an essential component of the membrane and as a precursor for other molecules (Michell 2008; Reynolds 2009). In the current study, MI was quantified in the jejunum, where fewer microbes are active (Mao et al. 2015). In wethers fed Diet RSM, there was a higher proportion of ruminally undegraded InsP isomers, which provided more substrate for phosphatases and phytases in the upper small intestine, and thus might have led to the numerically higher MI concentration compared to Diet SBM. This is supported by the fact that the lowest InsP₆ concentration was found in the digesta of the jejunum.

The MI concentration in the blood plasma tended to be higher in wethers fed Diet RSM than in those fed Diet SBM (p = 0.060). Considering the higher amount of post-ruminally degraded InsP₆ for Diet RSM, it is likely that a certain proportion of the released MI from InsP₆ degradation in the lower digestive tract of wethers was absorbed and reached the bloodstream, contributing to a higher MI concentration in the blood plasma compared with Diet SBM. An association between ileal and blood MI concentrations was found in pigs and chicken (Sommerfeld et al. 2018; Rosenfelder-Kuon et al. 2020; Klein et al. 2021). To the best of our knowledge, studies on MI metabolism in ruminants have not yet been published. The degree to which blood MI is indicative of dietary InsP₆ degradation in ruminants requires further investigation. Notably, microorganisms in the digestive tract can both take up and synthesise MI (Reynolds 2009), which may interfere with the blood MI concentration.

5. Conclusion

The degradation of InsP₆ in wethers differed markedly when either RSM or SBM was included in the feed. The high content of InsP₆ in RSM resulted in a higher amount of InsP₆ degradation for Diet RSM compared to Diet SBM, whereas more easily degradable InsP₆ in SBM rendered the extent of ruminal and total tract InsP₆ disappearance for Diet SBM higher than that for Diet RSM. Compared with ruminal degradation, post-ruminal InsP₆ degradation was negligibly low. Along with the observed consistency between ruminal InsP₆ disappearance in the wethers and in situ calculated InsP₆ED₂, this suggests that InsP₆-P from feed available to animals is almost entirely derived from InsP₆ degradation in the rumen at a low rumen passage rate. Further research regarding phytase-producing microbiota may be helpful in understanding the effect of adaptation to InsP₆ degradation in different diets.

Acknowledgments

This study was supported by the Hohenheim University Foundation following a donation from alumni Dr Gerster. We would like to thank Heike Trapp and Lisa Uhland for their help in carrying out the experiments. We also appreciate the chemical analyses conducted by the laboratory team at the Animal Nutrition Department.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This project was funded by the Hohenheim University Foundation following a donation from alumni Dr Gerster, which is gratefully acknowledged.