Red clover grown in a mixture with grasses: yield, persistence and dynamics of quality characteristics

Red clover (Trifolium pratense L.) was grown in mixtures with timothy and meadow fescue in field experiments at four sites in Finland to broaden knowledge on it’s potential as a forage crop. The effects of cutting frequency, nitrogen fertilization in the spring and sward density were investigated. Forage yield quality was analyzed using standard methods. Red clover produced well in all swards during the two first seasons. In the third summer the proportion of red clover was greatly diminished except on sandy soil at the northernmost locality (64 ̊40'N) where it remained productive. Linear regression adequately described the dependence of crude fibre content and crude protein content in dry matter of the primary growth and regrowth, both of red clover and grass, on accumulated temperature sum. The contents of P, K, Ca and Mg in clover and grass are given as a function of accumulated temperature to describe their changes during crop growth. The results give new knowledge about possibilities to grow red clover in the northern livestock region of Finland. They proved that soil type is more important for good persistence of red clover than latitude.


Introduction
Mixtures of red clover and timothy have been grown as the principal components of hay swards in Finland since the last decades of the nineteenth century when cultivation of grass in rotation with cereals began.In the early 1960s the sward area was at its greatest, 52%, corresponding to 1.4 million hectares: 1.1 million hectares were harvested for dry hay (Official Statistics of Finland 1961) and 74% of seed mixtures sown included red clover (Raatikainen and Raatikainen 1975).
The focus of studies on red clover has been improvement of its persistence.Under Finnish conditions red clover content in a sward decreases rapidly because of abiotic and biotic stresses incurred during winter after the second harvest Mela, T. Red clover grown in a mixture with grasses season (Paatela 1953, Ylimäki 1967).A considerable number of studies were directed at breeding (Ravantti 1980, Multamäki andKaseva 1983) and testing (Valle 1958, Paatela 1962, Mela et al. 1980) new persistent red clover genotypes and improving the hardiness of plant material.Bjursele in particular, a local variety from northern Sweden, and some newer varieties, demonstrated increased persistence in Finland.A Finnish tetraploid variety was durable but had too low a seed yield (Valle 1959, Mela 1969).
Following development of improved methods of harvesting and storage, as well as generation of new knowledge on the benefits of increased N-fertilization on grass yields and protein content, the area allocated to grass-only swards cut for silage began to increase rapidly in the 1960s.Most farmers also stopped growing red clover in their hay fields because it did not fit with mechanized hay drying in the field.Scattering of leaflets resulted in it losing much of its feeding value.As a consequence, the decreased research on red clover was directed at its suitability for intensive methods of cultivation.Red clover-grass mixtures proved to be profitable because they gave higher crude protein yields without N-fertilization than abundantly N-fertilized grass swards, although dry matter yields were lower (Salonen andHiivola 1963, Hakkola andNykänen-Kurki 1994).Irrigation, another component of intensive cultivation, increased red clover yields more than yields of grasses, but did not decrease their crude protein content as it did for grasses (Raininko 1968).Thus red clover could have represented a feasible option for producing protein rich feed.However, most farmers opted for N-fertilized pure grass swards that they considered to provide reliable forage yields.
During the last two decades interest has increased in forage legumes.Currently, the area devoted to organic farming in Finland is about 160 000 hectares and red clover is commonly grown in rotation for forage and to provide nitrogen-rich green manure on-farm (Mela 1988).Recent studies on red clover have concentrated on feed quality.The greatest advantage of red clover-grass silage over pure grass silage has proved to be increased intake and enhanced milk yield (Heikkilä et al. 1992, Vanhatalo et al. 1995, Heikkilä et al. 1996).Red clover forage is not associated with bloat or hypomagnesia because of its high magnesium content.Disadvantages are oestrogenically effective compounds in red clover (Kallela 1974) and high liquid loss if not predried (Syrjälä-Qvist et al. 1984).
In spite of occasional increased interest in red clover, large research projects on its cultivation under the range of growth conditions that exist across Finland have not been organized except for official variety trials.This investigation addressed this shortcoming.The effects of cultivation methods on yields, sward development and dynamics of common yield quality characteristics as a function of accumulated temperature sums were studied for red clover-grass mixtures across a range of growth conditions at four research sites in Finland.

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The trials were established as split-split plot designs.Treatments were allocated as follows: Main plots, cutting time = A: a 1 = two harvests, for hay and regrowth, a 2 = two to three harvests at silage stage, The area of the subplot was 15 m 2 .There were four replicate blocks.
Soil analysis was done at the start of the experiments at all sites, and also at the end in Jokioinen.According to the results P-and K-fertilizers were applied at establishment and during the course of the trials.The barley cover crop was fertilized only with small amounts of nitrogen to preclude lodging.For the split plots, the N-treatment was given once per season in the spring when grass growth had begun.
Plants were harvested from different parts of the subplots of each replicate and combined to provide a sample representing a treatment, but not a replicate.Botanical analysis was done to separate red clover, grasses and weedson a 0.5 kg subsample.In Jokioinen these species fractions were dried and analyzed separately.At the other localities 2 × 100 g subsamples of the original mixed sample were chopped fresh, dried and analyzed.Samples were dried in oven at 105˚C for two hours and then at 60˚C overnight.Total drying time was 14 to 16 hours.
The amount of reduced nitrogen was measured using a Kjeltec Auto 1030 analyzer (Tecator, Höganäs) and the Kjeldahl procedure (Tecator Kjeltec Auto, 1030Analyzer Manual, 1987).The crude protein concentration was calculated by multiplying the nitrogen concentration by 6.25.Crude fibre concentration was measured using the Fibertec System M (Tecator, Höganäs) and the Weende method (Tecator Fibertec System and Manual, 1978).Ca, Mg, K and P were analyzed with a Perkin Elmer 2100 flame atom absorption spectrophotometer.
Data on grass yields were analyzed using standard two-way analysis of variance: PROC GLM of SAS.Tukey's Studentized Range Test was used to compare statistically significantly different means among treatments.For regression analysis, t-tests and standard supplementary statistical calculations were used.

Performance of red clover
Red clover comprised the main part of the dry matter yields during the first year's harvest at Jokioinen and Sotkamo (Figures 1-3).In 1990 in the Jokioinen A experiment red clover dominated regrowth and gave as high a yield of regrowth as for the initial harvest (Fig. 1).
The proportion of red clover in the yields of the other experiments during the first year was also high, except for the first harvest of the Jokioinen B experiment.The red clover content of the second year's harvest of Jokioinen A trial, excepting the first cut of sward for pasture, and the second year's harvest at Maaninka were substantial.The red clover contents in the trials at Ruukki were high, not only during the first and second years but also during the third year of harvest, except for the pasture sward in the A trial of the second year.
As a rule red clover content of the initial harvest was smaller than that for regrowth.There are two reasons for this: the first is the innate slower start of growth of red clover than that of grasses in the spring, the second is N-fertilization given before the first growth in the spring, which promoted grass growth but not that of clover.
The red clover content in the Jokioinen A experiment was 80% for the second year autumn regrowth over all cutting treatments.Red clover was low 8 to 20 in the Jokioinen A experiment in the third year autumn regrowth except for in the hay sward, which included about 40% red clover in the regrowth.Similarly as for the Jokioinen trial, red clover was low in the Sotkamo experiment in the third year.Red clover content in the Maaninka experiment was at its peak of 50 to 80% at autumn harvest of regrowth in the first year and decreased by 20% in the second year.
Red clover is not drought hardy.In the spring, soil is wet and water is not a limit to growth, but the amount of precipitation can strongly affect regrowth after a cut for silage or pasture.Thus drought (Table 1) decreased growth in Jokioinen in June 1990, July 1991, July 1994and August 1994 (Figures 3-4).Abundant rainfall increased growth in Jokioinen in July and August 1993, in Maaninka in July and August 1992 and in Ruukki in July and August 1992.
Jokioinen B 1992-1995 7.6. 28.6. 6.8. 4.9. 24.6. 18.7. 26.8. 9.6. 30.6. 24.8. 1990 1991 1992 Ruukki Heavy clay characterising Jokioinen soil and silt in Sotkamo (Table 2) did not favour the persistence of red clover.In Ruukki well-aerated sandy soil allowed red clover to develop deep and strong roots and form a long-lived sward.pH values for all soils of the trials were satisfac-tory for red clover and the concentration of minerals was maintained through annual fertilizer application.In Maaninka and Sotkamo heavy and long-lasting snow cover (Table 1) created conditions favouring low-temperature fungal development, which probably killed red clover.

Effect of the number of cuttings and age of sward on yields
In most experiments the highest yield was recorded in the first year (Tables 3-7).Jokioinen A, Maaninka and Sotkamo experiments produced more than 10 t ha -1 yield in the first season, in Ruukki yields were somewhat smaller.An exception was the Jokioinen B experiment that failed in 1992.Swards harvested as hay and regrowth yielded most, swards harvested three times per season as silage yielded less and swards harvested three to five times per season as pasture yielded, as a rule, less than grass for silage.Yields from Jokioinen A trial (Table 3) decreased rapidly over time, to a half in the second and to a fifth to fourth in the third year compared with the yield from the first year.Jokioi-   4) yielded most in the second year and remained productive up to the fourth year.In the Sotkamo experiment (Table 5), which included only one cutting treatment, grass cut for hay and regrowth, yields decreased rapidly in subsequent years, similarly as in the Jokioinen A experiment.In Maaninka (Table 5) and Ruukki A (Table 6) experiments yields were quite stable up to the third year.The yields in Ruukki B (Table 7) experiment remained high throughout the trial period.

Effect of nitrogen fertilization on yields
N-fertilization generally increased dry matter yield.However, the effect was small when content of red clover was high, as in the first year swards of Jokioinen A, Maaninka and Sotkamo experiments.N-fertilization given in the spring variously affected the cutting treatments depending on the relative proportions of primary growth and regrowth and their different red clover and grass contents in the sward.This was demon-

Effect of growth density on yields
The ability of red clover and grass species to compete with each other and to fill empty space in a sward was tested through the row distance treatment.In nine years out of fifteen, the 12.5 cm inter-row distance promoted a statistically significantly (P ≤ 0.05) larger yield than 25.0 cm.In six other cases the yields were equal.Red clover content of dense swards was higher than that of thin swards.Red clover contents by sward density and degree of N-fertilization were as follows: 12.5cm/25.0cm(0N) 59a/51b, (30) 54a/ 47a, (60) 46a/40a, (90) 43a/37a.Statistically sig-

Yield quality
Crude fibre and crude protein contents Linear regression described well both the crude fibre (CF) and crude protein (CP) contents of red clover and grass yields as a function of accumulated temperature for the Jokioinen experiments  (Figs 4 and 5).Results of the second harvest were not included in the regression calculations because the number of culms, which increases in a sward after the primary growth, depends much on the time of the harvest and is not possible to predict.These culms have a substantial effect on the quality of the second harvest.
Based on the corresponding regression values reported in Figures 4 and 5, estimates were made of dependence of organic matter digestibility (OMD) and crude protein digestibility (CPD) of red clover and grass on temperature sum.The Kivimäe (1959) function (OMD = 94.3 -1.01 × CF) was used to calculate OMD values for red clover and the Korva and Tuori (1986) function (OMD = 92.0-139.3/CP-0.01261 × CF 2 ) to calculate OMD values for grass in Fig. 6.Crude protein digestibility values for both red clover and grass in Fig. 7 were calculated using the Korva and Tuori (1986)  Based on models developed by Kivimäe (1959) for red clover and Korva and Tuori (1986) for grass, and crude fibre (CF) and crude protein (CP) values from functions indicated in Figures 4 and 5.According to the calculations, OMD of red clover and grass primary growth were 80.2% and 74.8%, respectively, and at 200˚C accumulated temperature (AT), which was the average time of the first cut of sward for pasture (Fig. 6).At 400˚C AT, the time just preceding hay harvest, OMD of the primary growth red clover was 75.6% and that of grass 66.3%.OMD of regrowth red clover was 80.7% at 200˚C AT and 78.4% at 400˚C.OMD of regrowth grass was 75.6% at 200˚C and 72.8% at 400˚C AT.
CPD of the primary growth red clover (Fig. 7) was 79.7% at 200˚C AT and 76.1% at 400˚C AT.CPD of the primary growth grass was 70.9%

Minerals
Ca content of the primary growth red clover decreased but Ca content of the regrowth increased, when the sward aged (Table 8).Mg content did not change over time.P content of both the primary growth and regrowth as well as K content of regrowth red clover decreased over the growth period.Ca and P content of the primary growth and Mg content of grass regrowth increased and K content of the grass primary growth decreased over time.Contents of other minerals studied were unchanged over time.N-fertilization decreased Ca content of the spring harvested red clover (y = 13.42 -0.0115x; r = 0.213 * ; SE = 1.58; n = 128), but it did not affect Ca content of red clover regrowth or other mineral contents of red clover.N-fertilization increased Ca content (y = 2.93 + 0.0048x; r = 0.254 ** ; SE = 0.56; n = 151), P content (y = 2.77 + 0.0036x; r = 0.181 * ; SE = 0.64; n = 144) and K content (y = 24.28+ 0.052x; r = 0.388 *** ; SE = 3.48; n = 152) of the first harvest of grass species, and increased Ca content (y = 4.74 + 0.0039x; r = 0.117 * ; SE = 1.07; n = 297) of regrowth of grass species.

Discussion
According to previous results, red clover survives for only two to three years in the field (Paatela 1953, Williams 1982).This was the case also in the present study.In the Ruukki experiment, one reason for the good survival through three years could have been attributable to the variety grown,

Mela, T. Red clover grown in a mixture with grasses
Bjursele, which originates from northern Sweden and has been shown to be hardy in variety trials (Mela et al. 1980).Björn, another winterhardy Swedish variety, was grown in the other experiments of the study.Obviously the most important reason for the good survival at Ruukki was the sandy soil, which was well-aerated and allowed red clover taproots and Rhizobium to develop optimally.Furthermore, a thin snow cover is typical for the western coast region of Finland where Ruukki is located, and as a result soil is well frozen in winter, which prevents lowtemperature fungi from developing and damaging plants (Mäkelä 1981).Maaninka and Sotkamo are located in the less favourable deep snow region in eastern Finland.The heavy clay soil of Jokioinen fields and the summer dry periods did not favour red clover growth, and explain its lack of persistence there.
The growth of the experimental swards was based very much on red clover as nitrogen fertilizer was applied only in the spring at the start of growth and mainly favoured the growth of grass in the initial harvest.N-fertilizer applied in the spring has only a small effect on regrowth capacity (Agerberg 1943, Julén 1954).Some nitrogen becomes available through mineralization of plant material, harvest litter and dead roots, but it only partly satisfies the need of grasses for nitrogen through the summer (Ingebrigtsen 1959).To increase grass growth significantly, adequate N-fertilizer should be applied for regrowth even in grass-clover mixtures.However, it should not stimulate excessive competition between the grass and red clover.
At the time of this study estimated feed digestibility values were generally based on crude fibre and crude protein analysis, at least when the number of forage samples to be analyzed was large as is usually for field experiments.Other available methods were too expensive.Use of NIR (Near Infrared Reflectance) for organic matter digestibility determinations was developed but the required equipment became generally available in laboratories later during the 1990s (Hellämäki 1992).
Models based on CF and CP values were used for calculating the digestibility of organic mat-ter and crude protein (Kivimäe 1959, Korva andTuori 1986).In the present study, OMD and CPD contents based on these models are presented as a function of accumulated temperature sum.
The calculated OMD and CPD contents are close to the corresponding values from other studies: for OMD (Salo et al. 1975, Mela and Poutiainen 1975, Sanderson and Wedin 1989, Rinne and Nykänen 2000): for OMD and CPD (Kivimäe 1965, Feed tables and feeding recommendations 2002 by MTT Agrifood Research Finland (Tuori et al. 2002), based on plant material from different sources.
The digestibility of herbage can be predicted through observation of the phenological stage of plants (Kivimäe 1959, Deinum et al. 1968, Mela and Poutiainen 1975, Rinne and Nykänen 2000), accumulation of temperature degrees (present study) and even by determining the proportion of leaves in dry matter (Rinne and Nykänen 2000).
During primary growth the phenological stage of a single species is easily identified from the stage of ear and flower development.However, when there is a mixed sward of different species their relative proportions influence forage quality.Consequently, due to their differential development and growth rates it is difficult to forecast forage quality.Another difficulty is in estimating the digestibility of regrowth after the first cut as it includes a varying number of culms depending on the temperature during primary growth, the growth stage of grass at cutting, grass species and probably genotype.
Regrowth includes tillers at different stages of growth and it is difficult to determine its phenological stage.Regrowth consists mainly of leaf mass, the chemical composition of which changes relatively slowly.Therefore, specific knowledge of the dependence of the quality characteristics on accumulation of temperature degrees combined with botanical analysis can be considered a suitable method to predict the digestibility for practical purposes.
N-fertilization does not affect digestibility much, if at all (Mela and Poutiainen 1975, Fagerberg and Ekbohm 1995, Bélanger and McQueen Vol. 12 (2003): 195-212. 1998), but it affects protein content significantly.Except for N-fertilization there are several factors that affect the protein content of herbage, as the amount of free nitrogen in soil, soil organic matter and its decomposition, it is changes in air and soil temperature and soil aeration that affect the rate of decomposition and control soil N reserves available to plants.
There is close correlation between plant development and protein content.Quality of the tillers at the same phenological stage and established at the same time is similar.On the other hand, tillers at the same stage but established later have lower protein content.This depends on changes in competition in the sward for light and nutrients.Thus, the way a sample for analyses is taken is of great importance (Thorvaldsson 1989).More tillers and leaves develop in a low density sward than in a dense sward, which also affects quality (Wivstad 1997).
Weather conditions affect development of a sward and both quality and yield.A cold period slows down or stops growth and development of both red clover and grass: a large part of a grass stand remained in the node stages and budding of red clover was delayed one to two weeks (Fagerberg 1988b, Thorvaldsson andFagerberg 1988).After a long dry period precipitation stimulates growth of high quality new stems and leaves (Mela and Youngner 1976).Rising temperature hastens maturity and reduces carbohydrate content and digestibility (Deinum et al. 1968, Ruegg and Nösberger 1977, Fagerberg 1988a).High temperatures increase respiration more than photosynthesis and promote lignification of the cell walls.This results in reduced digestibility.
Similar to other results, those of the present study showed that an increase in N-fertilization increased K and Ca (Hiivola et al. 1974, Rinne et al. 1974).In contrast with previous results Nfertilization increased Mg content and decreased P content of grass.Differences in mineral content of grass among the experiments depend greatly on the various experimental treatments and different soil mineral contents.In the study of Rinne et al. (1974) there was only one cutting treatment, three cuts for silage and N-fertilization treatments ranged from 0 to 600 kg ha -1 .The mineral contents in the present study correspond quite well with those of Feed tables and feeding recommendations 2002 (Tuori et al. 2002).The mineral content of plants varies greatly according to soil mineral content (Jokinen 1979).Advantages of red clover for feeding dairy cattle have been demonstrated in several experiments.Better animal performance is achieved by feeding cattle with red clover-grass silage compared to pure grass silage of similar digestibility.Positive effects of red clover on milk output and composition were evident when silage included 30 to 70% red clover (Heikkilä et al. 1992).Milk output was increased and the protein/fat ratio of milk was improved compared with meadow fescue-timothy grass mixture for silage.Cows consumed more red clover-grass silage than grass silage and milk output was increased (Vanhatalo et al. 1995, Heikkilä et al. 1996).This resulted both from increased microbial N and decreased rumen N degradability for the red clover diet (Vanhatalo et al. 1995).Disadvantages of red clover are marked effluent losses (Syrjälä-Qvist et al. 1984) with high plant oestrogen content (Kallela 1974, Mela et al. 1993) and risk of bloat with cattle on pasture.

Fig. 2 .
Fig. 2. Development of swards and their red clover content cut for silage.Effects of age of sward, date of cutting and Nfertilization in spring.Results of row distances are combined.Filled sections of columns indicate proportion of red clover and unfilled sections the proportion of grass.

Fig. 5 .
Fig.5.Crude protein (CP) content of red clover and grass as a function of temperature sum.Experiments conducted atJokioinen in  1990Jokioinen in   -1995.   .

Fig. 7 .
Fig. 7. Crude protein digestibility (CPD) of red clover and grass as a function of temperature sum.Based on model developed by Korva and Tuori (1986), and crude protein (CP) values from functions indicated in Fig. 5.

Table 1 .
Monthly mean temperature and precipitation for those growing seasons at research stations when trials were performed.Snow cover duration and maximum depth during winter before each growing season are indicated.

Table 2 .
Analysis of soils at trial sites.In Jokioinen samples were collected both in spring when the trial was begun and in autumn when it was finished, at other sites samples were collected only in spring at trial initiation.

Table 3 .
Dry matter yields (kg ha -1 ) of red clover grass mixture as influenced by number of cuts and N-fertilization inJokioinen in 1990Jokioinen in  -1992.   .

Table 4 .
Dry matter yields (kg ha -1 ) of red clover grass mixture as influenced by number of cuts and N-fertilization in Jokioinen in 1992-1995.

Table 6 .
Dry matter yields (kg ha -1 ) of red clover grass mixture as influenced by number of cuts and N-fertilization inRuukki  in 1990Ruukki  in  -1992.   .

Table 7 .
Dry matter yields (kg ha -1 ) of red clover grass mixture as influenced by number of cuts and Nfertilization inRuukki in 1992Ruukki in  -1994.   .

Table 8 .
Mean mineral contents (g kg -1 ) of primary yield and regrowth of red clover and grass as a function of temperature sum.