Influence of urea feeding duration on nitrogen metabolism of rumen bacteria and their host sheep

.In,,,talI;eedScien~euund Trchnologv. 40 (1993) 117-189 ElsevierSciencePublishers B.V.. Amslerdam

177

Influence of urea feeding duration on nitrogen metabolism of rumen bacteria and their host sheep Y. Kobayashi, M. Wakita and S. Hoshino Faculry of Bior~x~urces. Me lQ~n,wrsic~~. Tsrr. .Mw 514. Japan (Received

4 October

1991: accepted

2 July 1992)

ABSTRACT Kobayashi, Y.. Wakita.M. and Hoshina,S.. 1993.Inlluenceof ureafeedingdurationon nilrogen metabolism

of rumen bacterm

and their host sheep. .&~un. FeedSci. Techno!.. 40: 177-l 89.

I

Two trials, usins I shce? in total. were conducted to investigate mminal reswnses 10 long-term urea feedingin the dia. In Tnal I isomtrogcnous semipurified duets wth soyabean meai or urea as a main nimxen source were offered to eight sheep for 60 days in a sinalecro~soverdesign. Nitrogen retention o?urea-fed sheep was improved as a &ion of ;ime cf f&ding. while fhat the control was Trot. Thiswasthe result largely of decreased nitrogen output in the urine. Ureolytic activity of mixed bacteria tended 10 decrease gradually when urea was fed. Ruminal axwcia and volatile fatly acids delennined one sampling day in every 10 days were not adequate parameters 10 explain the increase in niwogen rewntion. In Trial 2 using. three sheep, all of which were allotted the soyabean meal diet (for IDdays) followed by the urea diet (for 65 daysl. more frequent analyses in vitro and in wvo were performed m monitor poslprandial changes in ruminal parameters and their varialion with duration of urea feeding. With in viwo analysis bacterial urmlysiswas rapidlydepressed when urea feedingwasstarted and thedepression became moreapparcnt in laterstagesofurea feeding. This decreased ureolysis was also observed in the in viva experiment ofTrial 2. Sheep fed urea inilially had a lower density of bacteria in Leir rumen. bul the density recovered 10 the same level as that of control sheep in later stages of urea feeding. These resulfs suggest thl bacteria play an important role in the long-term adaplation to urea in mmen funclmn.

of

INTRODUCTION

Urea, an important nitrogen (N) source for ruminants, has been investigated extensively to characterize its metabolism in the rumen and host animals. Rapid hydrolysis of urea in the rumen, often inducing more N loss via ammonia absorption across the rutnen wail, resiricts etXcient use of dietary urea by nlminants. However, sheep (Ludwick et al., I97 1) and cattle (Qltjen and Putnam, 1946) receiving a urea-containing diet tend to retain more N as Correspondcwc

0 1993 Elsevier

m: Y. Kobayashi.

Science: Prbiishers

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of Rioresources.

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the duration of feeding increases; this trend has been termed ‘the adaptation to urea feeding in ruminants’. Using regression acaiysis, McLaren et al. ( 1965) showed that retention of absorbed N was increased by Z-3% units for each 10 days up to 55 days. This adaptive response may include metabolic moditications attributable to rumen microbes (Clifford et al., 1968) and host tissues (Salem et al., 1973; McLaren et al., 1976). However, these possibilities have not been completely assessed. Studies on the mechanisms underlying the adaptive phenomenon would be helpful in improving the utilization of urea in the diets of ruminants. The present experiment was conducted to determine long-term responses of rumen bacteria and the host to urea as a main source of N in the diet. MATERIALS

AND METHODS

Animals, diets, samplings and incubations Trial I

Eight wethers weighing an average 40 kg were employed. They were randomly divided into two groups of four and supplied either one of the semipurified diets containing soyabean meal or urea as the main N source (75% of the total N). Ingredients and chemical composition of the diets are shown in Table 1.The treatmen?s were carried out for 60 days and consisted of a 10 day preliminary period followed by five successive 10 day collection periods. Each diet was offered twice daily at 09:OOand 2 1:00 h at a level of 450 g-. per feeding with free access to water: Total fecal and urinary collections were made during the first 5 days of each cohection period. Ten percent of daily feces was sampled, sprayed with 10% HCl, dried in an air oven (60°C) and ground for analysis. Urine collected daily in a bottle containing 5% HzS04 (20 ml) was pooled and frozen until analysis. On the last 2 days of each collection period, ruminai fluid samp!es were taken 3 h after the morning feed via a stomach tube. Harvesting of mixed rumen bacteria and preparation of a ceil-free extract for enzyme assays were performed according to Baldwin and Paimquist (1965). Trial 2

Three wethers, weighing an average 42 kg, were fitted with ruminai cannuiae and used in the following in vitro and in vivo experiments. Experimental semipurified diets, feeding level and feeding time were the same as those employed in Trial 1.Sheep were fed a soyabean meal diet for the first IO days (Period C) and then the diet was changed to a urea diet for the following 65 days of feeding [Period U). Ruminai digesta were taken through the cannuiae just before the morning feed on the last 2 days of Period C and on Days 1,2, 4, 8, 42, 17, 26, 38, 50 and 65 of Period U (Ui, U2, U4, .... respectivdy).

Diet Urea (U)

Soyabran

40 30 20

40 20 14 19

-3 7 Trace

mean IC)

7 Trace

lo.8 13.4 12.6 27 s ‘Percmtage composition: CaHPO,. 48.9: K2COI. 3I S: MgSO,. 10.8; N&I. 7.j: F&O,. 0.91: N&B+O,. 0.19: ZnSO,, 0.14; MnS04.0. lo: CuCOI. O.O_: ’ KI. U.Sx Moo,. 0.008; CaCI>. 0.0003. ‘IU kg-‘: A.4404 D. 551): E. 88. TABLE 2 Changes

m N balance

of sheep (Trial Diet’

N intake &day-‘!

N loss (%ofN into feces

I) Colleclion

period’

I

2

3

4

5

IS.6 15.6

14.8 15.6

14.4 15.6

15.2 15.6

15.3 15.5

0.1

30.3 33.7 50.9*b 48.5

32.2 32.6 49.8” 49.2

29. I 30. I 48.0ab 49.6

28.8 31.7 47.lb ‘u.3

0.3 0.4 I.0 0.3

-

SE

iotnke) U C :

29.4 31.8 55.4’ 50.2

N digested (%ofNmtake)

U C

70.6 68.2

69.7 66.3

67.8 67.4

70.9 69.9

71.2 68.3

0.5 0.5

N retained (% of N intake)

u C

15.1’ 18.0

18.8’~ 17.8

18.0ab 18.2

22.gb 20.3

24.2”’ 20.0

I.1 0.4

into urine

Means ofeight ammals itn presrnied. ’ ?vleans within row that share no camnon superscript direr rrgdficantly ‘Means differ si.enificant!> from the corresponding control (PcO.05). ‘Sheep were fed the respective xmipuriiied diet for a IO da) preliminary succcssivc lOday collection peri& ‘See Table I.

(PcO.05). period followed by live

The digesta were strained through two layers of surgical gauze and employed in in vitro and in viva experiments. In the in vitro experiment the supematant of centrifuged ( IOOOxg, 5 mitt) strained ruminal fluid was centrifuged again (2! OOOxg, 20 min), washed twice with buffer (pH 6.8; Maeng et al., 1976) and bacteriai cells were harvested. The cells were suspended in the same buffer and used for in vitro batch culture incubation (6 i .8 ? 2.7 mg dry matter (DM ) of bacteria per bottle) with urea (33.8 mg) and soluble starch (337.5 mg; Merck, Rahway, NJ, USA) for 1, 2, 3, 4, 6 and 8h at 37°C under an Oz-free CO2 atmosphere. Duplicate tubes were employed in each incubation. Rates of urea degradation, ammonia and volatile fatty acid (VFA) production and bacteriaI growth were monitored. In the in viva experiment straiaed ruminal fluid samples were obtained at 0, I, 1.5,2,2.5,3,4,6 and 8 h after the morning feed on individual sampling

Fig. I. CLnges in activilies of urease (UR). glutamine synthetase (GS) and glummale dehydrogenase (GDH) in ruwen bacterial extract (Triai I ). 0-O(A-A ), U: l - -O(AA). C. A vertical bar rcprusents standard crrot of mean (n=X. IJR; n=6, GS and GDH). # Refer to Table 2.

LONG-TERM EFFECT OF UREA

cmSHEEP RUMEN

BKTERIAL

181

YEThBGLlSM

days and used to assess postprandial changes in ruminal levels of urea, ammonia, VFA and bacteria. On these sampling days urea was removed from the diet, dissolved in water and infused through the ruminal cannulae via falling drop equipment for 20 min; in this way the same rate of urea ingestion was attained for all the animals. Analyses The nitrogen contents of feeds, feces and urine were analyzed by the Kjeldahl method (Association of Official Analytical Chemists (AOAC), 1975). Other components of feeds were determined by standard AOAC methods (AOAC, 1975) anddetergenttiber (VanSoestandWine, 1967;AbeandHorii, 1978) procedures. Activities of urease (EC 3.5. IS), glutamate dehydrogenTABLE 3 Changes

in ruminal

hem

parameters’ Diet’

and NDFdigeslibility Collecnon

I U C

period2 4

3

2 6.81 6.75

I)

(Trial

6.80 6.71

SE

5

6.78 6.72

6.75 6.74

6.82 6.70

0.03 0.02

16.1. 7.8

14.9. 7.5

i5.96.3

1.2 c5

10.7 10.6

10.2

9.9

9.7

0.3 0.2

66.0 67.4

65.2 69.8

65.7 68.7

0.2 0.5

l?l’ 8.0

15.i 7.8

Tot:11 VFA’

9.4

(mmoldl-‘)

9.5

10.3 10.7

Acetate’ (M%)

65.6 67.6

65.0 68.7

Propion:&

25.4 23.1

23.9

26.

24.4

22.9 22.1

25.7 23.5

0.6

25.9

6.8 9.4

7.5 a.5

6.2 a.9

7.0 11.2

5.8 9.5

0.3 3.5

27.7 27.4

28.4 27.0

23.8 26.7

26.6 34.6

31.0 32.3

I.2 1.7

Ammonia (mgdl-‘)

N’

(M%) Butyntc’ (M%) NDFdigestibiliry (%)’

U C

‘Analyzed using the samples taken 3 h after feeding. ‘Refer 10 Table 2. ‘Mean of eight animals. ‘Mean of fouranimals. ‘See Table I ‘.“Means differsignificantly from the corresponding

I I.2

control

I

(Pc0.W

PcO.01).

0.5

182

Fig. 2. Changes in urea and mmcma levels in vitro culture (Trial 2). X Samples mxl were taken on Days 9 and IO of feeding soyabem meal diet IC) and the appropriate days of feeding urea diet (U4. U8. U 12. U 17, U26, U38, US0 and U65). A value in each graph shows disappemnce rate of urea (mg h-l). “,%lues witn no common superscript differsignificantly (PcO.05). A vertical bar represents standaruerror of mean (n=3).

ase (ET 1.4.1.2 and EC 1.4.!.4) andglutamine synthetase (EC 6.3.1.2) were determinedaccordingto Suto (1973), JoynerandBaldwin ( 1966), and Hubbard and Stadtman ( 1967), respectively. Specific activities were calculated by dividing total acrivity by the protein concentration as determined by the Folin merhod (Lowry et al., 195I ). Ammonia, urea ;.nd VFA in the rumen and in vitro culture were measured using a phenol-typochlorite reaction (Weatherburn, 1967), a diacetylmonoxime reaction (Yonemura, 1973) and gas chmmatography (Suto, 1973), respectively. Hexose disappearance in the in vitro culture was monitored by a phenol-sulfuric acid reaction (Dubois et ai., i956). Bacterial counts in the ruminal samples wrq’edetermined by a direct microscopic procedure (Suto,

LONO.TERM

EFFECT OF UREA ON SHEEP RtJMEN B.xTEtuAL

183

MElABOLtSM

1973 ) and also enumerated as amylolytic and cellulolytic organisms with selective carbohydrate media (Anderson et al., 1987). In Trial 1 the effect of treatment at each sampling time and tlzz effect of the feeding period within each treatment were examined by Student’s t-test. The same test was applied to data in Trial 2 to make comparisons between samples taken in a different period. RESULTS

Trial 1 Changes in N balance with feeding duration are shown in Table 2. Throughout the experiment sheep fed soyabean meal retained N at an almost uniform efficiency. In urea-fed sheep, however, the ratio of N retention to N intake became higher as the period of urea feeding lengthened (Pt0.05, Ul vs. U4 and U5). The value in Period 5 of uren feeding was higher (P-zO.05) than that ofthe control. Nitrogen output through the urine in urea-fed sheep gradually decreased (P~0.05, Ul vs. U5). Figure 1 illustrates changes in urease, glutamate dehydrogenase and glutamine synthetase activities of rumen bacterial extract. Urease activity in ureafed sheep tended to decrease with the feeding duration, while that in the control showed no apparent change. Although NADH-linked glutamate dehydroTABLE 4 Changes in rater of bancria~ growth. VFA production and hexose disappearance in in vitro culture ( irial2) --Sample’

Bacterial N (mgh-I)

Bacterial DM (msh-‘)

VFA production (pmol b- ‘)

Hexose disappearance (n&h-‘)

C tJ4 UB

0.57* OSl’b 0.49’b

10.ba 9.3”b 8.P

lula 99’ 82”

40.2’ 32.P 40.6’b

lJ1: u17 U26 U38 US0 Ub5 SF

li.431b 0.53’ 0.43b 0.4.P 0.59’ 0.51’ 0.01

6.9’ 8.6’” 8.1”c 7.7’bC 7.3b’ 9.7’b 0.3

73bC 87’” 6@ 67’b 79b 65” 4

33.lb 38.7’b 31.7=b 33.9’b 39.7’ 37.2” 1.0

Means of three animals are presented. ‘Wasbed bacterial incwla for in vitro culture were prepared from ruminal fluid taken on the sampling day (abbreviationndeli~ed in Fig. 2). ,‘,b,‘Mea..;.ktLi: I*??: wlumn that share no common superscript differ significantly (PcO.05 ).

184

Y.kOB*YSHtETAL.

activity was slightly higher than that of the NADPH-linked enzyme, no significant difference was observed between the treatments and treatment periods. Glutamine synthttase activity showed a tendency to decrease with the duration of urea feeding, while its activity remained constant in the control. Chemical characteristics of rumen fluid and neutral detergent fiber (NDF) digestibility are given in Table 3. Sheep fed urea showed a slightly higher ruminal PI-I attributable to more ammonia production, but the duration of urea feeding did not influence either parameter. Diets and feeding periods did not significantly affect total VFA level, its molar proportion or NDF digestibility.

genase

Trial 2 In vitro experiment Ureolysis and ammonia releasing capacity of mixed rumen bacteria in a batch culture are shown in Fig. 2. Ureolysis and ammonia production rate in

Fig. 3. Postprandial ch;nps A ruminal urea and ammonia levels (Tnal 2). ir Refer 10 P!P. ! A vertical bar represems standard error at mean (u= 3).

LONG-TERM

EFFECT OF “REA ON SHEEPRUMEN IUcTERlAL

METABOLISM

185

bscteria from sheep not receiving urea were extremely rapid, i.e. the urea kr the tubes disappeared within 3 h and a high ammonia accumulation was recorded. The bacteria from sheep receiving urea, however, degrade? mea more slowly (PcO.05, Ccntrol (C) vs. U4, U8, U38, US0 and U65), and the degradation rate tended to be lower after Day 12 of Period U. Rates of bacterial growth, VFA production and hexose disappearance in a batch culture are shown in Table 4. Bacterial growth rate in terms of dry matter (DM) h-r or N h-’ was depressed once in Period U (PcO.05, C vs.U12 or U26) but recovered later, as was the case with the hexose disappearance rate (P-cO.05, C vs.U12). VFA production rate became lower as the urea feedinglasted (PcO.05, U4 vs. U12, U50andU65).

Invivoexperimenl Figure 3 illustrates postprandial changes in ruminal urea and ammonia levels. On the first day of urea feeding, urea was not degrakd qu’.ckly and a large amount of urea still remained at 1 and 1.5 h after feeding, but this lag time in

Fig. 4. Postprandial changes in ruminal density of total bacteria (Trial 2). S Refer to Fig. 2. A wlue in each graph shows a pooled value in the mdividual sampling day. “,b,‘.dValueswith no common superscript differ significantly (P-=0.05 ).A vertical bar represents standard error of mean (n=3).

Y. KOBAYASHIET AL.

186 TABLE 5 Changes in mmen amylolytic and cellulolytic bacterial counts (Trial 2) Sample’

AmylolyIic

C UI u2

2.0

us u17 U38 UbS SE

1.2

2.8 7.6 4.1 1.5 3.1 0.8

(x IO-~ x111-l

)

Cellulolytic

( X IO-‘ml-‘)

1.8’ 0.3b 0.2b I.lab 1.9’b 1.7” I .2’b 0.2

Means of three animals are presented. ‘Samples for inoculation were taken just before morning feed on the sampling day (abbreviations defined in Fig. 2). “~Weans withm column that share no common superscript differ significantly (PcO.05).

degradation gradually disappeared towards Day 12. After Day 12 ureolysis tended to be depressed again and a large amount of urea remained at l-2 h after feeding in sheep on the urea diet for 50 and 65 days. Figure 4 shows changes in total counts of rumen bacteria with the time of urea feeding. The ruminal level of bacteria measured by the direct count method was depressed in the initial period of urea feeding (PC 0.05, C vs. U4 and U8) but was gradually restored. The level was higher in later periods of urea feeding (PxO.05, U4 vs. U65, U8 vs. U17, U38 and U65). Culturable bacterial density showed almost the same trend, i.e. amylolytic and cellulolytic bacteria were decreased in number at the very beginning of Period U (PcO.05, C vs. UI and U2) but soon recovered (Table 5). D’SCUSSION

Our results on N balance (Table 2) again confirmed that long-term feeding of urea gradually improves N retention by sheep, as proposed by previous researchers (Smith et al., 1960; McLaren et al., 1965; Ludwick et al., 1971). The improvement in the present experiment was largely the result of decreased N loss in the urine (Table 2), in good agreement with the observation by Ludwick et al. ( 1971). When linear regression analysis was attempted on the present data, the retention of absorbed N was increased by 3.0% units with each consecutive 10 day feeding period. This value agrees with that of Smith et al. ( 1960). However, it is not apparent how long the improvement continues, because the experiment was ceased before the N balance levelled off. Ludwick et al. ( 197I ) suggest that the improvement lasts up to 30-50 days, white Smith et al. ( 1360) give no clear statement. Assuming from our results that N loss via the urine tends to decline less with time in later periods

(Table 2), the improvement in N retention might be close to the end of Period 5. This adaptation may be related to metabolic regulation by rumen microbes and their host animal. The decreased urinary N could be induced via the mechanism that decreases ruminal ammonia production (Clifford et al., 1968) on the one hand, and increases its assimildion (Caffrey et al., 1967) on the other, ana s&r
McLaren et al. ( 1976) have shown that urea-fed sheep have a higher ability to fix ammonia in their muscles and this elevation can induce the increased N retention of sheep. Evidence in favor of enzymz,tic adaptation in liver and ruminal epithelium to a urea diet has also been obtained (Salem et al., 1973). In the present experiment we did not assess the above possibiiiiied in ilo& tissue adaptation. Such considerations are essential to conclude what factors cause the adaptive increase in N retention and how they interact. The present results, however, demonstrate that rumen microbial responses to the urea diet, decreased ureolysis and increased ammonia assimilation could be partly responsible for the improvement of N balance as a function of time of urea feeding. ACKNOWLEDGMENT’

This work was supported 62760237 (Y.K.) from the The authors ate grateful pan, for their generous gift

in part by a Grant-in-Aid for Scientific Research Ministry ofEducation, Science and Culture, Japan. to Shikishima Starch Co. Ltd.. Silzuka, Mie, Jaof experimental corn starch.

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LONG-TERM EFFECT OF “FE*

ON SHEEP RU.ME-4 RKrERIAL

METInOLtSM

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