Experimental Outline

This work is now published in the Journal of Dairy Science: Rojas-Gómez, Paula, Raghunath Pariyani, Lorraine M. Bateman, Denis Lynch, Mark Timlin, Michael Dineen, Noel A. McCarthy et al. "Effect of proportion of pasture in the cow diet and seasonality on the milk metabolome as determined by 1H-NMR." Journal of Dairy Science 108, no. 5 (2025): 4659-4673.

Perennial Ryegrass Pasture system (GRS)

Grass involves cows grazing primarily on pasture, relying on fresh grass as the main nutrient source. This system is low-cost, environmentally sustainable, and supports natural cow behavior. For our diet study, the GRS diet was composed of grazed pasture (95% annual average) through rotational grazing of perennial ryegrass (Lolium perenne L.) and parlor concentrates (5%).

Total Mixed Ration system (TMR)

TMR is a fully controlled feeding strategy where all dietary components—such as silage, hay, grains, protein sources, vitamins, and minerals—are mixed into a single ration and delivered to cows. For our diet study, the TMR concentrates had 93.9% DM, with 22% CP, 23.9% NDF, and 7.2% ash. In comparison, the parlour concentrates fed to each herd (sole concentrates for GRS herd) contained 89% DM, with lower CP at 13.3%, and higher fiber content at 30.9%

Partial Mixed Ration system (PMR)

PMR combines aspects of GRS and TMR, offering a partial mixed ration indoors while allowing cows to graze during part of the day. For our diet study, the PMR system involved cows grazing perennial ryegrass outside during daylight hours, whereas they were provided with a TMR diet at night. At night, cows were housed indoors and fed with the same diet as the TMR group but at half of the quantity. The PMR diet consisted of 45% grazed grass (9 kg), 22% concentrates (4.5 kg), 22% maize silage (4.5 kg), and 11% grass silage (2.25 kg).

Metabolites Detected

Metabolites

Major Findings

The proportion of pasture in cow diet was demonstrated to have a significant effect on the milk metabolome. Multivariate analysis using PLS-DA revealed clear distinguishing effects between the diets, as shown here, with a clear evolution of the relationship between samples in line with the proportion of pasture. As such, GRS-derived milks are segregated clearly away from that of TMR, whereas PMR-derived milks demonstrate a similarity with both cohorts.

Variable importance in projection analysis highlighted 12 metabolites, in particular, driving this observed separation. The key metabolites for differentiation of milks from different feeding systems are represented here.

Full Lactation Breakdown by diet

| **Metabolite

(mMol)**	Spring - GRS	Spring - PMR	Spring - TMR	Summer - GRS	Summer - PMR	Summer - TMR	Autumn - GRS	Autumn - PMR	Autumn - TMR	p-value Diet	p-value Season	p-value Diet × season
2-Oxoglutarate	0.100 ± 0.032	0.129 ± 0.019	0.105 ± 0.013	0.117 ± 0.022	0.145 ± 0.035	0.103 ± 0.016	0.132 ± 0.033	0.154 ± 0.023	0.115 ± 0.012	<0.001	0.004	0.373
3-Hydroxybutyrate	0.030 ± 0.016	0.038 ± 0.018	0.038 ± 0.009	0.021 ± 0.004	0.018 ± 0.003	0.023 ± 0.003	0.024 ± 0.003	0.024 ± 0.008	0.026 ± 0.004	<0.001	<0.001	<0.001
Acetate	0.076 ± 0.021	0.079 ± 0.021	0.081 ± 0.016	0.082 ± 0.017	0.066 ± 0.013	0.074 ± 0.010	0.083 ± 0.036	0.067 ± 0.010	0.070 ± 0.011	0.059	0.44	0.136
Acetone	0.074 ± 0.081	0.147 ± 0.110	0.067 ± 0.019	0.024 ± 0.006	0.083 ± 0.037	0.048 ± 0.010	0.028 ± 0.015	0.058 ± 0.027	0.031 ± 0.014	<0.001	<0.001	0.161
Alanine	0.028 ± 0.003	0.036 ± 0.004	0.038 ± 0.004	0.030 ± 0.003	0.039 ± 0.005	0.034 ± 0.003	0.031 ± 0.003	0.038 ± 0.006	0.034 ± 0.004	<0.001	0.953	0.003
Aspartate	0.016 ± 0.003	0.016 ± 0.003	0.016 ± 0.003	0.015 ± 0.004	0.018 ± 0.005	0.013 ± 0.004	0.011 ± 0.003	0.012 ± 0.004	0.011 ± 0.003	0.07	<0.001	0.13
Betaine	0.080 ± 0.027	0.099 ± 0.019	0.078 ± 0.014	0.067 ± 0.010	0.080 ± 0.021	0.074 ± 0.011	0.081 ± 0.016	0.106 ± 0.013	0.101 ± 0.011	<0.001	0.004	0.03
Butyrate	0.0430 ± 0.01	0.075 ± 0.054	0.047 ± 0.018	0.039 ± 0.011	0.062 ± 0.019	0.039 ± 0.011	0.069 ± 0.045	0.084 ± 0.059	0.039 ± 0.009	<0.001	0.256	0.384
Caprate+Caprylate	0.039 ± 0.006	0.057 ± 0.029	0.040 ± 0.012	0.035 ± 0.010	0.055 ± 0.013	0.039 ± 0.011	0.050 ± 0.030	0.063 ± 0.032	0.037 ± 0.009	<0.001	0.638	0.55
Carnitine	0.053 ± 0.012	0.054 ± 0.008	0.055 ± 0.010	0.037 ± 0.014	0.043 ± 0.016	0.055 ± 0.009	0.058 ± 0.009	0.043 ± 0.016	0.052 ± 0.011	0.026	0.043	0.002
Choline	0.280 ± 0.080	0.265 ± 0.056	0.273 ± 0.063	0.384 ± 0.054	0.333 ± 0.069	0.367 ± 0.046	0.478 ± 0.099	0.407 ± 0.053	0.442 ± 0.057	<0.001	<0.001	0.419
Citrate	6.278 ± 0.852	7.102 ± 0.991	6.345 ± 0.608	5.869 ± 0.558	5.943 ± 0.830	5.532 ± 0.455	6.251 ± 0.769	5.970 ± 0.661	5.545 ± 0.651	<0.001	0.004	0.019
Creatine	0.486 ± 0.040	0.587 ± 0.045	0.500 ± 0.051	0.498 ± 0.039	0.625 ± 0.095	0.572 ± 0.052	0.565 ± 0.056	0.670 ± 0.060	0.629 ± 0.064	<0.001	<0.001	0.241
Creatine phosphate	0.134 ± 0.079	0.154 ± 0.063	0.149 ± 0.043	0.020 ± 0.011	0.039 ± 0.019	0.055 ± 0.018	0.011 ± 0.004	0.024 ± 0.008	0.037 ± 0.011	<0.001	<0.001	<0.001
Creatinine	0.073 ± 0.011	0.086 ± 0.013	0.075 ± 0.012	0.067 ± 0.007	0.073 ± 0.009	0.067 ± 0.007	0.067 ± 0.007	0.069 ± 0.007	0.069 ± 0.006	<0.001	0.008	0.068
Dimethyl sulfone	0.029 ± 0.013	0.017 ± 0.004	0.015 ± 0.003	0.024 ± 0.007	0.014 ± 0.003	0.014 ± 0.002	0.014 ± 0.003	0.015 ± 0.002	0.017 ± 0.003	<0.001	0.009	<0.001
Dimethylamine	0.022 ± 0.006	0.022 ± 0.005	0.017 ± 0.003	0.014 ± 0.002	0.020 ± 0.004	0.016 ± 0.003	0.015 ± 0.003	0.017 ± 0.006	0.015 ± 0.002	0.004	0.001	0.028
Ethanolamine	0.045 ± 0.020	0.041 ± 0.01	0.037 ± 0.011	0.075 ± 0.022	0.068 ± 0.019	0.064 ± 0.014	0.072 ± 0.023	0.081 ± 0.022	0.070 ± 0.014	0.09	<0.001	0.426
Formate	0.009 ± 0.001	0.009 ± 0.002	0.007 ± 0.002	0.009 ± 0.001	0.008 ± 0.003	0.006 ± 0.001	0.009 ± 0.001	0.010 ± 0.001	0.007 ± 0.001	<0.001	0.083	0.297
Fucose	0.022 ± 0.004	0.025 ± 0.005	0.025 ± 0.006	0.016 ± 0.003	0.019 ± 0.005	0.019 ± 0.003	0.026 ± 0.005	0.027 ± 0.004	0.025 ± 0.004	0.027	<0.001	0.277
Fumarate	0.025 ± 0.008	0.036 ± 0.006	0.029 ± 0.004	0.022 ± 0.005	0.034 ± 0.007	0.022 ± 0.003	0.023 ± 0.006	0.033 ± 0.004	0.022 ± 0.003	<0.001	0.011	0.214
Galactose	0.208 ± 0.083	0.184 ± 0.046	0.187 ± 0.065	0.295 ± 0.031	0.323 ± 0.066	0.303 ± 0.036	0.323 ± 0.040	0.324 ± 0.029	0.328 ± 0.052	0.903	<0.001	0.189
Glucose	0.274 ± 0.085	0.279 ± 0.060	0.240 ± 0.046	0.312 ± 0.068	0.418 ± 0.065	0.307 ± 0.043	0.303 ± 0.067	0.312 ± 0.070	0.284 ± 0.043	<0.001	<0.001	0.013
Glucose-1-phosphate	0.047 ± 0.029	0.059 ± 0.034	0.061 ± 0.035	0.011 ± 0.006	0.018 ± 0.010	0.025 ± 0.009	0.004 ± 0.002	0.009 ± 0.003	0.014 ± 0.004	<0.001	<0.001	<0.001
Hippurate	0.213 ± 0.048	0.133 ± 0.021	0.102 ± 0.014	0.231 ± 0.051	0.101 ± 0.019	0.078 ± 0.010	0.179 ± 0.044	0.105 ± 0.023	0.075 ± 0.008	<0.001	<0.001	0.001
Isobutyrate	0.001 ± 0.000	0.001 ± 0.000	0.002 ± 0.000	0.001 ± 0.000	0.001 ± 0.000	0.001 ± 0.000	0.001 ± 0.000	0.001 ± 0.000	0.001 ± 0.000	<0.001	0.545	0.025
Lactate	0.030 ± 0.008	0.044 ± 0.012	0.046 ± 0.010	0.034 ± 0.007	0.058 ± 0.009	0.059 ± 0.027	0.040 ± 0.005	0.070 ± 0.013	0.052 ± 0.005	<0.001	<0.001	0.061
Lactose	96.473 ± 5.928	100.275 ±6.134	100.105 ±6.419	92.783 ± 5.571	94.366 ±10.710	94.882 ± 5.753	93.214 ± 6.138	92.215 ± 9.011	97.054 ± 7.18	0.109	0.047	0.603
Leucine	0.002 ± 0.001	0.002 ± 0.001	0.003 ± 0.001	0.003 ± 0.001	0.004 ± 0.002	0.004 ± 0.002	0.003 ± 0.001	0.004 ± 0.002	0.003 ± 0.001	0.002	0.001	0.462
Malate	0.120 ± 0.039	0.167 ± 0.033	0.135 ± 0.025	0.109 ± 0.032	0.155 ± 0.025	0.110 ± 0.019	0.110 ± 0.035	0.159 ± 0.027	0.105 ± 0.015	<0.001	0.077	0.601
Methanol	0.006 ± 0.001	0.007 ± 0.002	0.008 ± 0.001	0.007 ± 0.002	0.007 ± 0.001	0.008 ± 0.001	0.005 ± 0.001	0.007 ± 0.001	0.009 ± 0.002	<0.001	0.379	0.03
O-Acetylcarnitine	0.050 ± 0.019	0.056 ± 0.017	0.043 ± 0.009	0.037 ± 0.006	0.034 ± 0.007	0.031 ± 0.004	0.045 ± 0.008	0.037 ± 0.006	0.036 ± 0.004	<0.001	0.001	0.001
Orotic acid	4.183 ± 0.839	4.086 ± 0.854	4.172 ± 1.016	5.010 ± 0.585	5.301 ± 0.695	5.472 ± 0.409	5.026 ± 0.535	4.867 ± 0.746	4.774 ± 0.783	0.777	0.001	0.057
Propionate	0.003 ± 0.001	0.003 ± 0.000	0.004 ± 0.000	0.003 ± 0.000	0.004 ± 0.000	0.004 ± 0.000	0.002 ± 0.000	0.003 ± 0.001	0.003 ± 0.000	<0.001	<0.001	0.17
Pyruvate	0.028 ± 0.007	0.029 ± 0.007	0.031 ± 0.008	0.013 ± 0.003	0.018 ± 0.003	0.017 ± 0.003	0.012 ± 0.002	0.015 ± 0.003	0.014 ± 0.003	<0.001	<0.001	0.25
Succinate	0.019 ± 0.006	0.025 ± 0.004	0.025 ± 0.003	0.016 ± 0.002	0.024 ± 0.005	0.022 ± 0.002	0.018 ± 0.003	0.022 ± 0.003	0.022 ± 0.004	<0.001	0.068	0.246
Tyrosine	0.003 ± 0.001	0.003 ± 0.001	0.003 ± 0.001	0.004 ± 0.001	0.003 ± 0.001	0.004 ± 0.001	0.004 ± 0.000	0.004 ± 0.001	0.004 ± 0.001	0.628	0.011	0.354
Urea	1.612 ± 0.401	1.611 ± 0.268	1.499 ± 0.366	1.952 ± 0.718	2.327 ± 0.400	1.972 ± 0.216	2.767 ± 0.883	2.379 ± 0.414	2.224 ± 0.376	0.085	<0.001	0.056
Uridine	0.023 ± 0.010	0.022 ± 0.010	0.022 ± 0.007	0.012 ± 0.002	0.012 ± 0.002	0.013 ± 0.002	0.011 ± 0.001	0.010 ± 0.001	0.011 ± 0.001	0.284	<0.001	0.785
Valerate	0.004 ± 0.002	0.004 ± 0.001	0.004 ± 0.001	0.003 ± 0.001	0.004 ± 0.001	0.005 ± 0.002	0.004 ± 0.002	0.004 ± 0.001	0.003 ± 0.001	0.014	0.287	0.153
Valine	0.009 ± 0.001	0.012 ± 0.003	0.013 ± 0.004	0.010 ± 0.001	0.011 ± 0.001	0.012 ± 0.002	0.010 ± 0.002	0.011 ± 0.001	0.013 ± 0.002	<0.001	0.94	0.46
cis-Aconitate	0.031 ± 0.007	0.036 ± 0.008	0.031 ± 0.007	0.031 ± 0.004	0.036 ± 0.007	0.032 ± 0.006	0.029 ± 0.005	0.034 ± 0.007	0.031 ± 0.006	<0.001	0.527	0.962
sn-Glycero-3-phosphocholine	0.592 ± 0.114	0.559 ± 0.089	0.540 ± 0.082	0.590 ± 0.068	0.685 ± 0.102	0.752 ± 0.079	0.642 ± 0.079	0.697 ± 0.113	0.779 ± 0.080	<0.001	<0.001	<0.001

Terms & Conditions of Database