Fertilizer-nitrogen: effects on dairy cow health and performance

N.B. COOMBE and A.E.M. HOOD

Imperial Chemical Industries Limited, Agricultural Division, Jealott's Hill Research Station, Bracknell, Berkshire RG12 6EY, England

Key words: nitrogen fertilization, herbage nitrate nitrogen content, dairy cow health, milk yield, milk composition, blood composition

Abstract. The effects on daffy cow health and performance of applying very high rates of inorganic fertilizer nitrogen to grassland have been studied. Two comparable areas of grassland provided the grazing and silage requirements for two separate herds of Friesian daffy cows. These two areas received 250 and 750kg fertilizer nitrogen ha -1 yr -1 . The higher rate was chosen to represent a rate well in excess of the requirements for maxi- mum grass production. The performance and health of the two herds were monitored over five years by measuring milk yield and milk and blood composition for each animal every three weeks.

The only difference in milk yield and composition between the two herds was a consistently higher concentration of NPN in the milk from the N750 herd, but the values recorded were still within the ranges quoted in the literature for daffy cows in commercial herds.

There was some evidence of slightly higher incidences of milk fever and infertility problems in the N750 herd, although in both herds, the incidences were so low that any differences were of little practical significance. Both herds showed an increase in general reproductive disorders as the trial progressed and this may have been associated with a decline in tile copper status of both herds.

The most consistent significant differences between the herds in blood composition was that in serum urea-N values. These were higher in the N750 herd but no conse- quential clinical effects were observed.

There was some evidence to suggest that cows in the N750 herd were able to adapt to a high intake of nitrate by increasing the amount of functional haemoglobin circulating in the blood.

The results of this trial demonstrate that grazing cattle are able to tolerate much higher levels of nitrate-nitrogen in herbage than those commonly quoted as causing acute toxicity; levels of up to 0.52% nitrate-nitrogen were measured in the dry-matter of the N750 herbage. Furthermore, they demonstrate that the potential effects of increased herbage nitrate levels on the health and performance of dairy cows do not impose con- straints on the use of nitrogen fertilizer rates considerably in excess of the current economic optimum. Dairy farmers are in a position therefore to apply the rate of nitrogen fertilizer to grassland that will produce the optimum financial reward, without consideration of the possibihty of adverse effects on herd health and performance.

The in tens i f i ca t ion o f dairy fa rming tha t has occur red in the Un i t ed K i n g d o m

in recen t years can be a t t r i bu t ed largely to the use o f increasing a m o u n t s o f

fert i l izer n i t rogen on grassland. A l though the average appl ica t ion rate o f N

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Fertilizer Research 1:157-1 76 (1980) 016 7-1731/80/0013-0157 ¢03. O0 © 1980 Martinus NijhoffPublishers by, The Hague. Printed in the Netherlands.

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fertilizer on grassland in England is currently only about 100kgNha -~ yr -1 , many successful dairy farmers are using in excess of 300kgNha -1. The average amount used in the Netherlands is about 250 kg ha -1 .

It is well known that high rates of nitrogenous fertilizer application can lead to the presence of high levels of nitrate in grass. For example, concen- trations of up to 0.52% nitrate-N were recorded in the dry-matter of herbage receiving 585kgNha -~ [14]. It is also well known that the addition of nitrate to the diets of ruminant animals, or infusion of nitrate directly into the rumen, can lead to the death of the animals [4, 10, 11, 34]. Nitrate, although relatively non-toxic to most monogastric animals, is reduced to nitrite in the rumen of ruminant animals. Nitrite, when absorbed into the bloodstream, oxidizes the iron of haemoglobin to form methaemoglobin. The latter is incapable of transporting oxygen and the animal may die from oxygen starvation. As a result of these experiments it has been suggested that the LDso for nitrate in cattle is 33 g per 100kg bodyweight [4]. This has been equated to a concentration of 0.92% nitrate, or 0.21% nitrate-N, in the feed. This figure of 0.21% nitrate-N in herbage dry-matter has become widely accepted as the level likely to cause acute nitrate-poisoning in cattle grazing that herbage. In view of the levels of nitrate-N that can accumulate in grass treated with fertilizer nitrogen it is not unexpected that animals grazing herbage that has received high rates of nitrogen application may be con- sidered to be at risk. In fact during the last ten years, there have been many suggestions that heavy dressings of nitrogen applied to grassland can cause acute nitrate poisoning, leading to abortion in pregnant animals, and, in more severe cases, death of the adult animal. It has further been suggested that many of the so-called °production diseases' of present-day dairy herds, are merely the clinical manifestations of sub-acute or chronic nitrate poison- ing, and are the result of the increased rates of nitrogen application to pastures used for grazing. These "production diseases' include disturbances in calcium, magnesium, iodine, copper and vitamin A metabolism, and detri- mental effects on growth, reproductive performance and milk yield. It is perhaps surprising, therefore, to find that in the U.K. in the last fifteen years or so, there appear to be no authenticated, well-documented cases of acute nitrate-poisoning in cattle, which have led to either death or abortion.

This trial was designed to investigate the effects on the health and per- formance of dairy cows of applying high rates of nitrogenous fertilizers to the grassland that provided their complete requirements for grazing in the summer and silage in the winter. The trial was carried out at Jealott's Hill Research Station under normal farm management conditions, using two levels of fertilizer application. The health and performance of the animals were monitored by reference to milk yield and composition, reproductive performance, veterinary examination and blood composition. The first three of these criteria are of obvious practical importance; the regular monitoring of blood composition of the animals on the other hand, was seen as a possible

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means of identifying long-term sub-clinical effects. A brief account of the animal health results for the first four years of the trial has already been published [20].

Methods and materials

Trial outline

Three fields totalling 23 .5h were used for the trial. Two of these fields, totalling 18h, were subdivided into two comparable areas which provided grazing and silage for two separate herds of Friesian dairy cows. One of the areas received a total of 250 kg fertilizer-Nha -1 yr -1 ; the herd supported by this area will subsequently be referred to as the 'N250 herd'. The other area received a total of 750kg fertilizer-Nha -1 yr -1 and the herd supported by this area will be referred to as the 'N750 herd'. The third field provided an additional conservation area for the N750 herd. It must be emphasized that this 750 k g N h a -1 rate of nitrogen application was deliberately chosen to be far in excess of that required to produce the maximum economic production of grass. The 250 kg Nha -1 was chosen to represent a moderately intensive rate of nitrogen application at which, from past experience, no untoward health problems were likely to occur. The two herds were grazed rotationally in paddocks and on the aftermaths following two successive silage cuts each year.

Fertilizer treatments

The two areas of land were subjected to the fertilizer treatments shown in table 1. Nitrogen was applied to the grazing paddocks in six approximately

Table 1. Fertilizer treatments

Treatment k g h a -~ y r -1

N P K

N250 - grazed areas 250 30 57 silage areas 250 55 188

N750 - grazed areas 750 30 57 silage areas 750 55 188

Fertilizer constituents were ammonium nitrate, mono- and di- ammoniun phosphate and potassium chloride

equal dressings between March and mid-August. The first dressing, in March, was applied about one month before the first grazing. The remaining five dressings were applied immediately after the cows had finished grazing a particular paddock, i.e. approximately three weeks before the cows returned to that paddock. Dressings of P and K were applied in mid-season to all grazing areas. The silage areas received N, P and K for each of two cuts and a

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third and fourth application of N for aftermath grazing. Calcium carbonate (5 t ha -1 ) was applied to all areas in 1970 and, during the trial to any field or paddock that was found to have a soil pH-value of below 6.0. Farmyardmanure, collected throughout the winter, was not returned to any of the fields.

Animals

During the year preceding April 1971, all the land to be used for the trial received 250kgNha -1 yr -1 and all the cows in the Jealott's Hill dairy herd grazed this area. Older cows in the herd were replaced with heifers and all cows were paired at the end of the pre-trial year to form two balanced herds for the trial. Once assigned to their herds, animals were not changed from one herd to another for the duration of the trial. Pairings were on the basis of age, lactation number, lactation stage and peak milk-yield. The average num- bers of cows in the N250 and N750 herds throughout the trial were 26 and 30 respectively. Of these, an average of 22 cows in each herd were sampled for blood and milk analysis.

Veterinary record

The disorders recorded in table 3 were diagnosed by the consultant veterinary surgeon to the trial, Mr F A Edgson.

Sampling

Blood and milk samples were taken from each individual cow every three weeks. Samples of blood were taken from the jugular vein using 'Vacutainer' syringes (Becton Dickenson (UK) Ltd., Wembley, Middlesex). Two samples were taken from each animal. A 10ml sample was taken into a tube con- taining 20 mg potassium oxalate and 25 mg sodium fluoride as anticoagulants. This sample was used for packed cell volume, haemoglobin and glucose deter- minations. A 20 ml sample was taken into a tube without anticoagulants and the serum obtained from this sample was used for all other determinations.

On the same day as blood samples were taken, the morning and afternoon milk from each cow was sampled and individual milk yields recorded. For each cow, these samples of morning and afternoon milk were composited (in proportion to morning and afternoon yields) to give a single sample per cow for analysis.

Herbage samples were taken one day before the cows entered each' paddock. Six strips of grass, 0.9 m wide and 9 m long, were cut from each paddock. The grass was weighed, mixed and sub-sampled for analysis.

Analytical methods

Milk analysis

Total nitrogen was measured by the Kjeldahl method [2]. Non-protein nitrogen (NPN) was measured as follows: a weighed sample of milk was deproteinized by mixing with approximately 1.5 times its own

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volume of 15% (w/v) trichloroacetic acid (TCA). After being left to stand for 10 rain, the mixture was filtered under vacuum. The nitrogen content of the filtrate was then measured within 1 hr as described above. Butter-fat content was measured using a 'Milko-tester' (Foss-Electric (UK) Ltd., York). Total solids content was calculated according to British Standard 734, Part 2 (1979), from the density as measured by hydrometry.

Blood analysis

Packed cell volume (PCV) was measured using an Adams 'Autocrit' centri- fuge (Clay Adams, Parsippany, NJ 07054). Haemoglobin was measured by the cyanomethaemoglobin method [38]. Standard solutions of cyanomethaemoglobin, supplied by C F Boehringer and Soehne, Mannheim (Germany), corresponding to known haemoglobin contents were used. This method measures all forms of haemoglobin normally found in blood with the exception of sulphaemoglobin. Mean corpuscular haemoglobin concentration (MCHC) was calculated as follows:

measured haemoglobin (g/100 ml) MCHC (%) = x 100.

PCV

Glucose was measured by an automated glucose oxidase/peroxidase method [371. Urea-nitrogen was measured by the carbamidodiacetyl reaction as described in the Technicon Autoanalyser Method File N-1 c. Inorganic phosphorus was measured by the production of phosphomolybdic acid and its subsequent reduction to give a coloured product, as described in the Technicon Autoanalyser Method File N-4. Albumin was measured by an automated technique involving its binding with bromocresol green [31 ]. Calcium, magnesium and copper were measured using a Perkin Elmer 306 atomic absorption spectrophotometer. Sodium and potassium were measured using a Coming-Eel 170 flame pho- tometer. Asparate aminotransferase (SGOT) activity was measured by monitoring the conversion of NADH to NAD at 340nm on an LKB 8600 reaction-rate analyser using Boehringer test kit, catalogue number 15762. Alkaline phosphatase was measured by monitoring the rate of increase in absorbance at 410nm due to the release of p-nitrophenol. An LKB 8600 reaction-rate analyser and the Boehringer test kit, catalogue number 15987, were used for this determination.

Herbage analysis

Total nitrogen was measured by the Kjeldahl method [2]. Nitrate-nitrogen was measured by the magnesia/Devarda's alloy method [23 ].

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Phosphorus was measured by the production of phosphomolybdic acid [15], after ashing at 500°C and extraction with hydrochloric acid.

Potassium was determined by flame emission and calcium, magnesium, manganese and copper by atomic absorption spectrophotometry, after ashing at 500°C and extraction with hydrochloric acid.

Results

Milk yield and composition

During the first four years of the trial, cows in the N750 herd produced on

average slightly more milk than those in the N250 herd (table 2). In year 5,

however, the converse was true, the yield for the N750 herd being substan-

tially reduced from that in year 4. Despite this, over the duration of the whole trial, the N750 herd produced about 4% more milk than the N250 herd.

No statistically significant differences were recorded in the fat, total solids or protein contents of the milk from the two herds, nor were there any

significant changes in the concentrations of these constituents of the milk

from either herd during the five years (table 2). The NPN concentrations in

the milk from the N750 herd, however, were consistently and significantly

higher than those in the milk from the N250 herd (table 2). It was also

noticeable that the NPN concentrations in the milk from both herds

decreased substantially over the first four years. In year 5, however, the

contents were again increased although not to the levels observed in year 1.

Table 2. Average milk yield and composition for the two herds r

Yield, kg Protein Year and cow -1 yr -1 Fat Total solids (N X 6.38) NPN treatment ± SE % ± SE % ± SE % +- SE % _+ SE

Year 1 N250 4213 ± 552 3.69 ± 0.58 12.36 +- 0.75 3.10 ± 0.31 0.038 +- 0.004 N750 4553 ± 643 3.61 ± 0.39 12.32 +- 0.72 3.14 +- 0.30 0.048 ± 0.007

Year 2 N250 4394 +- 682 3.61 -+ 0.50 12.17 -+ 0.84 3.08 +- 0.46 0.032 ± 0.006 N750 4574 -+ 705 3.68 +- 0.36 12.33 ± 0.84 3.12 +- 0.42 0.045 -+ 0.007

Year 3 N250 4522 ± 666 3.88 +- 0.53 12.43 ± 0.75 3.07 +- 0.29 0.029 +- 0.004 N750 4656 ± 703 3.77 +- 0.48 12.40 ± 0.65 3.11 +- 0.31 0.034 -+ 0.004

Year4 N250 4506 +- 646 4.02 ± 0.67 12.69 +- 0.85 3.04 ± 0.47 0.028 ± 0.004 N750 4956 +- 679 4.00 +- 0.46 12.68 ± 0.67 3.05 ± 0.33 0.034 ± 0.005

Year 5 N250 4709 +- 595 3.58 +- 0.55 12.21 +- 0.85 3.15 ± 0.36 0.034 +- 0.003 N750 4625 -+ 621 3.76 +- 0.60 12.48 ± 0.84 3.23 +- 0.37 0.038 +- 0.005

Reproductive and metabolic disorders

The incidence of various reproductive and metabolic disorders requiring at tention in the two herds is shown in table 3. Both the number of cases of a disorder and the number of cows involved in those cases have been recorded as certain cows suffered recurring disorders. It should also be noted that the total number of animals suffering from reproductive and metabolic disorders

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Table 3. The incidence of reproductive and metabolic disorders in the two herds during the trial

Number of cows Number of cases Item N250 N750 a N250 N750 a

Reproductive disorders Cystic ovaries 5 4 7 4 Retained placenta 11 7 13 10 Abortion 5 4 5 4 Metritis/endometritis 7 2 11 2 Infertility problems 16 20 25 30

Total reproductive disorders 28 26 61 50

Metabolic disorders Milk fever 5 10 12 14 Grass tetany 0 1 0 1 Ketosis 4 4 4 5

Total metabolic disorders 8 12 16 20

aThe numbers of cows and cases in the N750 herd for each disorder have been corrected to allow for there being 30 animals in the herd compared with 26 in the N250 herd

is not the summation of the numbers suffering from individual specific com- plaints because certain animals required attention for more than one problem.

There were differences between the herds in the incidence of certain specific reproductive disorders, in terms of both number of cows involved

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and number of cases. For example, there were more cows in the N250

requiring t reatment for metri t is /endometri t is but slightly fewer cows had inferti l i ty problems. The overall number of cows suffering from reproductive problems, however, was similar for bo th herds. In both herds, the incidence of reproductive problems increased quite markedly as the trial progressed (figure 1).

Very few cows suffered from metabolic disorders during the trial and the only difference between the herds was in the incidence of milk fever. Although there was a similar nu. mber of cases of milk fever in the herds,

there were twice as many cows affected in the N750 herd as in the N250 herd; three of the N250 cows involved each required treatment in three separate years.

Reproductive performance

Table 4 shows the percentage of cows in each herd that were in-calf after 1 ,2 , 3 or more inseminations. The percentage of cows in-calf after one insemin- ation, i.e. the non-return-to-service percentage, was slightly higher in the N750 herd and this contr ibuted to the slightly lower calving interval in the N750 herd. There were no significant differences between the herds in the numbers o f bull and heifer calves produced or their birthweights (table 4).

Table 4. Fertility data for the two herds during the trial

Item N250 N750

% cows in-calf after I insemination 47.2 54.4 % cows in-calf after 2 inseminations 69.2 74.1 % cows in-calf after 3 inseminations 78.8 82.3 Total % cows'in-calf 82.2 85.3 % cows culled 17.8 14.7

Calving interval, days 371 364

Calves produced a Number of bull calves 46 45 b Number of heifer calves 37 43 b Weight of bull calves 41.5 -+ 5.8 40.1 Weight of heifer calves 37.4 +- 8.2 39.4

+-7.1 -+ 4.7

a Data refer only to calves produced between April 1972 and March 1976 b Numbers of calves produced in N750 herd corrected to allow for there being 30 cows in the herd compared with only 26 in the N250 herd

Herbage composition

The concentrations of various constituents of the herbage dry-matter , ex- pressed as overall means from sample cuts taken throughout the five years, are given in table 5.

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Table 5. Mineral composition of herbage dry-matter 1971-1975 means

N P K Ca Mg Mn Cu Treatment % % % % % mg kg- t mg kg-

(1972-75) (1972-75)

N250 2.43 0.31 2.3 0.5 0.16 47 5.1 N750 3.26 0.29 2.3 0.6 0.19 61 6.1

Potassium concentration was unaffected by fertilizer treatment. C~lcium, magnesium, manganese and copper concentrations were consistently higher, and phosphorus concentrations consistently lower, in the N750 herbage.

The most significant differences in herbage composition were in the total nitrogen (table 5) and nitrate-nitrogen (table 6) concentrations. On average, the nitrogen concentration in the N750 herbage was 30% higher than in the N250 herbage. The nitrate-nitrogen concentrations, on the other hand were increased by up to eight times, the highest measured concentration being 0.52% nitrate-nitrogen in year 3.

Table 6. The nitrate-N concentrations in the herbage dry-matter during the trial years

Nitrate-N concentration, %

N250 N750

Year Mean Range Mean Range

Year 1 0.056 0.017-0.157 0.205 0.028-0.392 Year 2 0.039 0.014-0.107 0.286 0.123-0.451 Year 3 0.039 0.008-0.161 0.310 0.108-0.518 Year 4 0.046 0.006-0.112 0.231 0.071-0.476 Year 5 0.057 0.015-0.176 0.188 0.052-0.445

Blood composition

Blood samples were taken from both herds on a total 6f83 sampling occasions throughout the trial. Mean values for each constituent in each year of the trial are presented in table 7. In addition, for each blood constituent, the number of sampling occasions when the mean concentration for one herd was greater than that for the other herd and the number of occasions when the difference between the herd means attained statistical significance, are presented in table 8.

In all five years of the trial, the yearly mean PCV values for the N750 herd were higher than those for the N250 herd (table 7). The sampling occasions when the difference between herd means was statistically signifi- cant (table 8) were spread randomly throughout the five years and occurred regardless of whether the cows were being grazed or fed on silage. The haemo- globin concentrations (table 7) mirrored those for the PCV quite closely. On those sampling occasions when the mean PCV for the N750 herd was significantly greater than that for the N250 herd, the mean haemoglobin

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concentration was almost significantly increased. There was no consistent significant difference between the mean corpuscular haemoglobin concen- trations for the two herds (table 7).

The glucose concentrations measured in both herds were all within the commonly accepted range of 3 7 - 5 4 m g per 100ml [33] (table 7) and although there were statistically significant differences between the herd means on about one third of the sampling occasions, these were not con- sistently in favour of one herd.

On all 83 sampling occasions, the mean value for the serum urea-nitrogen concentration of the N750 herd was higher than that of the N250 herd (table 7) and on the majority o f occasions, the difference between the herd means was highly significant (table 8). All the yearly mean values for the N750 herd were above the normal range of 9.5-19.5 mg per 100ml [33] and on the majority of sampling occasions during the grazing periods the urea-N mean concentration of the N750 herd exceeded this upper limit. The highest value recorded for the N750 herd at any sampling date was 38.9 mg per 100ml and for any individual cow in the N750 herd it was 54.4mg per 100ml. The highest values for both herds occurred in the spring soon after turnout. The yearly mean values for the N750 herd increased throughout the five years (table 7). For the N250 herd, the majority of the mean values for urea-N were within the range quoted above. In the fifth year, however, the mean urea-N values for the N250 herd were considerably higher and the number of occasions when there was a significant difference between the herds was greatly reduced.

There was no consistent difference between the serum albumin values for the two herds (table 7).

Serum calcium concentrations for both herds were within the accepted range of 8 .7-10.3mg per 100ml [33], and although on a number of occasions there were statistically significant differences between the mean values for the herds, these were not consistently in favour of one herd (table 8). Similarly, inorganic phosphorus concentrations were all within the published range of 4.3 to 7.7mg per 100ml [33], andwhilst there were eighteen occasions when the mean for the N750 herd was higher than that for the N250 herd (P < 0.05) there were seven occasions when the reverse was true (table 8).

Serum magnesium concentrations for both herds were within the accepted range of 2 .0 -3 .0mg per 100ml. Whilst there were some occasions when the mean for the N250 herd was significantly higher than for the N750 herd, the levels generally were higher in the N750 cows throughout the trial as a whole.

On nearly half the sampling occasions, there was a significant difference between the mean serum sodium concentrations for the two herds, but again no consistent effect was noted. Serum potassium levels, on the other hand, tended to be higher in the N250 herd. Both sodium and potassium levels were high in both herds in year 4 of the trial.

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Serum copper concentrations and serum enzyme activities were only measured from about halfway through year 2 onwards (table 7). No con- sistent difference between the copper levels in the herds was noted but the concentrations in the blood of both herds declined markedly as the trial progressed (table 7). There was a very large variation in serum alkaline phos- phatase activity between cows in the same herd and therefore, despite the large difference in mean values in year 3 (table 7), there were very few sampling occasions when the difference between means attained statistical significance (table 8). There was similarly no consistent difference between the serum aspartate aminotransferase activities of the two herds.

Discussion

One of the major difficulties in designing a trial of this nature lies in the choice of a suitable 'control' treatment. The lower rate of fertilizer nitrogen used in this study, 250kgNha -1 yr -1, is about twice the overall average rate of application used on grassland in England and Wales. It could there- fore be argued that cows grazing on herbage receiving 250kgNha -I yr -1 do not represent the 'norm' and that any changes in the susceptibility of the dairy cow to metabolic and reproductive disorders may already have occurred in a herd grazing grass treated with this level of fertilizer appli- cation. It was decided at the start of the trial, however, that the disadvantages of comparing an N750 treatment with an N250 treatment were less than those that would arise from a comparison of a high nitrogen application rate with a 'no-N' treatment either in the presence or absence of clovers. In addition, any such disadvantages could be overcome to some extent by com- paring the data from the two herds with the appropriate 'national averages'.

It has been previously demonstrated that ingestion of repeated daily doses of sodium nitrate by dairy heifers induced an erythropoietic response [22] ; as haemoglobin was oxidized to methaemoglobin, the production of red cells was increased. Thus the total oxygen-carrying capacity of the blood was not significantly reduced in these animals and there was no evidence of hypoxia. Although no measurements of either true haemoglobin or methaemoglobin were made in the present trial, the slightly higher total haemoglobin and PCV values observed in the N750 cows, suggest that a siinitar adaptation to the prolonged ingestion of nitrate may have occurred in these animals. The complete absence of any clinical signs of respiratory distress or hypoxia suggests that the extent to which the animals could adapt in this manner was not exceeded.

By far the most significant difference in blood conposition between the two herds was that in serum urea-N levels. The increased levels measured in the N750 cows are entirely expected in view of the increased levels of nitrate and total nitrogen in the herbage (tables 5 and 6), which would lead to increased ammonia absorption from the rumen and, in turn, to increased

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urea synthesis in the liver. No direct measurements of blood or rumen ammonia levels were made, but again the absence of any symptoms of ammonia toxicity indicates that the liver was well able to detoxify this additional loading of ammonia. Additional evidence of the absence of liver damage is provided by the absence of an increase in serum alkaline phos- phatase and aspartate aminotransferase activities. Experimental work has demonstrated that serum alkaline phosphatase [17] and particularly aspartate aminotransferase [16, 18] activities are substantially elevated in cattle suffering a wide variety and degree of liver tissue damage.

No explanation for the gradual cumulative increase in serum urea-N concentrations throughout the trial can be offered but the absence of any rapid build-up suggests that the capacity of the kidneys to excrete urea was not exceeded. Obviously there must be some point at which the concen- tration of urea in the blood interfers with cellular function and produces some pathological condition. The level at which this occurs and the nature of the resulting disorder are not clear from the literature. Higher levels of blood urea-N than those measured in the N750 herd are reported in the literature with no mention of the occurrence of any symptoms of toxicity

i n the animals, e.g. plasma urea-N concentrations of up to 57.5 mg per 100 ml were measured in cows consuming rations containing 5.2% urea pelleted in corn meal [29].

The reason for the substantial increase in serum urea-N levels in the N250 herd in year 5 is not known. Whilst the nitrate-N levels in the N250 herbage were higher in this year than in previous years, they were still considerably lower than the levels measured in the N750 herbage.

The average milk yield of cows in both herds, 4470 and 4672 kg cow -1 yr -1 for the N250 and N750 herds respectively, may be regarded as rather low by certain standards. For example, the average yield for all the herds in the Milk Marketing Board LCP Costed Farms Scheme in 1976 was 4833 kg cow -1 yr -1 [2]. One of the main contributory factors to these lower yields may well have been the low concentrate level of 1.2 tonnes cow -1 used com- pared with 1.7 tonnes cow -1 used on average by the FMS Costed Farms. The reason for adopting a low concentrate usage was to avoid the possibility of diluting any effects of the fertilizer treatment on cow health and performance.

The non-protein nitrogen (NPN) concentrations in the milk from the N750 herd, although consistently higher than those in the milk from the N250 herd, were within the ranges found in the literature for commerical dairy herds, e.g. 0.016-0.047% [6], apart from during year 1. The reason for the high levels of milk NPN in both herds, but particularly in the N750 herd, during the first year of the trial is not apparent. It may be that a period of adaptation to the high levels of NPN in the blood was necessary since during the next two years there was a decrease in milk NPN levels in both herds.

The NPN in milk is mainly absorbed directly from the bloodstream rather than being synthesized by the mammary gland. It is, therefore, likely that the

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increased NPN levels in the N750 milk were due to an increase in urea-N rather than other NPN fractions. This view is supported by work carried out at the National Institute for Research in Dairying (NIRD), Shinfield, which showed that milk from cows grazing herbage that had received 670 kg Nha -1 had an NPN content of 0.035-0.040%, compared with an average of 0.026% in the milk from the rest of the herd; this increase was due almost entirely to an increase in urea content [7]. In the same experiment, the nitrate-N concentration in the milk of the 'High-N' cows was almost undetec- table (less than about 0.4#g1-1). In the present experiment, no measure- ments were made of the nitrate-N concentrations in the milk of the N750 cows but it is unlikely (in view of the NIRD findings) that nitrate-N in milk would present any health hazard.

It has been suggested that diets high in calcium or low in phosphorus encourage the onset of milk fever [32]. It has also been recommended by the ARC that the calcium to phosphorus ratio should be 1.3:1 for a lactating dairy cow [1] although ratios of between 1 and 2 to 1 are generally con- sidered acceptable. Although the calcium content was consistently higher and the phosphorus content consistently lower in the N750 herbage than in the N250 herbage, individually these values were quite normal for pasture grass [26]. Increased calcimn levels in the herbage treated with high rates of nitrogen fertilizer have been reported previously [12], although in the present trial additional limestone applied to the N750 paddocks to control soil acidity may also have contributed to the increased herbage calcium levels.

These changes in herbage calcium and phosphorus contents did produce ratios of calcium to phosphorus in the N750 herbage that were often higher than that considered acceptable. The ratios varied between 1.9 and 2.5 to 1 compared with 1.5 and 1.9 to 1 in the N250 herbage. This may have con- tributed to the larger number of cows requiring treatment for milk fever in the N750 herd. In practical farming terms, however, the incidence of milk fever in both herds was extremely low considering the total numbers of cows involved in each herd for a five-year period. There was cert~/inly no indication from the blood calcium results of a prevailing sub-clinical hypocalcaemia problem in the N750 herd.

Hypomagnesaemia occurs most frequently on pastures with a high grass/low dover content. This increased susceptibility to tetany shown by animals grazing such swards is thought to result from the lower levels of magnesium that are generally present in grass species. Heavy nitrogen appli- cations to grass/clover swards will encourage dominant growth of the grass species, and may also lower the apparent availability of the herbage magnesium [36]. Consequently, use of heavy dressings of nitrogenous fer- tilizers has often been implicated as a causal factor in outbreaks of grass tetany [3, 5, 13], particularly when the N applications are accompanied by dressings of potash [21, 24]. In practice, however, dressings of either nitrogen alone or potassium alone did not induce a reduction in serum

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magnesium concentrations in animals grazing the pasture but combined dressings did have this effect which was often followed by the onset of tetany [35]. During the whole five years of this trial, only one case of clinical hypo- magnesaemic tetany was recorded and the serum magnesium concentrations did not suggest the existence of a subclinical problem in either herd. In fact, blood magnesium concentrations were higher in the N750 herd than in the N250 herd, reflecting the higher percentages of magnesium found in the N750 herbage. These results suggest that by delaying the application of potassium to pasture for grazing until after the first fllash of spring grass, the incidence of hypomagnesaemic tetany can be extremely low even when excessively high rates of nitrogen application have been used.

It is surprising that the mean annual concentration of serum potassium in the N250 animals were slightly higher in all years except year 3, because the average potassium concentrations in the herbage from the two treatments were similar. The high serum sodium and potassium values recorded in both herds during year 4 are difficult to explain. The grazing season during this year was very dry but the yalue for other blood constituents do not indicate any degree of dehydration at this particular time.

Reproductive problems in dairy herds have often been attributed to excessive consumption of nitrates and, by inference, to the use of nitrogenous fertilizers on grassland. Abortions have been produced experimentally by infusing nitrate directly into the tureen [34], and by adding nitrate to the feed [11], of pregnant heifers. In an extensive review of nitrate toxicity, however, it was conlcuded that the level of nitrate causing abortion in the ruminant animal cannot be distinguished from that which would cause the death of the animal [40]. It is impossible to say whether or not the abor- tions that occurred during the trial were a result of the high intake of nitrate by the cows. Neither herd was declared brucellosis-free until March 1975 and use of the Brucella 45/20 vaccine was not stopped until the spring of 1973. The total number of abortions that occurred during the five years was not very great in either herd (five in the N250 and four in the N750 herd). The additional stress on a pregnant cow of being loaded into a crush and blood-sampled every three weeks cannot be dismissed as a possible cause of some of these abortions.

Several veterinary observations have indicated that, in herds suffering from copper deficiency, an increase in the frequency of returns to service was observed [30]. It has also been demonstrated that the copper status of cattle declines during the grazing season and that 'lush' herbage (as would be produced by the application of nitrogenous fertilizer) promotes a more rapid decline in hepatic copper reserves than older herbage or hay of similar copper

content [19]. Although no consistent difference was observed between the serum copper concentrations of the two herds, a decline in the serum copper levels of both herds did occur during the last three years of the trial. Blood copper levels may not necessarily be the most sensitive indicator of copper

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status in the dairy cow; for example, it has been found that whilst blood copper levels in cattle remained constant, liver copper levels decreased signifi- cantly [12]. The guidelines given in the Committee on Mineral Nutrition, TNO [8] would tend to indicate that, in year 5 of this study, the copper status of the two herds was barely adequate to prevent the appearance of clinical symptoms of copper deficiency. This hypothesis is supported by the copper levels in the herbage being lower than those generally considered normal, i.e. 7 -30 mg kg -~ of dry matter [39]. The part that nitrogen fertilizer may have played in creating this potential problem cannot, however, be determined from this experiment because no differences in blood copper concentrations between treatments were recorded. From the plant nutrition viewpoint 'available' copper in the soil, which averaged around 3.0 mg kg -~ of dry soil, was adequate in the fields grazed by both herds.

Although the incidence of certain reproductive disorders, particularly those associated with infertility, was slightly higher in the N750 herd than in the N250 herd, the figures were not considered to be of practical significance in either herd when expressed as percentages of the number of 'cow-years' over which these data were collected. The increase in the incidence of these problems in both herds as the trial progressed, however, may have some significance in relation to the possible association between blood copper status and fertility referred to previously. The mean non-return-to-service percentages for both herds over the five-year period (54% for the N750 herd and 47% for the N250 herd) were lower than that usually considered satis- factory (between 55 and 60%). It is impossible to say to what extent this slightly low fertility and increasing incidence of reproductive problems can be attributed to the use of high rates of nitrogen fertilizer application. Factors other than high nitrogen application in the trial could presumably have had adverse effects on fertility. For example, in order to retain as many of the original cows as possible in the two herds throughout the trial, culling was less stringent than in a commercial herd. The actual culling-rates were 18% and 15% for the N250 and N750 herds respectively compared with a more normal culling-rate of 20-25% for commercial dairy herds in this country. The type of problem for which a cow would normally be culled from a herd, but in this trial was not, would be for example, pseudo-pregnancy (a cow returning to heat after missing two or three oestrus cycles after insemination). The low culling rate would have allowed any effect of increasing herd age on fertility to express itself. The only definite conclusion that can be drawn from these fertility data is that the additional nitrogen applied to the herbage grazed by the N750 animals did not further adversely effect the fertility of the N750 herd in comparison with the N250 herd. The percentage of cows in-calf after one insemination for the N750 herd was higher than that for the N250 and the numbers of calves produced by the two herds were similar.

Experiments in which single doses of nitrate have been either infused directly in to the rumen or added to the feed, have indicated that the LDso

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for nitrate for the ruminant is about 33g per 100kg bodyweight [4]. This figure has been equated to a concentration of 0.21% nitrate-nitrogen in herbage dry-matter. More recent work has demonstrated that the acceptable dose of nitrate can be appreciably increased firstly by spreading the dose over a 24-h period and secondly by allowing a period of adaptation to a nitrate-rich feed [25]. Both of these factors are obviously relevant to any discussion of nitrate poisoning in the grazing animal. Despite this 0.21% is still widely quoted as the level of nitrate-N in herbage dry-matter that is likely to cause acute nitrate-poisoning in cattle, possibly resulting in death. On certain occasions during the present trial, the N750 cows ingested herbage containing over twice the amount of nitrate-N alleged to cause acute toxicity without any apparent harmful effect. This confirms the above observation [9], that if ingestion of nitrate-containing feed or herbage is spread over an extended period, as in the grazing animal, the amount of nitrate that can be tolerated is substantially greater than that indicated by single-dose LD5o experiments.

The results of this trial identified some differences between the two herds in blood and milk composition and in the incidence of certain reproductive and metabolic disorders. These differences, however, were of little signifi- cance in practical farming terms. The general health status and overall performance of both herds was more than satisfactory in view of the con- straints imposed by the trial design as discussed above. This investigation and a similar one carried out in the Netherlands [12] demonstrate quite con- clusively that increased herbage nitrate levels and their potential effects on dairy cow health and performance do not impose any constraint on the use of nitrogen fertilizer rates considerably in excess of the rates currently recommended for optimum grass production [28]. The absence of any such constraint allows the choice of rate of fertilizer nitrogen application to be made solely on the basis of economic considerations.

Acknowledgments

The authors are indebted to the following individuals for their part in this work: Mr. F.A. Edgson Veterinary Consultant, Dr P. Jackson and ICI col- leagues, J.H. Bailie, A.J. Golightly, F.A.T. Holmes, F.G. Banks, D.W. Lock, C. Sealey, M.J. Smith, Mrs C. Wootton.

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