Effect of rate of inclusion of grass and maize silage fed without or with dietary copper antagonists on the performance, health and indicators of copper status in dairy cows. Research Partnership: Cattle health, welfare and nutrition Work Package FS3: Mineral and trace-element requirements of dairy cows

Report prepared for DairyCo September 2012

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Contents Section Page

1. 1.1 Farmer recommendations 3

1.2 Executive summary 4 2. Background 5 2.1. Hypothesis 5 2.2. Objectives 5 3. Materials and method 3.1. Animals 5 3.2. Diets 6 3.3. Housing 6 3.4. Experimental procedure 6 3.5. Chemical analysis 6 3.6. Statistical analysis 7 4. Results 4.1 Forage and diet analysis 7 4.2 Intake and animal performance 8 4.3 Mastitis and reproductive performance 8

4.4 Liver mineral concentration 8 4.5 Plasma minerals, Cu mediated enzymes and 9

metabolites 5. Conclusions 9 6. References 10 7. Appendices 12

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1.1. Farmer recommendations

At sulphur and molybdenum concentrations likely to be found in the majority of dairy cow rations in the UK there is little difference in Cu metabolism in dairy cows when fed a maize or grass silage based ration

For dairy cows fed a grass silage based ration, high dietary levels of sulphur and molybdenum reduce intake, milk yield and body condition, increase somatic cell count and decrease liver copper levels

For dairy cows fed a maize silage based ration, high dietary levels of sulphur and molybdenum have significantly less of an effect on performance or liver copper levels

For the majority of dairy cow diets, feeding 20 mg Cu/kg DM will increase liver concentrations substantially, and a lower level of 15-17 mg/kg DM may be adequate

Blood samples are not a good indicator of Cu status in dairy cows

Feeding a high maize compared to a high grass silage based ration will increase intake and milk protein yield, but may not always increase milk yield

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1.2 Executive summary

An experiment was conducted to determine the effect of rate of inclusion of grass and maize silage in the absence or presence of additional S and Mo on the performance, health and indicators of Cu status in dairy cows. Fifty six Holstein-Friesian dairy cows (8 primaparous and 48 multiparous) that were 35 d into lactation were allocated to one of four dietary treatments in a 2 x 2 factorial design:

G- 0.75 grass silage:0.25 maize silage (DM basis), no addition antagonists G+ 0.75 grass silage:0.25 maize silage (DM basis), with additional Mo and S M- 0.25 grass silage:0.75 maize silage (DM basis), no addition antagonists M+ 0.25 grass silage:0.75 maize silage (DM basis), with additional Mo and S

Copper (in the form of CuSO4) was added to each diet to provide a final concentration of 19 mg/kg DM. All diets were formulated to be isoenergetic and supply a similar level amount of metabolisable protein. Blood samples were collected via jugular venepuncture during weeks 0, 1, 2, 4, 8 and 14 of the study, whilst liver biopsy samples were collected from each cow during weeks 0 and 14. Milk yield and intake were recorded daily with samples collected weekly for subsequent analysis of fat, protein and lactose. Milk samples were also collected fortnightly for determination of somatic cell counts. Cows were weighed and condition scored fortnightly. Summary results are presented in Table S1. Table S1. Intake, performance and indicators of Cu status of dairy cows fed diets high in maize (M) or grass (G) silage fed either without (-) or with (+) added S and Mo.

Diets Significance1

M- M+ G- G+ s.e.d. F A Int

Intake, kg DM/d Milk yield, kg/d

23.5 38.1

24.0 40.6

22.6 38.9

20.5 37.9

0.74 1.22

<0.001 0.225

0.111 0.373

0.012 0.034

Fat , g/kg 37.8 36.6 38.2 37.4 1.92 0.656 0.475 0.889 BCS change 0.35 0.13 0.27 0.09 0.114 0.470 0.019 0.801 Milk SCC (log10) 1.72 1.50 1.39 1.67 0.138 0.381 0.714 0.017 Liver minerals Final Cu, mg/kg DM

587

437

490

357

58.0

0.038

0.002

0.837

Cu change, mg/kg DM 65 11 82 -61 35.0 0.275 0.001 0.078 Final Mo, mg/kg DM 3.92 4.19 3.79 4.71 0.312 0.377 0.011 0.149 Plasma Cu, µmol/l 13.3 13.7 14.3 13.7 0.77 0.340 0.889 0.332 Plasma Mo, µmol/l 0.33 0.50 0.27 0.50 0.041 0.271 <0.001 0.375 Ceruloplasmin, mg/dL 17.9 15.9 20.3 18.1 1.12 0.006 0.010 0.909 Ceruloplasmin:Cu 1.37 1.22 1.41 1.36 0.081 0.096 0.090 0.377 1P values; F= main effect of forage source, A = main effect of antagonists, Int = interaction between forage and antagonists

In conclusion:

A high inclusion rate of maize silage increased DM intake and milk protein yield

Added S and Mo decreased intake and milk yield in cows when fed grass but not maize silage based rations

Added S and Mo decreased liver Cu status, with the effect being greater in cows when fed the grass than the maize silage based ration

Feeding 20 mg Cu/kg DM to cows on grass or maize based rations will increase liver concentrations by 100 mg/kg DM over 140d

Plasma copper and superoxide dismutase are not sensitive indicators of Cu status. Ceruloplasmin and Cu:CP are more sensitive but are affected by the basal ration

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2. Background Copper (Cu) is one of the most important trace elements for normal health and performance in dairy cattle. It is required by approximately 300 different proteins whose function range from antioxidants, efficient iron metabolism, hair pigmentation, synthesis of connective tissue and release of hormones (Suttle, 2010). Clinical Cu deficiency is a problem of cattle in general throughout GB which results in economic and production losses due to effects on fertility, performance and health. However, clinical symptoms in GB dairy cows are rarely caused by a dietary deficiency of Cu but are more often related to interactions with antagonists such as molybdenum (Mo) and sulphur (S) which form thiomolybdates in the rumen and adversely affect absorption and normal Cu metabolism (Phillippo et al., 1987; Suttle, 1991). Reports of an increase in fertility of dairy cattle receiving appropriate Cu supplementation (Mackenzie et al, 2001; Black and French, 2004) has brought Cu to the attention of nutritionists and farmers as a means of improving sub-fertility. As a consequence, Cu has routinely been supplemented well above NRC (2001) recommended levels, with the result that there has been a concerning increase in the number of animals being diagnosed with Cu toxicity (Laven et al., 2004; ACAF, 2010). We recently reported that supplementing Cu at below the recommended level (17 mg/kg DM) was sufficient to maintain Cu status in high yielding cows (Hart et al., 2011). In agreement with these findings, ACAF (2010) have recently proposed a reduction in the maximum level of Cu in dairy cow diets from 40 to 20 mg/kg DM. However, under these lower dietary Cu levels the effects of interactions with S, Mo and Fe become more important. It is recognized that the degree of thiomolydate formation is dependent on the basal forage being fed (Suttle 1983 a,b) although our understanding of the exact mechanisms still remains poor. For example, Mackenzie et al. (2001) reported a response to Cu supplementation in dairy cows fed grass silage with a Mo level of 3.1 mg/kg DM, whereas Hart et al., (2011) supplemented cows fed maize based diets with additional S and Mo and found no effect on liver or plasma Cu levels. Consequently there is a need to provide dairy farmers with sound advice on the effect of forage type and antagonist level when Cu is supplemented at a lower level.

2.1. Hypothesis Feeding high levels of Mo and S affects Cu metabolism, irrespective of the relative content of grass and maize silage in the diet. 2.2. Objectives To determine the effect of inclusion level of grass and maize silage either without or with added sulphur and molybdenum on the performance, health, and indicators of copper status in dairy cows.

3. Materials and method 3.1 Animals Fifty-six Holstein-Friesian dairy cows (8 primaparous and 48 multiparous) that were 35 (SE +/- 2.2) days post calving and yielding 38.9 kg/d (SE +/- 0.91) of milk were used. Based on recordings taken in week 4 of lactation the animals were blocked and allocated to one of four dietary treatments according to lactation number (prima or multi), calving date, milk yield, milk composition, condition score and live weight. Cows remained on study for 14 weeks (98 days).

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3.2 Diets Based on the mineral analysis of the forages (Table 1) and standard values for the straight feeds, fours diets were formulated to contain 19 mg Cu/kg DM and contain a grass to maize silage ratio of 0.25:0.75 (G) or 0.75:0.25 (M: DM basis; Table 2). To evaluate the effects of dietary antagonists on Cu metabolism, cows received either no supplementary S and Mo (-) or additional S and Mo (+), to result in a total dietary concentration of 3.5 g S/kg DM or 7.5 mg Mo/kg DM. The level of inclusion of S and Mo were chosen to reduce the efficiency of Cu absorption by approximately 60%. There were therefore 4 dietary treatments:

G- 0.75 grass silage:0.25 maize silage (DM basis), no addition antagonists G+ 0.75 grass silage:0.25 maize silage (DM basis), with additional S and Mo M- 0.25 grass silage:0.75 maize silage (DM basis), no addition antagonists M+ 0.25 grass silage:0.75 maize silage (DM basis), with additional S and Mo

Additional Cu was supplied as CuSO4 and the diets were supplemented with straight feeds to support a milk production of approximately 38 kg of milk per day according to Thomas (2004). All dietary ingredients were mixed and fed as a total mixed ration (TMR) using a Keenan Compact forage mixer calibrated to ± 1kg and fed through Insentec roughage intake feeders (RIC feeders) fitted with an automatic animal identification and forage weighing system calibrated to ± 0.1 kg (Sinclair et al., 2005; Sinclair et al., 2007). Fresh feed was offered daily at the rate of 1.05 of ad libitum intake with refusals collected twice weekly on a Tuesday and Friday. 3.3. Housing The cows were housed in the same portion of a cubicle building containing Super Comfort free stalls fitted with mattresses. The passageways were scraped using automatic scrapers and the stalls bedded twice weekly. All cows had continual access to water. 3.4. Experimental routine

Cows were milked twice daily at approximately 05:30 and 15:30h through a Westaflia 40-point internal rotary parlour. Milk yield was recorded at each milking with samples taken weekly for subsequent analysis of fat, protein and lactose. Samples for somatic cell count were collected at consecutive am and pm milkings fortnightly. The cows were weighed and body condition scored using a 1-5 scoring system on a quarter point scale (Lowman et al., 1976) after the evening milking in the week prior to allocation and then weekly. Forage samples were collected weekly: half the sample was oven dried and the ratio of grass to maize silage adjusted to achieve the desired ratio. The other sample was frozen and bulked for subsequent analysis. Samples of each of the four diets were collected immediately following feeding once per week and stored at -20ºC. Blood samples were collected at weeks 0, 1, 2, 4, 8 and 14 of the study via jugular venipuncture for subsequent analysis of superoxide dismutase, ceruoplasmin, plasma Cu, β-hydroxybutyrate and urea. Liver samples were taken by biopsy through the 11th intercostal space using the procedure described by Davies and Jebbett (1981) from all animals during week 0 and 14 of the study and stored at -80ºC prior to subsequent analysis. Incidence of mastitis and fertility parameters during the study period were recorded for each cow.

3.5. Chemical analysis Weekly forage and diet samples were bulked within month and analysed according to AOAC (2000) for DM (934.01) and CP (988.05) whilst NDF and ADF were determined according to

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Van Soest et al. (1991). NDF determination was conducted without sodium sulphite, with alpha-amylase and was corrected for ash. In addition, forage samples were analysed for ammonia-N, ADF, water soluble carbohydrates, starch, ethanol and volatile fatty acids by Eurofins UK (Wolverhampton, UK). Forage and dietary Cu, Fe, Zn, Mn and Mo were extracted using the DigiPREP digestion system (Qmx Laboratories, Essex, UK) and analyzed by inductively coupled plasma-mass spectrometry (ICP-MS; Thermo Fisher Scientific Inc., Hemel Hempstead, UK) as described by Cope et al. (2009). Whole blood samples were analyzed for haemoglobin (Hb) using a Vet Animal Blood Counter (Woodley Equipment Company Ltd., Bolton, UK). Serum samples were analyzed for ceruloplasmin according to Henry et al. (1974) and plasma samples for superoxide dismutase (SOD; Randox Laboratories, kit catalogue no. SD 125), 3-OHB, glucose and urea (Randox Laboratories, County Antrim, UK; kit catalogue no. RB 1007, GL1611 and UR221 respectively) using a Cobas Miras Plus autoanalyser (ABX Diagnostics, Bedfordshire, UK). Additionally, plasma samples were analysed for Cu, Fe, Mn and Mo by ICP-MS by diluting 1:20 in 0.5% HNO3. Liver samples were analyzed for Cu, Mo, Zn, Mn and Fe by ICP-MS after digestion overnight at 60°C in concentrated nitric acid. Milk samples were analyzed using a Milkoscan Minor (FOSS, Warrington, UK) calibrated by the methods of AOAC (2000) and SCC was determined by Eurofins UK(Wolverhampton, UK). 3.6. Statistical analysis Milk yield parameters, live weight, body condition score and blood parameters were analysed as a 2 x 2 factorial design with main effects of forage source (F), without or with antagonists (A) and their interaction (Int)) using repeated measures analysis of variance. All analysis was conducted using Genstat 14. Milk SCC was transformed to log10 prior to analysis. The performance in the week prior to allocation was used as a co-variate where appropriate. Indicators of reproductive performance were analysed by ANOVA and mastitis incidence by Chi-square.

4. Results 4.1. Forage and diet analysis The grass silage was 85 g/kg lower in DM content than the maize silage, but was 82 g/kg DM higher in crude protein and 0.4 MJ/kg DM higher in ME (Table 1). The two forages had a similar fibre content but the grass silage was 43.2 g/kg DM higher in lactic acid than the maize silage. Mineral levels were higher in grass than in the maize silage apart from Mg concentrations which were similar between both forages The content of Ca, P, S, Cu, Mo, Fe were 5 g/kg DM, 1 g/kg DM, 2.2 g/kg DM, 3.3 g/kg DM, 84 mg/kg DM and 94 mg/kg DM respectively higher in the grass silage than the maize silage. The DM content of the maize based rations (M- and M+) was 47 g/kg higher than the grass silage based ration (G- and G+), whereas crude protein content was on average 11 g/kg DM higher in the grass than the maize silage based ration (Table 2). The content of NDF was higher in the maize than the grass silage based ration, but ADF concentration was similar in all four rations. All four study diets had a similar P and Mg concentration, but the grass silage based rations (G- and G+) contained approximately 2 g/kg DM more Ca. The mean concentration of Cu was 20 mg/kg DM, which was close to the predicted value of 19 mg/kg DM. The two diets with added Cu antagonists (M+ and G+) had concentrations of S and Mo of 3.3 g/kg DM and 7.8 mg/kg DM respectively, which were close to the predicted values of 3.5 g/kg DM and 7.5 mg/kg DM respectively. In contrast, the two diets with no added S and Mo (M- and G-) had considerably lower concentrations of S and Mo at 1.3 g/kg DM and 1.3 mg/kg DM respectively.

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4.2. Intake and animal performance Cows fed the maize based ration had a daily DM intake that was approximately 2.2 kg/d higher (P < 0.001) than those fed the grass silage based ration (Table 3), an effect that was evident from week 1 of the study (Fig 1). There was an interaction (P < 0.05) between forage source and Cu antagonists; adding S and Mo reduced DM intake by 2.1 kg/d in cows fed the grass silage based ration, but increased DM intake by 0.5 kg/d in cows fed the maize silage based ration. There was also an interaction between forage source and Cu antagonists on milk yield, with yield decreasing in cows when fed the grass silage based ration but increasing in those offered the maize silage based ration (Fig 2). There was no effect (P > 0.05) of dietary treatment on milk fat, protein or lactose content or daily fat yield, but daily milk protein yield was 0.05 kg/d higher (P < 0.05) in cows fed the maize silage based ration. There was no effect (P > 0.05) of dietary treatment on live weight or daily live weight change, but there was an effect of antagonist on body condition and body condition score change (P < 0.05), with cows fed added S and Mo (M+ and G+) having a lower body condition score and gaining less condition over the study period than those not fed added S and Mo (M- and G-; Fig 3). 4.3. Mastitis and reproductive performance There was an interaction (P < 0.05) between forage source and Cu antagonists on milk SCC count, with the addition of S and Mo decreasing SCC in cows fed the maize based ration, but increasing in cows fed the grass silage based ration (Table 4). There was however, no effect (P > 0.05) of dietary treatment on cow or quarter cases of mastitis. Caution should be exercised when interpreting the reproductive performance of the cows on study: cows commenced the study on average at day 35 of lactation and remained on study for 14 wk. Therefore any differences in reproductive performance may have been due to factors before or after the treatment period. There was a trend (P = 0.05) for cows fed the grass silage based ration to have a lower days to first service, although the difference was small (4 d). There was no other effect of dietary treatment on any of the reproductive parameters measured. 4.4. Liver mineral concentration There was no difference between dietary treatments (P > 0.05) in initial liver Cu concentration, which averaged 443 mg/kg DM (Table 5). There was an effect of forage source on final Cu concentration, which was higher (P < 0.05) in cows fed the maize compared to the grass silage based ration. There was also an effect of Cu antagonists on final liver Cu concentration, with the final concentration being 142 mg/kg DM lower (P < 0.01) in cows fed added S and Mo. The addition of S and Mo resulted in a decrease in liver Cu concentration of 50 mg/kg DM compared with an increase of 74 mg/kg DM in cows not receiving added Cu antagonists. There was also a trend for an interaction between forage source and Cu antagonists on the rate of change in liver Cu concentration (P = 0.08), with a net decrease of 61 mg/kg DM over the study period in cows fed added S and Mo in combination with the grass silage based ration (G+), but an increase in cows fed the maize silage based ration of 11 mg/kg DM (M+). There was no difference between treatments in initial liver Mo concentration (P > 0.05), whereas final Mo concentration was higher (P < 0.05) in cows fed added S and Mo (M+ and G+). There was no effect (P < 0.05) of dietary treatment on liver Mn concentration or on initial liver Fe concentration. Final liver Fe concentration was lower in cows when fed added S and Mo (P < 0.01) and there was a trend (P = 0.06) for the final liver Fe concentration to

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be higher in cows fed the maize compared to the grass silage based ration. The addition of S and Mo resulted in a net decrease in liver Fe concentration over the study period of 19 mg/kg DM compared to an increase in cows not receiving the additional Cu antagonists of 50 mg/kg DM. Most of this difference could be explained by the trend for an interaction between forage source and liver Fe concentration (P = 0.08), with cows fed the grass silage based ration and added S and Mo (G+) having the greatest decrease in liver Fe concentration (42 mg/kg DM), compared to an increase in cows fed any of the other dietary treatments. 4.5. Plasma minerals, Cu mediated enzymes and metabolites There was no effect (P > 0.05) of dietary treatment on plasma Cu concentration with a mean value of 13.7 µmol/L (Table 6). There was an effect of time on plasma Cu levels, with the concentration increasing in the first wk of the study, and then fluctuating between weeks (Fig 4). There was an effect (P < 0.001) of dietary treatment on plasma Mo concentrations, which were increased in cows when fed added S and Mo, but there was no effect (P > 0.05) of basal forage. There was also no effect (P > 0.05) of dietary treatment on plasma Fe or Mn concentrations. Ceruloplasmin (CP) concentrations were higher (P < 0.01) in cows fed the grass silage based ration or with added S and Mo. In contrast, there was no effect of dietary treatment on the CP:Cu ratio, although there was a trend (P < 0.1) for a lower ratio in cows fed the maize silage based ration or in animals receiving added S and Mo. There was no effect (P > 0.05) of dietary treatment on plasma SOD concentration, with a mean value of 2918 U/gHb. Plasma 3-OHB concentrations decreased over the study period from approximately 0.6 mmol/L in wk 0 to 0.3 mmol/L in wk 14, but all values were well below the 1.2 mmol/L considered to be the upper limit. There were no effects (P > 0.05) of basal forage or added S and Mo on plasma 3-OHB concentrations. There was also no effect (P > 0.05) of dietary treatment on plasma urea levels, with mean values being consistent over the study period and well below the 7 mmol/L generally considered to be the upper limit.

5. Conclusions In conclusion, a higher inclusion rate of maize compared to grass silage increased DM intake and milk protein yield. The addition of S and Mo had no effect on DM intake or milk yield in cows when fed the maize silage based ration, but when the grass silage based ration was fed intake and milk yield were reduced. The addition of the antagonist also reduced body condition score in cows when fed the grass or maize silage based ration. In the absence of additional S and Mo, a diet containing 20 mg/kg DM resulted in a net increase in liver Cu concentration of 0.75 mg/kg DM/d in cows when fed either the grass or maize silage based ration. At this rate of increase a cow fed either of these rations would increase from a low liver concentration of 100 mg/kg DM to above the upper limit of 500 mg/kg DM in approximately 18 months. The addition of S and Mo reduced the net increase in Cu deposition in cows fed a maize based ration to almost neutral, whereas in animals fed the grass silage based ration there was a net decrease in liver Cu concentration of 0.6 mg/kg DM/d. There was little effect of added Cu antagonists on plasma Cu or SOD, but ceruloplasmin concentrations were decreased and there was a trend for the ceruloplasmin to Cu ratio to be decreased. Milk SCC was increased in cows when S and Mo were added to the grass, but not the maize silage based ration, but there was no effect of treatment on the incidence of mastitis or reproductive performance.

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6. References Advisory Committee on Animal Feed (2010). Presentation on copper supplementation in

animal feed. Minutes of the meeting held on 15th Dec 2010, Aviation House, London. AOAC (2000). Official Methods of Analysis. 17th ed. Assoc. Offic. Anal. Chem., Arlington,

VA. USA. Black , D.H. and French, N.P. (2004). Effect of trace element supplementation on the

fertility of dairy herds. Vet. Rec. 155: 812-812. Cope, C. M., Mackenzie, A. M., Wilde, D. and Sinclair, L. A. (2009). Effects of level and

form of dietary zinc on dairy cow performance and health. J. Dairy Sci. 92:2128-2135.

Davies, D.C. and Jebbett, I. H. 1981. Liver biopsy of cattle. In Practice, 3:14-15. Gould, L., and N. R. Kendall. (2011). Role of the rumen in copper and thiomolybdate

absorption. Nutr. Res. Rev. 24:176-182. Hart, K.J., Mackenzie, A.M. and Sinclair L.A. (2011). Effect of dietary copper sulphate or

organically complexed copper (Bioplex® Cu) fed either without or with dietary Cu antagonists on the intake, performance and mineral status of early lactation dairy cows. Proc. British Soc Animal Science.

Laven , R.A., Livesey, C.I., Offer, N.W. and Fountain, D. (2004). Apparent subclinical

hepatopathy due to excess copper intake in lactating Holstein cattle. Vet. Rec. 155: 120-121.

Lowman, B. G., Scott, N. A., and Somerville, S. H. (1976). Condition scoring of cattle.

ESCA bulletin No. 6. Mackenzie, A.M., Moeini, M.M. and Telfer, S.B. (2001) The effect of a copper, cobalt and

selenium bolus on fertility and trace element status of dairy cattle. In: Fertility in the High-Producing Dairy Cow Occasional Publication No. 26 Vol.2 pp 423–427 Ed. MG Diskin. British Society of Animal Science, Edinburgh.

NRC (2001). Nutrient requirements of dairy cattle. 7th revised edition. National

Academy Press.

Phillippo, M., Humphries, W.R. and Garthwaite, P.H. (2007). The effect of dietary molybdenum and iron on copper status and growth in cattle. J. Agric Sci (Cambs) 109:315-320.

Sinclair, L.A., Jackson, M.A., Huntington, J.A., and Readman, R.J. (2005). The effects of processed and urea-treated whole-crop wheat, maize silage and supplement type to whole-crop wheat on the performance of dairy cows. Livestock

Production Science 95: 1-10. Sinclair, L. A., Lock, A. L., Early, R. and Bauman, D. E. (2007). Effects of trans-10, cis-12

conjugated linoleic acid on ovine milk fat synthesis and cheese properties. Journal of Dairy Science 90: 3326-3335.

Suttle, N.F. (1991). The interactions between copper, molybdenum and sulphur in ruminant

nutrition. Ann. Rev. Nutr. 11:121-140

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Suttle, N.F. (2010). Mineral Nutrition of Livestock, 4th Edition. CABI Publishing.

Thomas, C. (2004). Feed into milk. Nottingham University Press, Nottingham. Van Soest, P. J., Robertson, J. B. and Lewis, B. A. (1991). Methods for dietary fiber,

neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597.

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7. Appendices

Table 1. Chemical composition of the grass and maize silage

Grass silage Maize silage

Corrected dry matter (g/kg)1 256 341 Crude protein (g/kg DM) 157 75 Ash (g/kg DM) 46 Ammonia-N (g/kg total N) 8.39 9.03 pH 3.9 3.6 ME (MJ/kg DM) 11.2 10.8 Water soluble carbohydrate (g/kg DM) 68.8 26.2 Neutral detergent fibre (g/kg DM) 439 449 Acid detergent fibre (g/kg DM) 246 229 Volatile fatty acids (g/kg DM) Lactic Acetic Propionic Butyric

105.3 22.6 1.06 <0.6

62.1 16.1 0.92 <0.6

Ethanol (g/kg DM) Minerals Ca (g/kg DM) P (g/kg DM) Mg (g/kg DM) S (g/kg DM) Cu (mg/kg DM) Mo (mg/kg DM) Fe (mg/kg DM) Zn (mg/kg DM)

28.1

7.3 3.3 1.7 3.1 8.0

1.43 159.4 37.8

1.84

2.3 2.3 1.5 0.9 4.7 0.59 65.0 23.6

1Corrected for volatiles

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Table 2. Diet composition and chemical analysis of diets high in maize silage (M) or grass silage (G) fed either without (-) or with (+) added Mo and S.

M- M+ G- G+

Diet composition (g/kg DM) Grass silage

133

134

398

399

Maize silage 400 401 133 134 Urea-treated wheat 111 111 167 167 Soy hulls 89 89 89 89 Rapeseed meal 58 58 31 31 Soyabean meal 96 96 31 31 Malt distillers dark grains Sopralin Molasses Protected fat Urea

58 --- 33 13 2

58 --- 33 13 ---

31 58 33 20 2

31 58 33 20 ---

Mins/vits1 7 7 7 7

Total 1000 1000 1000 1000 Chemical analysis (g/kg DM) DM, g/kg Ash Crude protein NDF ADF Ca P Mg S Cu, mg/kg DM Mo, mg/kg DM Zn, mg/kg DM

404 71

181 407 222 5.40 3.57 2.72 1.20 19.9 1.17 49.2

421 71 185 403 224 5.45 3.82 2.84 3.15 19.1 7.94 46.3

364 92 193 381 228 7.84 3.96 2.92 1.32 20.7 1.48 51.8

368 93 194 387 224 7.49 3.69 2.79 3.45 20.5 7.70 48.8

1Mineral/vitamin premix (Rumenco, Staffordshire, UK). Major minerals (g/kg): Ca 240, P 80, Mg 120; Trace minerals (mg/kg): Cu 0, Zn 7,000, Mn 2,000, I 400, Co 80, and Se 50; vitamins (mg/kg) were: retinol 105, cholecalciferol 1.75, and all rac α-tocopherol acetate 5,000. Diets received M+ and G+ also received additional ammonium sulfate and sodium molybdate dihydrate.

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Table 3. Intake and performance of early lactation dairy cows fed diets high in maize (M) or grass (G) silage fed either without (-) or with (+) added Mo and S.

Diets Significance1

M- M+ G- G+ s.e.d. F A Int

Intake, kg DM/d Milk yield, kg/d

23.5 38.1

24.0 40.6

22.6 38.9

20.5 37.9

0.74 1.22

<0.001 0.225

0.111 0.373

0.012 0.034

Fat , g/kg 37.8 36.6 38.2 37.4 1.92 0.656 0.475 0.889 Protein, g/kg 32.5 32.6 31.6 32.5 0.77 0.173 0.901 0.646 Lactose, g/kg 46.5 46.4 46.7 46.3 0.45 0.975 0.328 0.680 Fat yield, kg/d 1.43 1.43 1.47 1.39 0.083 0.944 0.484 0.468 Protein yield, kg/d 1.23 1.30 1.22 1.21 0.034 0.049 0.242 0.142 Lactose yield, kg/d 1.77 1.93 1.80 1.73 0.089 0.185 0.434 0.060 Lwt, kg 651 653 646 639 11.1 0.237 0.818 0.587 Lwt change, kg/d 0.43 0.30 0.20 0.25 0.185 0.309 0.738 0.518 Condition score 2.49 2.35 2.49 2.31 0.066 0.803 0.001 0.744 Condition score change 0.35 0.13 0.27 0.09 0.114 0.470 0.019 0.801 1P values; F= main effect of forage source, A = main effect of antagonists, Int = interaction between forage and antagonists

Table 4. Mastitis and reproductive performance in early lactation dairy cows fed diets high in maize (M) or grass (G) silage fed either without (-) or with (+) added Mo and S.

Diets Significance1

M- M+ G- G+ s.e.d. F A Int

Milk SCC (log10) 1.72 1.50 1.39 1.67 0.138 0.381 0.714 0.017 Cow cases of mastitis 2 3 3 4 NS1 NS NS Quarter cases of mastitis 5 6 3 7 NS NS NS Days to first service2 54 (11) 63 (13) 53 (12) 56 (13) 4.3 0.053 0.214 0.243 Days to conception2 135 (11) 132 (13) 138 (12) 148 (13) 27.3 0.637 0.852 0.738 Services per conception2 3.5 (11) 3.4 (13) 2.9 (12) 3.4 (13) 0.81 0.542 0.706 0.654 1P values; F= main effect of forage source, A = main effect of antagonists, Int = interaction between forage and antagonists 2NS = not significant (P > 0.05) 3Numbers in brackets indicate cow numbers per treatment

15 Report prepared by Harper Adams University on behalf of DairyCo

Table 5. Liver mineral concentrations in early lactation dairy cows fed diets high in maize (M) or grass (G) silage fed either without (-) or with (+) added Mo and S.

Diets Significance1

M- M+ G- G+ s.e.d. F A Int

Initial Cu, mg/kg DM 522 426 407 418 66.4 0.201 0.372 0.262 Final Cu, mg/kg DM 587 437 490 357 58.0 0.038 0.002 0.837 Cu change, mg/kg DM 65 11 82 -61 35.0 0.275 0.001 0.078 Initial Fe, mg/kg DM 378 313 288 295 51.7 0.150 0.422 0.334 Final Fe, mg/kg DM 411 319 352 253 45.0 0.057 0.005 0.908 Fe change, mg/kg DM 33 6 64 -42 30.9 0.690 0.005 0.079 Initial Mn, mg/kg DM 10.20 9.60 9.15 10.41 0.799 0.839 0.560 0.109 Final Mn, mg/kg DM 10.18 10.38 9.96 9.84 0.431 0.223 0.895 0.610 Mn change, mg/kg DM -0.02 0.78 0.81 -0.57 0.790 0.641 0.605 0.060 Initial Mo, mg/kg DM 3.90 3.50 3.39 4.12 0.503 0.878 0.636 0.120 Final Mo, mg/kg DM 3.92 4.19 3.79 4.71 0.312 0.377 0.011 0.149 Mo change, mg/kg DM 0.02 0.68 0.40 0.59 0.640 0.750 0.356 0.600 1P values; F= main effect of forage source, A = main effect of antagonists, Int = interaction between forage and antagonists

Table 6. Plasma mineral concentration in early lactation dairy cows fed diets high in maize (M) or grass (G) silage fed either without (-) or with (+) added Mo and S.

Diets Significance1

M- M+ G- G+ s.e.d. F A Int

Plasma Cu, µmol/L 13.3 13.7 14.3 13.7 0.77 0.340 0.889 0.332 Plasma Mo, µmol/L 0.33 0.50 0.27 0.50 0.041 0.271 <0.001 0.375 Plasma Fe, µmol/L 43.2 40.5 40.7 40.9 2.28 0.519 0.446 0.384 Plasma Mn, µmol/L 0.25 0.24 0.27 0.25 0.014 0.124 0.239 0.740 Ceruloplasmin, mg/dL 17.9 15.9 20.3 18.1 1.12 0.006 0.010 0.909 Ceruloplasmin:Cu 1.37 1.22 1.41 1.36 0.081 0.096 0.090 0.377 SOD2 U/gHb 2960 2841 2954 2915 127.0 0.710 0.387 0.657 3-OHB, mmol/L 0.42 0.38 0.44 0.48 0.067 0.210 0.963 0.406 Urea, mmol/L 5.22 5.44 5.70 5.39 0.268 0.265 0.802 0.172 1P values; F= main effect of forage source, A = main effect of antagonists, Int = interaction between forage and antagonists 2Superoxide dismutase

16 Report prepared by Harper Adams University on behalf of DairyCo

Figure 1. Weekly DM intake in early lactation dairy cows fed diets high in maize silage and fed without (■) or with (□) added S and Mo, or diets high in grass silage fed without (▲) or with () added S and Mo.

Figure 2. Weekly milk yield in early lactation dairy cows fed diets high in maize silage and fed without (■) or with (□) added S and Mo, or diets high in grass silage fed without (▲) or with () added S and Mo.

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17 Report prepared by Harper Adams University on behalf of DairyCo

Figure 3. Fortnightly body condition score in early lactation dairy cows fed diets high in maize silage and fed without (■) or with (□) added S and Mo, or diets high in grass silage fed without (▲) or with () added S and Mo.

Figure 4. Plasma copper concentrations in early lactation dairy cows fed diets high in maize silage and fed without (■) or with (□) added S and Mo, or diets high in grass silage fed without (▲) or with () added S and Mo.

2.0

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18 Report prepared by Harper Adams University on behalf of DairyCo

While the Agriculture and Horticulture Development Board, operating through its DairyCo division, seeks to ensure that the information contained within this document is accurate at the time of printing, no warranty is given in respect thereof and, to the maximum extent permitted by law, the Agriculture and Horticulture Development Board accepts no liability for loss, damage or injury howsoever caused (including that caused by negligence) or suffered directly or indirectly in relation to information and opinions contained in or omitted from this document. © Agriculture and Horticulture Development Board 2012. No part of this publication may be reproduced in any material form (including by photocopy or storage in any medium by electronic means) or any copy or adaptation stored, published or distributed (by physical, electronic or other means) without the prior permission in writing of the Agriculture and Horticulture Development Board, other than by reproduction in an unmodified form for the sole purpose of use as an information resource when DairyCo is clearly acknowledged as the source, or in accordance with the provisions of the Copyright, Designs and Patents Act 1988. All rights reserved. AHDB® is a registered trademark of the Agriculture and Horticulture Development Board. DairyCo® is a registered trademark of the Agriculture and Horticulture Development Board, for use by its DairyCo division. All other trademarks, logos and brand names contained in this publication are the trademarks of their respective holders. No rights are granted without the prior written permission of the relevant owners.

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