For all blood metabolites measured, diet did not have an effect (P > 0.05; Table 35). Table 35 Mean blood plasma metabolites for dairy cows fed diets that contained (36:64 forage DM basis) lucerne and maize silage; lucerne plus tannin and maize silage, red clover and maize silage and red clover plus tannin and maize silage.
Treatment
Lucerne Red Clover P value
-Tannin +Tannin -Tannin +Tannin s.e.d Forage Tannin FxT
n 192 192 192 192 Total protein, g/l 75.1 75.3 75.9 74.3 1.298 0.929 0.474 0.328 Albumin, g/l 35.5 35.8 36.1 35.9 0.476 0.347 0.823 0.468 Glucose, mmol/l 3.24 3.20 3.22 3.25 0.072 0.835 0.910 0.426 Urea, mmol/l 4.79 4.64 4.59 4.44 0.159 0.081 0.190 0.996 BHB1, mmol/l 0.574 0.602 0.657 0.626 0.043 0.079 0.966 0.330 1 β-hydroxy butyrate
141 Table 36 Blood plasma metabolites over four time points for dairy cows fed diets that contained (36:64 forage DM basis) lucerne and maize silage; lucerne plus tannin and maize silage; red clover and maize silage; and red clover plus tannin and maize silage.
Treatment
Lucerne Red Clover P value
-Tannin +Tannin -Tannin +Tannin s.e.d Treatment Time TxT
n Time 12 12 12 12 Total protein, g/l 0700 74.4 76.0 76.7 74.8 2.595 0.900 0.316 0.008 0900 76.4 74.7 70.8 74.7 1100 74.6 76.2 77.2 74.8 1300 74.9 74.4 78.9 73.0 Albumin, g/l 0700 35.1 35.3 36.0 35.8 0.953 0.880 0.309 0.252 0900 36.3 35.7 35.0 35.8 1100 35.3 36.5 36.4 36.5 1300 35.3 35.9 36.8 35.6 Glucose, mmol/l 0700 3.36 3.31 3.25 3.28 0.143 0.931 <0.001 0.940 0900 3.44 3.37 3.65 3.53 1100 3.16 3.08 3.12 3.09 1300 3.00 3.02 3.10 3.08 Urea, mmol/l 0700 4.33 4.25 4.05 3.93 0.319 0.632 <0.001 0.878 0900 4.58 4.57 4.45 4.35 1100 4.82 4.61 4.82 4.55 1300 5.42 5.12 5.05 4.93 BHB1, mmol/l 0700 0.492 0.500 0.510 0.518 0.086 0.513 <0.001 0.512 0900 0.404 0.414 0.440 0.364 1100 0.612 0.715 0.830 0.737 1300 0.787 0.791 0.850 0.909 1 β-hydroxy butyrate
142 33.5 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 0700 0900 1100 1300 P la sm a a lb u m in g /L Hours of day 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 0700 0900 1100 1300 P la sm a B HB m m o l/L Hours of day
For all blood metabolites measured, diet did not have an effect (P > 0.05; Table 36), and there were no interactions between diet and time except for total protein (P < 0.05). Plasma albumin did not change during the day, (P > 0.05) with mean values of 35.5, 35.7, 36.2 and 35.9 g/L for 07:00, 09:00, 11:00 and 13:00 hours respectively (Figure 28).
Figure 28 Plasma albumin in dairy cows fed diets that contained lucerne silage (; n = 12); lucerne silage with tannins (●; n =12); red clover silage (; n =12) and red clover silage with tannins (●; n =12). Error bar indicates pooled s.e.d.
Plasma BHB decreased (P < 0.001) from 07:00 h to 09:00 h before increasing to 13:00 h (Figure 29 and Table 36), but there was no effect of dietary treatment (P > 0.05).
Figure 29 Plasma BHB in dairy cows fed diets that contained lucerne silage (; n = 12); lucerne silage with tannins (●; n = 12); red clover silage (; n =12) and red clover silage with tannins (●; n = 12). Error bar indicates pooled s.e.d.
143 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 0700 0900 1100 1300 P la sm a g lu co se m m o l/ L Hours of day 3.00 3.50 4.00 4.50 5.00 5.50 6.00 0700 0900 1100 1300 P lasm a ur ea m m ol /L Hours of day
Plasma glucose increased (P < 0.001) from 07:00 h to 09:00 h before decreasing to 13:00 h (Figure 30 and Table 36) but there was no effect of dietary treatment.
Figure 30 Plasma glucose in dairy cows fed diets that contained lucerne silage (; n = 12); lucerne silage with tannins (●; n = 12); red clover silage (; n = 12) and red clover silage with tannins (●; n =12). Error bar indicates pooled s.e.d. Plasma urea increased (P < 0.001) with time from 07:00 h to 13:00 h (Figure 31 and Table 36) but there was no effect of dietary treatment.
Figure 31 Plasma urea in dairy cows fed diets that contained lucerne silage (; n = 12); lucerne silage with tannins (●; n = 12); red clover silage (; n = 12) and red clover silage with tannins (●; n = 12). Error bar indicates pooled s.e.d.
144 64 66 68 70 72 74 76 78 80 0700 0900 1100 1300 P la sm a to ta l p ro te in g /L Hours of day
Plasma total protein did not change during the day, (P > 0.05) with mean values of 75.5, 74.1, 75.7 and 75.3 g/L for 07:00, 09:00, 11:00 and 13:00 h respectively (Figure 32 and Table 36) and there was no effect of dietary treatment.
Figure 32 Plasma total protein in dairy cows fed diets that contained lucerne silage (; n = 12); lucerne silage with tannins (●; n = 12); red clover silage (; n = 12) and red clover silage with tannins (●; n = 12). Error bar indicates pooled s.e.d. 5.3.2.7 Whole tract digestibility
Dry matter intake was highest (P = 0.009) in cows fed diets containing lucerne silage and lowest in cows fed diets containing red clover silage (Table 37). Tannin inclusion had no effect (P > 0.05) on dry matter intake. Similarly, faecal DM output was highest (P = 0.041) in cows fed diets containing lucerne silage. However, there was no effect (P > 0.05) of tannin inclusion on faecal DM output. Both
organic matter (P = 0.004) and nitrogen (P <0.001) intake was highest in cows fed diets containing lucerne silage with mean values of 20.61 kg/d and 582 g/d
respectively. Tannin inclusion had no effect (P > 0.05) on faecal OM output or nitrogen output. Dietary treatments had no effect (P > 0.05) on DM, OM or nitrogen digestibility and had mean values of 0.678, 0.688 and 0.613 kg/kg respectively.
145
Table 37 Digestibility of DM, OM, and N in dairy cows fed diets containing (36:64 forage DM basis) lucerne and maize silage, lucerne plus tannin and maize silage, red clover and maize silage and red clover plus tannin and maize silage.
Treatment
Lucerne Red Clover P value
-Tannin +Tannin -Tannin +Tannin s.e.d Forage Tannin FxT
n 12 12 12 12 Dry matter, kg/d Intake 21.4 22.9 20.2 19.6 1.15 0.009 0.533 0.206 Faecal output 6.78 8.14 6.32 6.10 0.837 0.041 0.341 0.189 Digestibility, kg/kg kg/kg 0.683 0.647 0.687 0.695 0.031 0.250 0.521 0.317 Organic matter, kg/d Intake 19.8 21.3 18.8 17.8 1.06 0.004 0.774 0.101 Faecal output 6.08 7.32 5.69 5.47 0.768 0.046 0.353 0.189 Digestibility, kg/kg 0.695 0.660 0.699 0.699 0.0309 0.333 0.421 0.430 Nitrogen, g/d Intake 564 601 499 480 29.0 <0.001 0.674 0.177 Faecal output 204 245 191 192 24.6 0.066 0.220 0.253 Digestibility, kg/kg 0.640 0.594 0.617 0.604 0.036 0.803 0.260 0.531
146 5.4 Discussion
5.4.1 Feed analysis
This is the first study to investigate the addition of hydrolysable tannins to lucerne and red clover silages in a maize silage based diet as dietary treatments to
compare effects on animal performance, milk quality and apparent digestibility in high yielding dairy cows. The dry matter content of all the forages in the current study fell between ranges of 215 – 456 g/kg, however, the lucerne silages had the largest DM content while red clover silages were 238 g/kg lower. The DM content of lucerne and red clover silages differed to previous studies by Hymes – Fecht et al. (2013) and Broderick et al. (2000) in which the DM content averaged 52 g/kg lower for lucerne silage and 308 g/kg higher for red clover silage than the silages in the current study. In the study of Broderick et al. (2000), both the lucerne and red clover silages were a mixture of first, second and third cuts whereas in the current study both silages were second cut. Similarly Hymes – Fecht et al. (2013), also fed second cut lucerne and red clover silages, however the difference in DM content may reflect the different varieties of lucerne and red clover used. In the current study, varieties Daisy (lucerne) and Corvus (red clover) were used whereas Hymes - Fecht et al. (2013) used Forecast 1001 and Rebound 4.2
(lucerne) while the red clover variety was Marathon. Alternatively, wilting time and weather conditions may have influenced the DM content between studies.
The crude protein content of lucerne and red clover silages in the current study were similar to values observed in previous studies (Broderick et al., 2001; Broderick et al., 2000 and Hoffman et al., 1997), with red clover silage having a lower CP content. For all the forages, pH values were below pH 4.6 suggesting the forages had fermented, which was reflected by the rapid drop in pH between days 1 and 7 post ensiling. The lactic acid content of the lucerne silages indicated good fermentation and preservation as the content was above 60 g/kg DM, similar to previous study by Dewhurst et al. (2003). However, in the current study, the red clover silages had high levels of butyric acid and ammonia nitrogen indicating poor fermentation and preservation. The poorer quality of red clover silage may have been influenced by the DM content at ensiling and weather conditions during the wilting period as there were periods of rainfall prior to harvesting. Fibre levels
147 observed in the current study were higher in the red clover than the lucerne silage, but were similar to that reported by Hymes - Fecht et al. (2013).
Tannin content in the current study for lucerne and red clover silage without tannin had values of 16.2 and 23.5 g/kg DM. Hymes-Fecht et al. (2013) reported lucerne silage to have no naturally occurring tannins while red clover silage had a tannin content of 2.3 g/kg DM. These variations in tannin content between the current study and Hymes-Fecht et al. (2013) may reflect the sensitivity of the methodology used as there was an intra-assay CV of 22.5%. This high CV indicates the
presence of a large variation between samples suggesting that the tannin content was not consistent throughout the clamps of silage. This intra-assay CV may also indicate that the method used to determine tannin content in the current study had poor sensitivity for the level of tannins present in the silages. Although, the
addition of tannins did increase the tannin content of lucerne and red clover silage slightly by 5.8 and 1.6 g/kg DM, respectively, this was lower than expected, as hydrolysable tannins were added at a rate of 25 g/kg DM. It may indicate that the tannins were not fully homogenised throughout the clamps, resulting in variable samples which may be attributable to the application and mixing method of the tannin to the forage prior to ensiling. The reliability of these results require further assessment, the current method used was the butanol-HCl assay. An alternative method that could be used to investigate tannin levels in lucerne and red clover silage is the acidified vanillin assay. However, a previous study (Broadhurst and Jones, 1978) investigated the effects of temperature and light on the sensitivity of the acidified vanillin assay and observed varying results. It was observed that both light and temperature influenced the assay and therefore impacted on the tannin concentration measured. As time increased, it was observed that there was a decline in absorbance when the reaction was exposed to light. However, this decline could be prevented by completing the reaction in the dark with the samples remaining stable for up to 60 minutes. The ambient temperature also affected the rate of the reaction. Where the assay was incubated at a temperature of 25°C it took 12.5 minutes to reach maximum absorbance, whereas at lower temperatures of 20°C and 15°C it took longer (Broadhurst and Jones, 1978). Therefore,
temperature and light were potentially affecting the butanol-HCl assay used in the current study thus impacting the estimation of tannin concentrations. This would suggest that the butanol-HCl assay needs to be further investigated to optimise
148 the temperature and light to enhance the sensitivity of the technique. Therefore, it can be concluded that multiple variables have potentially influenced the tannin concentrations observed in this experiment.
5.4.2 Degradation characteristics
Forage had an effect on the soluble DM fraction (a) where lucerne silage had higher content, and the potential degradable fraction (b), where red clover silage had higher content. The soluble protein fraction (a) was increased by the
supplementation of tannins in the current study. In contrast, Tabacco et al. (2006) reported a decrease in the soluble protein fraction as the inclusion rate of
hydrolysable tannin increased from 0, 20, 40 to 60 g/kg DM in lucerne silage. Rate of degradation (c) for DM and CP was higher in lucerne than red clover silage which is consistent with Coblentz et al. (1998) and Tabacco et al. (2006). These observations are similar to that reported by Broderick et al. (2004) when protein degradation was conducted in vitro, with lucerne silage having a faster rate of degradation compared to red clover silage. Effective degradation was also
affected by dietary treatment for DM and CP with lucerne silage being higher than red clover silage and for DM, supplementation of tannin increased the effective degradation of red clover silage but not lucerne silage. In contrast, Tabacco et al.
(2006) reported a decrease in effective protein degradability as the
supplementation of hydrolysable tannin increased in lucerne silage. Tabacco et al.
(2006) concluded that the addition of tannins could reduce the high rumen degradable protein that is associated with forage legumes like lucerne and red clover silages, although the results from the current study, where hydrolysable tannin was included at 25 g/kg DM does not support this.
5.4.3 Animal performance
In the mid-lactation dairy cows used in the current study, DM intake was improved by the inclusion of lucerne silage in a maize based diet. In previous studies
(Hoffman et al., 1998; and Dewhurst et al., 2003) improvements in DM intake have been reported when cows were fed lucerne silage compared to feeding grass silage alone. In contrast, in studies (Arndt et al., 2015 and Sinclair et al., 2015) comparing different inclusion rates of lucerne silage in a maize silage based TMR, DM intake did not differ between treatments except in Sinclair et al. (2015) who reported a drop in DM intake when lucerne silage was included in the TMR at a
149 rate of 60 % forage DM. Dry matter intakes have been shown to be improved by red clover silage when fed as a sole forage or at high inclusion rate in a grass based diet (Moorby et al., 2009; and Bertilsson and Murphy., 2003). Similar to the current study, Broderick et al. (2000) reported a higher DM intake 1.2 kg/d in cows offered lucerne silage over red clover silage. Broderick et al. (2000) concluded this difference in DM intake may be attributable to the higher CP content of 36 g/kg DM in the diets containing lucerne silage. However in the current study, the differences in DM intake may be attributable to the quality of the red clover silage which had high levels of butyric acid and ammonia nitrogen indicating poor fermentation and preservation. In addition, the high difference in DM content of the lucerne and red clover silages (238 g/kg DM) may have influenced DM intake.
Although DM intake was improved by lucerne silage, cows fed lucerne and red clover silages had a similar milk yield. Similarly, Broderick et al. (2000) and Hymes – Fecht et al. (2013) reported no effect on milk yield when cows were offered lucerne or red clover silage. In contrast, Hoffman et al. (1998) reported increased milk yields of 1.6 kg/d when comparing lucerne silage to grass silage. Previous studies have reported improvements in milk yield when red clover silage was compared with grass silage (Moorby et al., 2009); or as part of maize based diet (Moorby et al., 2016). The similarity in milk yields in the current study may be attributed to the similar crude protein content of all four treatment diets.
There was no effect of dietary treatment on milk fat, milk protein or milk lactose content in the current study. Arndt et al. (2015) also reported no effect of dietary treatment on milk fat or lactose content. However a decrease in milk protein was reported as lucerne silage in the diet increased from 20 to 80 % forage DM. Similarly, Moorby et al. (2009) and Moorby et al. (2016) reported a decline in milk protein content as the amount of red clover silage in the diet increased from 0 to 100 % forage DM. These changes in milk protein content as the amount of
legume silage in the diet increases may reflect the high rumen degradable protein that is present in legume silages, therefore reducing the amount of metabolisable protein available for digestion in the small intestine. The responses in milk
composition observed in the current study may have differed if the inclusion rate of hydrolysable tannins had been higher allowing an increase in rumen undegradable protein being available in the small intestine. However, in a previous study, Taha, (2015) fed hydrolysable tannin at four inclusion rates (0, 25, 50 or 75 g/kg DM) to
150 lactating ewes and found that there was no effect on milk protein suggesting that there was no increase in availability of RUP in the small intestine when tannin was added to the diet. Similarly, Lui et al. (2013) found that the inclusion of
hydrolysable tannins at 10 g/kg DM in the diet of early lactating dairy cows had no effect on milk protein. Therefore, the current study used an inclusion rate of 25 g/kg DM of hydrolysable tannins as milk yields were highest in ewes fed 25 g/kg DM hydrolysable tannin in the study by Taha, (2015). The inclusion of tannins however, had no effect on N efficiency in the current study, however forage source had a significant effect with N efficiency improved in cows fed red clover compared to lucerne silages principally due to the lower DMI in red clover fed cows. The higher N efficiency with red clover silage may be related to the presence of PPO binding with the protein (Lee, 2014), increasing the amount of RUP available for digestion in the small intestine. The lack of an effect of tannin on N efficiency is consistent with Dschaak et al. (2011) when lucerne hay was supplemented with 30 g/kg DM of condensed tannin. In contrast, Gerlach et al. (2018) reported a decline in N efficiency when grass silage was supplemented with condensed tannins at a rate of 30 g/kg DM. Dschaak et al. (2011) suggested that these observations in N efficiency may be due to no change in milk protein yield which is consistent with the current study.
5.4.4 Milk FA composition and blood metabolites
Inclusion of tannins had no effect on milk FA composition, however forage source altered a number of FAs. Milk fat content of C4:0, C18:2 n-6 and C18:3 n-3 were all higher in milk from cows fed red clover compared to lucerne silage. The
changes in linoleic acid (C18:2 n-6) and α-linolenic acid (C18:3 n-3) are consistent with previous studies where red clover silage has been compared to grass silage (Halmemies-Beauchet-Filleau et al., 2014; Dewhurst et al., 2003; Vanhatalo et al., 2007). Moorby et al. (2009) suggested that changes in linoleic acid and α-linolenic
acid in milk may be a result of lowered biohydrogenation of forage fatty acids in the rumen possibly influenced by the polyphenol oxidase (PPO) enzyme system that is present in red clover silage. Similar to the current study, Lee et al. (2009) observed an increase in milk fat content of C18:2 n-6 and C18:3 n-3 in cows fed fresh red clover compared to cows fed fresh grass. Lee et al. (2009) suggested that the linoleic acid and α-linolenic acid are provided protection from rumen metabolism by the presence and binding of PPO in the red clover. Lucerne silage
151 improved milk fatty acids C12:0, C15:0, C17:0 and C22:0 compared to cows fed red clover silage. Similarly, Leduc et al. (2017) reported increased levels in milk fatty acid concentrations of C12:0, C15:0 and C17:0 in cows fed lucerne silage compared to red clover silage as part of a TMR based diet. Leduc et al. (2017) suggested these increases in FA content of C15:0 and C17:0 may be due to higher concentrations of propionate and valerate available in the rumen for elongation during rumen fermentation by the microorganisms to form C15:0 and C17:0. Similarly, Sinclair et al. (2015) observed an increase in concentration of C17:0 from 0.48 to 0.51 g/100g when lucerne silage was increased in the diet of dairy cows from 20 to 60%, respectively. However, in contrast to the current study, Sinclair et al. (2015) observed an increase in C18:2 n-6 and C18:3 n-3 as lucerne in the diet increased, suggesting that biohydrogenation was reduced in the rumen. Dietary treatment had no effect on fatty acids of chain length more than C16, indicating uptake from the diet or less than C16, indicating de novo synthesis, in