Citation of this paper

Impact of level of rumen undegradable protein on in-vitro methane production and in-sacco degradability of concentrate mixtures

J S Lamba, M Wadhwa and M P S Bakshi

Department of Animal Nutrition, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, India
bakshimps2@gmail.com

Abstract

This study was taken up to assess the impact of low, medium and high levels of rumen undegradable protein (RUDP) on the in-vitro methane production and in-sacco degradability of iso-nitrogenous concentrate mixtures containing cotton seed cake (CSC) or spent brewer’s grains (SBG). The results revealed that irrespective of source of RUDP (CSC/SBG) the digestibility of NDF increased (p<0.01), while net gas production (NGP) and ME availability decreased linearly, with the increase in level of RUDP. The true OM digestibility of concentrate mixtures was not affected by the level of RUDP. The methane production expressed either as percent of NGP, ml/100mg of substrate or in mM decreased significantly with the increase in level of RUDP in concentrate mixtures. The total volatile fatty acids (VFAs), acetate and butyrate production decreased and that of propionate increased with the increase the level of RUDP in the concentrate mixture. Irrespective of level of RUDP, the NGP was significantly higher in SBG based concentrate mixtures in comparison to CSC based concentrate mixtures. The higher NDFD and NGP resulted in higher (p<0.01) availability of ME from SBG based concentrate mixture in comparison to CSC based concentrate mixture. The methane production expressed as above units was significantly lower in CSC based concentrate mixtures as compared to SBG based concentrate mixtures. The total and individual VFAs production was significantly higher from SBG based concentrate mixtures as compared with that of CSC based concentrate mixtures.

The digestion kinetic parameters for DM and CP of concentrate mixture irrespective of source of RUDP (CSC/SBG), the rapidly soluble fraction decreased, while insoluble but potential degradable fraction increased (p<0.01) with increase in the level of RUDP. The rate of degradation of potential degradable fraction of DM and for CP decreased with the increase in level of RUDP, reverse trend was observed for rumen undegradable fraction of DM and CP. The effective degradability (ED), true digestibility, potential and apparent extent of digestion (PED and (AED) of DM and CP declined (p<0.01) with the increase in RUDP level. The RUDP as per cent of CP increased (p<0.05) with the increase in level of RUDP in concentrate mixtures. Irrespective of level of RUDP, the concentrate mixture containing CSC had significantly higher rapidly soluble fraction and lower potentially degradable fraction than the concentrate mixture containing SBG. The ED, true digestibility, PED and AED of DM and CP were higher (p<0.01) in CSC than that in SBG based concentrate mixture. The results conclusively revealed that the in-vitro methane production was mitigated significantly with the increase in the level of RUDP of concentrate mixtures. Further cotton seed cake based concentrate mixture was more effective in mitigating methane production as compared to that containing spent brewer’s grains.

Key words: concentrate mixtures, cotton seed cake, in-vitro, in-sacco, methane, nutrient degradability, spent brewer’s grains, rumen un-degradable protein

Introduction

Though ruminants provide milk and meat for human consumption, but simultaneously emit greenhouse gases (GHGs), which are estimated at 7.1 Gt CO₂-equivalents per annum accounting for 14.5% of all anthropogenic emissions (Gerber et al 2013). Emissions of methane (CH₄) and nitrous oxide (N₂O) increased globally by nearly 17% each from 1990 to 2005 (Smith et al 2007). Between 1990 and 2008, there were continuous increases in enteric methane emission (111 to 129 kg/cow/year) in average Dutch dairy cow (Bannink 2011). Such projections are region specific e.g. enteric CH₄ from cattle has shown a downward trend from 1990 to 2014 in the EU-28 countries (EEA 2016). Efforts are afoot throughout the world to mitigate the enteric methane production, so as to check global warming.

The amount of methane produced depends largely on the amount of food consumed (Reynolds et al 2009), and the characteristics and composition of the feed. The enteric methane production depends on dietary factors like soluble sugars, dietary lipids, level of feeding, roughage to concentrate ratio, rate of passage of digesta, efficiency of feed conversion, processing and supplementation (Benchaar et al 2001, Bakshi and Wadhwa 2009). It is positively correlated with forage proportion and cell wall constituents of diet (Mills et al 2001, Ellis et al 2007, Kebreab et al 2010). To a lesser extent lipids also act as alternative hydrogen sinks and compete with methanogens for hydrogen in the rumen (Kebreab et al 2006). Similarly, ionophores such as monensin also reduce methane emissions, although the effect is normally short lived (about 4 -6 weeks) before the microbial community adapts to the treatment (Odongo et al 2007).

Kidane et al (2018) reported that gradual increase in dietary CP level (130, 145, 160, and 175 g/kg DM) did not affect DM intake, milk and milk component yield in Norwegian red dairy cows. Further the authors did not observe any reduction in daily enteric CH₄ emission by increasing dietary CP. Similarly, Hynes et al (2016) observed that by reducing concentrate protein content (18.1 to 14.1%) did not affect methane production, energy utilization or partitioning in lactating dairy cows. But, a negative relation between dietary protein and expected CH₄ emission was observed from the meta-analysis of calorimetric data on sheep (Pelchen and Peters 1998). Yan et al (2006) also observed similar relation. However, little information is available on effect of rumen undegradable protein (RUDP) in concentrate mixture/complete feed on enteric methane production. This study was therefore, taken up to assess the impact of different levels of RUDP on the in-vitro methane production and in-sacco degradability of concentrate mixtures.

Materials and methods

Formulation of concentrate mixtures

Six iso-nitrogenous concentrate mixtures were formulated by using either cotton seed cake (CSC) or spent brewer’s grains (SBG) supplemented with other protein and energy sources like tomato pomace and corn gluten meal etc. Both the CSC and SBG based concentrate mixtures had low, medium and high RUDP levels (Table 1).

In-vitro studies

The nutritional value of above concentrate mixtures with low, medium and high RUDP levels was evaluated by in-vitro gas production technique (Menke et al 1979, Menke and Steingas 1988).

Rumen contents were collected from three buffalo calves fitted with permanent rumen fistulae, which were maintained on 10 kg green fodder and 4 kg wheat straw supplemented with 2 kg concentrate mixture (wheat 25, mustard cake 10, de-oiled mustard cake 20, rice bran 15, de-oiled rice bran 15, wheat bran 12, mineral mixture 2, common salt 1 part each). The rumen contents collected before feeding were blended for 2-3 min in a blender and strained through four-layers of muslin cloth. The solution, containing 960 ml distilled water, 0.16 ml micro-mineral solution, 660 ml bicarbonate buffer, 330 ml macro-mineral solution and 1.6 ml resazurine (0.1%) were mixed in a Woulff flask (3 Litres capacity) with magnetic stirrer in a water bath at 39ºC. The mixture was continuously flushed with CO₂. Then strained rumen contents (SRC) was added to the buffer media in the ratio of 1:2. Three sets of samples were incubated, each in triplicate.

Table 1. Composition of concentrate mixtures containing different levels of rumen undegradable protein
Parameters	CSC-CM			SBG-CM
Parameters	L-RUDP	M-RUDP	H-RUDP	L-RUDP	M-RUDP	H-RUDP

*Ingredient composition, %*
Wheat	28	13	-	26.5	13	-
Maize	-	12	30	-	16	30
Tomato pomace	-	3	3	-	-	-
Mustard cake	29	17	-	28.5	17	-
Cotton seed cake	3	13	30	-	-	-
Spent brewers grains	-	-	-	3	13	30
Wheat bran	27	13	-	29	13	-
Deoiled rice bran	10	22	23.5	10	19	24
Corn gluten meal	-	4	10.5	-	5	11
Bypass fat	-	-	-	-	1	2
Mineral mixture	2	2	2	2	2	2
Salt	1	1	1	1	1	1
*Chemical composition, % DM basis*
Total ash	7.10	6.90	6.45	7.05	6.90	6.80
OM	92.90	93.10	93.55	92.95	93.10	93.20
CP	20.52	20.66	20.76	20.46	20.64	20.73
EE	3.70	3.70	3.75	3.65	3.70	3.70
Cellulose	8.80	14.10	15.70	5.20	16.60	18.00
NDF	27.20	36.20	47.20	48.00	53.60	51.20
ADF	11.60	17.40	21.30	12.10	13.40	13.30
Hemicellulose	15.60	18.80	25.90	35.90	40.20	37.90
L, Low; M, Medium; H, High; RUDP, Rumen undegradable protein; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture

About 375 ± 5 mg ground concentrate mixture samples (DM basis) were incubated at 39^oC for 24h in triplicate in 100 ml calibrated glass syringes (Haberle Labortechnik, Germany) with buffered rumen fluid for assessing the net gas production, digestibility of nutrients, VFA production and ME availability. Blank and sample of standard hay were run in triplicate with each set. If the volume of gas in the syringe exceeded 70 ml after 8 h the volume was recorded and the gas was expelled. After 24 h, the volume of gas produced in each syringe was recorded and the contents of syringes were transferred to spout-less beaker, boiled with neutral detergent solution for assessing the true OM and NDF digestibility.

Methane estimation

For CH₄ estimation, 200 mg of concentrate mixture was incubated for 24 h with buffered rumen liquor in triplicate. After the stipulated period, total gas production was measured. For CH₄ estimation, representative gas was sampled from the headspace of syringe in an airtight syringe and injected into Netchrom 9100 gas chromatograph equipped with flame ionization detector (FID) and stainless steel column packed with Porapak-Q. The gas flow rates for N₂, H₂ and air were 15, 30 and 300 ml min-1, respectively. Temperature of injector oven, column oven and detector were 70, 50 and 100ºC, respectively. A 50/50 mixture of CH₄ and CO₂ (Spancan; Spantech Products Ltd., England) was used as a standard.

Estimation of volatile fatty acids

After 24 h of incubation, a 5 ml aliquot of fluid from each syringe was mixed with 1 ml of 25% meta-phosphoric and kept for 1 h at ambient temperature. Thereafter, it was centrifuged at 5500 rpm for 10 min and clear supernatant was collected and stored at -20ºC until analyzed. The volatile fatty acids were estimated using Netchrom 9100 gas chromatograph equipped with glass column (packed with chromosorb 101) and flame ionization detector (Cottyn and Boucque 1968). Temperature of injection port, column and detector was set at 250, 175 and 270ºC, respectively. The flow rate of carrier gas (N) through the column was 15 ml min-1; and the flow rate of H₂ and air through FID was 30 and 300 ml min-1, respectively. Sample (2 μl) was injected through the injection port using a Hamilton syringe (10 μl). Individual VFA’s of the samples were identified on the basis of their retention time and their concentration (mmol) and calculated by comparing the retention time as well as the peak area of standards after deducting the corresponding blank values.

In-sacco degradability

The in-sacco degradability and kinetic parameters were determined (Mehraz and Ørskov 1977) by using the above mentioned rumen fistulated buffalo calves. Five grams of finely ground (1mm) concentrate mixture sample was placed in the nylon bag (8x17cm) stitched with monofilament polyester thread with pore size of 50±10µ. The bags were incubated in the rumen for 3, 6, 9, 12, 24, 36, 48 and 60h in triplicate. The bags were removed after the stipulated period, washed until rinsing water became colorless; bags were dried in a forced air oven at 60°C for 48h. The 'zero' h samples were not incubated in the rumen but the bags containing samples as such were washed in the same manner as incubated bags. The disappearance of DM was assessed as the loss in weight of the bag content. The residue was analyzed for DM and CP content. The different physical constants characterizing extent and rate of ruminal degradation, i.e., 'a' rapidly soluble fraction, 'b' insoluble but potentially degradable fraction and 'c' degradation rate constant of 'b' were worked out for DM and NDF. The effective degradability (ED) was calculated by using the equation of McDonald (1981).

Chemical and statistical analysis

The finely ground samples of different concentrates were analyzed for DM, CP and total ash (AOAC 2000), cellulose (Crampton and Maynard 1938) and other cell wall constituents like NDF, ADF, and ADL (Van Soest et al 1991). Hemicellulose was determined by difference in NDF and ADF. The data were analyzed by 3 x 2 factorial design (Snedecor and Cochran 1994), taking levels of RUDP (Low, medium and high) as one factor and source of RUDP (CSC or SBG based concentrate mixture) as second factor, by using SPSS (2007) version 16.0 and the means were tested for the significant difference by using Duncan’s multiple range test.

Results and discussion

The chemical composition of different concentrate mixtures revealed that the CP content ranged between 20.5 to 20.8% thereby indicating that all the concentrates were iso-nitrogenous (Table 1). The OM and EE contents were also similar in all the concentrates. The cellulose, NDF and ADF content increased linearly with increase in RUDP in both CSC and SBG based concentrate mixtures.

Irrespective of source of bypass protein (CSC/SBG), the net gas production declined (p<0.01) with the increase in level of RUDP (Table 2), without any significant impact on the digestibility of true OM. But, the digestibility of NDF increased (p<0.01) linearly with the increase in level of RUDP in concentrate mixtures. Gidlund (2017) reported that by replacing crimped barley with solvent-extracted, heat-moisture-treated rapeseed meal increased total neutral detergent fibre digestibility and potentially digestible detergent fibre digestibility. The methane production expressed either as percent of NGP, ml/100mg of substrate or in mmoles decreased (p<0.05) linearly with the increase in level of RUDP in concentrate mixtures. Gidlund (2017) also replaced soybean meal with solvent-extracted, heat-moisture-treated rapeseed meal and reported that methane yield (g CH₄/kg dry matter intake) and methane intensity (g CH₄/kg energy-corrected milk) decreased quadratically with increased dietary crude protein concentration. The main reason for this is probably that ruminal fermentation of protein produces less CH₄ than fermentation of carbohydrates. Bannink et al (2006) and Sveinbjörnsson et al (2006) showed by stoichiometric calculations that fermentation of protein produces 30-50% less CH₄ than fermentation of carbohydrates. The protein supplement with high rumen undegradable protein (RUDP) content produced less methane as compared to the ones that had low RUDP content (Lamba et al 2014). Lee et al (2003) also reported that the methane production was negatively correlated with CP and EE content of feedstuffs. Norwegian red dairy cows with higher feed use efficiency (FUE) showed improved apparent nitrogen use efficiency and decreased enteric CH₄ emission intensity compared with their low-efficiency contemporaries regardless of the level of dietary CP. This would imply that enteric CH₄ emission intensity and UN excretions can be reduced by selecting dairy cows with higher FUE (Kidane et al 2018). The partitioning factor was not affected by the level of RUDP.

Table 2. In-vitro digestibility, availablility of ME and methane production from concentrate mixtures containing different levels of rumen undegradable protein
Parameter	Irrespective of source of RUDP			PSE	p-value	Irrespective of level of RUDP		PSE	p-value
Parameter	L-RUDP	M-RUDP	H-RUDP	PSE	p-value	CSC-CM	SBG-CM	PSE	p-value

NGP, ml/g DM/24h	209.30^c	191.69^b	169.70^a	1.41	0.003	184.28^a	196.19^b	1.15	<0.001
NDFD,%	29.80^a	44.82^b	47.12^b	2.37	0.004	29.12^a	52.04^b	1.94	0.005
TOMD, %	74.18	73.18	72.90	1.12	0.711	73.67	73.17	0.92	0.715
CH₄ as % of NGP	32.90^c	31.88^b	29.13^a	0.21	<0.001	29.12^a	33.48^b	0.17	0.007
CH_4, ml/100mg DM	3.68^c	3.06^b	2.43^a	0.024	0.004	2.68^a	3.43^b	0.019	0.002
CH_4, mM	3.77^c	3.50^b	3.13^a	0.003	0.002	3.31^a	3.62^b	0.003	0.006
NH₃-N, mg/dl	35.25^c	30.46^b	25.36^a	0.27	0.006	28.26^a	32.45^b	0.22	0.003
PF	2.06	1.99	1.99	0.08	0.835	2.18^b	1.85^a	0.07	0.014
ME, MJ/kg DM	9.39^c	8.84^b	8.14^a	0.073	0.005	8.60^a	8.98^b	0.06	0.004
L, Low; M, Medium; H, High; RUDP, Rumen undegradable protein; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture; PSE, Pooled standard error; Figures with different superscripts in a row differ significantly.

The availability of ME from different concentrate mixtures followed the trend of net gas production and decreased (p<0.01) with increase in the level of RUDP in the concentrate mixture. As the level of RUDP in the concentrate mixture increased the release/ production of ammonia decreased (p<0.05) which was obvious as the amount and availability of quickly degradable protein decreased.

Irrespective of level of RUDP, the net gas production was significantly higher in SBG based concentrate mixtures in comparison to CSC based concentrate mixture (Table 2). The higher NGP production in SBG based concentrate mixture was due to higher (p<0.05) NDFD in comparison to that of CSC based concentrate mixture. The true OM digestibility of both the concentrate mixture was comparable. The digestion rate of potentially digestible detergent fibre also increased. The methane production expressed either as percent of NGP, ml/100mg of substrate or as mmoles was significantly low in CSC based concentrate mixtures as compared to SBG based concentrate mixtures. The contribution of methane in total net gas produced by SBG based concentrate mixture was 15% higher (p<0.05) than that contributed by CSC based concentrate mixture. The comparative evaluation of conventional and non-conventional protein supplements revealed that the in-vitro methane production (expressed as ml/g DM or ml/g digestible OM at t-half) was the lowest from tomato pomace, cotton seed cake and corn gluten meal, while the highest was observed from soybean meal and maize oil cake (Lamba et al 2014). The higher (p<0.01) PF in CSC based concentrate mixture in comparison to SBG based concentrate mixture was due to low net gas production with same digestibility of OM. The higher NDFD and NGP resulted in higher (p<0.01) availability of ME from SBG based concentrate mixture in comparison to CSC based concentrate mixture. Though both the concentrate mixtures were iso-nitrogenous the degradability of protein from SBG based concentrate mixture was higher than that from CSC based concentrate mixture, resulting in 14.8% higher (p<0.05) levels of ammonical nitrogen in SBG based concentrate mixture.

Irrespective of source of bypass protein, the total VFAs, acetate and butyrate production decreased (p<0.05) as the level of RUDP increased, which could be due to low availability of fermentable nutrients (Table 3). The propionate production increased (p<0.05) with the increase the level of RUDP in the concentrate mixture. Higher concentration of acetate and butyrate yield H₂, a precursor of methanogenesis, in comparison to higher volumes of propionate, a reaction which utilizes H₂, thereby resulting in low CH₄ yield (Janssen 2010, Dijkstra et al 2011). The acetate to propionate ratio followed the trend of TVFAs i.e decreased with the increase in the level of RUDP. The relative proportion of acetate in low RUDP concentrate mixture was lower (p<0.05) than that in other concentrate mixtures containing medium or high RUDP content. The relative proportion of propionate increased (P<0.05) while that of butyrate decreased with the increase in the level of RUDP in the concentrate mixture. Gidlund (2017) also reported that the low rumen degradability of the solvent-extracted, heat-moisture-treated rapeseed meal reduced the amount of substrate fermented in the rumen, the proportion of propionate in rumen VFAs increased, a reaction which utilizes H₂ resulting in decreased CH₄ yield.

Table 3. In vitro volatile fatty acid production (mM/dl) from concentrate mixtures containing different levels of rumen undegradable protein
Parameter	Irrespective of source of RUDP			PSE	p-value	Irrespective of level of RUDP		PSE	p-value
Parameter	L-RUDP	M-RUDP	H-RUDP	PSE	p-value	CSC-CM	SBG-CM	PSE	p-value

TVFA	11.02^c	10.66^b	10.19^a	0.006	0.008	10.08^a	11.17^b	0.005	<0.001
Acetate (A)	6.84^c	6.72^b	6.42^a	0.004	<0.001	6.41^a	6.91^b	0.003	0.005
Propionate (P)	2.32^a	2.44^b	2.62^c	0.004	0.005	2.31^a	2.61^b	0.003	0.002
Butyrate	1.87^c	1.50^b	1.14^a	0.004	0.002	1.36^a	1.65^b	0.004	0.008
A:P ratio	2.95^c	2.77^b	2.45^a	0.005	0.009	2.79^b	2.66^a	0.004	0.003
*Relative proportion, %*
Acetate	62.02^a	63.11^b	63.07^b	0.032	<0.001	63.63^b	61.83^a	0.026	0.009
Propionate	21.05^a	22.84^b	25.72^c	0.040	0.004	22.95^a	23.46^b	0.03	0.004
Butyrate	16.93^c	14.02^b	11.19^a	0.038	0.007	13.38^a	14.71^b	0.03	0.003
L, Low; M, Medium; H, High; RUDP, Rumen undegradable protein; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture; PSE, Pooled standard error; Figures with different superscripts in a row differ significantly.

The total and individual volatile fatty acids (VFAs) production was significantly higher from SBG based concentrate mixtures as compared with that of CSC based concentrate mixtures (Table 3), it could be due to higher digestibility of NDF. The concentration of acetate, propionate and that of butyrate in SBG based concentrate mixture was 7.8, 13.0 and 21.0% higher (p<0.05) than that in CSC based concentrate mixture. The A:P ratio in CSC based concentrate mixture was higher (p<0.05) than that of SBG based concentrate mixture. The relative proportion of acetate in CSC based concentrate mixture was higher (p<0.05), whereas that of propionate and butyrate was higher in SBG based concentrate mixture.

The digestion kinetic parameters (assessed by in-sacco studies) for DM of concentrate mixtures with different level of RUDP, irrespective of source of RUDP (CSC or SBG) used, revealed that the rapidly soluble fraction decreased (p<0.01), while insoluble but potential degradable fraction increased (p<0.01) linearly with increase in the level of RUDP (Table 4). The rate of degradation of potential degradable fraction of DM decrease p<0.01) with the increase in RUDP level, It was mainly due to decrease in rapidly soluble fraction with increase in the levels of RUDP. The true digestibility, potential extent of digestion and apparent extent of digestion of dry matter also followed the similar trend (p<0.01) as that of effective degradability.

Table 4. Digestion kinetic parameters for DM (%) of concentrate mixtures containing different levels of rumen undegradable protein
Parameter	Irrespective of source of RUDP			PSE	p-value	Irrespective of level of RUDP		PSE	p-value
Parameter	L-RUDP	M-RUDP	H-RUDP	PSE	p-value	CSC-CM	SBG-CM	PSE	p-value

‘a’	41.80^c	31.95^b	15.55^a	0.81	<0.001	39.64^b	19.89^a	0.66	<0.001
‘b’	42.39^a	50.37^b	64.31^c	0.78	0.005	42.50^a	62.22^b	0.64	0.005
‘c’, /h	0.105^b	0.064^a	0.060^a	0.003	0.001	0.081^b	0.072^a	0.002	0.017
UDF	15.81^a	17.68^b	20.13^c	0.18	0.008	17.86	17.89	0.14	0.899
ED	76.19^c	67.16^b	60.75^a	0.14	0.002	71.49^b	64.58^a	0.11	0.003
TD	80.72^b	70.69^a	70.37^a	0.59	0.005	75.86^b	71.99^a	0.48	0.007
PED	78.73^b	68.94^a	68.63^a	0.58	<0.001	73.99^b	70.21^a	0.47	0.004
AED	69.67^c	59.64^b	57.62^a	0.44	0.007	63.96^b	60.66^a	0.36	0.009
L, Low; M, Medium; H, High; RUDP, Rumen undegradable protein; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture; a, Rapidly soluble fraction; b, Potentially degradable fraction; c, Degradation rate of b; UDF, Undegradable fraction; ED, Effective degradability; TD, True degradability; PED, Potential extent of digestion; AED, Apparent extent of digestion; PSE, Pooled standard error; Figures with different superscripts in a row differ significantly.

The digestion kinetic parameters for DM of concentrate mixtures irrespective of level of RUDP revealed that the concentrate mixture containing CSC had higher (p<0.01) amount of rapidly soluble fraction and lower (p<0.01) amount of potentially degradable fraction than the concentrate mixture containing SBG (Table 4). The rate of degradation of potentially degradable fraction of SBG-CM was lower (p<0.01) than that of CSC-CM. The rumen undegradable fraction however was almost similar in both the concentrate mixtures. The effective and true degradability, potential and apparent extent of digestion of DM was higher (P<0.01) in CSC than that in SBG based concentrate mixture.

The digestion kinetic parameters for CP of concentrate mixtures revealed that with the increase in the level of RUDP in concentrate mixture, irrespective of source of ingredients (CSC/SBG) used, the rapidly soluble fraction decreased (p<0.05), while insoluble but potentially degradable fraction increased (p<0.05) linearly (Table 5). The rate of degradation of potentially degradable fraction and ED decreased (p<0.05) with the increase in the level of RUDP. The comparative evaluation of individual conventional and non conventional protein supplements also revealed that supplements like corn gluten meal, cotton seed cake and spent brewer’s grains had the lower degradation rate and higher RUDP as compared to that of mustard and groundnut cake (Lamba et al 2014). The true digestibility; potential and apparent extent of digestion of CP decreased (p<0.05) with increase in level of RUDP in the concentrate mixtures. The RUDP as per cent of protein increased (p<0.05) with the increase in level of RUDP in concentrate mixtures.

The digestion kinetic parameters worked out for CP of concentrate mixtures irrespective of level of RUDP, revealed that the CSC-based concentrate mixtures had 75% higher (p<0.05) rapidly soluble fraction and 39% lower (p<0.05) insoluble but potentially degradable fraction than that in SBG based concentrate mixtures (Table 5). The potentially degradable fraction of CSC based concentrate mixtures was degraded at a much faster rate (p<0.05) than that of SBG based concentrate mixtures. The effective degradability of CSC concentrate mixtures was 9.7% higher (p<0.05) than that of SBG concentrate mixtures. The RUDP in CSC based concentrate mixtures was 20% lower (p<0.05) than that in SBG based concentrate mixtures. The proportion of RUDP as percent of crude protein in SBG based concentrate mixtures was 30% higher (P<0.05) in comparison to CSC based concentrate mixtures. The true digestibility, potential and apparent extent of digestion of CP of concentrate mixture containing CSC was higher (p<0.05) than that of SBG based concentrate mixture.

Table 5. Digestion kinetic parameters for CP of concentrate mixtures containing different levels of rumen undegradable protein, %
Parameter	Irrespective of source of RUDP			SE	p-value	Irrespective of level of RUDP		PSE	p-value
Parameter	L-RUDP	M-RUDP	H-RUDP	SE	p-value	CSC-CM	SBG-CM	PSE	p-value

‘a’	53.08^c	43.39^b	24.68^a	0.28	0.003	51.41^b	29.36^a	0.23	0.009
‘b’	41.14^a	46.71^b	58.27^c	0.26	0.001	36.67^a	60.75^b	0.21	<0.001
‘c’, /h	0.101^c	0.054^b	0.042^a	0.001	0.007	0.070^b	0.061^a	0.001	0.001
ED	86.11^c	74.38^b	61.04^a	0.10	<0.001	77.26^b	70.42^a	0.08	0.008
TD	80.11^c	67.33^b	62.51^a	0.47	0.002	71.94^b	68.02^a	0.38	0.003
PED	78.13^c	65.66^b	60.97^a	0.46	0.005	70.16^b	66.34^a	0.38	0.007
AED	77.39^c	62.20^b	53.15^a	0.34	0.001	65.39^b	63.10^a	0.28	0.004
RUDP	3.02^a	5.56^b	8.44^c	0.02	0.004	4.70^a	6.65^b	0.02	<0.001
RUDP as % of CP	14.74^a	26.92^b	40.69^c	0.10	<0.001	22.76^a	32.27^b	0.08	0.001
L, Low; M, Medium; H, High; RUDP, Rumen undegradable protein; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture; a, Rapidly soluble fraction; b, Potentially degradable fraction; c, Degradation rate of b; UDF, Undegradable fraction; ED, Effective degradability; TD, True degradability; PED, Potential extent of digestion; AED, Apparent extent of digestion; PSE, Pooled standard error; Figures with different superscripts in a row differ significantly.

The summarized results in Table 6 revealed that with the increase level of RUDP in the concentrate mixture, the propionic acid production increased by 12.93%, while the methane production decrease by 33.97% in H-RUDP level as compared to L-RUDP level. Further, in comparison to SBG based concentrate mixture, both propionate and methane production were lower by 12.98 and 27.98% respectively in CSC based concentrate mixture. Phongphanith et al (2016) also reported that in-vitro methane production per unit of digestible DM was dependent on the N-solubility of the substrate.

Table 6. Effect of level of rumen undegradable protein (RUDP) and of Cottonseed meal versus Spent brewers’ grains based concentrate mixture on: in-vitro methane and propionic acid production
Parameter	Per cent increase/decrease over
	L-RUDP		SBG
	M-RUDP	H-RUDP	CSC

Propionate, mM/dl	+5.17	+12.93	-12.98
CH_4, ml/100mg DM	-16.85	-33.97	-27.98
L, Low; M, Medium; H, High; CSC-CM, Cotton seed cake based concentrate mixture; SBG-CM, Spent brewer’s grains based concentrate mixture

Conclusion

The results conclusively revealed that the in-vitro methane production can be mitigated significantly with the increase in the level of RUDP of concentrate mixtures.
Cotton seed cake based concentrate mixture was more effective in mitigating methane production as compared to that containing spent brewers' grains.

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Received 7 February 2019; Accepted 9 February 2019; Published 4 March 2019

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